Note: Descriptions are shown in the official language in which they were submitted.
~ 207~25~
TITLE OF THE INVENTION
SYNTHESIS OF SULFIDE-LINKED DI- OR OLIGONUCLEOTIDE ANALO&S ~ND
INCORPORATION INTO ANTI-SENSE DNA OR RNA
FIELD OF THE INVENTION.
The invention relates to a nucleoside analog for insertion in DNA
or RNA molecules. Particularly, the invention relates to sulfide-,
sulfoxides-, or sulfone-linked di- or oligo- nucleoside units for
inserting into anti-sense DNA or RNA.
BACKGROUND OF THE INVENTION
2'~Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are
linear, polymeric molecules consisting of nucleoside units joined
together by phosphodiester linkages. DNA is the blueprint chemical
supplying instructions for the synthesis of proteins. These
proteins play a central role in all aspects of cell function,
growth and reproduction. The key properties which allow nucleic
acids to store and coordinate the expression of genetic information
are the presence of four nitrogenous bases (designated A, C, G, and
T or U) found along the polymer which serves to store information.
The ability of complementary bases to recognize each other through
base-pairing allows for transfer of the information. ~he
availability of molecules capable o~ binding sequence-specifically
to particular nucleic acid species may allow for the design of
biological probes and therapeutic ayents.
,
Complementary nucleic acid strands are of choice as sequence
specific binding agents for both single-stranded and double-
stranded recognition. These agents make use o~ the molecular
recognition properties inherent in the natural system. Naturally
i occuring strands of unmodified RNA (anti-sense) have been found to
regulate expression of certain genes in a variety of living
systems.
'.
.. ...
- .
2~78256
, ~ ,
since the discovery, a decade ago, that bacterial gene expression
is naturally regulated by the binding of specific messenger RNA
species by complementary RNA strands, the anti-sense strategy has
developed into a major field of investigation. Much work has been
devoted to both natural and modified oligonucleotides which are
capable of forming stable double-helices with complementary mRNA,
thus blocking translation to the protein product. Many of these
agents bear uncharged phosphodiester group analogs which facilitate
the uptake of the oligonucleotide strands into cells and confer
increased stability towards degradation by cellular nucleases.
A ~actor which makes these agents attractive is the fact that they
can be prepared in a relatively straightforward manner by small
modifications to existing automated DNA synthesis technigues.
However such phosphate-modified agents, which include
methylphosphonate, phosphoramidate, and phosphorothioate-linked
oligodeoxynucleotide analogs, possess some inherent drawbacks.
Modification or replacement of one of the phosphoryl oxygens (or
both by different groups) gives rise to a chiral phosphorus center
which results in a mixture of diastereoisomeric oligonucleotide
products. In addition, the removal of the negative charge on the
internucleoside linkage results in the loss of the high chemical
stability associated with phosphodiesters. The absence of the
charged phosphodiester group has also been implicated in the
reduced stability exhibited by hybrid (P-modified ~ natural)
helices.
There have also been a number of examples of DNA analogs in which
the phosphodiester groups have been replaced altogether with non-
chiral linkages stable to hydrolysis. The prior art includes a
variety of modifications to the natural structure which can be
roughly divided into three categories:
1) modifications to the sugar moiety;
2) alteration of the phosphodiester linkage; and -
, . ': ' :' . ' ' ' : ' . '
` 20782~
3) replacement of the phosphodiester with a non-phosphorus
containing group.
Notable examples of the third category, the "dephospho" analogs,
are those containing sulphur atoms. Benner (Z. Huang, K.C.
Schneider, S.A. Benner J.Org. Chem. 56, 3869 (1991) ha~ ~eported
details of the syntheses of 3',5'-bis-homo-thionucleosides, short
polysulfone-oligomers of which were found to bind to complementary
DNA. Furthermore, Musicki et al. (B. Musicki & T.S. Widlanski
Tetrahedron Lett. 32, 1267 (1991)~ have recently described a
dinucleoside analog exhibiting chemical stability, in which the
phosphodiester is replaced with a sulfonyl ester. In addition, his
group prepared systems in which short modified regions are
incorporated into natural DNA. Such mixed molecules are of interest
since it has been shown that the addition of backbone-modified
units to the 3'- and 5'-ends of otherwise unaltered DNA greatly
increases the strands' stability towards nuclease degradation.
Protection and activation of sulphur-containing fragments can allow
for straightforward incorporation of such pieces into DNA by
standard automated techniques.
~he present invention provides for nucleotide analog units
~oligomers, homopolymers and dimers) incorporated into DNA or RNA
molecules. In these modified DNA strands, all or some of the
phosphodiester groups are replaced by non-hydrolysable sulphur-
containing linkages. These new complexes have the same number of
atoms joining adjacent sugar rings (ribose or deoxyribose) as in
natural systems. The sulfide linkage, however, varies from the
natural phosphodiester in many respects:
1) the new linkage does not bear a charge and is therefore
much less polar. The polari~y of the group can be altered by
oxidizing the sulfide to either the corresponding sulfone or
sulfoxids;
2) the sulphur-containing linkages are not subject to chemical
hydrolysis; and
~,
.
. ., .. , . , ; :, :, ,- ~ ~ "
,,, , : ., ~ , .
,: : . ,. . . , , j :
. ..
~7825~
: `
3) the sulphur-containing linkages are not subject to
enzymatic hydrolysis by phosphodiesterases. :
SUMMARY OF THE INVENTION
Therefore, the invention provides for an oligonucleotide_analog of
formula I in which one or more of the internucleoside
phosphodiester groups are replaced by non-hydrolysable dialkyl
sulfide, sulfoxide or sulfone linkages such as in: .
~B :.
[ Y ~
: R~ ~ :
.~
where R and R' are independsntly selected from the group consisting
of: H, DNA or RNA unsubstituted or substituted with oligonucleotide
analog of formula I, nucleoside, nucleotides and analogs thereof;
B is a base having a heterocyclic ring, each B being independently
selected from the group consisting of purine, pyrimidine,
azapyrimidine, azapurine, pyrrolopyrimidine, pyrazolopyrimidine,
triazolopyrimidine, imidazolopyrimidine, pyrrolopyridine,
pyrazolopyridine, and triazolopyridine~ where the ring may be
functionalized with amino groups, hydroxyl groups, halogen groups
or acylated derivatives of amino or hydroxyl groups;
each X is independently selected ~rom the group consisting of: O,
CH2 and S;
each Y is independently selected from the group consisting of: H
21~782~
, ~
and OR~, wherein R! is selected from hydrogen and alkyl, preferably
allyl;
Y' is selected fxom the group consisting of: H and ORI, wherein R~
is an alkyl;
n is O, 1 or 2; and
m is O or an integer.
It will be understood that the terms oligo- covers from 2 to
several monomer nucleoside units (eg. dinucleotides). Preferably,
the invention provides for a dinucleotide analog where m is O.
A further preferred embodiment of the invention provides for a
nucleotide analog wherein the base is selected from adenine,
guanine, cytosine, uracil or thymine. Most preferably, the base is
thymine.
The invention further provides for DNA and RNA molecules
incorporating the oligonucleotide of formula I at either end
thereof or internally. More preferably, the invention provides
anti-sense DNA and anti-sense RNA moleculesi including the
nucleotide of formula X.
Furthermore, the invention provides for a method o~ producing the
oligomer of formula I comprising the step of:
a) condensing a compound of formula II:
'Y ~
II ~ y
MsO
wherein P is a hydroxyl protecting group (preferably, when Y is H,
the protecting group is TBDMSi, whereas when Y is OH, the
1, , , , . ' ' " ~ ~ . , "' ' . '
~ 21~7825~
protecting group is an acetyl residue);
with a compound of formula III, this step being performed m times.
III ~ B
: pMeOC6H40 ;: :
to obtain a compound of formula-IV:
~ B
~ IV r ~
L ~ ~
wherein P, B, X, Y, and m have thE same meanin~ as define~ above.
:The process preferably further comprises the steps of:
i) deprotecting the 3'-end hydroxyl group to obtain a compound ~:~
of formula V: :
~B
L ~lm
Ho
6 .~ .:
",
207825~
ii) mesylating the 3'-end hydroxyl yroup to obtain a compound
of formula VI:
~B
_ y_
VI M ~ B m `~
and iii) condensing the compound of formula VI with a compound of
formul~ VII:
HS ~ B
VII TsDMSio ~ Y'
:~ to obtain a compound of formula VIII:
~-B - :
TBDNSil~y ~ `
wherein B, X, Y, Y', P and m have the same meaning as defined above ~-
and n is 0.
, ~'',
-.~ 2~7~2~6
The process further comprises the step~ of-
b) deprotecting sequentially the 5'-end and 3~-end hydroxyl
groups, and
c) treating the resulting free 5'-end hydroxyl group with
dimethoxytrityl chloride to give a compound of formula X:
DMTrO~,
[ ~im
OnS
~yx>-B
HO~~Y ~ :
:
and d) treating the compound of formula X with 2-cyanoethyl N, N-
dii~opropylchlorophosphoramidite in dichloromethane containing ::
triethylamine to give a compound of formula XI: ~.
." ,~.
DMTrO
y~,B .. :.
[ ~i
XI OnS Y m
iPr2N~ ,
o :
--CN
where B, X, Y, Y', and m have the same meaning as defined above,
and n is 0.
Alternatively, the compounds of formula VIII or IX (where n is O)
, 8 ~ ~.
:.
20782~6
.: ~
can be oxidiæed before further treatment to give the corresponding
sulfoxide- or sulfone- oligonucleotide analogs of formula I wherein
n is 1 or 2 respectively.
Also, preferably, the compound of formula III may be omitted in the
condensation step to condense simply compounds II and VII and yield
a dinucleotide of formula I where m is O.
The invention also provides for intermediates used for the
production of the oligomer of formula I, having the formulas II,
III, VII, VIII, I~, X, XI, ~II and ~III.
PO ~ III ~ B HS ~
TsDMSiO Y'
MsO~ pMeOC6H40
~B HO~
~OnS~ Or.S~
OnS~X OnS~
TBDMSiO>~ ~ HO~y .
; ~ DMTrO~X
~;rO~_ Xl ~ ~ m
OnS~ B iPr2N\ ,
HO>~ ~ CN
.' ' : '
~ 207~256
H~ DMTrO~ B
TBDMSi ~ TBDMSiO y~
:
where P, B, X, Y, Y', m, and n have the same meaning as defined ~:-
above.
~: :
D~TAILED DESCRIPTION OF EXAMPLES OF PREFERRED ~MBODIMENTS
The activated/protected sulfide-linked oligomer required for
incorporation into DNA is prepared according to the method outlined
in Schemes la and lb. Intermediates in the preparation of the
suIfide-linked oligomer I are: the 5'-0-tert-butyldimethylsilyl-3J-
deoxy-3'-C-(2"-hydroxymethyll-2l'-0-(methanesulfonyl~ purine or
pyrimidine II or "5'-end unit"; the 3'-5'-dideoxy-3'-C-(2"-
hydroxyethyl) 2"-O-par2-methoxyphenyl-5'-thionucleo~ide III or
"middle-unit'i; and the (3'-0-tert-butyldimethylsilyl)-5'-deoxy-5'-
thionucIeoside VII or "3'-end unit" ~hown in Scheme la. The
coupling of the units is carried out in a dimethylformamide (DMF)
solution containing cesium carbonate (1~5 equivalent). The sulfide-
linked ollgomer is obtained in approximately 90% yield.
:.
r. , ' , ~ - ~. .; ' ; .
~ 207~25~
Scheme la (n=O, X=O, Y=H, Y/=H, P= TBDMSi~
TBDMSiO B TBDMSio o B
TBDMSiO~ B HS~ a ~ b \~
II pMeOC6H4 ~ CAN [
PMeOC6H40 IV H V
. . MSC1
(m-l times ) Et3N
HS B
+ TBDMSio o
~ DMTrO~O~ B pMeOC6H40~ III ~B
~y RO~O B o r r ~ ~ -
r ~ ~ ~ d_
HS~ ~ B ~ m
L ~ ~m ~ `rB TBDMSiO - MS
. ~ ~ S m VI I VI
lPr2N~P~O~CN ~B
R~o
~, ~ XI e VIII R=R'=TBDMSi
f F~ IX R = R' = H
X R=DMTr R'=H
11
I
~` 2~782~
The process as outlined in Scheme la can be described as follows: :
Step a) The sulfide-linked oligomer IV is formed by displacement
of the 2"-mesyl group in the 5'-0-tert-butyldimethylsilyl-3'-
deoxy-3'-c-(2"-hydroxymethyl)-2"-0-(methanesulfonyl) pu~ine or
pyrimidine ~I by the thiolate obtained by deprotonation of the
5'-thiol group in the 3'-5'-dideoxy-3'-C-(2"-hydroxyethyl)- 2"-
0~ para- methoxyphenyl- 5'- thionucleoside ~ This is carried
out in N,N~dimethylformamide using cesium carbonate as bass;
Step b) The 2"-para-methoxyphenyl protecting group is removed
from IV by ceric ammonium nitrate oxidation to yield an alcohol
V, and
Step c) the resulting hydroxy group is mesylated in methylene
chloride containing pyridine and triethylamine yielding a 2"-
mesylated dimer VI;
Steps a), b), and c) are repeated as many times (m-1) as
necessary to elongate the dimer to a trîmer, or the trimer to a
tetramer, etc..;
Step d) In the case of the last nucleos:ide unit to be added to
the growing oligomer, the thionucleoside VII is used rather than
III. The resulting chain VIII will have a lenght of m + 2.
Step e) The final product of this condensation VIII is then
deprotected in a tetrahydrofuran ~olution by treatment with a
stock solution of tetra-n-butylammonium fluoride in
tetrahydrofuran which provides the corresponding diol I~;
Step f) The 5'-hydroxyl group of the nucleoside unit at the 5'-
end of the oligo-nucleoside analog is selectively protected by
forming the dimethoxytrityl ether. This is carried out by
12
20782~
treating a solution of the diol IX in methylene chloride
containing pyridine and triethylamine with dimethoxytrityl
chloride;
Step g) The resulting alcohol X is then reacted with 2-
cyanoethyl-N,N-diisopropylchlorophosphoramidite in --
dichloromethane containing a triethylamine, diisopropylethylamine
and/or pyridine. This reaction provides the fully activated /
protected sulfide-linked oligonucleotide analog phosphoramidite
Xl for incorporation into DNA or RNA.
Alternatively, an oligomer may be constructed from a plurality of
similar or different dimers that are attached to each cther via
phosphodiesters. For this purpose, no "middle-unit" III is
required. Units II and VII are coupled directly to give,
eventually, Xl (where m=O) for insertion into DNA or RNA, or for
joining together.
As shown in Scheme ~b, the resulting dimer VIII ~where P=TBD~Si,
m=o. n=0, X=O, Y=Y'=H, B=Thy) may be treated with Oxone~ prior
to, or after deprotection with a fluoride salt such as tetra-n-
butylammonium, ammonium or cesium fluoride in an organic solvent
such as THF, to give the corresponding sulfone IX (where n=2).
': 2~7g2~6
Scheme lbo (P=TBDMSi, m=O, X=O, Y=Y'=H, B=Thy)
TBDMSO ,~,
TBDMSIO ~ l<c>~
~ ~ ~ cS2cO~MF r
J ~ S
rTSDMSIO ,`
OM~
1~ ~, \~/ ;
b / oTeDMS
/D8u~NF ~
HO~ r / THF ( n_o)
~ .
d Oxons
3~ 1"
:~ ~ . ;
GH
C/ ~' `"
HO~ o ~ /Oxone (~-o~ TeDA~so~
~J ~' ~ .
J ~4NFITHF J
e, "~r
o~1~O J 4`l~o~ ~:
~J -
OH OTDDMS
: ~ ~Ç (n æ) ~ ~_z)
DMTrCI
Et3NUpyridine
DMTrO
DMTrO~ 1' )~J
\~ R~2NP(CI)CI 12CH2CN ~ r
0~ Et3N/CH2cl2 0~ ¦O J
s~ ~ q ~~
o ~ J o
OH ~
2 ) ~ CN
. .
2078~
SYNTHESIS OF SULFIDE-CONTAINING DNA OLIGOMERS
Sulfide dimer or oligomer phosphoramidites XI are freely soluble
in acetonitrile, which is the solvent of choice for
oligonurleotide synthesis by solid~phase methods. A bottle
containing a 0.08 M acetonitrile solution of phosphoramidite ~I
is attached to an automated synthesizer. DNA or RNA oligomers I
incorporating the sulfide-linked di- or oligonucleotide analogs
are prepared by minor alterations to the standard coupling cycle.
As is known in the art, specifically, the length of the
tetrazole-mediated phosphoramidite coupling step is increased to
5 minutes. The coupling efficiency of the phosphoramidite XI is
routinely greater than 95% as monitored by the release of the
dimethoxytrityl cation.
once prepared, the oligomers I are cleaved from the controlled-
pore glass support by treatment with ammonium hydroxide solution.
The resulting 5'-O-tritylated oligomers ~ can then be easily
purified (and deprotected) by reverse-phase chromatography (OPC~,
Oligonucleotide Purification Column, Applied Biosystems). The
purity of the DNA or RNA strands is demonstrated by analytical
polyacrylamide gel electrophoresis (PAGE3.
PROPERTIES OF THE SU~FIDE-CONTAINING DNA OLIGOMERS
The ability of the sulfide-~ontaining oligomers to complex to
complementary DNA is conclusively demonstrated by native PAGE
(non-denaturing gel run at low temperature and current). A new
band appears that corresponds to the complex when complementary
sulfide-containing and fully natural DNA strands are combined.
Thermal denaturation (melting) studies show cooperative binding
between the strands which is indicative of helix formation.
~ .
The sulfide-containing strands also exhibit resistance to
,, , . . ., , . ~ , . ., ,, , .. , .. . . , .. " , . -.. . . .
20782~i~
degradation by phosphodiesterases, enzymes that break down DNA
and RNA in the cell. An oligom~r bearing, internally, three
consecutive sulfide-linked dimers (NsN) flanked by natural
regions of DNA three nucleotides long (i.e.
NpNpNp~pNsNp~pNpNpN) is then treated with calf-spleen
phosphodiesterase (CSPDE)o This enzyme sequentially cleaYes
nucleotide units from the 5'-end of a DNA strand. In this case,
the progress of the enzyme is halted when a sulfide-linkage is
reached.
The strand is also treated with snake-venom phosphodiesterase.
This enzyme sequentially cleaves DNA from the 3'-end. Again this
appears to result in progressive cleavage of the phosphodiester
linkages until a sulfide-linkage is reached. However, the enzyme
appears to jump over the sulfide-containing linkages of the
strand and break all phosphodiester linkages present, releasing
only the remaining NsN dinucleotides. The internal cleaving
activity of this particular enzyme has been previously observad
in a number of cases.
As described above, the presence of sulfide-linkages in place of
the natural phosphodiester groups in DNA strands can protect the
olig~mer~ from at least certain types of enæymatic degradation -
while not disrupting their ability to complex to complementary
DNA. Thus, biologically stable anti-sense analogs are made by the
proper placement of sulfide (or sulfoxide, or sulfone) -linked
dimers, trimers or oligomers within DNA strands (likely at the
ends of the oligomers). Such molecules can be used to bind a
number of complementary targets and, through this, be useful in
the treatment o~ genetic disease; in the treatment of viral,
bacterial and parasitic infections. They can also be used as
biological probes. Some suitable binding targets include:
1. messenger RNA;
2. single-stranded RNA (e.g. RNA viruses);
3. single-stranded DNA;
16
''., . ' ~ ' ' . ' ' . " ' ' .. ' ' ' " ' ',.' . "
. '~': . ' ' : ' '. ' ' ' . ' . ' " ' ' ' ' ' '
', ' ,': ' ' ' ~ ' ' ' ' ~ ' : . '' . ' , '
2~82~
4. double-stranded DNA (i.e. triple helix formation); and
5. Other suitable polynucleotides.
The non-ionic nature of the sulfide-linkage lowers the polarity
of the oligonucleotide as a whole. This increases the ability of
the strands to cross biological barriers. This activity~nay be
modulated by varying the ratio of sulfide to phosphodiester
linkages. Alternatively, the sulfide linkages can be converted to
either stereoisomer of the sulfoxide or to the sulfone. The
binding properties of partially-modified DNA can also be altered
in a similar manner.
Since the sulfide (sulfoxide, sulfone) linkages are not subject
to hydrolysis, such modified systems may be used to carry
catalytic groups capable of hydrolysing the phosphodiester bond.
As persons skilled in the art would recognize, this would result
in an artificial enzyme able to cleave DNA or RNA in a sequence
specific manner. The linkage of the invention would circumvent
the problem of self-cleavage when such catalytic group~ are
attached to natural DNA or analogs containing hydrolyzable
groups. Phosphodiester cleaving molecules, especially metal
complexes, are often very efficient at c:leaving esters, amides ;~
and presumably also phosphate-group analogs. ~`
SYNTHESIS OF THE "5'-end" II, "middle;' III, AND "3'-end" VII -
UNITS.
:,
The mesylated derivative II can be synthesized by two routes as
shown in Schemes 2 and 3. The first approach is applicable mainly
to pyrimidine derivatives, in particular to thymidine, whereas
the second (from compound 12) is routinely applicable to all four
bases.
... -. ~ . . . . .. . . . . .. , ... , . - ; . . . . . . . . . .
', - ~ . : ! i: . ~ ` ` - :, ~. -
`-` 2078256
Scheme 2 (X=O, Y=H, B=Thy)
o o o
~NH ~I~H ~N~I
~ b l~o J C ~o N~o
\y \, ~ \,~_
k ~J :
(1~ Y~/ GH2 (3) \ ~,~FJ (4J or(~ )
(a) o~ f~la104 / Et20 / HzO; (b) NaBI~ / M~OH; (c) Msa / p~ / c~;
\
Scheme 3 (X=O, Y=H)
.
r~o r~o ~ rsr~o ~,
~ot~ ~OR.
OR' P~O ~ .' ~
~$) R-Tr ~.. H ~ ~ Jq_A~z.. o~ ) y~
d~;;(6~ R-Tr ff~R-lbzs~ 1) Y~
--~D) R.. H z=~ (12) y--H
--J m 1~~ ~~a~l
~ __ ~ i ~ '
p~OC~O ~ ~O
(13) ~ 4~ t~
I----(15) Y -OCSiOPh
c-r J~ (~8~ y--H
(~)
(~q p MeOC~H,OH /
DEAD / PPh3 / THF; ~ul I~OH 1~ / camphorsullonk acid I ~0C: ~fl ~MSi~2Thy / TMSiOT~ /
ClCI12CH2Ci / r011ux; (~ I MeOH: (h) T~iDMSiCI / irnid~l7ol~ / DMF; (q PhOSCCl / pyfidin~ / .
CH2Ck; 0 ~Bu3SnH / totuono / 7~iC; (k~ AcSH / DIAD / PPh3 / THF; (I) ~Bu~NF I THF; (m~ . . .
co~nions (9~ or NaOH / M~OH. ~ . .
.
.,,. ; . ... .. . .
' ' ' . ! ., , ., . ,; !
,, . i ~. . . i! i , . ' . '. . ' i:: j , .
.. ' ; ' ` " " ', '', '` ~ , ' ' . ','~ . . '"., ' .
207g25~
The first approach as described in Scheme 2, consists of the
treatment of the 3'-C-allyl 3'-deoxythymidine derivative 1 with
osmium tetroxide and sodium periodate following the two-phase
"ether-waterl' method as described by R. Pappo (D.S. Allen, Jr.,
R.V. Lemieux, W. S. Johnson, J. Org. Chem. 21, 478 (195~-. The
reaction provided aldehyde 2 in 74% yield. Subsequent reduction
with sodium borohydride in methanol provided a ~5% yield of 3'-C-
(2"-hydroxyethyl) nucleoside 3. Mesylation af~orded the thymidine
"5'-end unit" 4 in 90% yield.
However, this first approach does not appear to be applicable to
all the required purines and pyrimidines. Radical allylation of
2'-deoxy-3'-0-phenoxythiocarbonyl(cytidine) derivatives resulted
in complex mixtures. In addition, 2'-deoxynucleosides other than
thymidine are very expensive starting materials. However, uridine
derivatives may be transformed to cytidine derivatives so ~-
indiri~ict methods may be used for the formation of the latter.
.1 .
Therefore, as an alternative embodiment of the invention, there
is provided an alternate route to the branched-chain nucleosides
II following a second approach as described in Scheme 3, i.e. the
attachment of the hydroxyethyl group prior to base attachment.
This second approach commences from 3'-deoxy-3-C-(2'-
hydroxyethyl)-1,2-0-isopropylidene-5-0-trityl-D-ribofuranose 5.
The ribofuranose ~ is ef~iciently prepared in large scale, in
four steps, from monoacetone xylofuranose in 61~ overall yield as
described in S~H. Kawai, J. Chin, G. Just, Carbohydrate Res. 211,
245 (1991).
The 2"-hydroxyl group of the sugar can be protected as the p-
methoxyphenyl ether. Thi~ ether was formed under Mitsunobu
conditions using diethyl or diisopropyl a~odicarboxylate-and
triphenylphosphine in tetrahydrofuran (THF), to give the para-
.
, . . .
-~"` 2~782~
methoxyphenyl derivative 6 with a 91~ yield. The sugar was
subjecte~ to acetolysis in a mixture of acetic acid and acetic
anhydride containing camphorsulfonic acid heated to 70Co This
resulted in simultaneous cleavage of both the trityl and
isopropylidene groups, affording the B-triacetate 7 in 69% yield.
The B-triacetate was accompanied by a small amount of the
expected acetylated aldehydrol-derivatives.
The B-triacetate 7 and bis- (trimethylsilyl) base are coupled in
refluxing dichloroethane by the Vorbruggen coupling (H.
Vorbr~ggen, K. Krolikiewicz. Angew. Chem. internat. Edit. 14, 421
(1975) using trimethylsilyl tri~late as catalyst. This gives
diacetate nucleoside ~ in 91% yield (when B=~hy). Subsequent
deacetylation using methanolic ammonia affords diol g in a
quantitative manner. The diol 9 is selectively monosilylated to
give the 5'-TBDMSi ether 10 in 86% yield (when B=Thy).
The 2'-0~ of 10 was removed by first treating with phenyl
thiochloroformate/ pyridine/ dichloromethane to form the 2'-0-
phenoxythiocarbonate 11. Treatment o~ 11 with tributyltin hydride
~AIBN initiation) in hot toluene yieldecl the 2'-deoxy compound 12
in 81% yield for the two steps (when B=Thy). The stereo- and
regioselectivity of nucleoside formation was conclusively
verified by comparing this product to the p-methoxyphenyl ether
prepared from alcohol 3 obtained via the 3'-radical allylation
route (Scheme 2). The two samples of nucleoside 12 were
indentical in all respects. The product was also identical to
material prepared by a completely different asymmetric synthesis
commencing from 4-0-benzyl-3,4-dihydroxybutyne. (G. Just, J.F.
Lavallée, Tetrahedron Lett. 32, 3469 (1991).
The nucleoside "middle-unit" 13 was prepared from either
compounds 9 or 12, shown in Scheme 3. Alcohol 9 and thiolacetic
acid are coupled by the Mitsunobu coupling (R.P. Volante
Tetrahed. Lett. 22, 3119 (1931)) employing diisopropyl
. . - .
,., , :, . , : . ~ .: .
78256
azodicarboxylate and triphenylphosphine. This proceeds
regioselectively to give the thiolester 14 in 85% yield (when
B=Thy). The tributyltin hydride reduction of the
phenoxythiocarbonate ~5 effected removal of the remaining 2'-
hydroxyl to give a fair yield of 5'-S-Acetyl-3'-5'-Dideoxy-3'-C-
~2"-hydroxyethyl)-2"-o-para-methoxyphenyl-5"-thionucleoside 16.
This low yield is due presumably to interference with the radical
reaction by the 5'-thiolester functionO The target nucleoside is
much more efficiently obtained by desilylation, using tetra-n-
butylammonium fluoride in THF, of the 2'-deoxynucleoside 12 to
give the corresponding alcohol 17. This was followed by Mitsunobu
coupling with thiolacetic acid to give the thiolester 16 in 87%
yield for the two steps ~in the case of B=Thy~. The S-acetate was
deprotected in methanolic sodium hydroxide followed by
neutralization with acidic resin. Careful deoxygenation of all of
the solutions proved necessary to prevent the rapid oxidation of
the resultant thiolate to the symmetrical disulfide. The reaction
can also be performed using methanolic ammonia, again carefully
removing and excluding any oxygen. The thiol 13 is obtained in
98% yield (when B=Thy).
The "3'-end unit" i5 prepaxed in three steps from the nucleoside
as shown in Scheme 4. The Mitsunobu coupling of the free
nucleoside and thiolacetic acid in a mixture of THF and DMF gives
i the 5' monothiolester 18 in 52% yield which is converted to the
3'-silyl ether 19 in 92% yield for thymidine derivatives. ~he
deacetylation is again performed using methanolic hydroxide,
; which gives the thiol 20 in quantitative yield. Care must be
exeroised in order to exclude oxygen from the reactlon.
21
2~782~6
,. ...
Scheme 4
c
051 ao ~80
bC:.(10) R TBO'MS (O~' X~C~ Y`~~)
~a) ACSH / DlAD l PPI~ / DMF l THF, (b) rSDMSiCl l im~azole l DMF: (c) NaOH l MeOH.
'`:
':
` .
I
.
'
;:
22
~,
:` 207~2S6
EXAMPLES
General Nethods.
Melting points (m.p.) o~ nucleosides of the invention were
determined using an Electxothermal ~P apparatus _and are
uncorrected. Optical rotation measurements were carried out in the
indicated solvents employing a Jasco~ DIP-140 digital polarimeter
and a l~dm cell. W spectra were recorded on a Hewlett-Packard~
8451 diode array spectrophotometer. Low-resolution chemical
ionization mass spectra (CI) were obtained on an HP 5980A
quadrapole mass spectrometer in the direct-inlet mode.
High-resolution CI and fast atom bombardment (FAB) mass spectra
(~RMS) were obtained on a VG~/ZAB-HS sector mass spectrometer in
the direct-inlet mode. The measurements were generally carried out
at a resolving power (res.) of 10000 unless otherwise indicated.
~lemental analyses were performed by Guelph Chemical Labora~ories
Ltdo (Guelph, Ontario). All compounds were shown to be homogeneous
by thin layer chromatography (t.l.c.) and high-field nuclear
magnetic resonance (NMR), and/or to have a purity of >95% by
elemental analysis.
IH-NMR spectra were recorded on either Varian~/XL200 or Varian~
XL300 spectrometers and the assignments are based on homonuclear
decoupling and / or rosy experiments. When deuteriochloroform was
employed as solvent, internal tetramethylsilane (TMS) was used as
the reference. The residual proton signal of methanol, assigned a
value of 3.30 ppm, was used as the reference. The multiplicities
are recorded using the following abbreviations: s, singlet; d,
doublet; t, triplet; q, quartet; h7 heptet; m, multiplet; mn,
symmetrical sig~al of n lines; br, broad. 13C-NM~ spectra were all
obtained at 75.4 MHz using a Varian~ XL300 spectrometer. The
l3CDCl3, 13CD3OD signals, assigned values of 77.00 and 49.00 ppm
respectively, were used as references in these solvents. Peak
"~ 2~782~6
assigments were, in some cases, made with the aid of APT or HETCOR
experiments.
~; , .
Tetrahydrofuran was distilled from sodium benzophenone ketyl.
Dichloromethane methylene chloride and 1,2-dichloroethane were
distilled from P~05. Toluene was dried over sodium wire._ Pyridine
was distilled from calcium hydride. N,N-Dimethylformamide was
dried by shaking with KOH followed by distillation, at reduced
pressure, from BaO. Thin-layer chromatography (t.l.c.) was
performed using Kiaselgel~ 60 F2~ aluminium-backed plates (0.2 mm
thickness) and visualized by W and / or dipping in a solution of
ammonium molybdate r2.5 g~ and ceric sulfate ~1 g) in 10 % v/v
aqueous sulphuric acid (100 mL), followed by heating. Kieselgel0
(Merck 230-400 mesh) silica gel was employed for column
chromatography.
.
Examples 1 to 3 relate to the reactions described in schemes 2, 3
& 4, wherein the sugar ring is deoxyribose (i.e. X i5 0; Y is H;
and B is ~hymine). Example 4 relates to scheme Ia, and example 5
relates to scheme Ib.
:'
EXANPLE 1
SYNTHESIS OF _5'-O-tert-butyldimethylsilyl-3'-deoxy--3'-C-(2''-
hYdroxyethyl~-2"-O-¢methanesulfonyl)thymidine _4 (or _IY where
P=TBDMSio, X=O Y-H~ B=Thy) ~5'-end" unitL~ (Scheme 2)
a) Aldehyde 2.
Osmi~m tetroxide (100 mg, 0.393 mmol~ was added to a two-phase
system comprised of ethyl ether (8 mL) and water (8 mL) containing
the thymidine derivative 1 (1.00 g, 2.63 mmol). Sodium
metaperiodate was added (1.24 g, 5.80 mmol) in small portions over
0.5 h to the resulting brown mixture. The reaction was stirred at
1 ambient temperature under nitrogen in the dark. After an
additional 1 h the mixture was extract~d with dichloromethane (2 x
24
.: . ,, . . : : ,
; , .. ' ' , ,. - ,, :: . : ' .:
, . : ' ' : ,.
207~2~ ~
200 mL) and washed with aqueous sodium bicarbonate (5 % wtv, 300
mL) and water (300 mL). The combined organic phase~ were then
dried (Na2S0~), filtered and the solvent removed in vacuo yielding
a colorless glass. Chromatography over silica gel (2:1 to 4:1
ethyl acetate / hexanes, v/v) afforded aldehyde 2 as a white solid
(740 mg, 74 % yield) with IH-NMR (CDCl3, 200 MHz) ~ 0.120-and 0.122
(two s, 6H,SiM~2), 0.93 (s, 9H, t-butyl), 1.94 (fine d, 3H, J = 1.2
Hz, 5-Me), 2.08 (A of ABXY, 1H, H2'A), 2.32 (B of ABXY, lH, H2'~,
2.50-2.84 (m, 3H, H1"A~ and H3'), 3.73-3.84 (m, 2H, H5~A and H4');
3.97 (dd, lH, H5~B), 6.14 (dd, lH, H1'), 7.54 (fine q, lH, J = 1.2
Hz, H6), 8.72 (br s, lH, N~), 9. 80 (S, 1H, CHO) JHI~-H2~A = 6-7 Hz, JHI~-
H2 ~ 5.2, JH2'A-H2'B 13.5, JH2'A-~3' = 6 8, JH2'~H3' = 7.8;
3C-NMR (CDCl3) 12.58 ppm (5-Me), 18.40 (CMe3), 25.90 (CMe3 and
SiMe2), 32.24 (C3'), 38.57 (C2 ~ ), 46.72 (Cl"), 63.25 (C5' ), 84.65
and 85.36 (C1' and C4~), 110~58 (C53, 135.40 (C6), 150.50 (C2),
164.04 ~C4), 199.80 (CH0); MS (CI - NH3) m/e 383 (~MH+~, 100 %),
274 (~M ~ NH4+ - ThyH], 17), 257 (tMH+ - ThyH], 68), 127 ([ThyH +
H~], 10); HRMS (CI - NH3) m/e calcd. ~or C~8H3IN2o5Si: 383.20022,
found: 383.20018.
b) 5'-O-tert-Butyldimethylsilyl-3'-deoxy-3'-C-(2"-hydroxvethyl) :
thymidine 3.
Sodium borohydride (34 mg, 0.90 mmol) was added to a stirred
solution of aldehyde 2 (690 mg, 1. 80 mmol) in methanol (16 mL) and
the reaction was stirred at ambient temperature. Glacial acetic
acid (200 mL) was added after 1.5 hour to the solution and the
solvent evaporated in va~uo affording a colorless syrup which was
extracted with methylene chloride/ dichloromethane (150 + 100 mL)
and washed with aqueous sodium bicarbonate (sat., 300 mL) and brine
(300 mL). The combined organic phases were then dried (Na2SO4) and
the solvent removed in vacuo affording essentially pure alcohol 3
as a colorless glass (593 mg, 85 % yield) which was used without
further purification: IH-NMR (CDC13, 300 MHz) ~ 0.12 and 0.13 (two
~" 20782~
s, 6H, SiMe2), 0.94 (s, 9H,t-butyl), 1.51-1.62 (m, lH, H1l'A), 1.74-
1.85 (m, lH, H1"D),1,92 (fine d, 3~, J = 1.3 Hz, 5-Me), 2.14 (A of
ABXY, 1H, H2~A)~ 2.25 (B of ABXY, 1H, H2'B), 2.37-2.49 ~m, lH, H3'),
3-65-3-81 (m~ 5H, H5~A; H4'; H2llAB and - 0~), 4.01 (dd, 1H, H5~B)~
6.08 (dd, lH, H1'), 7.57 (fine q, lH, J = 1.3 HZ, H6), 3.34 (br s,
lH, N~), JHI~ A 6-8 HZ~ JHI~ B = 3-9, J~A-~B = -13.5, J~-A-~B~ = 9.2,
J~ 3, = 7-6;
3C-NMR (CDCl3) 12.84 (5-Me), 19.42 (SiCMe3~, 26.53 (SiCMe3 and
Si~Q2), 35.57 (C3'), 35.8~ (Cl"), 39.76 (C2'), 61.31 (C5'), 64.02
(C2"), 86.32 and 87.94 (C1' and C4'), 110.78 (C5), 137.80 (C6),
152.29 (C2), 166.42 (C4); MS (CI - NH3) m/e 385 (~MH+], 17 %), 276
(~M + NH4+ - ThyH], 21), 259 ([MH+ - ThyH], 100), 127 (~ThyH + H+],
10); HRMS (CI - NH3) m/e calcd. for C~aH33N2o5Si: 385.21588, found:
385.21593.
c) 5'-o~ tert-Butyldimethyl~ilyl-3'-deoxy-3'-C- (2"-hvdroxyethyl)-
2"-0- methanesulfonYl-thvmidine 4.
Methanesulfonyl chloride (201 ~L, 2.60 mmol) was added dropwise to
a stirred solution o~ alcohol 3 (500 mg, 1.30 mmol) in dry
dichloromethane (5 mL) containing pyridine (946 ~L, 11.7 mmol) and
the resulting solution was stirred at room temperature under a
nitrogen atmosphere. After 5 h the reaction was extracted with
dichloromethane (2 x 100 mL) and washed with dilute sulphuric acid
(2.5 % w/v, 200 mL), aqueous sodium bicarbonate (sat., 200 mL) and
water (200 mL). The combined organic phases were then dried
(Na2S04), filtered and the solvent removed in vacuo. The resulting
syrup was chromatographed over silica gel (8:1 ethyl acetate /
hexanes, v/v) to afford mesylate 4 as a crystalline solid (556 my,
92 % yield): m.p. 72-76~C (dec); IH-NMR (CDCl3, 300 MHZ) ~ 0.12
(s, 6H,SiMe2), 0.93 (s, 9H, t-butyl), 1.74 (m~, lH, H1"A), 1.93
(fine d, 3H,J = 1.2 Hz, 5-Me), 1.97-2.06 (m, lH, H1"B), 2.16 (A Of
ABXY, 1H, H2'A), 2.27 (B of ABXY, lH, H2'B), 2.40-2.52 (m, lH, H3'),
3.03 (S, 3H, OMS), 3.74-3.81 (m, 2H, H4' and H5~A); 4-00 (dd, lH,
26
~ 20782~ -
. .
H5~B), 4023-4.35 (m, 2H, H2"AE~), 6.12 (dd, lH, Hl'), 7.53 (fine q,
lH, J = 102 HZ, H6), 8.73 (br S,1H, N~) JHI"H2'A - 6-8 HZ, JH1'-H2'B = 4-4
JH2'A-H2'B 13.5, JH2~A-H3~ = 8 3, JH2~B-H3~ 8.0;
3C~ (CDCl3) 12.54 ppm [5-Me), 18.36 (SiCMe3), 25.86 (SiCMe3 and
Si~2), 31.72 (C1"), 34.39 (C3'), 37.42 (OME) ~ 38.46 (C~'-), 62.81
(C5'), 67.67 (C2"), 84.70 and 85.60 ~C1' and C4'), 110.40 (C5),
135.40 (C6), 150.51 (C2), 164.04 (C4); MS (CI - NH3) m/e 480 ([M
^t NH4+] ~ 23 96i~ ~ 463 ( [MH+], 87 %), 354 ( [M + NH4+ -- ThyH], 51~, 337
( [MH~ - ThyH], 100); HRMiS (CI - NH3~ m/e calcd. for Cl9H35N207SSi
463.19342, found: 463 .19343.
EX~P~E 2.
SYNTHESIS OF 3'-5'-dideoxy-3'-C-(2"-hvdroxyethyl)-2l'-O-
paramethoxyphenyl-5'-thiothymidine 13 ( or III where X=O, Y=H. B =
Thy)("middle" unit). (Scheme 3) :
:
d ) 3 ~Deoxy~ 3 -C- (2 '-hydroxyethyl)-1,2-O-iEiopropylidene-2'-O-para-
methoxyphenyl_5-trityl-~-D-ri~ofuranose 6.
A ~;olution containing alcohol 5 (2.96 g, 6.43 mmol), para-
methoxyphenol ~2.39 g, 19.29 mmol), triphenylphosphine (2.19 g,
8.36 mmol) and diethyl azodicarboxylate (1.32 mL, 8.36 mmol) in dry
tetrahydrofuran was refluxed for 20 mi.n. The solvent was then
removed in vacuo yielding a violet syrup. Chromatography over
silica gel (3:1 hexanes / ethyl acetate, v/v) afforded ether 6 as
a white solid (3.30 g, 91 % yield). Recrystallization from
dichloromethane gave white crystals: m.p. 117-118C; IH NMR (300
MHZ, CDC13) ~ 1.34 and 1.51 (two s, 6H, c~e2), 1.69-1.95 (m, 2H,
H1'A8), 2.39 (h7, 1H, H3), 3.10 (A Of ABXI 1H, H5A)I 3-40 (B Of ABX,
lH, H5B)~ 3.76 (S, 3H, O~e), 3.9~-3.98 (m, 3H, H4 and H2~"~B), 4.70
(t, lH, H2), 5.90 (d, lH, H1), 6.76 (apparent d, 4H, MeOPhO-),
7 21--7 47 (tWO m~ 15H~ CPh3) ~ JH1-~2 = 3 8, JH2-H3 = 4 2, Jll4-HSA = 3 9 ~
JH4-HSB = 3 0~ JH5A-H5B = --10-6; C_NMR (CDC13) 2d,.64 ppm (Cl'), 26~42
and 26.76 (CMe2), 41.87 (C3), 55.69 (OYe), 63.15 (C5~, 66.34 (C2'),
27
-" 2~1782~6
80.90 and 81.03 (C2 and C4), 86.45 (CPh3), 104.99 (C1), 111.42
(CMe2), 114.55 and 115.27 (CH of MeOPhO-), 126.90; 127.76; 128.70
(CH of Tr), 143.92 (4 of Tr), 152.83 and 153.69 ~4 of MeOPhO-);
MS (~I - NH3) m/e 567 ([MH+], 4.9 ~, 243 ([CPh3+], 100) HRMS (CI -
NH33 m/e calcd~ for C36~39O6: 567.27466, found: 567.274~9; Anal.
calcd. for C36H38O6: C:76.30; H:5.76, found: C:76.04; H:7.14.
e)l,2,5-Tri-O-acetyl-3-deoxy-3-C-(2'-hydroxyethYl)-2'~O-para-
methoxyphenyl-~-D-ribofuranose 7.
Acetonide 6 (576 mg, 1.00 mmol) was dissolved in glacial acetic
acid (15 mL) containing acetic anhydride (2.37 mL, 25.0 ~mol) and
the solution was heated to 70 C and stirred for 15 min.
Camphorsulfonic acid (464 mg, 2.00 mmol) was then added and the
resulting solution was stirred under nitrogen for 12 min. The
solution was cooled to 0C and carefully poured into a solution of
sodium carbonate (27.2 g) in water (170 mL) and the resulting
suspension swirled occasionally over 30 min. The product was
~xtracted with ethyl ether (2 x 200mL) and washed with aqueous
sodium bicarbonate (sat., 200 mL) and water (200 mL). The combined
ether extracts were then dried (MgSO4) and the solvent evaporated
in vacuo affording a colorless syrup which was chromatographed over
silica gel (3:1 hexanes / ethyl acetate, v/v) to give triacetate 7
as a colorless syrup (282 mg, 69 % yield): ~H-NMR (300 MHz, CDCl3)
~ 1.72-2.16 (m, 2H, H1'AB), 2.06; 2.07 and 2.09 (three s, 9H, OAc),
2.49 (h7, lH, H3), 3.76 (S, 3H, ON~), 3.86-4.05 (m, 2H, H2~A~),
4.05-4.34 (m, 3H, H4 and H5A~)~ 5-30 (d, lH, H2), 6.09 (s, lH, H13,
6.81 (s, 4H, MeOPhO-), J~l~ - 0, J~3 = 4.8; l3C-NMR (CDCl3) 20.16;
20.26 and 20.64 ppm (OCOMe), 2~.46 (C1'), 38.98 (C3), 55.15 ~OMe),
64.98 (C5), 66.13 (C2'), 76.68 (c2), 82.36 (C4), 98.42 (C1), 11~.2
and 114.76 (CH of MeOPhO-), 152.14 and 153.52 (4 of MeOPhO-),
168. 62; 169. 42 and 170.17 (COMe); MS (CI - NH3) m/e 42~ ( tM +
NH4~, 16 %), 411 ([MH4+], 1.2), 351 ([MH+ - AcOH], 100); HRMS (CI
- NH3) m/e calcd. for C2~27O9: 411.1655, found: 411.1657.
28
~0782~6
f)2',5'-Di-O-acetyl-3'-deoxY-3'-C-(2"-hydroxvethyl)-2"-0-para-
methoxyphenyl-B-D-ribofuranosyl-thymine 8.
Trimethylsilyl trifluoromethanesulfonate (0.75 mL, 1.56 mmol~ was
added dropwise to a stirred solution of triacetate 7 (6.41 g, 15.64
mmol) and bis-(trimethylsilyl)-thymine (4.10 g, 17.20 mmol) in dry
1,2-dichloroethane (120 mL~ and the resulting solution was heated
to reflux under nitrogen atmosphere (82 C). After 6 hours, the
solution was cooled in an ice bath and poured into ice-cold aqueous
sodium bicarbonate solution (5% w/v,500 mL) and extracted with
dichloromethane (500 ml). The organic phase was dried (Na2SO~),
filtered and the solvent removed in vacuo yielding a brown foam
which was chromatographed over silica gel (2:1 ethyl acetate /
hexanes, v/v) to give 8 as a white foam (6.7~ g, 91 ~ yield):
H-NMR (300 MHZ, CDC13) ~ 1. 93 (fine d, 3H, 5-Me, J = 0.7), 2.11 and
2.13 (two s, 6H, OAc), 2.55 (h7, lH, H3'), 3.76 (S, 3H, ONe),
3.89-4.00 (m, 2H, H2~A~), 4.~0 (ddd, lH, H4'), 4.38 (A Of ABX, 1H,
H5~A) ~ 4.45 (B of ABX, lH, H5'~), 5.51 (dd, lH, H2'), 5.72 (d, lH,
H1'), 6.80 (s, 4H, MeOPhO ), 7.26 (fine q, lH, J = 0.8 Hz, H6),
8.74 (br s, lH, NH), J~ -~2 = 1 . 3, JH2~-H3~ = 6-1, JH3~-H4~ = 10 2/ J~4'-H5'A
4 . 8, JH4'-H5'B = 2 . 2, JH5'A-H5'B = - 12 .6, C-NMR (CDCl3) 12.48 ppm
(5-Me), 20.57 and 20.64 ~CONe), 24.48 (C1"), 39.00 (C3'3, 55.53
(OMe), 63.20 (C5~, 66.06 (C2"), 77.31 (C2'), 82.00 (C4'), 91.23
(C1'), 110.63 (C5), 114.52 and 115.08 (CH of MeOPhO-), 135.64 (C6),
149.95; 152.35 and 153.80 (C2 and ~ of MeOPhO-), 163.85 (C4),
169.50 and 170.30 (COMe); MS (CI - NH3) m/e 477 ([MH+], 100 ~), 417
([MH+ - ACOH], 4.6), 351 ([MH~ - ThYH], 86); HRMS (CI - NH3) m/e
calcd. for C23H~N209: 477.18730, found: 477.18742. Anal. calcd. for
C23H28N2O~: C:57.98; H:5.92; N:5.88, found: C:57.g9; H:6.27; N:5.75.
g)3'-Deoxy-3~-C-(2''-hydroxyethyl~-2''-O-para-methoxvPhenyl-B-D-
ribofuranosyl-thymine 9.
Nucleoside 8 (107 mg, 0.20 mmol) was dissolved in methanol (1.00
mL), cooled to 0C and a steady stream of NH3 gas was bubbled into
the solution under ice bath for 10 min. The solution was then
29
~.
,... . . . . ; . . . , ~ , , .: : : . . . ..
; ,- '. :, ' ''' ' .' ,", ' , ' '. ,. .~ -.,'''.. ' , . . .
.. . . .... . . .. . . .
, . , ., ~ . .
, ~ ,, . .. ,. " ... . . .
-` 20~25~
allowed to warm to ambient temperature and was stirred for 10 h.
The solvent was removed in vacuo and the residue was
chromatographed with silica gel (20:1 dichloromethane / methanolt
v/v) yielding the diol 9 as a white foam (92 mg, 100% yield);
IH_NMR (300 MHZ, CDC13) ~ 1.88 (S, 3H, 5-Me), 1.76-1.92 (m, 1H,
H1"A), 2.08-2.23 (m, lH, H1"B), 2O33-2.41 (m, lH, H3'), 3.15 (S, 3H,
O~e) ~ 3.82 (br d, 1~, H4 ' ), 4 ~ 04 (br t, 2~, H2"AB), 4.16 (br t, 2H,
H5~AB) ~ 4-39 (d, lH, H2'), 5.55 (br s, 2H, -0~), 5.77 (S, 1H, H1'),
6.81 (s, 4H, MeOPhO-), 7.89 (S, 1H, H6), 10-03 (S, 1H, N~) ~ JHI~
0, J~,~ = 4.6; l3C-NMR (CDC13) 12.38 ppm ~5-Me), 24.01 (C1"),
37.73 (C3'), 55.60 (OMe), 60.74 (C5'), 67.05 (C2"), 76.85 (C2'),
85.41 (C4'), 92.59 ~C1'), 109.85 (C5), 114.58 and 115.32 (CH of
MeOP~O-), 136.50 (C6), 150.88 ; 152.49 and 153.85 (C2 and 4 of
MeOPhO~ 64.70 (C4); MS (CI - NH3~ m/e 410 ( [M ~ NH4~ %),
393 ( [MH+], 100), 284 (tM ~ NH4+ - ThyH], 4.0), 267 ([MH+ - Thy~],
9.4), 127 ([ThyH + H+], 0.8); HRMS (CI - NH3) m/e calcd. for
CI~N207. 393.16618, ~ound: 393.16625.
h~ 5 ' -O-tert-BUtY1dimethY1Si 1Y1-3 ' -deOXY-3 ' -C- ( 2"-hydroxyethyl~-2"
~O-para-methoxyphenyl-B-D-ribofuranosyl-thymine 10.
Tert-butylmethylsilyl chloride (0.63 g, 4.20 mmol) and imidazole
(598 mg, 8.80 mmol) were added to a solution of alcohol 9 (1.91 g,
4.00 mmol) in freshly distilled N,N-dime.thylformamide (4 mL). The
reaction was stirred at room tempe:rature under a nitrogen
atmosphere for 18 h and the solvent was then removed in vacuo. The
residue was extracted with ethyl acetate (2 x 300 mL) and washed
with water ~300 mL). The combined organic layers were dried
(Na2SO4), filtered a~d evaporated in vacuo yielding a white foam :
which was chromatographed over silica gel (2:1 ethyl acetate /
hexanes) to give silyl ether 10 as white solid (2.12 g, 86 %
yield~ IH_NMR (300 MHz, CDCl3) ~ 0.12 and 0.14 (two s, 6H, SiNe2),
0.95 (s, ~H, CMe3), 1.91 (s, 3H, 5-Me), 1.71-1.81 (m, lH, H1"A~,
2.05-2.18 (m, lH, H1"B), 2.37 (h7, lH, H3'), 3.74-3.80 (overlapping :
s and m, 4H, H5~A and OMe), 4.01 (br t, 2H, H2"AB), 4.14-4.20 (m,
. -
:
:
-`` 2078256
2Ht H4~ and H5~B), 4.36 (apparent t, lH, H2'), 4.81 ~s, lH, -OH),
5.75 (S, lH, H1'), 6.80 (s, 4H, MeOPhO-), 7.81 (s, lH, H6), 10.08
(S, 1H, NH), JHI~ O; l3C-NMR (CDC13) 12.72 ppm (5-Me), 18.64
(CMe3), 23.76 (C1"), 26.03 (CNe3 and Si~e2), 36.71 (C3'), 55.69
(OMe), 61.47 (C5'), 66.33 (C2"), 76.58 (C2'~, 85.61 (C4'), 92.97
(C1'), 109.82 (C5), 114.59 and 115.16 (CH of MeOPhO-), 13~89 (C6),
150.74; 152.88 and 153.73 (C2 and 4- of MeOPhO-), 164.77 (C4); MS
(FAB-glycerol) m/e 507 (tMH+3, 1.8%), 171 (22), 127 ([ThyH + H+],
3.6); HRMS (FAB - glycerol) m/e calcd. for C~H3907N2Si: 507.25265,
fou~d: 507.25~77; Anal. calcd. for C2sH38o7N2Si: C:59.26; H:7.56;
N:5.53, ~ound: C: 58.87; H:7.83; N:5.30.
i)5'~0-tert-Butvldimethylsil~1-3'-deoxy-3'-C-(2"-hydroxyethyl~-2"
-O-para-methoxyphenyl-2'-O-phenoxythiocarbonyl~ -ribofuranosyl-
thymine 11.
Phenyl chlorothionoformate (595 ~, 4.30 mmol) was added dropwise
to a stirred solution of nucleoside 10 (1.98 g, 3.91 mmol) in dry
dichloromethane (10 mL3 containing dry pyridine (2.5 mL3 and the
reaction was stirred under a nitrogen atmosphere at 0C. After lh
the brown solution was refrigerated for 20 h. The solvents were
then removed in vacuo yielding a red syrup to which water (200 mL3
was added and the resulting suspension shaken vigorously~ The
product was extracted with ether (2 x 200 mL) and washed with
dilute sulphuric acid (2 % w/v, 200 mL), aqueous sodium bicarbonate
(sat., 200 mL), and water S300 mL). The combined ether phases were
then dried (Na2SO4), filtered and evaporated in vacuo to a orange
solid which was chromatographed over silica (2:1 hexanes / ethyl
acetate, v/v) to afford thiocarbonate 11 a~ a pale orange solid
(2.20 g, 87 % yield): 'H-NMR (300 MHz, CDCl3) ~ 0.13 (s, 6H, SiMe2),
0.94 (s, 9H, CMe3), 1.93 (fine d, 3H, J = 1 . 1 Hz, 5-Me), 1.84-1.94
(m~ lH, H1"A), 2.04~2.16 (m, lH, H1~'B), 2.91 (m9~ lH, H3'), 3.76 (s,
3H, OMe), 3.80 (dd, lH, H5/A)~ 3.98-4.14 (m, 4H, H5'B; H4' and
H2"AB), 5.94 (dd, lH, H2'), 6.08 (d, 1H, H1'), 6.83 (s, 4H,
MeOPhO-), 7.08-7.41 (three m, 5H, CSOPh), 7.43 (fine q, lH, J = 1.1
2~7~2~6
~ .
HZ, H6), 8.28 (br S, 1H, N~), J~ = 2-4, Jl-2~ = 6-4; l3~_NMR
(CDC13) 12O47 PPm (5-Me), 18.37 (CMe3), 24.88 (C1"), 25.87 (C~e3and
Si~e2), 38O41 (C3'~, 55.5~ (O~e), 62.33 (C5'), 66.38 (C2"), 84.33
(C4'), 86-36 (C1'), 89.14 (C2'), 111.02 (C5), 114.56 and 115.16 (CH
Of MeOPhO-), 121.64 (O-CH Of Ph), 126.60 (P-CH Of Ph), 129.44 (m~CH
Of Ph~, 135.69 (C6), ~50.08; 152.55; 153.20 and 153.81 ~E2 and 4
Of ~eOPhO- and Ph), 163.96 (C4), 193.69 (CSOPh); MS (FAB -
nitrObenZY1 a1COhO1) m/e 643 (CMH+~, 6.7 %), 527 ([NH~ - ThYH3,
14), 489 (tMH+ - PhOCSOH], 100), 163 (73~, 137 ([PhO=C=S+], 73),
127 ([Th~H + H+], 27~; Ana1. Ca1Cd. fOr C32H42O8N2SiS C 59-79;
H:6.58; N:4.36; S: 4.99, fOUnd: C:59.~7; H:6.7O; N:4.43; S:4.87.
j)5' 0-tert-BUtY1dimethY1Si1Y1-3~-deOXY-3~C-(2~-hYdrOXYethY1)-2
-O-Para-methOXYPhenY1-thYmidine 12.
Tri n-bUtY1tin hYdride (2.48 mL, 6.06 mmOl) WaS added tO a SO1UtiOn
Of thiOCarbOnate ~ .94 g, 3.03 mmOl) and AIBN (0.61 g, 2.42
mmOl) in drY tO1Uene (15 mL). A Stream Of nitrOgen WaS PaSSed
thrOUgh the reaCtiOn fOr 20 min and the SO1UtiOn WaS then heated tO
75C POr 4 h. The 5O1Vent WaS remOVed in vacuo and the reSidUe
ChrOmatOgraPhed OVer Si1iCa (1:1 heXaneS / ethY1 aCetate~ V/V) tO
Yie1d the 2~-deOXY nUC1eOSide 12 aS a White SO1id (1.38 g, 93 %
Yie1d): ~H_NMR (300 MHZ, CDC13~ ~ 0.116 and 0.120 (tWO S, 6H,
SiMe2), 0-94 (S, 9H, CMe3), 1.92 (d, 3H, J = 1.0HZ, 5-Me), 1.71-1.78
(m, lH, H1"A ), 1.95-2.06 (m, 1H, H1"~), 2.14-2.31 (m, 2H, H2'A~),
2.47 2.56 ~m, 1H, H3'), 3.75-3.83 and 3.92-4.06 (tW~ m, 5H and 3H,
~~; H5~AB; H4'; and H2~lAU)~ 6.12 (dd, 1H, H1'), 6.82 (aPParen~ d,
4H, MeOP~O-), 7.61 (fine q, lH, J = 1.0 Hz, H6), 8.77 (br s, lH,
NH)~ JHI~ = 3.9 and 6.5; l3C_NMR (CDCl3) 12.55 ppm (5-Me), 18.41
(CMe3), 25.87 (CMe3 and SiNe2), 31.65 (C1"), 34.32 (C3'), 39.12
(C2')~ 55-58 (ONe), 62-60 (C5'), 66.39 (C2"), 84.83 and 86.18 (C4'
and C1'), 110.07 (C5), 114.56 and 115.10 (CH of MeOPhO-), 135.56
(C6), 150.52; 152.59 and 153.77 (C2 and 4~ of MeOPhO~ 64.18
(C4); MS (FAB - nitrobenzyl a1COhO1) m/e 491 ([MH+], 16 %), 365
([MH+ - ThyH], 77), 163 (100), 127 ([ThyH ~ H+], 51; HRMS (FAB -
32
. .
:
2078256
glycerol) m/e calculated for C2sH3906N2Si: 491.25774, found:
491.25772.
k)5'-S-Acetyl-3'r5'-Dideoxy~3'-C-(2"-hydroxyethYl~-2"-0-para-
methoxvphenyl-5'-thiothymidine 16.
Diisopropyl azodicarboxylate (1.05 mL, 5.32 mmol) w~s added
dropwise to a stirred solution of triphenylphosphine (1.39 g, 5.32
mmol) in dry tetrahydrofuran (~0 mL) cooled to 0C under nitrogen
resulting in a milky suspension. After 0.5 h a solution of
nucleoside 17 (l.oo g, 2.66 mmol) and thiolacetic acid (0.38 mL,
5.32 mmol) in dry tetrahydrofuran (20 mL) was slowly added and the
stirring at 0C continued for 0.5 h after which time the reaction
was allowed to warm to room temperature. After 40 min the solvent
was removed in vacuo and the resulting yellow syrup was
chromatographed over silica (25:1 methylene chloride / methanol,
v/v) affording thiolester 16 as a white solid (1.00 g, 87 % yield):
IH-NMR (300 NHz, CDCl3) ~ 1.96 (s, 3H, 5-Me), 1.69-1.80 (m,lH,
H1"A), 2.02-2.12 (m, lH, H1~B)~ 2.16-2.31 (m, 3H, H3' and H2~AB)~
2.38 (s, 3H, SAc), 3.26 (A of ABX, lH, H5~A)~ 3.36 (B of ABX, lH,
H5~B~ 3.76 ~s,3H, ONe), 3.88 (m6, lH, H4'), 3.96 (apparent t, 2H,
H2~AB~ J = 5-9), 6-0?3 (dd, lH, H1'), 6.82 (s, 4H, MeOP~O-), 7.36 (s,
lH, H6), 9.16 (br s, lH, NH); J~l.~ = 4.2 and 6.0, J~4-~A = ~-5, J~-
= 3-5, 2J~A-~B = -14-4; l3C-NMR tCDCl3) 12.62 ppm (5-Me), 30.52
(SCOM~), 31.56 and 31.67 (Cl" and C2'), 38.96 and 39.06 (C3' and
C5'), 55.66 (ONe), 66.50 (C2l~), 83~74 and 84.75 (C1' and C4'),
110.67 (C5), 114.63 and 115.22 (CH O:e MeOPhO-), 135.40 (C6),
150031; 152.54 and 153.89 (C2 and 4 of MeOPhO-), 163.85 (C4),
194.71 (SCOMe); MS (CI - NH3) m/e 452 (~M + NH4+~, 8.3 %), 435
([MH+], 100), 326 ([M ~ NH4+ - ThyH], 3.6), 309 (~MH+ - ThyH], 53),
127 ~[ThyH + H~3, 2.8); HRMS (CI - NH3) m/e calcd. for C21H2,N206S:
435.158g8, found: 435.15889.
1)3~-Deoxy-3' C (2"-hydroxYethvl)-2"-o-(Para-methoxYphenylL
, ~ . ,, ." , . . . : , ~
',''' ' ' ,.:. '' . ,.' ' 'il' ', ,;: : . ' '
:
- 2~782~6
thymidine 17.
A stock solution of tetra-n-butylammonium fluoride (1 N in ~HF, 7.9
mL, 7.9 mmol) was added to a stirred solution of nucleoside 12
(1.29 g, 2.64 mmol~ in dry tPtrahydrofuran (5 mL) and the resulting
solution was stirred at room temperature under a nitrogen
atmosphere. After 1.5 h the solvent was evaporated in ~cuo and
the residue chromatographed over silica (20:1 methylene chloride /
methanol, v/v) affoxding nucleoside 17 as a white solid (0.99 g,
quantitative): ~H-NMR (200 MHz, CD30D) ~ 1.86 (s, 3H, 5-Me),
1.63-1.80 (m, lH, H1"A ), 1.91-2.07 (m, lH, H1"~), 2.14-2.35 (m, 2H,
H2'AB), 2.38-2.53 (m, lH, H3'), 3.69-3.81 and 3.89-4.00 (two m, SH
and 3H, -OMe; H5~A~; H4'; and H2~AB)~ 6.04 (dd, lH, H1'), 6.81 (s,
4H, MeOPhO-), ~.00 (s, lH, H6), JHI~ = 3.2 and 6.7; l3C NMR (CDCl3)
12.40 ppm (5-Me), 31.45 (Cl"~, 34.53 (C3'), 39.09 (C2'), 55.59
(o~e), 61.50 (C5'), 66.74 (C2"), 84.95 and 86.41 (C1' and C4'),
110.19 (C5), 114.57 and 115.23 (CH of MeOPhO-), 136.37 (C63,
150.45; ~52.48 and 153.80 (C2 and 4- of MeOPhO-), 164.22 (C4); MS
(CI - NH3) m/e 394 ([M ~ NH4+], 2.5 %), 37/ ([MH4+~, 87), 268 ([M
NH4~ - Thy~, 23), 251 ([MH+ - ThyH], 100), 127 ([ThyH + H~], 16);
HRNS (CI ~ NH3) m/e calcd. for ClgH25N2O6 377.17126, found:
377.17114; Anal. calcd. ~or ClgH~N2O6: C:60.63; H:6.43; N:7.44,
found: C:60.23; H:6.77; N:7.30.
,::
m)3',5'-Dideoxy-3'-C-(2~'-hYdroxyethyl)-2"-O-Para methoxyphenyl-5'-
thiothymidine 13.
A solution of thiolester 16 (455 mg, 1.05 mmol) in dry methanol (5mL) previously saturated with nitrogen was cooled to 0C and a
stream of ammonia gas was passed through for 15 min. The vessel was ;~
removed from the ice bath and the reaction was allowed to proceed
by stirring the mixture for 20 hours. The solvent was then
evaporated in vacuo yielding a white solid which was
chromatographed over silica (4:1 ethyl acetate / hexanes, v/v) to
afford the thiol 13 as a white solid (410 mg, 98 % yield): IH_NMR
(300 MHz, CDCl3) ~ 1.59 (t, 1H, -SN), 1.72-1.83 (m, lH, H1"A), 1.94
3~
20782~fi
. ,
(d, 3H, J = 1.2 Hz, 5-Me), 1.~6-2.03 ~m, lH, H1llB), 2.24-2.29 (m,
2H, H2 'AB), 2.38-2.52 (m, lH, H3'), 2.83 (A of ABX with an
additional coupling to SH, ~H, ~5~A), 3.01 (B of ABX with an
additional coupling to SH, lH, H5~B), 3.77 (s, 3H, OMe), 3.86-3.92
(m, lH, H4'), 3.94-~.00 (m, 2~, H2"AB), 6.16 (t, lH, Hl'), 6.93
(apparent d, 4H, MeOPhO-), 7.51 ~fine q, lH, J = 1.2 Hz,~6), 8.43
(br s, 1H, N~), J~ ~ ~ 5- 4, JH4'-~j'A = 5-0, J~-~jB 3-7, J~j'A-~j'B
14-4~ J~A~H = 8-0, J~.~ = 8.9; l3C-NMR (CDCl3) 12.45 ppm (5-Me3,
27.11 (C1"), 31.45 (C2'), 37~59 (C3'~, 38.61 (C5'), 55.47 (O~2),
66.35 (C2"), 84.03 and 84.77 (C1/ and C4'), 110.63 (C5), 114.49 and
115.11 (CH of MeOPhO-), 135.47 (C6), 150.48; 152.30 and 153.75 (C2
and 4 of MeOPhO-), 163.99 (C4); MS (FAB - nitrobenzyl alcohol)
m/e 393 (tMH~], 12 %), 267 (~MH+ - ThyH], 4.3), 154 (100), 137
([MeOPhOCH2+], 65), 136 (6i8), 127 (tThyH + H~], 25); HRMS (EAB -
glycerol) m/e calcd. for C~25O5N2S: 393.14842, found: 393.14849;
Anal. calcd. for Cl~24O5N2S: C:58~15; H:~16; N: 7.14; S:8.17, found:
C:57.84; H:6.54; N:7.17; So7.8i8.
EXAMPLE 3.
SYNTHESIS OF 3'-Q~tert-Butyldimethilsil~1-5'-deoxY-5'-thiothymidine
20 (or VIII where X=O Y'=H,_B-Thv~. (Scheme 4)
a) 5'-S-Acetyl-5'-d~oxy-5'-thiothymidine 18.
-
Diisopropyl azodicarboxylate (1.62 mLI 8.26 mmol) was addeddropwise to a stirred solution of triphenylphosphine (2.16 g, 8.26
mmol) in dry te~rahydro~uran (l~i mL) cooled ~o 0~C under nitrogen
resulting in a milky suspension. After 0.5 h a solution of
~hymidin~ (1.00 g, 4.13 mmol) and thiolacetic acid (0.59 mL, 8026
mmol) in dry N,N-dimethylformamide (15 mL) was slowly added and the
stirring continued for 0.5 h at 0~C after which time the reaction
was allowsd to warm to room temperature. After 0.5 h the solvent
was removed in vacuo and the resulting brown syrup pumped for 2 h.
The resulting glass was chromatographed over silica (25:1 to 15:1
dichloromethane / methanol, v/v) affording thiolester 16 as a white
:: ; .. .:, :: :: . ~ : . : .,: - . .j: . : . : :- . . . , i ,.
..... ,, ,;, :: ,; , :, , .. , i. ~ , ., . .: . .. , . ,: : .
:: - . : .. . .::: ~ . ,... . . ,: . . . ,,. ~,. , " . , , . ~ .
, :: . .:.:.. .... :.:, .: ., , ,- : .: , . . :; ; . -::: . , :.
.~ . , ., . ~ . : . , ": . ,,: ,j. . . . . .
" ~, , . ' ~,; :: ' ' .' ' , ' ' ' '
~`` 20782!i6
solid (650 mg, 52 ~ yield): IH-NMR (300 MHZ, CD30D) ~ 1.90 (fine d,
3H, J = 1.2 Hz, 5-Me), 2.25 (dd, 2H, H2~AB) 2.35 (S, 3H, SAc), 3.23
(d, 2H, H5~AB), 3.93 (td, lH, H4'), 4.20 (td, lH, H3f ), 6.19 (t, lH,
H1'), 7.44 (fine q4, lH, J = 1.2 Hz, H6), JH1'_~12'AD= 6-8 Hz, JH2ABH3.
5.0 ~ JH3'-H4' 3.5 ~ JH4' HS'AB = 6 2; 13~ CD3OD3 12038 ppm (5-Ma),
30. 43 (COMe), 32.30 (C5' ), 40.09 (C2'), 74. 37 (C3 ~ ), 86.45- (2C, C1'
and C4'), 111.83 (C5), 137.6~ (C6), 152~25 (C2), 166.26 (C4),
196.46 (COMe); MS (CI - NH3) m/e 318 ( [M ~ NH4+], 14 ~6), 301
([MH+~, 100), 175 (tMH+ - rrhyH]~ 1.1), 127 (LThyH ~ H+], 17); H~MS
(CI - NH3) m/e aalcd. for Cl2HI7N2O5S: 301.08582, found: 301.08574.
b) 5 '-S-Acetyl-3'-O-t:ert-butyldimethYlsilyl-5'-deo~ -5'-
thiothymidine 19.
tert Butylchlorodimethylsilane (309 mg, 2.05 mmol~ and imidaz~le
(280 mg, 4.10 mmol) were successively added to a stirred solution
of nucleoside 1~ (560 mg, 1.86 mmol) in dry N,N-dimethylfo~mamide
(5 mL) and the reaction was stirred at ambient temperature under a
nitrogen atmosphere. After 18 h the solution was poured into water
(300 mL) and the product was extracted with dichloromethane (2 x
200 mL) and washed with water ~2 x 300 mL). The combined organic
extracts were then dried (Na2SO4), filtered and the solvent removed
in vacuo yielding a yellow syrup. chromatography over silica gel
(25:1 dichloromethane / methanol, v/v) afforded the silyl ether 19
as a colorless solid (709 mg, 92 % yield)~ NMR (300 MHZ, CDCl3)
0.08 and 0.10 (two s, 6H, SiMe2), 0.90 (s, 9H, t-butyl), 1.96
(fine d, J = 1.2 Hz, 5-Me), 2.10 (A of ABXY, lH, H2~A), 2.28 (B of
ABXY, lH, H2'fl), 2.40 (s, 3H, SAc), 3.21 (d, 2H, H5~AB) ~ 3.99 (td,
lH, H4'), 4.17 (dt, 1H, H3'), 6.21 (dd, lH, H1'), 7.23 (fine ~, lH,
J = 1.2 Hz, H6), 8.62 (br s, lH, N~), JH~ 2'A = 7-5 Hz, JH1-H2B = 6-1,
2JH2'A 1~2'B = --13 - 8 ~ JH2 A ~B = 6.5, JH2 B H3 3.2, HB -H4 ~ H4 HS
5.8; l3C-NMR (CDCl3) 12.60 ppm (5-Me), 17.87 (SiCMe3), 25.64 (SiMe2
and SiC~e3), 30.56 (COMe), 31.13 (C5'~, 40.44 (C2'), 73.84 (C3'),
36
2078~
85.13 and 85.17 (Cl' and C4'), 111.10 (C5), 135.32 (C6), 150.16
(C2), 163.74 (C4), 194.48 ~COMe); MS (CI - NH3) m/e 432 (
NH4+], 4.9 ~), 415 ([MH-~], 100), 306 ([M + NH4+ -- ThyH], 1.8), 289
( tMH+ - ThyH], 11), 127 ( ~ThyH + H+], 13); HRMS (CI NH3) m/e
calcd. for Cl8H3~N2o5SSiili 415.17230, found: 415.17213.
c~ 3'-0-tert-Butyldimethvlsilyl-5'-deoxy-5'-thiothymidine 20.
Methanolic sodium hydroxide solution (0.50 N, 7.7 mL, 3.86 mmol),
previously saturated with nitrogen gas, was slowly added to a
stirred solution of thiolester 19 (800 mg, 1.93 mmol~ in methanol
(30 mL, deoxygenated) and the reaction allowed to stir at ambient
temperature under nitrogen. After one hour, the base was
neutralized with Amb~rlite~ H+ resin which was filtered and washed
thoroughly with methanol. Evaporation of the alcohol in vacuo
af~orded the thiol 20 a colorless gel which crystallized upon
standing (738 mg, quantitative): m.p. 116-119C (dec); IH-NMR (300
MHz, CDCl3~ ô 0.098 and o.lo (two s, 6H, si~qe2), 0.90 (S, 9H,
t-butyl), 1.95 (fine d, 3H, J = 1.2 Hz, 5-Me), 1.53 (t,
exchangeable, lH, S~I), 2.16 (A of ABXY, lH, H2~A), 2.30 (B of ABXY,
lH, H2~B) ~ 2.78 (A of ABXY, lH, H5~A), 2.89 (B of ABXY, lH, H5'8),
3.9~; (apparent q4, lH, H4'), 4.36 (dt, lH, H3~), 6.25 (t, lH, H1'),
7.34 (fine q4, 1H,J=1.2 Hz, H5), 8.41 (br s, lH~ N~ Hl~-H2~A = JHI~H2~B
6 - 7 Hz~ JH2~A H3' = 7.1 ~ JH2'11 H3' = 4 3 f JH2'A H2'B 13.7 ~ JH3 -H4
JH4'H5'A = JH4:H5'B = 46~ JH5~A~5~B = --14.2~ JSHH5AI~ = 8.3; l3C--NMR (CI~Cl3)
12i.65 ppm (5-Me), 17.87 (SiCNe3), 25.66 (SiMe2 and SiCMe3), 26.4
(C5'), 40.54 (C2'), 72.50 (C3'), 84.46 and 85.97 (C1~ and C4'),
111.24 (C5), 135.51 (C6), 150.20 (C2), 163.65 (c4); MS (CI -- NH3~
m/e 390 ( [M + NH,~+], 1.4 g6), 373 ([MHt], 100), 247 ([MH+ - ThyH],
2.7), 127 (~ThyH + H+], 28); HRMS (CI - NH3) m/e calcd. for
Cl6H29N2o4SSi: 373.161733, found: 373.161730; Anal. calcd. for
Cl6N27N2o4SSi: C:51.58; H:7.58; N:7.52; S:8.60, found C:51.19; H:7.59;
N:7.36; S:8.32.
It was found that chromatography of the thiol over silica,
immediately after the reaction (1:1 hexanes / ethyl acetate, v/v),
removed a small amount of impurity which accelerates the oxidation
of the product to the symmetrical disulfide, yielding: 1H_NMR (300
MHz, CD3OD) .delta. 0.096 and 0.101 (two s, 6H, SiMe2), 0.90 (s, 9H,
t-butyl) 1.94 (fine d, 3H, J = 1.2 Hz, 5-Me), 2.16 (A fo ABXY, 1H,
H2'?), 2.30 (B of ABXY, 1H, H2'n), 3.02 (A of ABX, 1H, H5'?), 3.07
(B of ABX, 1H, H5'n), 4.10 (td, 1H, H4'), 4.35 (dt, 1H, H3'), 6.19
(apparent t, 1H, H1'), 7.23 (fine q, 1H, J=1.2 Hz, H6), 8.95 (br s,
1H, NH), JH?H2? = 7.1, JH?H2B = 6.3, 2JH2'H2'B= -13.6, JH2'A-H3' = 6.7,
JH2'B-B3'= 3.5, JH3'-H4'= 3.8, JH4'-H5'?=6.0, JH4'H5'B=5.4, 2JH5'A-B5'B=-14.0;
13C-NMR (CDCl3) 12.59 ppm (5-Me), 17.85 (SiCMe3), 25.64 (SiMe2 and
SiCMe3), 40.21 and 41.88 (C2' and C5'), 73.56 (C3'), 85.01 and 85.54
(C1' and C4'), 111.11 (C5), 135.59 (C6), 150.26 (C2), 163.95 (C4);
MS (FAB-glycerol m/e 743 ([MH+], 8.4%) 437 (46).
EXAMPLE 4
SYNTHESIS OF ACTIVEATED/PROTECTED DIMER XI (where m=0; n=0; X=0,
Y=Y'=H, B=Thy) OF DEOXYRIBONUCLEOSIDE. (Scheme Ia)
d) Dimer VIII (where P+TBDMSi).
Cesium carbonate (547 mg, 1.68 mmol) was flame dried in vacuo. It
was then suspended in dry N,N-dimethylformamide (7mL). A solution
of mesylate thymidine derivative II (517 mg, 1.12 mmol) and
thiothymidine VIII (458 mg, 1.23 mmol) in dry N-N-dimethylformamide
(12 mL) was then added which resulted in a yellow solution. The
solution was stirred for 3 h at ambient temperature under a
nitrogen atmosphere. THe solvent was then removed in vacuo and the
product was extracted with dichloromethane (200 + 100 mL) and
washed with aqueous sodoum bicarbonate (5 % w/v, 200 mL) and water
(200 mL). The combined organic phases were dried (Na2SO4), filetered
and evaporated in vacuo yielding a yellow foam. Chromatography
over silica gel (4:1 ethyl acetate / hexanes, v/v) afforded the
sulfide VIII as a colorless solid (725 mg, 88 % yield) with 'H-NMR
(300 MHz, CDCl3) .delta. 0.092; 0.096; 0.115 and 0.120 (four s, 12H,
38
20~8256
SiMe2~, 0.90 and 0.93 ~two s, 18H, t-butyl), 1.53-1.67 (m, lH,
5H1"A), 1.75-1.88 (m, lH, sH1"~), 1.92 and 1.93 (two fine d, 6H, J
= 1.1 Hz, 5-Me's), 2.05-2.33 (two overlapping AB portions of ABXY,
4H, SH2'~ and 3H2'AB), 2.31 2.45 (m, 1H, 5H3'), 2.53-2.70 (m, 2H,
sH2"AB), 2.77 (A of ABX, 1H, 3H5'A)I 2.83 (B of ABX, 1H, 3H5~B)~
3.70-3.76 (m, 2H, sH4' and 5H5~A)~ 3.95-4.02 (m, 2H, 3~4' a~d sH5'B),
4.33 (dt, 1H, 3H3'), 6.08 (dd, lH, 5H1'), 6.21 (t, lH, 3H1'), 7.30
and 7.56 (two fine q, 2H, J = 1.1 HZ, H6'S), 8.96 and 9.01 Itwo br
, , N~), JOHI~ A = J~)H1~ B = 6-6 HZ, J(~H1~ (S)~'A = 6-7, J(~HI~ B =
4.3, JO~AO~ = 6~7, JO~O~ = JO~)H4 = 4.5, JOH4O~A 5-1,
JOH4~O~= 5-3, J~ AO~B - -13.8; ~3C NMR ( 75.4 MHZ~ CDC13) ~ 12.58
(2C, 5-Me), 17.80 and 18.40 (SiCMe3), 25.61; 25.73 and 25.88 (SiNa2
and SiCM~), 31.40; 32.15; 34.23 (3 X CH2), 36.60 (SC3~)J 38.76 and
40.19 (2 X CH2~, 62.90 (SC5~)~ 73.25 (SC3~)I 84.84 ~2C); 85.34 and
85.90 (2 X H1' and 2 x H4'), 110.22 and 111.0~ (2 X C5), 135.43 and
135.54 (2 x C6), 1~0.35 and 150.57 (2 X C2), 163.96 and 164.18 (2
X C4); MS (FAB - g1YCQrO1 / HFBA ) m/e 739 ([MH~], 3.7 %), 613
([MH+ - ThyH], 22), 355 (11), 157 ~100), 127 ([ThyH ~ H+], 85).
e) Diol I~ .
A solution of tetra-n-butylammonium fluoride in tetrahydrofuran (1
M, 947 ~L, 0.947 mmol) was added to a stirred ~olution of sulfide
VIII (280 mg, 0.379 mmol) in dry tetrahydrofuran (10 mL) and left
standing for 2 hour. ~he solution was evaporated in vacuo and the
resulting glass was chromatographed over silica gel (10:1
dichloromethane / methanol, v/v) to give the diol I~ as a colorless
glass in quantitative yield: NMR data indicated the disappearance
of the t-butyl-dimethylsilyl protecting groups. The compound was
used without further purification in the next reaction.
f) Dimethoxytritylation of diol IX to qive tritYlated dimer x.
4-4'-Dimethoxytrityl chloride (83 mg, 0.244 mmol) was added to a
stirred solution of diol IX (104 mg, 0.204 mmol) in dry pyridine
(2.5 mL) containing 4-dimethylaminopyridine (2 mg, 0.01 mmol) and
.:
39
. , : ;: - . - , . - - , ~ . . , .............. . :,
.. ..
2~782~6
then triethylamine (0.041 mL) added. After stirring for 8 h, the
reaction was poured into water (25 mL) and extracted with
dichloromethane (3 x 15 mL). The organic phases were dried over
Na2SO4 and evaporated in vacuo to a syrup which was then
chromatographed over silica gel (100:5:1 CH2Cl2/ MeOH/ Et3N, v/v)
giving the dimer X as a white foam (158 mg, 85% yield~: I~ NMR (200
Hz , CD30D) ~ 1.28-1.40 (m, lH, sHl"A), 1.46-1.75 (m, lH, 5H1"~), 1.34
and 1.76 (two s, 6H, 5-Me's), 2.00-2.31 (m, 4H, sH2'AB and 3H2'A~),
2.32-2.41 (m, lH~ 5H3'), 2.42-2.63 (m, 2H, 5H2"AB), 2.69-2.90 (m, 2H,
3H5'3, 3.09-3.28 (m, 2H, 5H5'), 3.73 (s, 6H, 2 x C~I3), 3.65-3.81 (m,
lH, 5H4'), 3.85-3~98 (m, lH, 3H4'), 4.22-4.34 (m, lH, 3H3'~, 6.02
(dd, lHI 5H1'), 6.17 (t, lH, 3Hl'), 7.41 and 7.80 (two s, 2H, H6's),
J(3)HI'-~H2'A= J~3)HI'-(3)H2'B= 6 8Hz, J(5)HI-(5)H2A= 4.4~ J(S)HI'-(5)~2'B= 2. 6; 13C--NMR (49.0
MHz , CD30D) ~ 10.26 (2C, ~;-Me), 32.03 ; 33.12 and 35.31 (3 x CH2),
37.73 (5C3'~, 40.08 (2 x CH2), 47.39 (Ph3C~, 55.77 (2 x OCH3), 63.80
(5C5'), 74.19 (3C3'), 86.22; 86.54; 86.70 and 87.21 (2 x Cl' and 2
x C4 ' ), 110.99 and 111.76 (2 x C5), 137.80 and 137.84 (2 x C6),
152.23 and 152.28 (2 x C2), 166.25 and 166.52 (2 x C4), 114.22 (CH
of MeOPhO~), 160.27 and 160.30 (4 of MeOPhO-); MS (FAB-glycerol /
NBA) m/e 813 (tMH+], 9.7 %), 687 ([MH+-ThyH], 3.7), 509 ([MH~-
DMTrH], 4.0), 304 (~DMTr + H+], 100).
g) Phosphoramidite ~I.
2-Cyanoethyl N,N-diisopropylchlorophophoramidite (55 ~L, 0.246
mmol) was slowly added to a stirred solution of tritylated dimer
(100 mg, 0.123 mmol) in dry dichloromethane (1.5 mL) containing
triethylamine (68 ~L, 0.492 mmol). After 18 h of stirring at
ambie~t temperature under a nitrogen atmosphere, the solution was
diluted with ethyl acetate (35 mL) and washed with brine (4 x 70
mL~. The organic phase was then dried (Na2SO4) filtered and
evaporated in vacuo yielding a pale yellow foam. This crude
material was dissolved in dichloromethane (0.6 mL) and precipitated
at -78 C with hexanes (-5 mL). The solvents were decanted off and
the residue redissolved in dichloromethane containing ethyl ether
20782~
and carefully evaporated (Rotovap~) to give the phosphoramidite 33
as a colorless foam (120 mg, 96 % yield) which was used as such in
the subsequent solid-phase synthesis: 3IP-NMR (CD2C12) 148.94 and
149.31 ppm; MS (FAB - nitrobenzyl alcohol) m/e 1013 ([MH+], 75 %),
1011 (tNH+ ~ ~2] ~ 100) 942 ( [MH+ - HOCH2CH2CN~, 16), 912 ([MH+ -
iPr2NH], 10), 887 ([MH+ - ThyH], 82), 795 ([MH+-iPr2NP(OH)QCH2CH2CN],
21), 709 ([MH~ - DMTrH], 45~.
EXA~PLB 5.
PRODUCTION OF THE ACTIVATED SULFONE-LINKED DIMER XI (where m=0,
n=2, X=O, Y=Y'=H, B=Thy~. (Scheme lb)
c) Diol IX (where m=O, na0, X=O, Y=Y'=H, B=Thy) (77mg, 0,151 mmole)
was dissolved in methanol (1.5 mL) and cooled to 0C. A solution of
KHSOs (1.206 mL, 0.452 mmole) in water was added. The resulting
slurry was stirred for 3 hours at room temperature. Then the
solvents were evaporated and dried in vacuo. The dried white
residue was washed with methanol (3x10 mL) and filtered. The
methanol was evaporated. The residue was chromatographed over
silica gel (5:1 dichloromethane / methanol) yielding the sulfone-
linked dimer I~ (where m=0, n=2, X=O, Y=Y'=H, B=Thy) as a white
powder in quantitative yield.
f, g) The dimer was converted to phophoramidite ~I (where m=0, n=2,
X=O, Y=Y'=H, B=Thy) via alcohol X 5where m=0, n=2, X=O, Y=Y'=H,
B=Thy) in the same manner as for the corresponding sulfide dimer IX
(where n=o).
EXAMPLE 6.
INCORPORATION OF THE SULFIDE-LINKED DIMER XI (where m=0, n=0, X=O,
Y=Y'=H, B=Thy) INTO DNA.
The possibility of oxidation of the sulphur atoms during the iodine
oxidation step of the coupling cycle was a major concern. To avoid
41
' :`' ,, .;. , , ' "~ . ' :
20~2~
.~
such an effect during the incorporation of the sulfide dimers into
natural DNA by stand~rd phosphoramidite chemistry, a sample of the
disilyla~ed sulfide dimer VIII (where m=0, n=0, X=O, Y=Y'=H, B=Thy)
was dissolved in the I2 containing xeagent (I2 / pyridine / THF /
H2O) and stirred for 15 min. Thin-layer chromatography demonstrated
that no oxidation had occured when compared to the chromatogram of
the corresponding sulfone.
Oligonucleotid~s a to D (Scheme 5) were synthesized by standard
solid-~upport methodology. Dimethylformamidine-protected
cyanoethylphosphoramidites (Applied Biosystem~) were used on an
Applied Biosystems automated DNA synthesizer. Four bottles
contained each a solution of 2-deoxyadenosine, 2-de~xyguanosine, 2-
deoxycytidine, and Thymidine. A fifth bottle containing a 0.08 M
acatonitrile solution of the sulfide dimer phosphoramidite ~I was
attached to the synthesizer. The coupling efficiency of the
sulfide dimer units was routinely greater than 95 % as monitored by
the release of the DMTr cation. After cleavage from the support,
the sulphur-containing oligonucleotides were easily purified by
reverse-phase chromatography using OPCTM cartridges.
Scheme 5
5'- GC G T p T p T pT pT p T G C T -3'
~3 5~- G (: G T S T p T S T p T S T G C T -3
C) 6~- T s T p T s T p T s T G C T -3~
s~- A G C A A A A A A C: G C -3~ :
42
21D78~
EXANPLE 7.
THERMAL DENATURATION STUDIE5.
Thermal denaturation studies in 10 mM sodium phosphate buffer, pH
6.5, lM NaCl, indicate that the replacement of phosphodiester
groups with the dialkyl sulfide linkages weakens but_does not
prevent binding to a complementary, fully natural DNA strand. The
Tm for the unmodified romplex A/D was 46 C, exhibiting a broad,
cooperative transition and 18 % hypochromicity. A meltiny
temperature of 26 C was observed for the mixed complex B/D. This
showed a sharper cooperative transition and a hypochromicity of
10 %. Complex formation between ~ and D was also observed using
native PAGE. When an excess of either oligomer ~ or D was used
with respect to the other, the identical slower running (complex)
band was observed in both cases, accompanied only by the
single-stranded species present in excess.
EXAMP~E 8.
STABILITY OF THE DEOXYRIBONIJCLEOSIDE SULFIDE- CONTAINING OLIGOMER
TO NUCLEASE DE&RADATION.
The stability of the sulfide-containing oligomers towards nuclease
degradation was also investigated. Oligomer C was found to be
completely stable to calf-spleen phosphodiesterase (CSPDE, a
5'-exonuclease) after incubation at 37~C for 60 minO The identical
treatment of oligomer B resulted in complete conversion to a new
band (PAGE~ which migrated identically with C, indicating that
CSPDE can cleave the external phosphodiesters until a sulfide
linkage is reached which protects the remainder of the strand (i.e.
oligomer C). The DNA natural strand ~ is completely degraded under
these conditions. The incubation of oligomers B and C with snake
43
2~78~
, -
venom phosphodiesterase (SVPDE, a 3'-exonuclease) for 60 min at
37~C resulted, in both cases, in a faster moving band which we
identified to be the (TsT) "core dimer". SVPDE apparently cleaves
the phosphodiesters of the strand from the 3'-end as expected, but
can by-pass tin the case of B) the sulfide-containing region and
continue to degrade the natural region on the 5'-end of the
molecule and the phosphodiesters flanked by the sulfide-linkages.
This endonuclease activity of SVPDE has been recognized in earlier
studies involving nucleotide phosphotriesters, phosphorothioates,
methylphosphonates and, more recently, oligonucleotides containing
1,3-propanediol and 1,3-butanediol sugar moieties.
Examples 9 and 10 relate to the following Scheme 6 where the
reactions are described for ribonucleosides and analogs (i.e. X=O,
Y-OH or OAc, and B=Thy).
44
.
.
' :' ... .: . . ' ' .. ' . ~ ' : ' ., ' .',' " ' " ' ". ' .'' :''.'.. ' . ' ' ' "
2~78256
Scheme 6 ~P=OAc, m=0, n=0, X=O, Y=OH or OAc, B=Thy)
AcO ~r Q AcO ~ b AoO -r ~ O T
~OAc CH~ 97~ ~ TBDMU~=Ir C5~ DMF
O ~ OCH~ OH OMs
~ y~C~3
"~, d ,,0~ ~ ~MT.~7r ~ :
~NH3,MhOH ~ H(1)DMTCI,py,EbN OAc (n-BU)4N~F
~ ~OAC 9p/O ~ (2~Ac~O,Py~ E~N S _~ DMF 94%
S ~ ~ ~ ~ 7
TBDMS~ TBDMS~
TBDMS~
~ Xll ~
'' .
~/DMTrO ._
DMTIO ~ lLGN ~OAc
Sl,o~r CH2CI2,ElaN 72Yo ~1
OH ~ ~ N
: ~ lLcN
~ :
,, ; " ,; . !,, ' , ' ' ~ ' ''' ' ' ` ' ' ' ' ' " ' ' ' ; ~ "" " ;' "
2078256
,~ .
EXAMPLE 9.
SYNTHESIS OF ACTIVATED/PROTECTED RIBONUCLEOSIDE-CONTAINING DIMER XI
(where m=O~ n-O, X=O Y=OAc or OH! Y'=H, B=Thy).
a)2'~-5'-Di-O-acetyl-3~-deoxy-3~-C- (2"-hydroxyeth~l~-B-D-
ribofuranosyl-thymine 21.
To a solution of (para-methoxyphenyl) thymidine derivative 8 (R=~c,
Z=Thy) (4 . 82 g, 10.1 mmole) in acetonitrile (56 mL) at O C was
added a eolution of ceric ammonium nitrate (11.85 g, 21.6 mmole) in
water (56 mL). The reaction was stirred at 0C for 30 min and
diluted with brine (116 mL)~ The mixture was extracted with ethyl
acetate (3 x 200 mL)O The organic extracts were washed with sodium
sulfite (10% w/v, until the aqueous layer remained colorless),
sodium bicarbonate (5% w/v, 100 mL), and dried (Na2SO4). Removal of
the solvent yielded a yellow foam which was chromatographed over
silical gel (20:1 dichloromethane / methanol, v/v) and afforded the
alcohol 21 as a colorless foam (2.83g, 76%). ~H-NMR (300 MHz, CDCl3)
~ 1.78-1.88 (m, 2H, H15'A~), 1.93 (s, 3H, 5-Me's), 2.07 and 2.09 (two
s, 6H, OAc), 2.55 (h7, lH, H3'), 3.68 (t, 2H, H2"AB), 4 . 19 ~ddd, lH,
H4'), 4.42 (d, 2H, H5~AB), 5.53 (d, lH, H2'), 5.56 (s, lH, H1'),
7.31 (s, lH, H6), 9.2 (br s, 1H, NH), JHI^~= 1.2, J~ ~= 600, J~.~=
~ ~4 -~ A 3-5, J~4 -~ B= 2- 5, J~j A-~ B= ~ 12.8; l3C_NMR (CDC13) 13.50
ppm (5-Me), 21.25 and 21.45 (COMe), 29.20 (C1"), 40.11 (C3'), 61.33
(C5'), ~4.92 (C2"), 84.34 (C4'), 93.13 (C1'), 112.00 (iC5) 138.61
(C6), 152.58 (C2), 163.85 (C4), 172.11 and 172.94 (COMe); MS (FAB-
Glycerol/NBA) m/e 371 ([NH+], 46%), 311 ([MH~-AcOH], 7.2), 245
([MH+-ThyH], 100).
b)2' 5'-Di-O-acetyl-3'-deoxy-3'-C-(2"-hydroxyethyll-2"-0-
methanesulfonyl-~-D-ribofuranosyl-thymine II (where P=Ac, Y=OA~).
Methanesulfonyl chloride (0.48 mL, 6.20 mmole) was added to a
,
~6
. :
.~ .. . . , ,:,. : : , ; ,. . :..... , : , . . "., ".:, , . ~, : . ,
~07825~
-
stirred solution of alcohol 21 (1.00 g, 2.70 mmole) and dried
triethylamine (0 68 mL, ~.88 mmole) in dry dichloromethane (19 mh)
at room temperature under nitrogen. After 1 h, the reaction was
diluted with dichloromethane (20 mL) and washed with hydrochloric
acid (5~ w/v, 7 mL~, saturated aqueou~ sodium bicarbonate (7 mL),
and brine (5% w/v, 7 mL). The organic layer was dried (Na2SO4), and
the solvent was removed yielding a yellow foam. Chromatography
over silica gel (20:1 dichloromethane / methanol, v/v) af~orded the
mesylate II as a colorless foam (1.17 g, 97%).
'H-NMR (300 MHz, CDCl3) ~ 1.72-1.98 (m, 2H, Hl"~), 1.93 (s,
3H, 5-Me's), 2.13 and 2.16 (two s, 6H, OAc), 2.55 th7, lH, H3'),
3.02 (s, 3H, SO~e~, 4.12 (dq, lH, H4'), 4.24~4.29 (m, 2H, H2"~),
4.37 (A of ABX, lH, H5'~, 4.40 (B of ABX, lH, H5'~), 5.51 (dd, lH,
H2'), 5.64 (d, lH, H1'), 7.23 (s, lH, H6), 9.05 (br s, lH, N~, JHI:
~2 ~ JSI2-H3 59, JH3~ N4 19 . 3, Jll4 -H5 A = 4 . 6, JH4 -H5 ~= 25, 2J~S~A HS'~I = ~
10 ~ 2 ; ~3C-NMR (CDCl3) lZ.51 ppm (5-Me), 20.57 (2 x COMe), 24.25
(C1"), 37.23 (SO2Me~, 38.34 (C3'), 62.94 (C5'),67.98 (C2"), 77.00
(C2'), 81.7~4 (C4'), 91.77 (Cl'), 110.72 (C5), 136.39 (C6), 150.31
(C2), 164.28 (C4), 160.99 and 170.~2 (COMe); MS (FAB-Glycerol/NBA)
m/e 449 ([MH+], 46%~, 389 ([MH+ - AcOH],5.7), 323 [MH+- ThyH], 100).
c) Dimer VIII !where P=TBDMSi, Y=OAc).
Cesium carbonate (549 mg, 1.68 mmole), previously flame dried in
vacuo, was suspensed in dry N,N-dimethylformamide (DMF) (9 mL) and
a solution of mesylate II (505 mg, 1.17 mmole) and 3'-0-tert-
butyldimethylsilyl-5'- thiothymidine VII (479 mg, 1.~9 mmole) in
dry DMF (14 mL) was then added resulting in a yellow solution.
After 1 h of stirring at room temperature under nitrogen, the
solvent was removed and the product was extracted with
dichloromethane (2 x 270 mL) and washed with a~ueous sodium
bicarbonate (5% w/v, 225 mL) and water 1225 mL). The combined
organic layers were dried (Na2SO4) and evaporated yielding a yellow
foam. Chromatography over silica gel (1 : 2.5 ethyl acetate /
47
, . ,, : , , ~: ~ .,
~,, , , . ~.. . .. . . .
;
2~782~
hexanes, v/v) afforded the dimer V (Y=OAc) as a colorless foam (711
mg, 84%).
NMR (300 MHZ~ CDC13) ~ 0. 092 (5~ 6~I~ SiNa2) ~ 0. 90 (5~ 9H, t-
butyl), 1.60 -1080 (m, 2H, 5H1llA;3), 1.92 and 1.93 (two ~i, 6H, 5-
3l Me's), 2.10 and 2.15 (two s, 6H, COMe), 2.18 - 2.26 (m, 2-H7 3H2~AB),
2.41 - 2.51 (m, lH, 5H3') ~ 2.51 - 2.70 ~m, 2H, 5H2llAB), 2.75 (A of
ABX, lH, 3H5~A), 2.78 (B Of ABX, 1H, 3H5/B), 3.94 (q, lH, 5H4/), 4.10
(dt, lH, 3H4~) 4.28 - 4.37 (m, 3H, 3H3' and 5H51~B), 5.45 (d, lH,
SH2~), 6.21 (S, lH, 5H1'), 6.21 (t, lH, 3H1'), 7.30 and 7.56 (two s,
2H, H6 ~ S), 9.01 (br s, 2H, N~) ~ JOHI OI~A= J(3)HI--OH2H = 6 - 6 Hz, J~5)~H3
6.0, J(5)H3 ~H4 7.5, J(3)H3:(3);14 4 . 5, JP)H4 .(3)H5 A 5 - 7, J(3)H4 -(3);U B 4 9, JO)HS A-
(3)H5~ = -13.8; 13C-NMR (75.4 MHZ, CDC13)~ 11.11 (2C,5-Me), 17-12 (~H2),
20.22 and 20.32 ~CONe), 24,31 (SiCMe3), 25.21 (SiMe2 and SiC~e~),
- 30.50, 33.49 and 39.87 (3 X CH2), 40.16 (SC3~), 62.88 (5C5~), 73.01
(3C31), 76.67 (5C2), ~1.69, 84.54, 85.18 and 91.27 (2 X Hl' and 2 X
H4'), 110.25 and 110.70 (2 x C5), 135.35 and 135.62 (2 X C6~,
149.82 and lSo.10 (2 x C2), 163.79 and 163.87 (2 x C4), 169.21 and
169.94 (COMe); MS (FAB - glycerol / NBA) m/e 725 ( [MH~], 7.5%), 599
([MH' - ThyH], 52), 473 ( [MHI - 2 x ThyH~, 12), 399 ( [MH+- 2 x ThyH-
MeCOO~e],33), 341(57), 295 (17), 213 (100).
,
dL Alcohol XII (where Y=OH).
Dimer Y (518 mg, 0.715 mmole) was suspended in dry methanol (10 mL)
and cooled to 0C. The mixture was then saturated with ammonia gas
and allowed to warm to room temperature. After 11 h the resulting
homogeneous solution was evaporated yielding a white foam.
Chromatography over silica gel (20:1 dichloromethane / methanol,
v/v) afforded the alcohol ~II as a white foam (442 mg, 97% yield).
'H~ R (300 MHZ, CDC13) ~ 0.052 (S, 6~I, SiMe2), 0.85 (S, 9H, t-
butyl), 1.55 - 1.70 (m, 2H, 5H1~AB), 1.70 and 1.78 (two s, 6H, 5-
Me's), 2.10 - 2.15 (m, 6H, 3H2'AB, and 5H3' and 5H2"AB), 2.95 - 3.12
48
` ~` 207~2~fi
(m, 2H, 3H5~AD)~ 3.72 - 3.77 (m, lH, 5H4'), 3.93 (t, llI, 3H4'), 4.00 -
4.10 (m, 2H, 5H5Aj3), 4.44 - 4.46 (m, 2H, 3H2' and 5H2'), 5.77 (S,
1H, 5H1'), 6.21 (t, lH, 3H1'), 7.51 and 7.75 (two s, 2H, H6~S), 9.01
(br s, 2H, NH), J(3)HI (3)H2A J(3)HI (3)H2D~ 54 Hz, J (3)H3-(3)H4 3 ; C NM~
(75.4 MHZ, C~C13) ~ 12.62 and 12.73 (2C, 5-Me), 18.19 (CH2), 25.73
(SiCMe3), 26.13 (SiMe2 and SiCMe3), 31.56 and 32.58 (2 x CH2), 39.80
(5C3~), 40.75 (CH2), 6170 (5C5/), 72.44 (3C3~), 76.12 (5C2), 84.00,
85.59 and 92.40 ~2 X Hl' and 2i x H4'), 109.31 and 111.53 ~2 x C5),
135.52 and 136.62 (2 x C6), 151.19 and 151.79 (2 X C2), 164.63 and
î64.79 (2 x C4); MS (FAB -glycerol / N8A) m/e 641 (~MH+~, 18%),
515 ( [M~+ -- ThyH], 31), 389 ([MH+ 2 x ThyH], 17) 257 (61), 213
(100).
e~ Dimethoxytrityl Ether of alçohol XIII (where Y=OAc).
Alcohol ~II (220 mg, 0.343 mmole) was dissolved in dry pyridine
(3. 4 mL) at room temperature. Dimethoxitrityl chloride (330 mg,
0.974 mmole) and triethylamine (0.18 mL, 1.291 mmole) were added in
portions (DMTCl: 110 mg, 0.325 mmole and Et3N: 0.06 mL, 0.430 mmole)
during an 8 h period. After the reaction was finished which was
tested by TLC, acetic anhydride (2O00 m:L, 21.1~7 mmole) was added
to the solution which was then kept overnight at room temperature
under nitrogen. Saturated sodium bicarbonate (30 mL~ was added and
the resulting solution was extracted with dichloromethane (2 x 30
mL). The combined organic layers were washed with water (30 mL),
dried (Na2SO4) and evaporated yielding a yellow foam.
Chromatography over the silica gel (100: 5: 1 dichloromethane /
methanol / triethylamine, v/v) afforded ths dimethoxytrityl ether
XIII as a colorless foam (0.246 mg, 76%).
'H-NMR (300 MHZ, CDCl3) 8 0.078 (s, 6H, SiMe2), 0~89 (S, 9H, t-
butyl), 1.36 - 1.65 (m, 2H, 5H1lIAB) ~ 1.42 and 1.89 (two s, 6H, 5
Me's), 2.14 (S, 3H, COMe), 2.08 - 2.26 (m, 2H, 3H2~AD), 2.41 - 2.63
(m, 3H, 5H3~ and 5H2",~B), 2.66 - 2.69 (m, 2H, 3H5~AB), 3.21 (A of ABX,
1H, 5H5~,~), 3. 66 (B of ABX, lH, 5H5/D), 3.78 and 3.79 (two s, 6H, 2
49
2~7g2~$ :~
:
x COMe), 3.92 (q, lH, 5H4'), 3.99 - 4.02 (m, lH, 3H4'), 4.31 (dt,
lH, 3H3~), 5.45 (dd, lH, sH2~), 5.86 (d, lH, 5Hl'~, 6.17 (t, lH,
3H1'), 6.84 (q, 4H, C6H6), 7.72 - 7.43 (m, 8H, MeOPhO), 7.30 and 7.56
(tWO S, 2H, H6~S), 9.01 (br S, 2H, N~), J(3)UI-~3)H2~ = 66HZ.J(5)H2(5)U3 = 5-6,
3(5)NI:(5)1U = 1- 5, JQ)H3-(3)~ 44; C NMR (75.D, M~Iz, CDCl3) ,5 10.15 (2C,
5~ Me), 16.13 (CH2), 20.99 (COMe), 24.83 (SiCMe3), 25.94 ~SiMe2 and
SiC~3), 31.29 and 34 . 13 (2 x C~2), 39.94 (5C3 ' ~, 40.50 (CH2), 46 . 13
(Ph3C~, 55.48 (2 x OMe), 62.02 (5C5'), 73.52 (3C3'), 77.41 (5C2),
83 . 84 , 85 . 13 , 85.87 and 89.95 (2 X Hl ' and 2 X H4 ' ), 111.17 and
111.44 (2 X C5), 113.51 and 127.44 (12 x CH of C6Hi6), 135.41 and
135.84 (2 X C6), 144. 37 (C of C6H6), 150.43 and 150.51 (2 x C2),
158.98 (2 X C of MeOPhO), 164.06 and 164.22 (2 x C4), 169.69
(COMe); MS (FAB - glycerol I NBA) m/e 985 ( [MH+], 18%), 859 ( [MH+ -
ThyH], 23), 681 ( [MH+ DMTrH], 66~ ( [MH+ - DMTrOH], 100).
f ~ Dimethoxytritylated_alcohol X (where Y=OAc).
A solution of tetra-n-butylammonium fluoride in tetrahydrofuran
(lM, 0.647 mL, 0.647 mmole) was added to a stirred solution of
dimethoxytrityl ether XIXI (255 mg, O. 259 mmole) in dry
tetrahydrofuran (7 mL). After 1 h the solution was ~vaporated and
the resulting foam was chromatographed over silica gel (100 : 5 :
1 dichlorome.thane / methanol / triethylamine, v/v) to give the
dimethoxytritylated alcohol X as a colorless foam (212 mg, 94%).
'H-NMR (300 MHz, CDC13) ~ 1 . 38 - 1 . 62 (m, 2H, 5H1t'A~3), 1 . 43 and 1 . 85
(two s, 6H, 5-Me's), 2.14 (S, 3H COMe), 2.14 - 2.19 (m, 2H, 3H2~A),
2.32 - 2.67 (m, 6H, 3H2~D, 5H3' and 5H2l~AD~ 3H5~AD)~ 3.21 (A of ABX, lH,
SH5lA), 3.64 (B of ABX, 1H, 5H5~D), 3.76 (S, 6H, 2 X COMe), 3.94 -
4.02 (m, 2H SH4,, 3H4~), 4.31 (dt, lH, 3H3~), 5056 (dd, lH, 5H2~),
5.81 (d, 1H, 5H1'), 6.20 (t, lHI 3H1'), 6.83 tq, 4H, C~H6), 7.72 --
7 .42 (ml 8H, MeOPhO), 7.67 (s, 2H, H6's), J~3)UI'-(3)H2'A = J(3)~ll'-(3)~l2B= 6-6 HZ,
J(5)H2 -(5)H3 5. 6, J(5)HI -(5)H2 = 1 5, J(3)H2 A O)H3 = 80, Ja)H2 D43)!13 71, J(3) H3
(3)H4~ = 6.6; '3C_NMR (75.4 MHZ, CDC13) ~ 10.63 (2C, 5-Me), 21.20
. , , ,: , . ' ~,,, . :: , , . ,............ " : . , .
207~2~6
(CO~e), 25.10, 31.10, 34.76 and 40.23 (4 x CH2), 40.32 (5C3'), 46.31
(Ph3C), 55.61 (2 x OM~), 62.16 (5C5'), 73.25 ~3C3'), 77.70 (5C2),
83.91, 85.30 and 90.31 (2 x H1' and 2 x H4'~, 111.14 and 111.38 (2
j x C5~, 113~53 and 127.40 (12 x CH of C6H6and MeOPhO), 135.67 and
135.90 (2 x C6), 144.37 (C of C~6), 150.60 and 150.74 (2 x C2),
158.88 (2 x C of MeOPhO;, 164.2~ and 164.49 (2 x C4~, 170.05
(COMe); MS (FAB - glycerol / NBA) m/e 871 ([MH~], 59%), 745 ([MH+ -
ThyH], 28~, 551 ([MH+ - DMTrOH], 87).
~RMS (FAB-Glycerol) m/e Calcd. for C4.jH~N4SI: 870.31459, found:
870.31421.
g) Phosphoramidite XI (where Y=OAc).
2-Cyanoethyl N,N- diisopropylchlorophosphoramidite (0.137 mL, 0.618
mmole) was slowly added to a stirred solution of tritylated alcohol
X (265 mg, 0.305 mmole) in dry dichloromethane (4 mL) containing
triethylamine (0.168 mL, 1.200 mmole). After 6 h of stirring at
room temperature under nitrogen, the solution was diluted with
ethyl acetate (87 mL) and washed with brine (4 x 173 mL?. The
organic layer was then dried (Na2SO4), and evaporated yielding a
pale yellow foam. Chromatography over silica gel (100 : 5 : 1
dichloromethane / methanol / triethylamine, v/v) afforded the
phosphoramidite ~I as a colorless foam (234 mg, 72~), which was
used as such in the subsequent solid-phase syntheses.
31~-NMR (CDCl3) 148.94 and 149.31 ppm; MS (FAB - Glycerol/NBA) m/e
1072 ~[MH+], 10~i), 946 ([MH+ - ThyH), 8), 853 ([MH+
iPr2NP(OH)OCH2CH2CN], 5~, 768 ([MH+ - DMTrH], 9), 5S1 (16), 457
( 100) .
..
~X~MRLE 10.
INSERTION OF THE THYMIDINE CONTAINING DIMER INTO DNA.
The phosphoramidite XI (where Y=OAc) was inserted in DNA strands
following the same method as presented in example 6 to yield the
51
~ 20782~
modified DNA strand as present,~d in Scheme 7. The modified DNA
sequence was then hybridized with complementary RNA sequence with
which thermal denaturation studies were performed and compared to
the duplex made with natural DNA sequence hybridized with its
complementary natural RNA sequence.
Scheme 7.
,~ ,
5- GCG T T T T T T GCT -3' , natural DNA sequence
' ¦ l I I I I I I I 11 I Tm=55~
3'- CGC A A A A A A ~;A -5' oomplementa~ natural
RNA s3qu3nce
" .
5'- GCG THTTH T THT GCT -3' modified DNA sequence
i 11 Tm=33~
3'- CGCA AA AA ACGA-S complementa~ynatural
.,
: .
E8AMPLE 11.
~! HYBRIDIZATION STUDIES.
Thermal denaturation studies (3mM oligomer / lM NaCl / 10 mM
phosphate buffer pH 6.5) showed cooperative binding between the
hydroxysulfide-containing oligomer (modified DNA sequence) and the
complementary natural RNA strand. However, when the hydroxysulfide-
containing oligomer was hydridized with complementary DNA sequence,
no binding was detectable. This indicates that this hydroxysulfide
modification may allow one to æelectively target complementary RNA
molecules without affecting the corresponding DNA sequences, a fact
which may have an impact in anti-sense treatment regimens for
diseases as well as in their use as biological probes.
52
