CA2078256A1 - Synthesis of sulfide-linked di-or oligonucleotide analogs and incorporation into antisense dna or rna - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

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:

in which R and R' are independently selected from H, DNA, RNA
unsubstituted or substituted by I, nucleoside, nucleotides and analogs thereof; B is a base having a heterocyclic ring, X is independently O, CH2 or S; Y is independently H, OH or O-alkyl; Y' is H or O-alkyl; n is 0, 1 or 2; and m is 0 or an integer. These compounds are useful for binding selectively to complementary DNA
or RNA strands (particularly mRNA strands) for use in regulating gene expression and as biological probes.

Description

~ 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

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 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;

''., . ' ~ ' ' . ' ' . " ' ' .. ' ' ' " ' ',.' . "

. '~': . ' ' : ' '. ' ' ' . ' . ' " ' ' ' ' ' ' ', ' ,': ' ' ' ~ ' ' ' ' ~ ' : . '' . ' , ' 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 . . - .
,., , :, . , : . ~ .: .

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.

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

.

' ;:

~, :` 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.
:' 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 .: . ,, . . : : , ; , .. ' ' , ,. - ,, :: . : ' .:
, . : ' ' : ,.

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, ~ 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'), -" 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.

~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 ~.

,... . . . . ; . . . , ~ , , .: : : . . . ..
; ,- '. :, ' ''' ' .' ,", ' , ' '. ,. .~ -.,'''.. ' , . . .

.. . . .... . . .. . . .
, . , ., ~ . .
, ~ ,, . .. ,. " ... . . .

-` 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 -. .
:

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'), 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).

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, 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 .:

. , : ;: - . - , . - - , ~ . . , .............. . :, .. ..

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 ' :`' ,, .;. , , ' "~ . ' :

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~ :

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 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).

.
.

' :' ... .: . . ' ' .. ' . ~ ' : ' ., ' .',' " ' " ' ". ' .'' :''.'.. ' . ' ' ' "

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
~ :

,, ; " ,; . !,, ' , ' ' ~ ' ''' ' ' ` ' ' ' ' ' " ' ' ' ; ~ "" " ;' "

,~ .

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 /

, . ,, : , , ~: ~ ., ~,, , , . ~.. . .. . . .

;
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 ` ~` 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 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 ~ 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.

Claims (41)

1. An oligonucleotide analog of formula I:

wherein R and R' is independently selected from the group consisting of: H, DNA or RNA unsubstituted or substituted with an oligonucleotide analog of formula I, nucleoside, nucleotides and analogs thereof;
B is a base having a heterocyclic ring 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 from the group consisting of: O, CH2 and S;
each Y is independently selected from the group consisting of: H
and OR1, wherein R1 is selected from hydrogen or alkyl;
Y' is selected from the group consisting of: H and OR1, wherein R
is an alkyl;

n is 0, 1 or 2; and m is 0 or an integer.

2. An oligonuclsotide analog of formula I according to claim 1, wherein m is 0.

3. An oligonucleotide analog according to claim 1 or 2, wherein B
is selected from the group consisting of: adenine, guanine, cytosine, uracil, and thymine.

4. An oligonucleotide analog according to claim 3, wherein n is 0.

5. A DNA molecule having an oligonucleotide analog according to claim 3 either internally or at either end thereof, wherein at least one of R and R' is DNA.

6. A DNA molecule according to claim 5, wherein each X is independently O; each Y is independently H or OR1 wherein R1 is allyl; and each B is independently selected from adenine, guanine, cytosine, uracil and thymine.

7. A DNA molecule according to claim 6, wherein R and R' is DNA; Y
is H; and B is thymine.

8. A DNA molecule according to claim 7, wherein n is 0.

9. A RNA molecule having an oligonucleotide analog according to claim 3 either internally or at either end thereof, wherein at least one of R or R' is RNA.

10. A RNA molecule according to claim 9, wherein X is 0; each Y is independently OH or OR1 wherein R1 is allyl; and each B is independently selected from the group consisting of adenine, guanine, cytosine, uracil or thymine.

11. An intermediate for the production of a compound of formula I
according to claim 1, said intermediate selected from the group consisting of:

VIII IX

X XI XII XIII
wherein B, X, Y, Y', n, and m have the same meaning as in claim 1, and P is a hydroxyl protecting group.

12. An intermediate according to claim 11, wherein n is 0.

13. An intermediate according to claim 11, wherein X is 0; Y and Y' is H; and B is thymine.

14. An intermediate according to claim 11, wherein, when Y is H, P is TBDMSi.

15. An intermediate according to claim 11, wherein, when Y is OAc or OH, P is OAc.

16. A process for producing a nucleotide analog of formula I
according to claim 1, comprising the step of:

a) condensing a compound of formula II:

II with a compound of formula III:

III
to obtain a compound of formula IV:

IV

wherein B, X and Y have the same meaning as in claim 1, P is a hydroxyl protecting group, and n is o.

17. A process according to claim 16, further comprising the step of: i) treating the compound of formula IV with ceric ammonium nitrate to obtain a compound of formula V:

V

wherein P, B, X, and Y have the same meaning as in claim 16.

18. A process according to claim 17, further comprising the step of: ii) mesylating the compound of formula V in methylene chloride containing pyridine and triethylamine to obtain a compound of formula VI:

VI

wherein P, B, X, and Y have the same meaning as in claim 17.

19. The process of claim 18, further comprising the step of:
iii) condensing the compound of formula VI with a compound of formula III (m-1) times, and iv) for terminating the reaction, condensing the resulting compound of formula IV with a compound of formula VII:

VII
to obtain a compound of formula VIII:

VIII wherein P, B, X, and Y have the same meaning as in claim 18, m is 0 or an integer, and Y' is selected from the group consisting of H and OR1, wherein R1 is an alkyl.

20. A process for producing an oligonucleotide analog of formula I, I

comprising the step of:
a) condensing a compound of formula II:

II

with a compound of formula VII:

VII
to obtain a compound of formula VIII:

VIII

wherein B, X, Y, Y', and P have the same meaning as in claim 1, P
is a hydroxyl protecting group, n is 0, and m is 0.

21. The process according to claim 19 or 20, wherein the compound of formula VIII is oxidized to give a compound of formula VIII
wherein B, X, Y, Y' and m have the same meaning as in claim 19 or 20 respectively and n is 1 or 2.

22. A process according to claim 19 or 20, further comprising the step of:
b) deprotecting sequentially the 5'-end and 3'-end hydroxyl groups, and c) treating the resulting free 5'-end hydroxyl with dimethoxytrityl chloride in triethylamine and pyridine to give a compound of formula X:

wherein B, X, Y, Y', m and n have the same meaning as in claim 20.

23. A process according to claim 22, further comprising the step of: d) treating the compound of formula X with 2-cyanoethyl N,N-diisopropylchlorophosphoramiditeindichloromethanecontaining triethylamine to give a compound of formula XI:

XI wherein B, X, Y, Y', m and n have the same meaning as in claim 22.

24. A process according to claim 19, 20, or 23 wherein X is O; Y' is H; and B is thymine.

25. A process according to claim 24, wherein Y is H.

26. A process according to claim 24, wherein Y is OAc or OH.

27. A process according to claim 21, wherein X is O; Y' is H; and B is thymine.

28. A process according to claim 27, wherein Y is H.

29. A process according to claim 27, wherein Y is OAc or OH.

30. A process according to claim 22, wherein X is O; Y' is H; and B is thymine.

31. A process according to claim 30, wherein Y is H.

32. A process according to claim 30, wherein Y is OAc or OH.

33. A process according to claim 24, wherein the compound of formula XI is incorporated into a DNA or a RNA molecule.

34. A process according to anyone of claim 25 to 32, wherein the compound of formula XI is incorporated into a DNA or a RNA
molecule.

35. An intermediate of formula II wherein P, B, X, and Y have the same meaning as in claim 11.

36. An intermediate according to claim 35, wherein X is O, Y is H, B is thymine, and P is TBDMSi.

37. An intermediate according to claim 35, wherein X is O, Y is OAc or OH, B is thymine, and P is OAc.

38. An intermediate of formula III:

III

wherein B, X, and Y have the same meaning as in claim 1.

39. An intermediate according to claim 38, wherein X is 0, Y is H, and B is thymine.

40. An intermediate of formula VII:

VII
wherein P, B, X and Y' have the same meaning as in claim 11.

41. An intermediate according to claim 40, wherein X is 0, Y' is H, and B is thymine.