Note: Descriptions are shown in the official language in which they were submitted.
CA 02434685 2003-07-04
LMPPZo
Btockwise approach to fragments of the O-specific polysaccharide of Sltigella
Jlexneri
serotype 2a: Convergent synthesis of a decasaccharide representative of a
dimer of the
branched repeating unit'
This paper discloses the synthesis of a decasaccharide corresponding to two
consecutive
repeating units of the 0-Ag of S Jlex~eri 2a, based on the condensation of two
key
pentasaccharide irztermsdiates. Several routes to these two building blocks
were investigated,
involving either blockwise strategies or a lutear one. The latter was the
preferred one based on
yields of condensation and the number of steps.
CA 02434685 2003-07-04
LMPPIO.fheo-bt'e~ct-dacaOMc
Bloclcwise approach to fragments of the O-specific polysaccharide of Shigella
flexneti
serotype Za: Convergent synthesis of a decpsaccharide representative of a
dimer of the
branched repeating unite
ABSTRACT
Introduction
i
Shigellosis or bacillary dysentery is a worldwide disease, occurring in humans
only, caused by
organisms of the genus Shigella. Responsible for an estimated 200 million
cases annually.
Shigella is increasingly resistant to antimicrobial drugs, ZShigellosis is a
priority target as defined
by the World Health Organization since this disease is a major cause of
mortality in developing
countries: especially among children under S years of age and in the
immunoeotnpromised
population. 3Although no vaccine is yet available against shigellosis, several
programs targeting
the eradi6ation of this bacterial infection are under development, with
emphasis on vaccination
strategies involving either live attenuated strains of Shigella4 or acellular
vaccines based on
lipopolysa.ccharide (LPS) antigens and derivatives thereof. SOf particular
interest in the later
approach is the design of glycoconjugate vaccines based on the use of
detoxified LPS. Indeed,
there is evidence that natural and experimental infections with Shigella
confer type-specific
immunityb which points to the O-specific polysaccharide (O-SP) moiety of the
LPS as the target
antigen of the host's protective immune response to infection. Besides, data
show that significant
levels of pre-existing antibodies specific for the O-SF corzelate with a
diminished attack rate of
shigellosis.' Furthermore, it was recently demonstrated in field trials that
protein conjugates of
i
1
CA 02434685 2003-07-04
LMPP10-then-~evet~ceaOMe
detoxified ~PS provided protection to human volunteers against infections
caused by S: sonnei.g
As was particularly emphasized in the case of S rlysenteriae type l,
conjugates incorporating
oIigosa~eeh~rid.e fragments of the native bacterial polysaccharides may be
even more
immunoge~ic than their counterparts made of the detoxified LPS.9
Of most concern amongst the different species of Shigella, is S flexneri
serotype 2a, the
prevalent '~fective agent responsible for the endemic form ofthe
disease.t° Indeed, major efforts
from diffqrent laboratories including the development of conventional
polysaccharide-protein
conjugates,l' aim at the development of a vaccine against the disease
associated with this
particular I serotype. In parallel, a program aimed at the design of
chemically defined
glycoconj~gate vaccines based on the use of synthetic fragments of the O-SP of
S flexneri 2a, is
under development in this laboratory. We adopted a rational approach,
involving a preliminary
study of the interaction between the bacterial O-SP and homologous protective
monoclonal
antibodies.
A B E C D
2)-a-L-~hap-(1--~2)-a-L-Rhap-(la3)-[a-D-Glcp-(1-~4)]-a-L-Rhap-(1 >3)-[i-D-
GIcNAcp(1-~~
The 0-Sl~ of S fIexneri 2a is a heteropolysa.ccharide defined by the
pentasaccharide repeating
unit Liiv~ It features a linear tetrasaccharide backbone, which is comtt~n to
alI S flexneri 0-
antigens end comprises a N acetyl glucosannine and three rhamnose residues,
together with an a-
D-glucopyranose residue branched at position 4 of one of the rhamnoses.
Besides the known
methyl g~ycoside of the EC disaccharide;t~,ls a set of di- to
pentasaccharidesl6'Lg and more
recently Vin. octasaccharidel9 representative of fragments of S. flexneri 2a 0-
SP have been
synthesi2~d recently. The use of these compounds as molecular probes for
mapping at the
molecular level the binding characteristics of a set of protective antibodies
against S flexneri 2a
infection~indicated that access to larger oligosaccharides would help the
characterization of the
carbohydrate antigenic determinants. For this purpose, methodologies allowing
a straightforward
access to!5,~1'exneri 2a oligosaccharides of larger size are under study in
this laboratory. We now
report the synthesis of the first decasaccharide in the series, namely the
D'A'B'(E')C'DtI.B(E)C
fragment; which was prepared as its methyl glycoside (1).
z
CA 02434685 2003-07-04
LMPP10-theo~Etcyct~decnoMe
Re9ults and discussion
Considerir4g its dimeric nature, a convergent synthetic strategy to the target
1 was considered.
Indeed, re'trosynthetic analysis, supported by previous work in the feld,~9''Z
indicated that
disconnections at the C-D linkage, thus based on two DAB(E)C branched
pentasaccharides
corresponding to a frame-shifted repeating unit I, would be the most
advantageous (Scheme 1).
Such a strategy would involve a pentasaccharide acceptor easily derived from
the known methyl
glycoside ZI' or from the corresponding N-acetylated analogue 3 and a
pentasaccharide donor
bearing a ~-0-acyl protecting group at the reducing residue (C) in order to
direct glycosylation
towards the desired stereoehemistry. Depending on the nature of the 2-N acyl
group in residue D,
the latter could derive from the allyl glycosides 4 or 5. Besides, bearing in
mind that the major
drawbacks of the linear synthesis of pentasaccharide Z reported so far" dealt
with the selective
deblocking of key hydroxyl groups to allow further chain elongation, we
describe herein various
attempts at a convergent synthesis of the fully protected DAB(E)C
pentasaccharide as its methyl
(2, 3) or allyl (4, 5) glycosides- Precedents concerning a related serotype of
S. flexneri have
indicated that disconnection at the D-A linkage should be avoided. zt.zZ
However to our
knowledge, disconnection at the B-C or A-)i3 linkages was never attempted in
the series. Both
routes were considered in the :following study. The nature of the repeating
unit I indicated that
any blockwise synthesis involving such linkages would rely on donors lacking
any participating
group at position 2 of the reducing residue, thus the relevance of t's
strategy may be questioned.
Nevertheless, although (3-glycoside formation was observed occasionally,'3 the
good a-
sterooselectivity reported on several occasions in the literature for
glycosylation reactions based
on mannopyranosyl'~~'3 and derivatives such as perosaminylz6~' fef KOVACI or
rhamnopyranosyl donors that were either glycosylated at C-2,Zg or blocked at
this position with a
non partidipating group,~9 encouraged the evaluation of the above mentioned
block strategies. To
follow up:the work developed thus far in the S. Jlexneri 2a series, emphasis
was placed on the use
of the use of trichloroaeetimidate (TCA) chemistry,3o
Stt~ategy based on the disconnection at the A-B linkage (Scheme l, rouge a):
Such 3 strategy
involves the coupling of suitable DA donors to an appropriate B(E)C acceptor.
Takzng into
account the glycosylation chemistry, two sets of disaccharide building blocks
(6, 7, 8), easily
obtained 'from known monosaccharide precursors which were readily available by
standard
3
CA 02434685 2003-07-04
LMPP1~-then-tirevet-deceOMc
As shown previously in the construction of the DA intermediate 17, the 7~~
ttichloroacetyl
trichloroacetimidate 16 appears to be a highly suitable prECUrsor to residue D
when involved in
the formation of the ~i-GIcNAc Linkage at the poorly reactive 2,~ position.
Indeed, reaction of 16
with the acceptor 24 in 1,2-dichloroethane in the presence of TMSOTf went
smoothly and gave
the trisaccharide 26 in 96% yield, However, conversion of the N-
trichloroacetyl gxoup to the N
acetyl derivative 27 was rather Less successful as the desired trisaccharide
was obtained in only
42 % yield when treated under conditions that had previously been used in the
case of a related
oligosaccharide (sodium methoxide, ht3N, followed by re-N, 0-acetylation).t~
This result led us to
reconsider the protection pattern of the glucosamine donor. The N
tetrachlorophthalimide gzoup
has been proposed as an alternative to overcome problems associated with the
widely spread
phthalimic~o procedure when introducing a 2-acetamido-2,deoxy-ji-D-
glucopyranosidic linkage.°'
Thus, the IV tetrachlorophthalimide trichloroacetimidate donor 25 was selected
as an alternative.
It was prepared as described from commercially available D-
glucosamine,°2 apart from in the
final imidate formation step, where we found the use of potassium carbonate as
base to be more
satisfactory than DBU. GlyeosyIation of 24 with 25 in the presence of TMSOTf
resulted itt the
trisaccharide 28 in 65% yield. The tetrachlorophthaloyl group was then removed
by the action of
ethylenediamine, and subsequent re-A' 0-aeetylation gave the trisaccharide 27
in 6S% yield. The
latter was next converted into the donor 13 in two steps, analogous to those
described for the
preparation of 6 from 17. Indeed, de-O-allylation of 27 cleanly gave the
hemiacetal 29 (83°/),
which was then activated into the required trichIoroacetimidate (94%), It is
worth mentioning that
although they involve a different D precursor, both strategies give access to
the intermediate 2'7
in closely related yields, 40 and 42%, respectively.
Initial attempts to form the pentasaccharide 5 from 13 and the previously
described acceptor llis
in the presence of TMSOTf as promoter were rather unsuccessful, resulting in
at best 17% of the
desired product, accompanied by decomposition of the donor into the hemiacetal
29 (75%).
Using BF3.OEt2 as the promoter in place of TMSOTf, reaction of l I with 13 at
mom temperature
provided 5 in 44% yield, with the acceptor 11 and hemiacetal 29 also recovered
in 54 and 29'/°
yield, res~ectiveIy. We considered that the poor reactivity of the acceptor
was responsible for
these results, as the "C NMR of 13, showing several distorted signals (notably
???), suggests
that there is considerable steric hindrance around the position 3c. For that
matter, the 2~-0-
acetylated disaccharide I2 was considered as an alternate aceepfor.
Analogously to the
6
CA 02434685 2003-07-04
L.~tPPI O-thco-brevet-decaOMe
preparation of 11, it was obtained from the known diol 30 through
regioscIective opening of the
intermediate orthoester. However, coupling of the potentially less hindered
acceptor 12 and the
trisaccharitle donor 13 resulted, at best, in the isolation of the
condensation product 31 in 42%
yield (not described).
The modest yield of 1 obtained by this route made the alteznative reaction
path (Scheme 4) worth
investigating, despite the mere numerous synthetic steps required. Indeed, it
was found rather
appealing twhen eva.l.uated independently in a closely related series
(unpublished results). By this
route, a tetrasaccharidc acceptor can be formed from two disaccharide building
blocks (EC and
AB), and coupled with an appropriate monosaccharide donor as precursor to D.
Considering that
selective deprotection of the ~A hydroxyl group would occur in the course of
the synthesis,
glycosylation attempts were limited to the 2-D-benzoylated acceptor 11. The
disaccharide donor
necessary for this path could be derived from the building block 24, already
in hand. The choice
of temporary protecting group at position 2A was deternined by our experience
of the stepwise
synthesis of the corresponding methyl pentasaccharide," where we noted that an
acetate group at
this position may not be fully orthogonal to the benzoate located at position
20. The chosen group
had also to support removal of the anomeric allyl group and the subsequent
conversion to the
trichloroaeetimidate. At first, a chloroacetate group was anticipated to
fulfil these requirements.
Thus, the disaccharide 24 was treated with chloroacetio anhydride and pyridine
to give the
derivative 32 (57%). Anomeric deprotection to give the hemiacetal 33 (B4%) and
subsequent
trichloroacetimidate activation of the latter into the donor 34 (83%) were
performed in the same
way as before. Coupling of 11 with 34, carried out in the presence of TMSOTf
at -40°C, yielded
a complex mixture of products. When the temperature was lowered to -
60°C, the condensation
product 3$ could be isolated in Z2% yield. The a-stereoselectivity of the
glycosylation was
ascertained from the value of the 'Jc,H coupling constant at C-1$ which was XX
Hz.43''~(A
faire ?) Alternative donor protection was attempted. Treatrr~nt of 24 with p-
methoxybenryl
chloride and sodium hydride gave the fully protected derivative 35 (97%),
which was cleanly
converted into the trichloroacetimidate donor 37 (82%) in two steps involving
the hemiacetal
intermediate 36 (73%). GIycosylation of 1I with 37 in the presence of TMSOTf
at -40°C gave
the desired tetrasaccharide 39 in 44% yield. Again, the s~tereochemistry of
the newly created
linkage vvas ascertained based on the IJc,H heteronuelear coupling constants.
When the
temperature was lowered to -GO°C, the yield of 39 fell to 34% anti a
second major product 40
7
CA 02434685 2003-07-04
LMPPlO-theo-brevet-dccaOMe
(21%) was observed in the mixture. Indeed. examination of the NMR spectra of
this product
revealed that the pMeOBn group had been lost. That 40 was the acceptor
required for the next
step brought the. estimated yield of condensation to 55%. Nevertheless, the
overall outcome of
this blockwise strategy did not match our ea-pectations, and this route was
abandoned.
Linear strategy to the fully protected pentasaccharide d.~ As preliminary
studies have
demonstrated, rapid access to suitable building blocks allowing the synthesis
of higher-order
oligosaccharides representative of fragments of the 0-SP of S. flex~eri 2a
remains a challenge.
Major conclusions drawn from our studies favour the design of a linear
synthesis of the target 4.
Indeed, when put together with our previous work, such as the synthesis of
tetrasaccharide 41
(95%)" or that of trisaccharide 42 (97%),~e all the above described attempted
couplings outlined
the loss of e~ciency of glycosylation reactions involving rhamnopyranosyl
donors glyeosylated
at position 2 in comparison to those involving the corresponding acetylated
donor. Thus,
matching the linear strategy of the methyl pentasaceharide 2 described
previously,t' a synthesis
of 4, based on donors bearing a participating group at 0-2, was designed.
Three key building
blocks were selected. These were the readily accessible )EC disaccharide
acceptor 11 benzoylated
at C-2 as required for the final condensation step leading to the fully
protected decasaccharide
intermediate; the rhamnopyranosyl trichloroacetimidate 22, which serves as a
precursor to
residues ~1 and B, and bears a both temporary and participating group at
position 2; and the
trichloroacetamide glueosatninyl donor 16 as a precursor to residue D. As
stated above, coupling
of 11 and 22 gave 42 in high yield. As observed in the methyl glycoside
series,l' de-D
acetylation using MeONa or methanolic HCl was poorly selective. Although,
guanidinelguanidinium nitrate was proposed as a mild and selective O-
deacetylation reagent
compatible with the presence of benzoyl protecting groups,'S none of the
conditions tested
prevented partial debenzoylation leading to diol 43, as confirmed from mass
spectroscopy and
NMR analysis (ct Aone-Laure). The required alcohol 10 was readily obtained in
an acceptable
yield of 84% yield by a five-day acid catalysed methanolysis, using HBFs in
diethyl
ether/tnet'hanol,l'~'6 of the fully protected intermediate 42 (Scheme 5).
Repeating this two-step
process using 10 as the acceptor and 22 as the donor resulted first in the
intermediate 44 (90%),
and next ~in the tetrasaccharide acceptor d0 (84%). Glycosylation of the
latter with 16 gave the
fully protected pentasaccharide 4 in high yield (98%), thus confirming that
the combinatuon of the
8
CA 02434685 2003-07-04
LMPP l0-then-brevet~decaOMc
trichloroachtamide participating group and the trichloroacetimidate activation
mode in 16 results
in a potent donor to be used as a precursor to residue D in the S flexnert
series, where low-
reactive glycosyl acceptors are concerned. Following the above described
procedure, selective
anomeric deprotection of 4 furnished the hemiacetal 45 wluch was smoothly
converted to the
trichloroacetimidate donor 46 (66% from 4). From these data, the linear
synthesis of 4, truly
benefiting from the use of 2z as a common precursor to residue A and B,
appears as a reasonable
alterriative.to the block syntheses which were evaluated in parallel.
Synthesfr of the targel decasacchnride l: Having a peniasaccharide donor in
hand, focus was
next placed on the synthesis of an appropriate pentasaccharide acceptor. In
our recent descxiption
of the convergent synthesis of the B'(E')C'DAB(E)C oetasaccharide,'9 the
pentasaccharide 48,
bearing a 4p,6p-O-isopropylidene protecting group, was found a most convenient
acceptor which
encouraged its selection in the present work. Briefly, 48 was prepared in two
steps from the
known Z. Thus, mild transesterification of 2 under Zemplen conditions allowed
the selective
removal of the acetyl groups to give triol 47, which was converted to the
required acceptor 48
(72% from 2) upon subsequent treatment with 2,2-dimethoxypropane. Relying on
previous
optimisation of the glycosylation step, i9 the condensation of 48 and 46 was
performed in the
presence of a catalytic amount of triftic acid. However, probably due to the
closely related nature
of the donor and acceptor, the reaction resulted in an inseparable mixture of
the fully protected 49
and the hemiacetal 4S resulting from partial hydrolysis of the donor. Most
conveniently, acidic
hydrolysis of the mixture, allowing the selective removal of the
isopropylidene group in 49, gave
the intermediate diol 50 in a satisfactory yield of 72% for the two steps.
According to the
deprotectlon strategy used for the preparation of the closely related
octasaccharide,t9 diol SO was
engaged in a controlled de-O-acylation process upon treatment with hot
methanolic sodium
methoxide. However, partial cleavage of the trichloroacetyl moiety, leading to
an inseparable
mixture, was observed which prevented further use of this strategy. Indeed, it
was assumed that
besides being isolated and therefore resistant to Zempl~n transacetylation
conditions,a~~9 the 2c-
O-benzoyl groups were most probably highly hindered which contributed to their
slow
deprotection. Alternatively, 50 was submitted to an efficient two-step in-
house process involving
fizst, hydrogenolysis under acidic conditions which allowed the removal of the
benzyl groups and
second, basic hydrochlorination which resulted in the conversion of the N
trichloroacetyl groups
9
CA 02434685 2003-07-04
LMPP10-tl~eo-brc~~et-deeaoMc
into the required N acetyl ones, thus affording 51. Subsequent
transESterification gave the final
target l in 37% yield from S0.
CONCLUSION
The decasaccharide 1, corresponding to two consecutive repeating units of the
O-Ag of S.
Jlexneri 2a was synthesized successfully based on the condensation of two key
pentasaccharide
intermediates, the donor 46 and acceptor 48. Several routes to those two
building blocks were
investigated, involving either blockwise strategies or a linear one. The
latter was the preferred
one based on yields of condensation and the number of steps.
ACKNOWL,EDt~EMENTS
The authors thank Pr. P.7. Sansonetti who is a scholar of the Howard Hughes
Medical Institute
fior his key input in the project. The authors are grateful to J. Ughetto-
Monfrin (unite de Chimie
Organique, Institut Pasteur) for retarding all the NMR spectra. The authors
thank the CANAi.'~I
and the Fondation pour la Recherche Medicate (predoctoral fellowship to C.
C.), the Bourses Mrs
Frank Howard Foundation for its postdoctoral fellowship to K. W. and fnancial
support, as well
as the Bourses Roux foundation (postdoctoral fellowship to F. B.).
CA 02434685 2003-07-04
LMPPIo~theo-6~wd-deca0~~te
(1) ~ Alme Part 10 of the series Synthesis of ligands related to the O-specif
c
palysacchdrides of Shigella flexneri serotype Za and Shigella flexneri
serotype 5a. For part 9, see
ref. .~C.
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11
CA 02434685 2003-07-04
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CA 02434685 2003-07-04
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(43) Bock, K.; Pedersen, C, Acta Chem. Stand Ser. B 1975, 29, 258-264.
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(45) Ellervih, U.; Magnusson, G. Tetrahedron Lett. 1997, 38, 1627-1628.
(46) Pozsgay, V.; Coxon, B. Carbohydr. Res. 1994, 257, 189-215.
(47) Liptak, A.; Szurrnai, Z.; Nanasi, P.; Neszmelyi> A. Carboltydr, Res.
1982, 99.
(48) Szurrnai, Z.; Liptak, A.; Snatzke, G. Carbohydt~. Res. 1990, 200, 201-
208.
(49) Szurmai, Z.; Ker~kgyarto, J.; Harangi, J.; Liptak, A. Carbohydr. Res.
1987, 174,
313-325.
13
LMPPtUa~pbrcvctdeceOhlr
General rriethods
CA 02434685 2003-07-04
Optical rotations were measured for CHCh solutions at 25°C, expect
where indicated
otherwise; with a Perkin-Elmer automatic polarimeter, Model 241 MC. TLC were
performed
on precoated slides of Silica Gel 60 F25~ (Merck). Detection was effected when
applicable,
with UV light, andlor by charring in 5% sulfuric acid in ethanol.
Preparative chromatography was performed by Elution from columns of Silica Gel
60 (particle
size 0.040-0.063 mm). For all compounds the NMR spectra were recorded at
25°C for
solutions in CDCl3, on a Broker AVI 400 spectometer (400 MHz for 'H, 100 MHz
for "C).
External references : for solutions in CDCIa, TMS (0.00 ppm for both 'H and
"C). Proton-
signal assignements were made by first-order analysis of the spectra, as well
as analysis of 2D
'H-'H correlation maps (COSY) and selective TOCSY experiments. Of the two
magnetically
non-equivalent geminal protons at C-6, the one resonating at lower field is
denoted H-6a and
the one at higher field is denoted H-6b. The "C NMR assignments were supported
by 2D'3C-
'H correlations maps (HETCOR). Interchangeable assignments are marked with an
asterisk in
the listing of signal assignments. Sugar residues in oligosaccharides are
serially lettered
according to the lettering of the repeating unit of the O-SP and identified by
a subscript in the
fisting of'signal assignments. Fast atom bombardment mass spectra (FAB-MS)
were recorded
in the positive-ion mode using dithioerythridol/dithio-L-threitol (4 :I, MB)
as the matrix, in the
presence of NaI, and Xenon as the gas. Anhydrous DCM, I,2-DCE and Et20, sold
on
molecular sieves were used as such 4 ~ powder molecular sieves was kept at
100°C and
activated before use by pumping under heating at 250°C.
Phenyl (3,4,6-tri.O-acetyl-2-deoxy-2-trichloroaeetaraido-/3-n-gtucopyranosyt)-
(la2)-
(3,4-di~O~benzyt-1-thin-a-trrhamnopyranoside) (8). A mixture of alcohol 15
(0.12 g, 0.27
mmol) and imidate 16 (0.245 g, 0.41 mmol) in anhydrous DCM ( 10 mL) was
stirred for 1 S
min under dry ar. After cooling at 0°C, Me3Si0Tf (28 p,L) was added
dropwise and the
mixture was stirred for 0.5 h. Triethylamine (60 pL) was added anrl the
mixture was
concentrated. The residue was eluted from a column of silica gel with 4:1
cyclohexane-EtOAc
to give 8 (227 mg, 97 %) as a colorless foam; [aJD -G3' (c 1; CHCl3). 1H NMR
(CDCl3): o
7.10-7.40 (m, 15H, Ph}, &.73 (d, 1H, J2,~.,= 8.5 Hz, NHb), 5.47 (d, 1H, J,,2 =
1.2 Hz, H-1~),
1
t~t~pioG~Ptrev~.e~or,~
CA 02434685 2003-07-04
5.07 (dd, 1H, Jz,~ = J,,4 = 10.0 Ha., H-3n), 4.99 {dd, 1H, J~,S = 10.0 Hz, H-
4n), 4.80-4.SS (m
4H, CH,Ph), 4.52 (d, 1H, J~,Z = 8.2 H2, H-lp), 4.13-3.95 (m, 2H, Js,s = 5.3
Hz, J6,,5~ = 12.2
Ha., H-GaD, 6ba), 4.I0 {m, IH, J~,S = 9.5 H~., Js,b = G.1 Hz, H-Sb), 4.00 (dd,
IH, Jz,3 = 3.0 Hz,
H-2A), 3.99 (m, IH, H-2D), 3.77 (dd, IH, J3,a = 9.4 Hz, H-3A), 3.50 (m, IH, H-
Sp), 3.39 (dd,
1H, H-4,;), 1.90, 1.93, 1.95 (3s, 9H, OAc), 1.23 (d, 3H, H-6A). '3C NMR
(CDCl3):8 1'11.1,
170.9, 169.6, 162.1 (C=0), 127-138 {Ph), 102.1 (C-1D), 92.7 (CCl3), 87.4 (C-
lA), 81.3 (C-
4A); 80.5 {C-3A), 79.I (C-2,~), 76.4, 74.1 (CH2Ph?, 72.4 (C-SD), 72.4 (C-3p),
69.8 (C-5"), 68.?
(C-4o), G2.3 {G-6n), SG.2 (C-2D); 21.0, 20.9, 20.8 (3C, OAc), 18.1 {C-6n).
FARMS of
CAOH4~C13N0iZS (M, 867), m1z 890 ([M+Na]"). Anal. Calcd for C~oH4aC~N0,zS : C,
55.27 ;
H,S.lO;N, 1.61. FoundC,55.1G;H,5.18;N, 1.68.
Allyl (3,4,6-tri-p-acetyl-2-deoay-Z-trichloroacetamido-ø-D-gtucopyraoosyl)-(1--
~Z)-(3,4-
di-O-benzyl-a-trrhamnopyranoside) (17). A mixture of alcohol 14 (1.86 g, 4.86
moral) and
imidate 16 (3.85 g, G.47 mmol) in anhydrous CH3CN (80 mL) was stirred for 15
min under dry
Ar. After cooling at 0°C, Me3Si0Tf (46 uL) was added dropwise and the
mixture was stirred
for 0.5 h. Triethylamine {150 uL) was added and tile mixture was concentrated.
The residue
was eluted from a column of silica gel with 7:3 eyclohexane-EtOAe to give 17
(4.0 g, 99 %) as
a white solid; [a]n -3° (e 1, CHC13). 1H NMR (CDC)3):b 7.18-7.32 (m,
IOH, Ph), 6.70 (d, 1H,
Ji,~ = 8.4 Hz, NHD); 5.78-5.82 (m, 1H, All), 5.05-5.20 (m, 2H, All), 5.00 (tn,
2H, JZ,3 = J~., _
Ja.$ = 9.5 Hz, H-3D, 40), 4.45-4.75 (m, 4H, CH,Ph), 4.76 (d, IH, J,,~ = 1.1
Hz, H-1~), 4.60 (d,
1H, J~,z = 8.5 Hz, H-lo); 4.05-4.15 (m, 2H, J5,6 = 4.8 Hz, Jba.6b = I2.2 Hz, H-
6ao, 6bD), 3.98
(m, I H, H-2o), 3.90 (m, 2H, Alt), 3.86 (dd, 1 H, JZ., = 3.2 Hz, H-2A), 3.81
(dd, 1 H, J3,4 = 9.5
Hz, H-3A), 3.G2 (m, 1H, J4.5 = 9.5 Hz, Js,s = G.1 Hz, H-5,,), 3.50 (m, 1H, H-
SD), 3.32 (dd, 1H,
H-4,,), 1.93, 1.97, 2.02 (3 s, 9H, OAc), 1.24 (d, 3H, H-6A). "C NMR (CDC13):b
171.0, 170.9,
169.6, I62.I (C=O), 117.1-138.5 {Ph, All), 101.8 (C-lo), 98.5 (C-1"), 92.6
(CCl,), 8I.4 (C-
4A), 80.4 (C-3~), 7?.I (C-2,,), 75.9, 74.1 (CHZPh), 72.7 {C-3p), 72.5 (C-Sp),
GB.G (C-4D), 68.3
(C-5~), 68.1 (All), G2.3 (C-6n), SG.1 (C-2n), 2i.1, 20.9, Z0.9 (OAcj, 18.2 (C-
6A). FABMS of
C~,Ha,CI,NOj; {M, 815), m/a 838 ([VI+Na]"). Anal. Calcd for C37H,sdCI3NO~3: C,
54.39 ; H,
5.43 ; N, 1.71. Found C, 54.29 ; H, 5.45 ; N: 1.72.
(3,4,6~tri-p-acet3rl-Z-deoxy-2-trichloroacetamido-[i-D-glucopyranoFyl)-(1->2)-
(3,4-di-O-
bearyl-a-t..rhamnopyranose) (18). 1,5-Cyclooctadiene-
bis(methyldiphenylphosphine)iridium
2
LMPPtObdrbrcYctti°_noMe
CA 02434685 2003-07-04
hexafluorophosphate (120 mg, 140 umol) was dissolved tetzahydrofuran (10 mL),
and the
resulting red solution was degassed in an argon stream. Hydrogen was then
bubbled through
the solution, causing the colour to change to yellow. The solution was then
degassed again in
an argon stzeam. A solution of 17 (1.46 g, 1.75 mmol) in tetrahydrofuran (20
mL) was
degassed and added. The mixture was stirred at rt overnight. The mixture was
concentrated.
The residue was taken up in acetone (27 mL.), and water (3 mL) was added.
Mercuric bromide
(949 mg, 2.63 mmol) and mercuric oxide (761 mg. 3.5 mmol) were added to the
mixture,
protected from light. The mixture was stizxed For 2 h at rt, then
concentrated. The residue was
taken up in CHzCIz and washed three times with sat. aq. KI, then with brine.
The organic phase
was dried and concentrated. The residue was purified by column chromatography
(Cyclohexane-AcOEt 4:I) to give 18 (1.13 g, 81 %) as a white foam. [a]n
+4° (c 1. CHCl3),
1H NMR (CDCl3):8 7.05-7.35 (m, 10H, Ph), 6.74 (d, 1H, J2,~ = 8.5 Hz, NHn),
5.10 (d, 1H,
J,,z = 1.1 Hz, H-1,,), 5.02 (m, 2H, Jz,~ = J3,4 = J,,,s = 9.5 Hz, H-3p, 4D),
4.50-4.80 (m, 4H,
CHzPh), 4.61 (d, IH, Jl,i = 8.5 Hz, H-lp), 4.08-4.15 (m., 2H, Js,b = 4.5 Hz,
Jsa,6b = 12.3 Hz, H-
6an, Gbn), 4.00 (m, 1H, H-2p), 3.90 (dd, 1H. Ja.s = 3.3 Hz, H-2~), 3.86 (dd,
1H, J,,a = 9.5 Hz,
H-3,,), 3.85 (m, 1H, J4_5 = 9.5 HZ, Js,~ = 6.2 Hz, H-5~), 3.50 (m, 1H, H-5n),
3.30 (dd, IH, H-
4A), 2.85 (d, 1H, J ,.oH = 3.5 Hz, OH), 1.94, 1.97, 2.02 (3s, 9H, OAc), I.23
(d, 3H, H-GA). "C
NMR (CDCl3):8 171.1, 170.0, 169.6, 162.1 (C=O), 127.1-138.5 (Ph), 101.7 (C-
In), 94.I (C-
lA), 92.6 (CCL), 81.4 (C-4A), 79.9 (C-2A), 77.3 (C-3"), 75.9, 74.1 (CHzPh),
72.7 (C-3p), 72.5
(C-Sn), 68.6 (C-4n), 68.4 (C-5~), 62.2 (C-6n), SG.I (C-2D), 21.1, 21.0, 20,9
(OAc), 18.3 (C-
6A), FABMS Of C3dHsOCI~NO,3 {VI, 775), m1z 789 ([M~-Na]+) ; Anal. Calcd for
C,4H~oC~N0,3: C, 52.55 ; H, 5.19 ; N, 1.80. Found G, 52.48 ; H, 5.37 ; N,
1.67.
(3,4,6-tti-O-acetyl-2-deoYy-2-trichloroaeetamido-[3-n-glucopyrsnosyl}-(I-~2}-
3,4.di-O-
benzyl-a-r,-rhamnopyranose trichloroacetimidate (6). The hemiacetal 18 (539
mg, 0.68
mmol) was dissolved in CHzCIz (50 mL), placed under azgon and cooled to
0°C.
Trichloroacetonitrile (0.6 mT..., 6.8 rnmol), then DBU (10 ~tL, 70 p.mol) were
added. The
mixtuze~ was stirred at 0°C for 30 mm. The mixture was concentrated and
toluene was co-
evaporated from the residue. The residue was eluted from a column of silica
gel with 7:3
eyclohekane-EtOAC and 0.2 % of Et3N to give 6 (498 mg, 78 %) as a colourless
foam; [a]D -
18° (e 1, CHCl3). iH NMR (CDCI3):8 8.48 (s, IH, N=H), 7,15-7.40 (tn,
lOH, Ph), 6.75 (d,
IH, JZ.NH = 8.5 Hz, NHo), 6.18 (d, 1H, Jl,z = 1.1 Hz, H-1~), 5.15 (dd, 1H,
Jz., = J3.a = 9.5 Hz,
3
Lt~FI Oexpbrevecaecav~~.~
CA 02434685 2003-07-04
H-3D), 5.07 (dd, IH, J4,s = 9.5 Hz, H-4p), 4.50-4.82 (n~, 4H, CI~zPh), 4.62
(d, 1H, J~,z = 8.5
Hz, H-ln), 4.03-4.20 (m, 2H, J5,6 = 4.5 Hz, Jsy,sb = 12.3 H~~, H-6aD, 6ba),
3.98 (m, 1H, H-2D),
3.85 (m, 1H, Ja,s = 9.5 Hz, J5,5 = 6.2 H~, H~5A), 3.84 (dd, IH, Jz,3 = 3.3 Hz,
H-2"), 3.83 (dd,
IH, J~,A = 9.5 Hz, H-3,,), 3.55 (m, 1H, H-Sp), 3.45 (dd, 1H, H-4A), 1.93,
1.96, 1.98 (3s, 9H,
OAc), 1.23 (d, 3H, H-6A). 13C NMR (CDC13):8 171.1, 170.0, 169.6, I62.I (C=Oj,
I27.2-
138.4 (Phj, 101.7 (C-lo), 97.2 (C-lA), 92,6 (CCI~), 80.5 (C-4A), 79.I (C-3A),
76.2 (C-2x),
7G.2, 74.1 (CHzPh), 74.4 (C-3n), 74.1 (C-Sn), 71.3 (C-5A), 68.6 (C-4D), 62.3
(C-6D), 56.3 (C-
2nj, 21.1, 21.0, 20.9 (3C, OAc), 18.2 (C-6A). Anat. Calcd for
C36I3,,oCI6NZO13: C, 46.93 ; H,
4.38 ; N, 3.04. Found C, 46.93 ; H, 4.52 ; N, 2.85.
Al~y1 (2-acetamido-3,4,5-tri-O-acetyl-Z-deory-~3-n-glucopyranosyl)-(1-~2)-(3,4-
di-0-
bcnryl-a;-1.-rhamnopyranoside) (19). A mixture ofthe protected disaccharide 17
(3.0 g, 3.61
mmol) in MeOH {50 mL) was cold to 0°C and treated by NHS gaz overnight.
The solution was
concentrated and the residue (2.02 g) was dissolved again in MeOH (50 mI,) and
treated by
AczO (3.98 mL,, 3G.1 mol). The solution was stirred for 2 h and then
concentrated. The residue
was eluted from a column of silica gel with 95:5 DCM-EtOAC to give the
intermediate trial
which was dissoh~ed in Pyridine (5 mL), cold to 0°C and treated by AciO
(2.4 mL). The
mixture was stirred overnight and concentrated. The residue was eluted from a
column of silica
gel with3:2 cyclohexane-fitOAC to give I9 (2.3 g, 90 %) was obtained as a
colourless foam [
a]p -12° (c 1. CHCI;). ~H NMR (CDC13):d 7.18-7.32 (m, lOH, Ph), 5.70-
5.80 (m, 1H, All),
5.40 (d, 1H, J2,~ = 8.1 Hz, NH), 5.10-5.20 (m, ZH, All), 4.9G (dd, 1H, J,,, =
J4,~ = 9.5 Hz, H-
4D), 4.90 (dd, 1H, Jz,3 = 9.5 Hz, H-3p), 4.52-4.76 (m, 4H, CHzPh), 4.80 (d,
1H, J,,z = 1.2 Hz,
H-I,,), 4.46 (d, 1H, Ji,2 = 8.5 Hz, H-In), 4.OZ-4.I0 (m, 2H, Js,b = 4.7 Hz,
J~a,6b = I I.2 Hz, H-
6aD, Gbo), 3.92 (m, 1H, H-2D), 3.87 (m, 2H, All), 3.86 (dd, IH, Jz,s = 3.5 Hz,
H-2A), 3.82 (dd,
IH, J3,a = 9.5 Hz. H-3n), 3.62 (m, 1H, Ja,s = 9.5 Hz, Js,s = 6.2 Hz, H-5A),
3.52 (m, 1H, H-5o),
3.30 (dd, 1H, H-4A), 1.92, I.94, 1.98 (3 s, 9H, OAc), 1.26 (d, 3H, H-G~). ~'C
NMR (CDC13):8
171.1, 171.0, 170.3, 169.6 (C=O), I 17-138 (Ph, All), 103.4 (C-1D), 98.5 (C-
lA), 8I.3 (C-4A),
80.4 (C-3A), 78.5 (C-2,,), 75.9, 73.9 (CHzPh), 73.6 (C-3n), 72.4 (C-5p), 68.7
(C-4p), 68.2 (C-
5,~, b8.1 (All), 62.5 (C-6p), 54.5 (C-2D), 23.4 (AcNH), 21.2, 21.1, 21.0
(OAc), 18.1 (C-G0.).
FABMS of C3,Ha,N0,3 (M, 713.3), rrt~'~ 736.2 ([M+Na]') Anal. Ca>cd for
Ca~He,N0~3: C,
62.26 ; H, 6.G4 ; N, 1.96. Found C, d2.I2 ; H, G.79 ; N, 1.87.
4
CA 02434685 2003-07-04
(2-acetam ido-3,4,d-tri-0-acetyl-2-deoay-[i-n-glucopyranosy 1)-(1-~2)-(3,4-di-
O-benryl-a-
L-rhamnopyranose) (20). 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridium
hexafluorophosphate (10 mg, 12 ~mol) was dissolved tetrahydrofuran (10 mL),
and the
resulting red solution was degassed in an argon stream. Hydrogen was then
bubbled through
the solution, causing the colour to change to yellow. The solution was then
degassed again in
an argon stream A solution of 19 (830 mg, 1.16 mmol) in tetrahydrofuran (40
mL) was
degassed and added. The mixture was stirred at rt overnight. The mixture was
concentrated.
The residua was taken up in acetone (90 mL), and water (10 mL) was added.
Mercuric
chloride (475 mg; 1.75 mmol) and mercuric oxide (504 mg, 2.32 mmnl) were added
to the
mixture, protected 'from light. The mixture was stirred for 2 h at rt, then
concentrated. The
residue was taken up in CHZCI? and washed three times W th sat. a.q. KI, then
with brine. The
organic phase was dried and concentrated. The residue was purified by column
chromatography (Cyclohexane-AcOEt 3:7) to give 20 (541 mg, 69 %) as a white
foam; [aJp
+16° (c 1, CHCl3).~H NMR (CDC)3):cS 7.05-7.35 (m, IOH, Ph), 5.50 (d,
1H, Jz,rrEt = 8.2 Hz,
NHD), 5.22 (d, IH, Ji,z = 1.1 Hz, H-lA), 5.06 (dd, 1H, J3,4 = Ja,s = 9.5 Hz, H-
4D), 5.00 (dd,
1H, Jz,~ ~ 9.5 Hz, H-3D), 4.60-4.85 (m, 4H, CHZPh), 4.56 (d, 1H, J,,~ = 7.0
Hz, H-1D), 4.13-
4.22 (m, 2H, J5,6 = 4.5 Hz, J6a,Eb = 12.3 Hz, H-6ao, Gbo), 4.03 (m, IH, H-2D),
4.00 (m, IH, J4,s
= 9.5 Hz, J5,6 = 6.2 Hz, H~5e,), 3.96 (dd, IH, J2,3 = 3.3 Hz, H-2A), 3.90 (dd,
IH, .1~,4 = 9.5 Hz,
H-3A), 3.60 (m, 1H, H-SD), 3.48 (d, IH, J,,ox = 3.5 Hz, OH), 3.40 (dd, IH, H-
4A), 2.01, 2.03,
2.08 (3s, 9H, OAc), 1.65 (s, 3H, AcNH), 1.30 (d, 3H, H-6~). "C NMR (CDCl3):8
171.2,
171.0, 170.4, 169.6 (C=0), 128.0-138.2 (Ph), 103.3 (C-1D), 94.1 (C-la,), 81.4
(C-4"), '19.9
(C-2,,), 78.7 (C-3"), 75.8, 73.9 (CHzPh), 73.6 (C-3n), 72.4 (C-5o), 68.7 (C-
4p), 68.2 (G5~),
62.4 (C-6p), 54.5 (C-2p), 23.3 (AcNH); 21.1, 21.0, 21.0 (3C, OAc), 18.3 (C-
6A). FMS of
C34HmNO,~ (M, 673.2), nzl6 696.3 ([M+Na)T) Anal. Calcd for C~,H4a'Iv'0,,: C,
60.61 ; H,
6.43 ; N. 2.08. Found C, G0.4G ; H, 6.61 ; N, 1.95.
(2-acetamido-3,4,6-tri-0-acetyl-2-deoxy-~-n-glucopyranosyl)-(1 ~2)-3,4-di-O-
bcuxyl-a-
L-rhamnopyranoge trichloroacetimidate (7). The hemiacetal 20 (541 mg, 0.80
mmol) was
dissolved in CHaCh (20 mL), placed under argon and cooled to 0°C.
Trichloroacetonitrile
(0.810 mL, 8 mtnol), then DBLT (10 ~tL, 80 pmol) were added. The mixture was
stirred at 0°C
for I h. The mixture was concentrated and toluene was co-evaporated from the
residue. The
residue was eluted from a column of silica gel with I : I cyclohexane-EtOAC
and 0.2 % of Et3N
CA 02434685 2003-07-04
to give 7 (5G0 mg, 86 %) as a colourless foam; [a]p +2° (c l, CHCIa).
'H NMR (CDCI3):8
8.5G (s, 1H, N-H), 7.20-7.50 (m, lOH, Ph), G.29 (d, 1H, J~,2 = 1.3 Hz, H-lA),
5.50 (d, 1H,
J2.,,.H = 8.3 Hz, NHD), 5.17 (dd, 1H, J2,3 = J;,A = 9.5 Hz, H-3D), 5.09 (dd,
1H, Ja,s = 9.5 Hz, H-
4D), 4.60-4.85 (m, 4H, CHzPh), 4.68 (d, 1 H, .I,,2 = 8.0 Hz, H-1D), 4.10-4.22
(m, 2H, Js,b = 5.0
Hz, J6a.Gb .= 12.2 Hz, H-Gao, Gbn), 4.00 (m, 1H, H-2p), 3.99 (dd, 1H, Jz,s =
3.5 Hz, H-2A), 3.90
(m, 1H, J4,s = 9.G Hz, Js,s = 6.2 Hz, H-5A), 3.89 (dd, 1H, J3,s = 9.5 Hz, H-
3w), 3.62 (m, IH, H-
Sp), 3.50 (dd, IH, H-4A), 1.98, 2.00, 2.02 (3s, 9H, OAc), 1.65 (s, 3H, AcNH),
1.32 (d, 3H, H-
6w), ~'C NMR (CDC13):5 171.2, 171.0, 170.4, 1G9.G (C=0), 160.5 (C=NH), 128-138
(Ph),
103.3 (C-1D), 97.3 (C-lA), 91.4 (CC13), 80.3 (C-4w), 79.9 (C-3"), 77.5 (C-2w),
7G.0, 73.8 (2C,
CH2Ph), 73.1 (C-3o), 72.2 (C-5D), 71.1 (C-5A), 68.8 (C-4D), 62.5 (C-6o), 54.8
(C-2D), 23.3
(AcNH), 21.4, 21.1, 21.0 (3C, OAcj, 18.4 (C-GA). Anal. Calcd for
C36H4;C13Na0,3: C, 52.85 ;
H, 5.30 ; N. 3.42. Found C, 52.85 ; H, 5.22 ; N, 3.47.
Altyl (Z-0-acetyl-3,4-di-O-benzy!-a-L-rhamnopyranosyl)-(1-~2)-(3,4-di-0-henryl-
a-L-
rhamnopyranoside) (23). The acceptor Z1 (1.78 g, 4.65 mmol) and the
trichloroacetitnidate
donor 2Z (2.96 g. 5.58 rnmol) were dissolved in anhydrous ether (100 mL). The
mixture was
placed under argon and cooled to -55°C. TMSOTf (335 ~L, 1.8G mmol) was
added dropwise.
The mixture was stirred at -55°C to -20°C over 3 h.
Triethylamine (0.75 mL) was added, and
the mixture was allowed to warm to rt. The mixture was concentrated. The
residue was
purified by column chromatography (solvent x, 80 :20) to give 23 as a
colourless syrup (3.21
g, 92 %) ; [a]D -1G° {c 0.55, CHC13). ~H NMR (CDC13):8 7.30-7.42 (m,
20H, Ph), 5.82-5.92
(m, 1H, All), 5.G2 (dd, 1H, Jl,z = 1.G Hz, J,_, = 3.2 Hz, H-2,~), 5.20-5.32
(m, 2H, All), 5.07 (d,
1H, H-lA), 4.82 (d, IH, J,,Z = 1.0 Hz, H~1B), 4.G0-4.95 (m, 8H, CH2Ph), 4.15-
4.20 (m, 1H,
All), 4.09 (d, IH, Jz,3 = 3.0 Hz, H~2B), 4.05 (dd, 1H. J~,n = 9.4 Hz, H-3A),
3.95-4.05 (m, 1H,
Allj, 3.9G (dd, 1H, J;,q = 9.5 Hz, H-3a), 3.89 (m, 1H, Ja.s = 9.5 Hz, J5,6 =
6.3 Hz, H-51), 3.7G
{dd, 1H, J,,s = 9.5 Hz, Js,b = 6.2 Hz, H-5s), 3.52 (m, 1H, H-4B), 3.50 (m, IH,
H-4,,), 2.18 (s,
3H, OAc), 1.39 (d, 3H, H-6,,), 1.36 (d, 3H, H-6H). ~3C NMR (CDCI,):S 170.8
(C=0), 117.1-
138.4 (Ph, All), 99.5 (C-lA), 98.4 (C-18), 80.5 (2C, C-4w, 4H), 80.0 (C-3H),
78.1 (C-3~), 75.8,
75.7 (CHZPh), 74.9 (C-2B), 72.5, 72.2 (CHZPh), G9.3 (C-2~), 68.6 (C-SA), G8,4
(C-5B), G8.0
(All), 21.5 (OAc), 18.4, 18.2 (2C, C-6~, Ga). CI-MS for C,sHszOio (M = 752)
rrJz 770 [M +
NH4]'. Anal. Calcd, for CasHs20~o : C, 71.79 ; H, G.9G. Found C, 70.95 ; H,
7.01.
6
7
CA 02434685 2003-07-04
FAH-MS for CS,H580~o (M = 830.4) mlz 853.5 [M -~ Na]''. An3l. Calcd. for
CS,HsaO,o ; C,
73.71;H,7.03.FoundC,73.57;H,7.21.
(3,4-di-D-penzyl-2-0-paramethoxy benzyl-a-L-rbamnopyranosyl)-(1-~2)-(3,4-di-O-
benzyl-a-L-rbamnopyranose) (36). I,5-Cyclooctadiene-
bis(methyldiphenylphosph.ine)iridium
hexafluorophosphate (50 mg, GO p.mol) was dissolved tetrahydrofuran (G mL),
and the
resulting rid solution was degassed in an argon stream. Hydrogen was then
bubbled through
the solutidn, causing the colour to change to yellow. The solution was then
degassed again in
an argon istream. A solution of 35 (4.23 g, 5.09 tntnol) in tetrahydrofuzan
(24 mL) was
degassed end added. The mixture was stirred at rt overnight. The ,mixture was
concentrated.
The residue was taken up in acetone (45 mL), and watEr (5 mL) was added.
Mercuric chloride
(2.07 g, 7.63 mmol) and mercuric oxide (2.2 g, 10.2 mmol) were added to the
mixture,
protected. from light. The rni?tture was stirred for 2 h at rt, then
concentrated. The residue was
taken up in CHZCIz and washed three times with sat, sq. KI, then with brine,
Ths organuc phase
was dr'u'd and concentrated. The residue was purified by column chromatography
(CyclohE~Cane-AcOEt 4:l) to give 36 (2.97 g, 73 %) as a white foam; [cz]D
+8° (c 1, CHC>3).
'H NMR'(CDC13):8 7.25-7.40 (m, 20H, Ph), 6.73-7.18 (~ 4H, Ph), S.I2 (d, IH,
J,,z < 1.0 Hz,
H-lA), Sa05 (d, IH, J1,2 < 1.0 Hz, H-IH), 4.40-4.80 (m, lOH, PhCHi), 4.08 (dd,
1H, Jz~ = 3.0
Hz, H-2~), 3.80-3.90 (m, 2H, J3,4 = Ja,s = 9.5 Hz, Js,b = 6.1 Hz, H-38, 5$),
3.78-3.80 (m, 2H,
J~,~ = 3.1: Hz, J4,s = 9.4 Hz, Js,° = G.1 Hz, H-2A, 5A), 3.73 (m, 1H,
J3,4 = 9.4 Hz, H-3A), 3.72 (s,
3H, OCH3), 3.60 (dd, IH, H-4,,), 3.33 (dd, 1H, H-4H), 1.34 (d, 3H, H-Gn), 1.24
(d, 3H, H-6a).
''C NM~t (CDCI,):cS 113.2-129.8 (Ph), 99.1 (C-lA), 93.8 (C-1H), 80.7 (C-4,,),
80.3 (C-4a),
79.7 (C 3H), 79.2 (C-3p), 75.5, 75.4, 72.6, 72.5, 72.4, (PhCH~), 74.2 (C-2"),
74.1 (C-2g), 68.5
(C-5,~, 68.1 (C-5H), 55.3 (OCH3), I 8,1 (2C, C-6~, 6H). FAB-MS for
C.aHs<Ot° (M = 790.4)
m/z 813.4 [M + i~a]+. Anal. Calcd. for C~gHs4O,G : C, 72.89 ; H, G.BB. Found
C, 72.86 ;
H, &.98:
(3,4.diO.benxyl-Z-O-paramethoxybenzyl-a-t.-rhamnopyranosyl)-(1-->2)-3,4-di-O~
benzyl-,or.-z- rhamnopyranose trichlorogcetimidate (37). The herniacetal 36
(2.1 g, 2.6G
mrnol) v~~as dissolved in CHzC)z (20 mL), placed under argon and cooled to
0°C.
Trichloroacetonitrile (2.7 mL, 2G mmol), then DBU (40 ~,L, 0.2G mmol) were
added. The
mixture was stirred at 0°C for 30 min. The mixture was concentrated and
toluene was co-
8
CA 02434685 2003-07-04
evaporated from the residue. The residue was eluted from a column of silica
gel with 8:2
cyclohexane-EtOAC and 0.2 % EtzN to give 37 (2.03 g, 82 %) as a colourless
foam; [a]a -I O°
(c 1, CHCl3). 'H NMR (CDC13):S 8.50 (s, 1H, C=NH), 7.05-7.25 (m, 20H, Ph),
6.62-?.OS (m,
4H, Ph), 6.08 (d, 1H, Jm < 1.0 Hz, H-la), 5.10 (d, 1H, J~,z < 1.0 Hz, H-1,,),
4.40-4.80 (m,
IOH, PhCHa), 4.10 (dd, IH, Jz,3 = 3.0 Hz, H-2B), 3.80-3.90 (m, 4H, H-38, 2",
3A, SA), 3.72-
3.80 (m. 1H, H-5H), 3.72 (s, 3H, OCH~j, 3.G3 (dd, 1H, J3.e = Je,s = 9.5 Hz, H-
4,,), 3.42 (dd,
IH, J,,e =~Ja,S = 9.5 Hz, H-4a), 1.30 (d, 3H, H-6s), 1.25 (d, 3H, H-6,~). '3C
NMR (CDCIJ):S
161.1 (C=NH), 113.4-129.5 (Ph), 99.6 (C-1"), 97.0 (C-19), 80.6 (C-4"), 79.6 (C-
4H). 79.3
(2C, C-3~;. 3H), 75.7, 75.5, 72.8, 72.3, 72.0, (PhCH~), 74.4 (C-2A), 72.6 (C-
28), 71.1 (C-S,J,
G8.9 (C-5g), 55.3 (OCHy), 18.1 (2C, C-6A, 6H). Anal. Calcd, for C.c,H54C13NO,o
: C, 64,21 ; H,
5.82; N, 1.50. Found C, 64.67 ; H, G.O1; N, 1.28.
Aryl (3;4-di-0-benzyl-2-O-chloroacetyl-a-z-rhamnopyranosy!)-(1-32)-(3,d-di-O-
benzyl-
a-~rhamnopyranoside) (32). To a mi~rture of 24 (3.8 g, 5.35 mrnol) in pyridine
(40 mL) was
added chloroactie anhydride (1.83 g, 10.7 mmol) at 0°C. The solution
was stirred overnight at
0°C. MeOH (10 mL) was added and the mixture was concentrated. The
residue was eluted
from a oolumn of silica gel with 95:5 cyclohexane-acetone to give 32 (2.4 g,
57 %) as a
colorless. syrup; [a]ø -15° (c l, CHC~). 'H NMR (CDCI3):8 7.15-7.30 (m,
20H, Ph), 5.71-5,81
(m, 1H, All), 5.49 (dd, 1H, J,,Z = 1.7 Hz, Jz,3 = 3.2 Hz, H-2A). 5.08-5.20 (m,
2H, All), 4.90 (d,
1H, H-I~), 4.50-4.84 (m, 8H, PhCHa), 4.65 (d, 1H, J,,2 < 1.0 Hz, H-Ie), 3.85-
4,04 (m, 2H,
All), 4.02 (m, 2H, CHaCI), 3.93 (dd, 1 H, Jz j = 3.0 Hz, H-2H), 3.88 (dd, 1H,
J~,d = 9.5 Hz, H-
3n), 3.81 (dd, 1 H, J3,4 = 9.5 Hz, H-38), 3.62 (m, 1 H, J,,S = 9.0 Hz, Js,s =
6.1 Hz, H-58), 3.73
(m, 1H, J4,s = 9.5 Hz, Js,B = 6.2 Hz, H-5,,), 3.34 (dd, 1H, H-48), 3.30 (dd,
IH, H-4,,), 1.22 (d,
3H, H-6A), 1.21 (d, 3H, H-Gs). "C NMR (CDC13):8 166.9 (C=O), 117.2-138.5 (Ph,
All), 99.2
(C-1"), 98.2 (C-IB), 80.4 (C-4.,), 80.3 (C-38), 80.2 (C-4a), 77.9 (C-3A),
75.8. 75.7. 72.6, 72.4
(PhCHz), 74.9 (C-2B), 71.2 (C-2A), 68.G (C-5A), 68.4 (C-58), 68.0 (All), 41.3
(CHZCI), 18.3
(2C, C-6A, 6B). FABMS of C45Hs,C10,o (M, 786.3), m/z 809.3 ([M+Na]'). Anal.
Calcd for
CrsHsiClO,o: C, 68.65 ; H, 6.53. Found C, 68.51 ; H, 6.67.
(3,4-di-O-benzyl-Z-0-chloroacetyl-a-t~-rhamnopyranosyl)-(1~2)-(3,4-di-0-bcnzyl-
a![i-L-
rhamnopyranose) (33). 1,5~Cyclooctadiene-bis(methyldiphenylphosphine)iridium
hexaflu.orophosphate (40 mg, 46 psnol) was dissolved tetrahydrofuran (7 mL),
and the
I
i
i
9
CA 02434685 2003-07-04
resulting red solution was degassed in an argon stream Hydrogen was then
bubbled through
the solution, causing the colour to change to yellow. The solution was then
degassed again in
an argon stream. A solution of 32 (2.39 g, 3.04 mmoI) in tetrahydrofuran (I8
mL) was
degassed end added. The mixture was stirred at rt overnight. The mixture was
concentrated.
The residue was taken up in acetone (30 mL), and water (S mL) was added.
Mercuric chloride
(1.24 g, 4.56 mmol) and mercuric oxide (1.3 g, 6.08 mmol) were added to the
mixture,
protected from light. The mixture was stirred for 2 h at rt, then
concentrated. The residue was
taken up in CHzCh and washed three times with sat, aq. KI, then with brine.
The organic phase
was dried and concentrated. The residue was purified by column chromatography
(Cyclohexane-AcOEt 4:1) to give 33 (1.91 g, 84 %) as a white foam. [a]p -
2° (r 1, CHCIa). 'H
NMR (CDCl3):8 7.10-7.40 (m, 20H, Ph), 5.49 (dd, 1H, J,,z = 1.7 Hz, JZ,3 = 3.2
Hz, H-2"),
4.99 (d, 1H, J,,z < 1.0 Hz, H-18), 4.90 (d, 1H, H-l,,), 4.45-4.85 (m, 8H,
PhCHa), 4.01 (m, 2H,
CHzCI), 3.93 (dd, 1H, Jz,3 = 3.0 Hz, H-Z$), 3.90 (dd, 1H, J3,, = 9.3 Hz, H-
3~), 3.84 (dd, 1H,
.7,,4 = 9.0 Hz, H-3B), 3.81 (m, 1H, J4,s = 9.0 Hz, JS,~ = 6.2 Hz, H-SH), 3.72
(m, 1H, J4,s = 9.5
Hz, J~,6 = 6.2 Hz, H-5A), 3.33 (dd, 1H, H-4H), 3.30 (dd, 1H, H-4A), 2.81 (d,
1H, Jz,oH = 3.4
Hz, OH), 1.22 (d, 3H, H-6,,), 1.20 (d, 3H, H~68). "C NMR (CDCI3):8 167.0
(C=O), 127.2-
138.5 (Ph), 99.1 (C-1"), 93.9 (C-1H), 80.3 (C-48), 80.2 (C-4A), 79.7 (C-3H),
77.8 (C-3A), 75.8,
75.7, 72.G, 72.4 (PhCHz), 75.0 (C-2a), 71.1 (C-2A), 68.6 (C-S"), 68.4 (C-SH),
41.3 (CHiCI),
18.1 (2C, C-6A, 6B). FARMS of C,,Ha,CIO,o (M, 74b.3), m/~ 769.3 ([M+Na]*).
Anal. Calcd
for CaiHd,ClO,o: C, 67.51 ; H, 6.34. Found C, 67.46 ; H, 6.39.
(3,4-di-D-benzyl-2-O-chloroacety!-a-Irrhamnopyranosy!)-(1->2)-3,4-di-0-6enry1-
a-L-
rhamnopyranose trichloroscetimidate (34). The hemiacetal 33 (1.80 g, 2.41
mmol) was
dissolved in CH~CIz (25 mL), placed under argon and cooled to 0°C.
Trichloroacetonitrile (2.4
mL, 24 mmol), then DBU (3S uL, 0.24 mmol) were added. The mixture was stirred
at 0°C for
40 min. The. mixture was concentrated and toluene was co-evaporated from the
residue. The
residue was eluted from a column of silica gel W th 4: I cyclohexane~EtOAC and
0.2 % Et3N to
give 34 (1.78 g, 83 %j as a colourless foam; [a]o -12° (c 1, CHC13). ~H
Tv'NLR (CDCI,):S 8.60
(s, 1H, C=NH), 7.30-7.S0 (nt, 20H, Ph), 6.21 (d, lI~i, J,,2 = 1.8 Hz, H-1H),
5.63 (dd, 1H, J,Z =
1.5 Hz, Ja.3 = 3.2 Hz, H-2"), 5.07 (d, 1H, H-1"), 4.65-5.00 (m, 8H, PhCH2),
4.19 (m, 2H,
CH~CI), 4.09 (dd, 1H, Jz,, = 3.2 Hz, H-28), 4.04 (dd, 1H, J~,e = 9.0 Hz, H-
3B), 3.95 (m, 3H,
H-3~, 5A, 58), 3.58 (dd, IH, H-4A), 3.48 (dd, 1H, H-4g), 1.39 (m, 6H, H-6A,
68). ~'C NMR
CA 02434685 2003-07-04
(CDC~):o 167.1 (C=O), 160.7 (C=N), 127.0-138.3 (Ph), 99.4 (C-lA). 97.5 (C-1H),
91.4
(CC13), 80.1 (C-4$), 84.0 (C-4A), 79.2 (C-3A), 77.9 (C-38), 75.9, 75.8, 73.0,
72.6 (PhCH2),
73.7 (C-2a), 71.4 (C-2,~, 71.2, 68.9 (2C, C-5A, 5g), 41.3 (CH2C1), 18.4, 18.2
(2C, C-6,,, 6H).
Anal. Calcd for C,aHq~CI.,NOIO: C, 59.27 ; H, 5.31 ; N, 1.57. Found C, 59.09 ;
H, 5.49 ; N,
1.53.
Altyl (3,4,6-tri-O-acetyl-2-deo~y-2-trichloroacetamido-~3-D-glucopyranosyl)-(1-
~2)-(3,4-
di-O-benzyl-a-L-rhamnopyranosyl)-(1--i2)-3,4-di-0-benzyl-a-L-rbamnopyranoslde
(26).
1,2-Dichloroethane (35 mL) was added to the trichloroacetimidate donor 16
(2.49 g, 4.20
mmol), the acceptor 24 (2.48 g, 3.50 mewl) anrl 4A-MS powder (4 g). The
mixture was
stirred for 1.5 h at rt under argon. The mixture was cooled to -20°C
and TMSOTf (230 ~L,
I.26 mmol) was added. The temperature was allowed to rise to 0°C over 1
h, and the mixture
was stirred for an additional 2 h at this temperature. Trietl~ylamine (0.5 mL)
was added and the
mixture was allowed to warns to rt. The mixture was diluted with CHZC12 and
filtered. The
.filtrate was concentrated. The residue was purified by column chromatography
with 3 :1
cyclohexane-AcOEt to give 26 (3.83 g, 96 %) as a colourless amorphous solid ;
[a]D --6° (c
0.5, CHCI,). 'H NMR (CDCIj) : b 7.28-7.52 (m, 20H, Ph), 6.83 (d, 1H, J1,NX =
8.4 Hz, NH),
5.85 (m, 1H, AII), 5.09-5.26 (m, 4H, H-3D, 40, A11), 4.98 (d, 1H, J~,~ = I.4
Hz, H-IA), 4.58-
4.98 (m, lOH, H-1H, lp, CHZPh), 4.08 (m, 4H, H-2", 2p, 6aD, All), 3.91 (m, 5H,
H-2B, 3A, 3H,
6bp, All), 3.79 (m, 2H, H-SA, 5B), 3.45 (m, 3H, H~4n, 4H, 5D), 1.97, 2.02,
2.04 (3s, 9H, OAc),
1.30 (m, GH, H-6A, 6H). "C NMR (CDC~) : 0 170.6, 170.3, 169.1, 163.2, 161.6
(C=0),
138.4-117.1 (Ph, All), 101.3 (C-1D), 100.9 (C-1~), 97.6 (C-1B), 92.0 (CCt~),
80.9, 80.4 (2C,
C-4A, 4g), 79.1, 79.0 (2C, C-3,,, 3B), 77.3 (C-2A), 76.5 (C-2B), 75.4, 75.2,
73.6 (CHzPh), 72.2
(C-3p), 71.9 (C-Sp), 71.6 (CH~Ph), 68.2 (C-5B*), 67.8 (C-4D), 67.5 (C-5~*),
67.5 (CHzO),
G1.3 (C-6D), 55.7 (C-2D), 20.5 (OAC), 17.9, 17.7 (2C, C-6A, 6s). FAB-MS for
Cs~H~sCbNO"
(M = 1141.3) miz 1166.3, 1164.3 [M + Na]+. Anal. Calcd. for CS~H66C1~N0~,: C,
59.87 ; H,
5.82 ; N. 1.22. Found C, 59.87 ; H, 5.92 : N, 1.16.
Attyl (3,4,6-tri-O-acetyl-2-deoxy-2-tetrachlorophthalimido-p-D-glucopyranosyl)-
(1-~2)-
(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1->2)-3,4-di-O-benzyl-a-L-
rbamnopyranoside
(Z8), Anhydrous ether (30 mh) and CHzCIz (15 mL) were added to the
trichloroacetimidate
donor 25 (3.34 g, 4.66 mmol), the acceptor 24 (2.20 g, 3.I0 mmol). The mixture
was cooled
CA 02434685 2003-07-04
to 0°C and TMSOTf (85 pL, 0.46d mmol) was added dropwise. The mixture
was stirred at
0°C for 1 h, then at rt for 3 h. Triethylamine (1 mL) was added and the
mixture was stirred for
min., then concentrated. The mixture was taken up in ether and the resulting
precipitate was
filtered o~ The filtrate was concentrated. The residue was purified by column
chromatography
with 7:3 cyclohexane-AcOEt to give 28 (2.57 g, 65 %) as a colourless amorphous
solid ; [a]o
+22° (c 1, CHC~). 'H NMR (CDCl3) : 8 7.16-7.42 (m, 20H, Ph), 5.91 (dd,
1H, H-3p}, 5.81
(m, 1H, All), 5.10-5.24 (m, 4H, H-lp, 4p, All), 4.93 (s, 1H, H-1"), 4.53-4.81
(m, SH, H-1H,
CH~Ph), 4.23-4.45 (m, 5H, H-2D, CHZPhj, 4.05 (m, 2H, H-Gap, All), 3.58-3.91
(m, 8H, H-2A,
28, 3A, 3B; SA, 5a, 6bD, All), 3.38 (m, IH, H-So), 3.13-3.21 (m, 2H, H-4A,
4H), 2.00, 2.02, 2.05
(3s, 9H, OAc), 1.24 (m, GH, H-GA, 6H). "C NMR ~ 170.4, 169.3 (C=O), 117.1-
138.4 (Ph,
All), 101.1 (C-lA), 99.9 (C-lo), 97.7 (C-1B), 80.6 (2C, C-4,,, 4g), 78.9, 79.7
(2C, C-3~, 3B),
78.2 (C-2A), 76.3 (C-2B), 75.2, 75.1, 72.6, 71.3 (CHZPh), 71.2 (C-5p), 70.1 (C-
3D), 68.4 (C-
5s*), 68.4 (C-4D), 67.6 (C-5A*), 67.6 (All), 61.3 (C-6D), 55.4 (C-2D), 20.6
(OAc), 18.0, 17.6
(2C, C-6~, 6g). FAB-MS for C63H~SChNO~A (M = 1263.3) mlz 1288.4, 1286.4 [M +
Na]".
Anal. Calcd. for C6~H65C)aNO~g : C, 59.77 ; H, 5.17 ; N, 1.11. Found C, 60.19
; H, 5.53 ; N,
1.18.
Allyl (2-acetamido-2-deoxy-~3-D-glucopyranosyl)-(1--~2)-(3,4-di-0-benzyl-a-L-
chamnopyranosyl)-(1 >2)-3,4-di-O-benzyl-a,-C,-rhamnopyranoside (27). The
trisaceharide
26 (1.71 g, 1.50 mmol) was dissolved in MeOH (20 mL). A 1M solution of sodium
methoxide
in methanol (9 mL) and triEthylamine (5 mL) were added, and the mixture was
stirred at rt for
18 h_ The mixture was cooled to 0°C and acetic anhydride was added
dropwise until the pH
reached 6. A further portion of acetic anhydride (0.4 tnL) was added, and the
murturc was
stirred at n for 30 min. The mixture was concentrated, and toluene was co-
evaporated from
the residue. The residue was purified by column chromatography with 95 :5 DCM-
MeOH to
give 27 (623 mg, 45 %) as a colourless amorphous solid ; [a]o -16° (c
0.5, CHCIz). 'H NMR
(CDC13) : 8 7.24-7.48 (m, 20H, Ph), 6.79 (d, IH, NH), 5.73 (m, 1H, All), 5.12
(m, 3H, H-la,
All), 4.52-4.86 (m, 9H, H-1B, CHzPh), 4.34 (d, 1H, H-lp), 3.79-4.08 (m, 6H, H-
2A, 28, 3A, 3H,
AIl), 3.53-3.74 (m, 3H, H-5a, 5B, 6ap), 3.24-3.45 (m, GH, H-2p, 3D, 4A, 4H,
4T,, Gbp), 3.20 (m,
1H, H-SD), 1.46 (s, 3H, OAc), 1.24 (m, 6H, H-G~, 6B). '3C NMR G 173.6 (C=0),
117.3-137.4
(Ph, All), 103.2 (C-lo), 100.3 (C-1~), 97.9 (C-1H}, 81.3, 80.4 (2C, C-4~, 4H),
79.9 (2C, C-3A,
3B), 79.9 (C-2$*), 78.9 (C-3o), 75.7 (C-So), 75.6 75.3, 74.5 (CHaPh), 73.6 (C-
2,,*), 72.5
12
CA 02434685 2003-07-04
(CHiPh), 71.9 (C-4D), G8.2. 68.0 (2C, C-5", 5H), 67.7 (CHiO), 62.5 (C-6n),
58.8 (C-2p), 22.3
(OAc), 18.0,17.8 (2C, C-6", 6s). FAB-MS for CsvHs3NO~a (M = 913.4) m/z 936.6
[M + Na]''.
Anal. Calcd, for CS;H6~NO;a.H~O: C, 65.72 ; H, 7.03 ; N, 1.50. Found C, 65.34
; H, 7.03 ; N,
I.55.
Altyl (2-acetamido-3,4,6-tti-O-acetyl-Z-deoxy-[i-D-glucopyranosyl)-(i--32)-
(3,4-di-0-
benzyl-a-L-rhamnopyranosyl)-(1 >2)-3,4-di-O-benzyl-a-L-rhamnopyranoside (27).
(a)
Pyridine (5 mL) was added to 27a (502 mg, 0.55 mmol) and the mixture was
cooled to 0°C.
Acetic anhydride (3 mL) was added. The mixture was stirred at rt for 18 h. The
mixture was
concentrated and toluene was co-evaporated from the residue. The residue was
taken up in
CHzC>~ and washed successively with 5% aq HCl and saturated aq NaHC03. The
organic
phase was dried and concentzated to give 27 (538 mg, 94 %) as a colourless
foam.
(b) Tetrahydrofuran (3 mL) and ethanol (3.3 mL) were added to 28 (384 mg,
0.303 mmol).
Ethylenedia.mine (90 ~L, I.3G mmnl) was added and the mia.-turE was heated at
55°C for 4 h.
The mixture was alloyed to cool to rt. Acetic anhydride ( 1.0 mL) was added,
and the mixture
was stirred at rt for 1.5 h. The mixture was concentrated. The residue was
taken up in pyridine
(5 mL) and the mixture was cooled to 0°C. Acetic anhydridE (2.5 mL) was
added. The mixture
was stirred at rt for 18 h. The mixture was concentrated and toluene was co-
evaporated from
the residue. The residue was taken up in CH~C1~, which caused the formation of
a whit
precipitate. The mi.~cture was filtered through a plug of silica geL eluting
with 7:3 Cyclohexane-
acetone. The filtrate was concentrated to give 27 (215 mg, 68 %) as a
colourless foam ; [a]n -
7° (c 0.5, CHCL). ~H NMR (CDCb) : 0 7.24-7.48 (m, 20H, Ph), 5.84 (m,
1H, All), 5.53 (d,
1H, NH), 5.19 (m, 2H, Ah), 5.03 (dd, IH, H-4n), 4.98 (m, 2H, H-1", 3D), 4.54-
4.95 (m, IOH,
H-I H, 10, CHzPh), 4.07 (m, 4H, H-2A, 2p, 6aD, All), 3.88 (m, 5H, H-28, 3A,
38, 6bp, AIl), 3.79,
3.68 {2m, 2H, H-SA, 5$), 3.42 (m, 3H, H-4A, 4a, 5n), 2.02, 2.01, 1.97, 1.G4
(4s, 12H, OAcj,
1.30 (m, 6H, H-6", 6B). t'C NMR (CDCI3) S 170.7, 170.4, 169.9, 169.1 (C=0),
117.1-138.5
(Ph, All), 102.9 (C-la), 101.2 (C-1,,), 97.7 (C-la), 81.0, 80.5 (2C, C-4A,
4B), 79.5, 79.1 {2C,
C-3A, 3s), 78.2 (C-2A), 76.1 (C-2B), 75.5, 75.2, 73.6 (CH2Ph), 73.3 (C-3D),
71.9 (C-5D), 71.7
(CH2Ph), 68.3 (C-5A''), 68.0 (C-4D), 67.G (C-5H'"), 67.6 (CHzO), 61.6 (C-GD),
54.1 (C-2p),
22.9 (AcNH), 20.6 (OAc), 18.0, 17.7 (2C, C-6", 6B). FAB-MS for Cs,Hb9N0;, (M =
1039.5)
rn/z 1062.4 [M + Na]+. Anal. Calcd. for Cs,Hs9N0": C, 65.82 ; H, 6.69 ; N,
1.35. Found
C, 65.29 ; H, 6.82 ; N, 1.29.
13
CA 02434685 2003-07-04
(Z-acetamid o-3,4,6-tri-O-acety )-2-deoxy-[i-D-glucopyranosyl)-(I ~2)-(3,4-di-
O-benzyl-a-
L-rhamnopyranosyl)-(1~2)-3,4-di-O-benzyl-a!(3-L-rhamnopyranose (29). I,5-
Cyclooctadiene-bis(methyldiphenylphosphine)iridium hcxafluorophosphate (30 mg,
35 pmol)
was dissolved tetrahydrofuran (5 mL), and the resulting red solution was
degassed in an argon
stream. Hydrogen was then bubbled through the solution, causing the colour to
change to
yellow. The solution was then degassed again in an argon stream, A solution of
Z7 (805 mg,
0.775 mmol) in tetrahydrofuran (10 mL) was degassed and added. The mixture was
stirred at
rt overnight. The mixture was concentrated. The residua was taken up in
acetone (15 mL),
and water (1.5 mL) was added. Mercuric chloride (315 mg, 1.16 mmol) and
mercuric oxide
(335 mg, 1.55 mmol) were added to the mixture, protected from light. The
mixture was stirred
for I h at rt, then concentrated. The residue was taken up in CHzCIz and
washed three times
with sat, aq. KI, then with brine. The organic phase was dried and
concentrated. The residue
was purified by column chromatography with 4 :6 AcOEt-cyelohexane to give Z9
{645 mg, 83
%) as a white foam. The'H NMR spectra showed the a :~3 ratio to be 3.3 :1 ;
[a]D +3° (c 0.5,
CHC13). 'H NMR (CDCIa) a-anomer : 8 7.30-7.47 (m, 20H, Ph), 5.53 (d, 1H, NH),
5.17 (d,
1H, J,,2 = 1.9 Hz, H-1H), 5.08 (m, IH, H-4D), 5.03 (d, 1H, J,,~ = 1.5 Hz, H-
lA), 4.99 (m, IH,
H-3o), 4.62-4.92 (m, 8H, CFI~Ph), 4.60 (d, 1H, J,,z = 8.4 Hz, H-ID), 4.01-4.18
(m, 3H, H-2A,
20, 6aD), 3.90-3.97 (m, SH, H-2H, 3A, 3H, 5A*, 6bD), 3.83 (m, 1H, H-SH*), 3.37-
3.45 (m, 3H,
H-4A, 4H, Sp), 2.04, 2.03, 1.99, 1.68 (4s, 12H, OAc, AcNH), 1.32 {m. 6H, H-6A,
6H). "C
NMR (CDC13) S 170.7, 170.4, 169.9, 169.1 (C=O), 129.3-138.5 (Ph), 103.3 (C-
I~), 101.6 (C-
In), 93.9 (C-1H), 81.5, 80.8 (2C, C-4", 4B), 79.9, 78.9 (2C, C-3", 3H), 78.6
(C-2a), 76.8 (C-
2H), 76.0, 75.5, 74.0 (CH~Ph), 73.7 (C-3o), 72.4 (C-5o), 72.2 (CHZPh), 68.7 (C-
SA*), 68.5 (C-
4D), 68.2 (C-58*), 62.0 (C-6D), 54.6 (C-2D), 23.4 (AcNH), 21.1 (OAc), 18.5,
18.1 (2C, C-6~,
6H). FAB-MS for C~H65N0,~ (M = 999.4) mlr. 1022.5 [M + Na]+. Anal. Calcd. for
CsaH6sN0» : C, 64.85 ; H, 6.55 ; N. 1.40. Found C, 64.55 ; H, 7.16 ; N, I.IS.
(2-acetamido-3,4,6-tri-O-acetyl-Z-deo~y-[i-D-glucopyranosyl)-(1--~2)-(3,4-di-O-
bcnzy 1-ac~
Irrhamnopyranosyl)-(1-~2)-3,4-di-O-benzyl-a/(3-L-rhamnopyranosyl
trichloroacetimidate (13). The hemiacetal 29 (595 mg, 0.59 mmol) was dissolved
in CH2Clz
(IO mL), placed udder argon and cooled to 0°C. Trichloroacetonitrile
(0.6 ml:,, 6 mmol), then
DBU (10 pL, 59 ~rmol) were added. The mixture was stirred at 0°C for 20
min., then at rt for
14
CA 02434685 2003-07-04
20 min. The mixture was concentrated and toluene was co-evaporated from the
residue. The
residue was purified Iry flash chromatography with 1:1 cyclohexane-AcOEt and
0.2 % of Et3N
to give 13 (634 mg, 94 %) as a colourless foam. The'H NMR spectra showed the a
:~3 ratio to
be 10 :1. [a)D -20° (c 1, CHC13). 'H NVIR (CDCl3) a-anomer : E 8.47 (s,
IH, C=NH), 7.20-
7.38 (m, 20H, Ph), 6.10 (d, 1H, J,,z = 1.3 Hz, H-lg), 5.40 (d, 1H, NH), 5,01
(m, 1H, H-4D),
4.95 (d, 1H, Jl,z = 1.2 Hz, H-1"), 4.89 (m, 1H, H-3o), 4.55-4,85 (tn, 9H, H-
1D, CHlPh), 4.07
(dd, 1H, H-6ap), 4.03 (m, 1H, H-2A), 3.97 (m, IH, H-2a), 3.91 (dd, 1H, H-6bo),
3.7I-3,85 (m,
5H, H-2B, 3A, 3B, 5A, SH), 3.31-3.45 (m, 3H, H-4A, 4s, 5n), 1.58, 1.91. 1.9G,
1.99 (4s, I2H,
OAc, AcNH), 1.26 (m. 6H, H-6A, 6H). '3C NIvIR (CDCl3) 0 171.1, 170.9, 170.3,
169.6 (C=0),
160.6 (C=NH), 128.1-138.6 (Ph), 103.3 (C-lp), 101.6 (C-lA), 96.9 (C-1g), 91.3
(CC~), 81.4.
80.2 (2C, C-4", 4H), 79.9, 78.5 (2C, C-3~, 3B), 78.3 (C-2A), 75.9 (CHzPh),
75.0 (C-28), 73.7
(CHZPh), 73.7 (C-3o), 72.4 (CHzPh), 72.4 (C-5n), 71.0, 69.0 (2C, C-SA, 5H),
68.5 (C-4D), G2.1
(C-6o), 54.6 (C-2n), 23.4 (AcNI-1), 21.1 (OAc), 18.5.18.0 (2C, C-6A, 6B).
Anal. Calcd. for
CssH65C~Nz0": C, 58.77 ; H, 5.72 ; N, 2.45. Found C, 58.78 ; H, 5.83; N, 2.45.
Allyl (2-acetamido-3,4,b-tri-O-acetyl-2-deo~y-]i-D-glucopyranosyl)-(1 >Z)-(3,4-
di-O-
benryl-a-L-rhamnopyranosyl)-(1-~2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(i-
~3}-
(2,3,4,6-tetra-O-benryl-a-D-glucopy ranosy 1-(1-->4)-]-2-O-benzoyl-a-L-
rhamnopyranoside
(5}. Anhydrous ether (5 mL) was added to the donor 13 (500 mg, 0.437 mmol) and
the
acceptor 11 (242 mg, 0.29 mmol) and powdered 4~-MS. The mixture was placed
under argon
and cooled to 0°C. Boron trifluoride etherate (415 p.L, 3.27 mmol) was
added. The mixture
was stirred at 0°C for 1 h, then at rt for 18 h. The mixture was
diluted with CHzCIz and
triethylamine ( 1 mL) was added. The mixture was filtered through a pad of
Celite and the
filtrate was concentrated. The residue was purified by column chromatography
with 3:2
cyclohexane-AcOEt to give, in order, the acceptor 11 (132 mg, 54 %), 5 (231
mg, 44 %) and
the hemiacetal 29 (129 mg, 29 %). The desired pentasaccharide S was obtained
as a colourless
foam ; (a]D +10° (c 1, CHCl3). 'H NMR (CDC)3): & 7.09-8.02 (gin, 45H,
Ph), 5.92 (m 1 H,
Atl), 5.65 (d, 1H, NH), 5.37 (m,IH, H-2c), 5.19 (m, 2H, All), 5.13 (bs, 1H, H-
l~), 4.35-4.96
(m, 15H, H-1B, lc, 1D, ls, 2e, 3n, 4D, CHZPh), 4.17 (n~, 2H, H-2A, All), 3.8?-
4.04 (m, 8H, H-
2D, 3A, 3c, 3E, 5A, SE, ban, All), 3.63-3.81 (m, 7H, H-3B, 4c, 4E, Sc, Gas,
6bE, 6bn), 3.59 (m
1H, H-58), 3.43 (tn, 3H, H-2E, 4~, 50), 3.28 (t, 1H, H-48), 1.66, 1.71, 1.99,
2.01 (4s, 12H,
OAc, AcNH), 1.34 (m, 6H, H-6A, 6c), 1.00 (d, 3H, H-6g).'3C NMR (CDCl3): b
170.5, 170.0,
CA 02434685 2003-07-04
169.3, 165.8, 163.5 (C=O), 117.6-138.7 (Ph, Allj, 102.7 (C-lp), 100.8 (2C, C-
1,,, 1H), 98.1
(GIE}, 95.9 (C-lc), 81.8 (C-3E), 81.2 (2C, C-2E, 4A), 80.0 (C-4B), 79.7 (2C, C-
3A, 3c}, ?8.2
(C-3a), 77,7 (C-2,,), 77.3 (2C, C-4~, 4E), 75.G, 75.4, 74.9 (CHZPh), 74.3 (C-
2$), 73.8
(CHZPh), ?3.7 (C-3p), 72.8 (CH,.Ph), 72.3 (C-2c), 72.1 (C-Sc), 7I.5 (C-5E},
70.2 (CHzPh),
G8.5 (C-SE), G8.4 (C-SA, CHz4), 68.2 (C-4D), 6?.9 (C-GE}, 67.4 (C-Sc), G1.8 (C-
GD}, 54.3 (C-
2D), 23.1 (AcNH), 20.7, 20.6, 20.4 (OAc}, 18.6 (C-6A), 18.0 (C-6C), 17.8 (C-
GH). FAB-MS for
C,o4Hi~,NOa~ (M = 1812.1) m/z 1836.2, 1835.2 [M + Na]t. Anal. Calcd. for
C,o~H»,NOz~: C,
68.90 ; H, G.50 ; N, 0.77. Found C, G8.G4 ; H, 6.6G ; N, I.OS.
Allyl (3,4-di-O-benryl-2-O-chloroaceCyl-a-L-rhamnopyranosyl)-(1-~2)-(3,4-di-O-
benzyl-
a-L-rhamnopyranosyl)-(1 >3)-[2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-(1~4)-]-
2-O-
benzoyl-a-G-rhamnopyranoside (38). A mixture of alcohol 11 (212 mg, 0.255
mmol) and
imidate 34 (270 mg, 0.33 mmol) in anhydrous EtiO (4 mL) was stirred fnr IS min
under dry
Ar. After cooling at -60°C, Me3Si0Tf (30 ~L, 0.166 mmol) was added
dropwise and the
mixture was stirred overnight and allowed to reach rt. Triethylamine ( 120
~,L) was added and
the mixture was concentrated. The residue was eluted from a column of silica
gel with 7:1
cyclohe-EtOAc to give 38 (8G mg, 22 %) as a foam; [a]D +5° (c 1,
CHC13). ~H NMR
(CDC13):8 6.95-8.00 (m, 45H, Ph), 5.80-6.00 (m, 1H, All), 5.56 (dd, 1H, H-2A),
5.40 (dd, IH,
J~,z < 1.0 Hz, Jz,3 = 3.0 Hz, H-2c), 5.20-5.37 (m, 2H, All), 5.08 (d, 1H, J,,Z
= 3.2 Hz, H~ls),
5.04 (d, 1H, J,,~ < 1.0 Hz, H-lAj, 5.00 (d, 1H, J~,Z < 1.0 Hz, H-18), 4.99 (d,
1H, H-lc), 4.30-
4.90 (m; I6H, CHZPh), 4.35 (dd; IH, J2,3 -- 3.0 Hz, H-2a), 4.14 (dd, 1H, J3,a
= 9.5 Hz, H-3c),
4.03 (dd, 1 H, Jz,s = J~,4 = 10.0 Hz, H-3E), 3.90-4.20 (m, 2H, All), 3.75-4.00
(m, 4H, CIAc, H-
6aE, 6bs), 3.96 (dd, I H, H-3"), 3.95 (m, 1 H, H-SA), 3.95 (dd, 1 H, H-Se),
3.83 (dd, 1 H, H-4~),
3.80 (m 1H, H-Sc), 3.72 (dd, 1H, H-4~, 3.64 (dd, 1H, H-3a), 3.60 (m, 1H, H-
SH), 3.52 (dd,
1H, H-2g), 3.39 (dd, 1H, H-4A), 3.30 (dd, 1H, H-48), 1.35 (d, IH, H-6A), 1.30
(d, 1H, H-6c),
1.00 (d, 1H, H-6H). 1'C NMR (CDC13):S 166.1, 165.? (C=O), 117.0-133.4 (Ph),
100.9 (C-1H),
98.9 (C-lAj, 97.8 (C-lE), 96.0 (C-1~), 8I.8 (C-3E), 80.9 (C-2E), 79.9 (G4n),
79.6 (C-48). 79.6
(C-3c), 78.9 (C-3g), 78.0 (C-4c), 77.5 (C-4E), 77.3 (C-3A), 75.6, 75.3, 75.0,
74.7, 73.9, 73.5,
72.8, 70.9, (G'HZPh, All), 74.9 (C-29), 72.5 (C-2~), 71.2 (C-5E), 70.9 (C-2"),
G8.8 (C-5~), G8.5
(C-6~), 68.3 (C-5,,), G7.5 (C-5c), 40.9 (CIAc), 18.8 (C-6,,), 18.2 (C-dc),
17.8 (C-68). FAB-MS
for C91H~9CIO~o (M =1558.6) m/z 1581.7 [M + Na]+. Anal Calcd, for C9aH99C1Ozo
: C, 70.82 ;
H, 6.40. Found C, 70.67 ; H, 6.58.
16
CA 02434685 2003-07-04
Allyl '(3,4-di-O-benzyl-2-0 pmetho~ybenzyl-a-L-rhamnopyranosyl)-(1-->2)-(3,4-
di-0-
benryl-a-L-rhamnopyranosyl)-(1->3)-[2,3,4,6-tetra-0-benzyl-a-D-glucopyranosyl-
(1~
4)-]-Z-0-benzoyl-a-L-rhamnopyranoside (39). A mixture of alcohol 11 (125 mg,
0.15
mmol) and 4~ molecular sieves in anhydrous EtzO (3 mL) was stirred for 45 min
under dry Ar.
After cooling at -40°C, Me3Si0Tf (20 ~.L, 0.112 mmol) was added
dropwise. A solution of the
donor 37 (210 mg, 0.225 mmo!) in anhydrous EtzO (2 mL) was added dropwise to
the solution
of the acceptor during I h. The mixture was stirred for 3 h at -40°C.
Triethylamine (100 uL)
was added and the mixture was filtered and concentrated. The residue was
eluted from a
column of silica gel with 85:15 cyclohexane-EtOAe to give 39 ( 107 mg, 44 %)
as a foam; [a]D
+12° (c 1; CHC~). 1H NMR (CDC~):8 7.1-8.1 (m, 45H, Ph), 6.50-7.00 (m,
4H, CHzPhOMe),
5.70-5.90 (m, IH, All), 5.32 (dd, 1H, J,a = 1_G, Jz,3 --- 3.1 Hz, H-2c), 5.10-
5.25 (m, 2H, All),
5.05 (d, IH, H-lB), 4.98 (d, 1H, J~~ = 3.2 Hz, H-Ifi), 4.85 (m, 2H, H-lA, Ic),
4.20-4.80 (m,
18H, CHzPh), 3.90-4.20 (m, 2H, All), 3.00-4.20 (m, 20H, H-2A, 28, ZF, 3,,, 3H,
3c, 3E, 4A, 4B,
4c, 4E, 5A, 5B, Sc, 5E, 6aE, Gbs, 0CH3), 0.82-I.30 (3 d, 9H, H-G,,, 6H, Gc).
~3C NMR (CDC)3):8
166.3 (C=O), 118.2-138.5 (Ph, All), 99.5, 99.3 (2C, C-IA, lg), 98.4 (C-IE),
96.4 (C-lc), 82.3,
81.4, 81.1. 80.5, 80.3, 79.5, 78.2, 77.6 (8C, C-2E, 3," 3B, 3c, 3E, 4~" 4H,
4c), 76.0, 75.5, 75.3,
74.9, 74.3, 73.3, 72.3. 71.8, 71.6, (CH~Ph), 72.5 (C-ZC), 72.0 (C-4E), 69.2,
69.0, 68.9 (3C, C-
SA> Se, 5~), 68.8, 68.6 (All, C-GE), 67.8 (C-5E), 55.5 (OCH3), 19.0, 18.8,
18.4 (3C, C-6~, G8,
6c). FAB-MS for C9BH,osOzo (M - 1603.8) mlz 1626.6 [M + Na]+. Correct elem.
analysis
could not be obtain for this compound.
Altyl (2-O-acetyl-3,4-di-O-benTyl-ac-I~rhamnupyranosyl)-(1-->3)-[2,3,4,b-tetra-
O-benzyl-
a-o-glucopyranosyl-(1-->4)]-2-0-benzoyl-a-L-rhamnopyranoside (42).
A mixture of alcohol 11 (G.5 g, 7.8 mmol) and imidate 2Z (G.5 g, I2.2 mmol) in
anhydrous
EtzO (86 mL) was stirred far 15 min under dry Ar. After cooling at -
50°C, MejSiOTf (560 p.
L, 3.1 mmol) was added dropwise and the mi,~cture was stirred and allowed to
rt overnight.
TriEthylamine (I.1 mL) was added and the mixture was concentrated. The residue
was eluted
from a column of silica gel with 6;1 cyclohexane-EtO.~c to give 42 (8.0 g, 84
%) as a colorless
foam; [aJo +2I° (c I, CHC13). ~H NMR (CDC13):8 7.1-8.2 (m, 35H, Ph),
5.95 (m, 1H, All),
5.72 (dd, I H, J,,z = 1.0, Jz,3 = 3.1 Hz, H-2B), 5.44 (dd, 1 H, J,,z = 1.6 Hz,
Jz,3 = 3.1 Hz, H-2e),
5.30 (m, 2H, All), 5.07 (d, 1H, J,,z = 3.05 Hz, H-lE), 5.05 (d, 1H, H-1H),
4.95 (d, 1H, Jt,z =
17
r.........~ _.
CA 02434685 2003-07-04
1.6 Hz, H-lc), 4.35-4.90 (m 12H, CHZPh}, 4.00-4.20 (m, 2H, All), 4.20 (dd, 1H,
J3,4 = 8.5
Hz, H-3c); 4.05 (dd, 1H, Jz,3 = 9-7, J3 4 = 10.0 Hz, H-3E}; 3.80-3.90 (m, 2H,
H-GaE, 6bE), 3.82
(m, 1H, Js.s = 6.0 Hz, H-5c), 3.80 (m, 2H, H-4~, 5E), 3.76 {m, 1H, H-4c), 3.75
(dd, 1H, J3,a =
8.SHz, H-3H), 3.69 (m, 1H, J4,s = 8.5, J5,6 = 6.1 Hz, H-5B), 3.53 (dd, 1H, H-
2E), 3.35 (dd, IH,
H-4B), 2.15 (s, 3H, OAc), 1.40 (d, 3H, H-6~), 1.01 (d, 3H, H-6B}. "C NMR
(CDCI3):8 170.3,
166.1 (C=0), 118.2-138.6 (Fh, All), 99.7 (C-1a), 98.6 (C-IE), 96.4 (C-1~),
82.2 (C-3E), 81.7
(C-2E), 80.2 (C-4H), 80.I (C-3~), 78.0 (C-4c), 77.8 (C-3H), 75.9; 75.4, 75.2,
74.3, 73.3, 70.9
(6C, Cl-IaPh), 72.5 (C-2c), 72.0 (C-4F), 69.0 (C-Sc), G9.0 (C-5$), 68.9 (2C,
All, C-2a), 68.0
(C-6E), 67.8 (C-5E), 21.1 {OAC), 19.0 (C-Gc), 18.1 (C-GB), FARMS of C,lH~eO~s
(M, 1198.5),
m/z 1221.4 ((M+Na~+).
Altyl (3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(la3)-[2,3,4,6-tetra-O-benzyi-a-D-
glucopyranosyl-(1~4)J-Z-O-benzoyl-a-L-rhamnopyranoside (10). A mixture of the
trisaccharide 42 (8.0 g, 6.5 mmol) in MeOH (128 mL) was treated with 5.7 mL of
HBF4/ExzO
at rt. The solution was stirred during 4 days. Et3N was added until
neutralization and
concentrated. The residue was diluted with DCM, washed with satd aq NaHC03 and
water.
The organic layer was dried on MgSO,, filtered and concentrated. The residue
was eluted from
a column of silica gel with 15:1 toluene-AcOEt to give 10 (6.31 g, 84 %) as a
foam; [a]p +14°
(c 1, CHC~);'H NMR (CDCIj):& 7.05-8.10 (m, 35H, Ph), 5.82 (m, IH, All), 5.25
(dd, IH, Jl.a
= 1.7 Hz, Jz,~ - 3.1 Hz, H-2c), 5.19 (m. 2H, All), 5.00 (d, 1H, J~,Z = 3.1 Hz,
H-Ir), 4.87 (d,
IH, Jt,a = 1.8 Hz, H-IB), 4.81 (d, IH, H-lc), 4.35-4.90 (m, 12H, CHzPh), 4.00-
4.20 (m, 2H,
All), 4.10 (dd, 1H, J~,,= 8.5 Hz, H-3c), 4.09 (dd, 1H, Ja.3 = 3.2 Hz, H-2s),
3.95 (m, IH, Ja,s =
9.5 Hz, H~5E), 3.92 (dd, 1H, Jz,~ = 9.5 Hz, J3,~ - 9,5 Hz, H-3s), 3.78 (m, 1H,
J5,6 = 6.0 Hz, H-
5c), 3.70 (m, 1H, H-4~}, 3.58-3.62 (m, 2H, H-GaE, bbfi), 3.59 (m, 1H. J4,5 =
9.0 Hz, J5,6 = 6.2
Hz, H-5g), 3.54 (dd, I H, H-4E}, 3.48 (dd, 1H, J;,4 = 8.5 Hz, H-3a), 3.45 (dd,
1 H, H-2~j, 3.31
(dd, I H, H-4B), 2.68 (d, I H, Jz,o,~ = 2.3 Hz, 0-H), 1.29 (d, 3H, H-6c), 1.09
(d, 3H, H-6H). ''~C
NMR (CDC13):d 166.2 (C=O), 118.2-137.5 (Ph, AII), 103.1 (C-18), 98.5 (C-la),
96.6 (GIc),
82.1 (C-3E), 81.4 (C-2a), 80.4 (G-4H), 79.7 (C-3H), 79.4 (C-4c), 78.9 (C-3c},
78.1 (C-4E),
76.0, 75.5, 74.5, 74.2, 73.6, 72.1 (CHaPh), 73.7 (C-2c), G8.9 (C-6E), G8.8 (C-
5B), 68.7 (All,
C-5s), 68.1 (C-5c), 19.1 (G-6c), 18.2 (C-6a). FABMS of C~oH»O~s (M, 1156.5),
m/z I179.5
([M+Na]'). Anal Calcd for C,oH~60,s: C, 72.64; H, 6.62. Found C, 72.49; H,
6.80.
18
CA 02434685 2003-07-04
Altyl (2-0-acetyl-3,4-di-O~benzyt-a-t,-rhamnopyranosyl)-(1 >2)-(3,4-dl-O-
benzyl-oc-L~
rhamnopyranosyl)-(1 >3)-j2,3,4,6-tetra-O-benzyt-a-D-glucopyranosyl-(1~4)]-2-0-
benzoyl-a-i,-rhamnopyranoside (44).
A mixture of alcohol 18 (5.2 g, 4.49 mmol), imidate 2 (3.58 g, 6.74 mtrwt) and
4A molecular
sieves in anhydrous Et20 (117 mL) was stirred for 1 h under dry ar. After
cooling at -30°C,
Me~SiOTf (580 EtL, 3.2 mmolj was added dropwise and the mi~.-ture was stirred
and allowed to
rt overnight. Triethylamine {1.2 mL) was added and the mixture was filtered
and concentrated.
The residue was eluted from a column of silica gel with 9:1 cyclohexane-EtOAc
to give 44
(G.1G g, 90 %); (a]D +13° (c 1, CHC)3). ~H NMR (CDC13):8 7,00-8.10 (m,
45H, Ph), 5.82 (m,
1 H, All), 5 .45 (dd, 1 H, J,,2 = 1.5 Hz, Js,~ = 2.5 Hz, H-2~), 5.29 (dd, 1 H,
Jl,z = I . 5 Hz, J23 = 2.5
Hz, H-2C), 5.19 (m, 2H, AII), 4.9? (d, 1H, J,,~ = 3.2 Hz, H-lE), 4.95 (d, 1H,
H-lA), 4.91 (d,
1H, Jt,z = 1.G Hz, H-Ie), 4.84 (d, 1H, H-lc), 4.35-4.90 (m, IGH, CHZPh), 4.29
(dd, 1H, Jz,3 =
2.6 Hz, H-2a), 4.00-4.10 (m, 2H, All), 4.02 (dd, 1 H, J~.4 = 8.5 Hz, H-3c),
3.90 (m, ZH, J~,3 =
Ja.4 = J4,s = 9.5 Hz, H-3E, Ss), 3.85 (m, 2H, J,,, = 9.3 Hz, J4,s = 9.5 Hz, H-
3", SA), 3.72 (m, 2H,
JS,h= 6.0 Hz, H-4c, 5c), 3.G2-3.6G (m, ZH, H-6aE, 6b~, 3.61 (dd, 1H, H-4E),
3.54 (dd, 1H, ,h,4
= 9.4 Hz, H-3Bj, 3.45 (dd, IH, J~,a = 9.5 Hz, J5,6 = 6.1 Hz, H-5B), 3.39 (dd,
1H, H-2s). 3.34
(dd, 1H, H-4,~, 3.21 (dd, 1H, H-4$), 1.89 (s, 3H, OAcj, 1.26 {2d, GH, H-G~,
Gcj, 0.89 (d, 3H,
H-6B). "C NMR (CDCl3):8 170.2, 166.1 (2C, C=0), 118.1-138.4 (Ph, Allj, 101.3
(C-18), 99.8
(C-lA), 98.2 (C-lE), 96.4 (C-lc), 82,Z (C-3s), 81.4 (C-2s), 80.G (C-4A), 80.5
(C-3~), 80.1 (C-
4g), 79.3 (C-3ej, 78.5 (C-4~). 78.1 (C-3A)> 78.0 (C-4E), 76.0, 75.9, 75.7,
75.2, 74.3, 73.3,
72.1, 71.1 (GH~Ph), 75.2 (C-2a), 72.9 (C-2o), 71.7 (C-5E), 69.5 (C-2p), 69.2
(2C, C-5,,, 5$),
68.9 (Atl, C-2H), 68.9 (C-GE), 67.9 (C-5C), 21.4 (OAc), 19.1 (C-GA), 18.7 (C-
Gcj, 18.1 (C-GH).
FABMS of C9oHiooOzo (M, 1524.7), m/z 1547.8 ([M+Na]+). Anal. Calcd for
C9~H,ooO~u: C,
72.42; H, 6.61. Found C, 72.31; H, 6.75.
Allyt (3,4-di-O-bcnzyl-a-~rhamnopyranosyl)-(1--~2)-(3,4-di-O-benzyi-a-t,-
rhamnopyranosyl}-(1-33)-[2,3,4,6-tetra-O-benzyl-a-n-glacopyranosyl-(1-~4))-2-0-
benzoyl-a-z-rhamnopyranoside (40),
A mixture of 44 (6.0 g, 3.93 mmol) in MeOH (200 mL) was treated with 10 mI, of
H8F4lEt~0
at rt. The solution was stirred during 5 days. Et3N was added until
neutralization and
concentrated. The residue was diluted with DCM, washed with satd aq NaHC03 and
water.
I
The organic layer was dried on MgS04, filtered and concentrated. The residue
was eluted from
i
I 19
CA 02434685 2003-07-04
a column of silica gel with 6:1 cyclohexane-AcOEt to give 40 (5.0 g, 84 %) as
a colorless
foam; [a]p +12° {c 1, CHCI~).'H NhiR (CDCh):o 7.00-8.00 (m, 45H, Ph),
5.83 (m, 1H, All),
5.29 (dd, 1H, J~,2= 1.8 Hz, J2,3= 2.9 Hz, H-2c), 5.19 (m, 2H, Alt), 4.99 {d,
1H, Jl~= 1.4 Hz,
H-1~), 4.97 (d, 1H, J ,,~= 3.3 Hz, H-lE), 4.94 (d, 1H, J,,a,= 1.7 Hz, H-18),
4.83 (d, 1H, H-lc),
4.35-4.90 (m, 16H, CHzPh), 4.30 (dd, 1H, J,"3 = 2.7 Hz, H-Zg), 4.00-4.I0 (m.
2H, Ah), 4.02
(dd, IH, Ja,3= 3.5 Hz, J3,q= 8.5 Hz, H-3c), 3.98 (dd, IH, H-2A), 3.91-3.95 (m,
3H, H-SE, 6aE,
Garb, 3.90 (dd, 1H, J,,, = 9.5 Hz, J3,q = 9.4 Hz, H-3E), 3.73-3.82 (m, 4H, H-
3A, 5", 4c, 5c),
3.GG (dd, 1H, J4,s = 9.6 Hz, H-4E), 3.53 (dd, IH, J~,4 = 9.5 Hz, H-3B), 3.48
(m IH, J4,s = 9.5
~~ Js,s= 5.1 Hz, H-SH), 3.40.3.44 (m, 2H, H-4A, 2E), 3.17 (dd, IH, H-4B), 2.18
(d, IH, JZ.os°
Z,0 Hz, 0-H), 1.26 (d, 3H, H-6c), 1.25 (d, 3H, H-6~), 0.90 (d, 3H, H-6H). "C
NMR (CDC13):
~ 1G6.2 (C=0), 118.0-138.3 (Ph, All). 101.5 (C-18), 101.4 (C-IA), 98.2 (C-lE),
96.4 (C-Ic),
82.2 {C-3s), 8I.4 (C-2~, 80.G (C-4A), 80.3 (C-48), 79.9 (2C, C-3c, 3~), 79.2
(C-3H), 78.3 (C-
4c), 78.0 (C-4E), 75.9, 75.6, 75.5, 74.8, 74.2, 73.5, 72.4, 71.0 (CHzPh), 75.3
(C-2B), 72.9 (C-
2c), 71.6 {C-2A), 69.2, G9.I, GB.G, 68.3, 67.9 (SC, C-5", 5a, Sc, SE, 6E),
68.9 (All), 19.1 (C-
6c), 18.6 {C-6A), 18.1 (C-6,~). FABMS of C9°H9g019 (VI, 1482.7), rrrlz
1505.8 ([M+Na]').
Anal. Calcd for CgpH98019~2H20: C, 71.12; H, 6.77. Found C, 71.21; H, 6.78.
Altyl (3,4,6-tri-O-acetyl-2-deoxy-Z-tric6loroacctamido-[3-D-glucopyranosyl)-(1-
-~2)-(3,4-
di-O-benzyl-a-t-rhamnopyranosyn-(1-~2)-(3,4-di-O-bcnzyl-oc-lfrhamnopyranosyl)-
(1-->
3)-[2,3,4,6-tetra-0-benzyl-a-n-giueopyranosyl-(1--~4)]-2~O-benzoyl-a-L-
rhamnapyranoxidt (4). A mixture of alcohol 10 (5.0 g, 3.37 mnwl), imidate 16
(3,0 g, 5.04
mmol) algid 4A molecular sieves in anhydrous DCM (120 mL) was stirred for I h
under dry Ar.
After cooling at 0°C, Me3SiOTf (240 uL, 1.32 mmol) was added dropwise
and the mixture
was stirred for 2.5 h while coming back to rt. Et3N (800 p.L) was added, and
the mi~cture was
filtered and concentrated. The residue was eluted from a column of silica gel
with 4:1 to 2:1
cycbhExane-EtOAc to give X4 (G.27 g, 98 %); [a]D +1.5° (c l, CHCIa). 'H
NMR (CDCl3):c5
7.00-8.00 (m, 45H, Ph), 6.68 (d, 1H, J2,~= 8.5 Hz, N-HD), 5.82 (m, 1H, All),
5.29 (dd, 1H,
J,,z = 1.0 Hz, J~,~ = 2.3 Hz, H-2c), 5.19 (m, 2H, All), 5.00 (d, 1H, J~,~ =
1.0 Hz, H-la), 4.96
(dd, 1 H, J2,3 = 10.5 Hz, J3,4 = 10.5 Hz, H-3D), 4.88 (d, 1H, J,,z = 3.3 Hz, H-
lc), 4.85 (d, 1H,
H-lc), 4.82 (d, IH, J,2= I.7 Hz, H-IB), 4.81 (dd, 1H, Je,s=10.0 Hz, H-4D),
4.72 (d, 1H, J~,~=
8.G Ha, H-ID), 4.35-4,90 (m, 1GH, CHZPh), 4.38 (m, 1H, H-2g), 4.00~4.10 (m,
2H, All), 4.05
(dd, 1 H. Ja,~ = 2.7 Hz, H-2A), 3.95 (dd, 1H, Ja,j = 3.5 Hz f3,4 = 8.5 Hz, H-
3c), 3.90 (m, 2H, H-
CA 02434685 2003-07-04
Se, 4E), 3.82-3.8G (m, 2H, H-ban, 6ho), 3.70-3.84 (m, 6H, H-3E, GaP, 6bs, 3,,,
SA, 2D), 3.68 (m;
1 H, H-Sc), 3.61 (dd, 1 H, Jd,$ = 9.0 Hz, H-4o), 3.SG (dd, I H, J3,4 = 9.5 Hz,
H-3H), 3.47 (rn, 1 H,
Ja,s = 9.5 Hz, Js,b = 6. I Hz, H-58), 3.33-3.35 (m, 3H, H-4~, Sp, 2F), 3.17
(dd, 1H, H-4g), 1.98,
2.00, 2.02 (3s, 9H, OA.c), 1.24 (d, 3H, J5,6= G.0 Hz, H-G,,), 1.23 {d, 3H,
Js.s= 5.9 Hz, H-6c),
0.90 (d, .3H, H-6g). '3C NMR (CDCl3):& 170.9, 170.7, 1G9.G, 166.1, 162.1
(C=0), 118.1-
138.3 (Ph, AII), IOLS (C-lD}, 102.4 (C-ls), 101.1 (C-IA), 98.5 (C-IE), 9G.4 (C-
lc), 92.G
(CC)3), 82.1 (C-3e), 81.7 (C-3c}, 8L6 (C-2s), 80.4 (C-4s), 80.1 (C-3,,), 79.1
(C-4c), 78,5 (C-
3a), 77.9 (C-4,~), 77.G {C-4E), 76.4 (C-2,,), 7G.1, 75.8, 75.4, 74.7, 74.3,
74.2, 73.2, ?0.4
(CHZPh), 74.9 (C-2H), 72.9 (C-3D), 72.7 (C-2G): 72.5 (C-So), 71.9 (C-5E), G8.4
(C-G~, 68.8
(A!1), G8.9, 68.7, 68.5, 67.7 {4C, C-4c, S", 5B, Sc), G2.2 (C-6p), 56.2 (C-
2D), 20.9, 20.9, 20.7
(3C, OAc), 19.0 (C-GA), 18.5 (C-G~), 18.2 (C-GH). FABMS of C,o4H"4C1,N0~~ (Vf,
1916.4):
m/z I938.9 [M+Na]+. Anal. Calcd for CIO4H, iaCIJNOz?: C, 65.18 ; H, 6.00 ; N,
0.73. Found C,
64.95 ; H, 6.17 ; N, 0.76.
(2,3,4-tri-O-acetyl-a-deoxy-Z-trichloroncetamido-~i-D-glucopyranosy I)-(1-->2)-
(3,4-di-O-
benzyl-a-C,-rhamnopyranosyl)-(1 >2)-(3,4-di-O-benzyl-a-z-rhamnopyranosyl)-(1--
~3)-
(Z,3,4,6-tetra-O-benzyl-a-D-glucapyranosyl-(1-~4)]-2-O-benzoyl-a-
crrhamnopyranosyl
trichloroacetlmidate (46). Compound 4 {3.S g, 1.8 mmvl) was dissolved in
anhydrous THF
(35 mL). The solution was degassed and placed under Ar. 1,5-Cyclooctadiene-
bis(methyldiphenylphosphine}iridium hcxafluorophosphate (81 mg) was added, and
the
solution was degassed again. The catalyst was activated by passing over a
stream of hydrogen
until the solution has turned yellow. The reaction mixture was degassed again
and stirred under
an Ar atmosphere, then concentrated to dryness. The residue was dissolved in
acetone (15
mL). then water (3 mL), mercuric chloride (490 mg) anti mercuric oxide (420
mg) were added
successively, 'The mixture protected from light was stirred at rt for 2 h and
acetone was
evaporated. The resulting suspensinnwas taken up in DCM, washed twzce with 50%
aq KI,
water and satd aq NaCI, dried and concentrated. The residue was eluted from a
column of
silica gel with 2:1 petroleum ether-EtOAe to give the corresponding hemiacetal
45.
Trichloroacetonitrile (G.5 mL) and DBU (97 p.L) were added to a solution of
the residue in
anhydrous diehloromethane (33 mL) at 0°C. After 1 h, the mixture was
concentrated. The
residue was eluted from a column of silica geI with 5:2 cyclohexane-EtOAC and
0.2 % Et3N to
give 46 (2.48 g, 6G °!°); [a]~ -~4° (e 1, CHCu). 'H NMR
(CDCl3):S 8.71 (s, 1H, N=H), 7.00-
21
l,rvW uvcvy-u.- ...
CA 02434685 2003-07-04
8.00 (m, 45H, Ph), 6.80 (d, IH, Jz,~,~., = 8,2 Hz, NHp), G.37 (d, 1H, J,,z =
2.6 Hz., H-lc), 5.59
(dd, 1H, Jz,; = 3.0 Hz. H-2c), 5.10 (d, 1H, Ji,z = I.0 Hz, H-lA); 5.05 (dd, I
H, H-3D), 4.98-5.00
(m, 2H, H-lE, ls), 4.97 (dd, 1H, H-4n), 4.00-5.00 (m I9H, 8 CHaPh, H-3c, 2A,
2$), 3.20-4.00
(m, 17H, H-2E, 3E, 4E, 5E, Gas. 6bE, 4c, Sc, 3B, 4a, 5s~ 3~, 4n, $na Sa~ 6ao,
6bn), 1.80, 2.02, 2.03
(3s, 9H, OAc), 1.39, 1.32 and I .00 (3d, 9H, H-6~" 6s, 6c). i3C NvLR (CDC~):8
169.7, 169.5,
168.3, 164.5, 160.9 (C=0, C=N), 126.2-137,5 (Ph), lOI.G (C-1D), 101.3 (2C, C-
1~, ls), 98.7
(C-IE), 94.8 (C-lc), 91.3 (CCI~), 82.1, 81.5, 80.4, 80.1, 78.4, 77.9, 77.6,
76.5 (IOC, C-2A, 2E,
3,,, 3B, 3c, 3E, 4A, 4B, 4c, 4E), 76.0, 75.9, 75.5, 74.9, 74.3, 73.3 (CH2Ph),
72.9, 72.6, 71.9,
70.9, 70.d, 69.I, 68.8, 68.5 (9C, C-2B, 2c, 3p, 4D, SA, 5a, Sc, SD, 5~), 68.3,
62.1 (2C, C-6D, 6E),
56.2 (C-2D), 21.0, 20.9, 20.8 (3 OAc), 19.1, 18.3, 18.1 (3C, C-6,,, 6B, 6c).
Anal. Calcd for
C103H110C~N2O27 C: 61.22, H: 5.49, N: 1.39. Found C: 6I.24. H. 5.50, N: 1.21.
Methyl (3,4,6-tri-O-acetyl-Z-deo~y-Z-trichioroacetamido-[i-n-glucopyranosyl)-
(1--~Z)-
(3,4-di-O-benryl-a-z-rhamnopyranosyl)-(1--~2)-(3,4.di-O-benzyl-a-z.-
rhamnopyranosyl)-
(1-a3)-[Z,3,4,6-tetr8-O-benzyl-a-n-glucopyrenosyl-(1-~4)]-2-O-benzoyf-a-L-
rhamnopyranosyl)-(1-~3)-(Z-dcory-4,6-O-isopropyliden~2-tricttloroacetamido-j3-
n-
glucopyranosyl)-(1-i2)-(3,4-di-O-benzyl-a-L-rhamnapyranosyl)-(1--~2)-(3,4-di-D-
bcnzyl-
a-L-rhamnopyranosyl)-(1-~3)-[2,3,d,6-tetra-O-benzyl-a-D-glucopyranosyl-(1-~4)]-
2-O-
benzoyl-a-Irrbamnopyranoside (49), A mixture of 46 (154 mg, 76 p.mol) and 48
(92 mg, 51
~.tnal). 4A molecular sier,~es and dry 1,2-DGE (3 mL), was stirred for 1 h,
then cooled to -
35°C. Triflic acid (6 ~.L) was added. The stirred mixture was allowed
to reach 10°C in 2.5 h.
Et3N (25 ~.L) was added and the mixture was filtered. After evaporation, the
residue was
eluted from a column of silica gel with 2:1 eyclohexane-EtOAC and 0.5 % of
Et3N to give 49
which could not be obtained as pure material at this stage, and was directly
engaged in the next
reaction.
Methyl (3,4,6-tri-O-acetyl-Z-deoay-Z-trichloroacetamido-[i~D-glucopyranosy~-(1-
~2)-
(3,4-di-O-benzyl-a-z-rhamnopyranosyl)-(I--~Z)-(3,4-di-D-benzyl-a-L-
rhamnopyranosyn-
(1--~3)-[2,3,4,6-tetra-O-benryl-a-v-glucopyt~tnosyl-(1-~4)]-(z-O-benzoy 1-a-L-
rhamnopyranosyt)-(1-~3)-(Z-deoxy-2-trichioroacetamido-[i-D-glucopyranosyl)-(1-
~3)-
(3,4-di-O-benzyl-oG-L-rhamnopy~nosyn-(1-~2)-(3,4-di-O-benzyl-a-z-
rhamaopyranosy ~-
(1-~3)-[Z,3,4,6-tetra-O-bcnzyl-a-n-glucopyranosyl-(x-~4)]-2-O-benzoyl-a-L-
22
LMYltvcay~um. _.._
CA 02434685 2003-07-04
rhamnopyranoside (50). To a solution of the residue 49 (I86 mg, 51 ~mol) in
DCM (3 mL)
was added dropwise, at 0°C, a solution of TFA (0.5 mL) and water (0.5
mL). The mixture was
stirred for 3 h, then concentrated by co-evaporation with water then toluene.
The residue was
eluted from a column of silica gel with 2:1 to 1:1 petroleum ether-EtOAC to
give 50 (134 mg,
72 %, 2 steps); [a]p +G° (c l, CHCl3).'H NMR (CDCI~); 8 7.10-8.05 (m,
90H, Ph), 6.82-6.8G
(2d, 2H, Jz,~ = 8.0 Hz, J~,r,~.~ = 8.5 Hz, NHo, NHb~}. S.I9-5.35 (m, 2H, H-2c,
2c~), 5.20, S_OS
(2s, 2H, H-lA, 1"~), 5.05 (dd, 1H, H-3p~), 4.99-4.80 (m, 9H, H-18, la~, lc,
Ic~, 1D, 1D-, IE, Is~,
4D~), 4.30-4.80 (m, 32H, OCHzPh), 3.15-4.10 (m, 44H, H-2A, 2A~, 2g, 28., 2D,
2D~. 2E, 2E~, 3w,
3AV 3B> 3s', 3c, 3c~, 30> 3s: 3s~: 4a~ 4w, 4s~ 4av 4ct 4cv 4p~ 4s~ 4EV Sn~ Sav
~s. Ssv Sc, Scv SD,
SD~, SE, SE~. 6ao, 6bo, Gao~, 6bD~, 6aa, 6bs, 6aE~, 6br~), 3.42 (3H, s, OMe),
2.02. 2.04, 2.08 (9H,
3s, OAc}, 1.40-0.96 (I8H, m, H-6A, G,,~, 6H, 68~, Gc, 6c.). 13C ~ (CDCIs) : S
171.5, 170.9,
170.8, 169.6, 166.2, 162.4, 162.1 (C=O), 127.2-139.5 (Ph), 10I.9, IOI.G,
101.5, 101.3, 99.2,
98.8, 98.2 (lOC, C-lA, 1,~~, la, la~, Ic, lc~, 1D, 1D~, lE, IE,), 92.7, 92.6
(2C, CCl3}, 82.1, 81.8,
81.7, 80.5, 80.3, 80.1, 79.3, 77.9, 77.8, 73.0, 72.6, 72.5, 72Ø 69.4, 69.0,
68.9, 67.4, (39C, C-
2n~ 2w. 2s, 2a~, 2c, 2r, 2s, 2s~, 3n~ 3av 3s~ 3av 3c~ 3cv 3D~ 3D°~ 3c,
3ev 4A, 4nv 4s~ 4ae 4c~ 4ce
40, 4D~, 4Ea 4E~, Sn, SA', Ss~ Sav 5c, 5c~, SD, 5p', 5E, Sa~, Gp-), 76,0,
75.9. 74.8, ?4.3, 73.6, 73.2.
68.6 (CHzPh), 62.3, 62.2, 60.7 (3C, C-Gp, GF, 6E~), SS_5, SG.2 (3C, C-2D, 2D,,
OCH~), 20.97,
20.94, 20.77 (OAc), 19.01, 18.72, 18.62, 18.15, 17.90 (GC. C-Ga, 6,,~, 6H,
6g~, 6c, 6~~).
FABMS for Cy9,H~,aCIaNzOso (M, 3622.5), mla 3645.3 [M+Na]+. Anal. Calcd for
C~97H114C~Ny050 C: 65.32, H: 5.95, N: 0.77. Found C: 65.20, H: 6.03, N: 0.78.
Methyl (2-acetamido-2-deoxy-[3-D-glucapyranosyl)-(I >2)-(a-L-rhamnopyrano9yl)-
(1-~
2)-(a-L-rhamnopyranosyl)-(1-->3)-[a-n-glucopyranosyl-(1~4)J-(a-L-
rhamnopyranosyl)-
(Z-~3)-(2-acetamido-2-deo~,y-(i-n-glucopyranosyl)-(1--~2)-(a-z-
rhamnopyratnosyl)-(1-->
Z)-(a-L-rhamnopyrvnosyl)-(r-~3)-[a-D-glucopyraaosyi-(1 >4))-a-t,-
rhamnopyranosidc
(1). A solution of 50 (183 mg, 50 ~.mol), in EtOH (3 mL), EtOAc (0.3 mL}, 1M
HCI (100 p.L)
was hydrogenated in the presence of Pd/C (2~0 mg) for 72 h at rt_ The mixture
was filtered
and concentrated. A solution of the residue in MeOH (4 mL) and Et3N (200 uL)
was
hydrogenated in the presence of PdIC (200 mg) for 24 h at rt. The mixture was
altered and
concentrated. A solution of the residue (50 mg, 25 p.mol) in MeOH (3 mL) and
DCM (0.5 mL}
was treated by MeONs until pH=10. The mixture was stirred overnight at
55°C. After cooling
at rt, IR 120 (H") was added until neutral pH, and the solution was filtered
and concentrated,
23
v,.,. , , .,...,. _. _ _
CA 02434685 2003-07-04
then was eluted from a column of C-18 with waterJCH3GN and freeze-dried to
afford
amorphous I (30 mg, 37 %), [aJp -1° (c 1, Hz0). 'H NMR (D20): 8 S.i3
(2d, 2H, J,,z =
3.5Hz, H-lE, IE~), 4.75, 4.95, 5.05 (m, 5H, H-lA, 1a, lA~, IB~, lc-),
4.62~4.64 (2d, 2H, J,,i= 7.0
Hz, J~,I= 8.0 Hz, H-la, ID~), 4.58 (d, 1H, J,,z= 2.2 Hz, H-lc), 3.20-4.10 (m,
S1H, H-2A, 2A~,
2e, 2a', 2c, 2cv 2n, 2av 2E, 2EV 3a, 3nv 3a, 3e', 3c, acv 3n, 3v~, 3e,
3e°, 4A, 4w, 4s, 4BV 4c, 4cv
4D, 4av 4E~ 4EV 5A, 5nr sa, SH~, 5c, 5cv 5D, Sov 5a, SE', 6aD, 61~. 6aw,
6tfi~, 6aE, 6bE, 6aE~, 61~~,
OCH3), 1.97, 1.99 (2s, 6H, 2 AcNH), 1.I5-I.33 (6d, 18H, J5,6 = 6.3Hz, H-6A,
68, 6c, 6n', 6sv
6c-). "C NMR (D~0): 8 175.2, 174.7 (C=0), 103.1 (2C, C-1D~, Ia), 102.6; 101.7,
101.3,
100.8 (6G, C-lA, 1B, lc, lA~, la~, lc,), 98.0 (2C, C-lE, lE~), 81.6, 79.7,
79.G, 79.1, 76.2,, 76.1,
73.9, 73.0, 72.7, 72.6, 72.5, 72.2. 72.1, 71.6, 70.1, 70.0, 69.7, 69.0, 68.5
(38C, C-2A, 2A., 2a,
2H~~ 2c~ 2c~, 2F, 2F.~, 3n, 3w, 3B, 38~~ 3c, acv 3a, 3a°, 3g, 3s~, 4n,
4w. 48, 4s~, 4c, 4c~, 4D, 4av 4e,
4E~, 5A, SA~, 5a, SB., 5c, 5c., 5p, Sp~, 5E, 5a.), 60.9 (4C, C-6E, GE~. 6a,
6p~), 56.20, SG.00, 55.31
(3C, C-2p, 2a~, OCH,), 22.7, 22.6 (2C, AcNH), 18.3, I8.1, 17.2, 17.1, 16.95,
16.90 (6C, C-
6A, 68, 6c, 6,,', 6H', 6c~)- HR~1LS: calculated for CssH~ioNaO<s+Na:
1661.6278. Found
1661.6277.
24
CA 02434685 2003-07-04
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CA 02434685 2003-07-04
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CA 02434685 2003-07-04
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CA 02434685 2003-07-04
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CA 02434685 2003-07-04
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CA 02434685 2003-07-04
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CA 02434685 2003-07-04
L, M~,Pl l
Synthesis of a tetra- and two pentaaaccharide fragments of the O-specific
polysaccharide
of Sfiigella Jtexneri serotype 2a
This paper discloses the synthesis of a methyl glycoside of the repeating unit
I of the S
~lexneri Za O-SP. together with that of a corresponding pentasaccharide and a
tetrasaccharide.
All the methyl glycosides of the di- to pentasaccharides obtained by circular
permutation of
the monosaccharide residues partaking in the linear backbone of I, and
comprising the EC
portion. are now available. Their binding to a set of available protective IgG
antibodies is
reported elsewhere
LM?Pll~tAeo~brcvet-pent~OMe cA o24s4ss5 zoos-o~-o4
Synthesis of a tetra- and two pentasaccharide fragments of the O-specific
polysaccharide
of Shigella Jlexneri serotype 2a
INTRODUCTION
Shigellosis, also known as bacillary dysentery, is a major enteric disease
which
accounts for some 165 million annual episodes, among which 1.1 million deaths,
occurring
mostly in developing countries. {Kotloff, 1999 #147} Young children and
immunocompromised individuals are the main victims. Occurrence of the disease
is seen as a
correlate of sanitary conditions, and those are not likely to improve rapidly
in areas at risk. The
financial status of the populations in which shigellosis stands in its endemic
or epidemic forms,
as well as the emerging resistance to antimicrobial drugs, {Khan, 1985 #3G7} *
; Salam, 1991
#368} * {Ashkenazi, 1995 #328} * {Iversen, 1998 #357} * {Iwalol''un, 2001
#358} * limit the
impact of the latter. Some 15 years ago, vaccination was defined as a priority
by the WHO in
its program on enteric. diseases.REF However, there is still no license
vaccine against this
bacterial infection although intensive research is ongoing in the field.{Hale,
1995 #102}(voir
si ref reeente PS) Shigellae are Gram negative bacteria. As for other
bacterial pathogens, their
lipopolysaccharide (LPS) is an important virulence factor. It is also a major
target of the host's
protective immunity against infection. Indeed, data from infected patients
indicated that
circulating anti-LPS antibodies were strong markers of acquired immunity.
{Cohen, 1988
#329} {Cohen, 1991 #52} It was also demonstrated in a marine model that the
presence
locally, preliminary to infection, of a secretory antibody of isotype A
specific for an epitope
located on the O-specific polysaccharide (O-SP) moiety of the LPS of
Shigella,~lexrteri Sa,
prevented any host's homologous infection.{Pha(ipon, 1995 #228) Importantly,
field trial of an
investigaiional Shigella sonnei 0-SP conjugate, which was shown to induce anti-
LpS secrewry
IgAs, thus suggesting mucosal stimulation,{CohEn, 1996 #360; demonstrated 7S%
efficacy.{Cohen, 199? #54}
Shigella flexneri 2a is the prevalent serotype in dEVeloping countries, where
it is
responsible for the endemic form of the disease. Based on the early hypothesis
that a critical
LMPP77-~1~0'D~OVM~p~tItBOMC ~ 02434685 2003-07-04
level of serum IgG antibodies specific for the 0-specific polysaccharide (0-
SP) moiety of the
LPS Was sufficient to confer protection against homologous
infections,{Robbins, 1992
#256} {Robbins, 1994 #2S7} several S. ,~lexneri 2a 0-SP-protein conjugates
were designed.
They were found safe and immunogenic in both. adults and children.{Ashkenazi,
1965
#362} {Passwell, 2001 #220}
Allowing a better cornrol of the various structural parameters possibly
involved in the
immunogenicity of glycoconjugate vaccines, oligosaccharide-protein conjugates
were
proposed as alternatives to polysaccharide.-protein conjugate vaccines against
bacteria. {Pozsgay, 2000 #247} Indeed, such constructs were found immunogenic
on several
occasions, including examples whereby the oligosaceharide portion was made of
one
repeating unit only.~Benaissa-Trouw, 2001 #363}{Mawas, 2002 #364} We reasoned
that
glycoconjugates incorporating chemically synthesized oligosaeeharides,
appropriately
selected for their ability to mimic the native O-SP in terms of both
antigenicity and solution
conformation, may offEr an alternative to the S ,~lexneri 2a O-SP-protein
conjugates currently
under study, Our approach relies on a rational basis. Indeed, in order to
select the best
oligosaccharide mimic, we have undertaken the characterization of the
antigenic determinants
of S. j<lexneri 2a 0-SP recognized by serotype-specific protective monoclonal
antibodies. The
synthesis of a panel of methyl glycoside oligosaccharides representative of
fragments of S.
flexneri 2a 0-SP was thus undertaken to be used as probes in the study of
antibody
recognition.
A B E C D
2)-a-L-Rhap-(1--~2)-a-L-Rhap-(1-~3)-[a-D-Glcp-(I-~4)J-a-L-Rhap-(1~3)-[i~D-
GIcNAcp(1 ~
The 0~SP of S ,flexnerf 2a is a heteropolysaccharide defined by the
pentasaccharide
repeating unit T.{Simnwns, 1971 #88; Lindberg, 1991 #46} It features a linear
tetrasaccharide
backbone, which is common to all f. ,~lexneri O-antigens and comprises a N
acetyl
glucosamine and three rhamnose residues, together with an a-D-glucopyranose
residue
branched at position 4 of one of the rhamnoses. We have already reported on
the synthesis of
the methyl glycosides of various fragments of the 0-SP, including the known EC
i
i disaccharide, {Henry, 1974 #224; Lipkind, 1987 #223 } f Mulard, 2000 #52}
the ECD (Mulard,
2000 #52} and B(E)C{Mulard, 2000 #52} trisaccharides, the ECDA{Segat, 2002
#225} and
AB(E)L{Costachel, 2000 #I01} tetrasaccharides, the B(E)CDA{Segat, 2002 #225}
and
DAB(E)C {Costachel, 2000 #101 } pentasa,ceharides and more recently the
B(E)CDAB(E)C
octasaccharide.{Belot, 2002 #314} In the following, we report on the synthesis
of the
2
LMPPt 1~the°-b«VCt~pCfl~lOM~ ~ 02434685 2003-07-04
ECDAH, AB(E)CD pentasaeeharides as well on that of the B(E)CD tetrasaccharide
as their
methyl glycosides, 1, a and 3, respectively.
RESULTS AND DISCUSSION
Analysis of the targets shows that all the glycosylation reactions to set up
involve 1,2-trans
glycosidie linkages except for that of the E-C junction which is 1,2-cis.
Consequently, the
syntheses described herein rely on key EC disaccharide building blocks as well
as on
appropriate A, H and D monosaccharide precursors.
Synthesis of the linear ECDAB-OMe pentasaccharide (l): Based on earlier
findings in the
series which have demonstrated that the C-D linkage was an appropriate
disconnection
site,{Segat, 2002 #283} a blockwise synthesis of 1 was designed (Scheme 1). It
is based on
the glycosylation of the known EC trichloroacetimidate donor XX,{Mulard, 2000
#19G}
obtained in three steps (69%) from the key intermediate XX,{Segat, 2002 #283}
and the DAB
trisaccharide acceptor XX. The latter was obtained by the stepv~~ise
condensation of known
monosaccharide precursors, readily available by selective protection,
deprotection and
activation sequences. Thus, TMSOTf catalysed condensation of the
rhamnopyranoside
acceptor XX{Pozsgay, 1987 #241 } with the trichloroacetimidate donor XX
{Castro-Palomino,
1996 #47} in diethyl ether to give the fully protected rhamnobioside XX, and
subsequent de-
O-acetylation under Zempl~n conditions gave the AB disaccharide acceptor XX in
XX%
overall yield, which compares favourably with the previously described
preparation. {Pozsgay, 1987 #, 241 } Conventional glycosylation o.f XX with
the known
glucosaminyl bromide,{Debenham, 1995 #63}chosen as the precursor to residue D,
under
base-deficient conditions in order to avoid orthoester formation, smoothly
afforded the fully
protected DAB trisaccharide (XX%). Removal of 'the tetrachlorophtalimide and
concomitant
deacetylation by action of XXX in XXX, followed by N acetylation furnished the
triol XX
(XX%), which was next protected at positions 4D and 6o by regioselective
intxoduction of an
isopropylidene acetal upon reaction with 2,2-dimethoxypropane under acid-
catalysis (XX%).
(mentiohner produit vert fluo) Indeed, data previously obtained when
synthESizing shorter
fragments in the series outlined the interest of using 4,G-D-isopropylidene-
glucosami,nyl
intermediates instead of the more common benrylidene analogs.{Mulard, 2000
#196} Once
the two key building blocks made available, their condensation was performed
in
dichloromethane in the presence of a catalytic amount of TMSOTf to give the
fully protected
pentasaccharide XX (XX%). Conventional stepwise deprotection involving (i)
acidic
3
LMPPII-thco~brevtt~pentaOMe cA o24s4ss5 zoos-o~-o4
hydrolysis of the isopropylidene acetal using 90% aq TFA to give diol XX
(XX%), (ii)
conversion of the latter into the corresponding tetraol XX under Zempl6n
conditions (XX%),
and (iii) final hydrogenolysis of the benzyl protecting groups, gave the
linear pentasaecharide
I in XX% yield.
Synthesis ojthe AB(E)C,D perttasaccharide 2 acrd of the B(E)CD tetrasaccharide
3 (Scheme
2). For reasons mentioned above, compound XX,{Mulard, 2000 #196} protected at
its 4 and 6
hydroxyl groups by an isvpropylidene acetal was the precursor of choice for
residue D. In the
past, inuoduction of residue B at position 3~ was performed on a 2~-O-
benzoylated EC
acceptor resulting from the regioselective acidic hydrolysis of the
corresponding 2,3-
orthoester intermediate.{Costachel, 2000 #55} ~Segat, 2002 #283} It rapidly
occurred to us
that opening of the required phenyl orthoester was not compatible with the
presence of an
isopropy'lidene a.cetal. For that reason, the trichloroacetimidate donor XX,
suitably
benzoylated at 7.c and orthogonally protected by a chloroacetyl group at
position 3c was used
as the EC building block instead of the previously mentioned XX. The choice of
protecting
group at position 2 of the rhamnosyl precursor to residue B was again crucial
in the synthesis
of 2. Indeed, most of our previous work in the series relied on the use of the
known 2-0-
acetyl rhamnopyranosyl donor XX, {Castro-Palomino, 1996 #47} as an appropriate
precursor
to residue B. In the reported syntheses.{Costachel. 2000 #55} selective 2a~0-
deacetylation
the presence of a 2c-O-benzoate was best performed by treatment with
methanolic IIBF4.OEtz
for 8ve days, Clearly, such deacetylation conditions are not compatible with
the presence of
isopropylidene group on the m~olecuIe either. To overcome this limitation, the
corresponding
2-0-chloroacetyl rhamnopyranosyl trichloroacetimidate XX was selected as an
alternate
donor. The latter could indeed also serve as an appropriate precursor to
residue A.
Regioselective conversion of diol XX into its 2-D-benzoylated counterpart XX
was performed
as described. {Segat, 2002 #283; Treatment of the latter with chloroacetic
anhydride and
pyridine gave the orthogonally protected XX (XX%), which was smoothly de-0-
allylated to
yield the corresponding hemiacetal XX (XX%) by a two-step process, involving
(i) iridium
(I)-promoted isomerisation{Oltvoort, 1981 #216} of the allyl glycoside and
(ii) subsequent
hydrolysis in the presence of iodine.(Re~ ef Nacira) The selected
trichloroacetimidate leaving
group was successfully introduced by treatment of XX with
trichloroacetonltrile in the
presence of DBU, which resulted in the formation of XX (3CY%). TMSOTf mediated
glycosylation of donor XX and acceptor XX furnished the fully protected ECD
trisaccharide
(XX%), which was as expected readily converted to the required acceptor XX
upon selective
4
CA 02434685 2003-07-04
LMPP1 i.theo.brwa-pa°taOMa
deblocking of the chloroacetyl protecting group with thiourea (XX%). Following
the two-step
protocol described above for the preparation of XX, the known allyl
rhamnopyranoside
XX, { Westerduin, 1988 #348 ) bearing a 2-O-choloacetyl protecting group, was
converted to
the hemia~cetal XX (XX%). Next; treatment of the latter with
trichloroacetonitrile and a slight
amount of DBU ga~~e donor XX in an acceptable yield of XX%. Glycosylation of
the ECD
acceptor XX and the B donor XX was attempted under various conditions of
solvent and
catalyst. Whatever the conditions, hardly separable mixtures of compounds were
obtained.
When using TMSOTf as the promoter and X.XX as the solvent, the expected
tetrasaccharide
XX was indeed formed, although it was often markedly contaminated with
glycosylation
intermediates such as the. silylated XX as well as the. orthoester side-
product XX, as suggested
from mass spectroscopy analysis and NMR data. In fact, the nature of the
latter was fully
ascertained at the next step in the synthesis. Indeed, treatment of a miuture
of the
condensation products XX and supposedly XX resulted in the expected
tetrasaccharide
acceptor XX contaminated by the trisaccharide a.eceptor XX, whereas the
corresponding ~3B-
tetrasaccharide isomer could not be detected at this stage, which indicated
that the
corresponding ehloroacetylated XX was not part of the initial mixture.
Formation of the
starting XX during the dechloroacetylation step is not unexpected as it may be
explained by
intramolecular rearrangement Leading to expulsion of the B residue, following
dechlorination
in the presence of XXX. Starting from XX, the isolated yield of the
tetzasaccharide acceptor
XX was XX%, which encouraged us to reconsider the use of the 2-O-acetyl
analogue XX as a
precursor to residues B and A in the synthesis of 2.
A suivre ...
CONCLUSION
The synthesis of the methyl glycoside (2) of the reputing unit I of the S
~l'exrreri 2a
O-SP, togethez with that of the corresponding pentasaccharide 1 and
tetrasaeeharide 3 were
described. All the methyl glycosides of the di- to pentasaccharides obtained
by circular
permutation of the monosaccharide residues partaking in the linear backbone of
I, and
comprising the EC portion, are now available in the laboratory. Their binding
to a set of
available protective IgG antibodies will be reported elsewhere.
LMPP1 I-exp-brevet-pentaOMe
EXPERIMENTAL
CA 02434685 2003-07-04
General Methods. General experimental methods not referred to in this section
were
as described previously.(REF) TLC on precoated slides of Silica Gel 60 FZSa
(Merck) was
performed with solvent mixtures of appropriately adjusted polarity consisting
of A,
dichloromethane-methanol; B, cyclohexane-ethyl acetate, C, cyclohexane-diethyl
ether, D,
water-acetonitrile, E, iso-propanol-ammonia-water. F, cyclohexane-diethyl
ether-ethyl acetate.
Detection was effected when applicable, with UV light, and/or by charring with
orcinol (3S
mM) in 4N aq HZS04. In the NMR spectra, of the two magnetically non-equivalent
geminal
protons at C-6, the one resonating at lower field is denoted H-6a and the one
at higher field is
denoted H-6b. Interchangeable assignments in the 13C NMR spectra are marked
with an
asterisk in listing of signal assignments. Sugar residues in oligosaccharides
are serially lettered
according to the lettering of the repeating unit of the O-SP and identified by
a subscript in
listing of signal assignments. Low-resolution mass spectra were obtained by
Either chemical
ionisation (CIMS) using NH3 as the ionising gas, by electrospray mass
spectrometry (ESMS),
or by fist atom bombardment mass spectrometry (FARMS).
Methyl (2-acetamido-2-deoay-4,6-O-isopropylidene-(i-D-glucopyranosyl)-(1-~2)-
(3,4.~di-O-
benzyi-ot-L-rhamnopyranocyl)-(1~2)-(3,4-dr-0-benzyl-a-L-rhamnopyranoside (XX).
2,2-
dimethoxypropane (4.9 mL. 39.8 mmol) and para-toluenesulfonic acid (18 mg, 95
pmol) were
added to a solution of the triol XX (964 mg, 1.09 mmol) in acetone (3 mL) and
the mixture was
stined at rt for lh. Et3N was added, and volatiles were evaporated. Column
chromatography of the
residu~ (solvent D, 99:1) gave the acceptor XX as a white solid (969 mg, 96%)
which could be
crystallized from AcOEt:iPrzO; mp XX°C; [a]D XX (c 1.0); NMR: ~ H, 8
7.45-7.31 (m, 20H, Ph),
6,98 (d, 1 H, JrrE.t,i = 2.4 Hz, NH), G.37 (bs, 1 H, OH), 5.07 (d, 1 H, Jm =
1.9 Hz, H-I,~, 4.90 (d, 1 H,
J = 10.8 Hz, OCHZ), 4.85 (d, 1H, J = 10.1 Hz, OCH~), 4.84 (d, 1 H, J = 10.8
Hz, OCHZ), 4.76 (d,
1 H, OCHZ), 4.69 (d, 1 H, OCHz), 4.68 (s, 2H, OCHZ), 4.G5 (d, 1 H, OCH?), 4.61
(d, 1 H, Ji,~ = 1.6
Hz, H-1 H), 4.48 (d, 1 H,1~ ~ = 8.3 Hz, H- l o), 4.09 (dd, 1 H, H-2,~, 4.01
(dd, 1 H, J2,3 = 3.2, J3,4 = 9.4
Hz, H-3,~, 3.91 (dd, 1H, H-28), 3.89-3.84 (m, 2H, J5,6 = 6.3, J4,s =
9.4,1i~,3~ = 3.3, J3,,d~ = 9.4 Hz, H-
5~,, 3H), 3.68 (dq, partially overlapped, Js,b = 6.2, Ja,s = 9.5 Hz, H-S$),
3.66-3.58 (m,. 4H, H-6aD,
6bn, 20, 4D), 3.44 (pt, 1H, H-4,~, 3.41 (pt, 1H, H-4$), 3.32 (s, 3H, OCH3),
3.16 (m, 1H, H-SD),
1.60 (s, 3H, C(0)CH3), 1.54, 1.48 (2s, 6H, C(CH3jZ), 1.35 (d, 6H, H-6A, 6B);
13C, S 173.9 (CO),
138.8-128.0 (Ph), 103.7 (C-lv), 101.3 (C-1~, 100.3 (C(CH3)2), 100.2 (C-1B),
81.9 (C-4,~, 80.8
(C-4H), 80.5 (C-3,~, 79.7 (C-3g), 79.4 (C-2,,), 76.2 (OCHZ), 76.0 (C-2H),
75.6. 75.I (2C, OCHZ),
74.4 (C-4D), 74.4 (C-3D), 72.6 (OCHZ), 68.6 (C-5~, 68.0, 67.9 (2C, C-Sg, SD),
62.2 (C-GD), 60.6
(C-2o), 55.1 (OCH3), 29.5 (C(CH3)2), 22.7 (C(0)CH3), 19.4 (C(CH3)2), 18.5,
18.2 (2C, C-6,~ 6B).
FABMS for C52H65N014 (Vi, 92'7.44) ml~ 950.5 [M+Na]+.
Anal. Calcd for C52H65N014~ C, 67.30; H, 7.06; N, 1.~1%. Found: C, 67.15; H,
7.24; N,
LMPP11-exp-brevet~penmOMe
1.44%.
CA 02434685 2003-07-04
Methyl (2,3,4,6-Tetra-0-benzyl-a-D-glucopyranosyl)-(1-~4)-(2,3-di-O-benzoyl-a-
L-
rhamaopyranosyl)-(1-~3}-(2-acetamido-2-deoaty-4,6-D-isopropylidcnc-[i-D-
glucopyranosyl)-
(1-~2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1-3Z)-(3,4-di-O-benzyl-a-1.-
rhamnopyranoside (XX). Activated powdered 4A molecular sieves were added to a
solution of
the trisaccharide acceptor XX (202 mg, 0.22 mmol) and the disaccharide donor
XX (263 mg, 0.25
mmol) in anhydrous CHzC)z (5 mL} and the suspension was stirred for 30 min at -
IS°C.
Trifluoromethanesulfonic acid (7 pL, 34 ~,mol) was added and the mixture was
stirred for 2 h
while the bath temperature was slowly coming back to 10°C. TLC (solvent
D, 49:1) showed that
no XX remained. Et3N was added and after 30 min, the suspension was filtered
through s pad of
Celite. Concentration of the filtrate and chromatography of the residue
(solvent B, 9:1 -~ 17:5)
gave the fully protected pentasaceharide XX (330 mg, 84%) as a white foam;
[a]D XX (c 1.0);
NMR: tH, 8 8.07-6.96 (m, SOH, Ph), 5.82 (d, 1H, JN~= 7.4 Hz, NH), 5.63 (dd,
1H, J2,3= 3.5, J3,a
= 9.5 Hz, H-3o), 5.43 (dd, 1H, 3»= 1.6 Hz, H-2o), 5.09 (bs, IH, H-18), 5.02
(d, 1H, J1,Z= 3.4 Hz,
H~IE}, 4.99 (d, 1H, J1,Z = 8.3 Hz, H-lo), 4.95 (d, 1H, J1~, = 1.1 Hz, H-1~),
4.94-4.63 (m, 13H,
OCHZ), 4.63 (s, 1H, H-1,~, 4.37 (d, 1H, J = 11.0 Hz. OCH2), 4.29 (dq, 1H,
J4,s= 9.5, Js,b= 6.2 Hz,
H-5~), 4.25 (d, 1 H, J = 9.5 Hz, OCHZ), 4.23 (pt, I H, J3,4 = J4,s = 9.5 Hz, H-
3D), 4.01 (m, 1 H, H-2H),
3.97-3.86 (m, 5H, H-3>;, 2A, 3fi, 4~, OCHZ), 3.82 (m, 1H, H-3A, 5a), 3.71-3.57
(m, 7H, H-SD, 4E,
5,,, 4p, 4E, 6aE, 6bE), 3.54-3.41 (m, 3H, H-2E, 4B, 2p) 3.38-3.31 (m, 2H, H-
4A, 6aD), 3.31 (s, 3H,
OCH3), 3.17 (m, 1H, H-5E), 3.08 (d, 1H, J6~,6b° 10.1 Hz, H-61~), 1.84
(s, 3H, NHAc), 1.46 (s, 3H,
C(CH3)z), 1.45 (d, 3H, J5,6 = 5.9 Hz, H-6~), 1.35 (m, 6H, Js,6 = 5.9 Hz, H-6B,
C(CH3)z), 1.31 (d,
3H, Js.b= 6.2 Hz, H-GA); 13C, 8 171.7, 165.9, 165.8 (3C, CO), 138.9-127.9
(Ph), 102.3 (C-IX, J =
167 Hz), 101.5 (C-1x, J = 170 Hz), 100.3 (C.lx, J = 170 Hz), 99.8 (C(CH3)2),
99.6 (C-lx, J = 172
Hz), 98.2 (C-lx, J =172 Hz), 82.0 (C-X~, 81.2 (C-Xg), 80.9 (C-X~, 80.7 (C-Xe),
79.7, 79.3 (3C,
OCHz), 78.1 (C-X~, 77.8, 77,4, 75.5, 7I,8 (SC, OCHZ), 71.7 (C-XB), 71.6 (C-X),
68.8 (C-X), 68.0
(C-6E), 67.6 (C-XD), 62.5 (C-do), 58.9 (C-2o), 55.0 (OCH3), 29.5 (C(CH3)2),
23.8 (C(O)CH3), 19.8
(C(CH3)Z), 18.6 (C-Go), 18.5 (C-G,~. 18.3 (C~6$). XXMS for Cio6HmNOzs (M,
1803.79) m/z XXX
[M+H]*.
Anal. Calcd for CI~HIi~NOzs: C, 70.53; H, 6.53; N, 0.78%. Found: C, XXXX; H,
XXX;
N, XXX%.
Methyl (2,3,4,6-Tetra-0-benxyl-a-D-glucopyranosyl)-(1--~4)-(2,3-di-0-benzoyl-a-
L-
rhamnopyranosyl)-(1-->3)-(2-acetamido-2-deoxy-[i-D-8lucopyrano9y~-(1 >2)-(3,4-
di-0-
benzyl-a-L-r6amnopyranosyl)-(1-~2)-3,4-di-0-benryl-a-L-rhamnopyranoside (XX).
90% aq
TFA (750 ~.L) was added at 0°C to a solution of the fully protected XX
(588 mg, 326 p.mol) in
CH2Cl2 (6.7 mL) and the mixture was stirred at this temperature for 1 h. TLC
(solvent B, 1.5:1)
showed that no XX remained. Volatiles were evaporated by repeated addition of
toluene.
Chromatography of the residue (solvent B, 4;1 --> 1:1) gave XX (544 mg, 95%)
as a white foam;
z
LMPP 1 I-erp-brevet-pentaOMc
CA 02434685 2003-07-04
[aJD X?C° (c 1.0); NMR: tH, b 8.06-7.06 (m, 50H, Ph), 5.82 (d, 1H,
J~,z= 7.1 Hz, NH), 5.65 (dd,
IH, Ja,3 = 3.8, J3,a = 9.0 Hz, H-3c), 5.53 (m, 1H, H-2c), 5.34 (s, IH, H-IBj,
5.04 (d, iH, J,,~ = 8.3
Hz, H-lo), 5.00 (m, 2H, H-lo, lF), 4.97-4.63 (m, 13H, OCHz), 4.48 (bs, IH, H-
1,~, 4.40 (d, IH, J
= 8.4 Hz. OCHZ), 4.29 (d, 1 H, J = 8.0 Hz, OCHz), 4.28-4.21 (m, 2H. H-3D, 5~),
4.10 (m. 1 H, H-
2,~, 4.04 (m, IH, H-2H), 3.99 (d, 1H, OCHZ), 3.95-3.89 (m, 3H, H-3H, 3E, 4c),
3.87 (dd, 1H, J2,3=
2.7, J3,a = 9.7 Hz, H-3,~, 3.81-3.64 (m, SH, H-5E, 5H, GaD, 4E, 5"), 3.54 (dd,
1 H, J~,2 = 3.2, J2,3 = 9.7
Hz, H-2E), 3.51 (pt, 1H, J3,a = Ja,s = 9.5 Hz, H-4H), 3.45-3.37 (m, 4H, H-4A,
4p, 6aE; 2p), 3.33 (m,
5H, H-5o, GbD, OCH3}, 3.12 (d, 1H, Jse,6b= 10.6 Hz, H-GbE), 2.28 (bs, 1H,
OH),1.97 (bs, 1H, OH),
1.84 (s, 3H, NHAc), 1.54 (d, 3H. Js,fi= G.1 Hz, H-G~j, 1.37 (m GH, H-GB, GA);
~3C, 8 171.5, 165.8,
IGS.G (3C, CO), 138.8-127.9 (Ph), 101.6 (C-1D), 100.8 (C-IH), 100.5 (C-1,~,
100.1 (C-lE~), 99.9
(C-lc*), 84.9 (C-3p), 82.1 (C-3E), 80.9, 80.7, 80.6, 80.5 (4C, C-4B, 3A, 4,,,
2E), 79.7 (C-4~), 79.3
(C-38), 77.8 (2C, C-2B, 4E), 76.0, 75.9 (2C, OCH2), 75.8 (C-5D), 75.d, 75.I,
74.6, 73.7, 73.1 (5C,
OCHZ), 72.8 (C-2,~. 72.6 (OCHz), 71.8 (C-5E), 71.6 (C-4D), 71.3 (C-3c), 71.1
(C-2o), 69.4 (C-5~),
68.8 (C-5B), 68.3 (C-5A), 68.1 (C-6E}, 63.0 (C-6D), 57.6 (C-2D), 55.0 (OCH3),
23.8 (M-iAc), 18.8
(C-do}, 18.6, 18.5 (2C, C-6,v 6H). XXNiS for C~o3H1~3N025 (M, 1763.76) m/zX30i
[M+HJ+.
Anal. Calcd for C,o3H1t3N023: C. XX; H, XX; N, XX%. Found: C. ~;XXX; H, XXX;
N,
XXX%.
Methyl (2,3,4,6-Tetra-0-benzyl-a-A-glucopyranosyl)-(1-->4)-a-L-rhamnopyranosyl-
(1-~3)-(2-acctamido-2-deoxy-[3-D-glucopyranosyl)-(I-~2)-(3,4-di-O-benryl-a-L-
rhamnopyranosyl)-(1 >2)-3,4-di-0-benzyl-a-L-rhamnopyranoside (XX). 1M
Methanolic
sodium methoxide was added to a solution of XX (277 mg, 157 lrmol) in a 1:1
mixture of CH2C12
and MeOH (6 mL) until the pH was 10. The mixture was stirred overnight at rt
and neutralized
with Amberlite IR-120 (H+). The crude material was chromatographed (solvent D,
49:1) to give
XX (211 mg, 86%) as a white foam; [aJp XX° (c 1.0); NMR; ~H, 68.07-7.06
(m, 50H, Ph), 5.82
(d, IH. JHH,1= 7.1 Hz, NIA, S. 65 (dd, IN, J~,3 = 3.8, J3,a = 9.0 Hz, l~ 3c),
5.53 (dd, IH, J,,1 = 1.6
Hz, H-2~), S. 34 (s, 1 H, H I ~, 5. 04 (d, 1 H, J~,1= 8. 3 Hz, H 1 D), 5. 00
(m, 2H, H 1 c, I ~, 9. 97 4. 63
(m, 13H, OCH~, 4. 48 (Us, I H, H I,~, 4. 40 (d, 1 H, J = 8. 4 Hz, OCH,~, 4. 29
(d, I H, J = 8. 0 Hz,
OCH~), 4. 28-4. 21 (m, 2H, H 3a Sc), 4.10 (m, ll~ H 2,~ j, 4. 04 (m, ll~ l~
2B), 3. 99 (d, 1 H, OCHZ),
3. 95-3.89 (m, 3I~ H 3r, 3B, 4c), 3.87 (dd, I H, J1,3 = 2. 7, J3,., = 9. 4 Hz,
H 3,~, 3.81-3. 64 (m, 5H, H
5B, 5E, 6ao, 5,,, 9,~, 3. 54 (dd, I H, J1,3 = 3. 2, J,~,a = 9. 7 Hz. H 2E),
3.51 (pt, 1 H, J4,s = Jj,d = 9.5 Hz,
l~ 4B), 3.45-3.37 (nr, 4H. H 4A, 4D. 6aE, 2D), 3.33 (m, 5H, H So, 6bD, OCHj),
3.12 (d, III J6a.ch =
10. 6 Hz, H 6b,~ 2. 28 (bs, I H, 01~, 1. 97 (bs, I H, Oll), 1. 8~ (s, 3H,
NHAc), 1.53 (d, 3H, J5,6 = 6.1
Ice, H 6~, 1.37 (m, 6H, H Ge, b,~; '3C, 0171. 5, 1 GS. 8, 165. 6 (3C, CO),
138. 8-127.9 (Ph), 101. d
(C-1 ~, 100. 8 (C-18), l 00. S (Gl,~, 100. l, 99. 9 (ZC, C-I E. 1 c), 84. 9 (C-
3D), 82.1 (C 3~, 80. 9,
80. 7, 80. 6, 80.5, 79. 7 (Sc, C-dg, 3A, 4A, 2E, 4C) (3C, OCHZ), 78. I (C-X,~,
7? 8, 77.4, 75. 5, i I. 8
(SC, OCH~j, 71. 7 (C-X~, 71.6 (C ~, 68.8 (C ~, 68. 0 (C-6~, 67. 6 (C-XQj, 62.5
(C-5D), 58.9 (C-
2D), 55.0 (OCH,~, 29.5 (C(CH3)~, 23.8 (C(O)CH~), 19.8 (C(CHa),~, 18.6 (C-d),
18.5 (C-6,~,), 18.3
3
LhlPPI t ~exp-brevet-pent~OMe
CA 02434685 2003-07-04
(C-d~.'XXMS for Ct03HI13N~25 (M. 1763.76) m1z XXX [M+H]+.
Anal. Calcd for C103HI13N~25~ C. XX; H, XX; N, XX%. Found: C, XXXX; H, XXX; N,
XXX%.
Methyl arD-Glueopyranosyl-(1~4)-a-L-rhamnopyrsnosyl-(1~3)-2-acctamido-2-
deoxy-p-D-glucopyranosyl-(1-~2)-a-L-rhamnopyranosyl-(1->,2)-a-L-
rhamnopyranoside
(1). The bensylaled tetrasaccharide 23 (484 mg, 394 lemol) was dissolved in a
mixture of
methanol (IO mL) and AcOH (1 mL), treated with 10% Pd-C catalyst (200 mgj, and
the
suspension was stirred overnight at rt. TLC (solvent D, 3:2) showed that the
starting material
had been transformed into a mare polar product. The suspension was filtered on
a pad of
Celite. The filtrate was concentrated and coevaporated repeatedly with
cyclohexane. Reverse
phase chromatography of the residue (solvent F, 100: 0 -~ 49: I), followed by
freeze-drying,
gave the target tetrasaccharide 1 as an amorphous powder (230 mg, 85%); (aJo
+3° (c 1.0,
water); NMR: t H, d 5. 04 (d, 1 H, Jt, ~ = 3. 8 Hz. H I ,~, 4. 87 (bs, 1 H, H
I C), 4. 84 (bs, ll~ H
I,~, 4. 7d (d, overlapped, 11~ H I D), 4.10 (dq, I H, J~, g = 9. S Hz, H SCj,
4. 01 (m, I H, H-2~,
4.00 (m, ll~ H S~J, 3.92 (dd, IH, J6a,6b = 12.0, J5,6a = 1.8 Hz, H dap), 3.87-
3.73 (m, 7H. H
3C, 3,i, Gag. 6bE, Zp, 2C, 6bD), 3. 73-3. dl (m. 3H, N 3g, 3D, 5~, 3.59-3.43
(m, SH, H 2~, 4D,
9C, Sp, 4~, 3.39 (s, 31~ OCHj), 3.32 (pt, IH, Jj,4 = J4,5 = 9.6 Hz, H ~4,~,
2.07 (s, 31~
C(0)CH3), 1.32 (c~ 3H, J,5,6 = 6.2 Ha, H dC), and 1.28 (d, 3H, J5,6 = d.2 Hz,
H d,~; ~jC,
d 175. 3 (C(O)), I 02. 7 (C-1 D, J = I d3 Hz), 102. 0 (C-I C, J = 170 H;),
100. 5 (2C, C-I,q, 1 ~, J =
170 Hz), 82.3 (C-3D), 81.8 (C-4C), 79.3 (C-2A), 7ti.7 (C-4~, 73. d (C-3~, 73.1
(C-4~, 72. 6
(C-S~, 72.4 (C-2~, 71.8 (C-2~, 70. 7 (C-3,~, 70. l (C-SD), 69. 7 (C-3C), 69.3
(C-5,~, 69.2 (C-
dD), 68.9 (C-SCJ, 61.4 (C-6p), 60.9 (C-d,~, 56.4 (C 2p), 55.6 (OCHj), 23.0
(C(O)CH3), 17.5
(C-d,~, and 17.3 (C-d~. FABMSfor CzTH.tyNOlg (ELI, 689.3) mlz 712.2 (M+NcrJ+.
3,4-Di-O-benzyl-2-0-chloroacetyl-a-L-rhamnopyranose (XX). 1,5-Cyclooctadiene-
bis(methyldiphenylphosphine)iridium hexafluorophosphate (lr(I), 25 mgj was
dissolved in dry
THF (5 mL) and the resulting red solution was degassed in an argon stream.
Hydrogen was
then bubbled tluough the solution, causing the colour to change to yellow. The
solution was
then degassed again in an argon stream A solution of XX (3.28 g, 7.12 mmol) in
THF (30 mL)
was degassed and added. The miarture was stirred overnight at rt, and a
solution of iodine (3.G
g, 14.2 mmol) in a mixture of THF (70 mL) and water (20 mL) was added. The
mixture was
stirred at rt for 1 h, then concentrated. The residue was taken up in CHZC12
and washed twice
with 5% aq NaHSOa. The organic phase was dried and concentrated. The residue
was purified
by column chromatography (solvent B, X:X) to give XX (2.53 g, 85%) as a
slightly yellow
. , (aJD +25° (c L0); IH NMR: S 7.40-7.28 (m, IOH, Ph), 5.57 (bd, 0.2H,
H-2[i), 5.45
(dd, O.8H, Jt,Z - 2.0 Hz, H-2a), 5.13 (bd, 0.8H, H-la), 4.92 (d, 1H, J = 10.9
Hz, OCHZa,
OCHZ/3), 4.79 (d, 0.2H, J = 11.2 Hz, OCH2(3), 4.74 (d, 1 H, J = 11.2 Hz,
OCHZa; H-1 [3), 4.65
(d, 0.8H, OCH2a), 4.64 (d, 0.2H, OCHZp), 4.58 (d, 0.8H, OCHza), 4.54 (d, 0.2H,
OCHz(i),
4
LMPPII-cxp~brcvn~penL~OMe ~ o24s4ss5 zoos-o~-o4
3H, CH2C1, H-5c, 3c), 3.84 (m, 1H, H-5E), 3.78-3.74 (m, 2H, H-6aE, 4E), 3.70
(pt, 1H, Ja,s =
J3 4 = 9.3 Hz, H-4c), 3.58-3.54 (m, 2H, H-6b~, 2E), and i .50 (d, 3H, J5,6 =
6.2 Hz, H-6o); '3C
NMR: 6167.0 (C=O, CIAc), 166.0 (C=O, Bz), 139.1-128.0 (Ph, All), 118.5 (All),
99.5 (C-1E),
96.8 (C-lc), 81.9 (C-3fi), 81.0 (C-2E), 79.7 (C-4c), 77.7 (C-4e), 76.0, 75.4,
74.1, 73.8 (4C,
OCHZ), 73.5 (C-3c), 71.8 (C-5E), 70.9 (C-2c), 68.8 (OCHypli), 68.1 (C-6E),
67.7 (C-Sc), 41.5
(CHZCI), and 18.6 (C-dc); FAB-MS far C52H55012 (M, 906.5) mlz 929.3 [M+Na]*.
Anal. Calcd for C52H5sC1O12: C, 68.83; H, 6.11%. Found: C, 68.74; H, 6.19%.
(2,3,4,6-Tetra-D-benzyl-a-D-glucopyranosyl)-(1-~4)-2-O-benzoyl-3-0-
chloroacetyl-a,/[3-L-
rh9mnopyranose (XX). A solution of XX (7.21 g, 7.95 mmol) in THF (80 mL)
containing
activated iridium complex (GO mg) was treated as describd for the preparation
of XX. The
mixture was stirred at rt for 3 h, at which point a solution of iodine (4.0 g,
15.7 mmol) in a
mixture of THF (90 mL) and water (24 mL) was added. The mixture was stirred at
rt for 30 min,
then concentrated. The residue was taken up in CHzCIZ and washed twice with 5%
aq NaHSOd,
then with brine. The organic phase was dried and concentrated. The residue was
purified by
column chromatography (solvent B, 4:1) to give XX (6.7 g, 97%) as a slightly
yellow foam,
(aJD +ZS° (c I. O); 1H NMR: 8 8.10~7.09 (m 25H, Fh), 5.47 (dd, 1 H,
J2,3 = 3.5, J3,a = 9.3 Hz, H-
30), 5.41 (bs, 1H, H-2c), 5.03 (bs, IH, H-lc), 4.94 (d, IH, J = 10.9 Hz,
OCHa), 4.87 (d, 1H, J~,~
= 3.4 Hz, H-lE), 4.85 (d, 1H, OCHz), 4.80 (m, 2H, OCH2), 4.64 (m, 2H, OCH~),
4.45 (d, 1H, J=
10.7 Hz. OCHi), 4.41 (d, 1 H, J = 12.1 Hz, OCHZ), 4.16 (dq, 1 H, J4,5 = 9.3
Hz, H-5c), 4.09 (d,
1H, J = IS.G Hz, CHzCI). 3.96 (d, 1H, CHiCI), 3.93 (pt, 1H, H-3E), 3,83 (m,
1H, H-5e), 3,77-
3.68 (m, 2H, H-4E, 6aE), 3.65 (pt, 1H, H-4c), 3.54 (m, 2H, H-6bE, 2E), and
1.48 (d, 3H, J5,6= 6.2
Hz, H-6c); ~~C NMR: E167.0 (C=0, CIAc), 166.0 (C=O, Bz), 139.1-127.9 (Ph),
99.5 (C-la),
92.3 (C-lc), 81.9 (C-3E), 81.0 (C-2s), 79.9 (C-4c), 77.6 (C-4E), 76.0, 75.6,
74.2, 74.1 (4C,
OCHZ), 72.1 (C-3c), 71.7 (C-4E), 71.1 (C-2c), 68.0 (C-GE), 67.5 (C-Sc), 41.5
(CHZCI), and 18.9
(C-6c); FAB-MS for C49HstC1Oli (M, 866.3) m/z 889.3 [M+Na]+.
Anal. Calcd for Ca9H3iC101Z: C, 67.85; H, 5.93%. Found: C, 67.72; H, 6.00%.
(2,3,4,6-tetra-O-6enzyl-a-D-glucopyranosyl)-(1 >4)-2-O-benzoyl~3-0-
chtoroscetyl-a-L-
rhamnopyranosyl trichloroacetimidate (XX). Trichloroacetonitrile (I.1 mL, 10.9
mmol) and
DBU (17 p.L) were added to a solution of the hemiacetal XX (950 mg, 1.09 mmol)
in dry DCM
(8 mL), and the mixture was stirred at 0°C for 1.5 h. Toluene was
added, and volatiles were
evaporated. Th,e residue was purified by flash chromatography (solvent B, 3:2
containing 0.1%
Et3N) to give XX (930 mg. 84%) as a ~XX~Cx: Further elution gave some
remaining starting
material XX (13G mg, 14°l). ~aJD +25° (c 1.0); 1H NMR: S 8.7G
(s, 1H, NH), 8.12-7.17 (m,
25H, Ph), 6.34 (d, IH, Jl,z= 1.5 Hz, H-lc), 5.67 (dd, 1H, H-2c), 5.54 (dd, 1H,
JZ,~= 3.4, J3,a= 8.8
Hz, H-3C), 4.98 (d, 1 H, OCHZ), 4.88 (d, 1 H, Jls = 3.4 H-1 E), 4.84 (d, 1 H,
J = 11,1 Hz, OCHz),
6
(d, 0.8H, OCH2a), 4.64 (d,
LMPPII-exp-brevd~penmOMc ~ o24s4ss5 zoos-o~-o4
4,82 (d, 1H, J = 11.2 Hz, OCHz), 4.65 (d, IH, OCHZ), 4.G2 (d, 1H, OCHz), 4.4d
(d, 1H, J = 11.4
Hz, OCHz), 4.41 (d, 1H, J=11.8 Hz, OCHZ), 4.14 (dq, 1H, Ja,s = 9.5 Hz, H-5c),
4.11 (d, 1H, J =
I5.5 Hz, CHZCI), 3.98 (d, 1H, CHZCI), 3.94 {pt, 1H, H-3E), 3.83-3.71 (m, 4H, H-
SE, 6aE, 4E, 4c),
3.56-3.51 (m, 2H, H-6bE, 2a), and 1.51 (d, 3H, J5,6 = 6.2 Hz, H-Gc); t3C NMR:
O I G7.1 (C=0,
CIAc), 165.7 (C=0, Bz), IG9.6 (C=NH), 139.0-127.9 (Ph), 99.9 (C-IE), 95.2 (C-
lc), 82.1 (C-
3E), 80.9 (C-2~, 79.0 (C-4o), 77.6 (C-4E), 76.0, 75.6, 74.2, 73.8 (4C, OCHz),
73.0 (C-3c), 71.9
(C-5E), 70_7 (C-5c), 69.2 (C-2c), 68.0 (C-6E), 67.7 (C-Sc), 41.4 (CHZCI), and
18.6 (C-6c).
Anal. Calcd for CslHstClaNOIZ: C, 60.54; H, 5.08; N, 1.38%. Found: C, 60.49;
H, 5.01;
N, 1.34%.
Methyl (Z,3,4,6-tetra-O-beazyl-a-D-glucopyranosy()-(1-->4)-(2-0-benzoyl-3-O-
chloroacetyLa-L-rhamnopyranosyl)-(1--~3)-2-acetamido-2-deoxy-3,4-O-
isopropylidene-
~i-D-glucopyranoside (XI~. The acceptor XX (500 mg, 1.82 mmol) was dissolved
in DCM
(5.5 mL) and 4A-MS (300 mg) were added. The mixture was cooled to -60°C
and stirred for
15 min. TMSOTf (35 ~L, mmol) and a solution of the disaccharide donor XX (2.39
g, 2.3G
mm~ol) in DC~f (7.5 mh) were added. The mixture was stirred for 45 min while
the cooling
bath was coming back to rt, and for more 3 h at rt. The mixture was then
heated at 65°C for 1
h 30 min. EtjN was added and the mixture was stirred at rt for 20 min, then
diluted with
CHzCIz and filtered through a pad of Celite. The filtrate was concentrated and
purified by
column chromatography (solvent B, 85:15 -> l:l) to give XX (1.64 g, 80%) as a
'.
(a~D +25° (c 3.0); tH NMR: S 8.06-6,93 (m, 25H, Ph), b.18 (d, 1H, JNH.z
= 7.3 Hz, NHD).
5.40 (dd, 1H, Jz,3= 3.5 Hz, H-3c), 5.38 (bs, 1H, H-Zc), 4.98 (d, IH, JS,~= 8.3
Hz, H-1D), 4.94
(bs. IH, H-Ic), 4.94 (d, 1H, OCHZ), 4.93 (d, IH, Jl,~= 3.4 Hz, H-IE), 4.83 (d,
2H, J = 10.7 Hz,
OCHi), 4.81 (d, 1H, J = 10.6 Hz; OCHz), 4.67 (d, 1H, J = 11.7 Ha, OCHZ), 4.62
(d, 1H; J =
11.4 Hz, OCHa), 4.47 (m, 3H, H-3D, OCHz), 4.22 (dq, 1H,14,s= 9.4, Js,s= 6.2
Hz, H-Sc), 4.10
(d, 1H, J = 15.5 Hz, CHzCI), 3.96 (m, 2H, H-Gap, CH2C1), 3.91 (pt, IH, H-3E),
3.82 {m, 2H,
H-SE, 6bD), 3.72 (m, 3H, H-GaE, 4E, 4c), 3.G2 (pt, 1H, J~,4 = Js,s = 9.4 Hz, H-
4D), 3.55 (m, 2H;
H-6bE, 2~), 3.SI (s, 3H, OMe), 3.41 (m, 1H, H-5D), 3.15 (m, 1H, H-2D), 2.04
(s, 3H, NHAc),
I.51 (s, 3H, CMez), I.42 (m, 6H, H-6c; CMea), and I.51 (d, 3H, JS,6 = G.2 Hz,
H-6C); t3C
NMR: 8 171.8 (C=O, NHAc), 167.3 (C=O, CIAc), 166.1 (C=O, Bz), 139.0-128.0
(Ph), IOI.I
(C-ID, JcH < 164 Hz~, 99.9 (CMez), 99.4 (C-lE, 1cH > 165 Hz), 98.2 (C-Ic, JcH
= 172 Hz),
81.8 (C-3E), 80.9 (C-2~, 79.0 (C-4c"'), 77.7 (C-4E*), 76.7 (C-3D), 75.9, 75.3,
?4.2, 73.9 (4C,
OCHz), 73.7 (C-4D), 73.4 (C-3c), 71.9 (C-5E), 71.2 (C-2c), 68.2 (C-G~), G7.8
(C-Sc), 67.4 (C-
SD), 62.7 (C-do); 59.6 (C-2D), 57.6 (OMe), 41.5 (CHzCI), 29.5 (CMe2), 27.3
(NHAc), 19.7
(CMez), and 18.G (C-6c); FAB-MS for C6tH~oCINOt~ (M, I 123.4) m/z 1146.5
[M+Na]+.
Anal. Calcd fvr CsiH~oCINOt~: C, 65.15; H, 6.27; N, 1.25%. Found: C, 65.13; H,
6.23;
N, 1.22%.
7
CA 02434685 2003-07-04
GMPP I 1-exp-brcva-penmOMc
Methyt (2,3,4,6-tetra-O-benzyha-D-gtucopyranosyl)-(1-->4)-(2-O-bepzoyl-a-L-
rhnmaopyranoayl)-(1-~3)-Z-acetamido-2-deoxy-3,4-O-isopropylidene-p-D-
glucopyranoside
(XX). To a solution of the fully protected XX (1.40 g, 1.25 mewl) in a mixture
of methanol (18
mL) and pyridine (18 mL) was added thiourea (951 mg, 12.5 mmol). The mixture
was stirred at
65°C for 5 h at which time no TLC (solvent D, 4:1) that no starting
material remained.
E~~aporation of the volatiles and co-evaporation of petroleum ether form the
residue resulted in a
crude solid which was taken up in a minimum of methanol. A large excess of DCM
was added
and the mixture was left to stand at 0°C for 1 h. The precipitate was
ftltzated on a pad of Celite
and the filtrated was concentrated. Column chromatography of the residue
(solvent C, 4:1) gave
the trisaccharide acceptor XX (1.28 g, 97%) as a XXXX. ~aJD +ZS° (c
1.0J; tH NMR: & 8.10-
6.96 (m, 25H, Ph), 6.09 (d, 1H, J~Z -- 7.9 Hz, NHD), 5.26 (dd, 1H, J~,~ = 1.6,
JZ,3 = 3.4 Hz, H-
2~), 4.97 (m, 3H, H-1~, lE, OCHZ), 4.86 (m, 3H, H-1D, OCH~), 4.81 (d, IH,
OCHZ), 4.72 (d, 1H,
OCl-h), 4.58 (d, 1H, J =12.2 Hz, OCHi), 4.51 (d, 1H, 3 = 10.9 Hz, OCH2), 4.48
(d, 1H, J = 12.2
Hz, OCHZ), 4.23 (pt, 1H; JZ,~ = J3,d = 9.4 Hz, H-3D), 4. I 8-4.10 (m, ZH, H-
So, Ss), 4.OG-3.95 (m,
3H, H-3c, 3E, 6aD), 3.80 (pt, 1H, Js,sb= Js°,sb= 10.4 Hz, H-6bn), 3.GG
(m, 2H, H-6aE, 6bE), 3.G2
(dd, 1 H, Jz,3 = 9.8, Jl s = 4.1 Hi, H-2E), 3.59 (pt, 1H, J3,4 = Ja.s = 8.9
Hz, H-4E), 3.55 (pt, 1H, J3,4 =
J4,s = 9.2 Hz, H-4p), 3.51 (pt, 1 H, J3,a = Ja,s = 9.3 Hz, H-4~), 3.49 (s, 3H,
OGH~}, 2.22 (s, 3H,
NHAc), 1.90 (bs, 1H, OH), 1.49 (s, 3H, CMe2), 1.43 (s, 3H, CMez), and 1.40 (s,
3H, J5,6 = 6.2
Hz, H-6c); 13C NMR: b171.8, 166.6 (2C, C=O), 138.9-128.1 (Ph), 101.6 (C-1D),
99.8 (CMe2),
98.6 (C-IE*), 98.3 (C-to*), 85.4 (C-4c), 82.0 (C-3s), 80.4 (C-2fi), 78.2 (C-
4E), 77.1 (C-3d), 75.9,
75.5, 74.2, 73,9 (4C, OCH2}, 73.6 (C-4D*), 73.5 (C-2~*), 7I.7 (C-SE}, 69.0 (C-
6E), 68.3 (C-3c),
G7.S (C-SD), 66.9 (C-Sc), 62.7 (C-6n), 58.9 (C-2o), 57.5 (OMe), 29.5 (CMeZ),
24.0 (NHAc),
19.7 (CMe2), and 18.2 (C-6~); FAB-MS for Cs9H69N016 (M, 1047,5) mla 1070.4
jM+Na)+.
Anal. Calcd for C7pH~6016: C, 67.61; H, 6.64; N, 1.34%. Found: C, 67,46; H,
6.78; N,
1.24%.
Methyl (3,4-Di-0-benzyl-2-D-cbloroacetyl-a-L-rhamnopyranosyl)-(1-~3)-j(2,3,4,G-
tetra-O-
benzyl-a-D-glucopyranosyi-{1-~4))-(2-O-benzoyl-3-O-chioroacetyl-a-L-
rhamnopyranosyl)-
(1~3~-2-xcetamido-2-dcoxy-3,4-0-isopropylideae-(3-n-glucopyranoside (XX). The
trisaccharide acceptor XX (GI5 mg, 0.58 mmol) was dissolved in EtzO (10 mL)
and the solution
was cooled to -60°C. TMSOTf (32 pL) and donor XX (497 mg, 0.88 mmol) in
EtZO (12 mL)
were added, and the mixture was stired for 1 h while the bath was slowly
coming back to -20°C.
The mixture was stirred for 4 h at this temperature, then at 0°C
overnight. More XX (SO mg, 88
pmol) was added, and the mixture was stirred at rt for 3 h more at 0°C.
Et3N was added, and the
mixture was concentrated. Column chromatography of the residue (solvent B, 9:1
--; 1:I) gave
the orthoester XX (44 mg, 5%) then the fully protected XX (445 mg, 52%}
contaminated with
the trimethylsilyl side product XX (Ji;~UXX: XX:XX) together with a mixture of
XX and XX (65
mg, 8°~a), and the starting XX (27 mg, 4%). Compound XX (alpha) had
('aJp +25° (e 1.0J; 1H
8
LMPP l 1-~cp-brevet-pentaOMc
CA 02434685 2003-07-04
NMR b 8.07-7.12 (m, 3 SH, Ph), 5.9G (d, 1 H, J~,2 = 7.9 Hz, NHo), 5.82 (m, 1
H, H-2c), 5.33 (dd,
IH, J2,3= 3.2 Hz, H-2B), 5.07 (m, 1H, JlZ= 3.2 Hz, H-lE), 5.05 (d, 1H, J~,2=
1.7 Hz, H-lc), 4.98
(d, IH, OCHZ), 4.97 (bs, 1H, H-1B), 4.91-4.78 (m, 5H, H~lp, OCH~), 4.64 (d,
1H, J = 11.6 Hz,
OCHz), 4.60-4.45 (m, 5H, OCH~, 4.36 (d, 1 H, J = 11.9 Hz, OCHi), 4.26 (pt, 1
H, JZ,3 = J3.a = 9.5
Hz, H-3D), 4.17 {dd, 1H, Jz,3 = 3.4 Hz, H-3B), 4.16 (d, 1H, J = 15.1 H~~
CHZCI), 4.11 (d, 1H,
CHzCI), 4.10 (dq, 1H, Ja,s= 9.1, Js,s= 6.3 Hz, H-58), 4.06 (m, IH, H-5E), 4.00
(pt, 1H, J3,a= Jz,;=
9.4 Hz, H-3F), 3.97 (dd, IH, J5,6~= 5.3, J6a,6b= 10.8 Hz, 6aa), 3.89 (m, 1H, H-
6aE), 3.88-3.68 (m,
4H, H-6bE, 6bD, 4g, 3c), 3.G7 (m, 1H, H-Sc), 3.58 (pt, 1H, J3,a = Ja,s
° 9.4 Hz, H-4D), 3.52 (dd,
1H, Ji,2= 3.3, JZ,3= 9.8 Hz, H-2E), 3.49 (s, 3H, OCH3), 3.39 (m, 1H, H-5D),
3.30 (nn, 2H, H-2v,
4c), 2.12 (s, 3H, NHAc), I.52 (s, 3H, CMez), 1.42 {s, 3H, CMe2), 1.33 (d, 3H,
J,,6 = 6.2 Hz, H-
6X), and 0.96 (s, 3H, J5,6 = 6.2 Hz, H-6X): '3C NMR: 0171.9 (C=O, NHAc), 167.0
(C=O,
CHZCI), 166.3 (C=O, Bz), 138.8-128.0 (Ph), 101.4 (C-1D,1cx = 164 Hz), 99.9
(CMe2}, 99.3 (C-
lc, Jcx = 168 Hz), 98.3 {C-lE, J~ = 168 Hz), 97.9 (C-1H, Jcx = 171 Hz), 82.1
(C-3E), 81.8 (C-
2E), 80.4 (bs, G3B), 84.0 (C-4c), 78.8 (bs, C-4E*), 78.3 (C-4B*), 77.7 (C-
3c*), 76.9 (C-3D), 75.9,
75.5, 75.3, 74.3 (4C, OCHz), 73.4 (C-4o), 73.2 (OCHz), 72.7 (C-2B), 72.1 (C-
5E), 69.1 (C-5c),
67.7(C-SDI), 67.6 (C-5B"'), 62.7 (C-6D), 59.1 (C-2p), 57.5 (OMe), 41.4
(CHzCI), 29.5 (CMez},
24.0 (NHAc), 19.7 (CMez), I8.8 (C-6~), and 18.2 (C-6~; FAB-MS for C8~H9zNC1021
(M,
1449.5) m/z 1472.7 [vI+Na]T.
Anal. Calcd for CBtH92NClOz,: C, 67.05; H, G.39; N, 0.97%. Found: C, 66.21; H,
6.46;
1.01%.
Compound XX (ortltoester) had (aJp +25° (c 1.0); 'H NMR: 8 8.07-7.I5
(m, 35H, Ph), 5.47
(d, 1 H, Juu,z = 7.4 Hz, NHD), 5.45 (bs, 1 H, H-2c), 5.42 (d, 1 H, JI,Z = 2.3
Hz, H-1 B), 5.24 (d, 1 H,
J~,z= 3.4 Hz, H-lE), 4.94 (d, 1H, Jt,z= 8.2 Hz, H-1D), 4.91-4.82 {m, 7H, H-lc,
OCHZ), 4.80 (d,
1H, J = 11 Hz, OCHz), 4.75 {d, 1H, J = 11.6 Hz, OCHz), 4.68 (dd, 1H, J~,i=
2.4, Jz,3 = 4.0 Hz,
H-2H), 4.65-4.4? (m, 4H, OCHZ), 4.44-4.32 (m, 4H, H-5E, 3D, 3c, OCHz), 4.15
(nn, 1H, H-5c),
4.05 (pt, 1H, JZ,3 = J3,., = 9.5 Hz, H-3E), 4.03 (pt, 1H, J3,4 = J.~,~ = 9.4
Hz, H-4C), 3.94 (dd, 1H,
Js,s, = 5.3, Js,,6b= 10.7 Hz, H-6ap), 3.83-3.75 (m, 4H, H-6aE, 6bo, CHZCI),
3.74-3.70 (m, 3H,
H-4E, GE, 3H), 3.65 (dd, IH, J1,2= 3.4, JZ,~= 9.4 Hz, H-2E), 3.48 (pt, 2H, H-
4B, 4D}, 3.46 (s, 3H,
OCH3), 3.38 (m, 1H, H-5D), 3.22 (dq, 1H, J4,~ = 9.5, JS 6 = G.2 Hz, H-SB),
2.88 (m, 1H, H-2D),
I.90 (s, 3H, NHAc), 1.42 (s, 3H, CMez), 1.36 (s, 6H, CMe2, H-6~}, and 1.30 (s,
3H, JS,s= 6.3
Hz, H-68); ~3C NMR: X171.8 (C=O, NHAc), 166.4 (C=0, Bz), 139.1-122.5 (Ph),
101.0 (C-ID,
JcH =165 Hz), 99.7 (CMea), 98.3 (C-lc, JcH =172 Hz), 97.8 (bs, C-1 E, Jcx =
170 Hz), 97.5 (C-
18, JcH = 176 Hz), 82.2 {C-3E), 80.7 (G-2~), 79.3 (bs, C-4H), 78.8 (C-3B),
78.1 (bs, C-4E), 77.3
(C-2H), 76.2 (bs, C-3~), 75.8, 75.6, 74.9, 74.6, 73.9 (6C, C-4~, OCHZ), 73.5
{2C, C-4D,2c), 71.4
(OCHz), 71.0 (C-3o), 70.7 (2C, C-SF, SH), G9.0 (C-5c), 68.8 (C-6E), 67.2 (C-
Sp}, 62.5 (C-6p),
60.0 (C-2D), 57.6 (OMe), 46.9 (CHzCI), 29.5 (CMeZ), 23.9 (NHAc), 19.7 (CMez),
19.0 (C-Gg),
and 18.4 (C-6~); FAB-MS for CelH9zNC10zt (M, 1449.5) rrt~a 1472.7 [M+Na]+.
9
LMPPI 1-exp~brevet-pcntnOMe
CA 02434685 2003-07-04
Anal. Calcd for Cg~II92NCIO~~~I-.T2O: C, 66.23; H, 6.34; N, 0.96%. Found: C,
66,11; H,
6.62; N. 0.85%.
1~'olr tentative dcrblocage orthoester seul ou biers mcrlange alorthoesler
issu du couplage data
des proportions eonnues au depart.(6~4 il-12 par ex)
Methyl (2-O-Acetyl-3,4-di-0-benryl-oc-L-rhamnopyranosyl)-(1--~3}-j(2,3,4,6-
tetra-O-
benzyl-a-n-glucopyranosyl-(1 >4)]-(2-O-benzoyl-a-L-rhamnopyranosyl)-(1 >3)-2-
acetamido-2-deoxy-3,4-0-isopropylldene-[3-D-glucopytanoside (XX). The
trisaccharide
acceptor XX (500 mg, 0.47 mm~ol) was dissolved in DCM (5 mL) and the solution
was cooled
to -40°C. TMSOTf (21 ~L) and donor XX (328 mg, 0.62 mmol) were added
and the mixture
was left under stirring while the bath was slowly conning back to rt. Afier S
h, more XX (50
mg, 94 ~Imol) was added and the mixture was stirred at rt for 1 h more at rt.
Et3N was added
and the mixture was concentrated. Column chromatography of the residue
(solvent B, 4:1 --
l:l) gave the fully protected XX (484 rrtg, 72%) slightly contaminated with
the corresponding
trimethylsityl side-product XX. The XX:XX ratio was estimated to be XX:XX from
the 'H
NMR spectrum
R. II~IV c? ai
FAB-MS for CglHg3NOz1 (M, 1415) m~'z XXXX [M+Na]+. Yoir si presence silyl
Anal. Calcd for CgIH93NOzIH20: C, 68.69; H, 6.57; N, 0.98%. Found: C, 67.64;
H,
G.G7; N, 0.88%.
Methyl (3,4-Di-O-benzyl-a-L-rhamnopyranosyl)-(1--~3)-j(2,3,4,6-tetra-0-benzyl-
a-D-
gtucopyranosyl-(1--~4)j-(2-O~benzoyt-a-L-rhamnopyranosyl)-(1--~3)-2-acetamido-
2-
deoxy-3,4-O-isopropylidene-~-D-glucopyrsnoside (XX). (a) Thiourea (362 mg,
4.76 tnmol)
was added to an unseparable mixture of XX and XX (689 mg, 0.48 mmol) in
MeOHlpyridine
(1/1, 16 mL), and the mixture was heated overnight at 65°C. Volatiles
were evaporated, and
the solid residue thus obtained was taken up in the minimum of MeOH. DCM was
added, and
the suspension was left standing at 0°C for 1 h The precipitate was
filtrated on a pad of
Celite, and the filtrate was concentrated. Column chromatography of the
residue (solvent B,
9:1 -» 1:1) gave the trisaccharide acceptor XX (107 mg, 22%) as the first
elating product.
Further elution gave the tetrasaccharide acceptor (419 mg, 63%) together with
a mixture of
XX and XX (G6 mg).
(b) The monoacetytated XX (52.3 mg, 37 letrwl) was dissolved in a mixture of
EtOH (10 mL)
and DCM (100 IIL). A freshly prepared 0.4M ethanolic solution of guanidine (92
pL, 37 pmolj
was added anrl the mixture was stirred at tt overnight. VoLatiles were
evaporated, and the residue
taken up in DCM was washed with water. The organic phase was dried and
concentrated.
Column chromatography of the crude product gave XX (42 mg, 83%). Compound XX
had
CA 02434685 2003-07-04
LMPP11~exp-taevet~p~rttaOMc
RMN ~ faire
FAH-MS for C7gHg~NOZO (M, 1373) m~z 1396.5 [M+Na]+.
Anal. Calcd for C~9H91NOao~ 0.5 H20: C, 68.56; H, 6.65; N, 1.01 %. Found: C,
58.53; H,
6.71; N, 1.01%.
Methyl (2-0-Acetyl-3,4-di-0-benzyl-a-L-rhamnopyranosyl)-(1~3)-[(2,3,4,6-tetra-
0-
benzyl-a-n-glucopyranosyi-(1~4))-(2-0-benzoyl-3-O-chloroacetyl-a.L-
rhamnopyranosyl)-(1~3)-2-acetamido-2-deoay-3,4-O-isopropylidene-p-D-
glucopyranoside (XX). 4A Molecular sieves and TMSOTf (16 ~L) were added to a
solution
of the tetrasaccharide acceptor XX (406 mg, 0.29 mmol) in EtzO (10 mL), and
the mixture
was stirred at -60°C for 30 min. The donor XX (234 mg, 0.44 moral) in
DCM (7 mL) was
added, and the mixture was stizred for 1 h while the bath temperature was
reaching rt. After a
further 1 h at this temperature, more XX (50 mg, 94 Etrrwl) was added, and the
mixture was
stirred for 1 h before Et3N was added. Filtration through a pad of Celite and
evaporation of
the volatiles gave a residue which was column chromatographed twice (solvent
B, 4:1; then
solvent D, 17:3) to give XX (262 mg, 52%); (aJp +25° (c 1.0); LH NMR: &
8.07-7.13 (m,
4SH, Ph), 6.03 (bs, 1H, NHo), 5.59 (bs, 1H, H-2,~, 5.35 (bs, iH, H-2c), 5.16
(bs, 1H, H-lE),
5.13 (bs, 1H, H-lA), 5.06 (bs, 1H, H-1B), 5.02-4.97 (m, 4H, H-1~, lc, OCH~),
4.91~4.50 (m,
12H, OCHz), 4.44-4.32 (m, 4H, H-Ze, 3D, OCHi), 4.20-3.96 (m, 7H, H-5F, 5A, 3c,
3E, 6aD, 5c,
3~, 3.87-3.68 (m, 6H, H-4g, dar, Gba, 6bD, 40, 3s), 3.64-3.47 (m, 7H, H-5H,
4D, 2E, 4A,
OCH3), 3.42 (m, 1 H, H-Sp), 3.34 (pt, 1 H, J3,4 = Ja,s = 9.3 Hz, H-4a), 3,17
(m, 1 H, H-ZD), 2.13
(s, 3H, Iv'HAc), 1.49 (s, 3H, CMez), 1.43 (s, 6H, CMe2, H-6c), 1.33 (d, 3H,
Js,b = 6.I Hz, H-
6,~, and 1.01 (s, 3H, Js,s= 5.8 Hz, H-6H); 13C NMR: cS i71.9 (C=O, NHAc),
170.3 (C=0, Ac),
166.3 (C=0, Bz), 139.2-127.6 (Ph), 101.5 (bs, C-la, JcH = 171 Hz), 101.2 (C-
1D, JcH = 163
Hz), 99.8 (CMe2), 99.7 (C-I A, JcH = 171 Hz), 97.9 (2C, C-lE, lC, JcH =172,
1G9 Hz), 82.4 (C-
3E), 82.1 (C-2E), 80.5 (C-4A), 80.2 (C-3c), 80.1 (C-4$), 79,4 (C-3$*), 78.1
(2C, C-4~*, 3,~,
78.0 (C-4c), 76.6 (C-3D), 75.9. 75.8, 75.4 (3C, OCHZ), 74.8 (2C, C-2B, OCH?),
73.5 (C-4D),
73.4 (OCFii), 73.2 (C-2c), 72.1 (OCHZ), 71.8 (C-5A), 71.2 (OCHZ), 69.4 (C-2,~,
G9.2 (C-5B),
68.9 (C-6~, 68.7 (C-5c), 67.8 (C-SE), 67.5 (C-SD), 62.7 (C-6p), 59.G (C-2p),
57.G (OIvte), 29.5
(CMe2), 24.0 (NHAc), 21.4 (O Ac), 19.7 (CMe2), 19.1 (C-6~), 18.8 (C-6c), and
18.2 (C-6B);
FAB-MS for CLOIHtISNOzs (M. 1741.7) m/z 1765.9 [M+Na]''.
Anal. Calcd for C,alHl isNO~s: C, 69.60; H, 6.65; N, 0.80%. Found: C, 69.56;
H, 6.75; N,
0.73%.
I Methyl arL-rhamnopyranosyi-(1-->3)-[(2,3,4,d-tetra-0-benzyl-a-n-
glucopyranosyl-(1~4))-
a-L-rhl~mnopyranosyl-(1--~3)-2-scetamido-2-dcoay-[3-D-gtucopyranoside (XX).
50% aq
TFA (1 mL) was added at 0°C to a solution of the fully- protected
pentasaccharide XX (155 mg,
j 89 ~mol) dissolved in DCM (4 mL,). After 1 h at this temperature, volatiles
were evaporated.
11
LMPPII~GXp-bftwtt-pClIrHOMC CA 02434685 2003-07-04
The residue was taken up in O.SM methanolic sodium methoxide (8 mL) and the
miscture was
heated overnight at 55°C. Neutralisation with Dowex X8 (Hi'),
evaporation of the volatiles, and
colunnn chromatography of the residue gave XX (171 mg, 98%). Compound XX (111
mg, 81
~mol) was dissolved in a mixture of ethanol (13 mL) and ethyl acetate (2.G mL)
containing 1N
aq HCl (130 ~L). Palladium on charcoal (130 mg) was added and the suspension
was stirred
under a hydxogen atmosphere for 2 h. Filtration of the catalyst and reverse
phase
chromatography gave the target pentasaccharide (60 mg, 88%) as a slightly
yellow foam. RP-
HPLC purification (solvent XX, XXX) followed by freeze-drying gave pure XX (36
mg).
RMN
FIRMS (MALDI) Calcd for C33HS~NOi3 + Na: 858.3219. Found: 858.3089.
12
CA 02434685 2003-07-04
LMPPI 1-Sheme-brevet-pecCsOMc
OTCA OMe OM8
OAC
OMe BnOe ~ 0 Bnbe O Ac0'~~"B~
BnOMe 0 Bn0 pAc Bn0 o Ac0 NPhtCla
8n0 pH ~ Bn0 a o ~~ -....
Bn
OR R
At
H
R3 ~ Ra Re
Ac Ac Ac
08n
01
Bn0 p Me O
Bzo pBz
OBn
Ben~~~ OAIf
Bno o Me p R O
HO~ pH
1
LMPP11~Shemc-btevet~pen~eOMe
CA 02434685 2003-07-04
OH ~0-~~0' ~OMe OAiI
HO ~0 0 H~_5~0Me HO~Ac Me 0
NHAc HO--_~
HO
Me O + 0
0
Ogn
O OH ~ g BnO~ OTCA ~ +
H~ Bn0 0 Me 0
O O8n
CIAcO Ogz
E~O~ '~ g B O~OTO~t
HO pH OTCA ~Oan
Bn0 Me O
Bn0 pAc
CA 02434685 2003-07-04
L PPl2
Preparation of chemically defined gtycopeptides as potential synthetic
conaugate
vaccines against Shigella flexneri secotype 2a disease.
This paper discloses the synthesis of three fully synthetic glycopeptides
incorporating a tri-,
tetra-, and pentasaccharide haptens representative of fragments of the 0-Ag of
S flexi~erl
serotype 2a covalently 1'ed to the PADRE-sequence, which acts as a universal T
cell
epitope is reported. The carbohydrate haptens were selected based on a
preliminary study of
the recognition of synthetic oligosaccharides with homologous protective
antibodies. They
were synthesized following a common block strategy, in a form allowing their
coupling by
chemical ligation onto a maleimido-activated PADRE. Evaluation of the
immunogenicity of
the conjugates in mice is ongoing.
LMPP12-thco-ixevdgp
CA 02434685 2003-07-04
Preparation of chemically defined glycopeptides as potential Synthetic
conjugate
vaccines against Shigella Jlexneri serotype 2a discascl
Abstract
INTRODUCTION
Since the discovery of Shigella dysereteriae type 1 (Shiga's bacillus) more
than a
century ago, 'shigellosis or bacillary dysentery has long been known as a
serious infectious
disease, occurring in humans only. 3In a recent survey of the litezature
published between
19GG and 1977. 4the number of episodes of shigellosis occurring annually
throughout the
world was estimated to be 164.7 million, of which 163.2 nnillion were in
developing
countries. Up to 1.1 million annual deaths were associated to shigellosis
during the same
period. Of the four species of Shigellae, Shigella Jlexneri is the major
responsible of the
endemic form of the disease, with serotype 2a being the most prevalent. The
critical
importance of the development of a vaccine against Shigellae infections was
first outlined in
1987. SDue to increasing resistance of all groups of Shigellae to antibiotics,
hit remained a
high priority as stated by the World Health Organization ten years later. 'In
the meantime,
several experimental vaccines have gone through field evaluation,
8'1°but there are yet no
licensed vaccines for shigellosis.
Shigella's lipopolysaecharide (LPS) is a major surface antigen of the
bacterium. The
corresponding 0-specific polysaccharide domain (0-SP) is both an essential
virulence factor
1
LMPPi 2~tbco~brevetgp
CA 02434685 2003-07-04
and the target of the infected host's protective immune response. it.iZlndeed,
using the
pulmonary marine model for shigellosis, it was recently demonstrated that
secretory IgA
specific for the O-SP of S. flexneri serotype Sa were protective against an
homogolous
infection when present locally prior to the challenge. i313ased on the former
hypothesis that
serum IgG anti-LPS antibodies may confer specific protection against
shigellosis, '4several
polysaccharide-proteine conjugates, targeting either Shigella sonnei, S.
dysenteriae 1 or S.
flexneri serotype 2a, were evaluated in humans. lo,isln the case of S sontrei,
recent field trials
ahowed Bobbins and co-workers to demonstrate the efficacy of a vaccine made of
the
corresponding detoxified LPS covalently linked to recombinant exoprotein A.
i6Conversion of
polysaccharide T-independent antigens to T-depend ones through their covalent
attachment to
a carrier protein had a tremendous impact in the field of ba.eterial wecines.
Several such
neoglycoconjugate vaccines are currently in use against Haemophih~s
influeniae, '~Neisseria
meningitidis, igand Streptococcus pneumoniae. i9These polysaccharide-protein
conjugate
vaccines are highly complex structures, whose immunogenicity depends of
several parameters
amongst which the length and nature of the saceharide component as well as its
loading on the
protein. It is reasonably admitted that control of these parameters is
somewhat difficult when
dealing with polysaccharides purified from bacterial cell cultures. As recent
progress in
carbohydrate synthesis allows access to complex saccharides, it was suggested
that the use of
welt-defined synthetic oligosaccharides may show a better control, and
consequently the
optimisation, of these parameters. Indeed, available data on S, dysenteriae
type 1 indicate that
neoglyeacanjugates incorporating di-, tri- or tetramers of the O-SP repeating
unit were mare
immunogenic than a detoxified LPS-human serum albumin conjugate of reference.
z°Besides,
recent reports demonstrate that short oligosa;ccharides comprising one
repeating unit or less
may be immunogenic in animal models. zi.2zAnother critical parameter in the
design of
neoglycoconjugate vaccines is the carrier protein. As potential applications
for these vaccines
are expanding, the need for new carrier proteins licensed for human uee is
growing. 23That
synthetic peptides representing immunodominant T-cell epitopes could act as
carriers in
polysaccharide and oligosaccharide conjugates has been suggested, ~~and latter
on
demonstrated. Zs,zeBesides, the use of T-cell epitopes offer several
advantages, including
potential access to well-defined conjugates with no risk of epitopic
suppression, as the latter
phenomenon appeared as a major drawback of protein carriers. 2~-3oPolypeptides
containing
multiple T-cell epitopes have been generated in order to address the extensive
polymorphism
of HLA molecules. ~i.3ZIn other strategies, universal T-helper epitopes
compatible with human
use have been characterized, for example from tetanus toxoid, 33or engineered
such as the pan
2
LMPP12~theo~brevetgp
CA 02434685 2003-07-04
HLA DR-binding epitope (PADRE). 34Recently, covalent attachment of the human
nnilk
oligosaceharide, facto-N fucopentose II, to PADRE resulted in a linear
glycopeptide of
comparable immunogenicity to that of a glycoconjugate employing HSA as the
carrier. 3s
Based on these converging data, we focused on the development of well-defined
neoglycopeptides as an alternative to polysaccharide~proteine conjugate
vaccines targeting
infections caused by S. flexneri 2a. The target neoglycopeptides were
constructed by
covalently linking a short peptide, serving as a T-helper epitope, to
appropriate carbohydrate
haptens, serving as B epitopes mimicking the S. "~texneri 2a 0-Ag. Our
approach is based on
rational bases involving a preliminary study of the interaction between the
bacterial O-SP and
homologous protective monoclonal antibodies, which helped to define the
carbohydrate
haptens.
RESULTS AND DISCUSSIO\T
A S E C D
2)-a-L-Rhap-( 1 ~2}-a-L-Rhap-( 1 ~3 )-(a-D-Glcp-( 1->4)]-a-L-Rh,a~p-( 1-33)-~i-
D-GIcNficp( 1 ~
I
The O-SP of S flexneri 2a is a heteropolysaccharide defined by the
pentasaccharide repeating
unit I. 3w~It features a linear tetrasaccharide backbone. which is common to
all S. flexneri O-
antigens and comprises a N-acetyl glucosaminc (D) and three rhamnose residues
(A, B, C}
The specificity of the serotype is associated to the a-D-glucopyranose residue
linked to
position 4 of rharnnose C. Besides the known methy~1 glycoside of the EC
disaccharide, 38°39a
set of di- to pentasaccharides corresponding to frame-shifted fragments of the
repeating unit I,
°o-~3an octasaccharide4~ and more recently a decasaccharide45
representative of fragments of S.
Jlexneri 2a 0-SP have been synthesized in this laboratory. Based on the use of
these
compounds as molecular probes for mapping at the molecular level the binding
characteristics
of a set of protective nwnoelonal antibodies against S. flexneri 2a infection,
~6fragments ECD,
B(E)CD and AB(E)CD were selected as haptens that will act as B-epitopes in the
conjugates.
Three fully synthetic linear neoglycopeptides 1, 2 and 3, corresponding to
haptens ECD,
B(E)CD, and AB(E)CD, respectively, were synthesized according to a strategy
built up on
the concept of chemoseiective ligation which allows the selective one-point
attachment of the
free B and T epitopes in aqueous media. All conjugates involve the peptide
PADRE as the
universal T-cell epitope.
3
LMPPLZ-theo-brcretgP
Scheme 1:
CA 02434685 2003-07-04
Retrosynthetic analysis o~"the saccharidic haptens (Scheme 1): Analysis of
S,'lexr~eri 2a 0-
SP suggests that, due to the 1,2-cis glyeosidic linkage involved, construction
of the EC
disaccharide is probably the most demanding. Besides, prior work in this
laboratory has
shown that the C-D glycosidic linkage was an appropriate disconnection site
when dealing
with the blockwise synthesis of oligosaccharide fragments of S flexrteri 0-2a
SP. 4o,a2,a~These
observations supported the design of a synthetic strategy common to all three
targets.
Basically, it relies on (i) the condensation of an EC (4), 4~H(E)C (5) 42or
A.B(E)C (6) donor to
a D acceptor (7), functionalized at the anomeric position with an azidoethyl
spacer; (ii)
elongation of the spacer with introduction of a masked thiol group to allow
its coupling onto a
PADRE peptide derivatized by a maleimido group on a C-terminal Lysine (8). The
carbohydrate synthesis relies on the trichloroacetimidate methodologya~ and
the use of known
building blocks whenever possible.
Scheme 2:
Synthesis of the aminoethyl LcCD building block IS (Scheme 2): The now easily
accessible
disaccharide donor 4, '~zwith a benzoyI participating gxoup at position 20,
was used as the
precursor to the EC moiety in the construction of 1. It was prepared, as
described, ~~in 5 steps
and 45% overall yield from 2,3,4,6-tetra-O-henry)-~i-D-glucopyranosyl
trichloroacetinudate
(9) dg'a9and ally) 2,3-O-isopropylidene-a-D-rhamnopyranoside (IO)
s°through Lhe key
intermediate diet 1I (69% from IO). Introduction of the azidoethyl spacer on a
glucosaminyl
intermediate was performed according to a known procedures) by coupling of
azidoethanolsz
onto the oxazoline53 12 to give the triaeetate I3. sl,s'4We have shown on
several occasions in
the S Jlexneri series, that regioselective protection of the 4- and 6-OH
groups of precursors to
residue D with an isopropylidene acetal was appropriate, especially when such
precursors are
involved in a blockwise synthesis based on the disconnection at the C-D
linkage. ~~,44Thus,
Zemplbn deacetylation of I3 gave the trio) I4 which was converted to the key
acceptor 7
(81% from 13) upon reaction with 2,2-dimethoxypropane under acid catalysis.
V~hen the
latter was glycosylated with the donor 4 in the presence of BF3.OEtz in
dichloromethane, the
fully protected trisaccharide IS was isolated in S8% yield together with the
diet 16 (30%),
resulting from partial loss of the isopmpylidene acetal. When 4 and 7 were
glycosylated in the
presence of a catalytic amount of TMSOTf, no side-reaction was observed, and
the
condensation product 15 was obtained in 86% yield. Quantitative conversion of
15 into I6
4
LMPP E 2-thco-brrvetgp
CA 02434685 2003-07-04
was more conveniently achieved by acidic hydrolysis of the former with 95% aq
TFA
Zemplen debenzoylation of 16 gave the tetraol 17 (94%) which was subsequently
transformed
into the aminoethyl-armed trisaccharide 18 (69%) by hydrogena.tinn in the
presence of
palladium-on-charcoal (Pd/C) and 1N aq HCl to convert the formed amine to its
hydrochloride salt. Indeed, others have pointed out that hydrogenoiysis using
Pd/C in the
presence of a free amine was sluggish and low-yielding. ss-s~In order to
prevent any side-
reaction at a latter stage of the synthesis, isolation of pure 18 was
performed by reversed-
phase HPLC (ItP-HPLC).
Scheme 3:
Synthesis of tlae aminoethyl B(Lr')CD building block 25 (Scheme 3): The known
rhamnopyranosyl tricholoracetimidate 20, SRacetylated at its 2-, 3-, and 4-OH
groups thus
acting as a chain terminator, was chosen as the precursor to residue C.
Benzoylation of diol
11 to give 19 was performed by regioselective opening of the cyclic orthoester
intermediate as
described. ~lGlyeosylati~on of the latter by donor 20, with activation by a
catalytic amount of
TMSOTf proceeded smoothly in EtZO to yield the fully protected trisaccharide
21 (89%),
which was de-O-allylated into the hemiacetal 22 (80%) following a two step
process
involving (i) iridium(I)-catalysed isomerisation of the allyl glycoside to the
prop-1-enyl
glyCOSldes9 and (ii) subsequent hydrolysis. so,s°The selected
trichloroacetimidate leaving
group was introduced by treatment of 22 with trichloroacetonitrile in the
presence of a
catalytic amount of DBU, which resulted in the formation of S (99%).
Condensation of the
latter with acceptor 7 was performed in CHZC1Z in the presence of a catalytic
amount of
trifluoromethanesulfonic acid (TfbH) to give the required tetrasaccharide 23
(76%). Acidic
hydrolysis of the latter using 95% aq TFA gave the intermediate diol 24 in 95%
yield.
Deacylation of the resulting diol under Zemplen conditions followed by
debenzylation and
concomitant conversion of the azide into the corresponding amine to give the
key aminoethyl-
armed tetrasaccharide 25 (77%) was performed by treatment of 24 with hydrogen
in the
presence of Pd/C under acidic conditions. Again, compound 25 vt~as purified by
Rl'-HLPG
before elongation ofthe spacer or conjugation.
Scheme 4:
Synthesis of !he aminoethyl AB(E)CD building block 37 (Scheme 4); The
synthesis of 37 is
based on the condensation of acceptor 7 and donor 6, which resulted from the
selective
deallylation and anomeric activation of the key intermediate tetrasaccharide
33. The latter ws
LMPP12~theo-Mevetgp
CA 02434685 2003-07-04
obtained according to two routes following either a block strategy (route 1)
based on the
condensation of an AB disaccharide donor (30) and the EC disaccharide acceptor
16, or a
linear strategy (route 2) involving the stepwise elongation of 16. The
construction of the
donor 30 was based on the use of the known allyl rhamnopyranoside 26, 6~having
permanent
protecting groups at position 3 and 4, as the precursor to residue B, and the
trichloroacetimidate chain terminator Z7, 62acting as a precursor to residue
A. Condensation of
the two entities in the presence of a catalytic amount of TMSOTf resulted in
the fully
protected 28 (96%), which was selectively de-O-allylated into 29 (84%)
according to the
protocol described above for the preparation of 22. Subsequent treatment of 29
with
trichloroacetonitrile and a catalytic amount of DBU gave the required 30
(96%).
Glyeosylation of 16 with the latter under TMSOTf promotion afforded the fully
protected
I tetrasaccharide 34 in 55% yield. No (3-anomer was detected. The
stcreochemical outcome of
this glycosylation step involving a rhamnosyl donor glycosylated at C-2, thus
lacking any
participating group at this position is not without precedent. Related
examples involving
rhamnopyranosyl donors may be found in the synthesis of oligosaccharides
representative of
the capsular polysaccharide of the ~i-hemolytic Streptococcus Group A, ~3or of
the O-Ag of
Serratia marcescens 0186" as well as in our own wroxk on S ~lexneri seroty~pe
2a. A'Route 1
was considered initially in order to prevent extensive consumption of the EC
disaccharide il.
C3iven the relatively low yield of coupling of 16 and 30, route 2 was
considered as well. Of all
precursors to 34, only that to residue B, namely the donor and potential
acceptor 31, differed
from those used in routs 1. Conventional glycosylation of disaccharide 16 and
31 and
subsequent selective deacetylation using methanolic IdBF4, gave the acceptor
32 in 70% yield
from 16. 45The trisaccharide 32 was glycosylated vvith trichloroacetimidate 27
in as analogous
fashion to its glycosylation with 30, yielding 34 (92%). Deallylation of this
key intermediate,
as described above fox the preparation of 22, gave the corresponding
hemiacetal 35 (94%)
which was converted into the required trichloroacetixnidate 6 ($8%) upon
treatment with
triehloroacetonitrile and DBU. Condensation of donor 6 with the glucosaminyl
acceptor 7 was
performed under promotion by TfOFi or TMSOTf, which resulted in the fully
protected
pentasaccharide 35 in 62% and 80% yield, respectively. Following the process
described for
the preparation of 25, compound 35 was submitted to acetolysis (97%) and
subsequent
ZemplEn deacylation to give the partially debloeked 36 (87%), which was next
converted to
the aminoethyl-spacer pentasaccharide 37 upon treatment with hydrogen in the
presence of
Pd/C. Final RP-HPLC purification resulted in the isolation of 37 in 53% yield.
6
LMPPllwlteo-httroetgp
Scheme 5:
CA 02434685 2003-07-04
Synthesis of the target neoglycopeptides 1-3 (Scheme S): In all casES,
chemoselective ligation
of the B and T epitopes was achieved through coupling of the carbohydrate
haptens pre-
functionalized wish a thiol function and a maleimido group properly introduced
at the C
terminus of the T helper peptide. Such a strategy was chosen i.n order to
exploit the high
reactivity and specificity of thiol groups towards the maleimide
functionality, dswhich shows
specific and high-yielding modification of the former in the presence of other
nucleophiles.
slit was used previously under various forms in the coupling of carbohydrate
haptens to either
proteins6~~6g or peptides. 2s,saTo our knowledge, in all the reported cases
the maleimide
functionality was introduced onto the carbohydrate hapten. On the contrary,
our strategy relies
on the introduction of this activating group on the T helper peptide. The
immunogenicity of
various maleimide-derived coupling reagents was evaluated in a model system.
69Based on the
reported data, 694-(N maleimido)-n-butanoyl was selected as the linker, and
incorporated by
coval~nt linkage to the side chain amino group of a Lysine residue added at
the C-terminus of
the PADRE sequence (PADRE-Lys). It is worth mentioning that the strategy
described herein
somewhat differs from that described by others when demonstrating the
usefulness of PADRE
in the construction of immunogenic neoglycopeptides. 3s
The Lysine-modified PADRE (8) was assembled using standard Fmoc chemistry for
solid-
phase peptide synthesis. ~°Standard side chain protecting groups were
used, except. fo.r that of
the C-ternzinal Lysine side chain which was protected by the 1-(4,4-dimethyl-
?.,b-
dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) group. "Indeed, this orthogonal
protecting
group strategy allows specific introduction of the maleimide group on the
C~terminat Lysine,
upon selective cleavage of the ivDde by hydrazine, The thiol functionality was
intzoduced
onto the carbohydrate haptens as a masked fhiol function (acetylthioester),
which is easily
generated in situ during the conjugation process Thus, reaction of IS, 25, and
27 with S-
acetylthioglycolie acid pentafluorophenyl ester (SAMA-offp) resulted in the
site-selective
elongation of their aminoethyl spacer via a thioacetyl acetamido linker.
Derivati7.ation could
be monitored by RP-HPLC with detection at 215 nm. Under these conditions, the
required
thioacetyl-armed intermediates, 38, 39 and 40 were isolated in 53%, 74%, and
7S% yield,
respectively. Their structure was confirmed based on MS and NMR analysis.
Conjugation of
the carbohydrate haptens to the maleimido activated PADRE-Lys (8) was run in
phosphate
buffer at pH G.0 in presence of hydroxylamine'Zand monitored by RP-HPLC.
Lastly, RP-
7
LMPP12-tho°-brevet~p
CA 02434685 2003-07-04
HPLC purification gave the target neoglyeopeptides 1, 2, and 3 as single
products, which
identity was assessed based on MS analysis, in yields of 58%, 48% and 46%,
respectively.
CONCLUSION
The synthesis of three fully synthetic glycopeptides incorporating a trl-,
tetra-, and
pentasaccharide haptens representative of fragments of the 0-Ag of S flexneri
serotype 2a
covalently linked to the PADRE-sequence, which acts as a universal T cell
epitope is
reported. The carbohydrate haptens were selected based an a preliminary study
of the
recognition of synthetic oligosaccharides with homologous protective
antibodies. They were
synthesized following a common block strategy, in a form allowing their
coupling by
chemical ligation onto a maleimido-activated PADRE. Evaluation of the
immunogenicity of
the conjugates in mice is ongoing.
ACKNO WLEDGEMENTS
The authors are grateful to J. Ughetto-Monfrin (Unite de Chimie Organique,
Institut Pasteur)
for recording all the NMR spectra. The authors thank the Bourses Mrs Frank
Howard
Foundatian for the postdoctora! fellowship awarded to K. W., and the Institut
Pasteur for its
financial support (grant no. PTR 99 j.
REFERENCES
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c
polysaccharides of Shigella flexnera serotype 2a and Shigella flexneri
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ll, see ref. XX. 2003.
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8
LMPPI2.thee-brevetgp
CA 02434685 2003-07-04
(8) Coster, T. S.; Hoge, C. W.; Verg, L. L. v, d.; Hartman, A. B.; Oaks, E.
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(17) Ellis, R W.; Granofl7 D. M. Development and clinical use of Haemophilus b
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(18) Richmond, P.; Borrow, R.; Miller, E.; Clark, S.; Sa.dler, F.; Fox, A.;
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Morris, R.; Cartwright, K. J, Infect. Dis. 1999, 179, 1569-1572.
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CA 02434685 2003-07-04
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Lett. 2002, 43, 8215-8218.
(45) Blot, F.; Wright, K; Costachel, C.; Phalipon, A.; Mulard, L. A. J. Org.
Chem.
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Phalipon, A, in
preparation 2003.
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1981, 305-308.
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LMPP 12-then-brevetgp
CA 02434685 2003-07-04
(61) Westerduin, P.; Haan, P. E. d.; Dees, M. J.; Boom, J. H. v. Carbohydr.
Res.
1988, I80, 195-205.
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Glycoconjugate .I. 1998,15, 217-221.
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Immunol.
Methods 1989, l20, 133-143.
(70) Chan, W. C.; White, P. D. Fmoc solid phase peptide synthesis; Oxford
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Char, W.
C. Tetrahedron Lett. 1998, 39, 1603-1606.
(72j Brugghe, H. F. Ins J. Peptide Protein Res. 1994, 43, 166-172.
LMPP 12exp-brevet-gp
CA 02434685 2003-07-04
General Methods. General experimental methods not referred to in this section
were as
described previously.(REF) TLC on precoated slides of Silica Gel GO Fzsa
(Merck) was
performed with solvent mixtures of appropriately adjusted polarity consisting
of A,
dichloromethane-methanol; B, cyclohexanc-ethyl acetate, G, cyclohexane-diethyl
ether, D,
toluene-acetone. Detection was effected when applicable, with UV light, and/or
by charring
with orcinol (35 ttLM) in 4N aq HzSOa. NMR Spectra were measured in CDC13
unless stated
otherwise. In the NMR spectra, of the two magnetically non-equivalent geminal
protons at C-
6, the one resonating at lower field is denoted H-da and the one at higher
field is dcnr~ted H-6b.
Interchangeable assignments in the 1'C NMR spectra are marked with an asterisk
in listing of
signal assignments. Sugar residues in oligosaccharides arc serially lettered
according to the
lettering of the repeating unit of the O-SP and identified by a subscript in
listing of signal
assignments. Low-resolution mass spectra were obtained by either chemical
ionisation (CIMS}
using NHj as the ionising gas, by electrospray mass spectrometry ($SMS), or by
fast atom
bombardment mass spectrometry (FABMS), High-resolution mass spectra were
obtained by
MALDI-MS.
Solid phase peptide synthesis was performed using standard Fmoc chemistry
protocols on a
Pioneer peptide synthesiser (AppliedBiosystem). Fmoc-Lys{iv-Dde)-OH, Fmoc-Cha-
OH,
Fmoc-D-Ala-OH, Fmoe-eAhx-OH and Boc-D-Ala-OH were purchased from Noval3iochem
(VWR). All others reagents and amino acids were purchased from Applied
Biosystem-
2-A~idoethyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-~-D-glucopyranoside (7).
Camphorsulfonic acid (200 mg, 0.9 mmol) was added to a solution of triol 14
(1.31 g, 4.52
mmol) in a mixture of DMF (4 mL) and 2,2-dimethoxypropane (4 mL). After 3 h at
rt, low
boiling point solo~ents were evaporated under reduced pressure and more 2,2-
dimethoxypropane (2 mL, 15.8 mmol) was added. The mixture was stirred for 2h
at rt, Et3N
was added; and the mixture was concentrated. The crude product was purified by
column
chromatography (solvent A, 19:1) to give 7 as a white solid (1.21 g, 81%),
[a]p -89.8; 'H
NMR: b 6.15 (d, 1H, J = 5.9 Hz, NH), 4.70 (d, 1H, Jt,2 = 8.3 Hz, H-1), 4.05
(m, 1H, OCHZ),
3.97-3.89 (m, 2H, H-6a, 3), 3.79 (pt, 1H, Js,6b = 3s~,sb = 10.5 Hz, H-Gb),
3.70 (m, 1H, OCHz),
3.62-3.46 (m, 3H, H-2, 4, OCHZ), 3.35-3.ZG (m, 2H, H-5, CH2N3), 2.05 (s, 3H,
Ac), I.52 (s,
3H, C(CH3)z), 1.44 (s, 3H, C(CH3~); "C NMR: 8 100.9 (C-1), 74.3 {C-4), 81.8 (C-
3), 68.6
(OCHZ), 67,3 (C-5), 62.0 (C-G}, 58.7 {C-2), 50.7 (CHzN3), 29.0 (C(CH3)Z), 23.G
(CH3C0),
19.1 (C(CH3)x). CIMS for C,3H~I~406 (330) mla 331 [M+H]+. Anal. Calcd. for
Cs~H~aN40'~~O.SHZO: C, 46.OI; H, 6.83; N, 16.51. Found C, 46.37; H, G.69; N,
16.4.6.
2-Azidoethyl (2,3,4,6-Tetra-O-bearyl-a-D-glucopyranosy>)-(1--;~4)-(2,3-di-0-
be~nzoyl~a-L-
rhamnopyranosyl)-(I >3)-2-acetamido-Z-deo~,y-4,6-0-isopropylidene-(3-D-
LvIPPl2cxp-brevet-gp
CA 02434685 2003-07-04
glucapyranoslde (15). (a) The disaccharide donor 4 (1.425 g, 1.37 mmol) and
the acceptor 7
(377 mg, 1.14 mmol} with 4A-MS (2 g) were pla;eed under argon and CHzCl2 (15
mL) was
addod. The mixture was stirred for 1 h at rt, then cooled to -40°C. A
solution of BF3.OEtz (0.5
mL, 4.11 mmol) in CHZCh (5 mL;) was added dropwzse. The mix-hue was stirred at
-40°C to
-15°C over 3 h. Triethylaminc (2.5 mL) was added and the mixture
stirred for ZO min. The
mixture was filtered through a pad of Celite, and the filtrate was
concentrated. The mixture
was purified by column chromatogaphy (solvent A. 2:3) to give 15 (803 mg, 58%)
as a
colourless foam. Further elution (solvent B, 9:1) gave 16 (395 mg, 30%) as a
colourless foam.
(b) 4 A Molecular sieves (560 mg) were added to a solution of donor 4 (565 mg,
0.54 mmol)
arid acceptor 7 (150 mg, 0.45 mmol) in DCM (3 mL) and the suspension was
stirred for 15
min-40°C. Triflic acid (1G pL) was added and the mixture was stirred
for 3h at rt once the
cooling bath had reached rt. Et3N was added and after 15 min, the mixture
v~~as filtered
through a pad of Celite. Volatiles were evaporated and the residue was column
chromatographed (solvent B, 9:1) to give 15 (475 mg, 87%). [oc]n +87.7 (c
0.32);'H NM1Z: 8
6.99-8.07 (m, 30H, Ph), 6.21 (d, 1H, NH), 5.58 (dd, 1H, H-3~), 5.44 (m, 1H, H-
2c), 5.13 (d,
1H, Jl,z = 8.3 Hz, H-lp), 5.02 (d, 1H, J,,z = 3.4 Hz, H-lE), 4.97 (d, 1H, Jl,z
= 1.5 Hz, H-lc),
4.64-4.90 (m, SH, CHzPh), 4.45 (t, 1H, H-3n), 4.27 (m, 3H, H-Sc. CHzPh), 3.79-
4.05 (m, 7H,
H-3E, 4c, So, Gap, 6bD, CHzO, CH2Ph), 3.60-3.76 (rrt, 4H, H-4D, 4E, 5E, CHzO),
3.37-3.51 (m,
3H, H-2s, 5n, CHzN3), 3.16-3.34 (tn, 3H, H-2n, GaE, CHzN3), 3.04 (d, 1H, H-
6bE), 2.01 (s, 3H,
CH3C=0), 1.43 (s, 6H, (CH3)zC), 1.36 (d, 3H, H-6c); 13C NMR: cS 171.7, 165.6,
163.4 (C=O),
127.3-138.6 (Ph), 99.6 (C-1D), 99.1 (C-lE), 97.7 (C-lc), 91.9 ((CH3)zG~, 81.4
(C-3E), 80.3 (C-
2E), 79.4 (C-4~), 77.1 (C-4p), 76.0 (C-3d), 75.3, 74.6, 73.9, 73.2 (4C,
CH2Ph), 73.1 (C-4E),
71.2 (2C, C-2o, 3c), 71.1 (C-5E), 68.6 (CH20), 67.5 (C-Sc), 67.4 (C-6a), 67.1
(C-5D), 62.1
(C-6D), 59.0 (C-2D), 50.5 (CH2N3), 28.9 ((CH3)zC), 23.4 (CH3C0), 19.2
((CH3)zC), 18.1 (C-
G~). FAB-MS for C6~H~4NdOu (1206) m/z 1229 [M+Na]+.
Anal. Calcd. for C6~H7~N401~: C, 60.41; H, 5.66; N, 4.82. Found: C, 60.36; H.
5.69; N, 4.78.
Z-Azidoethyl (2,3,4,G-Tetra-O-benzyl-a-D-glucopyranosyl)-(1~4)-(Z,3-di-O-
benzoyl-a-L-
rbamnopyranosyl)-(1~3)-Z-acetamido-Z-deo~y-p-D-gtueopyranoside (16). Compound
15
(95 mg, 79 umol) was dissolved in 80% aq AcOH (2.5 mI,), and the mixture was
heated at
GO°C for 1 h. After cooling to rt and repeated co-evaporation with
toluene, the crude residue
was column chromatographed (solvent B, 1:4 -~ 0:1) to give 16 (80 mg, 87%) as
a white
foam. [a]D +91.5 (c 0.18); tH NMR: & 6.99-8.02 (m, 30H, Ph), 6.10 (d, 1H, NH),
5.60 (dd,
1H, H-3c), 5.52 (m, 1H, H-2c), 5.20 (d, 1H, J,~ = 8.3 Hz, H-1D), 5.00 (d, 1H,
Jl~ = 1.9 Hz. H-
2
L~lPPt2exp-brcva-gp
CA 02434685 2003-07-04
Ic), 4.95 (d, 1H, Jt,z = 3.4 H~, H-lE), 4.63-4.89 (m, 5H, 5 CHzPh), 4.47 (pt,
1H, H-3D), 4.25
(d, 1H, CHzPh), 4.19 (m, 2H, H-5c, CH2Ph), 4.06 (rn, IH, CHzO), 3.87 (m, 5H, H-
3E, 4c, 6aD,
Gbn, CHzPh), 3.58-3.74 (m, 4H, H-4E, 5n, 5E, CHzO), 3.50 (m, 3H, H-2E, 4D,
CH~N3), 3.29
(m, 2H, H-6aF, CHzN3), 3.04 (d, 2H, H-2p, 6bE), 2.02 (s, 3H, CH3C0), 1.SI (d,
3H, H-6c);
'3C NMR: 8 171.5, 165.6, 165.2 (3C, C=0), 127.3-138.6 (Ph), 99.G (C-lc), 99.5
(C-lF), 99.0
(C-ln), 83.4 (C-3n), 81.6 (C-3E): 80.1 (C-2E), 79.2 (C-4c), 77.2 (C-4F), 75.5
(CH~h), 75.1
(C-4p), 74.7, 74.0, 73.2 (3C, CHlPh), 71.3 (C-5o*)> 70.9 (C-5$*), 70.8 (C-3c),
70.4 (C-2c),
69.0 (C-Sc), 68.8 (CH20), 67.5 (C-GE), 67.6 (C-GD), 57.9 (C-2d), 50.5 (CHZNa);
23.4
(CH3C0), 18.2 (C-Gc). FAH-MS for C~yH~GN4Ol7 (11GG) m/z 1185 [M+Na]+.
Anal. Calcd. for C6~H~oNaO~,~HzO: C, 64.85; H, 6.12; 1~', 4.73. Found: C,
64.71; H, 6.01; N,
4.83.
2-Azidocthyl (2,3,4,6-Tetra-0-benzyi-a-D-glucopyranosyi)-(1--~4)-a-L-
rhamnopyrnnosyl-(1--~3)-2-acetamido-2-deo~.y-[i-D-glucopyranoside (17). An ice
cold
solution of95% aq TFA (1.5 mL) in CHzCl2 (13.5 mL) was added to the
trisaccharide 15 (730
mg, 0.60 mmol). The mixture was kept at 0°C for 15 min, then diluted
with toluene and
concentrated. Toluene was co-evaporated from the residue. The residue was
dissolved in
MeOH (20 mL), and a 1M solution of sodium methoxide in MeOH (1.5 mL) was
added. Ths
mi.~cture was left to stand at rt for 3 h. The mixture was neutralised with
Amberlite IR-120
(H~ resin and filtered. The filtrate was concentrated. The mixture was
purified by column
chromatography (solvent A, 9:1) to give 17 (S48 mg, 94%) as a colourless foam
(a]n +9.7 (c
0.48, MeOH); 1H NMR: 8 7.13-7.31 (m, 8H, Ph), 5.99 (d, 1H, NH), 4.79-4.97 (m,
7H, H-lc,
lo, lE, CHZPh), 4.35-4.74 (m, 4H, CHZPh), 3.91-4.10 (m, 7H, H-2c, 3n, 3E, 5c,
5E, ban,
CH?O), 3.80 (m, 2H, H-3E: Gbp), 3.73 (m, 1H, CH20), 3.40-3.63 (m, 8H, H-2E,
4c, 4n, 4E, 5p,
6aE, 6bE, CHzN3), 3.27 (m, 2H, H-2D, CH2N3), 1.99 (s, 3H, CH3C0), 1.41 (d, 3H,
H-dc); 13C
NMR 8 170.7 (C=O), 127.6-138.4 (Ph), 101.2 (C-1~), 99.7 (C-lFj, 99.0 (C-ln),
84.7 (C-4c),
84.3 (C-3o), 81.5 (C-3E), 79.G (C-2F), 77.6 (C-4D*), 75.G (CHzPh), 75.3 (C-
4s*), 74.9, 73.5,
73.4 (3C, CH2Ph), 71.2 (C-SE), 70.8 (C-5c), 70.8 (C-5n), G9.4 (C-3c), 68.6 (C-
6E), 68.4
(CH20), 67.6 (C-2c), G2.G (C-6n), 5G.4 (C-2n), 50.5 (CHzN3), 23.5 (CH3C0),
17.6 (C-6c).
FAB-MS for CsoHszNaO~s (958) rn/z 981 [M+Na)~'.
Anal. Calcd. for C5oH62N,~O~s.HZO : C, 61.46; H, G.GO; N, 5.73. Found: C,
61.41; H, 6.61; N,
5.97.
3
LMPP I ~exp-brevd~gp
CA 02434685 2003-07-04
2-Aminoethyl a-D-Glucopyranosyl-(1-~4)-a-L-rhamnopyrnnosyl-(1--~3)-2-acetamido-
2~
deo~,y-[i-D-glucopyranoside (I8). The trisaccharide I7 (368 mg, 0.38 mmol) was
dissolved
in a mixture of EtOH (10 mL) and EtOAc (1 mL). A 1N solution of aqueous HCI
(0.77 mL)
was added. The mixture was stirred under hydrogen in the presence of 10% Pd/C
(400 mg) for
24 h. The mixture was diluted with water and filtered. The filtrate was
concentrated, then
lyophilised. The residue was dissolved in a solution of NaHC03 (75 mg) in
water (1 mL) and
purified by passing first through a column of C~8 silica (eluting with water),
then through a
column of Sephadex Gio (eluting with water) to give, after lyophilisation, 18
(151 mg, 69%).
HPLC (230 nm): Rt 4,09 min (Kromasil 5 p.m C18 100 A 4.Gx250 mm analytical
column,
using a 0-20% linear gradient over 20 min of CH3CN in O,O1M aq TFA at 1 mLlmin
flow
rate).'H NMR (Dz0): 8 5.03 (d, 1H, Jl,z = 3.8 Hz, H-1~), 4.84 (bs, 1H, H-lc),
4.58 (d, 1H, Jt,z
= 8.5 H~ H-1D), 4.10 (m, IH, H-Sc), 3.98 (m, 3H, H-5E, 6I~, CHzO), 3.79 (m,
6H, H-?c, 2D,
3p, 6aE, 6bE, CHzO), 3.68 (pt, 1H, H-3E), 3.42-3.G0 (m, 6H, H-2E, 30, 4c, 4D,
4E, SD), 3.03 (m,
2H, CHZNHz), 2.OG (s, 3H, CH3G0), 1.31 (d, 3H, H-Gc); '3C S 175.2 (C=O), 101.9
(C-lc),
101.0 (C-lo), 100.3 (C-lE), 82.4 (C-3B), 81.G (C-4c), 76.5 (C-2E), 73.3 (C-
3E), 72.4 (C-56),
?2.1 (C-4D), 71.G (C-2c), 69.9 (C-4E), G9.5 (C-3c), G9.0 (C-Sp), G8.7 (C-5c),
68.7 (CHzO),
61.2 (C-6D), G0.7 (C-GE), 55.8 (C-2D), 40.3 (CHzNHz), 22.7 (CH3C0), 17.3 (C-
Gc).
Electrospray-MS for CzzHaoNz0ls (572) »~/z 573 [M+H]f. Manqae 6anel3~; 2x30
HRMS (MALDI] Calcd for CZZHaoNsOis+Na: 595.2326. Found: ~;XXXX.
Altyl (2,3,4-tri-0-acetyl-a-L-rhamnopyrranosyl)-(1-r3)-[(2,3,4,6-tetra-0-
benzyl-a-D-
glucopyranoayl)-(1-->4)]-2-0-ben~.oyl-a-L-rhamnopyranoside (21). TMSOTf (100
~.L) was
added to a solution of donor 20 (2.5 g, 5.78 mmol) and acceptor 19 (4.0 g,
4.80 mmol) in EtzO
(40 mL) at -SO°C. The mixture was stirred for 2.5 h, at which time the
cooling bath had
reached rt. Et3N was added and after 15 min, volatiles were evaporated. Column
chromatography (solvent C, 4:1) of the crude product ga~~e the folly protected
21 (4.74 g,
89%) as a white solid. 'H NMR: 8 8.00-6.90 (m, 25H, Ph), 5.92 (m, 1H, CH=),
5.53 (dd, 1H,
H-28), 5.40-5.20 (m, 4H, H-lE, 20, CHz=), 5.18 (dd, 1H, Jz,~ = 3.2, J3,4 =
IO.X Hz, H-31s), 5.10
(d, IH, H-1H), 5.00-4.40 (m, 10H, H-4B, lc, OCHz), 4,30-4.00 (m, SH, H-3s, 3c,
SE, OCHz),
4.00-3.50 (m, 7H, H-2r, 4F, 6aE, 6bE, Sa, So, 4c), I.90 (s, 3H, Ac), 1.G0 (s,
3H, Ac), 1.22 (s,
3H. Ac), 1.20 (d, 3H, J5,6 = X.X Hz, H-Gc), 0.80 (d, 3H, JS,~ = X.X Hz, H-Gn);
"C NMR: b
16.89-16.55, 1GG.1 (4C, C=O), 133.4-127.3 (Ph), 117.5 (=CHz), 9.8 (C-1B), 96.9
(C-lE), 95,7
(C-lc), 81.4 (C-3E), 80.7 (C-2~), 7.3 (C-3c), 77.7 (C-4E), 77.5 (C-4c), 75.6-
72.6 (4C,
OCHzfh), 72.7 (C-2c), 7.9 (2C, C-5E, 48), G.0 (C-2H), 68.7 (C-GE), 68.6 (C-
3H), 68.2 (OCHz),
4
LMPPI2exp~brevct-Rp ~1 02434685 2003-07-04
G7.2 (C-Sc), 6G.8 (C-5a), 20.7 20.2 {3C, C(0)CH3), 18.5 (C-6c), 16.8 (C-6B).
CI-MS for
C62H70018 (1102) m/z 1125 [M+Na]+.
Anal. Calcd. for C62H~o~~s: C, 67.50; H, G.40. Found: C, 67.51; H, 6.52.
(2,3,4-Tri-0-acetyl-a-L-rhamnopyranosyl)-(1->3)-((2,3,4,G-tetra-0-benzyl-a-D-
glucopyranosyl)-{1---~4)]-2-O-benzoyl-a-L-rhamnopyranose (22). 1,5-
Cyclooctadiene-
bis(methyldiphenylphosphine)iridium hexafluorophosphate (33 mg) was dissolved
in THF
(10 mL) and the resulting red solution was degassed in an argon stream
Hydrogen was then
bubbled through the solution, until the colour had changed to yellow. The
solution was then
degassed again in an argon stream. A solution of 21 (4.59 g, 4.1 G mmol) in
THF (30 mL) was
degassed and added. The mixture was stirred at rt overnight, then
concentrated. The residue
was taken up in a mixture of acetone (10:1, 44 mL). Mercuric bromide (1.78 g,
$.32 mmol)
and mercuric oxide (1.69 g, G.24 mmol) were added to the mixture, which was
protected from
light. The suspension was stirred at rt for 1 h, then concentrated. The
residue was taken up in
CH2Clz and washed three times with sat aq KI, then with brine. The organic
phase was dried
and concentrated. The residue was purified by column chromatography (solvent
B, 3:1 ) to
give 22 (3.52 g, 80%) as a colourless foam; [a]D -~-17.7; ~H NMR: 0 7.15 (m,
25H, Ph), 5.50
(dd, 1H, H-2H), 5.30-5.27 (m, 2H, H-lo, H-Zc), 5,23 (d, 1H, Jl~ = 3.3 Hz, H-
lE), 5.18 (dd, 1H,
1i,3 = 3.2, J3,4 = 10.0 Hz, H-3n), 5.10 (d, 1H, J~a = 1.2 Hz, H-la), 5.00-4.35
(m, 9H, H-4B,
OCH2), 4.28 (dd, IH, Jz,3 = 3.2, J3,4 = 8.G Hz, H-3o), 4.20-4.00 (m, 3H, H-3~,
5s, Sc), 3.80-
3.50 (m, GH, H-2s; 6aE, 6bs, 5H; 4E, 40), 3.05 (d, 1H, JoH,, = 4.0 Hz, OH),
2.09, 1.81, 1.44 (3s,
9H, CH3C=0), 1.37 (d, 3H, J5,6 = 6.2 Hz, H-Gc), 0.95 (d, 3H, J5,6 = 6.2 Hz, H-
6B); 13C NMR:
0169.9-169.6, I6G.2 {4C, C=O), 138.9-127.5 (Ph), 99.8 (C-1B), 97.3 (C-le),
91.3 (C-lc), 81.7
(C-3~, 80.7 (C-2E), 78.8 (C-3c), 78.1-78.0 (2C, C-4E, 4c), 7G.b, 75.5 (2C,
CHzPh), 74.9 (2C,
C-2E, CHzPh), 73.8 (CHzPh), 73.3 (2C, C-4B, 5E), 72.9 (C-2B), 71.2 (2C, C-3a,
GE), 67.5 (C-
5c), G7.1 (C-5a), 21.0-20.6 (3C, CH3C=0), 18.9 (C-6c), 17.1 (C-6B). FAB-MS for
C59Hss0is
(1042) »t/z 1085 [M+NaJ+.
Anal. Calcd. for G59H66~18'H2~~ C, 65.54; H, G.34. Found: C, 65.68; H, 6.41.
(2,3,4-Tri-0-acetyl-o~-L-rhamnopyranasyl)-(1-~3)-((2,3,4,6-terra-0-benzyl-a-n-
glucopyranosyt)-(1-~4)]-2-O-benzoyl-a-L-rhamnopyranose trichloroacetimidate
(S).
DBU (100 ~.L) was added at 0°C to a solution of the hemiacetal 22 (3.8
g, 3.58 mmol) in
DCM (40 mL) containing trichloroacetonitrile (4 mL). The mixture was stirred
for 30 min at
0°C, and volatiles were evaporated. Flash chromatography (solvent B,
7;3 + 0.2% Et3i~ of
the etude material gave the donor S (3.9 g, 90%) as a white solid; [a]D +2.8
(c ?);
h'MR ???
Anal. Calcd. for C61H6sC13NOts: C, 60.67; H, S.SI; N, 1.16. Found: C, 60.53;
H, 5.48; N,
1.38.
LMPPIZB%p-brevet-gp C~1 02434685 2003-07-04
2-Azidoethyl (2,3,d-Tri-O-acetyl-a-L-rhamnopyranosyl)-(1-~3)-[(2,3,4,6-tetra-D-
benzyl-
a-D-glucopyranosyl)-(1-~4)]-(Z-0-benzoyl-a-L-rhamnopy ranosyl)-(Z-~3)-2-
acetamido-2-
dco~y-4,6-O~isopropylidene-~-p-glucopyrnnoside (23). The trisaccharide donor 5
(1.86 g,
1.54 mmol) and the acceptor 7 (712 mg, 2.16 mmol) were dissolved in 1,2-
dichloroethane (15
mL) and 4~-MS (2 g) were added. The mixture was stirred at rt for I h. The
mixture was
cooled to 0°C and triflic acid (34 p>r, 0.385 mmol) was added. The
mixture was stinted at 0°C
for 30 min, then at rt for 30 min. The mixture was then heated at GS°C
for 1 h. The mi~rture
was allowed to cool, Et3N (0.5 mL) was added, and the mixture was stirred at
rt for 20 min.
The mixture was diluted with CI-IzClz and filtered through a pad of Celite.
The filtrate was
concentrated and purified by column chromatography (solvent B, 1:1) to give 23
(1.61 g,
76%). 1H NMR: b 7.90-G.90 (m, ZSH, Ph), 5.92 (d, IH, J = 7.5 Hz, NH), 5.53
(dd, 1H, Jl,a=
1.8 Hz, H-2a), 5.29 (d, 1H, H-lE), 5.19 (m, 2H, H-2x, 3H), 5.09 (m, 2H, H-lx,
1D), 4.97 (bs,
1H, H-IB), 4.9G-4.70 (m, 9H, CHaPh, H-4a), 4.54-4.41 (m, H, CHaPh), 4.34 (pt,
1H, J3,4 = J4,s
= 9.3 Hz, H-3D), 4.19-3.89 (m, 6H, H-3~, 5x, 5E, 3E, 6aD, OCHz), 3.79-3.60 (m
5H, H-6ba,
4~, 5H, 2E, OCHa), 3.SG-3.33 (m, 4H, H-5n, 4E, 4n, CH2N3), 3.27-3.12 (m, 2H,
GHaN3, H-2D),
2.10, 2.09 (2s, 6H, C(CHa)a), 1.78 (s, 3H, OAc), 1.73 (s, 3H, NHAc), 1.42,
1.35 (2s, 6H,
OAc), 1.30 (d, 3H, J5,6 = 6.2 Hz, H-Go), 0.90 (d, 3H, Js,6 = 6.2 Hz, H-6H);
~3C NMR: 8 171.4,
169.7. 169.6, 169.5, 166.0 (5C, C=0), 138.7-127.2 (Ph), 99.8, 99.7 (C-lp, lc),
97.1 (C-1B),
96.4 (C-lp), 81.5 (C-3E), 81.1 (C-2E), 79.5 (bs, C-3c), 77.9 (C-4o), 77.0 (bs,
C-4x), 75.4 (C-
3,~), 75.3, 74.7, 73.6 (3C, CHzPh), 73.0, 72.9 (2C, C-2c, 4E), 72.9 (CHaPh),
71.2 (C-Sa), 71.1
(C-48), 69.9 (C-2$), 69.2 (C-6E), G8.8 (C-3H), 68.7 (OCHz), 67.2, 67.1 (3C, C-
5~, 5H, 5p), 62.2
(C-6D), 59.0 (C-2p), 50.G (CHaN3), 29.0, 23.4 (2C, C(CH3)a), 20.9, 20.4 (3C,
OAc), 19.0
(NHAc), 18.4 (C-6x),17.0 (C-GB). FAB-MS for ClZHa6N4023 (1374) m/~ 1397
[M+Na)+.
Anal. Calcd. for C~zHBsNa0z3: C, 62.87; H, 6.30; N, 4.07. Found: C, ?P; I~,
??; N, P?.
Z-Azidocthyl (2,3,4-Trl-0-acetyl-a-L-rhamnopyranosyl)-(Ia3)-[(2,3,4,6-tetra-O-
benryl-
a-D-glucopyranosyl)-(I~4))-(Z-O-bcnzayl-a-L-rhamnopyranosyl)-(1-~3)-2-
acetamido-2-
deoxy-[i-D-glucopyranoside (24). 50% aq TFA (I.3 mL) was added to a solution
of the fully
protected tetrasaccharide 23 (210 mg, 11 I pmol) in DCM (6 mL). The mixture
was stirred at
0°C for 1 h. Volatiles were evaporated and toluene was co-evaporated
from the residue.
Column chromatography (solvent B, 7:3 -~ 1:1) of the crude product gave 24
(195 mg. 95%).
[a)D-6.9 (c 0.5, MeOH);
NMR?
FAB-MS for C(,9HgaNqOZ3 (1334) m/z 1357_5.
Anal. Calcd. for C69HgaN4021'H20~ C, 60.43; H, 6.32; Iv', 4.09. Found: C,
60.56; 6.22, 3.92.
C.MPPI2cxp-brtvet-Sp
CA 02434685 2003-07-04
2-Aminocthyl a-L-Rhamnopyranosyl-(1-->3)-(a-D-glucopyranosyi-(1-~4)J-o~.-L-
rhamnopyranosyl-(1--~3)-2-acetamido-2-deogy-~-D-glueopyranoside (25). An ice
cold
solution of 95% aqueous trifluoroace~tic acid (2.4 mL) in CHZCIz (21.6 mL) was
added to the
te2rasaccharide 23 (1.93 g, 1.40 mmol). The mikrture ws kept at 0°C for
5 min., then diluted
with toluene and concentrated. Toluene was co-evaporated from the residue. The
residue was
dissolved in MeOH (6S mL), and a 1M solution of sodium methoxide in MeOH (3
mL) was
added. The mixture was left to stand at rt for 18 h. then neutralised with
Amberlite IR-120
(F~ resin, and filtered. The filtrate was concentrated, and the residue was
purified by column
chromatography (solvent B. 9:1) to give 24 (1.38 g, 89°/) as a
colourless foam The
tetrasaccharirie 24 (1.38 g, I.25 mmol) was dissolved in a mixture of EtOH (35
mL) and
EtOAc (3.5 mL). A 1N solution of aq HC1 (2.S mL) was added. The mixture was
stirred under
hydrogen in the presence of 10% Pd/C (1.5 g) for 72 h, then diluted with water
and filtered.
The filtrate was concentrated, then lyophilised. The residue was dissolved in
a solution of 5%
aqueous NaHC03 and purified by passing first through a column of Cts silica
(eluting with
water), then through a column of Sephadex Glo (eluting with water) to give,
after
lyophilisation, 25 (693 mg, 77%). HPLC (234 nm): Rt 4.78 min (Kromasil S pm
C18 100 A
4.6x250 mm analytical column, using a 0-20% linear gradient over 20 min of
CH3CN in
O,O1M aq TFA at 1 mL/min flow rate). ~H NMR (Dz0): 8 5.10 (d, IH, J,,Z = 3.7
Hz, H-lE),
4.89 (d, IH, J,,Z = 1.1 Hz, H-1B), 4.73 (d, 1H, Ji,~ = 1.0 Hz, H-lc), 4.50 (d,
1H, Jl,z = 8.6 Hz,
H-lo), 4.08 (m, IH, H-5~), 3.96 (m, IH, H-2H), 3.9I (m, ZH, H-GaD, CHZO), 3.68-
3.88 (m,
12H, H-2~, 2p, 3H, 3c, 4$, 4~, 5s, SE, 6bn, 6aE, 6bE, CHaO), 3.59 (pt, 1H, H-
3E), 3.52 (pt, 1H,
H-3D), 3.33-3.48 (m, 4H, H-2E, 4D, 4E, 5D), 3.OI (m, 2H, CH2NH2), 1.99 (s, 3H,
CH3C=O),
1.28 (d, 3H, H-6c), 1.18 (d, 3H, H-GH); 13C ~ 174.8 (C=O), 103.2 (C-1$), 101.4
(C-lo), 100.9
(C-ID), 98.G (C-lE), 81.9 (C-3p), 79.0 (C-4g), 76.6 (C-4~), 76.3 (C-2E), 72.9
(C-3fi), 72.3 (C-
5E), ?2.3 (C-4D), 71.8 (C-3~), 71.1 (C-2~), 70.5 (C-2B, 3B), 69.7 (C-4H), 69.5
(C-4E), G9.2 (C-
Sp), 68.8 (2C, C-SB, 50), 67.9 (CHZO), 61.0 (C-6D), 60.8 (C-6~), 55.5 (C-2D),
40.0 (CHZNHZ),
22.6 (CH3C=O), 18.0 (C-6~). I 7.0 (C-6B). XXMS for CZBHSONZ019 (718) mlz 741
[M + NaJ+.
HRUIS (MALDI) Calcd for C2sH5oNZO19: 741.2905. Found: XXX?i.
Altyl (2,3,4-Tri-0-benxoyl-a-L-rhamnopyranosyi)-(1-+2)-3,4-di-0-benzyl-a-L-
rhamnopyraboside (28j. TM50Tf (11 uL, 59 wmol) was added to a solution of the
rhamnoside 26 (2.26 g, 5.88 mmol) and the trichloroacetimidate 27 (4.23 g,
6.82 mmol) in
7
LMPPI2exp-bteve!-gp
CA 02434685 2003-07-04
anhydrous EtzO (G0 mL) at --70°C. The reaction mixture was stirred For
8 h while the cooling
bath was slowly coming back to rt. Et3N (100 pL) was added, and the mixture
was stirred at rt
for 15 man. Solvents were evaporated, and the crude material was purified by
column
chromatography (solvent B, 49:1 --~ 9:1), to givE 28 as a white foam (4,78 g,
9G%). 1H NMR:
8 8.17-7.12 (m, 25H, Ph), 5.97-5.85 {m, 3H, H-2~, 3A, CH=), 5.67 (pt, 1H, J3,4
= 9.6 Hz, H-
4A), 5.34-5.19 (m, 3H, H-1,," CHZ=), S.OI (d, 1 H, J = 9.0 Hz, CHzPh), 4.92
(d, 1 H, J~,2 = 1.3
' Hz, 1-I-1 g), 4.82-4, 74 (m, 2H, CHZPh), 4. 71 (d, 1 H, J = 11. 8 Hz, OCHz),
4.31 (dq, I H, Ja,s =
9.7 Hz. H-5,~, 4.21 (m, 1H, OCHz), 4.10 (dd, 1H, H-2B), 4.02 (m, 1H, OCHz),
3.97 (dd, 1H,
Jz,3 = 3.0, J3,4 = 9.2 Hz, H-3H), 3.82 (dq, 1 H, Ja,s = 9.4 Hz, H-5$), 3.71
(pt, IH, H-4$), 1.43 (d,
3H, Js,6 = G.1 Hz, H-6B), 1.37 (d, 3H, Js,6 = G.2 Hz, H-G,~}; ~3C NMR: &
166.3, 165.9, 165.7
(3C, C=O), 139.0-127.9 (CH=, Ph), 117.8 (CHz=), 99.9 {C-1,~, 98.3 (C-IB), 80.6
(C-48), 80.2
(C-3a), 76.5 (C-2$), 76.0, 72.9 (2C, CHZPh), 72.3 (C-4,~, 71.0 (C-ZA*), 70.4
(C-3A*), 68.7
(C-SH), 68.1 (OGHz), G7.5 (C-5,~, 18.4 (C-GB), I8.1 (C-6,~. FAB-MS far Csol-
lso0~a (M =
842.3) m/z 8G5.1 [M+Na]'.
Anat. Calcd for CsoHso0J2: C, 71.21; H, 5.98. Found C, XX; X, XX~IG
(2,3,4-tri-O-Benzoyl-a-L-rhamnopyraaosyt)-(1-~2)-3,4-di-O-beezyt-a-t~-
rhamnopyranose (29). 1,5-Cyclooctadiene-bis(methyldiphenylphosphinejiridium
hekafluorophosphate (25 mg) was dissolved in THF (10 mL) and the resulting red
solution
was degassed in an argon stream. Hydrogen was then bubbled through the
solution, until the
colour had changed to yellow. The solution was then degassed again in an argon
stream. A
solution of 28 (4.71 g, 5.59 mmol) in THF (40 mL) was degassed and added. The
mixture was
stirred at rt overnight, then concentrated. The residue was taken up in
acetone (350 mL) and
i
water (82 mL). Mercuric bromide (3.23 g, 8.9G mmol) and mercuric oxide (2.64
g, 12.3
mtnol) were added to the mixture, which was protected from light. The
suspension was stirred
at rt fox 1 h, then concentrated. The residue was taken up in CHiCIz and
washed three times
with sat aq KI, then with brine. The organic phase was dried and concentrated.
The residue
was purified by column chromatography (solvent B, 3:1) to give 29 (3.87 g,
84%) as a
i
colourless foam ~H NMR: 8 8.15-7.12 (m, 25H, Ph), 5.94-5.88 (m, 3H, H-2A, 3A,
CH=), 5.70
(pt, 1H, J3,4= 9.7 Hz, H-4,~, 5.31 (dd, 1H, Jl.ort=3.0 Hz, H-1H), 5.28 (bs,
1H, H-1,~, 4.98 (d,
1H, J = I1.0 Hz, CHzPh), 4.82-4.G8 (m, 3H, CHZPh), 4.31 (dq, 1H, J4,5= 9.8 Hz,
H-5~, 4.13
(dd, 1H, Jl,z = 2.1 Hz, H-2B), 4.06-3.99 (m, 2H, H-3B, Sa), 3.72 (pt, 1 H,
J3,4 = Jd,s = 4.4 Hz, H-
48), 2.79 (bs, 1H, OH-1B), 1.41 (d, 3H, Js,6= 6.2 Hz, H-GB), 1.37 (d, 3H,
JS,~= 6.3 Hz, H-6,~;
i
LM~PI~C?t~-hICVE(-~1
CA 02434685 2003-07-04
"C NMR: s 166.2, 165,9, 165.7 (3C, C=0), 138.9-127.9 (Ph), 99.7 (C-1,~, 94.2
(C-1H), 80.5
(C-4g), 79.6 (C-3a), 7'7.6 (C-2B), 76.5, ?2.5 (2C, CI~Ph), 72.3 (C-4~, 7I-0 (C-
2A*), 70.4 (C-
3A~), 68.8 {C-SH), 67.6 (GSA), 18.5 (C-6s*), 18.1 (C-6a*). FAB-MS for Ca~H4sOu
(M =
802.3) »r/z 825.1 [M+Na]+.
Anal. Calcd. for C4~H,s011: C, 70.31; H, 5.78. Found C, XX; H, XXX.
i
{2,3,4-Tri-O-bcnzoyl-a-L-rhamnopyranosyl)-(1-->2)-3,4-di-O-benzyl-a-Lr
rhamnopyranosyl Trlchloroacetimidate {30). The hemiacetal 29 (3.77 g, 4.71
mmol) was
dissolved in CHzCIZ (15 mL) and the solution was cooled to 0°G.
TrichloroacetonitriIe (2.5
mL) was added, then DBU (200 pL). The mixture was stirred at rt for 2 h
Toluene was
added, and co-evaporated twice from the residue. The crude material was
purified by flash
chromato~aphy (solvent B, 4:1 + 0.1% Et3N) to give 30 as a white foam (4.29 g,
96°.0). Some
hydrolyzed material 29 (121 mg, 3%) was eluted next. The trichloroa,cetimidate
30, isolated
i
as an alb mixture had 1H NMR (a anomer): 8 8.62 (s, 1H, NH), 8.20-7.18 (m,
25H, Ph), 6.31
(s. 1 H, H- I e), 5.94 (dd, 1 H,11,~ = 1.6 Hz, H-2,~, 5. 89 (dd, 1 H, Ja,3 =
3.4, 33,4 = 9.9 Hz, H-3,~,
5.71 (pt, IH, H-4,~, 5.27 (bs, 1H, H-1,~, 5.02 (d, 1H, J = 14.8 Hz, CH2Ph),
4.84 (d, 1H, T =
11.9 Hz, CH~h), 4.79 (d, 1H, CHzPh), 4.72 (d, 1H, CH~h), 4.3G (dq, IH, J4,5=
9.8 Hz, H-
' S~, 4.13 (dd, IH, H-2B), 4.03-3.97 (m, 2H, H-3s, 5B), 3.80 (pt, 1H, J3,a =
9.5 Hz, H-4B), 1.45
(d, 3H, Js,s = 6.1 Hz, H-6H), 1.40 (d, 3H, Is,s= G.2 Hz, H-6,J; 13C NMR (a
an~omer): b 166.2,
IG5.9, 165.7 (3C, C=O), 160.8 (C=NH), 138.6-128.2 (Ph), 99.9 (C-1~, 97.2 (C-
18), 91.4
(CCI~), 79.9 (C-4B), 79.1 (C-3$), 76.2 (CH2Ph), 74.9 (C-2B), 73.3 (CH2Ph),
72.1 (C-4B), 71.7
' (C-Ss), 71.0 (C-2,~, 70.2 (C-3.~, 6?.8 (C-5~, 18.4 (C-6H),18.0 (C-6,~.
Ana6 Calcd, for Ca9HasC13NO,a: C, 62.13; H, 4.89; N, 1.48. Found C, XX; H,
XXX, N,
X.XX.
I Aryl (2,3,4-Tri-0-bcnzoyl-arrL-rhamnopyranosyl)-(1--~2)-(3,4-di-0-benzyl-arL-
rhamnopyranosyl)-(1-~3)-[(2,3,4,6-tetra-0-benzyl-a-D-glueopyranosyl)-(la4)]~2-
0-
benzoyl-a-L-rhamnopyranoside (33). (a) The acceptor 16 (465 mg, 0.56 mmol) was
dissolved in ether (3 mL). The solution was cooled to -GO°C and TMSOTf
(GS ~L, 0.3G
mmol) was added. The donor 30 (G90 mg, 0.73 mmol) was dissolved in ether (6
mL) and
added to the acceptor solution in two portions with an interval of 30 min. The
mixture was
stirred at -GO°C to -30°C over 2 h. Et3N (100 1rL) was added.
The mixture was concentrated
9
LMPP1 aexp-brevet-gp
CA 02434685 2003-07-04
and the residue was purified by column chromatography (solvent B; 7:1) to give
33 (501 mg,
55°/'0).
(b} A solution of the donor 27 (1.41 g, 2.25 mmol) and the acceptor 32 (1.07
g, 1.79 mmol} in
anhydrous EtzQ (88 mL) was cooled to --60°C. TMSOTf (63 ~L} was added,
and the mixture
was stirred at -60°C to -20°C over 2.5 h. Et3N was added (I00
pL). The mixture was
concentrated and the residue was purified by column chromatography (solvent D,
49.1) to
give 33 (2.66 g, 92%); (a]p +74.1 (c 0.5); 'H R'MR: S 7.06-8.11 (m, 50H, Ph),
5.88-6.05 (m,
3H, H-2~, 3,,, CH = ), 5.71 (t, 1H, H-4,~. 5.51 (dd, 1H, H-2~), 5.22-5.41 (m,
3H, H-1,4, CHz =
), 5.14 (d, 1H, l~,z = 0.9 Hz, H-1B), 5.10 (d, 1H, J1,2 = 3.Z H~~ H-lp), 4.97
(bs, 1H, H-lo),
4.35-5.00 (m, 14H, H-28, Sa, 12 x CHZPh), 3.98-4.19 (m, SH, H-3C, 3r, 5E,
OCHZ), 3.43-3.87
(m, 9H, H-2p, 38, 4a, 40, 4x, SB, 50, 6E, 6',~, 1.44 (d, 3H, H-G,~, 1.40 (d,
3H, H-6o), 1.13 (d,
3H, H-6B); 13C NMR: 8 165.9, 165.4, 165.1 (C=O), I27.1-138.7 (CH=, Ph), 117.8
(CHz=),
101.3 (C-1H), 99.d (C-1,,~, 97.9 (C-lE), 96.1 (C-lo), 81.9 (C-3E), 81.0 (C-
2E), 80,1 (C-3o},
79.8 (C~4aj, 78.9 (C-3~), 77.9 (C-4~), 77.4 (C-4E), 75.9 (C-2B), 75.G, 75.0,
74.9, 73.9, 72.9
(CHzPh), 72.4 (C-2o), 71.9 (C-4,~, 71.2 (C-SE), 70.9 (CHzPh), 70.7 (C-2,,*),
70.0 (C~3,,'"),
69.2 (C-5$), 68.5 (OCHz), 68.1 (C-6E), 67.6 (C-So), 67.2 (C-5,~, 18.8 (C-d,~,
18.1 (C-6c),
17.8 (C-GB). FAB-MS for Cg7H9gO?~ (1614) rrllz 1637 [M+Na]-.
Anal. Calcd, for C9~H9gOzz: C, 72.10; H, 6.11. Found: C, 71.75; H, G.27.
(2,3,4-Tri-O-benzoyl-a-L-rhamnopyraaosy!)-(1-->2)-(3,4-di-O-bcnryl-a-L-
rhamnopyranosylj-(1~3)-((2,3,4,6-tetra-O-benryl-a-D-glucopyranosyi)-(1-~4)]-(2-
O-
benzoyl-ocl(i-L-rhamnopyranose (34). 1,5-Cyclooctadiene-
bis(methyldiphenylphosphine)iridium hexafluorophosphate (12.5 mg) was
dissolved in THF
(5 mL) and Lhe resulting red solution was degassed in an argon stream.
Hydrogen was then
bubbled through the solution, causing the colour to change to yellow. The
solution was then
degassed again in an argon stream. A solution of 33 (1.138 g, 0.70 mmol) in
THF (15 mL)
was degassed and added. The mixture was stirred at rt overnight. The miucture
was
concentrated. The residue was taken up in acetone (7 mL) and water (0.7 mL}.
Mercuric
chloride (285 mg, 1.05 rnmol) and mercuric oxide (303 mg, I.4 mmol) were added
to the
mixture, which was protected from light. The mixture was stirred at rt for 1
h, then
concentrated. The residue was taken up in CHzCIz and washed three times with
sat. aq. RI,
then with brine. The organic phase was dried anti concentrated. The residue
was purified by
column chromatography (solvent B, 7:3) to give 34 (992 mg, 90%) as a
colourless foam jH
to
CA 02434685 2003-07-04
LMPPI2cxp.brevct~gp
NMR: $ 7.05-8.16 (m, SOH, Ph), 5.88-5.93 (m, 2H, H-2~. 3A), 5.73 (pt, IH, H-
4,~, 5.55 (m,
1 H, H-2c), 5.3 7 (6s, 1 H, H-1,~, 5.28 (bs, 1 H, H-1 c), 5.14 (bs, 1 H, H-1
H), 5.07 (d, 1 H, ,I ~ ,z =
3.1 Hz, H-lE}, 4.78-4.99 (m, 6H, CHzPh), 4.31-4.68 (m, 8H, H-2a, 5A, CHzPh),
4.24 (dd, 1H,
H-3~), 3.99-4.09 (m, 3H, H-3E, Sc, SE), 3.82 (pt, 1H, H-4c), 3.57-3.76 (m, 5H,
H-3a, 4E, 5B,
6aE, .6bE), 3.48 (dd, IH, H-2s), 3,17 (d, 1H, OH), 1.43 (d, GH, H-G", Gc),
1.14 (d, 3H, H-6H)
"C NMR: b 166.0, 165.6, 165.2 (4C, C=O), 127.2-138.9 (Ph), 101.1 (C-18), 99.7
(C-1,~, 98.1
(C-IE), 9L6 (C-Ic), 81.9 (C-3H), 81,1 (C-2E), 79.9 (C-4B): 79.4 (G3c), 78.9 (C-
3$), 78.3 (C-
4c), 77.6 (C-4E), 76.1 (C~28), 75.8, 75.3, 75.1, 74.0, 73.1 (XXC, CH~T'h),
72.7 (C-2c), 72.1
(C-4~, 71.4 (C-5E), 71.1 (CH2Ph), 70.8 (C-2A*), 70.2 (C-3A*), 69.4 (C-5B),
68.3 (C-6E), 67.7
(C-5c), 67.3 (C-5,~, 19.0 (C-6,~, 18.2 (C-6c), 17.9 (C-6B). FAB-MS for
Cg4H94072 (1$74) m1z
1597 [M+Na]t.
Anal. Calcd. fnr CgqH~sOI2: C, 71.65; H, 6.01. Found: C, 71.48; H, 6.17.
(2,3,4-Tri-O-benzoyl-a-L-rhamnopyranosyl)-(1--~2)-(3,4-di-O-benzyi-a-L-
rhamnopyranosyl)-(1-~3)-((2,3,4,6-tetra-O-benzyl-a-n-glueopyranosyl)-(1-34)]-
(2-O-
benzoyl-a![i-L-rhamnopyranosyl trichloroacetimidate (35). The he.miacetal 34
(412 mg,
0.26 mmol) was dissolved in CHzCIz (5 mL) and the solution ws cooled to
0°C.
Trichloroacetonitrile (0.26 mL) was added, then DBU (4 ~L). The mixture was
stirred at 0°C
for 1 h. The mixture was concentrated and toluene was co-evaporated from the
residue. The
residue was purified by flash chromatography (solvern B, 4:1 + 0.1% Et3N) to
give 34 (393
mg, 88%). 'H NMR: & 8.74 (s, 1H, NH), 7.03-8.10 (m, 50H, Ph), 6.42 (d, 1H,
J~,z = 2.3 Hz,
H-Ic), 5.87 (m, 2H, H-2~, 3a). 5.67 (m 2H, H-2c, 4~. 5.30 (bs, 1H, H-l~, 5,14
(bs, 1H, H-
1H), 5.08 (d, 1H, J,,2 = 3.1 Hz, H-lE), 4.74-4.98 (m, Gl-I, CH~'h}, 4.23-4.69
(m, 9H, H-2H, 3c,
5," CHzPh), 3.88-4.07 (m, 3H, H-3E, 5H, 5E), 3.57-3.74 (m, 7H, H-2c, 4g, 4c,
4E, Sc, Gae, 6GE),
3.50 (dd, IH, H-3H), 1.38 (d, 6H, H-6A, 6H), 1.07 (d, 3H, H-bc); 1'C NMR: 8
165.9, 165.5,
165.4, 165.1 (4C, C=O), 160.1 (C=NH), 127.2-138.7 {Ph), 101.2 (C-Ix), 99.7 (C-
1,~, 98.3 (C-
lE), 94.3 (C-lc), 90.9 (CCl3), 81.7 (C-3E), 80.9 (C-2E), 79.6 (C-3c, 4B), 78.5
(C-3a), 77.2 {C-
4c), 77.5 (C-4E), 75.9 (C-2H), 75.6, 75.1, ?5.0, 74.0, 72.9 (CH~h), 71.8 (C-
2c), 71.3 (C-4~,).
71.0 (CHzPtt), 70.7 (C-5E): 70.5 (C-2~*), 70.3 (C-3~*), 70.0 (C-SB), 69.5 (C-
5c), 67.9 (C-6~),
67.2 (C-5,~, 18.7 (C-GA), 17.8 (C-6c), 17.7 (C-6g).
Anal. Calcd. for Cg6Hg~C13NO2z: C, 67.03; H, 5.5I; N, 0.81. Found: C, 63.14,
H, 5.14; N,
1.00.
LMPPI2expbcevet~gp CA 02434685 2003-07-04
2-Azidoethy! (2,3,4-Tri-O-benzoyt-a-L-rhamaopyranosyl)-(1-a2)-(3,4-di-O-benzyl-
a-L-
rhamnopyranosyl)-(1-->3)-[(2,3,4,6-tetra-O-benry!-a-D-8lucopyranosyl)-(1~4)]-
(2-O-
benzoyl-ac-L-rhamnopyranosyI}-(la3)-2-xcetamido-2-deoxy-4,6-O-isopropylidene-
[3-D-
glucopyranoside (35). (a) The tetrasaccharide donor 6 (500 mg, 0.29 mmol) and
the acceptor
7 (140 mg, 0.42 mmol) were dissolved in I,2-dichloroethane (5 mL) and 4A-MS
(d00 mg)
were added. The mixture was stirred at rt for 2 h. The mixture was cooled to
0°C and triflic
acid (7 ~L, 0.072 mmol) was added. The mixture was stirred at 0°C to rt
over 1 h 30 min. The
mixture was then heated at 65°C for 1 h 30 min. The mixture was allowed
to cool, Et3N (0.5
mL) was added, and the mixture was stirred at rt for 20 min. The mixture was
diluted with
CHZC~ and filtered through a pad of Celite. The filtrate was concentrated and
purified by
column chromatography (solvent B, 4:3) to give 35 (340 mg, G2%).
(b) The tetrasaecharide donor 6 (250 mg, 145 ltmol) and the acceptor 7 (G7 mg,
204 pawl)
were dissolved in DCM {1.5 mL) and 4~1-MS (200 mg) were added. The mixture was
stirred
at -40°C For 30 min and triflic acid (5 p.L) was added. The. mixture
was stirred at rt over 3 h,
triethylamine was added, and the mixture was stirred at rt for IS min. The
mixture was diluted
with CHzCIz and filtered through a pad of Celite. The filtrate was
concentrated and purified
by column chromatography (solvent B, 9:1 -~ 1:1) to give 35 (219 mg, 80%).
[a)n +29.0 (c
0,25, MeOH); 'H NMR: b 7.04-8.06 (m, 50H, Ph), 6.24 (d, 1 H, NH), 5.90 (m, 2H,
H-2A, 3~,
5.70 (t, 1H, H-4~, 5.42 (m, 1H, H-2c), 5.35 (bs, 1H, H-lA), 5.13 (m 3H, H-1B,
lo, lr), 4.77-
5.00 (m, 5H, H-lo, CH2Ph), 4.29-4.66 (m, 11H, H-2B, 3n, 5~. CHzPh), 3.80-4.11
(m, 6H, H-
3c, 3s~ Sc. 5E, 6aD~ CHiO), 3.45-3.78 (m, 12H, H-2E, 3e~ 4e~ 4c~ 4p~ 4p~ 5B,
So~ dbn. 6aF, Gbe
CHZO), 3.39 (m, 1H, CHaN3), 3.23 (m, 2H, H-2D, CH2N3), 2.13 (s, 3H, CH3C0),
1.43 (d, 9H,
H-6A, (CH3)2C), 1.29 (d, 3H, H-Gc), 1.1 I (d, 3H, H-6g); "C NMR; S 171.8, 1
G5.9, 165.5,
155.0, 163.5 {SC, C=O), 127.1-138.7 (Ph), 101.3 (C-Ie), 99.8 (C-ID), 99.3 (C-
I,~, 97.7 (C-
Ic), 97.6 (C-1~, 91.8 (C{CH3)~J, 81.6 (C-3E), 81.0 (C-2a), 80.0 (C-3c), 79.7
(C-4D), 78.9 (C-
48), 77.5 (C-3H, 4c), 76.4 (G3D), 75.6 (C-28), 75.5, 74.9, 74.8, 73.8, 73.0
(SC, CHzPh), 72.9
(C-4s), 72.7 (G-2c), 71.8 (C-4,~, 71.3 (C-SE), 71.0 (CH~Ph), 70.6 (C-2A*),
70.0 (C-3A*), 69.3
(C-5B), 68.6 (OCH2), 68.3 (C-6s), 67.5 (C-Sc), 67.3 (C-5,~, 67.1 (G5p), 62.2
(C-6D), 58.9 (C-
2n), 50.6 (CHzN3), 29.1 (CH3C), 23.6 (CH3C=O), 19.2 (CH3C), 18.6 (C-6A), 18.0
(C-6c), 17.6
(G6H). FA.B-MS for C'o~H' 14N40a~ (1886) nt/z 1909 [M + Na]+.
Anat. Calcd. for CIO~W laNaOi~: C, 68.07, Fi, 6.09; N, Z.97. Found: contlent
du CCL3CN
12
LMPPI2exp-brevet-&p
CA 02434685 2003-07-04
2-Aminoethyl a-L-Rharnnopyranosyl-(1-->2)-a,-L-rhamnopyranosyl-(I--~3)-[a-b-
gtacopyranoeyl)-(1->4)~-a-L-rhamnopyranosyl-(1 >3)-2-acetamido-2-deny-[i-b-
glucopyranoside (37). An ice cold solution of 95% aq TFA (I mL) in CHZCIz {9
mL) was
added to the pentasaccharide 35 (645 mg, 0.34 mmol). The mixture was kept at
0°C for 10
min, then diluted with toluene and concentrated. Toluene was co-evaporated
from the residue.
The residue was dissolved in MeOH (20 mL), and a iM solution of methanolic
sodium
methoxide (3.5 mL) was added. The mixture was stirred at 50°C for 18 h.
The mixture was
neutralised with Amberlite IR-120 (I-l~ resin and filtered. The filtrate was
concentrated. The
mixture was purified by column chromatography (solvent A, 9:I) to give 36 (374
mg, 77%) as
a colourless foam The crude pentasaccharide 36 (3G0 mg, 0.25 mrrwl) was
dissolved in a
mixture ofEtOH (IO mL) and EtOAc (1 mL). A IN solution of aq HCl (0.5 mL) was
added.
The mixture was stinted under hydrogen in the presence of 10% PdIC (400 mg)
for 18 h. The
mixture was diluted with water and filtered. The filtrate was concentrated,
then lyophilised.
The residue was dissolved in a solution of NaHC03 (75 mg) in water (1 mL) and
purifed by
passing fast through a column of Cte silica (eluting with water), then through
a column of
Sephadex Glo (eluting with water) to give, after lyophilisation, 37 ( 138 mg,
64%). HPLC (230
nor): Rt 5.87 min (Kromasil 5 pm C18 I00 A 4.6xZ50 mm analytical column, using
a 0-20%
linear gradient over 20 min of CI-~CN in O,O1M aq TFA at I mL/min flow rate).
~H NMR
(Di0): S 5.15 (d, 1H, J,s = 3.7 Hz, H-IE), 5.00 (bs, 1H, H-1,~, 4.92 (d, 1H,
J1,2 = I.1 Hz, H-
1H), 4.7G (bs, IH, H-lc), 4.53 (d, 1H, Jl,z = 8.6 Hz, H-lp), 4.10 (m, 1H, H-
5c), 4.03 (m, 2H,
H-2,~, 2g), 4.01 (m, 3H, H-4A, 4H, CHZO), 3.83-3.88 (m, 7H, H-2c, 2D; 3,~,
GaD, Gbn, 6aE:
CHZO), 3.69~3.76 (m, 7H, H-3a, 30, 3E, 40, 5,~, SB, 6ba), 3.52 (pt, 1H, H-3D),
3.33-3.54 (m,
SH, H-2E, 4n, 4a, So. SE), 3.09 (m, 2H, CHZNHZ), 1.98 (s, 3H, CH3C=0), 1.28
(d, 3H, H-Gc),
1.22 (tn, 6H, H-6A, GB); 13C Iv'MR (Di0): ~ I75.3 (C=0), 103.4 (C-1H j, I O
I.9 (C-1 ~, 101.4
(C-lc, ID), 98.4 (C-lE), 82.3 (C-3D), 80.2 (C-2H), 79.9, 76.7 (C-2a), 72.9,
72.4, 72.4, 72.2,
j 71.8, 71.6, 70.5, 70.4, 70.I, 70.0, 69.7, 69.6, 69.4, 68.7, 66.7
(???"?CHsO), 61.0 (2C, C-6o,
6E), 55.5 (C-2D), 39.9 (CHzNHz), 22.6 (GH~C=O), 18.2 (C-. Gc), 17.2 (C-6,~,
I7.0 (C-GH). MS
for HRMS (MALDI) Calcd foz C34HHONzOz.3+H; 86S.36G5. Found: 865.3499.
Maleimido activated PADRE Lys (8).
Starting from 0.1 mm41 of Fmoc Pal Peg Ps resin, amino acids (0.4 mmol) were
incorporated
using HATU/DIEA (0.4 moral) activation. The N-terminal D-Ala was incorporated
as Boe-D-
Ala-OH. After completion of the chain elongation, the resin was treated three
times with
hydrazine nwnohydrate (2% solution in DMF, 2S mL/g of peptide resin) for 3min,
which
13
LMPPlZexp~brcvet-gp
CA 02434685 2003-07-04
allowed the selective deblocking of the Dde protecting group. To a solution of
maleimide
butyric acid (183 mg, 1.0 mmol) in DCM (2 mL) was added DCC (103 mg, 0.5
mmol). After
stirring for 10 min, the suspension was filtered, arid the filtrate was added
to the drained
peptide resin. DIEA (17 ~L, 0.5 mmol) was added. After 30 min, the peptide
resin was
washed with DMF (100 mL), MeOH (100 rnL), and dried under vacuum. After
TFA/TIS/H20
(95/2.5/2.5) cleavage (10 mL/g of resin, 1.5 h), the crude peptide (157 mg)
was dissolved in
16 mL of 15% CH;CN in 0,08% aq TFA, and purified by reverse phase Medium
Pressure
Liquid Chromatography (MPLC) on a Nucleoprep 20 yn C18 100 ~ column, using a
15-75%
linear gradient of CH3CN in 0,08% aq TFA over 60 min at 25 mLlmin flow rate
(2.14 nm
detection) to give 8 (107 mg, 61%). HPLC (214 nm): Rt 13_4 min (94% pure,
Nucleosil 5 um
C18 300 A analytical column, using a 15-45% linear gradient over 20 min of
CH3CN in
0,08% aq TFA at 25 mLlmin flow rate). Positive ion ESMS Calcd for
Cs3H~39NZZ0,9:
1759.18. Found: 1758.83 (SD: 0.40).
(S-Acetylthio methyl)carbonylaminoethyl a-D-Glucopyranosy I-(1--~4)-a-L-
rhamnopyranosyl-(1--~3)-2-acetsmido-2-deoxy-(i-D-glucopyranoside (38). The
trisaccharide 18 (58 mg, 0.1 mmol) was dissolved in DMF (I mL). SAMA-Pfp (33
mg, 0.11
mmol) was added, and the mixture was left to stand at rt for 40 min. Toluene
was added and
the mixture was concentrated. Ether was added to the residue. The resulting
precipitate was
collected and purified by passing through a column. of Cts silica (solvent D,
garadiont) to give
38 (36 mg, 53%). HPLC (230 nm): Rt 13.74 min (99% pure, Isromasil 5 pm C1$ 100
A
4.6x250 mm analytical column, using a 0-20% linear gradient over 20 min of
CH3CN in
0,01M aq TFA at 1 mL/min flow rate). t3C NMR (Dz0): S 200.3 (SC=0), 175.2,
171.9
(NC=O), 102.1 (C-1~), 101.2 (C-ln), 100.5 (C-lE), 82.7 (C-3n), 81.8 (C-4~.),
76.8 (C-2F), 73.6
(C-3s), 72.6 (C-5E), 72.4 (C-4D), 71.8 (C-2c), 70.2 (C-4E), 69.7 (C-3c), 69.4
(C-SD), 68.9 (C-
Sc), 68.9 (CHiO), 61.6 (C-6D), 60.9 (C-6fi), 56.1 (C-2D), 40.6 (CH2NH), 33.7
(CHZS), 30.4
(CH3C(O)S), 23.0 (CH3C(O)N), 17.5 (C-6c). ES-MS for C26H,~N2O17S (688) trtlz
689
(M+H]+.
HRMS (MALDn Calcd for C~6H.,4N~01~5 +Na: 7I1.ZZ58. Found: XXXXX.
(S-Acetylthiomethyl)carbonytaminoethyl a-L-Rhamnopyraaosyl-(1-~3)-[a-D-
glucopyra nosyl-(1-~4)]-ci-L-rhamno py raoosyl-( 1~3)-2-aceta mido-2-deoxy-[3-
D-
giueopyranoside (39). A solution of SAMA-Pfp (16.7 mg, 40 p.mo1) in
acetonitrile (150 pL)
was added to the tetrasaccharide 25 (20 mg, 28.8 umol) in O.1M phosphate
buffer {pH 7.4,
d00 pL). The mixture was stirred at n for 45 min and purified by RP-HPLC to
give 39 (17
14
LMPPI2expbrcvet~~
CA 02434685 2003-07-04
mg, 74%). HPLC (230 run); Rt 13.63 min (98% pure, Kromasii 5 ~m C18 100 e~
4.6x250 mm
analytical column, using a 0-20% linear gradient over 20 min of CH3CN in O,O1M
aq TFA at
1 mL/min flow rate). 'H NMR (Da0): 8 5.10 (d, 1H, Ji,z --- 3.7 Hz, H-IE), 4.91
(d, IH, J~,Z =
0.8 Hz, H-Ie), 4.73 (bs, 1H, H-Ic), 4.45 (d, 1H. J1,2 = 8.5 Hz, H-lD), 4.09
(m, 1H, H-5c), 3.97
(m, 1H, H-2s), 3.87 (m, 4H, H-2c, 3c, GaD, CHzO), 3.67.-3.78 (m, 8H, H-2n, 3H,
4c, 5B, GbD,
Gas, GbE, 1 x CHaO), 3.60 (m, 3H, H-3E, CHzS), 3,48 (pt, 1H, H-3D), 3.39-3.46
(m, GH, H.2E,
4s, 4D, 4E, 5n, 5E), 3.33 (m, 2H, CHzNH2), 2.35 (s, 3H, CH3C(O)S), I.98 (s,
3H, CH3C(O)N),
1.27 (d, 3H, H-Gc), 1.23 (d, 3H, H-6B): ~3C NMR (Dz0): ~S 199.8 (SC=O), 174.5,
171.3
(NC(O)), 103.2 (C-18), 101.4 (C-lc), 100.9 (C-lv), 98.6 (C-IE), 82,0 {C-3D),
79.0 (C-4B),
76.6 (C-4c), 76.3 (C-2E), 72.9 (C-3~), 72.3 (C-5E): 72.2 (C-4D), 71.8 {C-3c),
71.0 (C-2c), 70.5
(C-28, 38), 69.7 (C-4H), 69.5 (C-4E), 69.1 (C-5c, 5D), 68.8 (C-5g), 68.7
(CH?0), 61.1 (C-GD),
60.7 (C-6E), 55.5 (C-2D), 40.1 (CHlNH), 33.2 (CHzS), 29.9 (CH3C(O)S), 22.6
(CH3C(O)N),
17.9 (C-6c), 16.9 (C-da). MS for C3z~i54N20zrS (834) nrlz 857 [Ni + Na]+,
HRMS-MALDI Calcd for C3aHsaNzOziS+Na: 857.?-838. Found: 857.2576.
(S-Acetylthiomethyl)carbonylaminoethyl a-L-Rhampopyranosyl-(1--~Z)-a-L-
rhamnopyranosyl~(1-~3)-[c~A-glacopyranosyl)-(1~4)]-a-L-rhamnopyranosyl-(1 >3)-
Z-
acetamldo-Z-deoxy-[i-D-glueopyranoside (40). The pentasaccharide 37 (G.4 mg,
7.4 itmol)
was dissolved in O.1M phosphate buffer (pH 7.4, I.0 mL). SAMA-Pfp (6.6 mg, 22
~mol) was
added, and the mixture was stirred at rt for 5 h. More SAMA-Pfp (G.6 mg, 22
pmol) was
added end the mixture was stirred for I h more at rt. RP-HPLC of the mixture
gave 40 (5.4
mg, 75%). HPLC (230 nm): Rt 13.86 min (100% pure, Kromasil 5 gm C18 100 ~
4.6x250
mm analytical column, using a 0-20% linear gradient over 20 min of CH3CN in
O,O1M aq
TFA at 1 mL/min Ilow rate). ~H NMR (DZO): b 5,13 (d, 1H, J~,2 = 3.7 Hz, H-lE),
4.98 (bs, IH,
H-I,~, 4.90 (bs, 1H, H-1B), 4.74 (bs, iH, H-lc), 4.47 (d, 1H, J,~ = 8.5 Hz, H-
1D), 4.09 (m,
1H, H-5c), 4.00 (m, 2H, H-2A, 28), 3.79-3.85 (m, 8H, H-2c, 2p, 3a, 4A, 4B,
6aD, Gbn, CHzO),
3.65-3.74 (m, 9Fi, H-3g, 30, 3E, 40, 5A, 58, 6aE, 6bE, CH20), 3.60 (m, 2H,
CHZS), 3.53 (pt, 1H,
H-3D), 3.13-3.49 (m, 7H, H-2E, 4D, 4F, 5n, 5E, CHz~IH), 2.35 (s, 3H, CH3C=OS),
1.99. (s, 3H,
CHIC=ON), 1.28 (d, 3H, H-Gc), I.20 (m, 6H, H-6,~" GB); 13C NMR (DzO): 8 199.9
(SC=O),
174.5, 171.4 (NC=O), 102.8 (C-1H), 101.7 (C-lA), 101.4 (C-1~), 100,9 (C-1D),
97.9 (C-lE),
82.0 (C-3D), 79.7 (C-28), 79.0, 76.3, 72.9, 72.4, ?2.2, 71.8, 71,0, 70.5,
69.7, 69.5, 69.1, 68.8,
68.5 (XXXX, CHaO), 61.1 (2C, C-6p, 6~, 60.7 (C-6E), 55.6 (C-2n), 40.1 (CHZNH),
33.2
LMPPlzexp-brcvct~gp CA 02434685 2003-07-04
(CHZS), 29.9 (CHIC=OS), 22.7 (CH3C=ON), 18.2 (C-Go), 17.2 (C-G~, 17.0 (C-GB).
HRIvIS
(MALDI) Calcd for C38H6aNZOa55+Na: 1003.3417. Found: 1003.3426.
PADRE (thiomethy~carbonylaminoethyl a-b-glucopyranosyl-(1-->4)-a-L-
rhamnopyranosyl-(1-~3)-2-aeetamido-2~deozy-~-D-glucopyranoside (1). Compound
38
(5.0 mg. 7.3 pmol) was dissolved in watez (500 p,L) and added to a solution of
PADRE-Mal
{10 mg, 5.68 ptnol) in a mixture of water (900 ItL), acetonitrile (100 pL) and
O.1M phosphate
buffer (pH 6.0, 1 mL). 117 ~tL of a solution of hydroxylamine hydrochloride
(139 mg/mL) in
O.1M phosphate buffer (pH 6.0) was added and the mixture was stirred for 1 h-
RP-HPLC
purification gave the pure glycopeptide 1 ($.5 mg, 62%). HPLC (230 nm): Rt
10.40 min
(100% pure, Kromasil 5 pm C18 100 A 4.6x250 mm anal~2ical column, using a 0-
20% linear
gradient over 20 min of CH3CN in O,O1M aq TFA at 1 mL/min flow rate). ESMS
Calcd for
C~ogHIg,NZ343ss: 2405.85. Found: 2405.52.
PADRE (thiomethyl)earbonylaminoethyl a-L-rhamnopyranosy!-(I-~3)-[a~D-
glucopyranosy!)-(1~4)]-a-L-rhamnopyranosyl-(1 >3)-2-acetamido-2-dco~y-[3-n-
glucopyranoside (2). Compound 39 (4.9 mg, 5.8 pmol) was dissolved in water
(500 p.L) and
added to a solution of PADRE-Mal (13 mg, 7.4 p,mol) in a mixture of water (1
mL),
acetonitrile (200 ~L) and O.SM phosphate buffer (pH 5.7, 1.?_ mL). 117 pL of a
solution of
hydroxylaxnine hydrochloride (139 mg/mL) in O.SM phosphate buffer (pH 5.7) was
added,
and the mixture was stirred for 1 h. 1tP-HPLC purification gave the pure
glycopeptude 2 (G.7
mg, 48%). HPLC (230 nm): Rt 11.60 min (100°fo pure, Kromasil 5 Itm C18
100 A 4.6x250
mm analytical column, using a 20-50% linear gradient over 20 min of CH3CN in
0,01M aq
TFA at 1 mL/min flow rate). ESIvfS Calcd for C125H~gIN730395~ 2552. Found:
2551.90.
PADRE (thiomethyl)carbonylaminoethyl a-L-Rhamnopyranosyl-(1~2)-a-L-
rhamnopyranosyl-(1-~3)-[oc-D-glucopyranosyl)-(1-~4)]-a-L-rharnnopyranosyl-(1-
~3)-2-
acetamido-2-deoxy-[i-D-glucopyranoside (3). Compound 40 (5.59 mg, 5.7 pmol)
was
dissolved in water (500 uL) and added to a solution of PADRE-Mal (12.6 mg. 7.2
umol) in a
mixture of water (1 mL), acetonitrile (200 ~,L), which had been previously
diluted with 0.5M
phosphate buffer (pH 5.7, 1.2 mL). A solution of hydroxylamine hydrochloride
(139 mg/mL)
in 0.5M phosphate buffer (pH 5.7,117 p.L) was added and the mixture was
stirred for 1 h. RP-
HPLC purification gave the pure glycopeptide 3 (7.1 mg, 4G%). HPLC (230 nm):
Rt 10.33
16
LMPPI2exp-brevet-gp CA °2434685 2003-07-04
min (100% pare, Kromasil 5 ltm C18 100 A 4.Gx250 mm analytical column, using a
20-50%
linear gradient over 20 min of CH3CN in O.O1M aq TFA at 1 mL/min flow rate).
ESMS Calcd
for CiZiHzuiN~30a3S: 2698. Found: 2698.09.
17
LMPPI2.sehema6revet-gp CA 02434685 2003-07-04
T epilope
r
1
PADRE-Lys
8 ep'rtope
r
aKXVAAWTLKAAaZ-rvN
OH CONHz
O
HOO O p H b~p~ ,~,S I p
NNAc NH 1N.~--NH R
Ma p 1
o H
R O 2 a-L-Rha
a-L-Rha-(1..~ 2)-a-L-Rha
DAn
s~gdrto~ OTCA
Bn'oOMe O p O
RO aZ + HO~O~N~
NNAc
6 7
0
Bn0 MA O + ~.~"NH
R BZ Ac0 MA O BnO~ O
Ac0 p~ O
B20 Me O PqORE.L
B
08z
LMPP1Z-s<hertu-brtva-gp
CA 02434685 2003-07-04
/OAc OR°
ACQ 0 R°0 ~ 0
~c0~ R30 ' y O~~N9 . R . ._R° Rc
NHAc
t2 ~ ~ 13 ac Ac ac
~ 14 ~ H ~ H H
OAII ~.OBn ~OBn v 7 H IPr
HO MATO a BflO ~1 ~ QTCA B p~ OR'
BnO~ Bn,O I0 Me To
\0 Bn0 a
R0
9 0
1 I All H cc
4 , TC~1I Bz alp
-~r~ OH OOH
08n a
BnO~ R p~Q~./~Na Q Ho~ H O~O.~-wIJH
AnO~ NHAC H~-~~ NHac
Bn0 p Me , O HO O Me
Ro OR ~ R R4 RB H0~ off
p ~ 1~5 ~ Bz . - ~iPr ~ 18
16 Bz H H
1 17 H H N
2
LMPPI2.uheme-6revctgp CA 02434685 2003-07-04
oTCA Ogn
06n Ac0 M-.~/Bn0 p
B8 D ~ OAII AcO~ pAC 6n0 en0 Me~p DR
eno Me Q 20
O
HO OR ~ Ac0 ~'le O 08z b 21 All
° Aco c ~ x2 H
OAc 5 TCA ~ a/j3
h ~ 1t H
19 Bz
r w3
R Rz Ra R~
Ac 8z iPr
24 Ac ~ 8z ~ H H
9
~''~NHZ
1
3
LMPPIZ-~oherr~~b~evet.gp
CA 02434685 2003-07-04
OH OH O
OH ~~-Q ~ ~' ~
HHp O H 0~~~NHz HO ~0 O HO-0~ ,~.,,SAc
HO ~~NH'AC NH
HO Mo O
Ho Me O
RO
OH
R R
1& H 38 H
25 a.-L~Rha 39 u-L-Rhe
37 ot-L-Rha-('~~.,2)~a-t.-Rha do o.-~-Rh2-(1-.-2)-o.-t_-Rha
OH O O
OH HO O
H ~ p NHA ~NH~,r N~ NH
r~0 O Mo ~ f1
I .MJ~. ~ H RO OH 0 PADRE-Ly9
PADRE-Cy4
i H
2 ~ a-l-Rha
3 a-~-Rha-(1...,.2)-a~~.Rha
pADfiE-~ys-NHt
Sold phase
peptide synthexis
(Fmoc chemiBetty)
Fmoc PsI Pog P9 te6sn
CA 02434685 2003-07-04
LM_ PP13
Synthesis of a pentasaccharide building black of the O-specific polysaccharide
of
SlrigellaJlexneri serotype 2a.
This paper disclosed the preparation of oligo- or polysaccharides made of two
repeating units.
The inventors reasoned that it would best rely on the use of a pre-
functionalized building
block, representative of the repeating unit of the 0-Ag, or of a frame-shifred
sequence thereof,
and susceptible to act either as a donor and potential accEptor, or as an
acceptor and potential
donor. The synthesis of such a key synthetic intermediate is described,
together with its
conversion in the form of either a donor or an acceptor.
LMPP 13 ~thco~brevet-peritablock
CA 02434685 2003-07-04
Synthesis of a pentasaecharide building block of tl~e 0-specific
po>;ysaecharide of
ShigellaJhxneri serotypc 2a~~~
Abstract
INTRODUCTION
Shigellosis or bacillary dysentery is a sexious infectious disease,
responsible for some
200 million episodes annually, mostly in children and immunocompromised
individuals
living int areas were sanitary coruiitions are insufficient. ~Z~ Of the four
species of Shigellae,
Shigella ffexneri is the major responsible of the endemic form of the.
disease, with serotype 2a
being the most prevalent, Due to increasing resistance of all groups of
Shigellae to antibiotics,
X31 the development of a vaccine against shigellosis is of high priority as
stated by the World
Health Organization in its program against enteric diseases. ~4t However,
there are yet no
licensed vaccines for shigellosis,
Shigella's Iipopolysaccharide ()rPS) is a major surface antigen of the
bacterium. The
corresponding 0-antigen (O-Ag) is both an essential virulence. factor and the
target of the
infected host's protective immune response. ~5' 6~ Based on the former
hypothesis that serum
1gG anti-LPS antibodies may confer specific protection against shigellosis, ~~
several
polysaccharide-proteine conjugates, targeting either $higella sonnei,
Slaigella dysenteriae 1 or
S flexneri serotype 2a, were evaluated in humans. ~g~ 9~ Tn the case of S.
sonnei, recent field
trials allowed Robbins and co-workers to demonstrate the efficacy of a vaccine
made of the
corresponding detoxified LPS covalently linked to recombinant exoprotein A.
~y°t Even
though efficient, polysaccharide-protein conjugate vaccines remain highly
complex structures,
whose immunogenicity depends on several parameters amongst which the length
and nature
1
LMPP13-then-6ievet-pcntablock
CA 02434685 2003-07-04
of the saccharide component as well as its loading on the protein- It is
reasonably admitted
that the standardization of these parameters is somewhat difficult when
dealing with
polysaccharides purified from bacterial cell cultures. That short
oligosaccharides were
immunogetiic when conjugated onto a protein carrier was demonstrated on
several occasions.
~~ ~~ It may be assumed that the use of well-defined synthetic
oligosaccharides would allow a
better control, and consequently the optimisation, of the above mentioned
parameters. Indeed,
available data on S. dysenteriae type 1 indicate that neoglycoconjugates
incorporating di-, tri-
or tetramers of the O-Ag repeating writ were more immunvgenic than a
detoxified I,PS-
human serum albumin conjugate of reference. t~~~ Others have shown that
conjugates
incorporating oligosaccharides comprising one repeating unit or smaller
fragments were
immunogenic in mice. U3, ia~
Along this line, we recently prepared three neoglycoproteins as potential semi-
synthetic
vaccines against Shigella Jlexneri 2a infection. These incorporated short
oligosaccharide
haptens, representative either of part or of the whole repeating unit of the 0-
Ag of S ./lexneri
serotype 2a. Preliminary data indicate that two out ofthe three conjugates are
immunogenic in
mice.(Phalipon et al, unpublished results) However, parallel studies on the
recognition of
synthetic fragments of the O-Ag by protective homologous monoclonal antibodies
suggested
that sequences comprising more than one repeating unit of the O-Ag were more
antigenic,
thus probably better mimicking the natural polysaccharide. t"~ It is
anticipated that better
mimics of the O-SP would lead to conjugates of higher immunogenicity. Thus,
the
preparation of oligo~ or polysaccharidest'61 made of two repeating units or
more was
considered. We reasoned that it would best rely on the use of a pre-
functionalized building
block, representative of the repeating unit of the 0-Ag, or of a frame~shifted
sequence thereof,
and susceptible to act either as a donor and potential acceptor, or as an
acceptor and potential
donor. The synthesis of such a key synthetic intermediate is described in the
following,
together with its conversion in the form of either a donor or an acceptor.
RESULTS AND DISCUSSION
A B E C D
2)-a-L-Rhap-(1->2)-a-L-Rh~-(1~3)-[a-D-Glcp-(1 >4)]-a-L-Rhep-(1--~3)-[3-D-
GIcNAcp(1~
I
2
CA 02434685 2003-07-04
LMPP13-then-brovet.pcntablock
The O-SP of S flexneri 2a is a branched heteropolysaccharide defined by the
pentasaccharide
repeating unit I. tl~~ tsl It features a linear tetrasaccharide backbone,
which is common to all S.
flexneri 0~antigens and comprises a N acetyl glucosamine (D) and three
rhamnose residues
(A, B, C). The specificity of the serotype is associated to the a-D-
glucopyranose residue
linked to position 4 of rhamnose C.
As part of a study of the mapping at the molecular level of the binding of
protective
monoclonal antibodies to S. fTexrleri 2a 0-antigen, a set of of di- to
pentasaccharides
corresponding to frame-shifted fragments of the repeating unit h ti9-zz) an
octasaccharide~31
and more recently a deeasaccharide~~°1 have been synthesized in this
laboratory. The latter,
namely D'A'S'(E')C'DAB(E)C, was synthesized as its methyl glycoside by
condensing a
chain terminator pentasaccharide donor and a methyl glycoside pentasaccharide
acceptor. In
the following, the key intermediate is the DAH(E)C pentasaccharide 1, which is
protected in
an orthogonal fashion at position 0-3o with an acetyl group and at the
reducing end by an
allyl group. At this stage, the acetamido function is already present at
position 2p. Compound
1 may be converted to the corresponding alcohol 2, which acts as an acceptor
and a masked
donor, or to the trichloroacetimidate 3 which acts as an acceptor allowing
subsequent chain
elongation at the non-reducing end (Scheme 1 ). Previous work in the
laboratory has shown
that in order to construct the DAB(E)C sequence, the linear approach involving
stepwise
elongation at the non-reducing end, was more suitable than the blockwise one.
D-glucosamine uhit(D). In order w limit the number of steps at the
pentasaccharide level, we
reasoned that an appropriate precursor to residue D should have (i) permanent
protecting
groups at positions 4 and 6, (ii) a participating group at position 2 and
(iii) an orthogonal
protecting group at position 3, allowing easy cleavage. As they allow a wide
range of
protecting group manipulations previously to ultimate activation,
thioglycosides are highly
convenient masked donors. Recently, two sets of non-malodorous thioglycosyl
donors have
been proposed~ZS~Ref??, among which the thiododecanyl moiety was selected.
Thus, the
known peracetylated trichloroacetamide XX~~6~ was reacted with dodecanthiol in
the presence
of BF3.OEt2 to give thioglycoside XX in high yield (97%). Zemplen
deacetylation cleanly
afforded the corresponding triol XX, which was selectively protected at
position 4 and 6 upon
reaction with 2,2-dimethaxypropane (8~% from XX). Indeed, previous
observations in the
series have demonstrated that 4.6~O-isopropylidene-D-glucosaminyl derivatives
were highly
3
LMPP13-rheo-b~evct~pentablock
CA 02434685 2003-07-04
suitable precursors to residue D, t19, z3~ Next, conventional acetylation of
XX gave the required
donor thioglycoside XX.
G-Rhamnose units (A, B): Previous work in the series was mostly based on the
use of the 2-O-
acetyl trichloroacetimidate rhamrtopyranosyl donor XX. ~°. 24~
Condensation yields were
excellem. However, the acetyl protecting group not being fully orthogonal to
the benzoyl one,
the weak point of the strategy resides in the de-O-acetylation step which, in
fact, is required
twice. The levulinate on the contrary is fully orthogonal to either beniyl or
allyl ethers, and to
benzoates. The 2~O-levulinoyl trichloroacetimidate donor XX was thus evaluated
as an
alternative to XX. It was prepared from the known allyl rhamnopyranoside
XXtZ'~ in three
steps. Indeed, treatment of XX with levulinic acid gave the fully protected XX
(XX%,
ALGlGL), deallylation of which proceeded in two steps based on (i)
isomerisation of the ailyl
group into the propen-1-yI ether using an iridium complex, lasl and (2)
subsequent oxidative
cleavage of the latter to give the hemiacetal XX {XX%, ALGlGL). tz91 Reaction
of the latter
with trichloroacetonitrile un the presence of 1,8-diazabicyclo[5.4.OJundec-7-
ene (DBU)
resulted in the required donor XX (XX%, L.A GIGL). OnE should note that
several routes to the
known XX have been described including opening of the intermediate 2,3-O-
benzylidene
derivativet~'lor regioselective benzylation of the corresponding 2,3-diol via
the stannylidene
intermediate.(ref~) Alternatively, XX could be prepared from the orthoester
XX, readily
available from acetobromorhamnose XX upon reaction with allylic alcohol in the
presence of
lutidine (XX% from L-rhamnose, lI~IP et ???). Deacetylation of XX in
methanolic ammoniac
gave diol XX, which was next benzylated into the 1,2-orthoacetate XX (XX% from
XX~P et
???). Isomerisation of the latter to the corresponding glycoside in the
presence of TVfSOT~
analogously to that described in the mannose series, t3°' a O gave the
firlly protected XX (XX%,
GL, ~ together with the p-anomer XX (XX%, GL. MP). Zemplen deacctylation of
the
former gave XX quantitatively. Besides, XX is a convenient intermediate to the
2-O-
acetylated donor XX.
Synthesis of the pentasaccharide 1: The known allyl glycoside XX, acting as an
EC acceptor,
temporarily protected at the anomeric position and having a participating
group at position Z.c,
was prepared as described in 63% yield from allyl 2,3-0-ispropylidene-a-L-
rhamnopyranoside. tZll Its condensation with the trichloroacetimidate donor
XX, performed in
the presence of a catalytic amount of TMSOT'~ afforded the fully protected
trisaccharide XX
(XX%, ALG reproduire), and subsequently the knoum H(E)C acceptor XX~Z~1 upon
selective
4
LMPP13-theo~brcvet~p~tablock
CA 02434685 2003-07-04
removal of the O-levulinoyl group v4~ith hydrazine hydrate (XX%, ALG
reproduire). Starting
from XX, this two-step process was repeated to give first the fully protectEd
XX (XX%), then
the known AS(E)C acceptor XX~2'~~ in XX% yield. According to this strategy, XX
was
obtained in XX% overall yield from the key disaccharide XX, which compares
favourably
with the 6Z% yield obtained in the previously described strategy involving the
2-O-acetylated
trichloroacetimidate donor XX. ~z"~ Besides, considering that selective
deblocking at positions
2B and ZA was completed in overnight runs instead of the 5 days required for
each
corresponding chemoseleetive O-deacetylation steps, the use of the 2-O-
levulinoyl donor
appeared as a suitable alternative to that of XX, although its preparation,
may be somewhat
lower-yielding (XX'~ instead of XX% from XX, ALG/GL). Using a mixture of NIS
and triflic
acid as the promoter, condensation of the tetrasaeeharide acceptor XX with the
thioglycoside
donor XX gave the key intermediate XX in 58% yield. Although alternative
conditions in
terms of promoters and solvents (not described) were tested, this rather low
yield could not be
improved. Radical dechlorination of XX using Bu3SnH and a catalytic amount of
AIBN
readily afforded the corresponding acetamido key intermediate 1 (74%).
(attention schema
On one hand, compound 1 may be efficiently converted to the acceptor building
block 2 under
Zempl~n conditions. On the other hand, it was smoothly deallylated into the
hemiacetal XX,
following a two-step process as described above. Next, treatment of XX with
trichloroacetonitrile and DBU allowed its conversion to the building block 3
(82% from XX).
ACKNO WL.EDGEMENTS
The authors ate grateful to J. Ughetto-Monfrin (Unity de Chimie Organique,
Institut Pasteur)
for recording all the NMR spectra. The authors thank the Bourses Mrs Frank
Howard
Foundation for the postdoctoral fellowship awarded to K. W., and the Institut
Pasteur for its
financial support (grant no. PTR 99).
REFERENCES
[1] Almh, Part 13 of the series Synthesis of ligatzds related to the O-
specific
polysacclzarides of Shigella flexneri serotype 2a and Shigeha flexneri
serotype Sa. Fvr
part 12, see ref. XX, 2003.
LMPP13-then-bTCVa~penabloek
CA 02434685 2003-07-04
[2] K L. Kotloff, J. P, Winickoff B. IvanofF, J. D. Clemens, D. L. Swerdlow,
P. J,
Sansonetti, G. K. Adak, M. M. Levine, Bull. WHO 1999, 77, 651.
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Yavzori, N. Ort, C. Block, I. Ashkenazi, J. Shemer, D. N. Taylor, T. L. Hale,
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Poor. Natl.
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K.
Kraaijeveld, H. Snippe, Infect Immurz 2001; G9, 4698.
[14J F. Mawas, J. Niggcmann, C. Jones, M. J. Corbet, J. P. K.amerling, J. F.
G.
Vliegenthart, Infect. Immun. 2002, 70, 5107.
[15] L. A. Mulard, F. Nato, V. Marcel, A. Thuizat, P. Sansonetti, A. Phalipon,
irr
preparation 2003.
[16] oligosac, Eur, J. Biochem. 1982,126, 433.
[17] D. A. R. SlmRIDnS, Bacteriol. Reviewr 1971, 35, 117.
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[19] L. A. Mulard, C. Costachel, P. 1. Sansonetti, J Carbohydr. Chem. 2000,
19, 849.
[20] C. Costachel, P. J. Sanson~etti, L. A. Mulard, ,l. Carbohydr. Chem. 2000,
19, 1131.
[21] F. Segat, L. A. Mulard, Tetrahedron: Asymmetry 2002,13, 000.
[22] L. Mulard, C. Guerreiro, C. Costacheh A. PhaIipon, in preparatiorr 2003.
[23] F. B6lot, C. Costachel, K. Wright, A, Phalipon, L. A. Mulard,
Tetrahedron. Lett 2002,
000.
[24] F. Blot, K. Wright, C_ Costachel, A. Phalipon, L. A. Mulard; J. Org.
Clzem, 2003,
sumitted.
[25] H. Dohi, Y. Nishida, T.Takeda; K. Yobayashi, Carbolrydr, Res. 2002, 337,
983.
[26] (3, Blatter, J.-M. Beau, J.-C. Jacquinet, Carbohydr. Res. 1994. 260, 189.
[27] P. Westerduin, P. E. d. Haas, M. J. Dees, J. H. ~~, Boom, Carbohydr. Res.
1988, 180,
195.
[28] J. J. Oltvoort, C. A. A, v, Boeckel, J. H. d. Koning, J, v. Boom,
Synthesis 1981, 305.
[29] M. A. Nashed, L. Anderson, J. Chem. Soc. Chem. Common. 1982, 1274.
[30] T. Ogawa, K. Beppu, S. Nakabayashi, Carbohydr. Res. 1981, 93, C6.
[31] T. K. Lindhorst, J. Carbohydr. Chem. 1997, 162, 237.
6
LNlPP l3-e~ep-brevet~pentablock
General methods
CA 02434685 2003-07-04
Optical rotations were measured for CHC13 solutions at 25°C, expect
where indicated
otherwise, with a Perkin-Eliner automatic polarimeter, Model 241 MC. TLC were
performed
on precoated slides of Silica Crel GO Fz<,~ (Merck). Detection was effected
when applicable,
with UV light, and/or by charring in 5% sulfuric acid in ethanol.
Preparative chromatography was performed by elution from columns of Silica Gel
60 (particle
size 0.040-0.063 mm). For all compounds the NMR spectra were recorded at
2~°C for
solutions in CDCl3, on a Broker AM 400 spectometer (400 MHz for 'H, 100 MHz
for ''C).
External refotences : for solutions in CDCl3, TMS (0.00 ppm for both'H and
'3C). Proton-
signal assignements were mane by first-order analysis of the spectra, as well
as analysis of 2D
'H-'H correlation maps (COSY) and selective TOCSY experiments. Of the two
magnetically
non-equivalent geminal protons at C-6, the one resonating at lower field is
denoted H-6a and
the one at higher field is denoted H-Gb. The '3C N'vIR assignments were
supported by 2D "C-
'H correlations maps (HETCOR). Tnterchangeable assignments are marked with an
asterisk in
the listing of signal assignments. Sugar residues in oligosaccharides are
serially lettered
according to the lettering of the repeating unit of the 0-SP and identified by
a subscript in the
listing of signal assignments. Fast atom bombardment mass spectra (FAB-MS)
were recorded
in the positive-ion mode using dithioery~thridol/dithio-L-thrEitol (4 :1, MB)
as the matrix, in the
presence of NaI, anal Xenon as the gas, Anhydrous DCM, 1,2-DCE and Et20, sold
on
molecular sieves were used as such. 4 ~ powder molecular sieves vvas kept at
100°C and
activated before use by pumping under heating at 250°C.
Dodecyl 3,4,6-tri-O-acetyl-2-dcoxy-1-thio-2-trichloroacetamido-[i-D-
glucopyranoside (5).
A mixture of the peracetylated 4 (6.2 g, 12.5 pmol) and dodeeanthiol (2.5 mL,
94 Nmol), 4A
molecular sieves and dry 1,2-DCE (90 mL) was stirred for 1 h then cooled to
0°C. BF3.Et~0
(1.57 mI,, 12.5 ~mol) was added. The stirred mixture was allowed to reach rt
in 2h30. Et3N
was added until neutral pH and the mixture filtered. After evaporation, the
residue was eluted
from a column of silina gel with 2:1 cyclohexane-EtOAc to give 5 as a white
solid (7.5 g, 93
%); [a]p -20° (c l, CHCIs). 'H NMR (CDC)J);8 G.82 (d, 1H, ,IZ,Nh = 9.2
Hz, NH), 5.31 (dd,
CA 02434685 2003-07-04
LMPP13~dcPbrcvet-pentablock
1H, JZ,3 = 9.9 Hz, J3,a ' 9.6 Hz, H-3), 5.15 (dd, 1H, J,,,s = 9.6 Hz, H-4),
4.68 (d, 1H, JI,Z =
10.3 Hz, H-1), 4.28 (dd, 1H, Js,So = S.0 Hz, Jg~fib = 12.3 Hz, H-Ga), 4.17
(dd, 1H, JS,eb = 2.3
Hz, H-6b), 4.11 (dd, 1H, H-2), 3.75 (m, 1H, H-5), 2.70 (m, 2H,
SCHZ(CH~)loCH3). 2.10,
2.05, 2.04 (3s, 9H, OAc), 1.G5-1.20 (m, 20H, SCHZ(CH~),oCH3), 0.90 (t, 3H,
SCHz(CH~),oCH3). '3C NMR (CDCl3):o 171.0, 170.7, 169.3 (C=0), 161.9 (C=OCC1,),
92.3
(CC13), 84.2 (C-1), 76.5 (C-5), 73.4 (C-3), 68.6 (C-4j, G2.6 (C-6), 55,2 (C-
2), 32.3, 30.6,
30.0-29.1, 14.5 (S(CHz)iICH,), 21.1, 21.0, 20.9 (OAc). FABMS of Cz6H4~C13NOeS
(M,
635.0), m/z 658.1 [vI+Na]''. Anal. Calcd for Cz~H4zC13NOeS, C: 49.17, H: 6.67,
N: 2.21.
Found C: 49.16, H: 6.71, N: 2.13.
Dodecyl 2-deoxy-4,6-O-isopropylidene-1-thio-2-trichloroacetamido-~-D-
glucopyranoside
(7?.
A mixture of 5 (5 g, 7.87 mmol) in MeOH (15 mL) was deacetylazed by MeONa
overnight.
The solution was neutralized by 1R 120 (H+) and Filtrated. After concentration
in ~~acuo, the
residue 6 was treated by 2,2-dimethoxypropane (70 mL, 546 mtnol) and APTS (148
mg, 0.94
mmol) in DMF (20 mL). After stirring overnight, the mixture was neutralized
with Et;N and
concentrated. The residue was eluted from a column of silica gel with 3:1
cyclohexane-EtOAc
to give 7 as a white solid (3.45 g, 80 %); [a]p -35° (c 1, CHC)r).
1H NMR (CDCL):8 6.92 (d, 1H, .I,,~ = 8.0 Hz, NH), 4.77 (d, 1H, J,,2 = 10.4 Hz,
H-1), 3.98
(m 1H, Ja,3 = Ja.a = 9.2 Hz, H-3), 3.88 (dd, 1H, Js,c,q = 5.4 Hz, J~,,Eh =
10.8 Hz, H-6aj, 3.70
(dd, 1H, J5.6b = 0.5 Hz, H-db), 3.63 (tn, 1H, H-2), 3.53 (dd, 1H, J~,S = 9.2
Hz, H-4), 3.29 (m,
1H, H-5). 2.98 {s, 1H, OH), 2.G0 (m, 2H, SCH~(CHi),oCH3), 1.60-1.10 (m, 20H,
SCHa(CHi)loCHl), 1.45, 1.35 (2 s, 6H, C(CH3)Z), 0.80 (t, 3H, SCH2(CHZ)loCHa).
s3C NMR
(CDC13):8 IG2,5 (C=OCC13), 100.3 (C(CH3)i), 92.8 (CC13), 84.0 (C-1), 74.6 (C-
4), 72.3 (C-
3), 71.7 (C-5), 62.2 (C-G), 58.3 (C-2), 29.3, 19.5 (C(CH3)z), 32.3, 30.8, 30.1-
29.5, 29.1, 14.5
?-
CA 02434685 2003-07-04
LMPPI3~cpbrcvet-pent~block
(SCH,z(CHs),oCHs). FABMS Of Cz3HtoC13NO5S (M, 548.9), m/z 5'72_2 [~I+NaJ'.
Anah Calcd
for CZ,H4oCl~NOsS, C: 50.32, H: 7.34, N: 2.55. Found C. 50.30, H: 7.40, N:
2.36.
Dodeeyl 3-D-acetyl-2-deoay-4,6-O-isopropylidene-1-thio-2-trichlotroacetamido-
[i-D-
glucopyranoside (8).
A mixture of 7 (I.07 g, 1.94 mmol) in pyridine (10 mL) was cooled to
0°C. Ac~O (5 mL) was
added and the solution was allowed to reach rt in 2 h. The mixture was then
concentrated and
the pyridine coevaporated with toluene. The residue was eluted from a column
of silica gel
with 6:1 cyclohexane-EtOAc with 0.2% of Et3N to give 8 as a white solid (1.12
g, 97 %), [a]D
-62° (c 1, CHCIa)
'H NMR (CDC13):b 7.51 (d, IH, Jz,~c = 9.7 Hz, NH); 5.40 (dd, 1H, Jz,3 = J3.< =
I0.0 Hz, H-3),
4.62 (d, 1H, Ji,z = 10.4 Hz, H-1), 4.20 (m, 1H, H-2), 4.01 (dd, 1H, ,Isss =
5.2 Hz, J6a,6b = 10.7
Hz, H-6a), 3.84 (dd, 1 H, J,,s = 9.7 Hz, H-4), 3.70 (m, 2H, H-5, H-6b), 2.68
(m, 2H,
SCHz(Cl~),oCH3), 2.09 (s, 3H, OAc), 1.60-1.20 (m. 20H, SCHz(CHz),oCHa). 1.52,
1.38 (2 s,
6H, C(CH3)2), 0.90 (t, 3H, SCH2(CHz)~oCH,). ~3C NMR (CDCl3):8 171.4 (C=OCH3),
161.8
(C=OCCl3), 99.5 (C(CH,)z), 92.3 (CCl3), 84.6 (C-1), 73.6 (C-3), 72.0 (C-4),
71.9 (C-5), 42.2
(C-6), 55.0 (C-2), 29.1, 19.3 (C(CH3)z). 32.3, 30.7, 30.0-29.0, 14.5
(SCHz(CHz),oCH3).
FABMS of CzSH,,iCI31~065 (M, 591.0), m/z G14.1 [M+Na]+. Anal. Calcd for
CzsH4zC13N06S,
C: 50.80, H: 7.16, N: 2.37. Found C: 50.67, H: 7.32, N: 2.24.
3,4-Di-O-acetyl-1,Z-O-allyloxyethylidcne-(i-L-r6amnopyranose (12). A mixture
of L-
rhamnose mnnohydrate (50 g, 274 mmol) in pyridine (4I0 mL) was cooled to
0°C. AczO (I70
mL) was added and the solution was allowed to reach rt overni.ght. MeOH (100
mL) was
added and the solution concentrated. The resulting suspension was taken up in
DCM, washed
with water, satd aq NaHC03, water, and satd aq NaCI, successively. The organic
layer was
3
LMPP13-cx~brovet.pentabloclc
CA 02434685 2003-07-04
dried and concentrated to give the crude peracetylated rhamnose (quart,) as a
slightly yellow
oil. A solution of latter (21.1 S g, 63.7 mmol) in acetic acid (38 mL) and
acetic anhydride (6.7
mL) was treated by a 33% solution of HBr in AcOH (8G mL), then stirred for 15
h at rt. The
mixture was concentrated by repeated coevaporation with cyclohexane. The
resulting
suspension was taken up in DCM, washed with satd aq NaHC03 and water. The
orgatuc layer
was dried and concentrated to give 11 (quart.) as a brown oil. A solution of
the crude il
(22.29 g) in anhydrous 2,6-lutidine (37 mL) was treated by AllOH (9.6 mL, 142
mmol) at rt.
The solution was stirred overnight, then filtered and the solids were washed W
th EtOAc. The
liquid layer was concentrated anri the residuE was taken up in DCM, washed
with 1M HCl cold
solution, water and satd aq NaCI. The organic layer was dried and concentrated
by
coevaporation with toluene. Chromatography of the crude residue
(toluene:acetone, 49:1
containing 0.1% Et3N) gave orthoester 12 (18.5 g, 88%) as a slightly yellow
oil which
crystallized on standing. An analytical sample was recristallized from
isopropyl ether:petroleum
ether; mp XX°C, [a)o -XX° (c 1, CHCl3); 1H N1~IR (CDC13):8 5.88
(m, 1H, AIl), 5.42 (d, 1H,
J,,Z = 2.3 Hz, H-1), 5.25-5.40 (m, 2H, All), 5.10 (dd, 1H, J 2,3 = 3.3 Hz, H-
3), 5.05 (dd, 1H,
J4,s = 6.3 Hz, H-4), 4.60 (dd, 1H, H-2), 4.05 (m, 2H, All), 3.50 (qd, 1H, J5,6
=- 6.2 Hz, H-5),
2.12, 2.OG (2s, 6H, OAc), 1.7G (s, 3H, CHs), 1.23 (d, 3H, H-G); '3C NMR
(CDCI;): 8171.4
(C=OCHj), 1 bl. 8 (C=OCCI3), 99. S (C(CH;)a), 92.3 (CCI3), 84. 6 (C-1), 73. 6
(C-3), 72. 0 (C-
4), 71.9 (GS), 62.2 (C G), 55.0 (G2), 29.1, 19.3 (C(CH3)~, 31.3, 30.7, 30.0-
29.0, 14.5
(SCHi(CH~~oCH,~. FABMS of C2sH~ZCl3NOrS' (A~ 591.0) tnlz 614.1 (M+NaJ'. Anal.
Calcd
for C~sH4aCISN06S, C: 50.80, H.' 7.16, N.' 2.37. Found C.' SO. G7, H: 7.32,
N.' 2.24.
3,4-Di-0-benzyl-1,2-0-altyloxyethylidene-p-L-rhamnopyranose (14). A solution
of the
crude peraeetylated rhamnose (9.0 g, 27 mmol) was processed as described for
the preparation
i of 12. A solution of the crude lZ thus obtained in MeOH (GS mL) was cooled
to 0°C and
4
LMPPI3.exp-brovn-pent3block ~ 02434685 2003-07-04
treated with NI-13 until saturation. The solution was stirred for 6 h at rt,
then concentrated by
eo-evaporation with toluene to give 13. Column chromatography (DCM:M~eOH,
49:1) gave
pure 13 as a white solid. 'H NMR (CDC13):S 5.75 (m, 1H, All), 5.22 (d, 1H, H-
1), 5.00-5.10
(m, 2H, All), 4.60 (dd, 1H, H-2), 4.30 (d, 1H, H-3), 3,80 (m, 2H, All), 3.50
(m, 1H, H-5),
3.20 (t, 1H, H-4), 1.80 (s, 3H, CH3), 1,20 (d, 3H, J5,6 = 6.2 Hz, H-6).
A solution of crude 13 in anhydrous DMF {90 mL) was cooled to 0°C. NaH
(4.32 g, 108
rnrrwl) was added in 30 min then BnBr {8.5 mL, 71 mmol) was added dropwise at
0°C. The
solution was stirred overnight at rt, then MeOH (20 mL) was added dropwise at
0°C. The
solution was allowed to reach rt in 2 h, then concentrated. The residue was
taken up in DCM,
washed with satd aq NaHCO, until neutral pH, water and satd aq NaCI. The
organic layer was
dried and concentrated, After evaporation, the residue was eluted from a
column of silica geI
with 9:1 cycIohexane-EtOAc and 0.2 % of Et3N to give I4 as a white solid (8 g,
70%).
Crystallization of an analytical sample from isopropyl ether:petroleum ether
gave 13 as white
crystals; mp XX°C, [a]p XX° (c I, CHC13);'H NMR (CDC~):S 7.35
(m, IOH, Ph), 5.90 (m,
1H, All), 5.30 (d, 1H, Jr,2 = 2.2 Hz, H-1), 5.28-5.43 (m, 2H, All), 4.95-4.65
(m, 4H, CH~Ph),
4.40 (dd, 1 H, Jz a = 4.0 Hz, H-2), 4.10 {m, 2H, All), 3.70 (d, 1 H, J;,d =
9.0 Hz, H-3), 3.50 (t,
1H, J,,s = 9.0 Hz, H-4), 3.35 (m, 1H, Js,6 = 6.2 Hi, H-S), 1.77 (s, 3H, CHI),
1.33 (d, 3H, H-
6); ''C NMR (CDCl3): ~S 171.4 (C=OCH~, 161.8 (C=OCCl3), 99. S (C(CH;j~, 92.3
(CCI~,
84.6 (C-1), 73.6 (C-3), 72.0 (C-4), 71.9 (C-S), 62.2 (C-6), SS.O (C-2), 29.1,
19.3 (C(CH3)~,
32.3, 30.7, 30.0-29,0, 14.5 (SCH~(CH~taCH3). FABMS of CiJH.,IChNOsS (M, 591.0)
mlz
614.1 jM+NaJ~. Anal. Calcd for C~sH41C13N06S, C.' 50.80, H.~ 7.16, N: 2.37.
FouNd C:
50.67, H.' i.32, N.' 2.24
Altyl 2-O-acetyl 3,4-Di-0-benzyl-~i-L-rhamnopyranoside (15). A mixture of the
ort)zoester
LMPP13-cxp-b~avct-pentabiock
CA 02434685 2003-07-04
H-5), 3.43 (pt, 1H, H-4), 2.80 (m, 4H, levy. 2.19 (s, 3H, Ac), 1.37 (d, 3H, H-
G).i3C NMR
(CDCI3): b 124.0-125.1 (Ph), 1 I 8.0 (All ), 97.0 (C-1), 80.2 (C-4), 78.5 (C-
3), 75.2 (CHZPh),
72:0 (CHZPh), 70.2 (C-2), 68.5 (All ), G8.3 (C-5), 38.5 (Lev), 31.5 (Ae), 28,5
(lev), 20.1 (C~
G).
3,d-Di-O-benzyt-2-0-Icvulinoyl-a-L-rhamnopyranose (18). 1,5-Cyclooctadiene-
bis(methyldiphenylphosphine)iridium, hexafluorophosphate (25 mg, 20 p,mol) was
dissolved
THF (? mL), and the resulting red solution was degassed in an argon stream.
Hydrogen was
then bubbled through the solution, causing the colour to change to yellow. The
solution was
then degassed again in an argon stream A solution of 17 (1.4 g, 3.12 mmol) in
tetrshydrofuran
(? mL) was degassed and added. The mixture was stirred at rt overnnght, then
concentrated to
dryness. The residue was dissolved in a solution of Iz (1.37 g, 5.4 mmol) in
30 mL of
THF/Hz0 (15;4). The mixture was stirred at rt for 1 h and THF .vas evaporated.
The resulting
suspension was taken up in DCM, washed twice tenth water, said aq NaHS03,
water, satd aq
NaHC03, water and said aq NaCI, successively. The organic layer was dried and
concentrated.
The residua was eluted from a column of silica gel with 7:3 to 6:4 cyclohexane-
EtOAc to give
the corresponding hemiacetal 18 (1.3 g, 93 %). 'H NMR (CDC13): 'H b 7.3-7.4
(m, IOH, Ph),
5.40 (dq, 1H, J,,z = 1.8, Jz,3 = 3.4 Hz, H-2 ), 4.93 (d, 1H, CHzPh), 4.78 (d,
1H, J,,z = 1.6 Hz,
H-1), 4.78 (d, 1H, CHZPh), 4.G3 (d, 1H, CHZPh), 4.51 (d, IH, CHzPh), 3.99 (m,
IH, .I3_4 = 9.5
Hz, H-3), 3.78 (dq, 1H, Ja,s = 9.5, Js.s = G.2 Hz, H-5), 3.43 (pt, 1H, H-4),
2.80 (m, 4H, lev ),
Z.I9 (s, 3H, Ac), I.37 (d, 3H, H-6).
3,4-Di-O-benzyt-2-O-ievutinoyl-a-L-rhamnopyranosyl trichloroaeetimidate (19).
Trichloroacetonitrile (1.3 mL. 13 mmol) and DBU (51 pL, 0.3 mrrwl) wire added
to a solution
ofthe residue I8 (1.0 g, 2.3 rrunol) in anhydrous DCM (G mL) at 0°C.
After 2 h, the mixture
7
LhIpPI 3eapbrevct-pcntablock
CA 02434685 2003-07-04
was concentrated. The residue was eluted from a column of silica gel W th 3:1
eyelohexane-
EtOAc and 0.2 % Bt3N to give I9 as a white foam (1.0 g, 95 %); [a]D XX°
(c I, CHCl3). 'H
NMR (CDC.I~): 'H s 8.G7 (s, 1H, NH ). 7.3-7.4 (m, IOH, Ph), 6.19 (d, 1H, Jt.2
= 1.9 Hz, H-1),
5.48 (dd,, lH, J,,i = 2.0, Jz,3 = 3.3 Hz, H-2 ), 4.95 (d, 1H, CHaPh), 4.73 (d,
1H, CHaPh), 4.66
(d, 1H, CHZPh), 4.58 (d; 1H, CHzPh), 4.51 (d, 1H, CHzPh), 4.00 (dd, 1H, J3,a =
9.5 Hz, H-3),
3.95 (dq, 1H, J4,s = 9.G, Js,b -- 6.3 Hz, H-5), 3.52 (pt, 1H, H-4), 2.80 (m,
4H, levy, 2.20 (s, 3H,
Ac), 1.36 (d, 3H, H-6).
Altyl (2-O-levulinoyl-3,4-di-O-beniyl-a-z-rhamttopyranosyl)-(1-~3)-[2,3,4,6-
tetra-0-
benryl-a-D-glucopyranosyl-(1 >4)]-2-O-benzoyl-a-trrhamnopyranoside (22j. A
mixture
of alcohol 21 (300 mg, 0.3G mmol) and imidate 19 (320 mg, 0.54 mmol) in
anhydrous EtzO
(20 mh) was stirred for 15 min under dry Ar. A$er cooling at -75°C,
Me3Si0Tf (13 p,L, 70
pmoi) was added dropwise and the mixture was stirred 3 h. Trietlrylamine (G0
11L) was added
and the mixture was concentrated. The residue w-as eluted from a column of
silica gel with 9:1
cyclohexane-EtOAc to give 22 (440 mg, 92 %) as a colorless foam; [a]D
XX° (c 1, CHC13).'H
NMR (CDC13):8 7.1-8.1 (m, 35H, Ph), 5.95 (m, 11-l, All), 5.73 (dd, 1H, .1,,2 =
2.2, Ji,3 = 2.3
Hz, H-2B), 5.43 (dd, 1H, JI,2 - 2.0 Hz, JZ,3 = 3.0 Hz, H-2~), 5.30 (m, 2H,
All), 5.08 (d,1H, JI,~
= 3.2 Ha, H-ls), 5.03 (d, IH, Ji,z =1.7 Hz, H-lg), 4.97 (d, 1H, J~,~ = I .9
Hz, H-Ic), 4.30-5.00
(m, 12H, CHZPh), 4.20 (m, 2H, All, H-3c), 4.05 (m, 3H, All, H-3E, SE), 3.98
(m, 1H, H-6aE),
3.81 (m. SH, H-3H, 4c, 4~, Sc, GE), 3.G9 (dq, 1H, J~,s = 9.3, Js.6 = 6.0 Hz. H-
58), 3.52 (dd, 1H,
Ji,l = 9.7 Hz, H-2E), 3.29 (dd, 1H, J3 4 = J4,s = 9.4 Hz, H-4H), 2.71 (m, 4H,
Levy, (s, 3H, Ac),
1.40 (d, 3H, H-6c), 1.01 (d, 3H, H-6a).
Aliyl (3,4-dl-0-benryl-a-1.-rhsmnopyrnnosyl)-(I~3)-[2,3,4,6-tetra-0-ben2yl-a-D-
glucopyranosyl-(1--~4)]-2-O-benzoyl-a-L-rhamnopyrsmoside (23). The
trisa.ccharide 22
8
CA 02434685 2003-07-04
LJvfPPJ 3-agr.6rtwct-pcnusblack
(200 mg, 0.16 mmoI) was treated wJJth 0.4 mL of a solution 1 M of hydrazine
(I00 nng) diluted
in a mixture of pyridine (1.6 mL) and acetic acid (0.4 tnL) at rt. The
solution was stirred
during 20 min. Acetone (1.2 mL) was added and the solution was concentrated.
The residue
was eluted from a column of silica gel with 98.5:1.5 Diehlorometharre-AcOEt to
give 23 (174
mg, 92 %) as a foam; [a]p +14° (c 1, CHC13); 'H NMR (CDCI,):o 7.05-8.10
(m, 35H, Ph),
5.82 (m, 1 H, All), 5.25 (dd, 1 H, JJ,z = 1.7 Hz, Jz,3 = 3.1 Hz, H-2~), 5.19
(m, 2H, All), 5.00 (d,
1H, J~,z= 3.1 Hz, H-lE), 4.87 (d, 1H, J,,2= 1.8 Hz, H-IH), 4.81 (d, IH, H-Ic),
4.35-4.90 (m,
12H, CI~ZPh), 4.00-4.20 (m, 2H, All), 4.10 (dd, 1H, J;,4 = 8.5 Hz, H-3c), 4.09
(dd, IH, Jz,~ _
3.2 Hz, H-2B), 3.95 (m, I H, J4,s = 9_5 Hz, H-5E), 3.92 (dd, 1 H> Ja,3 = 9.5
Hz, J3,, = 9.5 Hz, H-
3E), 3.78 (m, 1H, Js,a = 6.0 Hz, H-5~), 3.70 (m, 1H, H-4c), 3.58~3.62 (m, 2H,
H-6aE, 6bE),
3.59 (m, 1H, J~,s = 9.0 Hz, -Is,s = 6.2 Hz, H-Se), 3.54 (dd, 1H, H-4E), 3.48
(dd, IH, J3,d = 8.5
Hz, H-3H), 3.45 (dd, 1H, H-2fi), 3.31 (dd, 1H, H-4H), 2.68 (d, 1H, JZ,oH=2.3
Hz, O-H), 1.29
(d, 3H, H-G~), 1.09 (d, 3H, H-6B). "C NMR (CDCu):8 166.2 (C=0), 118.2-137.5
(Ph, All),
103.1 (C-1B), 98.5 (C-ls), 96.6 (C-lc), 82.1 (C-3E), 81.4 (C-2E), 80.4 (C-48j,
79.7 (C-3B),
79.4 (C.4~), 78.9 (C~3~), 78.1 (C-4E), 76.0, 75.5, 74.5, 74.2, 73.6, 72.1
(CHZPh), 73.7 (C-
2c), 68.9 (C-6s), 68.8 (C-5e), 68.7 (All, C-5E), 68.1 (C-5c), 19.1 (C-6c),
18.2 (C-68). FABMS
of C~oH~60,s (M, 1156.5), mlz 1179.5 ([M+Na]+). Anal. Calcd for C~oH,EO,s: C,
72.64; H,
6.62. Found C, 72.49; H, 6.80.
Ally! (3-0-acetyl-4,G-0-isopropylidcne-.2-trichloroacetamido-2-deoxy-[3-n-
glucopyranosyt)-(1-~2)-(3,4-di-D-benzyl-a.-~rhamnopyranosyl)-(1--~Z)-(3,4-di-O-
benzyt-
a-r.-rhamnopyranosyl)-(1-~3)-[2,3,4,G-tetrn~D-benzyl-a-n-glucopyranosyl-(1-
~4)]-2-D-
benzoy!-a-L-rbamnopyranoside (Z6). A mixture of the donor 8 (294 mg, 357
~tmol) and the
acceptor 25 (313 mg, 211 pmol), 4~1 molecular sieves and dry DCM (4 mL) was
stirred for
9
CA 02434685 2003-07-04
LMPP13-cxp~brevebprntn6lock
1.5 h then cooled to -15°C. NIS (94 mg, 0.42 mmol) and Triflic acid (8
N.L, 0.1 mmol) were
successively added. The stirred mixture was allowed to reach 0°C in 1.5
h. Et3N (25 pL) was
added and the mixture filtered. After evaporation, the residue was eluted from
a column of
silica 8el with G:1 cyclohexane-EtOAc and 0.5 % of Et3N to give 26 as a white
foam (232 mg,
58 %); [oc]p -2° (c l, CHCl3); 'H NMR (CDCIs): 'H cS 7.04~8.00 (m, 45H,
Ph), 6.81 (d, IH,
J2_~ = 9.0 Hx, NHD), 5.82 (m, 1H, All), 5.30 (dd, 1H, J,~ = 1.0, JZ,~ = 3.0
Hz, H-2c), 5.10-
S.Z3 (m, 2H, All), 4.96 (bs, 1H, H-1,,), 4.91 (d, IH, Jl,~ = 3.1 I-Iz, H-IE),
4.87 (d, 1H, J,_i =
1.6 Hz, H-IB), 4.84 (bs, IH, H-lc), 4.79 (dd, 1H,.T~,3 =J3,a = lO.OHz, H-3p),
4.35 (d, 1H, H-
ln), 4.34 (dd, IH, H-Zg), 4.20-4.80 (m, 1GH, CH2Ph), 4.00 (dd, IH, H-2A), 3.90
(dd, 1H, H-
2D), 2.90-4.10 (m, 22H, All, H-2~,, 3~, 3g, 3c, 3F, 4A, 4H, 4c, 4D, 4E, SA,
58, Sc, 5~, SE, Gac, Gbu,
Gas, GbE), 1.93 (s, 3H, OAC), I.Z-0.9 (m, 15H, C(CH3)i, H-6A, GH, 6c). "C NMR
(CDCI..,):8
170.7, 165.5, 161.7 (C~O), 138.4-117.3 (Ph, All), 101.7 (C-1D), 100.8 (C-1H),
100.6 (C-IA},
99.5 (C(CH3)2), 97.9 (C-IE), 95.7 (C-lc), 92.0 (CC)3), 82.2, 81.7, 81.6, 80.3,
79.9, 78.8, 77.9,
77.9, 76.6, 76.0, 75.8, 75.4, 75.1, 74.7, 74.3, 74.1, 73.3, 72.8, 72.6, 71.9,
71.5, 70.8, 69.0,
d8.8, 68_5, 68.0, 67.8, 62.0, SG.7 (C-2D), 28.6 (C(CH,)~), 21.3 (OAc), 19.4
(C(CH3)~), 19.0,
18.5, 18.4 (3C, C-GA, 6B, Gc). FABMS of C1a31i,~aCl3NOis (M, 1872.3), m/z
1894.6 [vI+Na]+.
Anal. Calcd for C,°3H~~aCI3NOZ5, C: 66.07, H: 6.14, N: 0.75. Found C:
66.08, H: 6.09, I~':
a.81.
Atly1 (2-acetamido-4,G-O-isopropylidene-Z-deoxy-~-D-glucopyranoayl)-(Z-~Z)-
(3,4-di-O-
benzyl-a-t,-rhamnopyranosyl)-(Z.-~2)-(3,4-di-0-benry1-a-L-rhamnopyraaosyt)-(1-
a3)-
(2,3,4,6-tetra-0-benzyt-a-D-glucopyranosyl-(1--~4)-)-2-D-benzoyl-a-L-
rhamnopyranoside
(2). The pentasaccharide X (2.65 g, 1.47 mmol) was dissolved in MeOH {20 mL).
MeONa
was added until pH=10. The mixture was stirred for 25 min then treated by IR
120 (H') until
CA 02434685 2003-07-04
LMPP t 7-exp~breveipcutsblak
gel with 2:1 Cyclohexane-AcOEt and 0.2 % of Et3N to give 1 as a white foam
(1.99 g, 94 %);
[cc)b +1° (e I, CHC13).
(b) A mixture of26 (144 mg, 0.06 mmol), Bu3SnH (O.t mL, 0.37 mmol) and AIBN
(10 mg) in
dry toluene (3 mL) was stirred for 1 h at rt under a stream of dry Ar, then
was heated for 1.5
h at 90°C, cooled and concentrated. The residue was eluted from a
column of silica gel with
2:1 cyatohexane-1?tOAc and 0.2 % of Et3N to give 1 (100 mg, 74 %). 'H NMR
(CDC13): 5
6.95-8.40 (m, 45H, Ph), 5.82 (m, 1H, All), 5.46 (d, 1H, Jz,uH = 8.0 Hz. NHti),
5.29 (dd, 1H,
J~.z = 1.0, Ja,3 = 3.0 Hz, H~2c), 5.1 I-5.25 (m, 2H, All), 5.00 (bs, 1H, H-
lA), 4.90 (d, 1H, J~.z =
3.1 Hz, H-IE), 4.85 (d, 1H, J,,z = 1.6 Hz, H-1B), 4.83 (bs, 1H, H-1~), 4.70
(dd, 1H, Jz,3 =J3,, _
10.0 Hz, H-3o), 4.44 (d, 1H, H-1D), 4.34 (dd, IH, H-28), 4.20-4.80 (m, 1GH,
CHzPh), 4.02
(dd, 1H, H-2A), 3.37 (dd, 1H, H-2E), 2.90-4.10 (m; 21H, All, H-2o, 3A, 3B, 3c,
3E, 4A, 4B, 4c,
4p, 4F, 5,,, SH, Sc, SD, 5E, GaD, 6bi,, 6a~, Gbs), 1.92 (s, 3H, OAc), 1.57 (s,
3H, AcNH), 1.27-
0,90 (m, 15H, C(CH3)Z, H-6", 6a, Gc). t'C a 171.3, 170.3, 166.2 (C=0). 138.7-
117.9 (Ph, All),
103.9 (C-1D), 101.5 (C-lg), 101.4 (C-lA), 99.9 (C(CH3)?), 98.5 (C-ls), 96.3 (C-
Ic), 82.1,
81.7, 81.6, 80.3, 80.1, 78.8, 78.1, 77.8, 76.0, 75.8, 75.3, 75.1, 74,7, 74,2,
73.6, 73.3, 72.7,
71.9, 71.4, 70.8, 69.0, 68.8, 68,7, 68.4, 68,1, 67.8, 62.1, 55.0 (C-2D), 30.0
(C(C'F~,)~), 23.5
(AcNH), 21.6 (OAc), 19.2 (C(CH3)2), 19.0, 18.3, 18.2 (3C, C-6A, GB, 60). FAB-
ViS for
Cio3H,17N025 (M = 1769.0) m!z 1791.9 [M + Na)+. Anal. Calcd. for C,o3Ht,7N025
: C, 69.93 ;
H, 6.67 : N, 0.79. Found C, 69.77; H, 6.84; N, 0.72.
(2-acetamido-3-O-acetyl-4,6-0-isopropylideno-l-deoxy-[3-D-glucopyranosyl)-(1--
>2)-(3,4-
di-0-benzyl-a-L-rhamnopyranosyt)-(1~2)-(3,4-di-0-ben2yl-n-z-rhamnopyrsnosyl)-
(1-->
3)-[2,3,4,6-tetra-O-6enzy I-a-D-glucopyranosyl-(1 >4)-]-Z-O-beozoy I-a-L-
rhamnopyranosyl trichloroacctimidate (3). 1,5-Cyclooctadiene-
bis(methyldiphsnylphosphine)iridium hexafluorophosphate (50 mg, 58 pmol) was
dissolved
12
LMPP13-Schcmes-brc~wPcnG~block
OBn
BnBOCi ~ ORS
Bn0 0 Me O
0 OBz
Bn0 Me 0
Bn0 O
' OO O 0~ OBn
Rs~~ Me OBn
NHAc
R' R3
All Ac
2 All H
3 TCA Ac
CA 02434685 2003-07-04
L.htPPl3~Schcme,;-hrcvet-pentabtock
CA 02434685 2003-07-04
OAc
~A ~~S~OAc ~~~_~~S(CNZ)WH3
NHC(O)CCf3 NHC(0)CCI3
R
6 Ac
6 H
O t
--r R30 R
NHC(0}CCi3 r ~ S(CNZ)~tCH3 H
g 5(CHg}oCH3 Ac
g OH Ac
OTCA Ac
CA 02434685 2003-07-04
LMPP14
Synthesis of spacer-armed hexa-, deca-, and pentasaccharide haptens
representative of
the O-spccitic polysaccharide of Sh~gella Jtexneri serotype Za1
This paper disclosES total synthesis of fully defined aligomeric repeating
unit glycosides
mimicking the branched bacterial O-5Ps in the S. flexneri series. The strategy
disclosed herein
gives access to extended fragments of the O-SP of S flexneri serotype 2a in a
spacer-armed
form suitable for irnmunological studies. Indeed, amounts required for the
synthesis of fully
synthetic oIigosaccharide conjugates as potential vaccines targeting S
flexneri 2a infection
were made available
LMPPI dthco-brc..ct-synlong~
CA 02434685 2003-07-04
Synthesis of apa~er-armed hexa-, deca-, and pentasaccharide haptens
representative of
the O-specific polysaccharide of fhigello fl'exneri aerotype 2a1
Abstract
IN~'RODUGTION
Shigellosis or bacillary dysentery is a serious infectious disease,
responsible for some
200 million episodes annually, mostly in children and immunocompramised
individuals living
in areas were sanitary conditions are insuffrcient. 1 Of the four species of
Shigellae, Shigella
flexneri is the major responsible of the endemic form ojthe disease, with
serotype 2a being
the most prevalent. Due to increasing resistance of all groups of Shigellae to
antibiotics, j the
development of a vaccine against shigellosis is of high priority as stated by
the World Health
Organization in its program against enteric diseases. ' However, there are yet
no licensed
vaccitres for shigellosis.
As for other Gram negative bacteria, Shigella's Iipopolysaccharide (LPS) is a
major
surface antigen of the bacterium. The corresponding 0-specific polysaccharide
(O-SPj, a
polymer of less than 3Q lcDa, defines the serogroup and serotype of the
bacteria. Besides, it is
both an essential virulence factor and the target of the infected host's
protective immune
response. s'6 However, O-SPs are T-cell independent antigens, 7v which are not
immunogenic
by themselves. Nevertheless, benefiting from the successful conversion of
bacterial capsular
polysaccharides from T-independent antigens bo T-dependent ones through their
covalent
coupling to a protein carrier, it was shown that 0-SPs could be fumed into
immunogens.
Indeed, based on the former hypothesis that serum IgG anti-LPS antibodies may
confer
LMPPI4th~o-6revd~synlongc
CA 02434685 2003-07-04
specific protection against shigellosis, 9 several polysaccharide-proteine
conjugates, targeting
either Shigella sonnet, Shigella dysenteriae 1 or S flexneri serotype 2a, Were
shown to be safe
and immunogenic in humans. lo.n In the case of S, sonnet, recent field trials
allowed ).B.
Robbins and co-workers to demonstrate the efI'tcacy of a vaccine made of the
corresponding
detoxified LPS covalently linked to recombinant exoprotein A, ~Z Even though
e~cient,
polysaccharide-protein conjugate vaccines remain highly complex structures,
whose
immunogenicity depends of several parameters amongst which, the length and
nature of the
saccharide component as well as its loading on the protein. It is reasonably
admitted that
control of these parameters, and indeed standardization, are somewhat
difficult when dealing
with polysaccharides purified from bacterial cell cultures, or fragments
thereof resulting from
their partial hydrolysis. Mixture are often obtained, which may become a real
drawback in
terms of analysis of the products, particularly when multivalent wccines are
needed, as in the
case of shagellosis. It may be assumed that the use of well-defined synthetic
oiigosaccharides
suitable for single-point attachment on to the carrier would allow a better
control, and
consequently the optimisation, of the above mentioned parameters. That low
molecular
weight oligosaceharides mimicking antigenic determinants were immunogenic when
conjugated onto a protein carrier was demonstrated in the late 30s, l3,la and
since then
exploited;on several occasions. is Indeed, available data on S dysenteriae
type I indicate that
neoglycoconjugates incorporating di-, tri- or tetramers of the 0-SP repeating
unit were more
immunogenic than a detoxified LPS-human serum albumin conjugate of refierence.
16 In the
case of heteropolysaccharides, oligosaccharides made of at least two
contiguous repeating
units were originally considered to be necessary for the corresponding
oligosaccharide-protein
conjugates to induce anti-polysaccharide antibodies. ~~ However, more recent
data
demonstrated that neoglycoproteins incorporating oiigosaccharides comprising
one repeating
unit or smaller fragments were immunogenic in mice. ~B'1' Along this line, we
recently
reported the synthesis of three fully synthetic glycopeptides as potential
vaccines against
Shigella Jlexneri 2a infection. a° These incorporated short
oIigosaccharidc haptens,
representative either of part or of the whole repeating unit of the 0-SP of S.
flexneri serotype
2a. Preliminary data indicate that two out of the three conjugates are
immunogenie in
mice.(Phalipon et al, unpublished results) Besides, we found that the
corresponding
neoglycoproteins consisting of the oligosaccharides covalently linked to
tetanus toxoid via
single-point attachment were also immunogenic in mice.(Phalipon et at,
unpublished results)
Parallel studies on the recognition of synthetic fragments of the 0-SP by
protective
homologous monoclonal antibodies suggested that sequences larger than one
repeating unit
2
LMPPl4theo-bravo-synlongs
CA 02434685 2003-07-04
were more antigenic, thus probably better mimicking the natural polysaccharide
than shorter
ones. 21 Indeed, it is anticipated that better mimics of the O-SP, in terms o~
both antigEnicity
and conformation, would Iead to conjugates of higher immunogenicity. For that
reason, the
preparation of oligo- or polysaccharides22 made of two repeating units or
more, in a form
suitable for conjugation onto a caxtier, was undertaken.
RESULTS AND DISCUSSION
A B E C D
2)-a-L-Rhap-(1-a2)-a-L-Rhap-(1-~3)-[a-D-Gtcp-(1-~4)]-a-L-Rhap-(1 >3)-(i-D-
GIcNAcp(1-3
I
The 0-SP of S flexneri 2a is a branched h.eteropolysaccharide deFmed by the
pentasaecharide
repeating unit I. 23,ara It features a linear tetrasa~ccharide backbone, which
is common to all S.
,flexneri 0-SPs and comprises a N acetyl glucosamine (D) and three rhamnose
residues (A, B,
C). The spec~city of the serotype is associated to the a-D-glucopyranose
residue linked to
position 4 of rharnnose C.
Evaluation of the antigenicity of a panel of di- to pentasaccharides
representative of
frame-shifted fragments of I, had pointed out that the ECD portion was the
minimal sequence
required for binding, and that the B(E)C ramification had a great impact on
the recognition
process. zs Hosed on theses data, we described recently the synthesis of the
ECD, B(E)CD
and AB(E)CD fragments functionalized with an aminoethyl spacer at their
reducing end, and
demonstxated that the later could serve as a suitable anchoring point.
Z° As stated above,
subsequent work outlined the impact of chain elongation on the recognition
process. Taking
both sets of data into account, we report herein on the synthesis of the 2-
ami.noethyl
glycosides of a deco- (1) and a pentadecasaccharide (2), corresponding to
sequences
[AB(E)CD}Z and [AB(E)CD]s, respectively. The corresponding D'AB(E)CD
hexasaccharide
(3) was used as a model
Considering the target 1 and 2, a disconnection at the D-A linkage would
appear most
appropriate. However, others have shown that such a disconnection strategy was
not suitable
even when involving di- or trisaccharide building blocks, z6,Z~ and this route
was avoided.
More recently, disconnections at the A-B, B-C and C-D linkages were evaluated
in this
laboratory when synthesizing successfully the methyl glycoside of the
frame~shifted
decasaccharide D'A'B'(E')C'DAB(E)C by condensing a chain terminator
pentasaccharide
3
LMPPt4theo-brcvn-synlongs
CA 02434685 2003-07-04
donor and a methyl glycoside pentasaccharide acceptor. Z& It was demonstrated
on that
occasion that disconnection at the C-D linkage was indeed appropriate for the
construction of
large fragments of the S. flexneri 2a 0-SP. Based on oar experience in the
field, a blockwise
strategy to targets 1 and Z, implicating a DAB(E)C potential acceptor acting
as a donor, an
AB(E)C tetrasaccharide donor, and the recently disclosed acceptor XX~°
as a precursor to the
spacer-armed D residue (Scheme 1). Although permanent blocking of OH-4D and OH-
Gn with
an isopropylidene acetal may appear somewhat unusual, this choice was a key
feature of the
strategy. It was based on former observations in the methyl glycoside series,
demonstrating
that its use could overcome some of the known drawbacks of the cozresponding
benzylidene
acetal, Z9''° including its poor solubility. Compound XX was readily
obtained from the known
triacetate XX~1 (81%), by transesterification and subsequent treatment with
2,2-
dimethoxypropane.
S~nrhesfs of the hexasaccharide 3 (Scheme 2): In a preliminary study towards
the
target 3, the DAS(E)C building block bearing the required a,cetamido function
at position 2D
was used as the donor. It was obtained 5rom the recently described precursor
XX. ig Indeed,
reductive free-radical deehlorination of XX using Bu3SnH in the presence of
catalytic AIHN
allowed the conversion of the N trichloroacetyl moiety into N acetyl, to give
XX (G8%). The
latter was converted to the hemiacetal XX following a two-step process
including Iridium
complex promoted isomerisation of the aIlyl rr>Qiety into the propen-1-yl, 3Z
and hydrolysis of
the latter upon treatment with aqueous iodine. 33 Subsequent reaction of XX
with
trichloroacetonitrile in the presence of catalytic 1,8-
diazabicyclo[5.4.0)undec-7-ene (DgU)
cleanly gave the trichloroacetimidate donor XX (85% from XX). Previous
glycosidation
attempts in the series indicated that when run at low temperature or room
temperature,
reactions using the D acceptor XX occasionally resulted in a rather poor yield
of the
condensation product. This was tentatively explained by the still rather poor
solubility of the
acceptor XX. When using I,2-dichloroethane (DCE) as the solvent, the
condensation could be
performed at higher temperature, which proved rewarding. Indeed, optimized
coupling
conditions relied on the concomitant use of a catalytic amount of triflic acid
in the presence of
4th molecular sieves as the promoter and DCE as the solvent, while the
condensation was
perfiormed at 80°C. The fully protected hexasaccharide XX was isolated
in a satisfactory 78%
yield. That the hemiacetal XX, resulting from the hydrolysis of the excess
donor could be
recovered was of great advantage is one considers scaling up the process (not
described).
Acidic hydrolysis of the isopropylidene acctal smoothly converted XX into the
corresponding
4
LMPPI4theo~brevei-synlongs
CA 02434685 2003-07-04
dioI XX (94°/a). Resistance of isolated benzoyl groups to Zempldn
transesterification has been
reported, 3a.ys Ii was also observed previously in the series, upon attempted
removal of a
benzoyl group located at position 2~. 2$ Again; the 2~-O-benzoyl group in XX
was
particularly resistant to Zempl6n de-O-acylation, and in that case, successful
transesterification required a week. In that case, heating was avoided in
order to prevent any
potential migration of the aryl group which would lead to the N deacylated
product.
Conversion of the hexaol XX into the target 3 was successfully accomplished
upon
concomitant hydrogenolysis of the remaining benzyl protecting group and
reduction of the
azido moiety into the corresponding amine. As observed earlier, ~°~~
the latter was best
performed under acidic conditions. The target 3 was isolated in 77% yield
after reverse-phase
chromatography.
Synthesis of the decasaccharide f (5clzeme 3): Having the fully protected
hexasaccharide XX in hands, we reasoned that a convenient access to I could
involve the
condensation of an AB(E)C tetrasaccharide donor and a DAB(E)CD hexasaccharide
acceptor
prepared from XX. Preparation of the former was conveniently achieved from the
previously
described tetrasaccharide XX. 2g Removal of the anomeric allyl protecting
group involved a
two-step process as described above for the preparation of XX. The hemiacetal
was readily
converted into the trichtoroaeetimidate donor XX, which was isolated in an
unoptimized yield
of 56% over the two steps. Taking advantage of the stability of the 2~-O-
benzoyl group under
Zempl~n conditions, selective chemical modification at the D residue of XX was
anticipated
to give easy access to the selected acceptor XX. Indeed, transesterification
of the acetyl
groups in XX gave the expected triol XX, which was further regioselectively
protected at the
4D and 6o hydzoxyl groups when treated with 2,2-dimethoxypropane. However, the
key
acceptor XX was isolated in 50% yield only. Condensation of the latter and XX
was
performed in DCE using triflie acid as the promoter. One may note that
although the
condensation involves the construction of the C-D linkage, thus somewhat
resembling the
preparation of the hexasaccharide XX, heating was not required and the
glycosylation went
smoothly at low temperature to give the fully protected decasaccharide XX
(82%). Acidic
hydrolysis of the acetals gave the tetraol XX (?5%). 'I~ansesterif catioa of
the aryl groups was
best performed by overnight heating of XX in methanolic sodium methoxide.
Final
hydrogenolysis of the benzyl groups and concomitant conversion of the azido
group into the
corresponding amine gave the target 1 (71% from XX).
LMPPI4thco-brevet-synlonas
CA 02434685 2003-07-04
Synthesis of the pentadecasacel:aride Z: If the synthesis of 2 was to mimic
that of I,
the transformation of the non reducing 3,4,6-tri-O-acetyl D residue into the
corresponding
4,G-0-isopropylidene one was to be performed twice. Considering that besides
being rather
low, the yield of the transformation of XX into XX was also poorly
reproducible,
considerable loss of two costly intermediate, namely first the hexasaccharide
XX, then the
undeeasaccharide XX, was to be expected. The use of a pre-funetionalized
DAB(E)C
building block, that could act both as a donor and an acceptor based on
appropriate orthogonal
protection, was considered as an amactive alternative. Such an intermediate
(XX) was
recently prepared in the laboratory by condensation of an AB(E)C
tetrasaccharide acceptor
~(XX) 3a to a fully functionalized D thioglycoside donor (XX), and subsequent
free-radical
conversion of the N trichloroacetyl into the corresponding aeetamide (Scheme
4). 3a Since the
condensation of XX and XX was somewhat low-yielding, another route to XX is
disclosed
herein. Intakes advantage of the high-yielding condensation of the
tetrasaccharide acceptor
XX with the known trichloroacetimidate donor XX, 39 giving access to the fully
protected XX
(98%), za and subsequently to th:e corresponding acetamido derivative XX as
described above.
Controlled de-0-acetylation of XX under zemplGn conditions gave the triol XX,
which was
next converted to the corresponding alcohol XX upon reaction with 2,2-
dimethoxypropane
($1% from XX). Conventional acetylation at position 3D then gave the key
intermediate XX
(94%). Transformation of the latter into the trichloroacetimidate donor XX
(82%) was
performed as described for the preparation of XX via the hemiacetal
intermediate XX.
The rather satisfactory yields obtained all along the synthesis of the
building block XX
allowed the targeting of larger sequences. Indeed, when the newly formed
pentasaccharide
donor XX and the spacer-armed D acceptor XX were heated in DCE in the presence
of triflic
acid and 4A molecular sieves as described for the preparation of XX, the
condensation
product was isolated in 78%. The resistance of the two isapropylidene acetals
to the harsh
acidic conditions of the glycosidation reaction is noteworthy. Selective
deacetylation at the 3-
OH of the non reducing residue, then gave the D'AB(E}CD acceptor XX in a yield
of 76%,
confirming indeed than this route to XX was more appropriate than that
described above. This
two-step glycosidationldeacetylation process was repeated. However, whereas
the above
mentioned glycosidatinns required heating, condensation of the hexasaecharide
acceptor XX
and the pentasaccharide donor XX in the presence of triflic acid was run at
Iow temperature.
Under such conditions, the fully protected undecasaceharide XX was isolated In
an excellent
yield of 90%. Zempl~n transesterification at the non reducing 3p-OH of the
latter proved as
efficient, and gave the required acceptor XX (91%j. Condensation of this key
intermediate
6
LMPPI4theo-brevet-9yalonge
CA 02434685 2003-07-04
with the tetrasaccharide trichloroacetimidate donor XX was again perfornled at
low
temperature, using triflic acid as the promoter. The fully protected
pentadecasaecharide XX
was isolated in a satisfactory yield of 82%. Conversion of XX to the target 2
was performed
according to the stepwise sequence described for the preparation of 3. Acidic
hydrolysis of the
isopropylidene groups afforded the hexaol XX (83%). Again, running the
transesteriftcation
step at high temperature allowed to overcome the resistance of benzoyl groups
to Zemplen
conditions. Conventional hydrogenolysis of the intermediate XX, finally gave
the
pentadecasaccharide hapten 3 (65% from XX).
CONCLUSION
The synthesis of the 0-SP of S. flex»eri Y by way of polycondensation of a
tritylated
cyanoethylidene tetrasaccharide was reported by others. 4° However,
this is to our knowledge
the first report on the fatal synthesis of fully defined oligomeric repeating
unit glycosides
mimicking the branched bacterial O-SPs in the S, flexr:eri series. The
strategy disclosed herein
gives access to extended fragments of the O-SP of S. flexneri serotype 2a in a
spacer-armed
form suitable for immunological studies. Indeed, amounts required for the
synthesis of fully
synthetic oligosaccharide conjugates as potential vaccines targeting S
flexneri 2a infection
were made available. The preparation of such conjugates is in progress in the
laboratory.
ACKNOWLEDGEMENTS
The authors are grateful to J. Ughetto-Monfrin (Unitd de Chimie Organique,
Institut Pasteur)
for recording all the N1IR spectra. The authors thank the Bourses Roux
Foundation for the
postdoctoral fellowship awarded to F. B., and the Institut Pasteur for its
financial support
(grant no. PTR 99).
REFERENCES
(1) Aimi Part 14 of the sesies Synthesis of ligands related to the O-specific
polysaccharides ojShigella flexrteri serocype 2a and Shigella~lexneri serotype
Sa. For part
13, see ref. X.tr 2003.
(2) Kotloff, K. L.; Winickoff, J. P.; Ivanoff, B.; Clemens, J. D.; Swerdlow,
D. L.;
Sansonetti, P. J.; Adak, G. K, Levine, M. M. Bull. y~HO 1999, 77, 651-666.
7
CA 02434685 2003-07-04
LMPPL4theo~brcvet~synlongs
(3) Ashkenazi, S.; May-Zahav, M.; Sulkes, J.; Samna, Z. Antimicrob. Agents
Chemother. 1995, 39, 8 i 9-823.
(4) World; Health; Organisation WHO Weekly Epidemiol. Rec. 199?, 72, 73-80.
(5) Cohen, D.; Green, M. S.; Block, C.; Roua.ch, T.; Ofek, I. J. Inject. Dis.
1988,
157, 1068-1071.
(G) Cohen, D.; Green, M. S.; Block, C.; Slepon, R.; Ofek, I. J. Clin.
Microbiol.
1991, 29, 38G-389.
(7) Handing, C. V.; Kihlberg, J.; Elofsson, M.; Magnusson, G.; Unanue, E. R.
J.
Immunol: 1993, I51, 2419.
(8) Ishioka, G. Y.; Lamont, A. G.; Thomson, D.; Bulbow, N.; Gaeta, F. C. A.;
Sette, A.; Grey, H. M. J. Imrntrnol. 1992, 14~, 2446.
(9) Robbins, J. B.; Chu, C.; Schneerson, R. Clin. Infect. Dis. 1992,15, 346-
3GI.
(10) Taylor, D. N.; Trofa, A. C.; Sadoff, J.; Chu, C.; Bryla, D., Shiloach,
J.; Cohen,
D.; Ashkenazi, S.; Lerman, Y.; Egan, W,; Schneerson, R.; Robbins, J. B.
Infect. Immun. 1993,
dl, 3678-3687,
(11) Passweil. J. H.; Harlev, E.; Ashketuuizzi, S.; Chu, C.; Miron, D.; Ramon,
R.;
Far~an, N.; Shiloach, J.; Bryla, D. A.; hfajadly, F.; Roberson, R.; Robbins,
J. B.; Schneerson,
R. Inject. Immun. 2001, 69, 1351-1357.
(12) Cohen, D.; Ashkenazi, S.; Green, M. S.; Gdalevich, M.; Robin, G.; Slepon,
R.;
Yavzori, M.; Orr, N.; Block, C.; Ashkenazi, L; Shemer, J.; Taylor, D. N.;
Hate, T. L.; Sadoff,
J. C.; Pavliovka, D.; Schneerson, R; Robbins, J. B. The Lancet 1997, 349, 155-
159.
(13) Goebel, W. F. J. Exp. Med 1940, 72, 33.
(14) Goebel, W. F. J. Exp. Med 1939, 69, 353.
(15) Pozsgay, V. In Adv. Carbohydr. Chem. Biochcm.; Horton, D., Ed.; Academic
Press: San Diego, 2000; Vol. 56, pp 153-199.
(1G) Pozsgay, V.; Chu, C.; Panell, L.; Wolfe, J.; Robbins, J. B.; Schneerson,
R
Proc. Natl. Aced Sci. USA 1999, 96, S I 94-5197.
(17) Peeters, C. C. A. M.; Lagerman, P. R.: Weers, O. d.; Ooemn, L. A.;
Hoogerhout, P.; Beurret, M.; Poolmat~, J. T. In Vaccine Protocols; Robinson,
A.; Fatter, G.,
Wiblin, C., Eds.; Humarta Press Inc.: Totowa N, J., 1996, pp 1 I 1-133.
(18) Benaissa-Trouw, B.; Lefeber, D. J.; Kamerling, J. P.; Vliegenthart, J. F.
G.;
Kraaijeveld, K; Snippe, H. Inject. Immun. 2001, 69, 4698-4701.
(I9) Mawas, F.; Niggemanr~, J.; Jones, C.; Corbel, M. 3.; Kamerling; J. P.;
Vliegenthart, J. F. G. Infect. Immun. 2002, 70, SI07-5114.
(20) Wright, K. 2043, submitted.
(21) Mulard, L. A.; Nato, F.; Marcel, V.; Thui2.at, A.; Sansonetti, P.;
Phalipon, A. in
preparation 2003.
(22) oligosac Eur. J. Biochem. 1982,126, 433.
(23) Simmons, D. A. R. Bacteriol. Reviews 1971, 35, 117-148.
(24) Lmdberg, A. A.; Karnell, A.; Weintraub, A. Rev. Inject, Dis. 1991, 13,
5279-
5284.
(25) Mulard; L.; Guerreiro, C.; Costaehel, C.; Phalipon, A. in preparation
2003.
(2G) Pinto, B. M.; Reitner, K. B.; Mozissette, D. G,; Bundle, D. R- J. Org.
Chem.
1989, 54, 2650-2656.
(27) Pinto, B. M.; Reimer, K. B.; Morissette, D. G.; Bundle. D. R. J. Chem.
Soc.
Perkin Trans. I 1990, 293-299.
(28) Blot, F.; Wright, K.; Costachel, C.; Phalipon, A.; Mulard, L. A. J. Org.
Chem.
2003, sumitted.
(29) Bundle, D. R.; Josephson, S. Can. J. Chem, 1979, 57, 6&2-6G&.
L;~iPPl4.e~cQ6revet.syatongg ~1 02434685 2003-07-04
CH3C=O), 1.65 (s, 3H, GH3C=ONH), 1.32 (d, 3H, Js,s = 6.1 Hz, H-6,J, 1.30 (d,
3H, Js,6 = 6.0
Hz, H-6c), 0.97 (d, 3H, Js,6 = 6.0 Hz, H-6B). "C NMR (CDC13):8 171.1, 170.8,
170.2, 169.6,
166.2 (SC, C=0), 138.2-118.5 (Ph, All), 103.1 (C-1D), 101.4 (C-1H), 101.2 (C-
1,~), 98.5 (C-
IE), 96.4 (C-1~), 82.2 (C-3E), 81.7 (C-2E), 81.7 (C-4A), 80.4 (C-4B), 80.2 (C-
3c), 79.0 (C-3"),
78.6 (C-3$), 78.1 (C-2A), 77.8 (C-4c), 77.G (C-4E), 76.0, 75.8, 75.4, 74.7,
74.3, 74.2, 73.3,
70.5 (8C, CHaJ?h), 74.9 (C-2H), 72.7 (C-2~), 72.G (C-3D), 71.9 (2C, C-SE, SD),
69.1 (C-5H),
68.9 (2C, All, C-SA), 68.3 (C-6E), 67.8 (C-Sc), 62.3 (C-6p), 54.6 (C-2Dj, 23.5
(1C,
NHC=OCH3), 21.1, 21Ø 20.8 (3C, C=OCH~), 19.0 (C-Gc), 18.4 (C-6,,), 18.2 (C-
6B).
FABMS of GtoaHImNOz, (M, 1913.1), m/z 1936.2 [M+Na]*. Anal. Calcd. for
ClodH1"NO2~
C, 68.90 ; H, 6.50 ; N, 0.77. Found C, 68.64 ; H, 6.66 ; N, I .05.
2
LMPPI4.exp~btevct~s~'nlongs
CA 02434685 2003-07-04
C~";H~ ~~Chi~tOs.
E~cntt Me.s~ 1914,66
Mol. Wt. 1917,35
H 5,94, Ci 5,55; N l.dG; O 77,13
we; C:61.33%. H.G.IOSi, ; i: I a5?.
AIphaD-r10°, c=1, CHCI3
(2-aeetamido-3,4,6-tri-0-acetyl-2-deoxy-(3-D-glueopyranosyl)-(Z-~2)-(3,4-di-O-
benzyl-a-
L-rham>nopyraaosy!)-(1->2)-(3,4-di-O-benryl-a-L-rhamnopyranosyl)-(1-~3)-
[2,3,4,6-
tetra-0-benzyl-a-D-glucopyranosyl-(1-~4))-2-0-beazoyt-a-L-rhamnopyraaosyl
trichloroacetimidate (X).
1,5-Cyclnoctadiene-bis(methyldiphenylphosphine)iridium hexafluorophosphate (25
mg, 29 p.
mol) was dissolved tetrahydrofuran (5 mL), and the resulting red solution was
degassed in an
argon stream. Hydrogen was then bubbled through the solution, causing the
colour to change
to yellow. The solution was then degassed again in an argon stzeam. A solution
of 7 (I.0 g,
0.55 mmol) in tetrahydrofuran (10 mL) was degassed and added, The mixture was
stirred at rt
overnight, then concentrated to dryness. Ths residue was dissolved in acetone
(5 mL), then
water (1 mL), mercuric chloride (140 mg) and mercuric oxide (120 mg) were
added
successively. The mixture protected from Iight was stirred at rt for 2 h and
acetone was
evaporated. The resulting suspension was taken up in DCM, washed twice with
50% aq KI,
water and satd aq NaCI, dried and concentrated. The residue was eluted from a
column of
silica gel with 2:1 petroleum ether-EtOAc to give the corresponding
hemiacetal.
Trichloroacetonitrile (2.5 mL) and DBU (37 uL) wcre added to a solution of the
residue in
3
L1~P14txp-brevet-synlon6s
CA 02434685 2003-07-04
anhydrous dichlorom~ethane (12.5 mL) at 0°C. ARer 1 h, the mixture was
concentrated. The
residue was eluted from a column of silica gel with S:4 cyclohexane-EtOAc and
0.2 °!o Et3N to
give X as a white foam (0.9 g, 8S °!°); [a]D +10° (c 1,
CHC13).
'H NMR (CDCh):8 8.70 (s, IH, C=NH), 8.00-7.00 (m, 4SH, Ph), 6.36 (d, IH, J,,~
= 2.6 Hz,
H-lc), 5.59 (m 2H, N-HD, H~2~), 5.13 (d, IH, J,,Z = 1.0 Hz, H-I,,), S.O1-4.98
(m, ZH, H-le,
IH), 4.92 (dd, 1H, H-3p), 4.90 (dd, 1H, H-4p), 4.G8 (d, 1H, H-1D), 5.00-4.02
(m, 19H, 8
CHZPh, H-3c, 2,,, 2$), 4.01 (dd, 1H, H-2E), 4.00-3.20 (m, 16H, H~3E, 4E, SF,
GaE, Gbe., 4~, 5c,
38, 4s, SB, 3,,, 4~, 5~, SD, ban, 6bD), 2.02, 2.00, 1.75, 1.65 (4s, 12H,
C=OCH3), 1.40, 1.32 and
1.00 (3d, 9H, H-G~, 6s, Gc). "C NMR (partial) (CDCIz):8 170.2, 1b9.9, 169.3,
168.7, 164.9
(GC, C=O, C=N), 103.2 (C-1D), 101.4 (2C, C-lA, 1H), 99.0 (C-lE), 94.8 (C-lc),
21.1, 20.9,
20.8 (3C, CH3C=0), I9.1, 18.2 (3C, C-GA, GB, G~). FABMS of Clo3H»3C13Ni02, (M,
1917.4),
mla 1930.9 [M+Na]''. Anal. Calcd. for C,o3H~ nClzNiOa~ : C. 64.52 ; H, 5.94 ;
N, 1.46. Found
C,64.47;H,5.99;N, 1.45.
4
LMPP i Atopbrevet-synlongs
~OBn ~o-~
0
9n0~ O
An0 B~
~
~~
0
0 OAz
~.,~ CuaHmN~~3z
e~
ei,o p Exact Mose:
2AR3,R9
T~.~ Mol. W,.'
295,29
en0~ C 6 5,66; K 6;43,
:~: 3,3b;
O 14,55
tr°uot: C:G5.37, H 6.51, N 3.18
AIphaD='G 3°, C=1, CHC13
CA 02434685 2003-07-04
2-A~eidoet6yl (2-acetamido-3,4,6-trl-O-acetyl-2-dcoxy-(i-n-glucopyranosyl)-
(1~2)-(3,4-
di-0-benzyl-a-L-rhamnopyranosy 1)-(1~2)-(3,4-dl-O-beazyl-a-irrhamnopyranosyt)-
(1--~3)-[2,3,4,6-tetra-O-beaxyl-a-v-glucopyranosyl-(1-~4)j-(2-0-ben~.oyl-a-1.-
rhamaopyranosyl)-(1->3)-2-acetamido-2-deoxy-4,6-D-isopropylidene-[i-n-
glucopyranosidc (X).
A mixture of alcohol X (110 mg, 330 wnwl), imidate X (720 mg, 376 ~mol) and
4tl molecular
sieves in anhydrous DCE (6 mL) was stirred for 1 h under dry Ar. After cooling
at 0°C, Tfl~H
(16 ~L; 180 ~mol) was added dropwise and the mixture was stirred at
80°C for 2.5 h.
Triett>sylamirte (60 ~L} was added and the mixture was filtered and
concentrated. The residue
was eluted from a column of silica gel with 3:4 cyclohexane-EtOAc and Et3N
(0.2 %) to give
X as a colorless oil (540 mg, 78 %); [a]D +6.5° (c 1, CHCl3).
'H Ni~iR (CDCI3):8 8.00-7.00 (m, 45H, Ph), 5.95 (d, 1H, J2,~ = 7.1 Hz, NHDj,
S.SI (d, 1H,
J,.~., = 8.1 Hz, NHS), 5.20 (dd, 1H, Jl,~ = 1.7 Hz, J~,, = 3.0 Hz, H-2c), 5.08
(d, 1H, J1,2 = 1.0
H2, H-IA), 5.05 (d, 1H, J1,2 = 8.3 Hz, H-1D), 4.93 (d, 1H, JI,2 = 3.1 Hz, H-
lE), 4.87 (d, 1H,
J,,2 = 1.0 Hz, H-lg}, 4.82 (d, 1H, JI,2 = 1,7 Hz, H-lc), 4.80 (dd, 1H, J3,~
=J4,s = I0.0 Hz. H-
4~), 4.76 (dd, 1H, Ja,~ = 9.5 Hz, H-3o), 4.75-4.30 (m, 16H, CH2Ph), 4.57 (d,
1H, J,,~ -- 7.8
LMPPl4~xx~brevet-iynlongs
CA 02434685 2003-07-04
Hz, H-la~), 4.35 (dd, 1H, H-2a), 4.30 {dd, 1H, JZ,3 = 10.0 Hz, J3,a = 9.6 Hz;
H-3D), 4.02 (dd,
1H, JZ,~ = 2.0 Hz, H-2"), 4.00-3.60 (m, IGH, H-6ao, GbD, 3E, 4~, SE, 6aE, 61~,
3c, 4~, 5c, 3B,
3", 5~, 2a~, 6ao~, 6bo~), 3.48 (m, 1 H, J~,S = 9.5 Hz, H-SB), 3.46 (dd, 1 H. H-
4p), 3.40 (m, 1H, H-
5D), 3.36 (dd, 1H, H-2E), 3.35, 3.19 (m, 4H, OCH~CHzNz), 3.30 (dd, IH, H-4A),
3.I9 (dd, 1H,
J3,4 = 9.5 Hz, H-4B), 3.17 (m, IH, H-SD), 3.02 (m, IH, H-2D), 1.90-1.60 (6s,
18H, CH,C=0),
1.33, 1.26 {2s, GH, C(CH3)2), 1.27 (d, 1H, J5,6 = 6.2 Hz, H-G,,), 1.18 (d; 3H,
J5,6 = 6.1 Hz, H-
6c), 0.90 (d, 3H, Js,s = 6.lHz, H-6H). '3C NMR (CDCh):S 172.1, 171.1, 170.8,
170.1, 169.6,
166.2 (6C, C=0), 139.2-127.1 (Ph), 103.05 (C-1D.), 101.6 (C-1H), 101.0 (C-lA),
140.0 {C-1D),
98.1 (C-lE), 97.8 (C-1~), 82.0 (C-2E), 81.7, 81.5, 80.2, 78.6, 78.4. 77.9,
77.9 ($C, C-3s, 4E,
3c, 4c, 3s, 4~, 3n, 4,~), 77.8 (C-Z,,), 76.0, 74.6 (2C, C-3a, 3a~), 74.0 (C-
Ze), 73.4 (C-4a), 73.3
(C-2c), 72.2, 71.9 (2C, C-So, So~), 68.9, 68.8, 67.7 (3C, C-SA, 5B, SE), 68.6
(C-4a~), 68.5 (C-
6E), 67.5 (C-5~), 62.6, 62.2 (2C, C-6o, 6n~), 59.7 (C~2p), 54.6 (C-2D~), 51.0
(CH~N~), 29.5
(C(CH3)2), 23.9, 23.5, 21.1, 20.9, 20.7 (C=OCH3), 19.6 (C(CH3)z), 18.9 (C-G~),
18.4 (C-6n),
18.2 (C-6B). FARMS of C"~Hi3~N~03~ (M, 2085.3), m/z 2107.9 [M+Na]'
6
L~l4Qp-~1C112~8yhl0flg9 C~1 02434685 2003-07-04
N,
~mNI:nNsou
E :nu Mass: 2oaJ,eG
Mol. Wr.:20aS,2J
C 65,19: H 6,J6; N 3 42, 0 25,03
AlphaD=t9', c= I, CHCI3
2-Azidoethyl (2,3,4-tri-D-acetyl-2-deoxy-2-acetamido-~i-D-glucopyranosyl)-(1--
~2)-(3,4-
di-O-benryi-a-L-rhamnopyraaosyl)-(1-~2)-(3,4-di.0-benzyl-a-L-rhamnopyranosyl)-
(1-~3)-(2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-(1-~4)]-(2-O-benzoyl-a-L-
rhamnopyranosyl)-(1-~3)-2-acetamido-2-deoxy-[i-D-glucopyrnnoside (X).
To a solution of X (503 mg, 241 ~.mDl) in AcOH (6 mL) was added dropwise,
water (1.5 mL)
at rt. The mixture was stirred for 1 h at 60°C then concentrated by
successive coevaporation
with water and toluene. The residue was eluted from a column of silica gel
with 1:4
Cyclohexane-EtOAe to give X as a white foam (463 mg, 94 %); [aJD +9° (c
l, CHCh).
1H NMR (CDCI3):cS 8.00-7.00 (m 45H, Ph), 5.70 (d, 1H, NHD), 5.46 (d, 1H,
Jz,r~., = 8.0 Hz,
NFiD~), 5.25 (dd, 1H, H-2~), 5.05 (d, 1H, J ,,2 = 8.4 Hz, H-1D), S.Oa (d, 1H,
Jl,z = 1.0 Hz, H-
l,,), 4.86 (m, 3H, H-lc, 3n', 40~), 4.84 (m, 2H, H-la, lE), 4.56 (d, 1H, H-
ln~)~ 4.40 (dd, 1H, H-
3E), 4.35 (dd, 1H, H-2$), 4.15 (dd, 1H, H-3D), 4.80-4.00 (m, 1GH, C.H2Ph),
4.03 (dd, IH, H-
2a), 4.00-3.00 (m, 26H, H-4D, SD, 6aD, 6bn, 2E, 4E, Se~ 6a6, 6be, 3c. 4c, Sc,
3a, 4a, SH~ 3a~ 4n~
SA, 2D~, SD., 6aD~, 6tro~, OCH2CH2NJ), 2,99 (m, IH, H-2p), 1.85-1.60 (Ss, 1SH,
CHJC=O), 1.25
and 0.85 (3d, 9H, H-6A, 6H, 6c). 13C NMR (partial) (CDC1J):S 171.6, 171.4,
170.8, 170.1,
169.6 (C=0), 140,0-I27.I (Ph), 103.I (C-1~), 101.2 (C-l,,), 99.G (2C, C-IE,
1B), 99.4 (C-1D),
99.0 (C-1~), 23.8, 23.5 (2C, NHC=OCH3), 21.1, 20.9, 20.8 (3 CHJC=0), 19.1.
18.5, 18.2 (C-
7
LMPPI4~t~6t~~atttynlongs
CA 02434685 2003-07-04
6~, 6s, 6~). FABMS of C~ nHiz9Ns03a (M, 2045.2 j, m/z 2067.9 [M+Na]''. Anal.
Calcd for
C",H,i9N503z C: 65.19, H: 6.36, N: 3.42. Found C: 65.12, H: 6.51. N: 3.41.
L1~P14.ex~brNCt~synlcng~
CA 02434685 2003-07-04
Ho---1
OH H00
o~
~
HNO ~~ NN
HO NH7
~
~,~, ,,
0.
~ tow
C ,H N O
Ho Ho
Exact Mass 1067
4381
Mol. Wt.: Io68,U324
~
Ho
H C 47,23: H 6.89;
N 3,93; O 41,94
H0
~
HO
0
11
'NLH
~O
2-Aminoethyl (2-deoxy-2-acetamido-ji-D-glucopyranosyI)-(1-~Z)-(a-L-
rhamnopyranosyl)-(1~2)-( a-L-rhamnopyranosyt)-(I-~3)-[a-D-glucopyrauosyl-(1-
~4)j-
( a-L-rhamnopyranosyt)-(1--r3)-2-acetamido-2-deoxy-[i-D-glueopyranoaide (X).
A mixture of X (207 mg, 101 utnol) in MeOH (5 mL) was treated by MeONa until
pH=9. The
mixture was stirred 1 week at rt. IR 120 (Fib was added until neutral pH and
the solution was
filtered and concentrated. The residue was eluted from a column of silica gel
with 20:1 to 15:1
DCM-MeOH to give an am~oiphous residue. A solution of this residue in EtOH
(2.2 mL),
EtOAc (220 ~,L), IM HC1 (I72 il.L, 2 eq) was hydrogenated in the presence of
PdIC (180 mg)
for 72 h at rt. The trlikrture was filtered and concentrated , then was eluted
from a column of C-
18 with wated and freeze-dried to afford amorphous X (81 mg. 77 %); [a]D -
10° (c 1, Hz0).
'H NMR partial (D20):8 5.12 (d, 1H, Jt,? = 3.4 Hz; H-ls), 5.07 (d, 1H, J~,2 =
1.0 Hz, H-1R~),
4.94 (d, 1H, J,,Z = 1.0 Hz, H-1Rh"), 4.75 (d, 1H, J,,Z = 1.0 Hz, H-1R~), 4.63
(d, 1H, JI,Z = 8.35
Hz, H-lGlcNx), 4.54 (d, 1H, J,,i = 8.3 Hz, H-Ic,°,,u), 1.98 and I.96
(2s, 6H, 2 C'H3C=ONH),
1.28-1.20 (m, 9H, H-6,,, 68, 6~). "C NMR partial (Dz0):8 175.2, 174.8 (C=O),
103.1 (C-lp~),
101.6, 101.4 (3C, C-lA, 18, 1~) 100.8 (C-ID), 97.9 (C-lE), 56.2, 55.4 (2C, C-
2p, 2a), 22.7,
22.6 (2 NHC=OCH3), 18.2, 17.2, 17.0 (3C, C-6", dB, 6~). HRMS: calculated for
C4aH~3N30ie+Na: 1090.4278. Found 1090.428&.
9
CA 02434685 2003-07-04
LtvtPP 14-exp-brevet ;rynlongs
8z
~~nIHtISN~:a
Exact Masb. 1725.7845
Mol, l~'t.~ 1726,9861
70,24; H 6.71; N 0,81; 0 22,23
Aityl (2-acetamido-4,6-0-isopropylidene-Z-deoxy-~-n-glucopyranoayl)-(I~2)-(3,4-
di-0-
benzyl-a-L-rhamnopyranosyl)-(1~2)-(3,d-di-0-benzyl-a-L-rhamnopyranosyl)-(1~3)-
[2~,4,6-tetra-O-benzyl-a-la-glucopyranosyl-(1--~4)-]~2-0-benzoyl-a~L-
rhamnopyranoaide
(X).
The pentasaccharide X (2.65 g, 1.47 mmol) was dissolved in MeOH (20 mL). MeONa
was
added until pH=10. The mixture was stinted for 25 min, then treated by IR 120
(H'') until
neutral pH. The solution was filtered and concentrated. The residue was eluted
from a column
of silica gei with 9 :1 DCM-MeOH to give the expected triol which was then
treated by 2,2-
dimethoxypropane (11 mL,, 0.1 mot) and APTS (20 mg, 0.17 mmolj in DMF (20 mL)
overnight. Et3N was added and the solution evaporated. The residue was eluted
from a column
of silica gel with 1:1 Cyclohexane-AcOEt and 0.2 % of Et3N to give X as a
white foam (2.05
g, 81 % from X); [a)D +3° (c 1, CHCl3).
NMR (CDC~) :'H b 6.98-8.00 (m 45H, Ph), 6.17 (bs, 1H, NHD), 5.82 (m, 1H, All),
5.30 (dd,
1H, J1,2 = 1,0, J~,3 = 3.0 Hx, H-2~), 5.11-5.25 (m, 2H, All), 5.06 {bs, 1H, H-
I"), 4.92 (d, 1H,
J1,~ = 3.I Hz, H-lE), 4.88 (d, 1H, J1.2 = 1.6 Hz, H-IB), 4.84 (bs, 1H, H-lo),
4.35 (d, 1H, H-
1D), 4.34 (dd, 1H, H-2H), 4.20-4.80 (m, 16H, CHZPh), 4.05 (dd, 1H, H-2,,~,
3.36 (dd, 1H, H-
2e)t 2.90-4.10 (m, 22H, All, H-2D, 3n, 3>j, 3c. 30~ 3E~ 4n~ 4s~ 4c~ 4v. 4E,
Sn. Sa, Sc~ SD~ Sa, 6aD,
LMPPl4~ex~brrvct-sy~lengs
CA 02434685 2003-07-04
6bp, GaE, 6b~, 1.5 (s, 3H, FIcNH), 1.2-0.9 (m, 15H, C(CH3)2, H-6A, 6H, 6c). "C
8 ; 172.7
(C=0), 164.9 (C--0), 137.7-116.? (Ph, All), 102.3 (C-1D), 100.2 (C-1g), 100.0
(C-I,,), 98.9
(G(CH,)z), 97.2 (C-lE), 95.1 (C-1~), 82.1, 82.0, 81.8, 81.6, 80.6, 80.3, 79.0,
78.8, 78.3, 77.8,
77.6, 75.7, 75.6, 75.0, 74.3, 72.8, 71.8, 71.6, 70.8, 70.3, 69.0, 68.5, 67.8,
67.4, 61.9, 60.8,
60.5, 29.4 (C(GH,)2), 22.7 (AcNEi), 19.0 (C(C'H3)z), 18.9, 18.4, 18.2 (3C, C-
6,,, 68, Gc). FAB-
MS for C,orHrtsNOZa (M = 1726.9) »r/a 1749.7 [M + Naj'. Anal. Calcd. fnr
CrotHt rsNOza.H?O : C, 69.52 ; H, 6.76 ; N, 0.80. Found C, 69.59; H 6.71 ; N,
0.57.
11
LMPP la.cspbrevet-eynlanga
CA 02434685 2003-07-04
i
O°n
G 0
°8 0 enc 1 ~,..,,~
0
0~ IOBz
.~ 0~
BnO
Ctc:Hi i~NO~s
Exact Mss: 1767,7915
Mol. Wt.'. 1759,0225
C 69,93: H 6,67; N 4.79; O 22,61
Attyt (2-acetamido-3-O-acetyl-4,6-O-isopropylidene-2-deoxy-[i-D-
glucopyraaosyl)-(1-32)-
(3,4-di-O-benzyl-a-L-rhamnopyrunosyl)-(1->2)-(3,4-dl-O-benzyl-a-L-
rhamnopyranosyl)-
(l.-33)-[2,3,4,6-tetra-O-benzyl-a-D-gluoopyranosyl-(1-~4)-)-2-O-benzoy 1-a-t.-
rhamno.pyranoside (X).
a) A mixture of X (2.05 g, 1.19 mmol) in Pyridine (GO mL) was cooled to
0°C. AcaO (20 mL)
was added and the solution was stirred 2,5 h. The solution was concentrated
and coevaporated
with toluene. The residue was elutEd from a column of silica geI with 2:1
Cyclohexane-AcOEt
and 0,2 % of Et3\T to give X as a white foam (1.99 g, 94 %); [a]D +I°
(c I, CHCIJ).
b) A mixture of X (144 mg, 0.06 mmol), Bu3SnH (0.1 mL, 0.37 rnmol) and AIBN
(10 mg) in
dry toluene (3 mL) was stirred for 1 h at rt under a stream of dry Ar, then
was heated for I.5
h at 90°C, cooled and concentrated. The residue was eluted from a
column of silica gel with
2:1 cyclohexane-BtOAc and 0.2 % of Et3N to give X (100 mg, 74 %).
NMR (CDC13) : IH 5 6.95-8.00 (m, 4~H, Ph), 5.82 (m, 1H, All), 5.46 (d, 1H,
Jz,NH = 8.0 Hz,
NHD), 5,29 (dd, 1H, Jt,2 =' 1.0, Jz ~ = 3.0 Hz, H-2c), 5.11-5.25 (m, 2H, All),
5.00 (bs, 1H, H-
lA), 4.90 (d, 1H, J,,2 = 3.1 Hz, H-lE), 4.85 (d, 1H, Jl,z = 1.6 Hz, H-18),
4.83 (bs, 1H, H-lc),
4.70 (dd, 1 H, Jz.3 = .13,4 = 10.0 Hz, H-3 p), 4,44 (d, 1 H, H-1 a), 4. 34
(dd, 1 H, H-2B), 4.20-4. SO
(m, 16H, CHzPh), 4.02 (dd, 1H, H-2A), 3.37 (dd, 1H, H-2s), 2.90-4.10 (m, 21H,
All, H-2D, 3A,
3a, 3c, 3E, 4A, 4H, 4c, 4a, 4E, 5.,, 58, Sc, SD, 5fi, 6aD, 6ba, GaE, GbE),
1.92 (s, 3H, OAe), 1,57 (s,
12
LMPP l4~exp.breve4synlongc
CA 02434685 2003-07-04
3H, AcNH), 1.27-0.90 (m, 15H, C(CH3)Z, H-6A, 6g, Gc). '3C o 171.3, 170.3,
1GG.2 (C=0),
138.7-117.9 (Ph, All), 103.9 (C-1D), 101.5 (C-1B), 101.4 (C-1~), 99.9
(C(CH3)~), 98.5 (C-IE),
96.3 (C-lc), 82.1, 81.7, 81.6, 80.3, 80.1, 78.8, 78.1, 77.8, 76.0, 75.8, 75.3,
75.1, 74.7, 74.2,
73.6, 73.3, 72.7, 71.9, 71.4, 70.8, 69.0, 68.8, 68.7, 58.4, 68.1, 67.8, 62.1,
55.0 (C-2o), 30.0
(C(CH3)z), 23.5 (AcNH), 21.6 (OAc), 19,2 (C(CH3)a), 19.0, 18.3, 18.2 (3C, C-
dA, 6H, 6~).
FAB-MS for C,olH"~NO25 (M = 1769.0) mlz 1791.9 [M + Na]+. Anal. Calcd. for
C,o3H,l,NO~s : C, 69.93 ; H, 6.67 ; N, 0.79. Found C, 69.77; H, 6.84; N, 0.72.
13
CA 02434685 2003-07-04
LMPPIS-cxp~6revet.rynlengs
NH
~OBn ~
--~-~ p~ 0'r \CCia
BBnp
Bn0
o
~_~J ClntxtuCl~NaOts
~J~/ Exsct Mass: 1870,6698
Mol. Wt 187?.3~:2
~ C 65,A0; H 6,08; CI 5,68; N L50; 0 11,35
(Z-acetamido-3-O-acetyl-4,6-O-itoprapylideec-Z-deozy-(3-D-glucopyranosyl)-(1--
>2)-(3,4-
di-O-benzyl-a-L-rhamnopyranosyl)-(1~~2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-
(1--~
3)-jZ,3,4,6-tetra-0-benzyl-a-D-glucopyranosyi-(1--~4)-]-2-O-benzoyl-ac-L-
rhamnopyranosyl ttichlaroacetimidate (X).
1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridium hexafluorophosphate (50
mg, 58 a
mol) was dissolved tetrahydrofuran (10 mL), and the resulting red solution was
degassed in an
argon stream Hydrogen was then bubbled through the solution, causing the
colour to change
to yellow. The solution was then degassed again in an argon stream, A solution
of X (1.8 g,
1.02 mmol) in tetrahydrofuran (ZO mL) was degassed and added. The mixture was
stirred at rt
overnight then concentrated to dryness. The residue was dissolved in acetone
(9 mL), then
water (2 mL), mercuric chloride (236 mg) and mercuric oxide (200 mg) were
added
successively. The mixture protected from light was stirred at rt for 2 h and
acetone was
evaporated. The resulting suspension was taken up in DCM, washed twice with
50% aq K!,
water and Satd aq NaCI, dried and concentrated. The residue was eluted from a
column of
silica gel with 3:2 Cyclohexane-AcOEt and 0.2 % Et3N to give the corresponding
hemiacetal.
Trichloroacetonitrile (2.4 mL) and DBU (72 uL) were added to a solution of the
residue in
anhydrous dichloromethane (24 mL) at 0°C. After 1 h, the mixture was
concentrated. The
14
LI~ffP! 4.cxp-brevet-synlon~
CA 02434685 2003-07-04
residue vas eluted tom a column of silica gel with 3;2 Cyclohexane-AcOEt and
0.2 % Et3N to
give X as a colorless oil (1.58 g, 82 %); (a)a +2° (c 1, CHCI3).
NMR (CDC13) : 'H s 8.GZ (s, 1H, C=NH), 6.95-8.00 (rn, 45H, Ph), 6.24 (d, 1H,
Jl,i = 2.6 Hz,
H-lc), 5.48 (dd, IH, J2,3 = 3.0 Hz, H-2e), 5.41 (d, 1H, J,,N~r = 8.4 Hz, NHp),
4.99 (bs, 1H, H-
lA), 4.92 {d, 1H, Jt,~ = 3.2 Hz, H-lE), 4.88 (d, 1H, Jl,z = 1.6 Hz, H-lg),
4.G9 (dd, IH, Jz,3 =
J3.a = 10.0 Hz, H-3n), 4.44 (d, 1H, H-1D), 4.34 (dd, 1H, H-2H), 4.20-4.80 (m,
16H, CHZPh),
4.02 (dd, 1H, H-2~), 3.38 (dd, 1H, H-2E), 2.90-4.10 (m, 19H, H-2D, 3", 38, 3~,
3x, 4A, 4H, 4~,
4D, 4E, 5,,, 5B, Sc, Sp, 5E, 6aD, 6bn, 6a~, 6b~), 1.95 (s, 3H, OAc), 1.55 (s,
3H, AcNH), 1.30-
0.85 {m, 15H, C(CH3)z, H-6", 6B, 6c). "C b 172.4, 171.4, 166.9 (C=O), 140.2-
128.9 (Ph),
104.2 (C-1D), 101,4 (2C, C-IA, 1$), 101.1 (C(CH3h), 98.0 (C-1~), 94.8 (C-lc),
92.4 (CC13),
82.1, 81.5, 80.2, 80.1, 78.6, 78.1, 77.8. 77.6, 76.0, 75.8, 75.5, 75.0, 74,3,
74.2, 73.5 (C-3o),
73.4, 71.9, 71.4, 71.0, 70.5, 69.2, G8.8, 68.3, 68.1, 62.1, 54.9 (C-2D), 29.3
(C(CH3)i), 23.4
(AcNi-I), 21.4 (OAc), 19.2 (C{CH,)z), 19.0, 18.2, 18.1 (3C, C-GA, 6B, 6C). FAB-
MS for
C~ozHmCl3Naaas (M = 1873.3) m/z 1896.3 [M + Na]'". Anal. Calcd. for C,ozH~
oCl,No02s : C,
65.40 ; H, 6.08 ; N, 1.50. Found C, 65.26; H, 6.02; N, 1.31.
LMPP l4erpbcevet~synl°t~a
CA 02434685 2003-07-04
a lsHi33Ns0:°
t Mass; 2039.9035
',. Wt.: 2041,2808
~ G,57; N 3.43; 0 23,51
2-Azidoethyl (2-acetamido-3-O-acetyl-2-deoxy-4,6-D-isopropylidene-(i-D-
glucopyranosyl)-(1~2)-(3,4-di-0-benzyl-a-L-r6amnopyranosyl)-(1~2)-(3,4-di-0-
benzyl-
a-trrhamnopyranosyl)-(1~3)-(2,3,4,6-tetra-O-benzyl-a-u-glucopyranosyl-(1~4)j-
(2-O-
benzoyl-a-z-rhamnopyranosyl)-(1 >3)-2-acctamido-2-deoxy-4,6-O-fsopropylidene-
(3-n-
glucopyranoside (X).
A mixture of donor X (745 mg, 0.4 ~l) and acceptor X (170 mg, 0.51 mmol), 4 ~
molecular sieves and dry 1,2-DCE (12 mL), was stirred for 1 h then cooled to
0°C. Triflic acid
(25 NL) was added. The stirred mixture was allowed to reach rt in 10 min then
stirred again for
2.5 h at 75°C. After cooling to rt, Et,N (I00 p.L) was added and the
mixture filtered. After
evaporation, the residue was eluted from a column of silica gel with 1:2
Cyclohexane-AcOEt
and 0.2 % Et3N to give X as a white foam (615 m;, 76 %); [a]o +0° (c 1,
CHCI,).
NMR (CDCI,) : iH 8 6.95-7.90 (m, 45H; Ph), 6.02 (d, 1H, Ji,N,.i = 7.1 Hz,
NHa), 5.4G (d, 1H,
J2,NH " 8.6 Hz, NHD~), 5.20 (dd, IH, J,_Z = 1.0, J2,; = 3.0 Hz, H-2c), 5.03
(d, 1H, J,,, = 8.1 Hz,
H-1D), 5.02 (bs, IH, H-1,~), 4.92 (d, 1H, Jl,~ = 3.1 Hz, H-lE), 4.85 (d, 1H.
J,,Z = 1.6 Hz, H-
1B), 4.82 (bs, 1H, H-lc), 4.70 (dd. 1H, H-3o~), 4.44 {d, 1H, H-1D-), 4.30 (dd,
1H, H-2B), 4.20-
4.$0 (m, 16H, CI~Ph), 3.99 (dd, 1H, H-2,,), 3.37 (dd, 1H, H-2E), 2.90-3.95 (m,
29H, H-2D,
2nv 3~, 3s, 3c, 3ti, 3e. 4A, 48, 4c, 4n, 4D~, 4E, Sn. Sp. Sc, 5D, SD~, $e,
Gao, 6bo, GW, Glfi', 6aE,
6bE, OCHZCHZN3), 2.00 (s, 3H, AcNH), 1.92 (s, 3H, OAC), I .57 (s, 3H, AcNH),
1.27-0.90 (m,
1G
L;~3F I 4.exp-brevet~syTlongs
CA 02434685 2003-07-04
21H, 2 C(CH3)z, H-6A, 6~, 6c). 13C o 172.1, 171.5, 170.3, 1GG.2 (C=O), 139.0-
127,7 (Ph),
103.9 (C-1D.), 101.7 (C-1H), 101.2 (C-I~), 100.0 (C-la), 99.9, 99.8 (2C,
C(CH3)z), 98.3 (C-
lE), 97.8 (C-lc), 82.0, 81.7, 81.5, 80.8, 80.2. 80.1, 78.9, 78.6, 78.0, 77.9,
76.0, 75.9, 75.8,
75.3, 74:8, 74.6, 74.2, ?4.0, 73.6, 73.5, 73.4, 73.0, 71.9, 71.4, 70.8, 69.1,
69.0, 68.8, 68.6,
68.0, 67.7, 67.6, 62.6, 62.1, 60.8, 59.7 (C-2p), 55.0 (C-2D~), S 1.1
(0(CH~)~N~), 29.5
(C(CH3)Z), 29.3 (C(CHa)z), 23.9 (AcNH), 23.5 (AcNH), 21.3 (OAc), 19.7
(C(CH3)~), 19.2
(C(CH3)2), 18.8, 18.4, 18.2 (3C, C-6,,, 6$, 6~). FAB-MS for C113Hu3NsO3o (M =
2041.3) mla
2064.2 [M + Naj+. Anal. Calcd, for C"3H,~3NsO,o : C, 66.49 ; H, 6.57 ; N,
3.43. Found
C, 65.93; H, 6.57; N, 2.61.
17
LMPPId-apbre et-oynlongs
CA 02434685 2003-07-04
0
C:nHmVsOtq
tSact t.4acs: 1997.19
Mal. Wt.: 199924
C 68.fiB: H 6.60: N 3.50; 0 23,21
AIphwD=~t °, c t, CHCt?
2-Azidoethyl (2-acetamido-2-deoxy-4,6-D-isopropylidene-~-n-glucopyranosyt)-
(1~2)-
(3,4-di-D-benzyl-a-L-rhamnopyranosyi)-(1-~2)-(3,4-di-0-benzyi-a-
trrhamnopyranosyt)-
(1-->3}-(2,3,4,6-tetra-0-benzyl-ot-D-glucopyranosyl~(1--~4)]-(2-0-benzoyl-a-tJ-
rhamnopyranosyl)-(1->3)-2-acetamido-Z~deoxy-4,6-O-isopropylidene-(3-n-
glucopyranoside (X).
a) The hexasaccharide X (G15 mg, 0.30 mmol) was dissolved in MeOH (8 mL).
MeONa was
added until pH=9. The mixture was stirred for 3 h then treated by IR I20 (H')
until neutral pH.
The solution was filtered and concentrated. The residue was eluted fiom a
column of silica gel
with 25:1 DCM-MeOH and 0.2 % of 1?t?N to give X as a white foam (590 mg. 97
%); [a]ti
+1° (c l, CHCh).
b) To a mixture of X (770 mg, 370 ~mol) in MeOH (S mL) vras added MeONa until
pH=9.
The solution was stirred for 4U min, Amberlite IR 120 (Hf) was added until
neutral pH and the
mixture was filtered and concentrated. The residue was eluted fxom a column of
silica gel with
20:1 DCM-MeOH and Et,N to give a residue which was dissolved in DMF (2 mL).
The
mixture was treated by 2-methoxypropene (200 pL, 2.1 mmol) and CSA (20 mg) at
rt. After I
h, more 2-methoxypropene (200 pL) was added and the mixture was stirred 1 h.
Et?N (IGO
18
LMPPl4.c~cp~bm~ea~aynlongs
CA 02434685 2003-07-04
~L) was added and the solution was concentrated. The residue was eluted from a
column of
silica gel with 2:3 toluene-EtOAc and Et3N (0.2 %) to give X (400 mg, 54%).
~H NMR (CDCl3):o 8.00-7.00 (m, 45H, Ph), 6.10 (d, 1H, NHp~), 6.05 (d, 1H,
.h,~= 7.4 Hz,
NHo), 5.20 (dd, 1H, Jt,~ = 1.7 Hz, J2,3 = 3.0 Hz, H-2c), 5.10 (d, 1H, J,,z =
1.OHz, H-In), 4.99
(d, 1H, Jt,~ = 8.3 Hz, H-lnj, 4,96 (d, 1H, J,3 = 3.2 Hz, H-le), 4.90 (d, 1H,
J,,a = I.0 Hz, H-
1B), 4.86 (d, 1H, J,,z= 1.0 Hz, H-ic), 4.52 (d, 1H, J~,2= 7.5 Hz, H-lp~), 4.37
(dd, 1H, H-2a),
4.Z2 .(dd, 1H, H-3o), 4.02 (dd, 1H, H-2A), 4.80-4.00 (m, 16H, C~IzPh), 4.00-
2.95 (m 30H, H-
2D, 4D~ Sn, 6an~ ~bn~ 2E, 3s~ 4E~ Ss~ 6aE~ dbe., 3c~ 4c, Sc, 3a, 4B~ SH, 3A~
4n~ Sn~ 2D~, 3n~~ 4DV Snv
6a~, bb~, OCHzCHaN3), 2.00-0.92 (6s, 3d, 27H, 2 CH3C=0, 2 C(CH,)Z, H-6A, 68,
Gc). ~jC
NMR (CDCI3) partial: cS 173.9, 172.1, 166.3 (C=0), 140.0-125.0 (Ph), 103.6 (C-
lp), 101.7
(C-1$), 101.2 (C-IA), 100.2 (C(CH3),), 100.2 (C-1D), 99.9 (C(CH,3)Z), 98.2 (C-
lE); 97.8 (C-
lc), 29.4, 29.3, 23.9, 22.8, 19.6, 19.2, 18.9., 18.4, 18.2 (C-6A, 6B, ~c, 2
CH3C=0, 2 C(CH3)z).
FAB-MS for C,nH,3iNs0~9 (M = 1999.2) m/z 2021.8 [M + Na]'. Anal. Calcd. for
Ct~~H~31~'SO~9 : C, 66.68 ; H, 6.60 ; N, 3,50. Found C, 66.63 ; H, 6.78 ; ~',
3.32.
19
CMPPI4ccpbceve~synlangs
CA 02434685 2003-07-04
cl,
C91 H96C ~)N~Z4
Exact Mass; I627,56
Met. Wt.: 1630.09
? 5,94; Cl 6,5?.; h 0.86; 0 19.6'3
tphaD=T22' C=1. CHCf3
(2-0-acetyl-3,4-di-O-benzyl-a-L-r6amnopyrnnosyl)-(1 >2)-(3,4-di-0-benzyl-a-L-
rhamnopyranosyl)-(1->3)-(2,3,4,6-tetra-O-benry1-a-D-glacopyranosyt-(1-~4))-2-O-
benzoyi-a-L-rhamnopyranosyl trichloroacetimidate (X).
1,5-Cyclooctadiene-bis{rnethyldiphenylphosphine)iridium hexafluorophosphate
(80 mg, 93 ~
mol) was dissolved tetrahydrofuran (10 rnL), and the resulting red solution
was degassed in an
argon stream. Hydrogen was then bubbled through the solution, causing the
colour to change
to yellow. The solution leas then degassed again in an argon stream. A
solution of X (2.55 g,
1.G7 mmol) in te~trahydrofuran (20 mL) was degassEd and added. The mixture was
stirred at rt
overnight then concentrated to dryness. The residue was dissolved in acetone
(15 mL), then
water (3 mL), mercuric chloride (380 mg) and mercuric oxide (320 mg) were
added
successively. The mixture protected from light was stirred at rt for 2 h and
acetone was
evaporated. The resulting suspension was taken up in DCM, washed twice writh
50% aq KI,
water and satd aq NaCI, dried and concentrated. The residue was eluted from a
column of
silica gel with 3:1 petroleum ether-EtOAc to give the corresponding hemiacetaL
Trichloroacetonitrile (2.0 mL) and DBU (25 uL) were added to a solution of the
residue in
anhydrous dichloromethane (15 mL) at 0°C. After I h, the mixture was
concentrated. The
residue was eluted from a column of silica gel with 3:1 petroleum ether-EtOAc
and 0.2
LMPPI4~ecp~brcvet-synlon~
CA 02434685 2003-07-04
EtSN to give X as a white foam (1.5 g, 56 %); [a]D +22° (c 1,
CHC13).
'H NMR (CDC13):b 8.72 (s, 1H, C=NH), 8.00-7.00 (m, 45H, Ph), 6.39 (d, 1H, Ji,a
= 2.5 H2,
H-lc), 5.60 (dd, 1H, JZ,~ = 3.0 Hz, H-2~), 5.58 (dd, 1H, Jl,z = 1.7 Hz, Jz,3 =
3.0 Hz, H-2a),
5.12 (d, 1H, J~,z = 3.2 H~. H-lE), 5.08 (m, 2H, H-ln, Is), 5.00-4.00 (m, 16H,
CHzPh), 4.20
(dd, 1H, H-3c), 4.05 (dd, IH, H-3E), 4.00-3.35 (m, I4H, H-2E, 4E, SE, GaE,
6bE, 4c, 5c, 2B, 3a,
4B, SB, 3~, 4," S,J, 2.05 (s, 3H, C=OCHj), 1.42, 1.36 and 1.00 (3d, 9H, H-6n,
6s, 6c). '3C
NMR (CDCl3):b 170.3, 165.8 (G=0), 138-127 (Ph), 99.9 (zC, C-lA, 1B), 98.5 (C-
lE), 94.7
(C-lc), 82.1, 81.2, 80.4, 80.0, 79.1. 78.1, 78.0, 75.2, 71.7, 71.2, 70.7,
69.5, 69.4, 68.? (16C,
C-2n, 3n, 4n, 5", 2e~ 3s. 4B. 5a, 2c~ 3c. 4c~ ~c~ 2s, 3s. 4a. 5E), 76.0, 75.7,
75.5. 75.1, 74.3, 73.3,
?2.2, 71:2 (8C, PhCI-ia), 68.5 (C-6F), 21.4 (C=OGH~), 19.2, 18.5, 18.1 (C-6A,
6B, 6c). Anal.
Calcd. for C9,Hg6C~NO20 : C, 67.05 ; H, 5.94 ; N, 0.86. Found C, 66.44 ; H,
6.21 ; N, 0.93.
21
LMPPI4~cp~brc~ct-synlongs
CA 02434685 2003-07-04
0
N~
0
~cot1»s~:Qat
;t Mt9s. 3464,53
.l. Wt.:3466,93
6,54; N ~,a2; 0 22,15
, CHCt3
2-Azidoethyl (2-O-acetyl-3,4-di-0-benzyl-a-L-rhamnopyranosyl)-(1 >2)-(3,4-di-0-
benzyl-a-L-rhamnopyranosyl)-(1-a3)-[2,3,4,b-tetra-D-benzyl-a-D-glucopyranosyl-
(1-~4)J-(2-O-benzoyl-a-L-rhamnopyranosyl)-(1--~3)-(2-acetamido-Z-deoxy-4,6-0-
isopropylidene-[i-D-glucopyranosyl)-(1~2)-(3,4-di-O-benryl-a-L-
rhamnopyranosyl)-
(1--~2)-(3,4-di-O-benzyl-a-L-rhamaopyranosyl)-(1-~3)-[Z,3,4,6-tetra-O-benryl-a-
D-
gtucopyranosyl-(1-~4)]-(2-O-benzoyl-a-L-rhamnopyranosylj-(1--~3r2-acetamido-2-
deory-4,6-0-isopropylidene-[i-D-glucopyranoside (X).
A mixture of alcohol X (110 mg, 55 umol), imidate X (179 mg, I 10 1t1n41) and
4A molecular
sieves in anhydrous DCE (2.5 mL,) was stirred for 1 h under dry Ar. After
cooling at -35°C,
TfDH (5 uL, SD ~.m~ol) was added dropwise and the mixture was stirred for 2.5
h and allowed
to reach 10°C. Et3N (25 p.L) was added and tile mixture was filtered
and concentrated. The
residue was eluted from a column of silica gel with 4:1 to 3:1 toluene-EtOAc
and Et3N (0.2 °.'°)
to give X as a white foam ( 158 mg. 82 %); [a]D +18° (c I, CHC~).
22
LW(pPl4s~cøbrcve4symlongs
CA 02434685 2003-07-04
rH NMR (CDCis):& 8.00-6.90 (90H, m, Ph), 5.90 (d, 1H, Ji,,.~ = 7.0 Hz, N-HD),
5.58 (d, 1H,
Jz.uf, = 7.5 Hz, N-HD.), 5.45, 5.22 (m, 2H, J,,2 = 1.0 Hz, Jz,a = 2.0 Hz, H-
2c, 2c~), 5.12 (dd, 1 H,
H-2,,~), 5.I 1 (d, 1H, Ji,z = 8.3 Hz, H-ID), 5.05 (d, IH, Jl,~ = 1.0 Hz, H-
lA), 5.01 (d, 1H, J,,~
3.2 Hz, H-lE), 4.96 (d, 1H, J,,~ = 1.0 Hz, H-Ic), 4.94 (m, 2H, H-lp, 1H), 4.86
(d, IH, H-1B),
4,82 (d, 1H, H-lc), 4.72 (d, 1H, H-1~,), 4.70 (d, 1H, H-lA~), 4.90-4.20 (m,
36H, 16 OCXiPh,
H-2s, 2B', 3n, 3rr): 4.00-2.90 (m, 45H, H-2D, 4n, SD, Gao, 6UD, 3c, 4c~ 5c,
2a, 3s, 4s, 5s, Gas.
6be, 3a. 4e~ Sa. 2n, 3n: 4~, 5w: 2n~: 4a~ Sn~: 6a~. Gba, 3c~, 4c~, Sc~, 2s.,
3s~, 4E~, SE~, Ga~~, GbB~, 3g',
4H., SB~, 3A~, 4"~, 5"., OCHICHzN~), 2.00 (s, 3H, AcNH), 1,88 (s, 3H, OAc),
1.86 (s, 3H,
AcNH), 1,40-0.82 (m, 30H, G H-6R,~, 2 C(CH3)Z). "C NMR (partial) (CACI~):8
172.1, 171.4,
170.2, 166.2, 165.9 (C=O), 102.7 (C-1B~), 101.6, 101.2 (2C, C-1H, 1B~), 101.1
(C-ln), 99.8 (C-
1D), 99.7 (C-Ic), 98.2 (2C, C-lE, 1,,~), 97.2 (ZC, C-tc, IE), 63.3, 62.6 (2C,
C-6E, 6E'), 60.0,
57.8 (2C, C-2o, 2D~). S 1.0 (OCH2CHZNl), 29.5, 29.4 (2C, C(CH3)Z), 24.0 (2C, 2
AcNH), 21.3
(Ac0), I9.6, 19.5 (2C, C(CH~)2), 19.I, 18.9, 18.8, 18.5, 18.2, 18.1 (6C, C-6A,
68, 6c, 6n~, 68~,
6~~). FABMS of CzooHnsNsOaa (M, 3446.9), nr/z 3489.5 ([M+Na]'). Anal. Calcd
for
C2uoHiuNsOae +SHiO, C: 67.47, H: 6.65, N: I.96. Found C: 67.40, H: 6.57, N:
1.72.
23
LMPP 14-exPbmcMynt°n~
Ho
!pH HO
0
NOp HO , ~,.,~,,~
0..
_0' OOH
H0
HO
Hp H
OH HO~O,
CA 02434685 2003-07-04
Oa~W i:N30,s
Exact MQ$F; 1667.66dG
Mol, Wt ~ 1668,5966
C 47.St: H 6.93: N 2.52; 0 43,15
2-Aminoethyl (a-L-rhamnopyranosyl)-(1~2)-(a-L-rhamnopyranosyl)-(1~3)-[a-D-
glucopyranosyl-(1-~4)]-(oc-L-rhamaopyranosyt)-(1-~3)-(Z-acetamido-Z-deoxy-[i-D-
gtucopyranosyl)-(1-~2)-(a-L-rhamnopyranosyl)-(I~2)-(a-L-rhamnopyranosyt)-(1--
~3)-
[a-D-glucopyranosyl-(1--1~4)]-(a-L-rhamnopyranosy I)-(1-~3)-2-acetamid o-2-
deoxy-[i-D-
glucopyranoside (X)-
A mixture of X (130 mg, 38 p,nwl) in MeOH (4 mL) was treated by MeONa until
pH=9. The
mixture was stirred 1 h ai rt, then heated at 55°C and, stirred
overnight. After cooling at rt, IR
120 (H') was added until neutral pH and the solution was filtered and
concentrated. The
residue was eluted from a column of silica gel with 25:1 to 20:1 DCM-MeOH to
give an
amorphous residue. A solution of this residue in EtOH (1.5 mL), EtOAc (150
pL), 1M HCl
(66 ~L, 2 eq) was hydrogenated in the presence of PdIC (100 mg) for 72 h at
rt. The mixture
Llvg'Pl4~xpbrevec-syniongs
CA 02434685 2003-07-04
was filtered and concentrated , then was eluted from a column of C-18 with
wated and freeze-
dried to afford amorphous X as a white foam (41 mg, ?1 %); (a]D -7° (c
1, HBO). 'H NMR
partial (D~0):8 4.90 (m, 2H, J1,2 = 3.S Hz, 2 H-lE), 4.82 (bs, 1H, H-1R,,~,
4.76 (bs, 1H, H-
1R,~), 4.72 (bs, 1H, H-IRh~, 4.67 (bs, IH, H-lpr,), 4.52 (bs, 1H, H-
IAh°), 4.51 (bs, 1H, H-lp~,
4,41 (d, 1H, J 1,2 = 8.6 Hz, H-louhu), 4.29 (d, IH, JI,Z = 8.6 Hz, H-l~~cua~),
1.77 (s, 6H, 2
CH3C=ONH), I.15-0.96 (m, 18H, H-6Rh,). 1'C NMR partial (D,0):S 174.8, 174.7
(C=O),
102.9 (C-IRS), 102.6 (C-lQ,tNa~), 101,8 (2C, Z C-lRha), 101.6 (C-1R~), 101.4
(C-lRha), 101.3
(C-lah4), 100,8 (C-IotcNzc), 97.9 (2C, 2 C-Ipso), 56.0, 56.4 (2 C, 2 Co~cN~),
22.7, 22,6 (2
NHC=OCH3), 18.2, 17.2, 17.0, 16.9 (bC, 6 C-6R~,). HRMS: calculated for
C~sH~,3NsO4s+Na:
1690.6544. Found 1690.6537.
26
LMPPI4.cx~bmet~synlongs
CA 02434685 2003-07-04
~o
oen
o~
eBnD ~ ~ Ns
Bn0
0
OBI
Bn0 8~
0
~z i ~ Htaa'-'le~s~
Exact Mass: 3707,6426
Mot. Wt:3710,l878
3,3I: H 6.57: N 2,27; 0 22,86
2-Azidoethyl (Z-aeetamido-3-O-acetyl-2-deoxy-4,6-O-isopropylidene-[i-n-
glucopyranosyl)-(1--~Z)-(3,4-di-0-benzyl-a-L-rhamnopyranosyI)-(1-~2)-(3,4-di-0-
benzyl-
a-L-rhamnopyranosyl)-(1--~3)-[2,3,4,6-tetra-D-benzyl-a-n-giucopyranosy I-
(1~4)]-(Z-O-
benzoyl-a-L-rhamnopyranosyl)-(1 >3)-(Z-acetamido-Z-deoxy-4,6-O-Isopropylidene-
~i-n-
glucopyrano9yl)-(1-~Z)-(3,4-di-O-benzyl-oc-z-rhantnopyranosyl)-(1 >Z)-(3,4-di-
O-benzyt-
a.-c,-rhamnapyranosyl)-(I >3)-[2,3,4,6-tetra-O-benzyl-a-D.glucopyranosyl-(1--
~4)j-(2-O-
benzoyl-a-cr-rhamaopyranosy I)-(1-~3)-Z-acetumido-Z-deo~y-4,6-O-isopropylidene-
~-n-
glucopyranoside (X).
A mixture of donor X (835 mg. 0.44 mmol) and acceptor X (590 mg, 0.3 mm.ol), 4
~
molecular sieves and dry 1,2~DCE (12 mL), was stirred for 1 h then cooled to -
30°C. Triflic
acid (35 1cL) was added, The stirred mixture was allowed to reach 5°C
in 2.5 h. Et)N (150 pL)
was added and the mixture filtered. After evaporation, the residue was eluted
from a column of
27
Lfv~P I4.rxp.breve4synlongs
CA 02434685 2003-07-04
silica gel with 1:2 Cyclohexane-AcOEt and 0.2 % >;t3N to give X as a white
foam (990 mg, 90
%); [a]D +10° (c 1, CHC13).
'H NMR (CDCL,): partial b 6.95-7.90 (m, 90H, Ph), 5.98 (d, 1H, Jz,N,~ = 6.9
Hz, NHD), S.GO
(d, 1H, JZ,~ = 7.5 Hz, NHc), 5.45 (d, 1H, Jz,~ = 8.5 Hz, NHp), 5.22 (dd, IH,
,T~,Z = 1.0, Jz.a =
3.0 Hz, H-2c), 5.13 (dd, 1H, J,,z = 1.0, J2,3 = 3.0 Hz, H-2c), 5.08 (d, 1H,
J~,z = 8.3 Hz, H-lp),
5.07 (bs, 1 H, H-1 "), 5.04 (bs, 1 H, H-1,,), 4.97 (d, 1 H, J,,z = 3.0 Hz, H-1
E), 4.94 (d, 1 H, J,,i =
3.0 Hz, H-lE), 4.90 (bs, 1H, H-Is), 4.86 (bs, 1H, H-I$), 4.82 (bs, 1H, H-lc),
4.73 (d, 1H, H-
lp), 4.70 (bs, 1H, H-lc), 4.43 (d, 1H, H-1D), 4.20-4.80 (m IGH, CH2Ph), 2.00,
1.85, 1.58 (3s,
9H, AcNH), 1.95 (s, 3H, OAc), 1.37-0.85 (m, 36H, 3 C(CH3)z, 2H-GA, 2H-6H,. .
2H-6~). '3C
NMR (CDC13) partial: S 171.7, 170.8, 169.8, 165.8, 1b5.4 (C=0), 139.0-127.7
(Ph), I03.9
(C-1D), 102.8 (C-1D), 101.5 (2C, C-lg), 101.3 (C-1A), 101.1 (C-lA), 100.0 (C-
1D), 99.5, 99.3
(3 C(CH3h), 98.3 (C-1fi), 98.1 (2C, C-lc, lE), 97.8 (C-lc), 82.0, 81.7, 81.6,
81.4, 80.3, 80.2,
80.1, 79.5, 79.2, 78.9, 78.7, 78.4, 78.1, 77.9, 77.8, 77.6, 76.0, 75.8, 75.3,
75.2, 74.7, 74.4,
74.1, 74.0, 73.6, 73.5, 73.4, 73.3, ?3.0, 72.7, 71.9, 71.4, 70.9, 70.8, 69.1,
69.0, 68.9, 68.7,
68.6, GB.S, 68.1, 67.8, 67.7, 67.5, 62.6, 62.3, 62.1, 60.8, 59.9 (C-2D), 57.9
(C-2D), 55.0 (C-
2D), 51.1 (0(CH1)~N3), 29.5, 29.4, Z9.3 (3 C(CHz)Z), 24.0, 23.9, 23.5 (3
AcNH), 21.3 (OAc),
19.7, 19.6, 19.2 (3 C(CH?)z), 18.9, 18.8, 18.6, 18.5. 18.2, 18.1 (GC, 2 C-6A,
6H, Gc). FAB-MS
for Cz~,Hz~zNsOs3 (M ' 3710.2) m/z 3733.3 [M + Na]". Anal. Calcd. for
Cz,lHzazNsOss : C,
68.31 ; H, 6.57 ; N, 2.27. Found C, 68. i 7; H, 6.74; N, 2.12.
28
LMPPI4exp6tevehsynloBgs
CA 02434685 2003-07-04
Ha
~Bz
~OBn
0
Ba ° Bn0 '
0
Nrl
Cao9H2.eoNsOs2
Exacs Mass: 3665,6320
Mol. Wt.: 3668,1511
C 68,43; H 6,.9; N 2,29; O 22.68
2-Azidoethyl (2-acetamido-2-deozy-4,6-O-isoprapylidenc-ji-D-glucopyranosyi)-(1-
->2)-
(3,4-di-O-benzyl-a-L-rbamnopyrsrnosyt)-(1->2)-(3,4-di-O-benzyl-a-L-
rhamnopyranosyl)-
(1-~3)-[2,3,4,6-tetra-D-bevzyt-a-n-gtucopyranosyl-(1->4)1-(Z-0-benzoyl-a-it
rhamnopyranosyl}~(1 >3)-(2-acetamido-2-deoxy-4,6-O-isopropylidene-(3-D-
glucopyranosyl)-(1-~~)-(3,4-di-0-benzyl-a-trrhamnopyranasyl)-(1->2)-(3,4-di-0-
benzyl~
a-1,-rhamnopyranosyl)-(1 >3)-j2,3,4,6-tetra-0-bcnzyl-a-n-gIucopyranosyl-(1-
>4)~-(Z-0-
benzoyl-a-L-rhantnopy ranosyl)-(1-~3)-2-acetamido-2-deoxy-4,G-D-esopropylidene-
[3-n-
glucopyranoside (X).
The undecasaccharide X (990 mg, 0.27 mmol) was dissolved in MeOH (30 mL).
MeONa was
added until pH=9. The mixture was stirred for 3 h then treated by IR 120 (H")
until neutral pH.
The solution was filtexed and concentrated. The residue was eluted from a
column of silica gel
with 1:1 toluene-AcOEt and 0.2 % of Et3N to give X as a white foam (900 mg, 91
%); [a]D
+15° (c I, CHCl3).
29
L~Pl4cap-bm~t-synlonga
CA 02434685 2003-07-04
'H NMR (CDC)'): partial 0 6.95-8,00 (m, 90H, Ph), 6.19 (bs, 1H, NHD), 5.96 (d,
IH, Jz,~H =
6.8 Hz, NHo), 5.57 (d, 1H, JZ,uH = 6.8 Hz. NHp), 5.22 (dd, 1H, H-Z~), 5.13
(dd, 1H, H-2c),
5.10 (d, 1H, H-lv), 5.07 (bs, 1H, H-la), 5.04 (bs, 1H, H-la), 4.96 (d, 1H, H-
lE), 4.94 (d, 1H,
H-le), 4.85 (bs, 1H, H-1g), 4.84 (bs, 1H, H-1H), 4.82 (bs, 1H, H-l~), 4.70 (d,
1H, H-1~), 4.67
(bs, 1H, H-1D), 4.44 (d, 1H, H-1D), 4.20-4.80 (m, 16H, CHzPh), 2.00, 1.85,
1,58 (3s, 9H,
AcNH), 1.37-0.80 (m, 36H, 3 C(CH3)z, 2H-6a, 2H-6B, 2H-6~). ~3C NMR (CDCl3)
partial: 8
172.8, 170.9, 170.3, 165.1, 164.7 (C=O), 139.0-127.7 (Ph), 103.5 (C-1D), 103.1
(C-lp), 101.5
(2C, C-lB), 101.2 (C-la), 101.1 (C-lA), 99.9 (C-1D), 99.0, 98.8, 98.7 (3
C(CH~)Z), 98,3 (C-
lE), 98.I (2C, C-1~, lE), 97.8 (C-1~), 82.1, 82.0, 81.9, 81.7, 81.6, 81.5,
80.6, 80.3, 80.2, 80.1,
79.7, 79.1, 78.9, 78.5, 77.9, 77.6, 75.7, 74.9, 74.6, 74.3, 73.3, 73.0, 72.7,
71.9, 71.8, 69.1,
68.9, 68.7, 68.5, 68.0, 67.8, 67.7, 67.6, 67.5, 62.6, 62.3, 61.9, 60.5, 59.9
(C-2D), 57.4 (C-2o),
55.0 (C-2b), 51.0 (O(CH~)1N3), 29.51, 29.47, 29.3 (3 C(CH,)~): 24.0, 23.9,
22.7 (3 AcNH),
19.7, 19.6, 19.3 (3 C(CH3)z), 19.0, 18.9, 18.G, 18,5, 18.2, 18.1 (6C, 2 C-6a,
68, 6c). FAB-MS
for C=o9HaeoNsOsa (M = 3668.1) mla 3690.8 [M + Na]~. Anal. Calcd. for
C2[,H24~'6Os3 : C,
68.43 ; H, 6.59 ; N, 2.29. Found C, 68.28; H, 6.72; N, 2.11.
IHfPPI4.ap-6~evet.synionga C~1 02434685 2003-07-04
:2,12
2-Azidoethyl (2-O-acetyl-3,4-di-0-benzyl-a-tr-rhamnopyranosyl)-(I-a2)-(3,4-di-
0-
benzyl-a-z-rbamnopyranosyl)-(1->,3)-[2,3,4,6-tetra-O-benzyl-a-n-gtucopyranosyl-
(1-->
4)]-(2-0-benzoyl-a-~t,-rb amnopy ranosyl)-(1-~3)-(2-acetam ido-2-deoxy-4,6-0-
isopropylidene-[3-o-glucopyranosyl)-{1->2)-(3,4-di-O-bcnryl-a-L-
rhamnopyranosyl)-(1
2)-(3,4-di-0-benzyl-a-irrhamnopyranosyl)-(1~3)-[2,3,4,6-tetra-O-benzyl-a-D-
glucopyranosyl-(1-ad)]-(2-O-benxoyl-a-r~rhamnopyranosyl)-(1-~3)-(2-acetamido-2-
deoxy-4,6-O-isopropylidene-[i-D-glucopyranosyl)-(1->2)-(3,4-di-O-benzyl-a-~-
rbamnopyranosyl)-(1->Z)-(3,4-di-O-benzy 1-a-z-rhamnopyranosyl)-( 1 ~3)-
[2,3,4,6-tetra-
3I
LMPPI4tx~b~evetsyal°n~s
CA 02434685 2003-07-04
O-beazyl_a-n-glucopyranosyl-(1-~4)]-(1-0-benzoyl-a-z-rhamnopyranosyt)-(1-~3)-2-
acetamido-2-deoay-4,6-O-isopropylidene-[i-n-glucopyranoside (X).
A mixture of donor X (377 mg, 0.230 mmol) and acceptor X (427 rz>8, 0.115
mmol), 4 t~
molecular sieves and dry 1,2-DCE (10 mL), was stirred for 1 h then cooled to -
30°C. Triflic
acid (20 p.L) was added. The stirred mixture was allowed to reach 5°C
in 2.5 h. Et;N (150 p.L)
was added and the mixture filtered. After evaporation, the residue was eluted
from a column of
silica gel with 3:1 toluene-AcOEt and 0.2 % Et;N to give X as a foam (490 mg,
82 %); [a]p
+20° (c 1, CHC13).
'H NMR (CDC~): partial 8 6.90-8.00 (m, 135H, Ph), 5.95 (d, 1H, J?,~ = 6.6 Hz,
NHD), S.GO
(d, 1 H, Jz.~, = 8.0 Hz, NHc), 5.59 (d, 1 H, J1,~, = 7.5 Hz, NHD), 5.44 (dd, I
H, H-2c), 5.22
(dd, 1H, H-2c), 5.10 (dd, 1H, H-2~), 2.20 (s, 3H, OAc), 2.00, 1.85, 1.84 (3s,
9H, AcNH),
1.40-0.80 (m, 45H, 3 C(CH~)~, 3H-6A, 3H-6g, 3H-Gc). "C NMR (CDC13) partial: b
173.2,
172.6, 172_5, 171.3, 167.4, 167.0, 166.9 (C=O), 140.2-126.8 (Ph), 102.8,
102.7, 101.5, 101.3,
101.1, 99.9, 99.8, 98.1, 97.8, 82.0, 81.7, 81.5, 81.4, 80.2, 80.1, 79.6, ?9.4,
78.9, 78.6, 78.0,
77.9, 77.6, 75.5, 73.4, 73.3, 73.0, 72.8, 71.9, 71.G, 69.4, G9.1, G9.0, G8.G,
67.8, 67.7, 67.6,
67.5, 62.6, 62.3, 60.0, 57.9, 57.7, 51.0 (OCHZCHZN3), 30.5 (3C, C(G'Fi3)z),
25.0, 22.4 (3C,
AcNH), 22.9 (OAc), 20.7. 20.6, 20.2 (3C, C(CHy), 20.0, 19.9, 19.8, 19.7, 19.6,
19.3, 19.2.
19.1 (9C, 3 C-6A, 6H, 6~). FAB-MS for CzggH;3qN6O7, (M = 5135.8) m!z 5159.3 [M
+ Na]'.
Anal. Calcd. for C298H;;aN6O7t : C, 69.69 ; H, 6.55 ; N, 1.64. Found C, 69.74;
H, G.72; N,
1.49.
32
LMPPI4~x~bm~errynlon8.c
CA 02434685 2003-07-04
8n0 B~
0
J~
~1
Z
H
~~
eno 771
o 2
Exaa
50
I~i
s
e Mel. Wt.: 5015,6468
C 69.21; H 6,47;
N 1,68; 0 22,65
2-Azidoethyl (2-0-acetyl-3,4-di-0-benzyl-a-L-rhamnopyranosyl)-(1~2)-(3,4-di-D-
benzyl-a-t-rhamnopyranosyl)-(1->3)-[~,3,4,G-tetra-O-benryl-a-n-glucopyranosyl-
(1-~
4)]-(2-O-ben~oyl-a-z-rhamnopyranosyl)-(1 >3)-(Z-acetamido-2-deoxy-a-n-
glucopyranosyl)-(1->2)-(3,4-di-0-benzyl-a-i.-rhamnopyranosyt)-(1-~2)-(3,4-di-0-
benzyl-
a-L-rhamnopyranosyl)-(1->3)-[2,3,4,G-tetra-O-benzyl-a-n-glucopyranosyl-(1->4)]-
(Z-0-
benzoyl-a-L-rbamnopyranosy I)-(1-~3)-(2-acetamido-2-deoay-[3-n-gtucopyranosyl)-
(1~
2)-(3,4-di-O-benryi-a-irrhamnopyranosyl)-(1 >2j-(3,4-di-0-benzyl-a-ir-
rhamnopy ranosyl)-(1-~3)-[2,3,4,G-tetra-0-benzyl-a-n-glucopyranosyl-(1~4)]-(2-
O-
benxoyl-a-z-rhamnopyranosyl)-(1--~3)-2-acetamido-2-deo~y-[i-D-glucopyraaoside
(X).
To a solution ofthe pentadecasaccharide X (480 mg, 93 pmol) in DCM (14 mL) was
added
dropwise at 0°C, a solution of TFA (1.75 mL) and water (1.75 mL). The
mik~ture was stirred
for 3 h then concentrated by coevaporation with successively water and
toluene. The residue
33
LMPPI4.exp.brevet-syntongg
CA 02434685 2003-07-04
was eluted from a column of silica gel with 1:1 toluene-AcOEt to give X as a
white foam (390
mg, 83 %); [a]D +12° (c l, CHCl3).
FAB-MS for Czs9H3a2ycCm (M = 5015.6) m/z 5037.2 [M + lvTajy.
Anal. Calcd. for C2pgH322~6W1~81~0: C, 67.27 ; H, G.60 ; 1~', 1.63. Found C,
67.31; H, 6.45;
N~ 1.64.
34
CA 02434685 2003-07-04
LA?PPl4~x~brcvacynlon~
HO
Hi
HO
H
C98H1 bGV4~67
HO Exact Mass: 2470,9706
~
H Mol. Wt.: 2472,3534
"
C 47,61; H 6,77;
N 2,27; O 4336
NO O
OH
O
H00
Ho
0
N
HO
H
HO
Hd H
Z-Aminoethyt (a-1.-rhamnopyranosyt)-(I >2)-(a-z-r6amnopyranosyt)-{I~3)-[a-n-
glucopyranosyt-(1 >4)]-(a-c,-rhamnopyranosyl)-(1->3)-(2-acetamido-2-deoxy-(3-D-
gtucopyranosyt)-(I-32)-(a-t,-rhamnopyranosyl)-(1-->2)-(a.-L~rhamnopyranosyl)-
(1-33)-
a-n-glucapyranosyl-(1-~4)]-(a-L-rhamnapyranosyl)-(I-~3)-(2-acetamido-2-deoxy-~-
n-
gtucopyranosyt)-(I~2)-(a-~rrhamnopyranosyl)-(I-~2)-(a-z-rhamnopyranosyt)-(1-
~3)-[
a-v-glucopyranosyl-(I-~4)]-(a-z-rhamnopyranosyl)-(1-~3)-2-acetamido-2-deo~cy-
[3-D-
glucopyranoside (X).
A solution of the partially deprotected pentadecasaccharide X (390 mg, 77
ltmol) in MeOH
(10 mL) was treated by MeONa until pI-I=10. The mixture was stirred overnight
at 55°C. After
cooling at rt, IR 120 (H~ was added until neutral pH and the solution was
filtered and
concentrated, then was eluted from a column of silica gel with 20:1 DCM-MeOH
to give the
benrylated residue (252 mg). A solution of this residue in EtOH (3 mL), AcOEt
(250 p.L) and
LMPP t4txp.brcvel~synlongc
CA 02434685 2003-07-04
1M HCl (106 wL) was hydrogenated in the presence of Pd/C (300 mg) for 43 h at
rt. The
mixture was filtered and concentrated, then was eluted from a column of C-18
with
water/CH;CN and freeze-dried to afford amorphous X (127 mg, GS %): [aJD -
5° (c 1, H~0).
'H NMR (D2~): partial 8 5.13 (m, 3H, 3 H-lE), 5.07 (m, 2H, H-lRhn)~ 4.99 (bs,
1H, H-lRha)~
4.95 (m, 2H, H-lRh~), 4.90 (m, IH, H-IRU), 4.75 (m, 3H, H-1R~,), 4.63 (d, 2H,
J,,2 = 8.5 Hz, 2
H-1Q), 4.51 (d, 1H, Jl,s = 8.5 Hz, H-1D), 2.00 (s, 9H, 3 AcNH), L30-1.18 (m,
27H, 3H-6",
3H-68, 3H-6~). "C NMR (CDCh): 8 174.8, 174.7 (3C, C=O), 102.9, 102.6, 101.7,
101.3,
100.8, 97.9, 81.8, 81.7, 79.6, 79.0, 76.3, 76.2, 73.0, 72.7, 72.4, 72.1, 71.6,
70.5, 70.1, 70.0,
69.7, 69.6, 69.4, 68.7, 68.6, 66.0, 61Ø 56.0, 55.4, 39.8, 22.7, 22.6 (AcNH),
18.2, 17.2, 17.0,
16.9 (9C, 3 C-6h, 6s, dc). FAB~MS for C98H166N4067 (M = 2470.9706) tnla
2493.9660[M +
Na]'.
36
CA 02434685 2003-07-04
LMPPIb-schemca-brcv~-s't~loogs
~OBn cJ'"~y~yO.J'Na
o --~'0
eng p NHAc
9nOOMe
XX a_~gz OR~
Bn~ $n4' o O Q Rdg ~ O./~R~
R~ ~ NHAc
OBn ~'O O ~ ROpM-~
O O O~pBBn O OR'
9no~ , p~! _ 0
8
BnO-'~"'1 OR d ~"~~~t OR
Bn0 O RR ~ Me R 00 NHAc~OR
~ R
a0 O a~~09n ~ 0 Ra
R" NHAc OBn Me 0
RO R
R ORs
AC OR Rap O
R~0 ~ ta~ Ac O
M° 1~..' R~ R2 Rs Rd.~-
R
M O ORS gn N3 Bx Ac -- iPr -
RO R~ a Bn~ Ns 6z Ac H H
gn N3 H H H H
..s ~~na '~ H NH2 ~ ~ H M
1
LMPP14-nchcmcs~brcvet-synlongs
CA 02434685 2003-07-04
OBn
Bn0 O OR'
Bn0
Bno 0 Me O Rt R3 R4
Rs
O OBz a All H H H
Me O f All H - iPr
-
BnO
9 All Ac - iPr
Bn0 -
O OH Ac - iPr
-
~ORG ~ OTCA Ac - iPr
-
Bn
)Bn
N HAc
OBn
\O B6 O O O-~e~0~N3
O O NHAc
HO~~~O~N3 Bn0 0 Me O
NHAc
o OBz
BnOMe O R
Bn0 O a ~ Ac
N
O O O
O O Me OBn
OBn
NHAc
2
CA 02434685 2003-07-04
LMPPId.srhemes-Ixevet~synlong9
OR
OAc RO--~~S(CHz)~~CH3 ~ ~ ~ Ac
p RO H
Ac0 ~~OAC NHC(O)CC13
NHC(O)CCI3
00 O CH CH3
RCS( z)11
\NHC(O)CCI3 O6n
Dan R Bn0 ~ O OAfI
O OAII ( H BnO~ Me O
Ac
Bn< OBz
Bn0 M~
B Of Bn0 O
~O O p OSn
Me OBn
Ac R~
CC13C(O)NH
H
CA 02434685 2003-07-04
LMPP (4-schcmes~brcva-4yn(ongs
<OB n
OBn /OAc B~O~ 3 R~
BAnO O oAU ~O \ O
Bn0 Me 0 Ac0 B°OO'~~~c 0
O CChC(0)NH TCA " OBz
~Bz
Bn0 Me O Ma 0
Bn0 ""' B Bn0 O R~ R~
d
Bn0 Mo O ~OAC O a AA C(0)CCIs 0.
Bn0 R Ac0 t~~OB~ f All Ac a
OR c ~ ~ AcO~a OBn g H Ac an
TCA Ac a!p
ORe
OBn R°O 0 OH
Bn0 0 ~O~ OH
Bn0 O NHAc Ns HO-~S~ O~ HO~O~
Bn0 0 Me O H~ 0 NHAc NHa
t R~ H'O O M~0
Bn0 Me j H
O Bn0 O HO M-~../~~
HO 'NHAc~Ns OR3 HO
RaO~p OBn ~OH~
Rs0 Me Bn HO~O
NHAC -Ra Rs Ra Re HO O/ Me H
NHAc OH
OAe p Bz Ac - iPr
AeA 0 O O~Ns
Bt Ac H H
NHAc 9 H H H H
LMPPl4.~brcvctaynlongt
CA 02434685 2003-07-04
'OBn
p p~
BBnp Bn0
p
° OBa
An0 Bn0
C,o,H",N0~7
Exac~M~ss; tett,78
Bn0 Bnp Moh Wc: 1813,03
Acp~ C 88,90, H 6,50; N 0,77, U 23,9J
u~-p~.,\~~~J// AIphaD-+3°, C'1, CHC13
~VH
y~p~0
Allyl (2-acetamido-3,4,6-tri-D-acetyl-2-deoxy-[3-D-glucopyranosyl)-(1-~2)-(3,4-
di-O-
benzyl-a-L-rhamnopyranosyl)-(1 >2)-(3,4-di-O-bcnzyl-ac-L-rhamnopyranosyl)-(1--
1,3)-
[2,3,4,6-tetra-D-benzyl-a-D-glucopyranosyl-(1-r4)]-2-D-benzoyl-a-L-
rhamuopyranoside
{X).
A mixture of X (3.14 g, 1.6 mmol), Bu3SnH (2.5 rnL, 9.3 rnnwlj and AIBN (240
mg) in dry
toluene (40 mL) was stirred for 30 min at rt under a stream of dry Ar, then
was heated for 1 h
at 100°C, cooled and concentrated. The residue was eluted from a column
of silica gel with 3:2
petroleum ether-EtOAc to give X as a white foam (2.0 g, G8 %); [a]p +3°
(c 1, CHC13).
1H Ni~IR (CDC13):8 8,00-7,00 (m, 45H, Ph), 5.82 (m, 1H, All), 5.58 (d, 1H,
J2,NH = 8.0 Hz; N-
HD), 5.35 (dd, 1 H, J,,2 = I .0 Ha, J2,3 = 2.3 Hz, H-2c), 5.19 (m, ZH, All),
5. I 0 (d. I H, J,,2 = 1.0
Hz, H-lA), 4.92 (dd, 1H, J2,3 = 10.5 Hz, J3,4 = 10.5 Hz, H-3D), 4,92 (d, 1H,
J,,2 = 3.3 Hz, H-
1 E), 4.90 (d, 1 H, Jt.2 = 1.7 Hz, H-18), 4, 89 (d, 1 H, H-1 ~), 4.88 (dd, 1
H, J4,s = 10.0 Hz, H-4D),
4.62 (d, 1H, J,,2 = 8.5 Hz, H-1D), 4.90-4.35 (m, IGH, CH2Ph), 4.40 (m, 1H, H-
28), 4,10-4.00
(m, 2H, All), 4.08 (dd, 1H, J2,3 = 2.4 Hz, H-2A), 4.02 (dd, 1H, H-3c), 3.91
(m, 1H, H-2D),
3.90-3.70 (m, 11H, H-4~, 5~, 3A, 5A, 6aD, 6bD, 3~, 4E, SE, 6aE, 6bE), 3.61
(dd, 1H, J3,a = 9.S Hz,
H-38), 3.55 (m, 1H, H-5B), 3.41-3.40 (tn, 3H, H-4A, 5D, 2E), 3.47 (m, 1H, J4,s
= 9.5 H~, JS,s =
6.1 Hz, H-5B), 3.35-3.33 (m, 3H, H-4,,, 5D, 2E), 3.25 (dd, 1H, H-48), 1.95,
1.70 (3s, 9H,
LMPP14-cxp~brrva~ynlongs ~1 02434685 2003-07-04
CH3C=0), 1.65 (s, 3H, CH3C=ONH}, 1.32 (d, 3H, .~5,~ = 6.1 Hz, H-GA), 1.30 (d,
3H, J5,6 = 6.0
Hz, H-6~), 0.97 (d, 3H. J;,b = 6.0 Hz, H-6H). '3C NMR (CDCI,):8 171.1, 170.8,
170.2, 169.6,
166.2 (5C, C=0}, 138.2-118.5 (Ph, All), 103.1 (C-1D), 101.4 (C-lB), 101.2 (C-
1~), 98.5 (C-
lE), 96.4 (C-l~), 82.2 (C-3E), 81.7 (C-2E), 81.7 (C-4A), 80.4 (C-4B), 80.2 (C-
3~), 79.0 (C-3A),
78.6 (C-3B), 78.1 (C-2A), 77.8 (C-4~), 77.6 (C-4E), 76.0, 75.8, 75.4, 74.7,
74.3, 74.2, 73.3,
70.5 (8C, C'~hPh), 74.9 (C-2s), 72.7 (C-2o), 72.G (C-3D), 71.9 (2C, C-Se, 5n),
G9.1 (C-5B),
68.9 (2C, All, C-5,,), G8.3 (C-6E), 67,8 (C-5~), 62.3 (C-6o}, 54.6 (C-2D),
23.5 (1C,
NHC=OCHz), 21.1, 21.0, 20.8 (3C, C=OCH3), 19.0 (C-G~), 18.4 (C-6A), 18.2 (C-
Gs).
FARMS of C,o4Hi1~N0~~ (M, 1913.1), m/z 1936.2 [M~-Na]'. Anal. Calcd. for
C,naH,ml'TOz,
C, 68.90 ; H, 6.50 ; N, 0.77. Found C, 68.64 ; H, 6.G6 ; N, 1.05.
CA 02434685 2003-07-04
LMPPl4<xp-bcevet-synlongs
Cc o~Ht uChN~Oz?
EaAtt',~ta~,5: 1914,86
Mol. Wc: 1917,35
H 5,95: CI 5,55; N 1 a6; O 7.2.59
rc: C:61.3&°~. H fi.10!~, Id:Ls54o
AlDhanxlD', c-1, CHCt3
(Z-acetamido-3,4,6-tri-0-acetyl-Z-deoay-~3-D-glucopyranosyl)-(1-~2)-(3,4-di-O-
benryl-a-
L-rhamnopyranosyl)-(1--~2)-(3,4-di-O-benryl-a-L-rhamnopyranosyl)-(1->3)-
(2,3,4,6-
tetra-O-benzyl-a-D-glucopyranosyl-(1 >4)J-Z-0-benzoyl-a-L~rhamnopyranosyl
trichloroacetimidate (X).
1,5-Cyclooctadiene~bis(methyldiphenylphosphine)zridium hexafluorophosphate (25
mg, 29 1l
mol) was dissolved tetrahydrofuran (5 mL), and the resulting zed solution was
degassed in an
argon stream Hydrogen was then bubbled through the solution, causing the
colour to change
to yellow. The solution was then degassed again in an argon stream A solution
of 7 (1.0 g,
0.55 mrnol) in tetrahydrofitran ( 10 rnL) was degassed and added. The mixture
was stirred at rt
overnight, then concentrated to dryness. The residue was dissolved in acetone
(5 mL), then
water (1 mL), mercuric chloride (140 mg) and mercuric oxide (120 mg) were
added
successively. The mixture protected from light was stirred at rt for 2 h and
acetone was
evaporated. The resulting suspension was taken up in DCM, washed twice with
50% aq ~:I,
water and sold aq NaCI, dried and concentrated. Th>r residue was eluted from a
column of
silica gel with 2;1 petroleum ether-EtOAc to give the corresponding
hemiacetal.
Trichloroacetonitrile (2.5 mL) and DBU (37 p.L) were added to a solution of
the residue in
3
LMPPl4.c:cp.brcvec-eynlongs
CA 02434685 2003-07-04
anhydrous dichloromethane (12.5 ml.) at 0°C. Aver I h, the mixture was
concentrated. The
residue was eluted from a column of silica gel with 5:4 cyclohexane-EtOAc and
0.2 % Et3N to
give X as a white foam (0.9 g, 85 %); (a)o +10° (c I, CHC13).
~H NMR (CDC~):8 8.70 (s, IH, C=NH), 8.00-7.00 (m, 45H, Ph), G.3G (d, 1H, J,,~
= 2.6 Hz,
H~Ic), 5.59 (m, 2H, N-Ho, H-2c), 5.13 (d, 1H, Ji,2 = I.0 Hz, H-l,,), 5.01-4.98
(m, 2H, H-lr,
18), 4.92 (dd, 1H, H-3p), 4.90 (dd, 1H, H-4D), 4.68 (d, 1H, H-ID), 5.00-4.02
(m, 19H, 8
CHZPh, H-3c, 2A, 28), 4.01 (dd, 1H, H-2E), 4.00-3.20 (m, 1GH, H-3E, 4E, 5~,
GaE, GbG, 4c, 5c,
3e, 4H, Sg, 3A, 4,,, 5A, Sn, 6aD, 6th), 2.02, 2.00, 1.75, 1.G5 (4s, 12H,
C=OCH3), 1.40, 1.32 and
1.00 (3d, 9H, H-6~. 6B, 6~). "C NMR (partial) (CDC13):cS 170.2, 1 G9.9, 1
G9.3, 168.7, 164.9
(GC, C=O, C=N). 103.2 (C-ID), 101.4 (2C, C-1,,, 1H), 99.0 (C-lE), 94.8 (C-I~),
21,1, 20.9,
Z0.8 (3C, CH3C=O), 19.1, 18.2 (3C, C-6,,, 5g, G~). FABMS of C~o3Hi,3C13Nz02~
(M, 1917.4),
mfz 1930.9 [M+NaJ'. Anal. CaIcd. for C,o3H"3CI3Nz027 : C, 64.52 : H, 5.94 ; N,
1.46. Found
C, 64.47 ; H, 5.99 ; N, 1.45.
4
8~i~elln flcsenri Sa
ICS-ELISA d'Iobi6itCoe
CA 02434685 2003-07-04
AntigEnicitE des Oligosaecharldes SynthEtiques
(ICso - ELISA d'inhibition)
Shigella Jl'exrrerl Sa
La reconnaissance oligosaccharideslanticorps a etC waluee pas ELISA (Enzyme-
Linked
Immunosorbent Assay) d'inhibition, qui sans dormer acres ~ une constants
d'aflinite
absolue », permet de comparer aisement et rapidement un grand hombre de
ligands. Il
apparait que le rEsidu glucose E E5t indispensable a la reconnaissance quelque
soil 1'anticorps
etudie. En outre, les constantes d'inhibition (ICSO) les plus favorables sont
obtenues pour !es
pentasaccharides. Les donn6es disponibles indiquent que
- les sequences CDA(E)B-OMs et DA(E)BC-OMs sont les mieux reconnues par les
IgA TS
et IgA C5, aver des ICso de fordre de 25 mM.
- le trisaccharide A{E)B-OMs (ICso = 1,3 ~, correspondant au site de
ramification, semble
definir to structure minimale necessaire ~ la reconnaissance par fIgG C20.
Mais, le
pentasaccharide DA(E)BC-OMs (ICso = 25 ~Imol) correspond ~ la sequence la
mieux
reconnue parmi celles testes.
Ces resultats correlent parfaitement aver les donnees d'analyse
conformationnelle. Le
pentasaccharide DA(E)BC-OMc est, a ce niveau d'etude, le plus representatif de
fantig6nicite
du PS de S. flexrleri sErotype Sa. D'autre part, la difference d'afI'mite poux
Ies oligosaccharides
synthetiques observes entry IgG serique et IgA secretoires (sIgA) est
remarquable. Par
analOgie aver le mode du reconnaissance des antigenes polymeriques par Ies
immunoglobulines d'isotype M, fhypothese a ete emise que. le caractere
dimerique des slgA
impliquait un phenomene d'avidit6 compensant Ia faibIe affinite intrinsCque
des sites do
reconnaissance. Les donnees quant a la reconnaissance sIgA:LPS valident cells
hy~poth~se.
L'etude des complexes sIgA/oligosaccharides en solution a ete affinee par RMN
a (aide
de deux techniques particulierement petformantes et compl~mentaires (i) Ies
exp6ziences de
NOE-transferes et (ii) les experiences de transfert de saturation (STD-RMN),
compatibles
aver la faible afFmite des sIgA pour les pentasaccharides.
Les donnees de M.-J. Clement (These Mars 2003, Unite de Resonance MagnCtique
Nucleaire des Biomol6cules), obtenues sur les sequences DA(E)BC-OMs et CDA(E}B-
OMs,
indiquent que Ies pentasaccharides en interaction aver les IgA conservent la
conformation
moyenne qu'ils adoptent sons leur forrne fibre. II apparait egatement que les
atomes
d'hydrogene port6s par le glucose E et le rhamnose B sont tons en interaction
directs aver les
anticorgs. Daps leer ensemble, les resultats sont en faveur dune contribution
directs a
(interaction de ehacun des residus eomposant les pentasaccharides et sous-
entendent que les
slgA accommodent un bpitope relativement large. En accord aver les donnees de
TCso
obtenues pour fIgG C20, yes resultats confirment Egalement que le site de
ramification A(E)H
Shigtllo Jlexneri 9a
iCs. - ELISA d'ladihiNoa
CA 02434685 2003-07-04
represents to sequence critique pour la reconnaissance du PS de S. ,~lexneri
Sa par les anticorps
monocl.onaux protecteurs.
ShigellaJlexneri Za
Comme en ssrie S. flexneri ssrotyPe Sa, la reconnaissance
oligosa.ccharides/anticorps a tits
evaluee par EL1SA d'inhibition. Quatre des cinq IgG evaluss semblent avoir un
eomportement
tres proche en terms de reconnaissance. du LPS et different de celui du
quatrieme, l'IgG F22-
4, plus aff"m. Cependant quelque soit I'anticorps pris en consideration, le
residu D-glucose E
et le residu N acetyl-b-glucosarnine D semblEnt tous deux iruiispensables ~ la
reconnaissance.
Ainsi, la sequence minimale reconnue par I'IgG F22-4 est le trisacoharide
lineaire ECD-
OMe. Dune importance moindre puisque non reconnue en tent que tells ~. la
concentration de
1 mM (absence de reconnaissance du pentasaccharide DAB(E)C-OMs par exemplej,
la
ramification B(E)C joue sgalement un role critique. En comparaison, la
contribution du
residu A a la reconnaissance semble faible si celui-ci est introduit ~
fextremite reductrice des
sequences ECD-OMs et B(E)CD-OMs.
Comme illustre pour 1'octa- et le deeasaccharide. 1'slongation de la chains
~.1'extrCmitd
r6ductrice par introduction de la. sEquence B(E)C ameliore grossiLrement la
reconnaissance
pour quatre des anticoips drn facteur 5. Il est vraisemblable que la
contribution impliquee est
de type confotmationnel et non pas interaction directs. En revanche, anti
amelioration d'un
facteur 30 cst observes pour 1'IgG A-2, rl est possible que tie demier
aecommode. un epitope
plus long. La comparaison des donnses obtenues pour 1'octa- et le
pentasaccharide indique
quc dens 1'ensemble, la contribution do la sequence DA est minims, voice
negative daps le cas
de I'TgG F22-4.
En permettant la couverture complete des sequences obtenues par permutation
circulaire
des residue composant 1'unite repetitive de 1' Ag-0 de S. flexneri. 2a,
1'Evaluation des deux
pentasaccharides ECDAB-OMs et AB(E)CD-OMs apportera des elements d'information
comptementaires a notre etude. A ce stale, nos resultats suggerent que quatre
des cinq IgGs
disponibles reconnaissent, avec anti affinite plus ou moms bonne, anti
sequence minimale
commune (B(E)CD) qui peat titre qualifies d'u epitope immunodominant ». Dens
tous tee cas,
1'elongation de la chains ameliore Ia reconnaissance. Cette observation
suggere que lee
anticorps reconna.issent un spitope presents de faron multiple le long du
polymers (17 unites
rspstitive.s en tnnyenne) et non pas unique en bout de chains.
2
AntigEnicitE des olioecelurides synthetiques
(IC,o~EL1SA d'inhibitioa)
CA 02434685 2003-07-04
Antigenieit~ des oIiosaccharides synthefiques
(ICsa-ELISA d'inhibition)
Shigella Jlexneri 2a
L'etude de la reconnaissance, en ELISA d'inhibition, de 1'ensemble des di-,
tri-, tetra-
et pentasa.ccharides obtenus par permutation circulaire des r~sidus composant
1'unite
repetitive (AB(E')CD) de. fAg-O de S flexneri 2a, et specifiques de ce
s~rotype, par cinq
anticorps monoclonaux d'isoptype G, sprscifique de S. flex~eri 2a et
protecteurs a et~
completee (Annexe 3). Deux groupes d'anticorps peuvent titre distingues. Tout
d'abord F22-
4, anti IgGI qui est le soul anticorps ~ reconnaitre le.s oligosaccharides
lineaires E'CD,
E'CDA et E'CDAB. Pour les quatre autres anticorps, le residu B est requis pour
observer anti
inhibition a anti concentration en ligand inferieure ~ 1 mM. Ainsi, la
sequence B(E')CD
apparait comme la sequence minimale permeYtant d'observer anti inhibition ~
anti
concentration en ligand inferieure ~ 1 mM quelque snit 1'anticozps. Comparee a
la
contribution des residus B, E, C et D, (influence du rGsidu A sur la
reconnaissance, evaluGe
par elongation du fragment B(E')CD a son extrem3te reduetrice ou nan
rt;ductrice, semble
relativement minim~e. Toutefois, les pentasaccharides AS(E')CD et B(E')CDA
sorer les
sequences les mieux reconnue.s par (ensemble des anticorps.
Les hapt~nes selectionnes pour les etudes d'immunogc;nicite sorer les
stsquences E'CD,
B(E')CD et AB(E')CD qui reprEsente (unite reptstitive de 1'antigene 0 (Ag-0).
Afire d'analyser la contribution de residus additionnels dens la.
reconnaissance de la sequence
minimale par les anticorps, 1'octasaccharide B(E')CDAB(E')C et Le
decasaccharide
DAB(E')CDAB(E')C ont ere synthetises. En moyenne, 1'elongation a 1'extrEmite
reductrice
de B(E')CDA par B(E')C pour eonduire a 1'octasaccharide ameliore legeremcnt la
reconnaissance. Il en est de nc»me de 1'elongation de 1'octasaccharide ~ son
extremite non
reductrice par Ia sEquence DA pour conduire au dr'casaccharide. Le cumin des
deux
contributions correspond ~ un gain en reconnaissance superieur ~ un log pour
1'ensemble des
anticoxps t:xcepte 1'IgG FZZ-4. Pour ce dernier, la sequence la mieux reconnue
correspond a
1'octasa:echaride. L'influence de la longueur de la chauie est done manifesto,
elle doit titre
prise en compte duns les etudes d'immunogenicitE.
Proro°ole-IC50-S~JexSa
CA 02434685 2003-07-04
INHIBITION ELISA
PROTOCOLE
Obtention of IgGC20
Mice were immunized intraperitoneally with lOg killed S. ,~exneri Sa bacteria,
Those
developing an anti-S. flexneri Sa LPS antibody response were used for the.
obtention of
monoclonal antibodies as previously described (Kohler and Milstein). The
hybridoma
obtained were tested for the production of a monoclonal antibody specifte for
S. ,~I'exrreri Sa
LPS by ELISA using purified LPS from different S flexneri serotypes. Only
monoclonal IgG
recognizing exclusively LPS Sa were selected,
Inhibition ELISE.
First of all, a standard curve with IgGC20 was established. Different
concentrations of the
anti~dy was incubated at 4°C overnight and then incubated on microtiter
plates coated with
purified Shigella flexneri Sa LPS at a concentration of S~g/ml in carbonate
buffer at pH 9.6,
and previously incubated with PBSBSA 1% for 30 min at 4°C. After
washing with PBS-
Tween 20 (0.05%), alkaline phosphatase-conjugated anti-mouse IgG was added at
a dilution
of 1:5000 (Sigma Chemical C0.) for I h at 37°C. After washing with PBS-
Tween 20
(0,05%), the substrate was added (I2 mg of p-nitrophenylphosphate in 1.2 ml of
Tris, HCl
buffer ph 8.8 and 10.8 ml ofNaCl SM). Once the color developped, the plate was
read at 405
nm (Dinatech MR 4000 microplate reader). A standard curve OD= f(antibody
concentration)
was fitted to the quadratic equation Y= aX2+bX+c where Y is the OD and X is
the antibody
concentration. Correlation factor (r2) of 0.99 were routinely obtained.
Then, the amount of oligosaccharides giving 50% inhibition of IgGC20 binding
to LPS
(IC50) was then determined as follows. IgGC20 at a given concentration (chosen
as the
minimal concentration of antibody which gives the ma~nal OD on the standard
curve) was
incubated overnight at 4°C with various concentrations of each of the
oligosaccharides to be
tested, in PBSBSA 1%. Measurement of unbound IgGC20 was performed as described
above
1
fNHIHITION ELISA
CA 02434685 2003-07-04
INHIBITION ELISA
Shigella flexr2eri serotype Sa
IgG C20 / synthetic oligosaccharides
Only, those oligosaccharides which were recognized with an IC50 bElow 1 mmol/L
are listed.
A(E)B > I OOOfnM pas d'~cart type
A(E)BC 208 +/- I08 uM
A(E)BCD 389 +I- 84 uM
DA(E)B 242 +/- 25 uM
DA(E)BC 39 +/- 19 uM
CDA(E)B 268, 5 +/- 180 uM
Serum IgG pspiu~brevet
CA 02434685 2003-07-04
The serum immunoglobulin G-mediated response to serolype-specific determinants
of
ShigellaJlrxneri lipopotysaccharide profecta against experimental shigellosis
5ctumIgOpapier~brc~ct ~1 02434685 2003-07-04
Introduction
Shigellosis is a major cause of infant morbidity and mortality in developing
countries
but an increasing number of cases in industrialized countries has been
recently reported (33).
Shigella, the causative agent of bacillary dysentery, invades the human
colonic epithelial cells
by manipulating processes that control the host cytoskeletal dynamics (8).
Host response to bacterial infection is characterised by the development of an
acute
inflammation which is responsible for the destruction of the colonic mucosa
and accounts for
the symptoms observed at the early stage of the disease (53). Acquired humoral
immunity
induced upon primary infection confers protection against re-infection,
although the duration
of the disease~induced immunity seems to be limited. Antibody-mediated
protection is
species- and serotype-specific, pointing out LPS as the major protective
antigen (19, 22, 38).
In fact, species and sexotypes among a given species are defined by the
structure of the
repeated saccharide unit that forms the 0-Ag polysaccharide part of LPS (35).
Other bacterial
antigens, as for example the invasins IpaB and IpaC, are recognized by sera
from
convalescent patients (18, 45, 46, G3), but their contribution to protective
immunity is poorly
documented.
Both intestinal SIgA and serum IgG directed against the O-Ag are elicited (13,
28, 31,
69). However, the respective protective roles of local and systemic humoral
immunity remain
unclear. The ineffectiveness of parenterally injected inactivated whole-cell
vaccines in
inducing protection, despite the high level of anti-LPS serum IgG antibodies
raised, has led to
the belief that serum antibodies do not confer protection (21, 25). However,
several indirect
pieces of evidence suggest that anti-O-Ag serum IgG may confer protection
during natural
infection. A correlation was found between the level of anti-LPS IgG
antibodies and
resistance to shigellosis among Israeli soldiers (14, 15), and an inverse
relationship exists
between the age of incidence of shigellosis and the presence of IgG antibodiES
to Shigella
3
Serum IgG papicr~brc~n
CA 02434685 2003-07-04
LPS (47, 63). In addition, a detoxified LPS-based conjugate vaccine
administered parenterally
and eliciting mainly, if not only, serum antibodies has bean shown to induca
protective
immunity ( 1 G j.
The use of experimental models of shigellosis has allowed the study of; at
least in part,
Shigella specific humoral immunity. The rabbit ileal loop model has been used
to assess
SIgA-mediated antibody response (31, 32), and more recently, the mouse model
of pulmonary
infection has been developed (51, GG). Following i.n. administration of
bacteria, mice develop
an acute broncho-pneumonia leading to massive destruction of the lung tissues.
This response
mimics the acute inflammation developed in intestinal tissues in the course of
shigellosis.
This model has been used to assess the immunogenicity and protective capacity
of different
Shigella vaccine candidates, either live attenuated strains administered i.n.,
or subunit
vaccines administered parenterally (3, 34, 36, GS). Using this model, we have
demonstrated
that the IgA-mediated immune response specific for a serotype-specific
determinant is
sufficient to confer protection, (51), with an improved protective capacity of
the IgA when
bound to secretory component (52). In the current study, using the same
experimental model
and specific polyclonal serum or mIgG, we have addressed the protective role
of serum IgG
recognizing serotype-specific LPS determinants or peptide epitopes on the
invasins IpaB and
IpaC.
4
6crum 1gG pnpirnbrrvet ~1 02434685 2003-07-04
Materials and Methods
Bacteria! strai»s
M90T, an invasive isolate of S ,Jlexneri serotype Sa, an,d 454, an invasive
isolate of S. flexneri
setotype 2a, were the virulent strains of reference. For i.n. infection,
bacteria were. routinely
grown on Luria Bertoni agar plates at 37°C. They were recovered from
plates and bacterial
dilutions were performed in 0.9% NaCI with the consideration that, for an
optical density of 1
at 600 nm, the bacterial concentration was 5 x 108 c.fu.lml. Killed bacteria
for systemic
immunizations were prepared from bacterial cultures at stationary phase,
diluted to S x 108
c.f.u. /ml in 0.9% NaCI, and then incubated at 100°C for lh. They were
then kept at -20°C in
aliquots.
Production and characterization of mAbs specific for S. , fTexneri LPS
BALHIc mice were immunized i.p. with 10' c.f.u. of killed S. jlexneri Sa or S,
flexneri Za
bacteria three times at 3 week-intervals. Mice eliciting the highest anti-LPS
antibody response
were given an intravenous booster injection 3 days before being sacrificed for
splenic B cell
fusion according to Kohler and Mitstein (30). PTybridoma culture supernatants
were screened
for antibody production by ELISA using purified S, flexneri Sa or 2a LPS. We
selected only
the hybridoma cells secreting mlgG reacting specifically with LPS homologous
to the strain
used far immunization, i. e. recognizing serotype-specific determinants on the
LPS 0-Ag.
Those selected were then cloned by limiting dilution, and injected i. p, into
histocompa,tible
mice. for ascitis production. mlgG were precipitated with 50% ammonium sulfate
from ascitis
fluid, centrifuged, and dialysed against PBS before being purified using ion-
exchange
chromatography as previously described (2, 50). The avidity of anti-LPS mIgG
was
determined as follows: various concentrations of LPS were incubated in
solution overnight at
4°C with a defined amount of a given mlgG until equilibrium was
reached. Each mixture was
SaumI~(3(18Pt2T-b~°V2t CA 02434685 2003-07-04
then transferred to a microtiter plate previously coated with homologous
purified LPS. Bound
antibodies were detected by using peroxidase-conjugated anti-mouse
immunoglobulins
specific for IgG subclasses. ICso was defined as the concentration of LPS
required to inhibit
50% of mlgG binding,
Aclive arid passive imrnureiEation ojitrice
xo obtain polyclonal serum, mice were immunized i.p. with 5 x 10' killed
bacteria, three
times at 3 week-intervals. After bleeding, anti-LPS antibody titer in the
polyelonal sera was
measured by ELISA, as described below, and those ranging from low (1/4,000) to
high liter
(1164,000) were used fox i.n. passive transfer. Purified mAbs (20 or 2 fig)
w~ete also
administered intranasally. All i.n. administrations were performed using a
volume of 20pI and
mice previously anesthesized via the intramuscular route with 50p1 of a
mixture of 12.5%
ketamine (Merial , Lyon, France) and 12.5°,'o acepromazine
(~~etoquinol, Lure, France). Each
experiment was performed using I O mice per group and was repeated three
times.
Proleclion experiments
Intranasal challenge was performed using either 109 live virulent bacteria
when protection
was assessed by mortality assay or 108 bacteria when protection was assessed
by
measurement of the lung-bacterial load. Naive. mice were used as controls in
each experiment.
Mice immunized i.p. were challenged i.n. with virulent bacteria, 3 weeks after
the Last
immunization. Mice passively transferred i.n. with polyelonal sera or with
purified mAbs
were challenged I h after administration of the mt~.bs. Measurement of lung-
bacterial load was
performed at 24h post infection as follows. Mice were sacrificed by cervical
dislocation and
lungs were removed « en bloc » and ground in 10 ml sterile PHS (Ultra Turrax
T25 apparatus,
Janke and Kunkel IKA Labortechnik GmbH, Staufen, Germany). Dilutions were then
plated
on Trypticase Soy Broth plates for c.fu. enumeration.
I
6
Serum IgG papia-brcva
CA 02434685 2003-07-04
ELISA
Hybridoma culture supernatants were tested by ELISA for the presence of anti-
LPS
antibodies as previously described (2, SO) except that LPS purified according
to Westphal
(67) was used at a concentration of 5pg/ml in PBS. As secondary antibodies,
anti-mouse IgG-
or IgM- or IgA-alkaline phosphatase-Labeled conjugate (Sigma) were used at a
dilution of
1:5,000. To measure the anti-LPS antibody titer in polyclonal serum,
biotin~labeled Abs to
IgG and its different subclasses (IgGl,-Za, -2b, -3) (Pharmingen) aiui avidity
conjugated with
alkaline phosphatase (Sigm ) were used at a dilution of 1:5,000. Antibody
titers were defined
as the last dilution of the sample giving an OD at least twice that of the
control.
,~isdopathologicat studies
Mice were anesthesized, their trachea catheterized, and 4% formality injected
in order to fill
the bronchoalveolar space. Lungs were then removed and fixed in 4% formality
before being
processed for histopathologieal studies. Ten-micrometer paraffin sections were
stained with
Hematoxiline and Eosin (HE), and observed with a BX50 Olympus microscope
(Olympus
Optical, Europa, GmbH, Hamburg, Germany).
Slaifsfical analysis
Significant differences were compared using the Student's test. Probability
values < 0.05 were
considered significant.
7
$G'fllml~G, Jl9PlMWV~ CA 02434685 2003-07-04
Results
1) Protection conferred upon systemic immunization or lntranasal
administration of
specific immune serum.
Firstly, to address the role of the systemic anti-LPS IgG antibody response in
protection against the mueosal infection, we assessed the protection conferred
against i.n.
challenge with a lethal dose of S. flexneri 2a bacteria in mice immunized i.p.
with the
homologous killed bacteria. Antibodies induced upon such an immunization were
mainly
anti-LPS IgG antibodies (data not shown) with all the IgG subclasses similarly
elicited
(Figure 1 A). No mucosal response was elicited, as reflected by the absence of
anti-LPS
antibody response detectable in the bronchoalveolar lavage of immunized mice.
Only 40% of
the immunized mice survived the. i.n. challenge, whereas I00% of naive mice
succumbed. The
low efficacy of systemic immunisation in inducing protection could be due to
either the
inability of anti-LPS IgG to be protective or the absence of the protective
antibodies (or their
presence but in insufficient amount) in the mucosal compartment at the time of
i.n. challenge.
We, therefore, tested whether the anti-LPS IgG antibodies may confer
protection if
present locally prior to mucosal challenge. Polyclonal sera exhibiting
different anti-LPS
antibody titers were intranasally administered to naive mice 1 h prior to i.n.
infection with a
sublethal dose of S. flexr~eri 2a bacteria. Protection was assessed by the
reduction of the lung-
bacterial load in comparison to control mice and mice receiving preimmune
serum. In contrast
to control mice and mice receiving preimmune serum, naive mice receiving
anti~LPS IgG
serum showed a significant decrease of the lung-bacterial load. The reduction
was dependent
on the amount of anti-LPS IgG antibodies administered as reflected by the anti-
LPS antibody
titer of the immune serum used for passive transfer. Thus, the highest
reduction was obtained
with serum having the highest anti-LPS antibody titer ( I/G4,000) (Figure 1 B,
c) (p=5 x 10-6 in
comparison to mice receiving preimmune serum). However, in mice receiving
immune serum
8
CA 02434685 2003-07-04
Serum IgC papist-hrcvZ
with lower anti-LPS antibody titer (1/16,000 and 114,000) (Fig. IB, a and b),
even if less
efficient, the decrease of the bacterial load was still significant in
comparison to mice
receiving preinunune serum (p = 0, 027 and 0, 015, respectively).
These results demonstrated that, if present locally at the time of rnucosal
challenge, the
anti-LPS IgG antibodies were protective, thus limiting bacterial invasion.
2) Protective capacity of mAbs specific for S, flexrzeri Za serotype
determinants and
representative of the different IgG subclasses
Depending of the infecting strain, different subclasses of IgG specific for
LPS are induced
followizig natural Slzigella infection (28). To test whether the different
anti-LPS IgG
subclasses exhibit similar protective capacity, marine mIgG specific for
serotype determinants
on the O-Ag and, representative of each of the four marine IgG subclasses were
obtained, We
selected 5 mIgG specific for S. Jlexoeri 2a LPS : mIgG F22 (IgGl), mlgG D15
(IgGl), mIgG
A2 (IgG2a), mIgG E4 (IgG2b) and mIgG C1 (IgG3). The avidity of each mIgG for
LPS,
defined by ICSo, ranged from 2 to 20 ng/ml. To analyse the protective capacity
of the selected
mAbs, naive mice were administered i.n, with each of the purified mIgG prior
to i.n.
challenge with a S. fJexneri sublethal dose. Upon challenge, lung-bacterial
load in mice
passively administered with 20 ~g of Each of the mIgG specific for S. flexneri
2a LPS was
significantly reduced in comparison to mice receiving PBS (Fig. 2A). Upon
passive transfer
using 2pg ofmlgG, only mIgG D15, A2 and E4 were shown to significantly reduce
the lung-
bacterial load in comparison to control mice, but with much less efficiency
than that observed
using 20p,g (Fig. 2A), As shown in Figure 2H, reduction of lung-bacterial load
in mice
receiving 20 p.g of mIgG was accompanied by a reduction of inflammation and
therefore of
subsequent tissue destruction. In comparison to control mice showing an acute
broncho-
alveolitis with diffuse and intense polymorphonuclear cell infiltration
(Figure 2H, a, h)
9
Setuml&Crpapia-bcnvet ~1 02434685 2003-07-04
associated with tissular dissemination of bacteria (Figure 2B, c), only
restricted areas of
inflammation were observed, essentially at the infra- and peribronchial level
(Figure 2B, d, e),
where bacteria localized (Figure ZB,,~. Following passive administration with
2~g of mIgG,
inflammation resembled that of the control mice with a similar pattern of PMN
infiltration
and tissue destruction, in a,eeordance with the very loin, if any, reduction
in lung-bactezial
load (data not shown).
3) Serotype-specific protection induced by the anti-LPS mlgG
Antibodies specific for epitopes common to several serotypes of a given
species as well as
serotype-specific antibodies are elicited upon natural or experimental
infection (58, 64).
However, the serotype-specific protection observed following natwal or
experimental
infection suggests that the antibodies directed against serotype determinants
play a major
protective role (19, 38). For instance, mIgA specific for S. flexneri serotype
Sa has been
shown to protect only against homologous challenge (51). We, therefore, tested
whether the
protection observed with the anti-LPS mIgG obtained in this study was also
sErotype-specific.
Mice passively administered with 20 ug of m>;gG C1 specific for S. J).~2xneri
2a were protected
against homologous challenge, but not upon heterologous challenge with S
Jlexneri Sa
bacteria (Fig. 3A). Similarly, mice receiving 20 ~g of mIgG C20, a mAb
specific for S
flexrreri serotype Sa and, of the same isotype than mIgG C1, i.e. IgG3, showed
a significant
reduction of lung-bacterial load upon i.n. challenge with S flexneri Sa, but
not with S. flexneri
2a (Figure 3A). In mice protected against homologous challenge, inflammation
was
dramatically reduced with a slight infra- and peribronchial PNfhl infiltrate
remaining present
(Figure 3B, b and r). In contrast, in mice not protected upon heterologous
challenge (Figure
3B, a and c~, inflammation and tissue destruction were similar to those
observed in control
mice (Figure 2B, a and b).
sCfvln (gG pspier~brevet C~1 02434685 2003-07-04
4) Protective capacity of mIgG specific for S. fl'exneri invasins
The invasins IpaH and IpaC are essential to the expression of the Shigella
invasive
phenotype (39). Moreover, they are targets for the humoral response since
antibodies specific
for both proteins are detected in sera ofpatients convalescent from
shigellosis (18, 45, 46, 63).
To assess whether the anti-invasin antibody response may contribute to
protection, in addition
to the anti-LPS anfibody response, we used mlgG recognizing different epitopes
on IpaB or
IpaC (z, 50). Whatever the dose used, in contrast to mIgG C20, no reduction in
Iung-bacterial
load was measured upon challenge in mice treated with mIgG H16 and mIgG H4
recognizing
distinct epitopes in the central region of IpaB or with mlgG J22 and mlgG K24
recognizing
the N~ and the C-termini domain of IpaC, respectively (Figure 5). Protection
was also not
observed upon combining anti-IpaB and anti-IpaC mIgG (data not shown.
11
CA 02434685 2003-07-04
Seism I~Cr paDier-brevet
Dixcussion
To date, the respective roles of local and systemic humoral immune responses
specific for
LPS 0-Ag in protection against Shigella infection remained unclear, although
this question is
crucial for the design of accurate vaccine candidates, Indirect evidence has
suggested a
protective role for anti-LPS IgG (14, 15, 1G, 34, 47, 63). We demonstrate here
for the first
time, using poiyctonal serum and specific mAbs, that the systemic IgG-mediated
response
specific for serotype determinants carried by LPS 0-Ag confers protection
against mucosal
infection. if present locally at the time of bacterial challenge.
LPS has been recognised for a long time as the major protective antigen (19,
22, 38).
However, the question of the protective role of the antibody response to
bacterial proteins
remains unanswered. Among the proteins recognized by sera from patients
convalescent from
shigellosis, IpaB and IpaC, the invasins involved in the entry of bacteria
into enterocytes, are
two major antigens. Only indirect evidence suggested that the systemic
response to these two
virulence factors was not essential for protection ( 1 G). We show here that
mlgG specific for
IpaB or IpaC are not protective despite the fact that they are directed
against epitopes located
in different regions of these proteins (2, 50) and that they have been shown
to interfere with
their functional properties in in vitro studies (4, 40). The most likely
explanation is that these
invasins, that are secreted through the type III secretion apparatus, are
injected straight into
the host cell, upon contact of the bacterium with the cell membrane (G, 41).
Therefore, there is
probably very limited access, if any, for specific antibodies to interact with
their targets.
Although not tested. it is unlikely that the local SIgA-mediated response to
these proteins will
be protective.
In the past, several sets of mAbs of M or G isotype specific for Shigella
species have been
produced. They are directed against the 0-Ag of S sonnet (1 ), of S.
dysenteriae ( 20, 56, 60)
and, of S. flexneri (9, 10 1 l, 24, 26, GO). However, as the goal Was to
develop diagnostic. tests
12
Scrum IgG papler-breve~
CA 02434685 2003-07-04
for Shigella identification (12, 27), their protective properties have not
been investigated.
Except for a few (42, 43), the sequence of the VH and VL genes is unknown.
Similarly, the
oligosaccharide determinants they recognize have not been characterized,
except for 2 mAbs
specific for S dysenteriae 1 (43, 49). Thus, for a better understanding of
carbohydrate
antigenlantibady interactions, we are currently characterising the fine
specificities of
recognition between the mAbs obtained in this study and the 0-Ag they
recognize.
To obtain tnIgG, hybridoma cells were selected, upon cell fusion, on the basis
of their
secretion of mAb recognizing determinants specific for the S. flexneri
serotype used for
immunization, i.e. serotype 2a and serotype Sa, respectively. During the
screening, we
observed that most of the hybridoma cells tested (about 90%) were secreting
serotype-specific
mAbs. This result slightly differs from previous reports showing the obtention
of mAbs
directed to determinants common to several S. flexneri serotypes including 2a
and Sa (11, 24),
However, it may be explained by recent new insights on bacterial 0-Ag
conformation. For
instance, in the case of S. dyser~teriae l, the a-z-I2hap-(1-->2)-a-D-Galp
disaccharide
represents the major antigenic epitope on the O-Ag. Interestingly, in the
proposed
conformational model of S. dysenteriae 1 O-Ag, which is a left-handed helical
structure, the
galactose residues protrude radially at the helix surface, therefore resulting
in a pronounced
exposure of both the galactose and the adjacent rhamnose of each repeating
unit (44). A
similar result has been obtained in our hands with the O-Ag of S. .Jlexneri
Sa. In that case, the
branched glueosyl residue specifying this serotype and required for
recognition by serotype-
specific antibodies (L. Mulard and A. Phalipon, persona! communication) points
out of the
surface of the helix, which exhibits a right~handed three-fold helical
structure (M. J. Clement
and M. Delepierre, personal communication ). Therefore, we may reasonably
hypothesize that
these peculiar sugar residues repeatedly exposed at the O-Ag surface, and
therefore at the
13
Scrum IgG papier-brevet
CA 02434685 2003-07-04
bacterial surface, preferentially trigger B cell receptor-mediated
recognition, thus leading to
the induction of a predominant anti-serotype specific antibody response.
In humans, depending on the infecting strain, different subclasses of IgG
specific fox LPS
are induced following natural Shigella infection (28). For instance, S
flexneri 2a and S.
dysenxeriae 1 preferentially induce IgG2, whereas S. .sonnei mainly induces
IgGI. Similarly,
upon vaccination with glyeoeonjugate vaccines using detoxified LPS from S,
flexneri 2a and
S. sonnei, the same pattern is obsen~ed, IgG2 and IgGI, respectively. These
antibodies may
confer protection by different pathways involving or not the complement
cascade. In the
present study, all the different marine IgG subclasses were shown to be
protective, suggesting
that depending on the subclass, different mechanisms may be involved in IgG-
mediated
protection. Whereas antibody-dependant cellular cytotoxicity (ADCC) has been
reported for
Shigella-specific secretary IgA and lymphocytes from the gut-associated
lymphoid tissues
(61), Shigella IgG-mediated ADCC occurs in vitro with splenic T cells but not
with T
lymphocytes from the GALT (G2). Further studies using mice deficient for T
cells or for
proteins of the complement cascade will be required to analyze the IgG-
mediated protective
mechanisms in viva.
The protective role of the serotype-specific antibody response has been
firstly emphasized
in a study using a m~orwclonal dimeric IgA (mIgA) specific for a S. flexneri
serotype Sa
determinant (51). Here, we demonstrate that mIgGs specific for S, flexneri
serotype 2a or
serotype Sa also confer serotype-specific protection. It seems that whatever
the antibody
isotype and the bacterial strain, the serotype-specific antibody response is
protective against
homologous bacterial challenge. It should be noted that using the same amount
of mlgA and
mIgG specific for S. flexneri Sa, both exhibiting a similar ICs for LPS,
reduction in lung-
bacterial load was much more efficient with mIgA. Actually, in contrast to
mIgG, protection
was observed in the presence of 2pg of mIgA. The dijcrepancy between the two
isotypes may
14
Serum 7gG pnpier.brevet
CA 02434685 2003-07-04
be duE to the dimericlpolymeric (dlp) form of mIgA, which mimicks the IgA
response at the
mucosal surface. In contrast to monomeric IgG, interaction of d/p IgA
exhibiting at least four
antigen-binding sites with a specific determinant highly repeated on the
bacterial O-Ag
surface may lead to the formation of aggregates that are efficiently removed
by local physical
mechanisms (17). Also, quantitative assessment of IgG and IgA subclass
producing cells in
the rectal mucosa during shigellosis in humans has revealed the predominance
of the IgA
response. The IgG response which is about 50 times lower than the IgA response
is mainly
IgG2 and correlates with the presence of specific IgG2 in serum. This
correlation suggests
that the majority of the Shigella specific serum antibodies are derived from
the rectal mucosa
(29). Together, these results suggest that in the situation where both local
and systemic anti-
LPS antibody responses are induced, as for example upon natural infection, the
local SIgA-
mediated response will be the major protective response, with the IgG-mediated
response
possibly contributing to a lesser extent to local protection.
On the other hand, our data suggest that in the absence of local SIgA-mediated
response, as for example upon vaccination via the systemic route using
glycoconjugate
vaccines, the systemic anti-O-Ag response induced is effective in protecting
against
homologous Slaigella infection, if the effectors are present locally. Previous
reports have
shown that serwn IgGs may protect from gastrointestinal infections (7, 54).
Therefore, it
should be admitted that serum IgG efficiently gain access to the intestinal
barrier in ozder to
prevent bacterial invasion and dissemination. How IgG crosses the epithelial
barrielr to
function in mucosal immunity remains unclear. One possible pathway is passive
transudatinn
fxom serum to intestinal secretions (5, 37, 67). After its passage of the
intestinal barrier
through M cells and its interaction with resident macrophages and epithelial
cells, Slaigella
initiates an inflammatory response leading to infiltration of the infected
tissues with
polymorphonuclear cells (53). We may therefore reasonably envision that
specific serum IgGs
IS
Serum IgG papier-brevet
CA 02434685 2003-07-04
transudate to the intestinal tissue during this inflammatory process that
occurs very soon after
bacterial transloeation. Another explanation could be the involvement of the
FcRn receptor in
IgG transport. FcIW was firstly identified as the Fc receptor responsible for
transferring
maternal IgGs from mother's milk across the intestinal EC of the neonatal gut
of rodents.
Much evidence supports the concept that FcRn is ubiquitously expressed in
adult tissues and
plays a role in IgG homeostasis, dealing with IgG half life (23). It has been
recently reported
that this receptor is expressed by enterocytes in human adults and mediates
transcytosis of
IgG in both direction across the intestinal epithelial mnnolayer (57). Further
investigation is
required to improve our Imowledge on the role played by Fcltn in IgG-mediated
protection of
the intestinal barrier against enteropathogens. Nevertheless, the existence of
such a pathway
already enlarges the current view of the humoral response at mucosal surfaces.
To conclude, our data are in favor of the hypothetical concept proposed by
Robbins et
al. stating that protection against bacterial enteric diseases may be
conferred by serum IgG
antibodies to the 0-Ag of their bacterial LPS (59). The demonstration of the
protective role
of anti-LPS IgG-mediated systemic response against Shigella infection supports
vaccine
approaches based on detoxified LPS/protein glycoconjugate vaccines
administered
parenteTally (47). In addition, the serotype-specific protection suggests
that, upon their
characterisation, the protective serotype-specific determinants for prevalent
Shigella strains
could be suitably combined in order to develop a multivalent synthetic vaccine
for parenteral
vaccination, since promising results have been recently obtained with
synthetic
oligosaccharides as immunogenic conjugates (55).
1G
CA 02434685 2003-07-04
Serum IgG papier-brevet
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CA 02434685 2003-07-04
Serum 7 ~0 p~pierbrcvet
Legends of figures
Figure 1 : Protection conferred by immune serum specific for S.jdexnert 2a LPS
intranasa113~ administered prior to i.n. challenge.
A) Serum IgG subclasses elicited in mice upon i.p, immunization with killed S.
Jlexrreri 2a
bacteria. - represents the mean value of the antibody titer (n=10 mice).
B) Protection assessed by reduction of lung-bacterial load in mice receiving
anti-S.flexneri 2a
LPS immune serum raised upon i.p, immunization, lh prior to i.tt. challenge
with a sublethal
dose of S, flexneri 2a bacteria. a, b, c, correspond to immune sera exhibiting
an anti-S
,flexneri 2a LPS IgG antibody titer of 1/4,000, 1/16,000 and 1/64,000,
respectively. Standard
deviatifln is indicated (n-I O mice per group).
Figure 2 : Protection conferred by different subclasses of mIgG specific for
S. Jlexneri 2a
serotype determinants.
A : mice receiving intranasally 20pg and 2pg of purified mIgG, respectively,
lh prior to i .n.
challenge with a sublethal dose of S flexneri 2a. Lung-bacterial load was
expressed using
arbitrary units with 100 corresponding to the bacterial count in lungs of
control mice.
Standard deviations are represented (n=10 mice per group).
B : Histopathological study of mouse Iungs. Upper row : control mice. Lower
row : mice
receiving mIgG. HE staining : a and d magnification x 40 ; b and a
magnification x 100.
Itnmunostaining using an anti-LPS antibody specific for S. ~lexrreri serot3~pe
2a : c and f
magnification x100.
Figure 3: Serotype-specific protection conferred by the anti-LPS mIgG.
A : Mice were receiving i.n. 20Ng of each of the purified mIgG, C20 and C1, lh
prior to i.n.
challenge with a sublethal dose of S. ,/lexneri serotype 2a (.A) or serotype
Sa (B) bacteria.
I
27
CA 02434685 2003-07-04
....,...,~ ag.n y.nym.n-mvrw
Lung-bacterial load was expressed using arbitrary units with 100 corresponding
to the
bacterial count in lungs of contzol mice. Standard deviations are represented
(n=10 mice per
group).
H : Histopathological study of mouse lungs. a and b : mice receiving mIgGC20
specific for S.
~lexneri serotype Sa and challenged with S. flexneri serotype 2a and 5a,
respectively, c and d
mice receivir~ mIgGC1 specific for S flexneri 2a prior to challenge with S.
~exneri serotype
2a and Sa, respectively. HE staining, magnification x 100.
Figure 4 : Protection conferred by mIgG specific .for S. flexrreri IpaS or
IpaC invasins.
Mice were receiving i.n. 20wg of each of the purified mIgG, H4, H16, J22, KZ4,
and C20, lh
prior to i.n. challenge with a sublethal dose of S flexneri semtype 5a. Lung-
bacterial load was
expressed using arbitrary units with 100 corresponding to the bacterial count
in lungs of
control mice. Standard deviations are represented (n=10 mice per group j.
28
Serum Ig0 papia-brevet
CA 02434685 2003-07-04
Acknowledgements : We thank Nicole blusher and Michel Huerte (Unite de
reeherche et
d'expertise Histoteclmologie et Pathologie, Institut Pasteur) for their
unvaluable work in
histology, Verronique Cadet (Hybridolab, Institut Pasteur) for her help in
mAbs production,
and Josette Arondel for the mice experiments she did just before getting
retraited. We also
thank Isabel Fernandez and Maria Mavris for careful reading of the manuscript.
P. J. S. is a
Howard Hughes A~fedical Institute scholar,
29
CA 02434685 2003-07-04
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PREPARATION OF THE OLIGOSACCHARIDE-TETANUS TOXOID CONJUGATES
CGS0303-8-3: Tetanus toxoid-ECD conjugate
CGS0303-8-4: Tetanus toxoid-B(E)CD conjugate
CGS0303-8-5: Tetanus toxoid- AB(E)CD conjugate
EXPERIMENTAL SECTION
General procedures. N (y-maleimidobutiryloxy) sulfosuccinimide ester (sulfo-
GMBS) was
purchased from Pierce. Tetanus toxoid (TTY (MW 150 kDa) (batch n°FA
045644), was
purchased from Aventis Pasteur (Marcy fEtoile, France), and stored at
4°C in a 39.4 mg.mL't
solution.
Dialyses were performed with Slide-A-Lyzer~ Dialysis Cassettes (Pierce).
Derivatization of the tetanu9 toxoid
Protocol A
To a solution of tetanus toxoid (12 mg, 304 p,L, 0.08 mole) diluted in PBS 0.1
M, pH 7.3
(29G p,L), was added N (y-maleimidobutiryloxy) sulfosuccinimide ester (GMBS)
(3 x 1.53
mg, 3 x 29 pL of an 60 mg.mL-' solution in CH3CN, 3 x 50 equiv), in three
portions every 40
minutes. The pH of the reaction mixture was controlled (indicator paper) and
maintained at ?-
7.5 by addition of aq NaOH O.SM. Following an additional reaction period of 40
minutes, the
crude reaction mixture was dialy2ed against 3 x 2 L ofphosphate buffer 0.1 M,
pH 6.0 at 4°C
to eliminate excess reagent.
Preparation and characterization of the conjugates
Following dialysis, maleimide activated-TT in. phosphate buffer O.1M solution
was divided
into three portions which were fi~th~z reacted with reacted S-
acetylthioacetylated-tri-, tetra-
and penta-saccharide from Shige~Ia flexneri 2a antigen-O in a I:12 molar
ratio, respectively.
Reaction mixtures were buffered at a 0.5 M concentration by addition of
phosphate buffer
1M, pH 6Ø Then, NHzOH, HCl (7.5 pL of a 2 M solution in phosphate buffer 1
M, pH G),
was added to the different mixtures and the couplings were carried out for 2 h
at room
teperature. The conjugated products were dialyzed against 3 x 2 L of PBS 0.05
M, pH ?.4 at
4°C, and further purified by gel permeation chromatography on a
sepharose CL~6B column (1
I
Synthesis ofihe TT C°I~U~7~CS ~1 02434685 2003-07-04
m x 164 mm) (Pharma.cia .Biotech), using PBS 0.05 M, pH 7.4 as eluent at a
flow rate of 0.2
mL.miri ~, with detection by measuring the optical density at 280 nm and the
refractive index.
The appropriate fractions were pooled and concentrated over a dialysis tubing-
visking size 1-
8132" membrane. The conjugates were stored at 4°C in the presence of
thimerosal (0.1
mg.mL'i) and assessed for their total carbohydrate and protein content.
Hexose concentrations were measured by a colorimetric method based on the
anthrone
reaction, using pmLPS as a standard.
Protein concentrations were measured by the Lowry's spectrophotometric method,
using HSA
as a standard and/o total acidic hydrolysis (6N HCl at X°C for 20 h),
using norleucine as an
internal standard.
Determination of hexoses with anthrone
Reagents: The reagents are as follows
Stock sulfuric acid. Add 750 mL of concentrated sulfuric acid to 250 mL of
distilled water
and cool the solution to 4°C.
Anthrone reagent. Dissolve 1.5 g of anthrone in 100 mL of ethyl acetate and
cool the solution
to 4°C.
Standard pmLPS O1 Inaba solution: Prepare a solution at a concentration of $
mg.mL't in
water. Prepare serial dilutions of 800 to 25 p,Mol of pmLPS O1 Inaba standard
solution in
water.
Procedr~re:
Prepare serial dilutions of 800 to 25 pMol of pmLPS O1 Inaba standard solution
in water (1
mL) in screw-threaded tubes. Prepare similarly a reagent blank containing 1 mL
water and
control reagents containing a known amount of pmLPS O1 Inaba or glucose in 1
mL water.
Prepare samples and make up to 1 mL if necessary by adding water. Cool all
tubes in ice-
water.
To each tube, add ~ mL of the concentrated HzSOs and 0.5 mL of the anthronc
solutions. Heat
the tubes at 100°C, caps unscrewed for 3 minutes and then caps screwed
for 7 minutes. After
exactly 10 minutes, return the tubes to an ice-bath and when cool measure the
absorbances in
' a spectrophotometer (Seconam 5.750I), at a wavelength of 625 nm. The
quantity of
carbohydrate in the unknown samples can be read off from the standard curve
prepared with
the standard solution samples and the blank
Determination of~rotein content ~Lowry~
i Reagents: The reagents are as follows
2
Synthesis of the TT conjuggtcs
CA 02434685 2003-07-04
Stock solution A. Add 1 g of sodium carbonate to 50 mL of 0.1 N aqueous sodium
hydroxide
Stock solution B. Mix 1 mL of a stock solution of 1°/a (w/v) aqueous
cupper(II) sulfate with 1
mL of a stock solution of 2% (w/v) aqueous sodium tartrate;
Stock solution AB: Mix 1 mL of the stock solution B with 50 mL of the stock
solution A-
Standard BSA solution: Prepare a solution at a concentration of 1 mg.mL-I in
water.
Folin reagent
Procedure:
Prepare a blank and serial dilutions of BSA by adding 0, 10, 20, 30, 40 or 50
p.L of standard
BSA solution to 1 mL of stock solution AB in clean disposable tubes. Complete
to 1.2 mL by
adding water. Prepare similarly sample dilutiotls by adding 200 p,L of the
sample solutions to
1 mL of stock solution AB.
Incubate for 10 minutes at room temperature and add 100 pL of folin reagent in
each tube.
Incubate for 20 minutes at room temperature and measure the absorbance in a
spectrophotometer (Seconam 5.750I), at a wavelength of 660 nm. The quantity of
protein in
the unknown samples can be read off from the standard curve prepared with the
standard
solution samples and the blank.
RekrensProtemquattrFicaaonIsel~ed GluadeSWedhtGlucideslwdghtGluadc~Jprote
Cluades
y~Id~prxein protin(mgimg) (mmolehrvno
qrnnufiatrenratio
Lowry.'BSA T,AI-l,(adtronc)
I (Nla outcelmntmtl~iF)CD
AT-(E) 2.7y mg 2.1 G 70SZ~'o IL1 5.401, 51% !~
atg S mg
CGS0303-9-32.04 mg
AT-B~)CD2 57 mg 2,05 6d-47,~~ 7.d"/o 6.~0 13
mg 0 19 mg J
CG50303q41 89 m
Ar-na(ExDs.etmg z.7a mg nwGZ~r~ 031 , ~ro a,~~ r~
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