Process for preparing dithiophosphate oligonucleotide analogs via nucleoside thiophosphoramidite intermediates

A method for synthesis of oligonucleotide analogs having dithiophosphate internucleosidic linkages is described. Monohalohydrocarbylthiophosphoramidites are utilized to prepare nucleoside thiophosphoramidite intermediates which are activated for nucleoside coupling with tetrazole catalysts. Sulfur oxidation and dehydrocarbylation of the coupled thiophosphite intermediates provide oligonucleotide analogs having achiral dithiophosphate internucleosidic linkages.

FIELD OF THE INVENTION 
This invention relates to the synthesis of oligonucleotide analogs having 
dithiophosphate internucleosidic linkages. More particularly, this 
invention relates to an improved process utilizing nucleoside 
phosphoramidite intermediates in the synthesis of nucleoside 
phosphorodithioates. Novel monohalohydrocarbyl thiophosphoramidites are 
useful intermediates for the synthesis of 3'-O-hydrocarbyl 
thiophosphoramidite nucleosides activated for coupling with nucleosides or 
oligonucleosides or analogs thereof using mildly acidic nitrogenous 
catalysts such as tetrazole. 
BACKGROUND AND SUMMARY OF THE INVENTION 
One of the most widely employed methods of synthesizing oligonucleotides is 
known as the phosphate triester method. The phosphate triester method can 
be employed in solution, and generally involves coupling of a protected 
nucleoside 3'-phosphate with another protected nucleoside having a 
5'-hydroxyl group. The coupled reaction product is typically 
isolated/purified chromatographically and one of the protecting groups is 
removed to yield a dimer block. The dimer block can then be coupled with 
other selected oligomeric blocks having an unprotected 3'-phosphate or 
5'-hydroxyl terminus to yield oligomers having desired nucleoside 
sequences. 
The phosphate triester method for oligonucleotide synthesis can also be 
readily adapted for solid phase reaction conditions. Thus a 5'-O-protected 
deoxyribonucleoside, for example, can be covalently attached to a solid 
support, such as polystyrene, cellulose, or silica gel, and subjected to a 
sequence of reaction conditions which first effects removal of the 
5'-O-protecting group and thereafter couples the support bound nucleoside 
to other nucleosides. Repeating the process with selected reactive 
nucleosides, dimer blocks, or oligonucleotide intermediates enables 
extension of the nucleotide chain to a predetermined length/composition. 
In the final step the oligonucleotide is cleaved from the solid support 
and subjected to conventional purification techniques. 
The present invention allows application of such solid phase synthesis 
procedures to the synthesis of phosphorodithioate oligonucleotide analogs. 
Matsukura et al. , Proc. Natl. Acad. Sci., Vol. 84, pp. 7706-7710, 1987, 
have shown that phosphorothioate analogs of oligonucleotides are effective 
inhibitors of HIV replication and are cytopathic to virally infected T 
cells. They exhibit antiviral activity in vivo by binding with either the 
RNA template derived from a virus or duplex DNA derived from a virus that 
has integrated into the genome of the host. Both complementary "antisense" 
oligonucleotide phosphorothioates and homooligomer analogs exhibited 
potent anti-HIV activity. The most effective analog was reported to be a 
28-mer oligodeoxycytidine phosphorothioate (S-dC.sub.28) which exhibited 
anti-HIV activity at 1 .mu.M concentration and inhibited de novo DNA 
synthesis. The authors reported that the S-analogs of deoxyribonucleotides 
(phosphorothioates) showed no significant degradation over a period of 
weeks in their cytopathic assay and during incubation in human serum at 
37.degree. C. Hydrolysis of normal oligonucleotides indicated a half-life 
of .about.17 hr in the in vitro assay. The S-analogs also showed good 
permeability to the target cells. Using .sup.35 S-labeled 
phosphorothioates, S-dC.sub.28 showed significant amounts of radioactivity 
in the immortalized T4.sup.+ ATH8 and H9 cells within several minutes. 
Notably, however, the phosphorothioate linkages of oligonucleotide analogs 
as disclosed by Matsukura et al. are chiral structures. In the best of 
circumstances one might be able to achieve &gt;99% yields in the nucleotide 
chain assembly steps during solid-phase synthesis. Yet, if a coupling 
reaction proceeds with no stereospecificity, only 50% of the dimer product 
will have the correct stereochemistry. Thus, for example, in the synthesis 
of an n-mer having chiral phosphorothioate internucleosidic linkages, the 
best theoretical yield of diastereomerically pure n-mer, would be 
1/2.sup.n-1 (&lt;3% for a 6-mer and &lt;1.0% yield for an 8-mer). This 
highlights the importance of avoiding phosphorothioate or other chiral 
centers in oligonucleotide analogs. Use of achiral internucleosidic 
linkages in the construction of nucleotide analogs not only avoids yield 
loss due to unwanted diastereomeric by-products, but also eliminates need 
for complex separation of diastereomers. 
It is therefore an object of the present invention to provide a method for 
synthesis of achiral dithiophosphate oligonucleotide analogs. 
It is a further object of the invention to provide novel 
monohalohydrocarbyl thiophosphoramidites as intermediates for the 
synthesis of achiral analogs of oligonucleotides. 
Yet another object of the invention is the use of tetrazole or other acidic 
pKa nitrogenous compounds to catalyze the production of thiophosphite 
coupled nucleosides by the reaction of nucleoside 
3'-O-thiophosphoramidites with nucleoside 5'-hydroxyl groups. 
In accordance with the foregoing objectives a method is provided for the 
synthesis of intermediates useful for producing achiral phosphorodithioate 
analogs of oligonucleotides. The unprotected 3'-hydroxyl group of a 
nucleoside (hereinafter meaning nucleoside or deoxynucleoside) or 
oligonucleoside (that term hereinafter inclusive of oligonucleotides, 
oligodeoxynucleotides, and analogs thereof having internucleosidic 
linkages other than phosphate) is coupled with a 
halohydrocarbylthiophosphoramidite to provide a nucleoside 
3'-O-hydrocarbylthiophosphoramidite. That product is reacted with a 
nucleoside or an oligonucleoside having an unprotected 5'-hydroxyl group 
and a protected 3'-hydroxyl group in the presence of a weak nitrogenous 
acid having a pKa equal to or greater to the pKa of 1H-tetrazole to 
provide nucleosides coupled through a 3', 5' hydrocarbyl thiophosphite 
linkage. Oxidation of that coupled oligonucleside analog with sulfur 
converts the thiophosphite to a dithioate triester coupled 
oligonucleotide. Deprotection of the 3'-hydroxyl group, removal of the 
hydrocarbyl moiety, and repetition of the synthesis scheme allows 
construction of a nucleoside oligomers having achiral phosphorodithioate 
internucleosidic linkages. 
In a preferred embodiment of the present invention, a 
monochloro-N,N-dialkylaminohydrocarbylthiophosphine is used to form the 
reactive nucleoside 3'-O-hydrocarbylthiophosphoramidite. Most preferred 
mono-chloro-N,N-dialkylaminohydrocarbylthiophosphine intermediates are 
those wherein the N,N-dialkylamino group is N,N-diisopropyl, N,N-dimethyl, 
or morpholino, and the hydrocarbylthio group is methylthio, benzylthio, 
chlorobenzylthio, or dichlorobenzylthio. 
The intermediate nucleoside 3'-O-thiophosphoramidite can be coupled by 
reaction with a nucleoside or oligonucleoside having an unprotected 
5'-hydroxyl group and a protected 3'-hydroxyl group in the presence of 
1H-tetrazole. Oxidation of the coupled oligonucleoside analog with sulfur 
in the presence of a tertiary amine base, preferably pyridine or 
2,6-lutidine, followed by removal of the hydrocarbyl moiety through 
reaction with sulfur nucleophiles such as thiophenol provides dimers or 
oligomers having a phosphorodithioate internucleosidic linkage. 
Deprotection of the 5'-hydroxyl group of the resulting oligomer and 
repetition of the aforedescribed synthesis scheme allows for facile 
synthesis of dithiophosphate coupled oligonucleotide analogs having a 
predetermined oligonucleotide sequence. Thus by using appropriate reaction 
sequences of oligodeoxynucleotide analogs (again, including 
oligodeoxynucleotide analogs) in a predetermined combination of 
phosphorodithioate and, for example, natural phosphate or other linkages 
can be synthesized. 
Oligonucleotide analogs having a phosphorodithioate linkages are of 
potential use for both therapeutic and diagnostic applications. 
Phosphorodithioate linked oligonucleotides are isosteric and isopolar with 
normal phosphodiester linkages and are expected to have other biochemical 
and biophysical properties similar to natural DNA. Such DNA analogs are 
expected to be relatively nuclease resistant and easily derivatized with 
reporter groups, two very significant chemical properties important for 
numerous biochemical and biological applications. Therapeutically, 
phosphorodithioate oligonucleotide analogs can also be used as antisense 
agents directed against foreign or aberrant genetic elements such as 
virally derived nucleic acids or mRNA of oncogenic or other undesired 
genetic elements. Dithiophosphate analogs of ribozymes can also be 
produced that could catalytically cleave mRNA of viral, bacterial, or 
oncogenic origin in addition to mRNA derived from any other undesired 
genetic elements.

DETAILED DESCRIPTION OF THE INVENTION 
This invention provides novel halophosphoramidite intermediates and an 
improved method for preparing 3',5'-dinucleoside hydrocarbylthiophosphite, 
precursors to biologically significant achiral oligonucleotides having 
phosphorodithioate internucleosidic linkages. 
With reference to FIG. 1, there is provided a 
monohalohydrocarbylthiophosphoramidite of Formula I wherein X is halo, 
preferably chloro or bromo, R.sub.1 is a hydrocarbyl radical containing up 
to 10 carbon atoms, and A is a secondary amino group. Phosphoramidite I is 
typically prepared via a dihalophosphoramidite intermediate formed by 
reacting the corresponding phosphorus trihalide with two equivalents of 
the corresponding secondary amine. Conversion of the dihalophosphoramidite 
intermediate to I is accomplished by reacting it with a sodium salt of the 
corresponding thiohydrocarboxide in the presence of aluminum trichloride 
and potassium iodide. 
In Formula I, A is preferably a group of the formula --NR.sub.3 R.sub.4 
wherein R.sub.3 and R.sub.4, taken separately, each represents alkyl, 
aralkyl, cycloalkyl and cycloalkylalkyl containing up to 10 carbon atoms 
or R.sub.3 and R.sub.4, when taken together with the nitrogen atom to 
which they are attached, form a 5' or 6'-membered ring. Secondary means 
for which the group --NR.sub.3 R.sub.4 include a wide variety of saturated 
secondary amines such as dimethylamine, diethylamine, diisopropylamine, 
dibutylamine, methylpropylamine, methylhexylamine, methylcyclopropylamine, 
ethylcyclohexylamine, methylbenzylamine, methylcyclohexylmethylamine, 
butylcyclohexylam:ine, morpholine, thiomorpholine, pyrrolidine, 
piperidine, 2,6-dimethylpiperidine, piperizine and similar saturated 
monocyclic nitrogen heterocycles. Preferably A in Formula I is either 
diisopropylamino or morpholino, both of which groups have been found to 
enhance the stability of the halophosphoramidite of Formula I presumably 
due to the high degree of steric hindrance about the nitrogen atom. The 
compounds of Formula I wherein X is the chloro, and wherein A is either 
diisopropyl or morpholino, are easily purified by distillation and exhibit 
good stability in solution. In the compounds of Formula I, R.sub.1 is 
generally specified herein as a hydrocarbyl radical containing up to 10 
carbon atoms. Such hydrocarbyl radicals include alkyl, aralkyl, cycloalkyl 
and cycloalkylalkyl containing up to 10 carbon atoms. For use in 
accordance with the present invention, the nature of the group R.sub.1 is 
not critical to use of the intermediate. Thus R.sub.1 can be selected from 
the group consisting of C.sub.1 -C.sub.10 alkyl, C.sub.1 -C.sub.10 
alkenyl, benzyl, chlorobenzyl, dichlorobenzyl, methylbenzyl, 
chloromethylbenzyl, and cyclohexyl. Preferred R.sub.1 groups include 
C.sub.1 -C.sub.4 alkyl such as methyl, ethyl, propyl, and butyl, C.sub.3 
-C.sub.1- alkenyl, including allyl, 2-butenyl, benzyl, chlorobenzyl, and 
dichlorobenzyl. Most preferred groups, because of their ability to be 
cleaved from a derivative of the halophosphoramidite, a dinucleoside 
dithiophosphate triester (Formula IV in FIG. 1) are methyl, chlorobenzyl 
and dichlorobenzyl. 
Halothio-phosphoramidite I readily reacts with an unprotected 3'-hydroxyl 
group of a 5'-protected hydroxyl nucleoside such as represented by Formula 
II in FIG. 1. "Nucleoside", as it is used herein to define this invention, 
shall refer to deoxynucleosides, ribonucleosides, and 
deoxyribonucleosides, nucleosides with modified heterocyclic substituents 
at the ring nitrogen atoms, nucleosides with substituted exocyclic groups, 
ring analogs of purines or pyrimidines, and nucleotides having altered 
sugars or N-glycosidic linkages. 
In FIG. 1 G is selected from the group consisting of a hydroxyl protecting 
group, a solid support, a nucleoside, and a nucleotide; R.sub.2 is 
hydrogen (representing a deoxynucleoside) or a protected hydroxyl group; 
R.sub.5 is hydroxyl, protected hydroxyl or a covalently bound solid 
support. B is an amino protected base including purine, pyrimidine, 
purines and pyrimidines modified by exocyclic substituents, ring analogs 
of purines or pyrimidines, and purines or pyrimidines substituted at ring 
nitrogen atoms. Most preferably, "B" is an amino-protected base selected 
from the group consisting of adenine, thymine, cytosine, guanine, uracil, 
and hypoxanthine. 
"Amino-protected" as used herein indicates that the exocyclic amino groups 
on said bases are covalently bonded to a readily removable amino 
protecting group. Such groups, means for their application and removal, 
and their properties under various reaction conditions are known in the 
art. The benzoyl group is commonly used on adenine and cytosine while the 
isobutyl group is often used to protect the exocyclic amino group of 
guanine. The nature of amino protecting groups are not critical so long as 
they are stable under the reaction conditions of the present process and 
removable from the product oligonucleoside analogs under conditions not 
causing degradation of the product itself. 
Suitable hydroxyl protecting groups include those covalently bound groups 
which can be coupled and removed under art-recognized reaction conditions. 
Suitable hydroxyl protecting groups include trityl, methoxytrityl, 
dimethoxytrityl, dialkylphosphate, pivaloyl, isobutyloxycarbonyl, 
t-butyldimethylcylo, tetrahydropyranyl, trialkylsialyl and N-acetyl. As 
with the amino protecting groups discussed above, the nature of the 
hydroxyl protecting group utilized to protect the 5'-hydroxyl (or the 
2'-hydroxyl in the case of nucleosides) is not critical so long as the 
protecting group is stable under the conditions of the synthesis process, 
yet can be removed following completion of the coupling reactions under 
conditions which do not effect degradation of the coupled oligomer. 
As discussed above in FIG. 1, both G and R.sub.5 can represent a solid 
support. Many solid supports are known in the art and include polymers, 
silica or silica gel having functional groups capable of covalent bonding 
to the 5' or 3' positions. It should be understood that the group 
represented by G or R.sub.5 include art-recognized covalent linkers for 
covalent attachment to solid supports such as long-chain alkyl amino 
groups or other easily derivatized chemical coupling functionalities. 
Where the group G or R.sub.5 in FIG. 1 is a nucleoside or oligonucleoside, 
it is understood that such entities are bonded to the nucleoside sugar 
through natural phosphate diester bonds (and thus are nucleotides) or by 
other internucleosidic linkages reported in the art, including 
methylphosphonate, carbonate, oxyacetamide, carbamate, thiophosphate and 
dithiophosphate internucleosidic linkages. 
Further with reference to FIG. 1, 5'-protected nucleoside II is reacted 
with halohydrocarbylthiophosphoramidite I to form the nucleoside 
3'-O-hydrocarbylthiophosphoramidite III. That reaction is typically 
conducted at room temperature utilizing 2 to 3 fold molar excess of 
halophosphoramidite I in the presence of a 2 to 4 fold molar excess of a 
tertiary amine base. The nucleoside 3'-O-hydrocarbylthiophosphoramidite 
III can be reacted with a nucleoside, for example, a deoxynucleoside of 
Formula II wherein B is a purine or pyrimidine base, R.sub.2 is hydrogen, 
R.sub.5 is protected hydroxyl or solid support and G is hydrogen, to 
provide a 3'-O-5'-O-dinucleoside hydrocarbylthiophosphite which is 
subjected to sulfur oxidation to provide the corresponding 3', 
5'-dideoxynucleoside dithiophophpate triester illustrated as Formula IV in 
FIG. 1. 
Coupling of the deoxynucleoside (R.sub.2 =H) 3'-O-thiophosphoramidite III 
with a 3'-protected hydroxyl-5'-hydroxyl nucleoside can be accomplished in 
polar aprotic organic solvents, preferably under anhydrous conditions. 
Anhydrous acetonitrile is a preferred solvent for that coupling reaction. 
It has been found that the coupling reaction can be effected 
advantageously in the presence of a nitrogen acid having a pKa equivalent 
to or greater than tetrazole. The pKa of tetrazole is 4.9. Preferred 
agents for activating the coupling reaction include nitrogen acids having 
a pKa between about 4.9 and about 6.0, more preferably between about 4.9 
and about 5.5. The term "nitrogen acid" as used herein is an 
art-recognized term which refers to compounds having a nitrogen atom 
bonded to a proton which can disassociate with said nitrogen atom and 
impart acidity to solutions of said compounds. Such compounds include 
salts, for example, the hydrohalide salts, of tertiary amines including 
alkylanilines such as dimethylaniline, diisopropylaniline, 
methylethylaniline, methyldiphenylamine, and pyridine. Nitrogen acids also 
include nitrogen heterocyclic compounds such as tetrazoles, imidazoles, 
nitroimidazoles, benzimidazoles, triazoles such as 3,5-dichloro-1,2,4 
triazole, and benzotriazoles. Most preferred of the nitrogen acids 
suitable for use in accordance with this invention is tetrazole. 
Recent literature describing the preparation of oligonucleotides containing 
phosphorodithioate linkages (D. Brill and M. H. Crauthers, Aug. 1988) 
teach that activating agents more acidic than tetrazole must be used 
because the coupling reaction stops at the thiophosphoryl-tetrazolide 
intermediate. The use of more acidic coupling activators as taught by 
Brill and Crauthers enhances the probability of undesired side reactions 
such as premature deblocking of protected hydroxyl and amino groups. 
Subsequent sulfurization of the dinucleoside thiophosphite intermediate to 
produce a corresponding dinucleoside dithiophosphate triester IV is 
carried out typically using elemental sulfur in the presence of an amine 
base. Subsequent treatment of IV with a source of nucleophilic sulfur such 
as thiophenol results in removal of the hydrocarbyl group (R.sub.1) of 
Formula IV and production of the corresponding achiral 3',5' dinucleoside 
phosphorodithioate (not shown). The preparation of nucleoside oligomers 
having phosphorodithioate internucleosidic linkages using the method of 
the present invention in a solid phase synthesis scheme is illustrated in 
FIG. 2. 
Chloro-N,N-diisopropylaminothiomethoxyphosphine Ia is reacted with 
5'-O-dimethoxytrityl nucleoside IIa to form a 5'-O-dimethoxytrityl 
nucleoside-3'-thiomethoxyphosphoramidite IIIa. Where the targeted 
nucleoside oligomer comprises more than one nucleoside, for example, an 
oligomer IX wherein B in each coupled nucleoside is different, it is 
advantageous to prepare in advance of initiating the coupling reaction 
scheme, phosphoramidite intermediates IIIa corresponding to each of the 
component nucleosides. 
Nucleoside phosphoramidite IIIa is reacted with nucleoside V covalently 
bound to the surface of a solid support through the 3'-hydroxyl moiety. 
The support is a silica gel, a controlled pore glass, polystyrene, or 
other art-recognized oligonucleotide support having surface functional 
groups capable of forming covalent bonds to the nucleoside through its 
3'-hydroxy group. The nature of the surface functional groups used for 
nucleoside covalent bonding is such that the nucleoside can be released 
from the solid phase surface under reaction conditions selected to cleave 
the connecting covalent bonds. The reaction between support bond 
nucleoside V and intermediate nucleoside phosphoramidite IIIa in the 
presence of tetrazole provides 3',5'-dinucleoside methylthiophosphite VI. 
Oxidation of VI with sulfur converts the thiophosphite internucleosidic 
linkage to a dithiophosphate triester linkage VII. In addition, a 
so-called capping step is implemented to block any extant 5'-hydroxyl 
groups on support bound oligonucleoside V that failed to couple with 
nucleoside phosphoramidite IIIa. The capping step ensures that subsequent 
reaction cycles will only propagate oligomer chains having the desired 
nucleoside sequence. Preferred capping agents include acylating reagents 
such as anhydrides that acylate available unreacted 5' hydroxyl groups. A 
preferred capping agent is acetic anhydride, typically used in the 
presence of 4-dimethylaminopyridine. 
Detritylation of the support bound dimer VII utilizing a mild acid such as 
acetic acid, deblocks the 5'-hydroxyl group of the terminal nucleoside. 
The support bound deblocked dimer can then be subjected to additional 
nucleoside coupling cycles, each initiated by reaction of the support 
bound oligomer with a nucleoside thiophosphoramidite IIIa corresponding to 
the desired nucleoside oligomer sequence. Thus one repetition of the cycle 
produces support bond trimer IX. Additional repetitions of the coupling 
cycle can be implemented as necessary to produce the targeted oligomer. As 
desired, either thiophosphoramidite or phosphoramidite coupling reagents 
can be utilized to respectively couple nucleosides by dithiophosphate 
triester linkages or phosphodiester linkages. Treatment of the supported 
oligomer with thiophenol in the presence of a tertiary amine base converts 
the internucleosidic dithiophosphate triester linkages to the 
corresponding phosphorodithioate linkages as shown in Formula X. Isolation 
of the product oligonucleoside phosphorodithioate analog is accomplished 
by treatment first to remove hydroxyl and amino protecting groups and 
secondly to cleave the oligomer from the solid support. Purification of 
the product can be accomplished using art-recognized DNA purification 
techniques including gel electrophoresis, high pressure liquid 
chromatography, or affinity chromatography. 
The methods and intermediates in accordance with the present invention are 
further illustrated by the following examples. 
EXAMPLE 1 
The overall synthesis of the thiophosphoramidite intermediates 2 and 4, and 
deoxythymidine dithiophosphate 6, is outlined in FIG. 3. 
Dichloro-N,N-diisopropylaminophosphine 1 was prepared by the reaction of 
phosphorus trichloride with diisopropylamine (2 equiv). Distillation 
(56.degree.-59.degree. C./0.5 mmHg) afforded pure phosphine as shown by 
.sup.31 P NMR (.delta.ppm in benzene-d.sub.6, downfield relative to 
external 85% H.sub.3 PO.sub.4 :169.0). 
The synthesis of monochloro-N, N-diisopropylaminothiomethoxyphosphine 2 was 
accomplished by reacting dichloro-N,N-diisopropylaminophosphine 1 with 
sodium thiomethoxide (1 equiv) in the presence of aluminum trichloride and 
potassium iodide. The best yield of the reactive phosphine 2 was obtained 
by the following procedure. A 50 ml addition funnel was charged with a 
suspension of sodium thiomethoxide (22.8 mmol) and catalytic amount (2.28 
mmol) of potassium iodide in 40 ml of anhydrous dichloromethane. This 
suspension was added dropwise over a period of 10 hours at -65.degree. C. 
to a magnetically stirred solution of 
dichloro-N,N-diisopropylaminophosphine 1 (24.7 mmol) and a catalytic 
amount of aluminum trichloride (0.5 mmol) in 20 ml of anhydrous 
dichloromethane. After addition of sodium thiomethoxide the resulting 
suspension was allowed to stir for 3 hours at -35.degree. C., then 5 hours 
at -20.degree. C. and finally 10 hours at room temperature. The reaction 
mixture was then vacuum filtered and the sodium chloride salt was washed 
with 100 ml anhydrous ether. The filtrate was evaporated under a dry 
nitrogen atmosphere and reduced pressure (100 mmHg) at room temperature. 
The purity of the crude residue was checked by .sup.1 H and .sup.31 P NMR 
(benzene-d.sub.6 : chloro-N,N-diisopropylaminothiomethoxyphosphine 2 
.delta..sup.31 P 168.0 ppm; N,N-diisopropylaminodithiomethoxyphosphine 
impurity 3 .delta.118.2 ppm). The above procedure typically provides 2 in 
about 95% yield and less than 5% yield of 3 and the crude reaction product 
used for further reaction without purification. 
Crude chloro-N,N-diisopropylaminothiomethoxyphosphine 2 from the above 
procedure can be stored at -18.degree. C. under an inert, dry atmosphere 
for at least several months without any decomposition. In contrast, the 
N,N-diisopropylaminodithiomethoxyphosphine 3 undergoes a Michael-Arbuzov 
reaction to the extent of roughly 50% after 6 weeks at -18.degree. C. One 
problem manifested by all monofunctional phosphitylating agents including 
chloro-N,N-diisopropylaminothiomethoxyphosphine 2 is its sensitivity 
towards hydrolysis and air oxidation. 
Chloro-N,N-diisopropylaminothiomethoxyphosphine 2 was used to prepare the 
deoxynucleoside thiomethoxyphosphoramidite 4. Excess 2 (5.52 mmol) was 
added to a mixture of diisopropylethylamine (7.4 mmol), 
5'-O-dimethoxytrityl thymidine (1.84 mmol) in 4 ml dichloromethane at room 
temperature. The complete reaction course was monitored by tlc (silica 
gel) and by .sup.31 P NM spectroscopy. 
After completion of the reaction the mixture was transferred to a 
separatory funnel and diluted with ethyl acetate. The solution was washed 
three times with a saturated NaHCO.sub.3 solution (25 ml). This was 
followed by an additional washing with saturated NaCl aqueous solution (25 
ml; 3 times). The washing steps were performed under nitrogen at room 
temperature within 15 minutes. The organic layer was dried over magnesium 
sulfate overnight and the solvent was evaporated to a foam under reduced 
pressure. The residue was then taken up in a few ml of dichloromethane and 
precipitated with 500 ml of ether/n-hexane mixture (-78.degree. C.). This 
procedure provides 
N,N-diisopropylamino3'-O-(5'-O-(dimethoxytrityl)thymidine) 
thiomethoxyphosphine 4 in a 55:45 diastereomeric mixture as indicated by 
the .sup.31 P NMR spectrum of 4 showing two signals at 164.85 and 163.14 
ppm corresponding to a diastereomeric mixture of the thiophosphoramidite. 
The Cl (70 ev) MS analogs of 4 shows a prominent pseudo-molecular ion peak 
at m/z 722 (M+H).sup.+ . Additional intense ions are also observed at m/z 
692 (M-2xCH.sub.3 +H).sup.+, 674 (M-SCH.sub.3).sup.+ and 596 
(M-1-thyminyl).sup.+. .sup.1 H NMR (benzene-d.sub.6,.delta.ppm) 1.0 (12H, 
d, (CH.sub.3).sub.2 CH).sub.2 N), 1.55 (3H, s, thymine-CH.sub.3), 2.27 
(3H, d, CH.sub.3 S), 3.30 (6H, s, CH.sub.3 O), 3.52 (2H, m, 5',5"), 4.20 
(1H, br. s, H4'), 4.78 (1H, br. s, H3'), 6.50 (1H, br. m, H1'), 7.20 (13H, 
m, aromatic). 
In addition to the major peaks assigned to 4, there are some minor .sup.31 
P peaks at 146.3 ppm and 13.2 ppm, which are assigned to the 3'-3' 
nucleoside dimer and a phosphoamidous acid, respectively. 
Thiophosphoramidite 4 was coupled with methanol to test the feasibility of 
the reaction using 1H-tetrazole as a catalyst. The reaction was monitored 
by .sup.31 P NMR spectroscopy. Addition of excess methanol and the acid 
catalyst 1H-tetrazole (0.26 mmol) to a benzene solution of 4 (0.035 mmol) 
at room temperature resulted in the complete disappearance over several 
hours of the two .sup.31 P signals of 4 at 164.85 and 163.14 ppm. The 
signals are replaced by two new signals at 193.0 and 192.8 ppm (55:45 
ratio), which are assigned to a R.sub.p and S.sub.p diastereomeric mixture 
of 3'-O-methoxythiomethoxyphosphine derivative 5 of 
5'-O-(dimethoxytrityl)thymidine. 
Oxidation of 5 by sulfur in pyridine generated the dithiophosphate triester 
6. .sup.31 P NMR spectra indicated that the sulfurization reaction was 
rapid. After 10 minutes at ambient temperatures only about 5% of unreacted 
5 remained. The R.sub.p and S.sub.p diastereomeric mixture of 
dithiophosphate triesters 6 appears as a set of sharp .sup.31 P NMR 
resonances at 92.62 and 92.11 ppm (benzene-d.sub.6). This is similar to 
the .sup.31 P chemical shift for authentic samples of dimethyl and diethyl 
thiophosphoric acids (.sup.31 P (benzene d.sub.6), 90.05 and 90.67 ppm, 
respectively). Additional .sup.31 P signals are observed at 70.97, 68.97, 
68.38 ppm, and a number of peaks in the phosphate ester region around 0.0 
ppm, the latter presumably arising from desulfurization. Dithiophophpate 
triester 6 was relatively stable in solvents even at elevated 
temperatures. 
EXAMPLE 2 
Thiophosphoramidite 4 was prepared as illustrated in FIG. 3 followed the 
procedures set forth in Example 1. Thiophosphoramidite 4 was coupled with 
3'-O-(t-butyldimethylsilyl) deoxythymidine using 1H-tetrazole as an acid 
catalyst. The reaction was monitored by .sup.31 P NMR spectroscopy. 
Addition of excess 3'-O-(t-butyldimethylsilyl)deoxythymidine and 1H 
tetrazole (0.26 mmol) to a benzene/acetonitrile solution (1:3) of 4 (0.035 
mmol) at room temperature resulted in the complete disappearance over 
several hours of the two .sup.31 P signals at 164.85 and 163.14 ppm, and 
their replacement by two new signals at 190.7 and 191.3 ppm (55:45 ratio), 
which are assigned to a R.sub.p and S.sub.p diastereomeric mixture of 
3'-O-(t-butyldimethylsilyl)-5'-O-deoxythymidinethiomethoxyphosphine 
derivative of 5'-O-(dimethoxytrityl) thymidine. The best yield of the 
thiophosphite was obtained by reaction for 60 minutes. 
After sulfurization with a large excess of sulfur in pyridine the 
3',5'-dideoxythymidine methyl phosphorodithioate triester were observed as 
two .sup.31 P signals at 95.7 and 95.4 ppm (benzene-d.sub.6). The acetyl 
protected dithioate triester .sup.31 P signals appear at 96.8 and 97.2 ppm 
(benzene-d.sub.6). The sulfurization reaction was rapid, and after 10 min. 
at ambient temperature only about 5% of the unreacted dinucleoside 
thiophosphite was still present. 
S-demethylation of 3',5'-dideoxythymidine methyl phosphorodithioate was 
accomplished by reaction with thiophenol/diisopropylethyl-amine for 40 
hours at room temperature. Removal of the S-protecting methyl group yields 
the achiral 3',5'-dideoxythymidine phosphorodithioate 114.9 ppm). 
EXAMPLE 3 
Synthesis of thiophosphoramidites is performed according to the following 
procedure adapted from Brill et al., J. Am. Chem. Soc.. 111, pp.2321-2322 
(1989). Either dipyrrolidinylchlorophosphine or 
bis-(dimethylamino)chlorophosphine (0.6 mmol) is added to a 5'-O-protected 
deoxynucleoside (0.5 mmol) in 4 mL of acetonitrile:triethylamine(2:1, 
v/v). Immediately a precipitate appears. After 5 min, either 
4-chlorobenzyl- or 2,4-dichlorobenzylmercaptan (1 mmol) is added, and the 
reaction mixture is evaporated to a gum. CH.sub.2 Cl.sub.2 : ethyl 
acetate: TEA (47.5:47.5:5.0) was used to dissolve 0.5 mmol of the crude 
product. The crude thiophosphoramidite is purified by gravity or flash 
chromatography using a column of silica gel (320-400 mesh) 5 cm.times.1 
cm, with the above solvent as eluent. 1.5 ml fractions are collected. The 
product is observed on TLC as the fastest running compound. Yield was 
about 70-80% as monitored by P-31 NMR. The solvent of the purified 
fraction was evaporated under a stream of nitrogen and the last 
approximately 3 ml of liquid were triturated into n-pentane cooled to 
0.degree. C. Product yield can be enhanced by running the synthesis in an 
inert atmosphere dry box. 
EXAMPLE 4 
A tetrameric oligonucleotide analog d(Ap(S.sub.2)GCT) in which the AG 
phosphate linkage is replaced with a dithiophosphate linkage and a d(C 
Tp(S.sub.2) Tp(S.sub.2) G C T A C T C C Cp(S.sub.2) Cp(S.sub.2) A T) 
corresponding to an antisense oligonucleotide targeted to the arc oncogene 
were prepared using solid phase synthesis procedures and the reactions 
exemplified in Examples 1-3 above. 
Synthesis of both oligonucleotide analogs was accomplished utilizing the 
respective thiophosphoramidites (as prepared in Example 3) with a 50-fold 
excess of tetrazole. Coupling was performed twice to ensure high yields 
(greater than 98%). A capping acylation of unreacted 5'-hydroxy 
oligonucleosides, followed by detritylation and oxidation with 5% sulfur 
in pyridine: carbon disulfide completed the reaction cycle. Each double 
coupled reaction cycle took approximately 10 minutes. The product 
oligodeoxynucleotide dithiophosphite triester intermediates were reacted 
with 5% elemental sulfur in 2,6- lutidine/CS.sub.2 for 5-7 minutes 
followed by I.sub.2 oxidation. Repetitions of this cycle utilizing the 
respective thiophosphoramidites provided precursor dithiophosphite 
triester intermediates for the above designated oligonucleotide analogs. 
These dithiophosphite oligonucleotide analogs were reacted with 
thiopheno:: triethylamine: dioxane (1: 1: 2; v/v/v) for 24 hours followed 
by reaction with concentrated ammonium hydroxide for 15 hours to convert 
the internucleosidic dithiophosphite triester linkages to the 
corresponding phosphorodithioate linkages. The product analogs were 
purified to homogeneity by standard procedures (polyacrylamide gel 
electrophoresis and reverse phase HPLC). 
The overall yield of d(Ap(S.sub.2)GCT) was about 80% and that for d(C 
Tp(S.sub.2) Tp(S.sub.2) G C T A C T C C Cp(S.sub.2) CP(S.sub.2) A T) was 
&lt;10%.