Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
111~417
BACKGROUND OF THE INVENTION
It is well known that carbohydrate structures of various
complexities are the antigenic determinants for a wide range of substances.
It is also well established that relatively small molecules, known as
haptens, can correspond to the structure of the antigenic determinant.
The hapten, when attached to an appropriate carrier molecule,
provides an artificial antigen which,when administered to an animal under
appropriate conditions,will give rise to the production of antibodies
having a specificity for the hapten. Furthermore, in recent years, much
art has developed for the preparation of immunoabsorbents from haptens.
This art involves the attachment of the hapten, normally through covalent
.. I
~ bonding but at times through hydrophobic bonding, to a solid, latex or
; gelatinous support. Thus, the hapten is immobilized so that when the
resulting i~munoabsorbent is exposed to antibodies with combining sites
for the haptenic structure, the antibodies will attach themselves to
the surface of the immunoabsorbent and thereby be specifically removed from
solution.
Many varieties of solid, latex and gel supports for the
preparation of immunoabsorbents have been developed and many ways have
been devised for attachment of the hapten to these insoluble structures.
Although improvements in these matters are possible, the main problem remains
- of having simple access to the desired hapten in a form convenient for
- attachment to the carrier molecule.
It was the original purpose of our work to develop a practical
process for the synthesis of =D-galactosamine hydrochloride (XXXVII).
and of =D-lactosamine hydrochloride (XXXIX) and derivatives of these. I
. !
Both galactosamine and lactosamine, usually in the form of their N-
acetylated derivatives, are found widespread in nature. They occur in
glycoproteins, glycolipids and mucopolysaccharides. As such they are
important building units found in the blood group substance antigenic
determinants.
(- I
i
1111417
The main prior art source of D-galactosamine is the acid
hydrolysis of chrondroitin sulfate C which is obtained by extracting
; cartilaginous tissues such as tendons, trachea and nasal septa. These
yields are uncertain and it is difficult to obtain a crystalline product.
Numerous chemical syntheses exist which include the opening of 1,6:2,3,-
dianhydro-~-D-talopyranose with ammonia or with azide ion. However, these
methods involve six to eleven separate chemical transformations starting
from the simple sugars. Shorter methods depend upon rather rare sugars
as starting materials.
Inversion of the C-4 configuration of glucosamine through
displacement of a 4-0-sulfonate of 2-acetamido-2-deoxy glucopyranosyl
derivatives has also been utilized for the synthesis of D-galactosamine.
However, the elaboration of glucosamine to the necessary starting material
is tedious.
.
The synthesis of lactosamine is more difficult as it nec-
essarily involves a glycosylation of a galactosyl halide with an elaborate
. derivative of 2-acetamido-2-deoxy-glucose. The most recently published
method requires nine chemical transformations, starting from 2-acetamido-
, . .
2-deoxy glucosamine, prior to the glycosylation step.
In accordance with a feature of the present invention,
there is provided a reagent that allows efficient and high yield pre-
parations of glycosides which contain the 2-acetamido-2-deoxy-a-D-
galactopyranosyl group which is-found, for example, in the antigenic
determinant for the human A blood group and the Forssman antigen. The
~` 25 reagent thus claimed useful is 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-
galactopyranosyl chloride (XXIII) prepared simply from D-galactal triacetate
(I) in high yield.
It has long been anticipated that the use of a ~-glycosyl halide
would tend to yield the a-(1,2-cis)-glycosidic linkage through Walden
inversion of the reacting center under Koenings-Knorr reaction conditions
417
when the 2-substituent is so chosen as to not participate in a reaction
at the anomeric center. Thus, for example, Wolfrom, Thompson and Linebeck
(J. Org. Chem., 28, 860 (1963)) developed tri-0-acetyl-2-nitro-~-D-
glucopyranosyl chloride for the purpose of synthesizing a-D-
glucopyranosides. Indeed, several papers have appeared in the recent
literature which utilize 2-azido-2-deoxy-~-D-glycopyranosyl chlorides
such as is reported in processes of this invention leading to the
formation of 2-azido-2-deoxy-~-D-galactopyranosides. However, it must
be noted that the processes reported by Paulsen and co-workers (Angew.
Chem., Int. Ed., 14, 558 (1975); Tet. Lett., 1493 (1975) and 2301 (1976);
Angew. Chem., Int. Ed., 15, 440 (1975)) are of limited, if any commercial
value in view of the extreme difficulty in achieving the synthesis of
: the desired 2-azido-2-deoxy reagent; namely, 6-0-acetyl-2-azido-3,4-
- O-benzyl-2-deoxy-~-D-galactopyranosyl chloride.
;~ 15 This invention reports a novel process for preparing ef-
~i~ ficiently the compound 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-
galactopyranosyl chloride ( XXI I I ) and its engagement in reactions with
:: :
alcohol to form 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-galactopyranosides
(A) under appropriate Koenings-Knorr type conditions for the condensation.
The invention in AcO
AcO ~
OR
A
part concerns the discovery of processes that render compound XXIII a
readily available reagent for use in reactions leading to products of
type A. Thus, it has become commercially feasible to synthesize the
terminal trisaccharide antigenic determinant for the human A blood as
is present in structures B for the type 1 and type 2 antigenic determinants
for the human A blood group. The trisaccharide is synthesized in a form
useful for the preparation of artificial antigens and immunoabsorbents
related to the human A blood group.
` (-` - !
111~4~7
,
H~ _O
, f o~ '
OH
H
(Type 1 A Determinant)
HO
OH
HO NHAc
. AcNH ¦ ~ O \ HO
"', ~ 0~o~ I
- ~ OH
HO
(Type 2 Determinant)
.'` ~
B
The formation of a-azido-~-nitratoalkanes from the reaction
of olefins with sodium azide and ceric ammonium nitrate has been reported
by Trahanovsky and Robbins (J.Am. Chem. Soc., 93, 5256 (1971)). However
the extension of the above reaction to vinylic ethers or structures as
complex as D-galactal triacetate is not obvious. The base of this
invention was the discovery that the addition of the azide and nitrate
groups to 1,2-unsaturated sugars can be made to proceed in high economical
yield to form the 2-azido-2-deoxy glycosyl nitrate.
SUMMARY OF THE INVENTION
In accordance with the basic aspect of the present invention,
the treat~ent of protected glycals with azide-ion in the presence of
11.1~17
,;
ceric ammonium nitrate results in the addition of an azide group and a
nitrate group to the C-2 and C-l positions, respectively, of the glycal.
` These novel products, namely the anomeric mixture of 2-azido-2-deoxy
glycosyl nitrates, allow entrance into the following classes of compounds: I
(1) the 2-amino-2-deoxy sugars by hydrolysis of the nitrate group and 1,
reduction of the azido group,
(2) the 2-azido-2-deoxy glycosyl halides by displacement of the glycosyl
- nitrate,
(3) the 2-amino-2-deoxy glycosides by reaction of the 2-azido-2-deoxy
glycosyl halides.
The virtue of the azido group is that it is a non-participating progenerator
of an amino function and as such does not interfere with the synthesis of
the 2-amino-2-deoxy-~-D-glycosides.
In accordance with a feature of the present invention, the
2-azido-2-deoxy glycosyl nitrates can be converted to the corresponding
2-amino-2-deoxy sugars by hydrolysis of the nitrate and protecting groups,
and reduction of the azido group by methods well known to those skilled
in the art. Hydrolysis may precede reduction or vice versa. N-acetylated
; derivatives of the amino sugars can be obtained by conventional methods.
In accordance with a further aspect of the present invention,
- the 2-azido-2-deoxy glycosyl nitrates may be treated with a halide
salt to effect the displacement of the nitrate group and
to produce the 2-azido-2-deoxy glycosyl halides, which are novel compounds.
In a preferred procedure, by treating with iodide ion, an anomeric
mixture of the glycosyl nitrates produces the thermodynamically more
favorable anomer, 2-azido-2-deoxy-~-D-glycosyl iodide. The ~-glycosyl
iodide is readily displaced with one equivalent of chloride ion through
- inversion to give in high yields the 2-azido-2-deoxy-~-D-glycosyl chloride.
This route to the ~-halide is advantageous as it allows conversion of the
nitrates to a reaction product which comprises predominantly the 2-azido-
2-deoxy-~-D-galactosyl chloride, which is useful for the formation of a
417
:-
2-deoxy-~-D-glycosjde, an integral unit of the A blood group determinant.
The reagent thus claimed useful-is 3,4,6-tri-0-acetyl-2-azido-2-deoxy-
~-D-galactopyranosyl chloride (XXIII).
The 2-azido-2-deoxy glycosyl halides may be used to prepare
2-amino-2-deoxy glycosides under conditions for glycosidation, such as
those generally known in carbohydrate chemistry as Koenings-Knorr
conditions. These reactions involve the treatment of the glycosyl halide
with an alcohol in the presence of a promoter to effect the replacement
of the halogen by the alkoxy group of the alcohol. The 2-azido-2-deoxy
glycoside, thus obtained, is reduced by methods well known to persons
skilled in the art to obtain the 2-amino-2-deoxy glycosides. In addition
the protecting groups can be removed in order to deblock the glycoside.
Specifically, 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-galactopyranosyl
,.
chloride may be reacted with 8-methoxycarbonyloctyl-2-0-(2,3,4-tri-0-
benzyl-~-L-fucopyranosyl)-4,6-0-benzylidene-~-D-galactopyranosyl in the
presence of a promoter. The trisaccharidic product is deblocked and its
azido group is reduced to the amine which is subsequently acetylated
to give 8-methoxycarbonyloctyl-3-0-(2-acetamido-2-deoxy-a-D-galacto
pyranosyl)-2-0-(~-L-fucopyranosyl)-~-D-galactopyranoside. This latter
product corresponds to the antigenic determinant for the human A blood
. .; .
; group and can be used to prepare an immunoabsorbent specific for the
anti-A antibodies by attachment to an insoluble support. Also, this
latter product can be used to inhibit the reaction between anti-A
antibodies and human A erythrocytes. Furthermore, the product can be
used to prepare artificial antigens which allow the raising, through
immunization, of monospecific anti-A antibodies in test animals. The
subsequent isolation of these antibodies using the immunoabsorbent
then provides an important and useful reagent for cell and tissue typing.
L417
Broadly stated, the invention provides a process which comprises
reacting an acylated 2-azido-2-deoxy glycosyl nitrate with a halide salt
in a suitable solvent to form an acylated 2-azido-2-deoxy glycosyl halide~
The invention also broadly provides a process which comprises
reacting an acylated-2-azido-2-deoxy glycosyl nitrate with an iodide
salt in a suitable solvent to form an acylated-2-azido-2-deoxy-~-
glycosyl iodide; and reacting said ~-glycosyl iodide with a chloride salt
in a suitable solvent to produce the acylated-2-azido-2-deoxy-3-glycosyl
chloride.
DESCRIPTION OF THE DRAWINGS
Figure 1 is a formula sheet showing structure formulas and
: names for compounds referred to by number in the specification; and
Figure 2 is a reaction sheet showing examples of the reactions
described in the specification.
.' :
- 7a -
114~7
.
DESCRIPTION OF THE PREFERRED EMBODIMENT OF THE INVENTION
The Azidonitration Reaction:
The Formula Sheet provides structural formulas for compounds I
to LII. Reference is made to these compounds in the course of this des-
cription and in specific experimental examples which demonstrate the
invention.
Examples I through VII show the reaction of suitably protected
glycals with ceric ammonium nitrate and an azide salt to form the
I corresponding 2-azido-2-deoxy glycosyl nitrates.
:: j
; 10 The term glycal applies to 1,2 - unsaturated sugars which are
characterized by the structural entity
,...................................... ~o
:~. \~/
The term protected glycal denotes that the hydroxyl substituents have been
masked by blocking groups such as acetyl, propionyl, and benzoyl which, being
less reactive than the hydroxyl group, will not participate in subsequent
reactions. In this manner,the properties of the glycal other than those
of the unsaturation will be retained.
Examples of protected glycals are 3,4,6-tri-0-acetyl-D-galactal,
- I; 3,4,6-tri-0-acetyl-D-glucal, VI; 3,4,6-tri-0-benzoyl-~-galactal, X;
hexa-O-acetyl-D-lactal, XIII; and 3,4-di-0-acetyl-D-xylal, XVI.
In the azidonitration of glycals, demonstrated in Examples I
through VII, the protected glycals are reacted with an excess of a 2:1 (mole/
mole) mixture of ceric ammonium nitrate and an azide salt. It is known
that these two salts react to form nitrogen gas as a product. The slight
excess of reagent is used to compensate for this loss.
Without being bound by the same, the following mechanism is
suggested for the azidonitration reaction:
1~11417
Cet4 t N3 Ce 3 + N3
~,
O~c ~ OAc qAc
~ OAc A I QAc ~ I /---OAc
AcO ~ ~ ~ ~ L ~ J
~ACO ~ ~ ACO~ ~ OAc
,.ON02
Ce(IV) is a strong oxidizing agent and strips an electron from the negatively
- charged azide ion. The resulting azide radical adds across the 1,2-un-
saturated bond of the glycal to form an intermediate radical. A second
Ce(IV) ion may oxidize the intermediate radical to give an
oxycarbonium. The addition of a nitrate ion, to the C-l position, results
in the 2-azido-2-deoxy glycosyl nitrate.
The azide salt may be any of the common alkali metal azides.
Sodium azide is used, preferahly for reasons of cost and handling, but the
lithium or potassium azides are also suitable.
A 2-azido substituent is desirable as it will not interfere
in the subsequent formation of a ~-glycosidic linkage at the anomeric
(C-l) center and can be reduced to an amino function by well known methods
to produce 2-amino-2-deoxy sugars.
A solvent is- used which is able to dissolYe the three reagents,
the nonpolar glycal and the ionic salts, at a level to provide sufficient
concentrations of these in the reaction mi`xture. In additi`on, the solvent
should be substantially inert to reacti:on and resistant to oxidation by
the ceric salt. The preferred solvent is acetonitrile because of its
_ g
4~L7
resistance to oxidation and its ahility to proYide appropriate concentra-
tions of the reacting speci.es in s.oluti:on. ~ther solvents can be used
such as ethyl acetate or acetic'aci'd, but s.ide reactions are rather
severe in the case of the latter. The solvent is preferably dried prior
to use as the presence of water was found to support side reactions.
'. Due to the dissimilarity in the solubility of the reactants,
'. effective sti.rring is required to mai'ntain suffi.cient concentrations in the
reaction mixture and to ensure an efficient rate of reaction.
' The preferred reaction temperature range is from -25C to +25C.
' 10 The lower limit was determined by the freezing point of the acetonitrile,
the solvent preferentially used; while the upper li:mit was arbitrarily
'' chosen as a cutoff above which competing si.de reactions became significant.
Although the reaction kinetics were slower at low.er temperatures, giving
rise to longer reaction times, the yields of the desired products were
. 15 better.
'' Although the reaction can be performed in air, an inert
atmosphere, such as nitrogen, is preferably used.
Examples I and II illustrate two di.fferent techniques, within
~'' the scope of the present invention, for preparing the 2-azido-2-deoxy
nitrates of 3,4,6-tri-0-acetyl-D-galactal. The fi'rst is a process which
: is attractive to commercial production while the second describes the
experiment which led to the discovery. It is wi.thin the scope and spirit
of this invention to claim all those variations in the reaction conditions
and work-up procedures that are evident to chemists competent to consider
and to test the effectiveness of alternate procedures which would involve
such variations as changes in reaction and extractïng solvents, modes of
addition, stirring rates and temperature range.
EXAMPLE I
The reaction of 2,3,~-tri-0-acetyl-=-galactal.(I) with ceric ammonium
nitrate in the.presence.of.sodium azide
A three-necked, five liter, round bottom flask equipped with an
-- 10 --
`~
417
inlet tube, exhaust tube and an effic;ent mechanical stirrer was charged
with solid ceric ammonium nitrate (899.90 9, 1.64 mole) and solid sodium
azide ~53.37 9, 0.82 mole) and cooled to -15C under a nitrogen atmosphere.
2,3,4-tri-0-acetyl-D-galactal(I)(150 9, 0.551 mole) was dissolved in
anhydrous acetonitrile (3.4~) in a three-necked, four-liter flask equipped
with an inlet and an outlet tube. This soluti~on was cooled to -15C
while sweeping with nitrogen. By applying a positive pressure of nitrogen
"' the acetonitrile solution was pumped into the vessel containing the solid
'- reactants vi'a an inert tube. After camplete addition of the acetonitrile
solution (approximately 1 minute ), mechanical sti:rring was commenced and
' continued for approximately l5 to 20 hours or until such time as no glycal
remained on examinati'on of the reacti'on mi'xture by thin layer chromatography
(t.l.c.) on silica gel eluted with hexane-ethyl acetate (v/v~ 6:4. At
:
', that time toluene (1~) and cold water (1~) were added and the reaction
' 15 vessel was removed from the cooling bath. This mixture was transferred
to a ten-liter container and after addition of toluene (2~) the organic
layer was separated and transferred to a separatory funnel. This solution
was washed with cold water (3 x lR). The organic layer was filtered through
toluene-wetted filter paper and the filtrate was concentrated in vacuo
at a temperature below 40C to a syrup (200 9~. The proton magnetic
resonance (p.m.r.) spectrum of this syrup showed it to be composed mainly
of 2-azido-2-deoxy nitrates. The composition of the product was 37% of
3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-D-galactopyranosyl nitrate (II),
55% of 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-galactopyranosyl nitrate
(III) and 8h of 3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-P-talapyranosyl nitrate
(IV).
The low yield of compound IV indicates that the azidonitration
reaction is highly stereoselecti`ve at the C-2 positi`on.
Tri'turation of a portion of the syrupy product (21.0 9~ with
cold ethyl ether gave compounds II and ~V ~8.3 9) whi'ch co-crystallized.
The mother li'quor contained almost pure ~-D-nitrate, III, (12.6 9).
- - 11 -
-` 1111417
'~ Compound III could not he crystallized. The infrared (i r.) spectrum (film)
' of compound III displayed absorbances at 2120 cm 1(N3) and 1650 cm 1
(ON02); its partial p.m.r. spectrum in CDC13 was, ppm 5.71 ~d, 1, Jl 2
9.0 Hz, H-ll, 5.42 (q, 1, H-4), 5.08 (q, 1, J3 4 3.2 Wz, H-3), 3.87 (q,
1' J2 3 10.8 Hz, H-2), 2.18, 2.10, 2.03 (3s, 9, 3 OAC).
Compound II`, free of the talo azide (lV), was obtained by
anomerization of the ~-D-ni'trate, III, with ni~trate ion. A solution of
the syrupy ~-D-nitrate, III, (9.50 9, 25.5 mmole~ and anhydrous lithium
- nitrate (3.50 9, 50.1 mmole) in 4:1 (v/v) acetoni'trile:dimethylformamide
(35 ml) was stirred for 42 hours at ambi~ent temperature, after which time
it was diluted with dichloromethane (250 ml) and washed with ice cold water
(3 x 125 ml). The organic solution was dried and evaporated to give a
syrup (9.0 9). The p.m.r. spectrum of this syrup showed it to be a
mixture of 63% ~- and 37% ~-~-ni'trates, II and III. Crystallization from
ethyl ether gave the ~-D-nitrate, II, (6.2 g), m.p. 103 - 104C,
[~]D5 + 125 (c 1, chloroform). The infrared spectrum (film) of compound
II displayed absorbances at 2120 cm 1 (N3) and 1650 cm 1 (ON02); its partial
p.m.r. spectrum in CDC13 was, p.p.m. 6.34 (d, 1, Jl 2 4.1 Hz, H-3), 4.12
(q, 1~ J2 3 11.5 Hz, H-2), 2.18, 2.09, 2.02 (3s, 3 OAC).
A minor side product (<10%) of the reaction could be isolated i~
either by chromotography on si'lica gel of the reacti'on mixture or, in
some cases, by evaporation of the three aqueous washings obtained during
the reaction product workup described above. The compound readily crystallized
- from the washings by evaporation or upon tri'turati'on with ethyl ether, and
~: 25 was shown to be N-(3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-=D-galactopyranosyl)
acetamide (V); m.p. 142-143.5C, [~]D5 + 68.0 (c 1, chloroform). Its
' partial p.m.r. spectrum in DMS~-db was,p.p.m. 9.83 (d, 1. JNH 1 9-5 Hz, NH),
5.78 (q, 1~ Jl 2 5.5 Wz, H-l), 5~48 ~1~ q~ 32 3 11.3 Hz, H-3), 5.22 (1,
d, J3 4 3.5 Hz, H-4), 4.2~ (q, 1, H-2~.
- 12 -
`` 1111417
,
EXAMPLE II
The reaction of 3,4,6-tri-0-acetyl-D-galactal (I) with ceric ammon;um
`'' nitrate i'n the presence of sodium azi''de~
,,
Distilled 2,3,4-tri-0-acetyl-D-galactal (I~ ('21.1 9, 0.007 M)
(b.p. 147-155 at 0.1 mm) was dissolved in dry acetoni-trile (420 ml)
and cooled to -25C under a nitrogen atmosphere i'n the dark. A mixture
~- of solid ceric ammonium ni'trate (lOa.2 9, Q.182 mole) and soli-d sodium
'~ azide ~6.043 9, 0.092 mole) was added all at once and the resulting
; suspension was stirred for 15 hours at -25C~ At this time cold ethyl
~- ~ 10 ether (40Q ml) was added and the resulting mixture filtered to remove any
- .
' solids. The filter cake was washed with diethyl ether (2 x 100 ml) and
the combined filtrate was poured into i-ce water (500 ml). The organic
solution was separated and washed with i-ce cold water (3 x 500 ml), dried
- over anhydrous sodium sulfate, fi'ltered and evaporated to give a syrup
- 15 (21.0 9), which corresponded to a 73% yield of the crude nitrates (II and
III). Thin layer chromatography examinati-on on silica gel developed
; with 6:4 (v/v) hexane: ethyl acetate showed no remaining starting material.
P.m.r. examination showed the product to be essentially identical to the
-' syrupy product obtained in Example I.
Because of the reactivity of glycosyl nitrates in general,
'; care must be exercised in the handling of these compounds so as to not
~-' effect undesired decomposition or solvolytic reactions. The mixture of
a- and ~-nitrates obtained may vary since, as is demonstrated in Example I,
the compounds are readily interconverted in the presence of nitrate
ion. The mixture is as useful as either of the pure products for the
purposes of this invention, as will be demonstrated later. In general,
no effort is made to separate the compounds (I'I and III). However, it was
found that the ~-anomer (I-II`) i`s readily obtai`ned in the crystalline state
and if this substance i's desired, the yi`eld can be improved by anomerization
of the ~-anomer which is- the thermQdynaml~cally leas stable compound.
-
,- - 13 -
.
~: -`; 1111417
' The azidonitration reaction demonstrated in Example I is not
restricted to the acetylated galactal, I, but finds useful application with
suitably 0-protected glycals in general. This is demons;trated by Example
` III wherein the selected reactant is tri-0-acetyl-~-glucal, (VI), a different
hexal, and further exemplified through the use of hexa-0-acetyl-D-lactal
- (XIII), having a disacchari'de structure, i`n Example IV and of 3,4-di-0-
acetyl-D-xylal (XVI), a pental, in Example'Y.
Further, Example III illustrates that the temperature at which
the reaction is conducted may be varied although product puri'ty decreases
at reaction temperatures above 0C. TKe use of potassium azide is also
demonstrated.
EXAMPLE III
The reaction of 3,4,6-tri-0-acetyl-D-glucal (VI) with ceric ammonium
nitrate in the presence of potassium azide
!
Treatment of 2,3,4-tri-0-acetyl-D-glucal ~VI) ~5.86 g, 21.5
mmole) with ceric ammonium nitrate (27.8 9, 50.7 mmole~ and potassium azide
(2.39 9, 25.7 mmole) at 25C by the method of Example I for tri-0-acetyl-
D-galactal, gave a mixture of 2-azido nitrates in 60% yield. Of the azido
nitrate products, this mixture was shown to be composed of 3,4,6-tri-0-
- 20 acetyl-2-azido-2-deoxy-~-D-glucopyranosyl nitrate (VIII~ 42.~%, 3,4,6-
tri-0-acetyl-2-azido-2-deoxy-~-D-glucopyranosyl nitrate (VII) 24~,
' and 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-mannopyranosyl nitrate (IX~
33%. This composition was based on the relative intensities of the p.m.r.
'' anomeric signals assigned to compounds VII, IX, and VIII which were at 6.4
p.p.m., J=4.0 Hz, 6.28 p.p.m., J=1.8 Hz and 5.72 p.p.m., J-8.8 Hz, re-
spectively.
' EXAMPLE IV
The reaction of hexa-0-acetyl-D-lactal (XIII) with ceric ammonium nitrate
in the presence of sodium azide
The azidonitration of hexa-0-acetyl-D-lactal (XIII) serves as
a new route to the important disaccharide'known as a lactosamine and which is
a building block of oligosaccharides which form the core structure of
- 14 -
f~
41'7
' oligosaccharides found in human milk and the antigenic structures of
the human blood group substances.
. I
Treatment of hexa-0-acetyl-=D-lactal ~XIII) (1.0 9, 1.7g mmole)
with ceri:c ammonium n;trate ( 2.45 9, 4.48 mmole) and sodi'um azide (0.174 g,
; 5 2.685 mmole) by the method of Example ~I gave a mixture of the 2-azido
'- nitrates (0.89 9) in greater than 75% yield. P.m.r. examination showed
signals at 6.3a p.p.m. (d, 4.25 Hz) and 5.56 p.p.m. ('d, 8.5 Hz) which
were ass-igned to the anomeri`c protons of the 2-azi`do nitrates XIV and XV,
respectively. Trituration of this s~yrup w;tfi ethyl ether gave crystalline
3,6-di-0-acet~1-4-0-~2,3,4,6-tetra-0-acetyl-~-D-galactopyranosyl~-2-azido-
2-deoxyl-~-D-glucopyranosyl nitrate ~XV) (0.5 9) in 42% yield: m.p.
69 ~ 70; [~]D5 + 15 (c 1, chloroform). The infrared spectrum (nujol
mull) of compound XV di'splayed absorbances at 2120 cm 1~N3) and 1650 cm 1
(ON02); it partial p.m.r. i`n CDC13 was, p.p.m.' 5.56 ~d, 1, Jl 2 8.5 Hz,
lS H-l), 3.56 ~q, 1, J2 3 8.25 Hz, H-2).
Column chromatography of the mother liquor, after the removal of
crystalline compound XV, on si1i'ca gel developed with hexane-ethyl acetate-
ethanol (v/v) 10:10:1 afforded addi`ti`onal quantities of compound XV (0.05 9)
and 3,6-di-0-acetyl-4-0-(2,3,4,6-tetra-0-acetyl-~-~-galuctopyranosyl)-2-
- 20 azido-2-deoxy-~-D-glucopyranosyl nitrate ~XIV) (0.31 9) which was crystal-
lized from ethyl ether: m.p. 138 - 140; [~]2D5 + 69.7(c 1, chloroform).
The infrared spectrum (nujol mull) of compound XIV displayed absorbances
at 2120 cm 1 (N3) and 1650 cm 1 (ON02); its partial p.m.r. spectrum in
CDC13 was, p.p.m. 6.30 (d, 1, Jl 2 4.25 Hz, H-1),3.72 (q, 1, J2 3 10.5
Hz, H-2).
EXAMpLE y
' The reaction of 3, 4-di-0-acetyl-D-xylal'~XYI;) with ceric ammonîum nitrate
.
in the presence of sodium azide~'
Example V shows that the appli`cati`on of the process of azido
: 30 nitration can be extended to the pentopyranoglycals.
.
~ - 15 -
!
1~11417
;:'"'
~ Treatment of di-0-acetyl-D-xylal (XVI) (29~ (0,472 9, 2.36
., I
mmoles) with ceric ammonium nitrate ( 4.39 9, 8.0 mmoles) and sodium azide
(0.260 9, 4.0 mmoles) by the method des-cribed in Example II gave a
mixture of 2-azido-nitrates in 88% yield. P.m.r. exami'nation of the product
mixture showed signals at 5.70 p.p.m. (d, 7.5 Hz), 68%, 6.28 p.p.m.
(d, 4.0 Hz), ~16%, and 6.56 p.p.m. ~d,' 4.5 Hz~, ~16%. The major product
was shown to be 3,4-di-0-acetyl-2-azido-2-deoxy-~-D-xylopyranosyl nitrate
XVII by double i'rradiation experi'ments whi'ch showed the presence of a
quartet at 3.70 p.p.m., wi'th J2 3 ~' 8.75 Hz and Jl 2 = 7-5 Hz, which was
assigned to H-2 of compound XVII. The products comprising the remaining
32% of the mixture of 2-azido-2-deoxy-ni'trates must be the ~ and ~-=D-lyxo
anomers XVIII as anomerization of tne mi'xture of ni'trates, by the method
described in Example I for compound III, caused the appearance of a
new signal in the p.m.r. spectrum of this product mi'xture, at 6.31 p.p.m.
(d, Jl 2 3.65 Hz). This signal is attri'buted to the anomeric proton of
'-: 3,4-di-0-acetyl-2-azido-2-deoxy--=D-xylopyranosyl nitrate (XVIX).
EXAMPLE VI
' The reaction of 3,4,6-tri-0-benzoyl-=D-galactal (X) with ceric ammonium
, :, - - ............ ..
nitrate in the presence of sodium azi-de.
The azidonitration reacti-on i's not restricted to acetylated -
glycals but can be applied to any sui'tably protected glycal. For example,
the blocking groups may be propionyl or benzoyl. This is demonstrated in
:; this example wherein 3,4,6-tri-0-benzoyl-D-galactal (X) is used as the
-~ starting material.
Treatment of 3,4,6-tri-0-benzoyl-=-galactal (X) (7.18 9, 12.2
mmole) with ceric ammonium nitrate (20.2 9, 36.6 mmole) and sodium azide
(1.18 9, 18.1 mmole) by the method descri~ed i'n Example I for tri-0-
acetyl-D-galactal gave a mixture of 2-azido-2-deoxy-nitrates (7.5 9~ in
75% yield. Examination of the p.m.r. spectrum of tfie crude product in
CDC13 showed i-t to be composed of 2-azi`do-3,4,6-tri-_-benzoyl-2-deoxy-~-
- 16 -
.~
f , I
11114~7
`.'' I
D-galactopyranosyl nitrate (X11'(30%~ and 2-azido-3,4,6-tri-0-benzoyl-2-
deoxy-~-D-galactopyranQsyl nitrate ~XII~ (45%). The anomer;c si'gnal of
the a-D-ni'trate was observed at 6.67 p.p.m. with Jl 2 = 4.6 Hz.'
Although the anomeric signal of the ~-D-anomer was masked, the H-2 signal
was observed at 4.20 p.p.m. as a large tri'plet with Jl 2 = 9-5 Hz.
EXAMPLE VII
Reaction of 3,4,6-tri-0-acetyl-D-galactal wi`th sodium azide and ceric
,
ammonium nitrate in ethyl acetate.
Although acetoni'trile i's the preferred solvent, the azidonitration
; lO reaction is not restricted to the choice of this solvent. This is demon-
strated by this example wherein ethyl acetate is used as the solvent.
Treatment of tri-0-acetyl-D-galactal (I) ~0.30 9, l.09 mmole)
'~ with ceric ammonium nitrate (l.4l 9, 2.57 mmole) and sodium azide (0.084 9,
; l.29 mmole) in ethyl acetate (5 ml) by the method described in Example I
gave a mixture of the 2-azido nitrates in greater than 60% yield. P.m.r.
' examination of the product showed the 2-azido-nitrate composition to be
similar to that described in Example I. However, examination by thin layer
chromatography on silica gel, developed with 6:4 (v/v) hexane:ethyl acetate,
- gave evidence that more side reactions had occurred in this solvent.
- l7 -
417 '
; Conversion of Azidon;trates to Aminosugars
The acylated 2-azido-2-deoxy nitrates can be converted to the
corresponding 2-amino-2-deoxy sugars by hydrolysis of the nitrate and
acyl groups and reduction of the azido group by methods well known to those
skilled in the art. Hydrolysis may precede reduction or vice versa. The
aminosugars, in particular galactosamine and lactosamine and their
N-acetylated derivatives are important building units for the blood group
substance antigenic determinants. The N-acetylated derivatives are pre-
pared from the aminosugar by methods well known to those skilled in the art.
The aminosugars may also be used to prepare the 2-acetamido-2-deoxyglycoses.
Reduction ot azido groups to amino groups is well known and
can be conducted in virtually quantitative yield under a wide variety of
conditions including reductions with metals such as sodium or zinc,
reduction by catalytic hydrogenation using such catalysts as nickel,
platinum or palladium, reduction using hydrides such as sodium borohydride,
; borane and lithium aluminum hydride, electrolytic reductions and reduction
by hydrogen sulfide under alkaline conditions.
; Broadly stated, the invention provides a process for converting an
acylated 2-azido-2-deoxy glycosyl nitrate to a 2-amino-2-deoxy glycose
which comprises reducing the azido group to an amino group and hydrolyzing
- the acyl and nitrate groups.
- The nitrate group of the acylated 2-azido-2-deoxy-nitrate
may be displaced with an acyl group by conventional methods prior to
- hydrolysis or reduction. For example, the nitrate compound may be treated
with sodium acetate in acetic acid as illustrated in Examples XIV - XVI.
EXAMPLE VIII
Preparations of the anomeric 1,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-D-
galactopyranoses (XXVII) and (XXVIII).
A solution of the pure 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-
; 30 galactopyranosyl nitrate (III) (0.15 g, 0.40 mmQle) and sodium acetate
(0.65 g, 0.80 mmole) in glacial acetic acid (2 ml) was heated to 100
- 18 -
- !
1111417
for 15 minutes at which time examination by thin layer chromatography of
silica gel developed with 6:4 (v/v) hexane:ethyl acetate showed one
homogeneous spot of lower Rf than compound III. The solution was diluted
with dichloromethane (5 ml) and washed with ice cold water (5 ml).
Evaporation of the solvent, after drying over sodium sulfate and filtration,
gave a syrup (0.134 9, 90% yield), which spontaneously crystallized upon
trituration with ethyl ether.
Recrystallization from ethyl ether or cold ethanol gave an
analytically pure sample of l,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-~-D-
galactopyranose ~XXVII), m.p. 114 - 115, [~]D5 + 91.70 (c 1.05, chloroform),
i.r. (film) 2120 cm (-N3).
The p.m.r. spectrum of compound XXVII in CDC13 showed, in
part, p.p.m. 6.38 (d, 1, Jl 2 3-7 Hz, H-l), 5.50 (q, 1, J3 4 3 Hz, H-4),
5.36 (q, 1, J2 3 7 Hz H-3), 3.97 (q, 1, H-2).
" ~ .
A solution of the crude 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-
D-galactopyranosyl nitrate II (1.01 9, 2.70 mmole) and sodium acetate
(0.43 9 5.20 mmole) in glacial acetic acid (10 ml) was heated to 100 for
20 minutes. The reaction solution was then diluted with dichloromethane
- (50 ml) and washed with ice cold water (250 ml). Evaporation of the solvent,
after drying over sodium sulfate and filtration, gave syrup (1.0 9).
. . i
~; Inspection of this syrup by p.m.r. spectroscopy showed it to be composed of
, compound XXVII (30%) and 1,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-~-D-
. .
~ galactopyranose XXVIIr (60%). The anomeric proton of the ~-anomer
- (XXVIII) was assigned to a doublet, with J=8.5 Hz, at 5.61 p.p.m.
Compounds XXVII and XX~III were obtained in a near 3:1
`, mixture by acetolysis in acetic acid containi`ng sodium acetate of the
mixture of compounds II and I:II obtained by way of the process described
~:- in Example II.
,:~
-'''
:'
19
.
(~
1~11417
EXAMPLE IX
Preparations of 1,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-~-=D-gluco and
manopyranoses (XXIX and XXX).
A mixture of 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-=D-
mannopyranosyl nitrate (IX) and the ~- and ~- anomers of 3,4,6-tri-0-
acetyl-2-azido-2-deoxy-=D-glucopyranosyl nitrate (VII and VIII), obtained
as described in Example III, was treated with a solution of sodium acetate
(0.350 9, 4.26 mmoles) in acetic acid (10 ml) at 100C for one hour. Work
up of the product mixture by the method of Example II gave a foam (0.70 9).
Column chromatography (30 x 2 cm~ on silica gel (70 9) eluted with hexane-
; ethyl acetate-ethanol (v/v) 10:10:1 afforded the separation of the
gluco-(XXIX) and mano(XXX) 2-azido-2-deoxy acetates, 0.340 9 and 0.310 9
respectively.
Pure 1,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-~-=D-glucopyranose
(XXIX) (0.211 9, 21%) was obtained by recrystallization from ethyl ether;
m.p. 117 - 118C, [~]D5 + 128 (c 0.9, chloroform). The partial p.m.r.
spectrum of compound XXIX in CDC13 gave, p.p.m. 6.29 (d, 1~ Jl 2
3.5 Hz, H-l), 5.45 (t, 1, J3 4 9.0 Hz, H-3), 5.08 (t, 1, J4 5 9.Q Hz,
H-4), 3.65 (q, 1~ J2 3 9 0 Hz, H-2~.
Pure 1,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-~-=D-mannopyranose
(XXX) (0.220 9, 22%) ~as obtained by recrystallization from ethyl ether;
m.p. 131 - 132C, [~]D5 + 78.6 (c 1.02, chloroform). The partial p.m.r.
spectrum of compound XXX in CDC13 gave, p.p.m. 6.09 (d, 1, Jl 2 1.8, H-l).
EXAMPLE X
Preparations of the anomeric forms of 1,3,6-tri-0-acetyl-4-0-(2,3,4,6-
.-. i
tetra-0-acetyl-~-=D-galactopyranosyl)-2-azido-2-deoxy-=D-glucopyranose
(XXXI and XXXII)
Treatment of an anomeric mixture of 3,6-di-0-acetyl-d-0-
(2,3,4,6-tetra-0-acetyl-~-galactopyranosyl)-2-azido-2-deoxy-=D-
glucopyranosyl nitrate (XIV and XV) comprising about 70% of the
- 20 -
,r-
111 1417
~-anomer (XY) (3.50 9) with sodium acetate (2.16 9, 26.3 mmole) in acetic
acid by the method described in Example XV gaYe crystalline 1,3,6-tri-
' O-acetyl-4-0-(2,3,4,6-tetra-0-acetyl-g-=D-galactopyranosyll-2-azido-2-
deoxy-a-=-glucopyranose (2.48 9) (XXXI) in 73% yield. Recrystallization
from ethyl acetate-pentane gave the pure ~-anomer ~XXXI), m.p. 77 - 78C,
[~]2D5 ~ 55.4 (c 1, chloroform). The partial p.m.r. spectrum of compound
XXXI in CDC13 was, p.p.m. 6.22 (d, 1~ Jl 2 3.65, H-l), 3.46 (q, 1~ J2
10.5, H-2).
Similar treatment of the pure a-nitrate, XIV, in the manner
described above gave crystalline 1,3,6-tri-0-acetyl-4-n-(2,3,4,6-tetra-
; 0-acetyl-~-@-glucopyranose (XXXII) in good yield (70%).
- Excellent yields of compound XXXII were also obtained by the
treatment of 3,6-di-0-acetyl-4-0-(2,3,4,6-tetra-0-acetyl-~-=D-galactopyranosyl)-2-azido-2-deoxy-~-=D-glucopyranosyl chloride (XXV) (Q.264, 0.414 mmoles)
or of the corresponding ~-bromide, XXVI, with silver acetate (0.137 9,
; 1.656 mmoles) in acetic acid (5 ml) at ambient temperature for one hour.
At that time, the reaction solution was diluted with dichloromethane
(20 ml), filtered and washed with water (2 x 20 ml). The organic layer
was dried and evaporated to give a white foam (0.25Q 9~. Crystallization
of this material from hot methanol gave 1,3,6-tri-0-acetyl-4-0-(2,3,4,6-
tetra-0-acetyl-~-D-galactopyranosyl~-2-azido-2-deoxy-~-=-glucopyranose
`~, (XXXII). The partial p.m.r. spectrum of compound XXXII in CDC13 gave,
~ p.p.m. 5.51 (d, 1, Jl 2 8.75 Hz, H-l), 3.57 (q, 1~ J2,3
I EXAMPLE XI
Preparations of the 2-acetamido-1,3,4,6-tetra-Q-acetyl-2-deoxy-
~- and ~-D-galactopyranoses ~XXXIV and XXXV~
This example provides an effi:cient process, based on reduction
by zinc, for the conversion of the mixture of anomeric 1,3,4,6-tetra-0-
acetyl-2-azida-2-deoxy-D-galactoses, XXV M and XXVrII, o~tained in
, ,
- 21 -
11114~7
Example YIII to an anomeric mixture of the 1,3,4,6-tetra-0-acetyl-2-
acetamido-2-deoxy-galactopyranoses, XXIV and XXV, and how this m;xture
is useful for the preparation of D-galactosamine hydrochloride (XXXVII).
Glacial acetic acid (200 ml) and sodium acetate (8.2 9, û.l
mole) were added to the a- and ~-anomer;c mixture of 3,4,6-tri-0-acetyl-
2-azido-2-deoxy-a-=D-galactopyranosyl nitrates (II and III) (32 9, 0.08
mole) prepared by the method of Example II and the mixture was stirred
for one hour at 100. Zinc metal (12.8 9, 0.2 mole) was then added to
the solution cooled to 60 and stirred for 15 minutes. Acetic anhydride
(17 ml) was added and the mixture heated on the steam bath (100) for one
hour and filtered. The solution was poured into 100 ml of water and stirred
for one hour. Then 300 ml of water was added and the mixture extracted
; three times with dichloromethane (100 ml). The extracts were combined,
filtered through dichloromethane-wetted paper and evaporated to a thick
syrup which hardened to a crystalline mass on trituration with ether.
The p.m.r. spectrum of this product was in agreement with that expected
for a 4:1 mixture of the a- and ~-anomers of 2-acetamido-1,3,4,6-tetra-
0-acetyl-2-deoxy-=D-galactopyranose.
.~ .
-- Recrystallization from ether provided the pure a-anomer
(XXXIV) in 55% yield; m.p. 177 - 178; [a]D5 ~ 99 (c 1, chloroform).
The mother liquors were combined to provide 14 9 of a syrupy
product which was found to be a near 1:1 anomeric mixture of the
tetraacetates XXXIV and XXXV. The mixture was dissolved in 4N aqueous
hydrochloric acid (150 ml) and the solution heated at 100 for 7 hours.
The solution was decolorized with activated charcoal and diluted with
n-butanol (500 ml) prior to evaporation to a brownish syrup (10 9). The
product was found to be D-galactosamine hydrochloride XXXVII by comparison
of its paper chromatographic mobility and its p.m.r. spectrum in D20 to
those of an authentic sample. Pure D-galactosamine hydrochloride was
readily obtained by crystallization usi`ng ethanol-water-acetone as is
1111417
described in the literature for the purification of this compound. I
2-Acetamido-2-deoxy-D-galactose (XXXVI) can be prepared by
simple N-acetylation of D-galactosamine hydrochloride, by methods well
known to one skilled in the art, but is also available as an intermed;ate
in the acid hydrolysis of compounds XXXIV and XXXV.
; EXAMPLE XII
Preparation of 2-acetamido-1,3,4,6-tetra-0-acetyl-2-deoxy-a-D-galactopy-
ranose (XXXIV).
Hydrogenation of 1,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-a-
~-galactopyranose (XXYII) (0.20 9, 0.536 mmole) dissolved ;n ethanol (3
ml) containing acetic anhydride (0.25 ml~ and 5% palladium on charcoal
.~ (0.80 9) was complete in 1 hour at room temperature under 1 atmosphere
; of hydrogen. Filtration through diatomaceous earth and evaporation
J
of the solvent gave a white foam (0.206 9). Examination by their layer
chromatography on silica gel developed with 5:5:1 (v/v) benzene:ethyl
acetate:ethanol showed the presence of two compounds which were readily
-; separated by silica gel column chromatography (20 x 1 cm) eluted with the
same solvent. This afforded 2-acetamido-1,3,4,6-tetra-0-acetyl-2-deoxy-
; a-D-galactopyranose (XXXIY) (0.10 9, 5Q% yield) which was recrystallized
from ethyl ether, m.p. 177 - 178, [~]2D5 ~ 99 (c 1, chloroform).
The p.m.r. data for compound xxxrv were in excellent agree-
ment with those previously reported.
The second compound proved to be 2-(N-acetyl) acetamido-1,3,4,6-
tetra-0-acetyl-2-deoxy-a-@-galactopyranose ( 0.068 9) by inspection
of its p.m.r. spectrum and comparison of these data with those previously
reported.
EXAMPLE XIII
Reduction of 1,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-a-D-galactopyranose
(XXVII~ with hydrogen sulfide in the presence of triethylamine
This example provides an alternate method for reducing the
,. 1,
1~11417
azido group to amine. Hydrogen sulFide was bubbled through a solution of
compound XXVII (0.20 9, 0.53 mmole) and tri'ethylamine (0.135 9, 1.34
mmole) dissolved in dichloromethane (5 ml) at 0. After 20 minutes,
inspection of the reaction mixture by thin layer chromatography, developed
' 5 with lO:lO:l (v/v) hexane:ethyl acetate:ethanol, showed no remaining
starting material and one homogeneous spot of low Rf. A yellow precipi-
' tate was seen to appear upon standing. This suspension was evaporated to
dryness and the residue was dissolved in pyridine (2 ml) and acetic anhydride
' (0.5 ml). After 15 hours, the reaction solution was diluted with
dichloromethane (20 ml) and water ~la ml). The organic layer was separated,
dried and evaporated to give a brown syrup (0.17 9), which had the same
mobility on silica gel as 2-acetamido-1,3,4,6-tetra-0-acetyl-2-deoxy-a-
~-galactopyranose (XXXIV). The p.m.r. spectrum of this syrup in CDCl3
was identical to that of compound XXXIV.
- lS EXAMPLE XIV
Preparation of D-galactosamine hydrochloride (XXVII) from the a- and ~- ;
3,4,6-tri-0-acetyl-2-azido-2-deoxy-@-galactopyranosyl nitrates II and III
`' @-galactosamine hydrochloride (XXXVII~ can be obtained directly
from the anomeric mixture of 3,4,6-tri-0-acetyl-2-azido-2-deoxy
galactopyranosyl nitrates, II and III, by hydrogenation to produce 3,4,6-
tri-0-acetyl-@-galactosamine (XXXVIII) followed by acid hydrolysis, as is
illustrated in this example.
A solution of the anomeric mixture of the 2-azi'do-2-deoxy
' nitrates II and III (1.0 9, 7.68 mmole) was hydrogenated in acetic acid
(5 ml) containing 5% palladium on carbon (0.10 9~ at one atmosphere and
ambient temperature for 5 hours. After removal of the catalyst by
filtration and evaporation of the solvent gave 3,4,6-tri-0-acetyl-@-
galactosamine (XXXVIII) (0.85 9) as a foam. Treatment of this'foam'with
2N aqueous hydrochloric acid (lO ml) at ambient temperature for two to
three hours followed by dilution with n-butanol (5 ml) and evaporation
gave @-galactosamine hydrochloride XXXVII (0.50 9) which was recrystallized
from butanol-ethanol-water.
- 24 -
,
417
EXAMPLE XV
. .
Preparation of =D-galactosamine hydrochloride (XXXVII) from the a- and ~-
1, 3, 4,6-tetra-0-acetyl-2-azido-2-deoxy-~-galactopyranoses (XXVII) and
( XXVI I I ) .
=D-galactosamine hydrochloride can also be produced in high
yields from the anomeric mixture of the 2-azido-2-deoxy acetates XXVII
` and XVIII by acid hydrolysis followed by reduction. The process is
~~ demonstrated as follows.
'. ' ,
: A mixture of the anomeric compounds XXVII and XXVIII (1.0 9,
,
; 10 2.68 mmole) was dissolved in 2N hydrochloric acid (lO ml) and stirred
for two to three hours at room temperature. Dilution with n-butanol
(5 ml) and evaporation of the solvent gave a white solid (0.51Q 9).
Recrystallization of this solid from ethanol by evaporation gave pure
2-azido-2-deoxy-D-galactopyranose XL (0.40 9, 72% yield), m.p. 173 - 175
:~ 15 (decomposition), [a]D5 + 53.7 ~ 76.9 (c 0.98, water). Reduction of
compound XL under acidic conditions gave B-galactosamine hydrochloride
XXXVII.
- One can also obtain 2-azido-2-deoxy-galactopyranose (XL) by
.- similar treatment of N-(3,4,6-tri-0-acetyl-2-azido-2-deoxy-o~-B-
20 galactopyranosyl) acetamide (Yl.
EXA~lPLE XVI
Preparation of 2-deoxy-D-lactosamine hydrochloride (XXXIX)
:
Example XVI describes the synthesis of D-lactosamine
hydrochloride (XXXIX) from the 2-azido-2-deoxy lactosyl acetates, XXXI
25 and XXXII.
An anomeric mixture of the 2-azido-2-deoxy-lactose acetate,
compounds XXXI and XXXII, (5.0 9, 7.75 mmole) was dissolved in anhydrous
methanol which was 5% in hydrogen chloride (2a ml) and stirred for two to
three hours at room temperature. Dilution of this solution with n-butanol
:
''
- 25 -
,
( 111~.9~17 (
(10 ml) and evaporation of the solvent gave a light yellow syrup (1.40 9).
Reduction of this compound with hydrogen in the presence of palladium and
hydrochloric acid gave 2-deoxy-D-lactosamine hydrochloride (XXXIX).
'
.'. 1.
~', f
" i
. ' ,
'', ;
. - 26 -
.
f ~ 1.417 (-- !
. ~
Conversion of Azidonitrates to'Azidohalides
The nitrate group, being strongly electronegatfve,'serves as a
good leaving group and, especially when at the anomeri:c center of sugar
structures, is readily displaced'by nucleophiles. ~f special interest
is the preparation, from the aforementioned 2-azi:do-2-deoxyglycosyl
nitrates, of 2-azido-2-deoxyglycosyl halides since these latter substances
' can be used for the preparation of 2-azido-2-deoxyglycosides'under con-
ditions for glycosidation generally known in carbohydrate chemistry.
:: I
" The displacement reaction, illustrated in Examples'VIrI
., I
; 10 through XIII, involves the treatment of the novel 2-azido-2-deoxy glycosyl
f,."' nitrates with a halide salt to effect the displacement of the nitrate group
, ~
:1 by substitution by the halide. This reaction is well known to those
skilled in the art and by virtue of the novel starting material leads to the
formation of the novel 2-azido-2-deoxy glycosyl halides.
Similar to the nitrates, the ~-glycosyl halides are more stable
than their corresponding ~-anomers. This will be evident in Examples
XVII - XXI wherein the -anomer is the predominant product. The ~-
'~ glycosyl halides will anomerize to the more stable ~-form in the presence
'' of a large concentration of the halide ion. The rate of anomerization
; 20 for the halides decreases in the order
. . i
' iodide > bromide > chloride
:.~
As will be demonstrated in Example XXII, the ~-anomer can be produced
in high yield under conditions of kinetic control. The 2-azido-2-deoxy
glycosyl halides are useful in the preparation of 2-amino-2-deoxy glycosides.
The ~-glycosides, important building units in biological systems, can be
obtained in good yield by route of the ~-halides.
The preferred halide salts for the halogenation reaction
are the tetraalkylammonium halides and the alkali metal halides, but the
- process is not limited to these.
The preferred solvent is acetonitrile but other aprotic, inert
solvents, such as acetone, dimethylformamide and ethyl acetate, are
suitable.
,
- 27 -
4 1 7
Broadly stated, a process is provided for producing a 2-azido-
2-deoxy glycosyl halide which comprises reacting an acylated 2-azido-2-
deoxy glycosyl nitrate with a hal;de salt in a suitable solvent.
More specifically, an anomeric mixture of the 3,4,6-tri-0-
S acetyl-2-azido-2-deoxy-D-galactopyranosyl nitrate, II and III, is reacted
with tetraethylammonium chloride in acetonitrile to produce an anomeric
. . . I
'
,
- 27a -
-. `` 111~417
; mixture of 3,4,6-tri-0-acetYl-2-azido-2-deoxY-D-galactopyranOsyl chloride.
In a preferred embodiment, the ~-chlori-de is prepared in high
yield by reacting an anomeric mixture of 3,4,6-tri-0-acetyl-2-azido-2-
deoxy-D-galactopyranosyl nitrate, I I and I I I, with anhydrous lithium
S iodide in acetonitrile to obtain 3,4~6-tri-0-acetyl-2-azido-2-deoxy--
,. . .
D-galactopyranosyl iodide (XXIV) as the predominant product. The product
;' is immediately treated with a molar equivalent of tetraethylammonium
chloride in acetonitrile. The mi'xture is cooled, and extraction affords
- 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-galactopyranosyl chloride (XXIII)
in approximately 60% yield.
' The 2-azido-2-deoxy glycosyl halides are useful in the pre-
' paration of 2-azido-2-deoxy glycosi'des under conditions for glycosidation
~' generally known in carbohydrate chemistry as Koenings-Knorr conditions.
- These reactions will be discussed later.
EXAMPLE XVII
Preparation of 3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-D-galactopyranosyl
` bromide (XX)
3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-D-galactopyranosyl
nitrate (II) (0.50 9, 1.34 mmole) was dissolved in anhydrous acetonitrile
(4 ml) at room temperature containing li'thi:um bromide ~0.80 9, 9.38 mmole~.
After 40 minutes the solution was diluted with dichloromethane (25 ml)
' and washed with ice cold water (25 ml), dried over anhydrous sodium
sulfate, and evaporated to give a clear syrup (0.40 9). The p.m.r. spectrum
-` of this syrup had a doublet, J=4 Hz, at 6.51 p.p.m. which was assigned
.-.
'- 25 to the anomeric proton of 3,4,6-tri-0-acetyl-2-azi'do-2-deoxy-a-D-
galactopyranosyl bromide (XX). This compound could not be crystallized.
:'
:
. _
-~ - 28 -
111~417
:`
: EXAMPLE XYIII
Preparation of 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-=D-galactopyranosyl
chloride (XXII)
An about 1:2 mixture of the a- and ~- anomers of 3,4,6-
tri-0-acetyl-2-azido-2-deoxy-=D-galactopyranosyl nitrate (0.377 9, 1.01
mmole) was dissolved in acetonitrile (6 ml) containing tetraethylammonium
chloride (0-924 9, 5.05 mmole) and the solution was left at room temperature
for 48 hours. The reaction mixture was diluted with dichloromethane (25 ml),
washed with water (25 ml) and dried. EYaporati`on of the solvent ~n ~acuo
left a syrup (0.325 9) which showed doublets with spacings of 9.0 and 3.5
Hz at ~ 5.15 and 6.20 p.p.m., respectively in the p.m.r. spectrum measured
in CDC13. These signals are assigned to the ~- (XXIII) and ~-anomers (XXII)
for 3,4,6-tri-0-acetyl-2-azido-2-deoxy-=D-galactopyranosyl chloride,
respectively. Judging from the relative i:ntensitfes of the signals, the
product consisted of a near 10:1 mixture of the ~- and ~-anomers, (XXII)
and (XXIII) respectively.
EXAMPLE XIX
Preparation of 3,6-di-0-acetyl-4-0-(2,3,4,6-tetra-0-acetyl-~-=-
. ,
- galactopyranosyl)-2-azido-2-deoxy-~-=-glucopyranosyl chloride (XXV~
A mixture of the a- and ~-anomers of 3,6-di-0-acetyl-4-0-
- acetyl-~-=-galactopyranosyl)-2-azido-2-deoxy-=D-glucopyranosyl nitrate,
(XIV) and (XV), (l.Q 9, 1.5 mmoles) ~as treated with a solution of
acetonitrile (20 ml~ containing tetraethylammonium chloride (1.30 9, 7.8
mmoles) at ambient temperature for one hour. At that time the solution was
diluted with dichloromethane (50 ml) and washed with water (2 x 50 ml).
. The organic layer was dried over anhydrous sodium sulfate, filtered, and
evaporated to give a syrup which soon solidified. Recrystallization of
this solid from ethyl acetate-ethyl ether gave pure 3,6-di-0-acetyl-4-0-
(2,3,4,6-tetra-0-acetyl-~-=D-galactopyranosyl)-2-azido-2-deoxy-~-=D-
glucopyranosyl cKloride (XXV~, m.p. 167 - 168C, ~]D5 ~ 59-3 (c 1,
chloroform~, in 66% yield.
.,
- 29 -
.
`` 111~41~ ~ I
'
The partial p.m.r. spectrum of compound XXV in CDC13 was,
p.p.m. 6.08 (d, 1, Jl 2 3 9 Hz, H-l), 3 74 (q~ 1~ J2 3 10 Hz, H-2)-
EXAMPLE XX
Preparation of 3,6-di-0-acetyl-4-0-(2,3,4,6-tetra-0-acetyl-~-D-
galactopyranosyl)-2-azido-2-deoxy-a-=-glucopyranosyl bromide (XXVI)
Treatment of a mixture of the a- and ~- anomers of 3,6-di-0-
acetyl-4-0-(2,3,4,6-tetra-0-acetyl-~-D-galactopyranosyl)-2-azido-2-deoxy-
=D-glucopyranosyl nitrate, (XIV) and (XV), (1.0 g, 1.5 mmoles) with a
solution of acetonitrile (2 ml) containing lithium bromide (0.130 9, 1.5
- 10 mmoles) at ambient temperature for two to three hOurs~followed by workup
of the product mixture by the method described in Example XII,~gave a white
foam (0.850 9) on evaporation. Crystallization of this material from ethyl
acetate-ethyl ether gave pure 3~6-di-0-acetyl-4-0-acetyl-4-0-(2,3,4,6-
tetra-0-acetyl-~-=D-galactopyranosyl)-2-azido-2-deoxy-a-=D-glucopyranosyl
bromide (XXVI), m.p. 156 - 157C, [a]D5 + 87 (c 0.93, chloroform), in
41% yield.
The partial p.m.r. spectrum of compound (XXVI) in CDC13 was,
p.p.m. 6.36 (d, 1~ Jl 2 3-9~ H-l, 3.65 (q, 1~ J2 3 10.2, H-2)-
EXAMPLE XXI
Synthesis of 3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-=D-galactopyranosyl
.
iodide (XXIV) in acetone
3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-D-galactopyranosyl nitrate (II)
: (32.5 9, 0.087 mole) was treated with a solution of anhydrous sodium
iodide (64.29 9, 0.43 mole) dissolved in acetone (259 ml) at room
temperature for twenty minutes. At that time the reaction solution was
treated by the method of Example XIX , to give a syrup (37.6 9). Examination
of this syrup by p.m.r. showed it to be mainly 3,4,6-tri-0-acetyl-2-azido-
2-deoxy-a-=D-galactopyranosyl iodide (XXIV).
- 30 -
,~
1~11417
"
EXAMPLE XXII
Preparation of 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-galactopyranosyl
chloride (XXIII)
Although the 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-
galactopyranosyl bromides and chlorides can be prepared conveniently
for later use, the corresponding -iodide proved highly reactive and
not readily amenable to purification. However, its high reactivity
proved useful for the preparation of the ~- chloride (XXIII) under con-
ditions of kinetic control. That is, the -iodide could be reacted with
0 chloride ion to form the ~-chloride (XXIII) at a rate much greater than
the anomerization of the ~-chloride to the -chloride XXII. The pre-
paration of the pure ~-chloride is presented in the following example.
; The mixture of 3,4,6-tri-0-acetyl-2-azido-2-deoxy-- and ~-D-
galactopyranosyl nitrates (II and III) (0.781 9, 2.09 mmole) prepared as
described in either Example I or Example II was added to a suspension of
anhydrous lithium iodide (1.86 9, 14 mmole) in anhydrous acetonitrile (3 ml).
This mixture was stirred in the dark at room temperature for 15 - 17 min-
; utes and then poured into an ice cold 1% aqueous solution of sodium
thiosulfate. A dichloromethane (10 ml) extract was dried over sodium
sulfate, filtered and evaporated to give a white foam which discoloredupon standing. The p.m.r. spectrum of this compound in CDC13 showed no
; remaining starting material and contained a doublet with J=4.0 Hz at 6.93
p.p.m. which was assigned to the anomeric proton of 3,4,6-tri-0-acetyl-2-
azido-2-deGxy-~-D-galactopyranosyl iodide (XXIV). This -=D-iodide (XXIV)
(2.09 mmole) was immediately treated with a molar equivalent of either
tetraethylammonium chloride (0.344 9, 2.09 mmole) dissolved in anhydrous
acetonitrile (2 ml) or lithium chloride (0.081 9, 2.0 mmole) at ambient
temperature. After 1.5 minutes, the solution was poured into ice cold
; water (10 ml) and extracted with cold dichloromethane (lO ml). The
organic solution was dried and evaporated to give a light yellow syrup
, ~ I
L7
which, on trituration with ethyl ether, afforded crystalline 3,4,6-tri-
0-acetyl-2-azido-2-deoxy-~-=D-galactopyranosyl chloride (XXIII) in 50 - 60%
yield; m.p 102 - 104, [a]D5 - 16.5 (c 1, chloroform).
The p.m.r. spectrum of compound XXIII in CDC13 showed, in part,
p.p.m. 5.91 (q, 1, J3 4 3 Hz, H-4), 5.15 (d, 1~ Jl 2 9 Hz, H-l), 4-86
(q' 1' J2 3 10.5 Hz, H-3), 3.88 (q, 1, H-2).
: !
. ~
,' , ~
."
`,
.,
,
',.`',''
:
.. . .
, , .
417
Conversion of Glycosyl Halides tO Glycosides
Glycosidation, under Koenigs-Knorr conditions, involves the
- treatment of a glycosyl halide with an alcohol, ROH, in the presence of a
promoter. The promoter is commonly a salt or compound which contains a
heavy atom, such as silver, lead or mercury, which can coordinate with
the halogen atom so as to facilitate the cleavage of its bond with the
anomeric carbon. The halogen is replaced by the alkoxy group, -OR, to
produce the glycoside.
The novel a-glycosyl halides of 2-azido-2-deoxy-D-galactose,
XX and XXII, prepared as shown in Examples XVII and XVIII and of 2-azido-2-
deoxy-D-lactose, XXV and XXVI, as shown in Examples XIX and XX, can be used
for the preparation of the novel 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-
galactopyranosides (Examples XXIII, XXIV) and 2-azido-2-deoxy-~-D-lactosides
(Example XXV), respectively, under conditions of the Koenigs-Knorr reaction.
Broadly stated, a process is provided which comprises reacting
3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-galactopyranosyl chloride (XXIII)
with an alcohol in the presence of a promoter to produce 3,4,6-tri-0-
acetyl-2-azido-2-deoxy-a-D-galactopyranosides.
More specifically, 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D- '
- 20 galactopyranosyl chloride (XXIII) is reacted with 8-methoxycarbonyloctyl-
2-0-(2,3,4-tri-0-benzyl-a-L-fucopyranosyl)-4,6-0-benzylidene-~-D-
galactopyranoside (XLVII) in the presence of silver trifluoromethane
; sulfonate and silver carbonate in the solvent dichioromethane to produce
8-methoxycarbonyloctyl-3-0-(3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-D-
galactopyranosyl)-2-0-(2,3,4-tri-0-benzyl-a-L-fucopyranosyl)-4,6-0-
, benzylidene-~-D-galactopyranoside (XLVIII). This product was isolated
- and treated, by methods well known to persons skilled in the art, to
accomplish the following: deblocking, that is conversion of the acetyl,
benzyl, and benzylidene groups to hydroxyl groups, reduction of the azido
group to an amine and acetylation of the amine. The final product of these stepsis 8-methoxycarbonyloctyl-3-0-(2-acetamido-2-deoxy-a-D-galactopyranosyl)-2-
- 33 -
417
.-
0~ L-fucopyranosyl)-~-D-galactopyranoside, the terminal tri-
saccharide anti'genic determinant for the human A blood group.
EXAMPLE XXIrI
.
I Preparation of ~-butyl-3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-
galactopyranoside (XLI)
3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-galactopyranosyl
bromide (XX, 0.90 9, 2.28 mmole) prepared either by reaction of 1,3,4,6-
tetra-0-acetyl-2-azido-2-deoxy-~-galactopyranose (XXVII) with hydrogen
bromide in methylene chloride or by the method of Example XVII, was
added to ~-butyl alcohol (0.236 ml, 2.40 mmole) dissolved in methylene
chloride (3 ml) which contained silver carbonate ( 1.8 9, 6.74 mmole)
' and 4A molecular sieves. After stirring for 1 hour at room temperature,
.- i
' the product was isolated in the conventional way to provide a syrup.
`~ P.m.r. examination of this syrup showed a doublet at 4.64 p.p.m. with Jl 2=
' 15 9 Hz and a singlet at 1.31 p.p.m. which are assigned to the anomeric
proton and aglycon, respectively of ~-butyl-3,4,6-tri-0-acetyl-2-azido-2-
' deoxy-~-D-galactopyranoside (XLI~, obtained in 75% yield.
' EXAMPLE XXIV
Preparation of 8-methoxyoctylcarbonyl-3,4,6-tri-0-acetyl-2-acetamido-2-
:
deoxy-~-D-galactopyranoside (XL)
A solution of 3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-
. i
galactopyranosyl bromide (XXI) (1.0 9, 2.54 mmole) dissolved in dichloro-
methane (2 ml) was added to a mixture of 8-methoxycarbonyl octanol (0.565 9,
O
2.79 mmole), 4A molecular sieves, and silver carbonate (2.30 9, 8.37
mmole) in dichloromethane (5 ml) and stirred for 3 hours at room temperature.
At that time the solution was filtered and the filtrate evaporated to gi~e
a syrup (1.30 9). This syrup was dissolved in acetic acid 10 ml containing
acetic anhydride (10 ml) and zinc metal (1.17 9, 18 mmole) was added with
stirring. After 2Q minutes the solids were removed by~filtrati'on and the
filtrate concentrated to approximately 2 or 3 ml. This was diluted with
- 34 -
417
dichloromethane (25 ml) and washed with saturated aqueous sodium bicarbonate
; (20 ml) and water (10 ml). Dryi-ng of the organic solution and evaporation
gave a syrup (1.0 g, 75% yield) which was shown by examination of its
p.m.r. spectrum in CDC13 to be essentially pure 8-methoxyoctylcarbonyl-
2-acetamido-3,4,6-tri-0-acetyl-2-deoxy-~-D-galactopyranoside (XLII). A
partial p.m.r. of this compound in CDC13 gave; p.p.m. 6.40 (d, 1~ JNH 2
8.2 Hz, NH), 4.70 (d, 1~ Jl 2 8.0 Hz, H-l). The large coupling constant
of 8.0 Hz for H-l confirmed the formation of the ~-D-glycosyl linkage.
EXAMPLE XXV
; 10 Synthesis of t-butyl-3,6-di-0-acetyl-4-0-(2,3,4,6-tetra-0-acetyl-~-_-, :
galactopyranosyl)-2-azido-2-deoxy-~-D-glucopyranoside (XLIII)
Treatment of the 2-azido-2-deoxy-lactosyl bromide XXVI (1.0 g,
1.46 mmoles) (prepared by the method of Example XXIII for two hours gave)
after conventional work up, the compound XLIII (0.80 g, 80%).
Deacetylation and reduction of the azido group, followed by
N-acetylation, of compound XLIII by the method of Example XI gave the
corresponding 2-acetamido-2-deoxy-~-D-lactosyl glycoside (XLIV).
- An outstanding feature of this invention is the provision of
3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-galactopyranosyl chloride (XXIII) as
a reagent for the preparation of 2-amino-2-deoxy-~-D-galactopyranosides
as depicted in formula A.
`` In the following examples, compound XXIII is used to prepare
a simple glycoside (Example XXVI), a disaccharide (Example XXVII) and
the trisaccharide antigenic determinant for the human A blood group
(Example XXVIII).
EXAMPLE XXVI
Synthesis of t-butyl-3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-=-
galactopyranoside (XLV~
A solution of freshly prepared 3,4,6-tri-0-acetyl-2-azido-2-
deoxy-~-D-galactopyranosyl chloride (XXII, 0.160 9, 0.458 mmole) in
- 35 -
.
:
j dichloromethane (1 ml) was added dropwise to a mixture of silver tri- !
fluoromethanesulfonate (0.010 9, 0.039 mmole), silver carbonate (0.443 9,
1.61 mmole), 4A molecular sieves (0.150 9), and t-butanol (55 ~1, 0.583
mmole) in dichloromethane. This mixture was stirred for 2.5 hours
in the dark and then filtered and the resulting filtrate evaporated to
dryness to give a syrup (0.150 9). The p.m.r. spectrum of this material
in CDC13 indicated the presence of about 60% a-t-butyl glycoside (XLV)
by the presence of a singlet at .30 p.p.m. The anomeric proton was
obscured by signals for H-4 and H-3 near 5.1 p.p.m.
EXAMPLE XXVII
Preparation of 8-Methoxycarbonyloctyl-3-0-(2-acetamido-
2-deoxy-~-D-galactopyranosyl)-~-D-
: i
galactopyranoside (XLVII)
A solution of freshly prepared 3,4,6-tri-0-acetyl-2-azido-2-
deoxy-~-D-galactopyranosyl chloride XXIII (0.335 9, 0.96 mmole~ in
dichloromethane (1 ml) was added to a mixture of silver trifluoromethane-
sulfonate (0.022 9, 0.085 mmole), silver carbonate (1.06 9, 3.85 mmole),
, O
4A molecular sieves (0.70 9) and 8-methanoxycarbonyloctyl-4,6-0-
benzylidene-2-0-benzoyl-~-D-galactopyranoside XLVI (0.250 9, 0.461 mmole)
in dichloromethane (4 ml). After 4 hours at ambient temperature the
. i
mixture was filtered through diatomaceous earth which was washed with
dichloromethane (10 ml). This solution was evaporated to give a syrup
which was dissolved in a small amount of 1:1 (v/v) benzene:ethyl acetate
and chromatographed on neutral aluminum oxide (15 9), in a column (10 x 2
cm), eluted with the same solvent, to afford a syrup (0.524 9). Crystal-
lization of this syrup from ethyl acetate:pentane afforded crude 8-
methoxycarbonyloctyl-3-0-(3,4,6-tri-0-acetyl-2-azido-2-deoxy-~-D-
galactopyranosyl)-4,6-0-benzylidene-2-0-benzoyl -~-D-galactopyranoside
(0.324 9) in 79% yield. Recrystallization gave the pure compound
m p 175 - 176, [~]2D5 + 119.7 (c 1, chloroform), i.r. (film)
2120 cm 1 (-N3).
- 36 -
1~11417
The p.m.r. spectrum of this latter compound in CDC13 contained
in part at 4.62 p.p.m., a doublet with Jl 2 = 8.0 Hz which was assigned to
H-l. The signal for H-l' was obscured by signals for H-3' and H-4'.
That the newly formed ;ntersugar glycosidic linkage was a was shown by
the presence of the signal ;n the 13C-spectrum of the compound in CDC13
at 95.4 p.p.m. which was assigned to C-l'. The signal assigned to C-l
was observed at 100.7 p.p.m. Hydrogenation followed by N-acetylation and
removal of the acetyl and benzoyl blocking groups as described in Example
XXVII for the preparation of compound L gave crystalline 8-methoxycarbonyl-
0 octyl-3-0-(2-acetamido-2-deoxy-a-D-galactopyranosyl)-~-D-galactopyranoside,
(XLVII). Recrystallization from methanol-ethyl ether gave pure XLVIII;
m.p. 214 - 216, [a]2D5 + 126.3 (c 0.98, water).
EXAMPLE XXVIII
;~ Preparation of 8-methoxycarbonyloctyl-3-0-(2-acetamido-2-deoxy-a-D-
~ 15 galactopyranosyl)-2-0-(a-L-fucopyranosyl)-~-D-galactopyranoside L
=
In this example, the alcohol has a disaccharidic structure
; and the glycosidation product is treated to convert the acetyl, benzyl, and
': benzoyl groups to hydroxyl groups (deblocking) and to reduce the azido
"..,
group to amine which is then acetylated. The method used to perform the -
above deblocking, reduction and acetylation reactions are well known to
persons skilled in the art.
; A solution of freshly prepared 3,4,6-tri-0-acetyl-2-azido-2-
deoxy-~-D-galactopyranosyl chloride (XXIII) (0.588 9, 1.6 mmole), dissolved
in dichloromethane (2 ml), was added to a solution of silver trifluoro-
methanesulfonate (0.035 g, 0.136 mmole), silver carbonate (1.70 9, 6.18
mmole), 4A molecular sieves (1.12 9), and 8-methoxycarbonyloctyl-2-0-
(2,3,4-tri-0-benzyl-a-L-fucopyranosyl)-4,6-0-benzylidene-~-D-
galactopyranoside (XLVIII~ (0.787 9, Q.9 mmole) in dichloromethane (5 ml).
After 4 hours at ambient temperature the mi`xture was diluted with di-
chloromethane (10 ml) and filtered through diatom~ceous silica and the
; filtrate was then evaporated to give a syrup (1.25 9). This syrup was
417
chromatographed on a column (44 x 2 cm) of silica gel with 2:1 (v/v)
benzene:ethyl acetate as the eluent, to afford pure 8-methoxycarbonyloctyl-
3-0-(3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-D-galactopyranosyl)-2-0-(2,3,4-
tri-0-benzyl-a-L-fucopyranosyl)-4,6-0-benzylidene-~-D-galactopyranoside
XLIX (0.780 9, 75% yield), [a]2D5 + 15.5 (c 1, chloroform), i.r.
(film) 2110 cm 1 (-N3).
,:
',
~.,
'''
'~
,.. '~; .
'
'';
- 38 -
(~ ( !
417
The p.m.r. spectrum of compound XLIX in CDC13 had in part,
5-47 (d, 1~ Jl" 2~ 3-4 Hz, H-l"), 5.32 (d, 1, Jl' 2~ 3 Hz, H-l'). Its
13C-n.m.r. spectrum in CDC13 clearly showed the two -glycosidic inter-
sugar anomeric carbon atoms with signals at 97.9 p.p.m. and 94.0 p.p.m.
for C-l' of the fucosyl unit and C-l" of the 2-azido-2-deoxy galactosyl
unit, respectively. The signal assigned to C-l of the galactosyl unit
occurred at 100.5 p.p.m.
Compound L (0.10 9, 0.085 mmole) was dissolved in ethyl
acetate (2 ml) containing acetic anhydride (0.2 ml) and hydrogenated
~ 10 in the presence of 5% palladium on charcoal (0.06 9) at 100 p.s.i. and
-~ ambient temperature. After 23 hours the solution was filtered and
evaporated to give a foam. The infrared spectrum of this compound showed
the absence of an azide group. This compound was deblocked or deacetylated
, with sodium methoxide in anhydrous methanol (5 ml) at ambient temperature
, 15 for 15 hours. After deioni`zation and filtration, evaporation of the solvent
gave a foam (0.08 9). Hydrogenation of this material in ethanol (3 ml)
in the presence of 5% palladium on charcoal (0.065 9) at ambient temperature
and 100 p.s.i. for 40 hours followed by filtration and evaporation gave
8-methoxycarbonyloctyl-3-0-(2-acetamido-2-deoxy-a-=-galactopyranosyl)-2-
0-(a-L-fucopyranosyl)-~-=D-galactopyranoside (XLIX) (0.046 9, 78% yield)
as a white solid.
The p.m.r. spectrum of compound L in D20 was consistent
with the assigned structure and showed in part, p.p.m. 5.62 (d, 1~ Jl~ 2
1 Hz, H'), 5-46 (d, 1~ Jl" 2~ 3-5 Hz, H-l"), 2.24 (s, 3, NAc). This
compound is the trisaccharide antigenic determinant for the
human A blood group.
EXAMPLE XXIX
Preparation of an immunoabsorbent (LII) specific for anti-A antibodies
The trisaccharide antigenic determinant (L)
for the human A blood group can be used to prepare an artificial antigen by
attachment to a soluble carrier molecule such as proteins, red blood cells,
polypeptides and soluble aminated polysaccharides using known methods.
. .
- 38a -
417
The glycoside L can also be used to prepare an immuno-
absorbent specific for anti-A antibodies by attachment to an insoluble
support such as aminated glass, aminated polyacrylamide, aminated poly-
vinyl, aminated agarose and other insoluble aminated polysaccharides.
This process is demonstrated below.
8-Methoxycarbonyloctyl-3-0-(2-acetamido-2-deoxy-a-=D-
, galactopyranosyl)-2-0-(c~-L-fucopyranosyl)-B-=D-galactopyranoside (L)
(0.044 9, 0.063 mmole) was stirred with 85% hydrazine hydrate (2 ml) at
room temperature for 90 minutes. Examination by thin layer chromatography
of the reaction mixture, on silica gel developed with 7:1:2 (v/v)
. .
isopropanol: ammonium hydroxide:water, showed no remaining starting
material. This solution was diluted with 50% aqueous ethanol (1 ml)
and evaporated to dryness to give a white foam (0.044 9). The material
was dissolved in water (2 ml) and dialyzed against five changes of
distilled water in an ultrafiltration cell equipped with a membrane with
a molecular weight cut-off of 500 and freeze-dried to give the corresponding
hydrazide LI as a white solid (0.039 9).
The p.m.r. spectrum of compound L in D20 was consistent with
the assigned structure and had in part, p.p.m. 5.58 (d, 1, J > 1 hz,
H-l'), 5.44 (d, 1~ Jl~ 2~ 3.5 Hz, H-l"), 2.30 (s, 3, NAc)-
The hydrazide LI (0.35 9, 0.05 mmole),was dissolved in
; dimethylformamide (0.7 ml) and cooled to -25. A solution of dioxane(0.057 ml) which was 3.5 N in hydrochloric acid was added and this was
- followed by t-butyl nitrate (0.007 9, 0.069 mmole) dissolved in dimethyl-
- 25 formamide (0.1 ml). This mixture was stirred for 30 minutes at -25
at which time sulfamic acid (0.0049 9, 0.052 mmole) was added. After 15
minutes, this solution was added dropwise to silylaminated glass beads
- (5.0 9) suspended in a buffer solution (25 ml) 0.08 M in Na2B407 and 0.35 M
in KHC03 at 0. This suspension was tumbled slowly at 3 - 5 for 26
hours at which time the support was filtered and washed with water (5Qa ml).
The beads were then suspended in saturated sodium bicarbonate (30 ml)
- 39 -
111~417
and 5% aqueous acetic anhydride (30 ml) was added and agitated for 15
minutes. The beads were then filtered and washed with water (500 ml)
and suspended in phosphate buffered saline (pH 7) (25 ml) and subjected
to reduced pressure for 15 minutes. Filtration and water washing (100 ml)
gave the hydrated immunoabsorbent LIII (11.2 g). A phenol-sulfuric assay
for total hexose on this immunoabsorbent before acetylation indicated a
~; loading of 6 ~mole of hapten per gram of support.
The immunoabsorbent LIII was found to selectively remove anti-
A blood group antibodies from human sera Thus, for example, treatment
of 1 ml of a serum which effectively agglutinated human A blood cells with
200 mg of the immunoabsorbent LII removed those antibodies responsible
for the agglutination within 20 minutes. The use of the immunoabsorbent
in the form of a packed column was more efficient.
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