IL110600A - Nucleoside and polynucleotide phosphorodithioate compounds and their production - Google Patents

Description

Nucleoside and polynucleotide phosphorodithioale compounds and their production UNIVERSITY PATENTS, INC.

C: '. IS9/8 1 10600/2 This is a divisional application of Israel Patent Application No. 90359 which relates to phosphorus compounds which are particularly useful in the production of polynucleotides having analogs attached to phosphorus.

The invention described and claimed in Israel Patent No. 90359 relates to nucleoside thiophosphoramidite, polynucleotide dithioate phosphoramidi te and polynucleotide phosphor thioamidate phosphoramidite compounds as well as the processes v/hei:eby these compounds can be used for synthesizing new mononucleotides and polynucleotides having phosphorodi h io e , phosphorothioamida te , phosphoro hio tries ers , and phosphoro hioa e i n ternuc leo tide linkages. These new mononucleotides and oligonucleotides can be used for many biological, therapeutic, and diagnostic applications. Potential therapeutic applications include treating tumors, viral infections and bacterial infections. Additionally, these compounds can be used to deliver metal ions, toxins, intercalating agents and other reagents that alter the biochemical reactivity of polynucleotides and proteins to specific sites in cells and tissues. These compounds can also be used for diagnostic purposes. By attaching fluorescent or other chemically reactive reagents, antigens, antibodies, proteins, and metal ions to these compounds, they can be used for diagnosing diseases and the normal and abnormal biochemistry of cells, tissues, and body fluids such as blood and urine. There are also many uses in modern biology and chemistry as well. For example, these compounds can be used to develop improved methods for sequencing and cutting DNA, for imaging in X-ray crystallography, NMR , and electron microscopy, and for studying enzymic reactions.

High yielding methodologies are currently available for the rapid synthesis of sequence defined polynucleotides having the natural internucleo tide linkage (M. H. Caruthers, Science 230, 281-285, 1985; M. H . Caruthers and S. L. Beaucage, U.S. Patent 4,415,732; M. II. Caruthers and M. D. Matteucci, U.S. Patent 4,458,066). An important step in this process is oxidation of the intermediate phosphite triester to the naturally occurring phosphate triester with aqueous iodine. These phosphite triesters can also be oxidized under anhydrous conditions with amines or ammonia and iodine to yield variable reported amounts of phosphoramidates or with sulfur to yield phosphorothioates (B. Uznanski, M. Koziolkiewicz , W. J. Stec, G. Zon, K. Shinozuka, and L. Marzili, Chemica Scripta 26, 221,224, 1986; M. J. Nemer and K. K. Ogilvie, Tetrahedron Lett. 2_1 , 4149-4152 , 1980). Other methods employing H-phosphonate internucleotide linkages can also be used to synthesize phosphoramidates (B. C. Froehler, Tetrahedron Lett. 27 , 5565-5568, 1986). Unfortunately, none of these procedures can be used to synthesize polynucleotides containing the phosphorodithioate or the phosphorothioamidate internucleotide linkages.

The production of uridine 2 ',3 '-cyclic phosphorodithioate is described in the literature (F. Eckstein, J. Am. Chem. Soc. 92 , 4718-4732 , 1970). Unfortunately, the process cannot be used to 110600/ 3 .synthesize deoxynucleoside phosphorodi thioa tes or nucleoside phosphorodi thio tes useful for synthesizing polynucleotides containing the dithioate linkage. The procedure also yields a mixture of mononucleo ides having phosphorodi thioa e and phosphorothioate moieties. Additionally the yield of uridine 2' ,3'-cyclic phosphorodi thioa te in only 28% and the acidity of P2S5 anc tlie h^'"1 temper ures used in the synthesis of the cyclic phosphorodithioate would preclude the use of this procedure with protected deoxyadenosine which would undergo depur ina t ion .

Similarly adenosine cyclic 3 ' , 5 ' -phosphorodithioate can be synthesized by treating sui ably protected adenosine with 4 - ni trophenylphosphoranil idoc lor ido hioa te followed by cyclization with potassium t-butoxide and conversion to the dithioate in a reaction with sodium hydride/carbondisulf ide (J. Boraniak and W. Stec, J. Chem. Soc. Trans. I, 1645,1987) . Un ortunately these reaction conditions and the low synthesis yields preclude the use of this chemistry for synthesizing oligonucleotides having phosphorodi thioa te linkages.

The present invention provides oligonucleotides comprising at least two nucleotide moieties having an intemucleotide linkage between said moieties wherein at least one intemucleotide linkage is a phosphorodithioate linkage 0f the structure .

I RS - P = S I wherein R is H or a blocking group (as herein defined) .

In general, the compounds according to the present invention can be represented by the general formula wherein B is a nucleoside or deoxynuclcoside base (as herein defined); A is H, halo, OR2, SR2, N(R-,)2 or azido where R2 is independently a blocking group (as herein defined); R, and R3 are blocking groups (as herein defined) or one of R, and R3 may also represent a group -P(OR NR'6R'7 where R., is a blocking group (as herein defined) and R'6 and R'7 are the same or different substituted (as herein exemplified) or unsubstituted ary 1, alkyl or aralkyl moieties; R5 is a blocking group (as herein defined). 110600/2 As used herein the symbols for nucleotides and polynucleotides are according to the IUPAC-IUB Commission of Biochemical Nomenclature Recommendations (Biochem istry 9, 4022, 1970). Several chemical terms as used in this invention are further defined as follows : These definitions apply unless, in special cases, these terms are defined di fferently. alky 1 - a non-cyclic branched or unbranched hydrocarbon radical having from 1 to 20 (preferably 1 to 12) carbon atoms, optionally substituted by hydroxy, alkoxy, mercapto, alkylthio, amino, alkylamino or dialkylamino. aryl - an organic radical derived from an aromatic or heteroaromatic compound by the removal of one hydrogen atom. This radical can contain one or more hetcroatoms as part of the aromatic hydrocarbon ring system and may be optionally substituted by hydroxy, alkoxy, mercapto, alkylthio, ami no, alkylamino or dialkylamino. aralkyl - an organic radical in which one or more aryl groups, preferably 1 to 3, are substituted for hydrogen atoms of an alkyl radical. 110600/2 P phosphorodithioa te in ter nuc leo tide linkage - an internucleotide linkage having the general formula S'-nucleoside-O-PS^-O-nucleoside-B' which can be illustrated with the following structure where D and Λ are as defined previous- phosphorothioa linkage - an internucleotide linkage having the general formula 5 ' -nucleoside-0-POS-0-nucleoside-31 - 9 - which can be llustrated with the following structure where B and Λ are as defined previous- ~s phosphoro hioamida te in ernucleo ide linkage -an in e nucleo i.de linkage ha ing the general formula 5 ' -nucleos ide-0 -PSNI]Rr -0 -nucleos ide- 3 ' 6 and 5 ' - nucleos ide-0-PSMRr R-,-0 -nucleos ide-3 ' b / which can be illustrated with the following structures where B , Λ , Rr and R-. are as previously phosphoromida te in ernucleo tide linkage - an in ternucleo tide linkage having the general formulae 5 ' -nucleoside-0-PONIIRr ~0-nucleoside-31 6 and 5' ' -nucleoside-0-PONR,, R-,-0- nucleos ide-31 0 / which can be illustrated with the following structures where B,A,Rr and R., are as previously defined . - - phosphoro thio tries ter in ernucleo ide linkage - an in te nucleo ide linkage having the general formulae 5 ' -nucleoside-O-PSOR^-O-nucleoside-3 ' which can be illustrated with the following structure where 13 , Λ , and II Λ are as previously defined. ■ Amines from which the substituent group W can be derived include a wide variety of primary amines such as e thy larnine , e thy larnine , propy larnine , isopropyla.mine , aniline, cy clohexy larnine , benzy larnine , polycyclic amines containing up to 20 carbons, heteroatom substituted aryl or alkylamines having up to ten he teroa toins , preferably oxygen, sulfur nitrogen or halogen, and similar primary amines containing up to 20 carbon atoms. Amines from which the substituent group X can be derived include a wide variety of secondary amines such as dime thy larnine , die thy larnine , diisopropy larnine , dibutylamine , ruethylpropylamine , rue thylhexy larnine , me thy Icyclopropy larnine , ethy Icyclohexy larnine , me thy Ibenzy arnine , me thy Icyclohexy lme thy larnine , bu ty lcyclohexylamine , inorpholine, thiomorpholine , pyrrolidine, piperidine, 2 , 6-dimethylpiperidine , piperazine, and heteroatom substituted alkyl or aryl secondary amines containing up to 20 carbon atoms and ten heteroatoms from the group consisting of sulfur, oxygen, nitrogen and halogens.

- - The nucleoside and deoxynucleoside bases represented by B in the above formulae are well known and include purines, e.g., adenine, hypoxanthine and guanine, and pyiimidines, e.g., cytosine, uracil and thymine.

The blocking groups represented by R,, R, and R3 in the above formulae are moieties conventionally used to prevent the compounds from undergoing unwanted chemical reactions by "blocking" a reactive site on the molecule, e.g. a reactive hydroxyl (-OH) group. Such blocking grou ps include t ri phenyl methyl (trily I), p-a n isy Id i phenyl methyl (methoxy-(n'(yl), di-p-anisylphenylmethyl (dimelhoxylrityl), pivalyl, acetyl, 4-methoxytet ahydropy ra n-4-yl, tet r h yd ropy rany I, phenoxy acetyl, isobulyloxycarbonyl, i-bulyldimethylsilyl, triisopropylsilyl, alkyl or aryl c rbonyl, and similar blocking groups well known in the art.

The general reaction scheme A for synthesizing compound la and II is shown in (lie following overview:  The preferred reaction scheme A is represented llows: P X u cx. 14 wherein R^ , R^ , B, Λ, X, Z, L and Y are as previously defined; and M is sulfur single bonded to phosphorus and to R_ where RD is a heteroatom substituted or unsubsti tuted alkyl , aryl, aralkyl, cycloalkyl, cycloalkylalkyl , alkenyl, cycloalkeny1 , aralkenyl, alkynyl, aralkynyl or cycloalkynyl . Coinpounds VIII. and Villa are those in which phosphorus is linked through a single bond to Y and through a double bond to Z or L. Thus, it can be seen that compounds VIII and Villa are a subset of compounds II. Likewise, compounds V and Va are a subset of compounds la.

The process of reaction scheme Λ involves condensation of Ilia with IVa , which preferably is bis (dime thylamino) chlorophosphine or dipyrrolidinylchlorophosphine , to yield IXa in the presence of triethy lamine . Further addition of a mercaptan, which preferably is 2,4-dichlorobenzylmercaptan , in the presence of trieth lamine hydrochloride generated in the first step leads to the conversion of IXa to Va . Table 1 lists the 31 P-NM characterization data for a series of Va derivatives where the nucleoside base (B) , amine functionality (X), and mercaptan (M) are altered in a systematic manner. Reaction of Va with Via and an activator (e.g., tetrazole, 5-subs ituted tetrazoles and substituted triazoles, alkylammonium salts, arylalkylammonium salts, substituted and unsubstituted pyridinium salts of tetraf luoroborate , and substituted and unsubstituted pyridinium and imidazolium salts of acids, 5-substituted tetrazoles, halogenated carboxylic acids and N-hydroxybenzotriazole) yields Vila, the dinucleoside S- (2 , 4-dichlorobenzy 1 ) phosphite, which can be preferably oxidized with sulfur to yield Villa, the dinucleoside phosphorodithioate triester with P(Y,Z). Of course oxidation with t-butylperoxide yields the 15 corresponding dinucleoside phosphorothioate triester P (Y,L) · Table 1. 31P-NMR Characterization of Deoxynucleoside Phosphorothioamidites (Va) Base Amine Mercaptan 31p-NMRa (B) (X) (M) T pyrrolidinyl 2 , 4-dichlorobenzyl 164.8;161.8 T pyrrolidiny 1 4-chlorobenzyl 164.2;161.0 T dimethy lamino 4-chlorobenzyl 172.3;170.5 T dimethylamino 2, 4-dichlorobenzyl 172.1?170.4 ,Dz pyrrolidinyl 2 , 4 -dichlorobenzyl 165.1;162.6 ,Bz pyrrolidiny 1 4-chlorobenzyl 161.8; 159.9 ,Bz dime thy lamino 4-chlorobenzyl 171.9;170.7 ,Bz dime hylamino 2 , -dichlorobenzyl 172.0;171.0 Bz pyrrolidinyl 2 , 4 -dichlorobenzyl 163.8 162.7 Bz pyrrolidinyl 4-chlorobenzyl 163.5 162.3 Bz dimeth lamino 4-chlorobenzyl 171.8 170.9 Bz dimethyla ino 2 , 4 -dichlorobenzyl 171.7 170.9 pyrrolidinyl 2 , 4-dichlorobenzyl 163.9;160.9 ,iB pyrrolidinyl 4-chlorobenzyl 163.4;161.6 .iB dimethylamino 4-chlorobenzyl 171.5;169.5 .iB dimethylamino 2 , -dichlorobenzyl 171.9 169.6 P-N R were recorded in CDC1-. on a Brucker WM-250 Bz with 85% aqueous H-PO. as external standard. T, C , Bz iB A , and G refer to thymine, N-benzoylcy tosine , N-benzoy ladenine , and N-isobutyrylguanine respectively; R^ is dimethoxytrity 1 ; A is hydrogen.

A second general reaction scheme for synthesizing compounds la and II, scheme B, is shown in the following overview: 16  The preferred reaction scheme B is represented llows: 18 Thus it can be seen that the processes of reaction schemes Λ and B are identical except for the use of two different reagents, IVa or IVb, in order to generate V and Va. Reagent IVa is a bis (secondary amino) chlorophosphine whereas IVb is a bis (secondary amino) mercaptylphosphine . The use of IVa is a more general reaction leading to V and Va as these bis (secondary amino) chlorophosphines are more easily purified by distillation. Of course the use of IVa generates an intermediate diamidite, IX and IXa, to which the mercaptan is added to form V and Va . The use of IVb, leading directly to V and Va , is restricted to compounds IVb where the thiodiamidite can be purified by crystallization or distillation without decomposition. The process of reaction scheme B involves condensation of Ilia with IVb which is 4-chlorobenzylmercaptyl-bis (diisopropy 1amino) phosphi-ne to yield Va with tetrazole as catalyst. Reaction of Va with Via and an activator (e.g., 5-substi u ted tetrazoles and substituted triazoles, alky lammonium salts, arylalky lammonium salts, substituted and unsubstituted pyridinium salts of tetraf luoroborate , and substituted and unsubstituted pyridinium and imidazolium salts of acids, 5-substituted tetrazoles, halogenated carboxylic acids and N-hydroxybenzotriazole) yields Vila, the dinucleoside 5- ( -chlorobenzyl) phosphite , which can be preferably oxidized with sulfur to yield Villa, the dinucleoside phosphorodithioate triester with P(Y, Z) . Of course oxidation with t-butylperoxide yields the corresponding dinucleoside phosphorothioate triester, P (Y, L) . Activators that are more acidic than tetrazole, such as certain 5-substituted tetrazoles (e.g. -nitrophenyl) tetrazole) and pyridinium tetraf luoroborate , can be used with success to 19 activate Va . Certain side reactions, however, can lead to reductions in yields of the correct product. Λ third reaction scheme, scheme C, was also discovered for the purpose of synthesizing compound II. The general reaction scheme C for synthesizing Compound II is as follows: 20 The preferred reaction scheme C is represented 21 wherein R, , B, Λ, X, W, Z, Y, and V are as previously defined and Q is H. Compounds XII and Xlla are those in which Z is sulfur double bonded to phosphorus plus one other substituent from the group of substituents V, W X and Y which are single bonded to phosphorus. These are derived from XI or XIa. Compounds XII and Xlla can also be L which is oxygen double bonded to phosphorus plus Y which is single bonded to phosphorus. These are derived from XI or XIa.

The process of reaction scheme C involves synthesis of IXa from a protected nucleoside and a bis (secondary amino) chlorophosphine and then condensation with Via to yield Xa. Reaction of Xa with II2 S and an activator such as tetrazole .yields the dinucleoside Il-phosphonothioate , XIa, which can be chemically converted by oxidation with sulfur to dinucleoside phosphorodithioates, P (Z,Y) ; by oxidation with iodine in the presence of amines to phosphorothioamidates , P (Z, W or X) ; by alkylation of the corresponding dinucleoside phosphorodithioate to phosphorodi hioate triesters, P (Z,Y) ; by oxidation with iodine in the presence of alcohols to phosphorothioate triesters, P (Z,V) : and by oxidation with aqueous iodine to phosphorothioates, P (Z,V) . Compound Xa can also be reacted with a rnercaptan in the presence of an activator such as tetrazole to yield the dinucleoside phosphorothioite , Vila, which can be chemically converted to Xlla by oxidation with sulfur to dinucleoside phosphorodithioates, P (Z,Y) and by oxidation with t-buty lperoxide or aqueous iodine to phosphorothioates, P (L,Y) . Thus it can be seen that the compounds XII and Xlla, as synthesized by process C, can be derived either from two intermediates, XIa and Vila, or from one of these two intermediates. For example Xlla, where P (Z,Y) can be derived from either intermediate XIa or Vila. For 22 Xlla where P (Z and X or W) , XIa can be used to synthesize this class of compounds.

The present new compounds of structure II having different heteroatom containing substituents covalently linked to phosphorus can thus be prepared by processes Λ, B,and C. In some cases where Z and Y are linked to phosphorus and, therefore, yield a dinucleoside phosphorodi thioate , processes Λ, B and C can all be used to prepare the same compound. This is also the case for certain other compounds such as II where Y and L are linked to phosphorus. Alternatively, compound II having Z and X or W or V (where V is covalently linked to phosphorus and to some group other than hydrogen as defined previously) linked to phosphorus can be synthesized by process C. Thus it can be seen that these processes lead to the synthesis of all the compounds described by II.

Compounds I, as the subset defined by V and Va , and II can then be used to synthesize polynucleotides having phosphorodithioate , phosphorothioamidate and phosphorothioate internucleotide linkages. These processes can be completed either on art form polymer supports or in the absence of these supports.

Of course the nucleoside moiety of the present invention can include more than one nucleoside and may include a number of nucleosides condensed as oligonucleotides having one or more phosphorus moieties (as shown in II) in combination with additional internucleotide phosphate diester linkages. These oligonucleotides may also only contain phosphorus noieties as shown in II. Polynucleotides having a mixture of in ernucleotide linkages including the presently described linkages as in II or only linkages as described in II are prepared using the new processes comprising one aspect of the present invention in combination with preferably conventional phosphoramidite methodologies 23 for synthesizing the other polynucleotide linkages (although other methods such as phosphate triester and phosphate diester and H-phosphona te procedures can also be used to synthesize these additional linkages) . These condensation steps are best carried out on polymer supports although nonpolymer support procedures can also be used.

The present invention is par icularly useful in the chemical synthesis of any deoxyribonucleic acid (DMA) or ribonucleic acid (RNA) containing any deoxynucleotide , nucleotide, polynucleotide, or polydeoxynucleotide . Hybrid structures containing elements of deoxynucleotides and nucleotides in any combination as part of the same polynucleotide are also possible using compounds I and II. These new DNA or RNA compounds have analog substituents L, W. V, X, Y or Z covalently bonded to phosphorus at one or more internucleotide phosphorus containing linkages as found in DNA and RNA.

The synthesis of compounds according to general formula lb can be represented by the following general reaction scheme, scheme D: 24  The preferred reaction Scheme D is represented as follows: ( I Y wherein , B, A , Q, X r Z, Y , W, and V are as previously described. Compounds XVI and XVIa are those in which all compounds have phosphorus double bonded to Z and also single bonded to V plus Y. 26 The process of scheme D involves synthesis of XIV and XlVa from Ilia and XIII or XHIa. Reaction of XIV or XlVa with I-^S and an activator such as tetrazole yields a new compound, XVa , the nucleoside H-phosphonothioate , which can be chemically converted by oxidation v/ith sulfur to nucleoside phosphorodi thioates , P (Z, V, Y) and by alkylation of the nucleoside phosphorodithioate to the nucleoside phosphorodithioate triesters, P (Z, V, Y) .

The preferred novel compounds according to the present invention are those compounds of general formula la and II wherein (for la) Y is a substituent having sulfur single bonded to phosphorus and to R_. where R_ is a heteroatom substituted or unsubstituted 5 blocking group; A is II; ^ is a blocking group, B is a nucleoside or deoxynucleoside base having art form blocking groups; and X is a secondary amino group; and (for II) Z is sulfur double bonded to phosphorus; Y is a substituent having sulfur single bonded to phosphorus and to R,- where R,. is a heteroatom substituted or unsubstituted blocking group? A is II ; R^ is a blocking group; B is a nucleoside or deoxynucleoside base having art-recognized blocking groups; and R^ is H. These new compounds can then be used to prepare oligonucleotides having phosphorodithioate internucleotide linkages with P(Z,Y). These oligonucleotides are also preferred and novel new chemical entities.

The new compound II of the present invention can be prepared as shown in Scheme C from art-recognized starting materials such as IXa, a nucleoside 3 ' -phosphorodiamidi e . The initial reaction is accomplished by dissolving the nucleoside in an organic solvent such as dioxane or tetrahydrofuran containing triethy lamine to take up the liberated hydrochloric acid and adding a bis (dialkylamino) chlorophosphine . The resulting 27 nucleoside phosphorodiamidite is reacted without isolation with a second nucleoside. The isolated product of this reaction is a dinucleoside dialkylamino phosphoramidite , Xa, which can be reacted by two different pathways to form Xlla. The preferred pathway is to react Xa with a mercaptan in the presence of tetrazole to yield Vila which is further treated with elementary sulfur to form the deoxydinucleotide phosphorodithioate, Xlla, where P(Z,Y). Λ second pa hway is to treat Xa with hydrogen sulfide and tetrazole in an organic solvent such as acetonitrile to yield the dinucleoside H-phosphonothioate , XIa. Further reaction of the isolated dinucleoside II-phosphonothioate with elementary sulfur in an organic solvent such as a mixture of toluene and lutidine yields the dinucleoside phosphorodithioate, Xlla where P(Z,Y). Reaction of the dinucleoside phosphorodithioate with an alkyl or aryl halide capable of alkylating thiols yields the sulfur protected dinucleoside phosphorodithioate triester, Xlla. These new compounds of the present invention can then be used to synthesize polynucleotides having phosphorodithioate moieties at selected phosphorus internucleotide linkages. This is possible by first removing R._ by conventional methods from Xlla to yield II and then reacting this compound with preferably an art-recognized phosphorodiamidite which leads to the dinucleotide 3 ' -phosphoramidite for use as a synthon in preparing polynucleotides. Compound II can also be converted to a dinucleotide 3 ' -phosphate , 3 '-phosphate diester, or 3 ' -H-phosphonate . Synthesis of the polynucleotide can then proceed using any of these dinucleotide synthons on silica-based polymer supports using recognized procedures or in reaction solutions free of polymer supports. 28 As a further embodiment of the invention, the dinucleoside phosphorodithioa es are preferably prepared by either reaction schemes Λ or B with A being preferred over B. These two reaction schemes differ in the method of preparing V and Va, the nucleoside phosphorothioamidite . For reaction scheme Λ, a bis ( secondaryamino) chlorophosphine., which is prepared by standard procedures, is reacted with an appropriately protected nucleoside dissolved in acetonitrile and triethy lamine . The resulting nucleoside diamidite, IXa, is then reacted without isolation with a mercaptan to yield the nucleoside thioamidite, Va , which is isolated by aqueous extraction and precipitation. For reaction scheme B, the mercaptyl-bis (dialkylamino) phosphine , IVa, · is first formed and then condensed with the selected nucleoside in acetonitrile using tetrazole as an activator in order to form a nucleoside thioamidite, Va . Compound Va can then be condensed with a second nucleoside using an activator in order to form an S-aralky Idinucleoside phosphite, Vila, which, after oxidation with elementary sulfur, yields Villa with P(Z, Y) , the dinucleoside phosphorodi hioa e triester. These procedures shown in schemes Λ and B eliminate the requirement for dinucleoside phosphorodithioate triesters, as shown in scheme C, as synthons for preparing polynucleotides and are, therefore, preferred. Thus the nucleoside S-aralkyldialkylaminophosphoramidite or thioamidite (Va) and art-recognized nucleoside phosphoramidites can be used in any desired sequence in concert with either elementary sulfur or aqueous iodine oxidation procedures, respectively, to yield polynucleotides having any selected combination of phosphorodithioate and phosphate internucleotide linkages. By using only the S-aralky Idialkylaminophosphoramidite or thioamidite Va in concert with sulfur oxidation, 29 polynucleotides having only phosphorodithioa e linkages can be prepared.

The synthesis of aralk lmercaptyl-bis- (dialky lamino) hosphine , IVb, is effected in an organic solvent solution whereby the bis (dialkylamino) -chlorophosphine , IVa , is first synthesized and then further condensed with an aralky lmercaptan . The first step is reacting phosphorus trichloride in an organic solvent such as tetrahydrofuran or dioxane with a five-fold excess of the dialkylamine . The reaction proceeds smoothly at reflux in a dry atmosphere of nitrogen or argon. The solution of the product is separated from the precipitated hydrochloride salt of the added amine, and can be concentrated under reduced pressure to a solid, if the dialkylamine is at least as large as diisopropylamine . This solid can then be recrys-tallized from chemically inert solvents such as pentane, hexane and heptane. Distillation of the bis (dialkylamino) chlorophosphine is also possible, especially for lower molecular weight compounds. These bis secondaryamino chlorophosphines can then be used directly to form compound IXa (schemes Λ and C) or for synthesizing IVb. For the synthesis of IVb, the next step involves dissolving an aralkylmercaptan in an inert solvent such as ethyl ether, tetrahydrofuran or dioxane; adding an equivalent of sodium hydride in order to convert the mercaptan to the mercaptide; and finally adding the bis (dialkylamino) chlorophosphine to the reaction mixture. The S-aralkylmercaptyl-bis (dialkylamino) -phosphine is formed quantitatively over several hours at room temperature. Removal of sodium chloride followed by crystallization from solvents such as acetonitrile affords the desired product. If the product, IVb, cannot be crystallized then purification may be possible by vacuum distillation. 30 However; if distillation leads to decomposition, then the nucleoside thioamidite should be synthesized by the preferred method using scheme A which does not require the synthesis of IVb as an intermediate.

Synthesis of in ternucleo tide bonds containing phosphorodithioate linkages where IVb is used for this conversion requires activating agents which are proton donors. Thus, these phosphines are activated by acidic compounds through protonation which facilitates the formation of the desired in ernucleotide bonds containing initially a thiophosphite triester. The initial activation step involving IVb requires acidic species, preferably mildly acidic, and include tetrazole and 3-nitrotriazole . The resulting nucleoside thioamidite, Va, may be difficult to activate and require more acidic species such as aromatic amine salts of strong acids, para-nitrophenyltetrazole , pyridinium tetraf luoroborate , t i f luoromethy Iphenyltetrazole and trif luoromethy ltetrazolide salts. This is especially the case where X is diisopropy lamino . However, when the nucleoside thioamidite contains a less sterically hindered X such as dimethylamino or pyrrolidino, then activation with a much milder acid such as tetrazole is possible and is preferred. These less sterically hindered nucleoside thioamidites are most easily prepared via reaction scheme Λ.

The mercaptyl moiety can vary considerably in structure. The criteria are that it facilitate activation of Va and that it is easily removed after completion of the synthesis of a polynucleotide. Thus, the preferred mercaptans include benzyl and heteroatom substituted benzyl moieties such as 2 , -dichlorobenzy 1 , phenyl and heteroatom substituted phenyl, and heteroatom substituted or unsubstituted alkyl substituents such as -* cyanoethyl and methyl. 31 The secondary amino moieties as part of the phosphines IVa and IVb and the nucleoside thioami-dites, Va, are preferably substitutents that stabilize these intermediates toward storage and synthesis. These secondary amino groups should also preferably facilitate activation of the phosphine during the reactions leading to the formation of internucleotide bonds. These criteria are met most easily by substituents such as dimethy lamino , diethy lamino , diisopropy lamino , dipropy lamino , dibuty lamino , dipen tylamino , pyrrolidino, piperidino, various isomeric alkyl groups, and also aralkyl groups .

When the present new compounds are used to form polynucleotides, they are employed in combination with art recognized nucleoside phosphoramidites or in the absence of nucleoside phosphoramidites. Thus at sites where normal phosphate diester linkages are to be inserted into polynucleotides, art recognized procedures such as activation with tetrazole, oxidation with aqueous iodine, capping with acetic anhydride if synthesis is on art recognized polymer supports, and detrity lation with acid are used for synthesis. At the sites where phosphorodithioate linkages are to be incorporated into polynucleotides, a nucleoside thioamidite, Va, is activated with tetrazole, aromatic amine salts, pyridinium tetraf luoroborate , para-nitrophenyl tetrazole, trif luoromethylaryl tetrazole or similar reagents, and following coupling to the growing polynucleotide, the thiophosphite internucleotide linkage is oxidized, preferably with elementary sulfur to yield the dithioate. Other steps for utilizing Va in the polynucleotide synthesis are the same as with art recognized nucleoside phosphoramidites. When DNA containing only phosphorodithioate linkages is to be prepared, Va is activated, condensed, and oxidized with sulfur as described above, repetitively with a nucleoside preferably attached to a polymer support to yield polynucleotides having phosphorodithioate linkages. Dinucleoside phosphorodithioate triesters Villa or Xlla where P(Z,Y) can also be used as synthons for polynucleotide synthesis. These new compounds are prepared using the presently described new processes. After conversion to preferably protected dinucleoside phosphorodithioate 3 ' -phosphoramidites , they can be activated with tetrazole and used directly as dinucleotide synthons via normal art recognized polynucleotide synthesis procedures, either preferably on polymer supports or in the solution phase in the absence of polymer supports .

Of course once the internucleotide bonds of the polynucleotide have been synthesized, which includes both normal linkages and the phosphorodithioate linkages, or polynucleotides having exclusively phosphorodithioa e linkages, the product can, if desirable, be freed of blocking groups. Thus the first step is treatment with preferably trialky lammonium thiophenolate to remove the aralkyl blocking group from the dithioate moiety or, if methyl groups are used to protect either normal or phosphorodithioate internucleotide linkages, the methyl group from these triesters. The remaining blocking groups on sugars, bases or phosphorus, and also the linkage joining the polynucleotide to a support if the synthesis had been completed in this manner, can then be removed using art recognized procedures such as hydrolysis with aqueous ammonia. If blocking groups on sulfur are used that are labile to reagents other than thiophenolate (i.e., trichloroethy 1 or/? -cyanoethyl) , then the depro-tection protocol should be modified accordingly. 33 The following examples and procedures depicting the formation of the compounds according to the present invention are presented in order to provide a more complete understanding and illustration of the present invention. 34 EXAMPLE I Dis (dime thy lamino) chlorophosphine was prepared by adding (iris ( d li e thylamino) - phosph ine (36.3 ml, 32.6 g, 0.2 mole) and tr ich lorophosphine (0.7 ml, 13.7 g, 0.1 mole) to anhydrous ether (100 mol) . Af: t G i: stirring for 3 hours at room temper tu e, solvent was removed by concen ration in v.'n.-uo I. room tempe a ure. The product was then distilled (b.p. 72-75°C) at reduced pressure (approx. 16 mm llg) using a water aspirator to yield 30 y. of product. 31 P-NM ( CMC 12 ) S 163.06. This procedure is also used to produce d ipy r ol id i ny lchlorophosph i ne . Preparation of thiophosphoramidi tes of the formula P X M represented as Va (Reaction Scheme Λ) where B = 1 -Thymi ny 1 ; D = 1- ( Μ-Ί -benzoy Icy osiny 1 ; B = 9- (N-6-benzoyladeninyl) ; 13 = 9- (N-2-isobu tyrylguaniny 1) ; and DMT = di-p-anisylphenylme thyl ( d ime thoxy tr i ty 1 ) M = Ί-chlorobcnzyl hio or 2 , 'i-chlorobenzyl hio X = N , N-diine tlry lamino or pyrrolidinyl and the further use of these compounds to prepare oligonucleotides having phosphorodi thioate inter-nucleotide linkages.

The following example describes the synthesis of 5 ' -0 -dime thoxy tr i ty 1-N -benzoy Id oxycy tidy ly 1.-3 ' - Γ. (4·--chlorobenzyl) ; pliosphor thiopy rrolid.ini te . The same procedure can be used for the other suitably protected deoxynucleosidcs . Similarly the same procedure is 35 useful for the 2.4-dichlorobenzyl and 4 -chlorobenzyl protected sulfur derivatives and for the , N-dimethylamino and pyrrolidinyl amidites. Table 1 31 . . summarized the P-NMR data for all these amidites. 51 -0-Dimethoxytrityl-N4-benzoyldeoxycytidine (317 mg , 0.5 rnmol) was dissolved in acetonitrile (2 ml) and triethylamine (1 ml) under argon. Bispyr-rolidiny lchlorophosphine (124 mg , 0.6 rnmol) was added which was followed by the immediate formation of a 31 precipitate ( P-NMR of the reaction product was at 133.8 ppm) . After 5 minutes stirring at room temperature, 4-chlorobenzylmercaptan (159 mg , lmmol) was added to the reaction mixture and the solution, including the precipitate, was concentrated to a glass i_n vacuo at room temperature. The glass was 31 resuspended in acetonitrile (2 ml) . The P-NMR spectrum of the reaction mixture indicated that the major phosphorus containing product was the diastereoisomers of the thioamidite (161.5, 159.7 ppm) . Minor impurities were an adduct of: bispyrrolidiny lchlorophosphine and 4-chlorobenzylmercaptan (107.0 ppm) and hydrolysis products (12.4 ppm) . Triethylamine was next added to the reaction mixture. The solution was diluted with deacidified ethylacetate (50 ml) and extracted with aqueous saturated sodium bicarbonate (50 ml x 2) and brine. The combined aqueous solutions were back-extracted with deacidified ethylacetate (10 ml) . The organic solutions were combined, dried for 1 hour over sodium sulfate in the presence of 101 (volume) triethy lamine , filtered, and the filtercake washed with 5 ml deacidified ethylacetate. The organic solution was then concentrated χτλ vacuo to a white foam. This foam was dissolved in toluene (10 ml) containing 1% triethylamine and the product isolated by precipitation into n-pentane: triethylamine (999:1, v/v) . After filtration, the product was 36 dried _in vacuo over phosphorus pentoxide and potassium hydroxide and isolated in 83.1% yield (741 g) .

^ H-N R (CDC13) 8.76 (broad s, 1H, NH) , 8.37 (d, JIIEI = 7.47 Hz, 0.511 , 115 , cytosine) , 8.31 (d, JI1H = 7.48 Hz, 0.5II, H5, cytosine) , 7.94 (d, JHII = 7.37 Hz, 2H, 112 and 116 of benzoyl group) , 7.68-7.54 (m, 3H, H3, 114, 115 of benzoyl group) , 7.44-7.14 (rn, 1411, aromatic protons of 4 -chlorobenzyl group, H2, 116 of anisyl (DMTr) , ph-protons (DMTr) , 116 cytosine)) , 6.91 (d, JHH = 7.57 llz, 41-1, H3 , H5 of anisyl DMTr) , 6.33 (m, 111, I'll) , 4.72 (m, 111, 3Ή) , 4.22 ( , 111, 4Ή) , 3.84 (d of singletts, 611, methyl protons of anisyl DMTr) , 3.84-3.76 (m, 2H, methylene protons of 4-chlorobenzyl group) , 3.59-3.35 (m, 211, 5Ή) , 3.19-3.01 (m, 411, methylene protons of pyrrolidinyl group a to nitrogen) , 2.84-2.75^ 2.37-2.26 (m, 211, 2Ή) , 1.79-1.71 (m, 4H, methylene protons of pyrrolidinyl group b to nitrogen) . 31P-NMR (CDC1 ) 161.79, 159.97. Fab+: 923 (M + S ) + , 907 (M + 0)" . tic: .75 (chloroform: ethylacetae: tr iethy lamine (45:45:10, v/v/v) .

Using a deoxynucleoside attached covalently to a silica based polymer support through the 3'-hydroxyl (U.S. Patent 4,458,066) , synthesis of deoxyoligo-nucleotides containing phosphorodi hioate linkages proceeded according to the reaction sequence outlined in Figure 1. Synthesis began by reacting a dry acetonitrile solution of any compound Va (10 equivalents) and tetrazole (50 equivalents) with y * mole of deoxynucleoside on silica for 30 sec (step i) followed by a 400 sec oxidation with 5% sulfur in pyridine : carbon disulfide (1:1, v/v, step ii) . Coupling was performed twice to ensure high yields (greater than 98%. Acylation of unreactive deoxynucleoside (step iii) , detritylation (step iv) and various washes were the same as those described 37 previously for synthesizing natural DNA from deoxy-nucleoside phosphorarnidites (U.S. Patent 4,415,732 and Science 230, 201-285, 1985) . Multiple repetitions of this cycle then led to the synthesis of DNA containing exclusively phosphorodithioate linkages or, when used in combination with deoxynucleoside phosphoramidities , to deoxyoligonucleotides having both phosphorodithioate and phosphate internucleo tide bonds .

Synthetic deoxyoligonucleotides were isolated free of protecting groups via a two-step protocol ( thiophenol : triethy larnine : dioxane , 1:1:2, v/v/v for 24 h followed by cone, ammonium hydroxide for 15h) and then purified to homogeneity by standard procedures (polyacrylamide gel electrophoresis and reverse 31 phase hplc) . P-NMR spectra (Figure 2) of phosphorodithioate DNA indicated that this synthesis protocol yielded DNA containing exclusively phosphorodithioate internucleo ide linkages. No hydrolysis of these dithioates to phosphorothioates 31 ( P-NMR 56) or phosphate was observed. So far a pentadecarner homopolymer containing fourteen dithioate linkages, lac and cro operators (0 1) with multiple dithioates at defined sites, and a cro operator segment (0^.1) containing seventeen contiguous dithioates have been synthesized. 38 Figure 1. Synthesis of DNA on a Polymer Support. (P) , silica based polymer support. 39 Figure 2. P-NMR Spectra of a Polynucleotide Derivatives. Spectra of d(C)15 containing exclusively phosphorodithioate internucleotide linkages (113 3 ] ppm in D^O) . P-NMR spectra was recorded on a Varian VXR-500S. Aqueous 85% H3P04 was the external standard . 40 EXAMPLE II Preparation of thiophosphoramidites of the represented as Va (Reaction Scheme B) B = 1-Thyminyl; B = 1 - (N-4 -benzoylcy osiny 1) ; B = 9- (N-6-benzoy ladeninyl ) B = 9- (N-2-isobutyry Iguaninyl ) ; and DMT = dimethoxytrityl The synthesis of compounds Va begins with the preparation of 4-chlorobenzylmercaptyl-bis- (diisopropylamino) hosphine . Phosphorus trichloride (0.5 mole, 68,665 g, 43.6 ml) was dissolved in 300 ml anhydrous tetrahydrof uran (THF) . The P 13 solution was cooled to -18°C by a NaCl ice mixture. Diisopro-pylamine (2.5 mole, 252.983 g, 350.4 ml) was then added slowly via a dropping funnel. At first the reaction was violent and had to be carried out under vigorous stirring (mechanical stirrer) and cooling.

After the reaction to the bis- (diisopropylamino) chlorophosphine was complete, the reaction mixture was refluxed for 12 hours to afford the desired product. After 12 hours the reaction mixture was cooled to rt and the diisopropy lammonium chloride was removed by filtration through a Schlenk-fritt . After washing the salts with THF, the clear reaction mixture was refluxed again for 12 hours to afford the desired product as the only phosphorus containing 31 material in the reaction mixture ( P-NMR 132.4 ppm) . The newly formed diisopropy lammonium chloride 41 was removed by filtration and washed with anhydrous ether. The filtrate was evaporated under reduced pressure (rotary evaporator) to a yellowish solid which was recrystallized from hexanes to afford a colorless crystalline solid. This compound was air stable and moisture sensitive. -Chlorobenzyl-mercaptan (50 mmol, 7.93 g, 6.6 ml) was dissolved in anhydrous ether (300 ml) and an amount of a sodium hydride suspension in oil (50% Nail in oil) equivalent to 50 mmol (2.4 g) was added to the mercaptan solution. As the solution was stirred (magnetic stirrer) , hydrogen evolved indicating the formation of sodium 4-chlorobenzylmercaptide. After two hours, bis- (diisopropylamino) chlorophosphine (50 mmol, 13.34 g) was added and the reaction mixture was stirred 31 until gas evolution stopped (4 hours at rt) . P-NMR of the reaction mixture indicated quantitative conversion of the chlorophosphine to the desired 31 product without any side reactions ( P-NMR 91.4ppm) . The salt (sodium chloride) was removed by filtration through a Schlenk fritt and washed with anhydrous ether (50 ml) . The colorless filtrate was evaporated to a white foam (4-chlorobenzylmercaptyl-bis- (diisopropylamino) phosp-hine) which was dissolved in a minimum amount of hot acetonitrile (100 ml) and recrystallized from the same solvent to afford a white crystalline product.

The 5 ' -O-diniethoxytrityl nucleoside (5 mmol) and 4-chlorobenzylmercaptyl-bis- (diisopropylamino) phosph-ine (6 mmol, 2.33g) were suspended in dry acetonitrile (15 ml) . Tetrazole (10 mmol, 0.69 g) was added and the reaction was stirred for 16 hours at room temperature. The initially present solids (phosphine and nucleoside) dissolved during the reaction time and a crystalline solid (diisopropy lammonium tetrazo-lide) precipitates. After 16 hours, the reaction was quenched with pyridine (1 ml) and diluted into acid 42 free ethylacetate (100 ml). The solution was extracted twice with an aqueous saturated solution of sodium bicarbonate and once with brine, successively.

The organic layer was dried over sodium sulfate. After removal of this salt, the solvent was evaporated in vacuo to afford a glass which was redissolved in a mixture of chloroform, ethy lacetate and trie-thylamine (45:45:10, v/v/v) and chromatographed on silica gel with the same solvent. Column chromatography fractions containing the desired product were combined and the solvent evaporated in vacuo . The product was dissolved in toluene and precipitated into n-pentane. The nucleoside phosphor th.ioainidite was isolated after drying the precipitate i_n vacuo over P OJKOll (3.33 g, 80.1% yield) . 3 T P NMR 161.3 and 159,97 ppm (two diastereomers) with respect to external standard of H_PO. for the 1 thymidine derivative. H NMR 8.0 ( N-II ) , 7.59 and 7.58 (2 x d, J = 1.2 Hz), 7.42-7.19 (in), 6.83 (d, J = 8.7 Hz), 6.37 (q, H ' ) , 4.65-4.58 (m, H3 , ) , 2.05-1.83 (m, Ηβ , ) , 3.80-3.61 Cm, CH2 of p-chloro benzyl) , 3.78 (s, Hg) , 3.48-3.29 (m. H5 , ) , 2.45-2.24 (m, H2') 1.44 (CH3-T) , 1.17-1.04 (m, CH3 of isopropyl) . 43 EXAMPLE III Synthesis of Dinucleoside Phosphorodi thioate ters of the formula: represented as Villa (Reaction Scheme B) where B = 1-Thyminyl; B = 1- (N-4 -benzoylcy osinyl) ; B = 9- (N-6-benzoyladeninyl) ; B = 9- (N-2-isobutyry lguaninyl) ; and DMT = dimethoxytrityl 5 ' -O-dimethoxytritylthymidine-3 ' -S-( 4 -chlorobenzyl) diisopropy laminophosphor midite (compound Va , example II) (0.2 mmol, 166.3 mg) and 3 ' -O-acetylthymidine (0.2 mmol, 56.8 mg) were dissolved in anhydrous dimethylformamide (2 ml) . 4-Nitrophenyltetrazole (1 mmol, 191.2 mg) was next added to this solution. After 15 minutes the reaction to the dinucleoside thiophosphite was quenched v/ith sulfur (1 atomic equivalent, 32 mg) . The reaction mixture was then diluted with ethylacetate (50 ml) and the sulfur removed by filtration through a cotton plug. After removal of the solvents in high vacuo, the desired product was dissolved in 44 ethylacetate (10 ml) and extracted twice with an aqueous saturated solution of sodium bicarbonate and once with brine successively. The organic layer was dried over sodium sulfate. After removal of the salt, the product was chromatographed on silica with a mixture of 1.1.1-trichloroe thane and methanol (92.5:7.5, v/v) . The product fractions were combined and the solvent removed _in vacuo . The dinucleoside phosphorodithioate was dissolved in toluene and 31 precipitated into n-pentane ( P NMR 97.8, 96.2 with respect to 85% h^PO^ as an external standard) . FAB- mass spectrum, 1047 (M ) , 921 ( -p-chlorobenzy 1) , 743 ( -DMT) , 619 ( -DMT and 4 -chlorobenzy 1) , 519 (31 -O-acetylthymidine 5 ' -0-4 -chlorobenzyl phosphorodithioate) , 395 (3'-0- acetylthymidine 5'-0-phosphorodithioate) .

The 4-chlorobenzyl group was removed from the phosphorodithioate triester with a mixture of dioxane : triethy lamine : thiophenol (2:2:1, v/v/v) within 1.5 hours at room temperature.

These dinucleoside phosphorodithioate triesters can also be prepared by using pyridinium tetrafluoro-borate as an activating agent. Pyridinium tetra-fluoroborate was prepared by dissolving HBF^ (10 mmole, 1.9 g of a die hyletherate , Aldrich Chemical Co.) in dry dichloromethane (5. ml) and adding this solution with stirring to dry pyridine (791 mg, 10 mmole) in dry ethyl ether (50 ml) . After 2 h the salt was removed by filtration, washed with dry ethyl ether, and dried in a dessicator over V^O^. n a typical reaction, 3 ' -O-ace ylthymidine (142 mg. 0.5 mmole) was allowed to react with 5 ' -0-dimethoxy-trity lthymidine-3 ' -S (4-chlorobenzyl) diisopropyl-aminophosphoramidite (833 mg . 1 mmole) in the presence of pyridinium tetraf luoroborate (334 mg , 2 mmole) in dry acetonitrile (5 ml) . After ten minutes the reaction mixture was quenched by addition of 20 45 atomic equivalents of sulfur (640 mg) in pyridine (2 ml) , concentrated ijn vacuo to a gum, redissolved in ethylacetate (50 ml) , and the excess sulfur removed by filtration. Following the standard aqueous work-up, as described previously in this example, and column chroma ography (Ci^CCl^ CH..0H, 95:5, v/v) , the dinucleoside phorphorodithioate in protected form was isolated by precipitation into pentane (60% yield) . The following dinucleoside phosphorodi-thioates in approximately 60% yield have been prepared via this procedure. a . ) 5 ' -O-Dimethoxy tritylthymidine S- (4-chlorobenzyl) -3 ' -0- (5 ' -0- hymic^ylyl ) -phosphorod-ithioate. FAB mass spectrum, 1005 (M ) , 847 (M - 4-chlorobenzylmercaptyl) , 703 (M - DMT + H)+, 455 (M + - DMT-4 -chlorobenzy lmercaptyl + H) ; FAB mass spectrum, 879 (M - 4-chlorobenzyl) , 779 (M - 5 ' -thymidylyl) , 477 ( thymidine-S-4-chlorobenzyl-phosphorodithioate) , 355 (thymidine 5 ' -phorphorodithioate) ; 31P NMR (CDC13) 96.44 UV (EtOH) max 228, 268 nm. b. ) 5 ' -O-Dime hoxytritylthymidine S~(4-chlorobenzyl) -3 ' -0- (5 ' -0-N2-isobutyryldeoxyguan-osinyl)- phosphoroditliioate. FAB ' mass spectrum, 1277 (M - Na) + , 952 (M - DMTr ) ' ; 31p-NMR (CDC13) 95.8, 96.14; UV (EtOH) max 262 nm. c . ) 5 ' -O-Dimethoxytri yl-N6 -benzoy ldeoxya-denosine S- (4-chlorobenzyl) -3 ' -0- (51 -0-N -benzoyl- 31 deoxycy tidme) -phosphoroditliioate . P NMR (CDC13) 93.89, 93.31.

Synthesis of dinucleoside phosphorodithioates , especially with strong acid catalysts such as -nitrophenyltetrazole or pyridinium tetrafluoro-borate, should be carried out under an inert atmosphere. Handling in air leads to the formation of various amounts of the corresponding oxides. Also compounds tentatively assigned as the 46 4-chlorobenzylphosphonothioamidates are formed when phosphoro thioamidites are reacted with acidic catalysts. These reactions, however, do not necessarily interfere with coupling as complete conversion of the 3 '-protected deoxynucleoside to the dinucleoside thiophosphite can be achieved by using an excess of the thioamidite and high concentrations of both deoxynucleoside derivatives. Preliminary investigations have also revealed that the resulting thiophosphite triesters are stable toward nonnucleophilic base and undergo rapid acid catalyzed hydrolysis to hydrogen phosphonates . They are susceptible to rapid oxidation by air or ^ -butylhydroperoxide to yield phosphorothioates and by sulfur to the phosphorodithioate triester. 47 EXAMPLE IV Synthesis of Dinucleoside H-Phosphonothioate of the formula: represented as XIa (Reaction Scheme C) where B = 1-Thyniinyl ; B = 1- (N-4-benzoylcytosinyl) ; B = 9- (N-6-benzoyladeninyl) ; B = 9- (N-2-isobu yrylguaninyl) and DMT = di ethoxytrityl The first step was condensation of 5 ' -O-dimeth-oxytritylthymidine with bis (diisopropylamino) -chlorophosphine in dioxane containing trie thylamine . The resulting phosphorodiarnidite was reacted without isolation with 31 -O-acetylthymidine to yield a homogeneous dinucleoside amidite in 62% yield after silica gel chroma ography (5% triethylamine in ethylacetate ) . Synthesis of the dinucleoside H-phosphonothioate proceeds by dissolving the dinucleoside phosphoroamidite (470 mg , 0.5 iwnol) in acetoni-trile (5 ml) , bubbling tt^S through the solution for 1 min, adding tetrazole (35 mg, 0.5 mmol in 1 ml acetonitrile) , and finally stirring the sealed reaction flask for 16 h. The reaction mixture was 48 concentrated to a gum on a rotary evaporator, redis-solved in ethylacetate (50 ml) and extracted twice with 2 M triethylammonium bicarbonate (pll 7.4, 20 ml each) . After concentra ing iri vacuo to a gum, the product was dissolved in dichloromethane (5 ml) and isolated by precipitation into pentane (400 mg , 90%) . FAB+ mass spectrum, 527 (anhydro DMT dT) : FAB- mass spectrum, 890 (M~) , 623 (DMT dT-3 ' -PHO-,"" ) , 363 (M-527, 5 ' -PH02"-dT-3 ' -OAc) ; 31 NMR 71.7 and 70.7 ^Jjjp = 673.8 Hz and 676.3 Hz; 1H NMR 7.81 and 7.80 (P-H, '''J = 671.4 Hz and 676.7 Hz) , 7.55 and 7.53 (s, II ) , 7.37-7.20 (m, aromatic) ,. 6.82 (d, J = 8.8 6 Hz, DMT) , 6.49 and 6.26 (m, H ,) , 5.49 and 5.25 (m, H ) , 4.35 (m, H4,) , 4.19 (m, Hg , ) , 4.07 (m, H4 , ) , 3.76 (s, MeO-DMT) , 3.42 (m, H , ) , 2.54-2.32 (m, H2,) , 2.08 and 2.07 (2 x s, CH-j-acetyl) , 1.90 (m, CH3~T) , 1.43 (s, CH--T) . R , = 0.35 and 0.28 (methanol/- ' 3 f dichloromethane, 1:9, v/v) . 49 EXAMPLE V Synthesis of a Dinucleoside Phosphorodithioate of the formula : *?> ?1 TO B o H ς - P - s represented as XIIa,P(Z,Y), (Reaction Scheme C) where B = 1-Thyminyl; B = 1 - (N- -benzoy Icy osiny1 ) ; B = 9-N-6-benzoyladeninyl) ; B = 9- (N-2-isobutyrylguaninyl) ; and DMT = dimethoxytrityl Dithymidine phosphorodithioate was synthesized by stirring the dinucleoside H-phosphonothioate (104 mg . 0.1 mmol in 1 ml dichloromethane ) with elementary sulfur (1 mmol in 2 ml toluene : 2 , 6-lutidine , 19:1, v/v) for 0.5 h. Purification via silica gel column chromatography (0-12% methanol in dichloromethane and 0.5% triethylamine) afforded 70% isolated yield.

+ + - FAB mass spectrum, 303 (DMT ) ; FAB mass spectrum, 921 (M~) , 395 (5'-PS 0~-dT-3 ' -OAc ) ; 31P NMR 112.7; 1 H NMR 8.12 (s, NH) , 7.90 and 7.60 (2 x s, Hg), 7.40-724 (m, aromatic) , 6.80 (d, JHp = 8.8 Hz, DMT), 6.43 (m, Hj,), 5.46-5.36 (m, H3 * ) , 4.40 (m, ' ) , 4.16 (m, Hct) , 3.76 (s, MeO-DMT) , 3.52 (m, II-.'), 2.28 5 D (m, H2,) , 2.05 (Ch3-acetyl) , 1.97 (CH3T) , 1.58 (s, 50 CII-jT) . Rf = 0.14 (methanol/dichlorome thane , 1:9, v/v) .

The dinucleoside phosphorodi thioate was depro-tected by standard procedures and isolated in 86% yield after ether extractions ( 3x) , sephadex G10 gel filtration Π^Ο) , and lyophilization as the ammonium salt. FAB+ mass spectrum, 579 (M) 31P N R (D20) 113.3; Hi NMR 7.60 and 7.46 (2 x s, Hg), 6.11 and 5.99 (m, IT1 r ) r 5.17 (m, H3 ' ) , 4.85 (m, H3 , ) , 4.15 (rn, »4 ' ) , 4.03 and 3.62 (m, II ' ) , 2.21 (m, Π2 ' ) , 1-88 m, CII^-T) . Rf = 0.25 (methanoltriethylamine/chloroform, 15:1:84, v/v/v). When the dinucleoside phosphoro-dithioate was phosphory lated with T4-polynucleotide 32 kinase and [ ^ - Ρ]ΛΤΡ, the rate of kination was approximately one-half that of unmodified 3' -5' dithymidine phosphate under identical conditions. Further testing with snake venom phosphodiesterase (Crotalus adamanteus venon, Sigma) indicated that the phosphorodithioa te was stable using conditions where the natural dinucleotide was completely hydrolyzed (assayed by reverse phase HPLC) . This compound was also observed to be stable to cone, ammonium hydroxide at 55°C (16 h) as no degradation or isomerization 31 was observed ( P NMR, thin layer chromatography). 51 EXAMPLE VI Synthesis of Dinucleoside Phosphorodi thioa te 3 -Phosphoramidi te the formula: 0 represented as XVIIa where D = 1 -Thyininy 1 ; B = 1- (N-4 -benzoylcy tosinyl) ; B = 9- (N-6-benzoyladeninyl) B = 9- (N-2-isobutyrylguaninyl) ; and DMT = dimethoxy t ityl In order to introduce the phosphorodithj.oa te linkage into oligonucleotides, a protection/-deprotection scheme for the phosphorodi hioate inter-nucleotide linkage was developed. Thus the dinucleoside phosphorodi thioa te in protected form (XU.a) (57 mg , 0.06 mmol) was alkylated with c , 2 , -trichlorotoluene (50 Conversion to a synthon useful for DNA synthesis was a two step process. The dinucleoside phosphorodithioate trioster was first deacylated (the 3' acetyl group) using 0.15 M tert-bu ylamine in methanol (0°C, 10 h) and purified by silica gel chromatography to yield Ila. Less than 5% cleavage 31 of the internucleotide linkage ( P NMR, TLC) was observed. The deacylated compound was then reacted with bis (diisopropy lamino) -2-cyanoethoxy phosphine (1.5 eq) in the presence of tetrazole (1 eq , 1 h at rt) to yield the dinucleoside phosphorodithioate 31 triester as the 3 ' -phorphoramidite in 76% yield. P NMR 149.4, 149.2, 148.9 and 97.2, 95.7, 95.5. ' H-NMR 7.56 (s, HJ, 7.33-7.27 (rn, aromatic) , 6.84 D (d, J = 8.5 Hz, DMT) , 6.39-6.29 (ra, ll ^ ) , 5.44 (m, H3') , 3.79 (s, MeO-DMT) , 1.90 (s, CHg-T) , 1.45 (s, CH -T) ,1.18 (d, J = 6.6 Hz, CH3-iPr) . Rf = 0.29 and 0.17 (chloroform/ethylacetate/triethylamine , 45:45:10, v/v/v). The resulting dinucleotide phos-plioramidite , XVIIa, has been used successfully in combination with unmodified mononucleoside phosphora-midites for the synthesis of 26-mer DNA fragments containing the phosphorodithioate linkage (98.2% 53 coupling efficiency) . These syntheses were completed on silica based polymeric supports and in combination with phosphoramidite coupling methodologies (U.S. patents 4,458,066 and 4,415,732; also Science 230, 281-285, 1985) . The oligodeoxynucleotides had the following sequences where the phosphorodithioate linkage in each segment is marked x and the normal phosphate internucleotide linkage is marked p. d (TpGpTpGpGpApApTxTpGpTpGpApGpCpGpGpApTpApApCpApApTp-T) d (ApApTpTpGpTpTpApTpCpCpGpCpTpCpApCpApApTxTpCpCpAp-CpA) 54 EXAMPLE VII Synthesis of Dinucleoside Thioamidates , Thiotriesters , and Thioates of the formulae: where B = 1-Thyminyl; D = 1- (N-4-benzoylcytosinyl) ; B = 9- (N-6-benzoyladeninyl) ; B = 9- (N-2-isobutyrylguaniny 1) ; and DMT = dime thoxytrityl The dinucleoside H-phosphonothioate was also found to be useful as a versatile synthon for preparing several analogs rapidly (5 min) in quantitative 31 yield ( P NMR). Thus, when oxidized with iodine/n- butylamine the phosphoro thioamidate , XIIa,P(Z,W) , was isolated in 92% yield. FAB mass spectrum, 961 (M ) , 695 (DMT dT-3 ' -POSNHBU) , 434 ( 5 ' -POSNHBU-dT-3 ' -OAc ) ; "F MR 74.4 and 74.0; H NMR 8.36 and 8.34 (2 x s , NH) , 7.59 and 7.56 x s , Hg) , 7.44-7.24 (m, aromatic) , 6.82 (d, J = .7 Hz , DMT) , 6.41 and 6.28 (m, Ηχ ' ) , 5.28 and 5.23 n, H- ' ) , 4.21 and 4.13 ( , H3' H4' (2 x) 2.94 (m, CH2-N) , 2.41 (m, H2' ) , 2.09 and 2.07 (2 x s, 55 CH -acetyl) , 1.93 and 1.88 (2 x s, CH..-T) , 1.12 (s, CH -T) , 1.39-1.23 (m, CU?) t 0.90 and 0.83 (2 x t, J = 7.2 Hz and 7,1 Hz, CH..) . Rf = 0.56 (methanol/-dichlorome thane , 1:9, v/v) .

The dinucleoside H-phosphonothioa e was converted quantitatively to a phosphoro thioate triester by oxidation with iodine and 9-anthracenyl methanol (10 equivalents) under anhydrous conditions, Xlla, P(Z,V) . FAB+ mass spectrum, 527 (anhydro DMT dT) : FAB mass spectrum, 906 (in-an thraceny lmethy 1) , 639 (DMT dT-3 ' -PS02~) , 379 ( 5 ' -PS02~-dT-3 ' -OAc ) . 31p NMR 51.7 and 51.0. R^. = 0.41 (methanol/dichlorome thane , 1: , v/v) .

Treatment of the dinucleoside H-phosphonothioa e with an aqueous solution of iodine and pyridine using art form conditions gave the dinucleoside phosphorothioate , XIIa,P(Z,V) , in 87% yield. FAB~ mass spectrum, 906 (M~) , 603 (M-DMT) , 379 (5 ' -PS02~dT-3 ' -OAc) 31 P NMR 60.2 and 60.0. 56 EXAMPLE VIII Synthesis of Dinucleoside Phosphorodithioate Triesters of the formula: represented as Xlla, P(ZfY), (Reaction Scheme C) where B = 1-thyminyl; B = 1- (N- -toluoy Icy osinyl) ; B = 9- (N-6-benzoyladeninyl) ; B = 9- (N2-isobutyrylguaninyl) ; and DMT = dimethoxytrityl .

Ac = acetyl and the further conversion of the deoxydicytidine derivative to deoxycytidine oligodeoxynucleotides having phosphorodithioate internucleotide linkages at various positions.

A. Synthesis of a Thymidine Dinucleotide Having a Phosphorodithioate Internucleotide Linkage. 5 ' -O-dimethoxytritylthymidine (1.2 g, 2.21 mmol) was dried by co-evaporation with anhydrous THF and then dissolved in THF (10 ml) and triethylamine (0.46 ml, 3.3 mmol). Bis (diisopropylamino) chlorophosphine (650 mg, 2.44 mmol) was added and the solution stirred at room temperature. After 35 minutes, the precipitate was removed by filtration and washed with 57 T1IF (1 ml). The combined filtrates containing the deoxynucleoside phosphorodiamidite were pooled, concentrated in vacuo, and redissolved in acetoni-trile (5 ml) . 31 -O-ace tylthymidine (639 g, 2.25 mmol) and tetrazole (142 mg, 2.0 iratiol) were dried by co-evaporation with THF (10 ml), redissolved in acetonitrile (5 ml) , and added to the acetonitrile solution of the deoxynucleoside phosphorodiamidite. After stirring for 45 minutes at room temperature, the reaction mixture v/as diluted with dichloromethane (75 ml) , extracted with an aqueous sodium bicarbonate solution (5% w/v) , dried over sodium sulfate, filtered, and concentrated in vacuo to a gum. The product v/as then purified by column chromatography (100 ml silica, ethylacetate : dichloromethane : -triethylamine ; v/v/v) to yield 1.59 g of the doxydinucleoside phosphoramidite (1.66 mmol, 75%). 31P-NMR (CII3CN 148.5 , 148.1.

The deoxydinucleoside phosphoramidite was then converted to the deoxydinucleoside phosphorodithioate triester. The deoxydinucleoside phosphoramidi e (1.59 g, 1.66 mmol) was dissolved in acetonitrile (7 ml). 4 -Chlorobenzylmercaptan (1.0 ml, 1.20 g, 7.6 mmol) and tetrazole (281 mg , 4.01 mmol) were then added and the reaction mixture stirred at room temperature for 30 minutes. A solution of sulfur in toluene : 2.6-lutidine (19:1, v/v, 10 ml containing 4 mmol atomic sulfur) was added and the resulting solution stirred for 10 minutes. The reaction mixture was diluted with ethylacetate (75 ml) , extracted with an aqueous sodium bicarbonate solution (5%, w/v), dried over sodium sulfate, filtered and concentrated in vacuo to an oil. The oil was dissolved in ethylacetate (40 ml) and triturated with hexanes (200 ml) to give a crude product as a white powder. Purification by silica column chromatography (100 ml silica, 2-12^methanol in dichloromethane as 58 eluant) yields the deoxydinucieoside phosphoro-dithioate triester (1.59 g, 1.52 mmol, 91%) . 31P-NMR (C11C13) 97.9 , 96.4.

Removal of the 3'-0-acetyl group (0.15 tert-butylamine in methanol, 0°C, 10 h) yields a deoxydinucieoside phosphorodi thioate that can be used for DNA synthesis (1.26 g, 1.28 mmol, 84%) 31P-NMR (CIICl-j) 97.3 , 96.9. The deoxydinucieoside phosp horodithioate is converted to the 3 ' -phosphoramidi e (see example V) and then used to synthesize DNA on a polymer support.

B. Synthesis of Deoxycytidine Oligomers Containing Phosphorodithioate s 5 ' -O-Dimethoxy trityl-N-toluoyldeoxycy tidine was prepared by minor modification of a published procedure (H. Koster, K. Kulinowski, T. Liese, W. Heikens, and V. Kohli, Tetrahedron 1_, 363 , 1981) . Deoxycy-tidine hydrochloride (10 mmol, 2.64 g) was co-evapor ted twice with anhydrous pyridine and resuspended in pyridine (50 ml) . Trimethylchloro-silane (7.5 ml, 59 mmol) was added and the mixture stirred for 45 minutes at room temperature. o-Toluoyl chloride (1.44 ml, 11 mmol) was added and the reaction stirred for two additional hours. The reaction mixture was chilled in an ice bath, treated with methanol (10 ml) and 25% ammonium hydroxide (20 ml) for 30 min, and the suspension removed by filtration. The resulting solution was concentrated to dryness iri vacuo . The resulting solid was suspended in 40 ml dichloromethane : methanol (8:2) and the insoluble salts removed by filtration. The filtrate was concentrated .in vacuo to an oil, reconcentrated twice in vacuo after addition of pyridine and redis-solved in pyridine (50 ml) . After addition of 0.9 equivalents of dimethoxy trityl chloride (3.05 g) , the reaction mixture was stirred for 30 min at 0°C and 30 min at room temperature. Dimethoxytrity lchloride 59 (0.3 equivalents) was added and stirring was continued for 30 minutes. The reaction was quenched by addition of methanol (1 ml) and the solution concentrated iri vacuo . The resulting oil was dissolved in dichloromethane (75 ml) and extracted sequentially with aqueous 5% sodium bicarbonate (w/v) and brine. The combined organic phase was dried over sodium sulfate, filtered, concentrated to dryness in vacuo , dissolved in dichloromethane : pyridine (99.5:0.5, v/v) and the product purified by column chromatography (50 g silica, dichloromethane : methanol : pyridine gradient from 0 to 3% methanol; 400 ml each) . Fractions containing 5 ' -0-dime thoxytrity 1-N- toluoy Ideoxycy idine were pooled, concentrated iri vacuo , redissolved in ethylacetate and precipitated into pentane (5.01 g, 7.7 m ol, 77%) . 31 -O-Phenoxyacetyl-N-toluoyldeoxycy tidine was prepared by minor modification of a published procedure (C. B. Reese and J. C. M. Stewart, Tetrahedron Letters 4273, 1968) . 5 ' -O-Dirnethoxytrity 1- N-toluoyldeoxycy tidine (1.94 g, 3 mmol) and phenoxyacetic anhydride (1.72 g, 6 mmol) was dissolved in tetrahydrof uran (50 ml) . After addition of pyridine (0173 ml, 9 mmol) , the solution was stirred for 14 hours at room temperature and then concentrated iri vacuo . The resulting oil was dissolved in dichloromethane (75 ml) , extracted twice with 5% aqueous sodium bicarbonate (100 ml, w/v) and the combined aqueous phases extracted with dichloromethane (50 ml) . The product in the combined organic phase ; was dried over sodium sulfate, filtered, concentrated to dryness iri vacuo, redissolved in ethylacetate and precipitated in pentane. The solid corresponding to totally protected deoxycytidine was dissolved in dichloromethane : methanol (8:2, v/v) and chilled in an ice bath. A solution of 60 p-toluenesulfonic acid (2.28 g, 12 mmol) in dichloromethane :methanol (50 ml, 8:2, v/v) was added and the solution stirred for one hour in an ice bath. The reaction was then quenched by addition of 5% aqueous sodium bicarbonate. The organic layer was extracted with brine and the aqueous phase re-extracted with dichloromethane (60 ml) . The combined organic phase was dried over sodium sulfate, filtered and concentrated to dryness in vacuo . The resulting oil was dissolved in dichloromethane and the product purified by silica gel column chromatography (20 g of silica, elution with dichloromethane and dichloromethane : methanol (1 to 3% methanol) . Fractions containing 3 ' -O-phenoxyacety 1--N-toluoy ldeoxycytidine were pooled, concentrated to an oil, and the product isolated as a precipitate by addition of ethylacetate (1.20 g, 83%) .

Deoxydicy tidine phosphoroamidite in protected form was prepared using the following procedure. 51 -O-Dimethoxy trityl-N-toluoy ldeoxycytidine (647 mg , 1 mmol) was co-evaporated three times with THF, dissolved in THF (5 ml) and triethylamine (0.21 ml, 1.5 mmol) and reacted with bis (M ,N-diisopropy lamino) chlorosphosphine (320 mg , 1.2 mmol) . After 90 minutes under argon, the reaction mixture was filtered under argon pressure to remove insoluble salts. The salts were washed with THF (2 ml) . The filtrate was concentrated to dryness and the product redis-solved in acetonitrile (2 ml) . 3 ' -O-Phenoxyacety 1-N-toluoy ldeoxycytidine (527 mg , 1.1 mmol) and tetrazole (70 mg , 1 mmol) were suspended in acetonitrile (4 ml) and the above solution, including 1.5 ml acetonitrile used to wash the flask, was added. The reaction mixture was stirred under argon for 105 rnin. and then poured into e thylaceta te : trie thylamine (99:1, V/V, 50ml) . After two extractions with 2 trie thylammonium bicarbonate (20 ml each) and back 61 extraction of the aqueous phase with ethylacetate : triethylamine (99:1, v/v, 25 ml) , the organic phase was dried over sodium sulfate, filtered, and concentrated _in vacuo . Purification was achieved by silica gel column chromatography (25 g silica, elution with hexanes : dichloromethane : riethylamine ; 50:50:0.5, 400 ml; 45:55:0.5, 200 ml; 40:60:0.5, 200 ml; and 35:65:0.5, 100 ml) . Product fractions were pooled, concentrated iri vacuo, and precipitated into pentane (67%) . 31P-NMR (CHC13) 149.3, 149.1. 1H-NMR 8.22 and 8.19 (s, H6) , 7.54-6.80 (m, HAr) , 6.30 (m, III') , 5.39 (m, H31 ) , 4.67 (m, CH2 phenoxyacetyl + H iPr) , 4.25 (m, 114') , 3.78 (2s, Me DMT) , 3.5 (m, 115' , 5' ') , 2.8 and 2.3 (m, H2 ' , 2") , 2.47 (m, Me tol) , 1.14 (m, Me iPr) .

Deoxydicytidine phosphorodithioate was prepared using the following procedure. The deoxydicytidine phosphoramidite as prepared in the previous procedure (1.40 g, 1.12 mmol) was dissolved in acetonitrile (5 ml) (previously flushed with helium to avoid oxygen oxidation of thiophosphi e ) and 4-chlorobenzyl-mercaptan (0.5 ml, 3.7 mmol) and tetrazole (190 mg , 2.7 mmol) were added. The solution was stirred under argon for 30 min and, without isolation, the resulting thiophosphite (completely formed in 15 minutes as 31 shown by P-NMR, 193.4 ppm in the crude reaction mixture) was oxidized to the phosphorodithioate triester by addition of 5 ml of a 0.4 M solution of sulfur in 31 toluene : lutidine (19.1, v/v) . Based on P-NMR analysis ( f 94.9, 94.7) , oxidation was complete after 10 minutes. The reaction mixture was diluted with ethylacetate (75 ml) , extracted twice with 5% aqueous sodium bicarbonate (75 ml each) , and the combined aqueous phases back extracted with 62 ethylacetate (50 ml) . The combined organic phases were dried over sodium sulfate, filtered, and concentrated in vacuo to an oil. The oil was dissolved in a minimal amount of dichloromethane, diluted with ethylacetate to approximately 40 ml, and the product precipitated by addition of 200 ml hexanes. The white precipitate was filtered, redissolved in dichloromethane, and the solution concentrated to dryness. The product was purified by silica gel column chromatography (40 g silica gel, elution with dichloromethane : hexanes : triethy lamine , 66:33:0.03, 400 ml and dichloromethane : triethy lamine , 100:0.03,200 ml) . Fractions containing the completely protected product were pooled, concentrated in vacuo , redissolved in dichloromethane, and precipitated into pentane (60%). 31P-NMR (C11C13) 97.5, 96.7. ½-NMR 8.1 (m, 116), 7.6-6.8 (m, HAr ) , 6.25 (m, Hi'), 5.25 (m, H3 ' ) , 4.70 (m, CH2 phenoxyacety 1 ) , 4.5-4.0 (m, CM,, benzyl, 115', H4') , 3.79 (s, Me DMT) , 3.73-3.35 (m, 115'), 3.0-2.55 and 2.45-1.95 (m, H2', 2''), 2.50 (m, Me tol) .

The 3 ' -O-phenoxyacetyl protecting group was removed using the following procedure. The completely protected deoxydicytidine phosphorodi hioate triester (355 mg, .264 rrunol) was dissolved in acetonitrile (3 ml) and diluted with methanol (9 ml) . After chilling the solution in an ice bath, tert-butylamine in methanol (0.3 M, 12 ml) was added and the reaction mixture stirred for 90 min in an ice bath. The reaction solution was concentrated to dryness and the product purified by silica gel column chromatography (30 g silica, elution with dichloromethane : trie thylamine , 100 : 0.03 , 100 ml followed by 200 ml each of dichloromethane :methanol : -triethylamine, 99:1:0.03, 98:2:0.03 and 97:3:0.03). Product fractions were concentrated to dryness, 63 redissolved in dichloromethane, and precipitated into pentane (95% yield) . 31P-N R (CDC13) 96.5, 96.2. 1H-NMR 8.2-8.06 (m, 116) , 7.52-6.81 (m, HAr) , 6.25 (m, Hi') , 5.24 (m, 113') , 4.5-4.0 (m, C\\ benzyl, H3 ' , H4 ' , H5 ' ) , 3.79 (s, Me DMT) , 3.6-3.3 (m, H5 ' ) , 2.95-2.55 and 2.45-2.05 (m, M2' , 2" ) , 2.50 ( in , Me tol) .

The deoxydicytidine phosphorodithioate was next converted to the 3 ' -phosphoramidite which is useful as a synthon for synthesizing DNA containing dithioate internucleotide linkages. The deoxydicytidine phosphorodithioate having a free 3'-hydroxyl (304 mg , 0.251 rrunol) was dissolved in acetonitrile (5 ml) . Bis (diisopropy larnino) - cyandethoxyphosphine (121 mg , 0.402 mmol) and tetrazole (20 mg, 0.286 mmol) were added under argon and the solution stirred for 2 hours. After quenching with ethy lacetate : triethy lamine (19.5:0.5) and diluting further with ethylacetate (20 ml) , the reaction mixture was extracted twice with 2 M triethylammonium bicarbonate (13 ml each) and the aqueous phase back extracted with ethylacetate: triethylamine (19.5:0.5) . The organic layer was dried over sodium sulfate, filtered, and concentrated to an oil iri vacuo . The resulting oil was redissolved in dry ethylacetate and precipitated into pentane (87% yield) . 31P-NMR (dichloromethane) 149.5, 149.2, 149.0, 96.5, 96.0.

Deoxycytidine pentadecamers containing phosphorodi hioate internucleotide linkages at selected sites were synthesized using the deoxydicytidine phosphorodithioate synthons having a 31 -0- ( -cyanoethyl) -Ν,Ν-diisopropylphosphoramidite moiety as described above and 51 -O-dimethoxy trityl-N-benzoy ldeoxycytidine -3 ' -0- ( -cyanoethyl) -M ,N-diisopropylphosphoramidite . The standard 64 phosphoramidite synthesis methodology was used (M. II. Caruthers and S. L. Beaucage, U. S. Patent 4,415,732 and M. H. Caruthers and M. D. Matteucci, U. S. Patent 4, 458,066). The average coupling efficiency was 99% (3 minute coupling time, 0.2 mol deoxycytidine on controlled pore glass as a support) . The products were freed of protecting groups by treatment with a solution of thiophenol : triethylamine : dioxane (1:1:2, v/v/v) at room temperature for 6 hours (some product remains as the S-protected dithioate (5-10%) when analyzed by gel electrophoresis and concentrated ammonium hydroxide at 55°C (15 hours) . Purification of the final product was by either polyacry lamide gel electrophoresis or high performance liquid chromatography. Three pentadecamers having phosphoro-dithioate linkages at specific positions were synthesized and have the following sequence: d (CpCxCpCpCpCpCpCpCpCpCpCpCxCpC) d (CpCpCpCpCpCpCxCpCpCpCpCpCpCpC) d (CxCpCxCpCxCpCxCpCxCpCxCpCxCpC) where x represents a dithioate linkage and p represents the natural internucleotide linkage. 65 EXAMPLE IX Synthesis of Nucleoside 3 ' -Phosphorod hioa e of the formu l : o represented as I a: B = 1 -Thyminy 1 ; B = 1 - (N-4 -benzoylcy tosiny 1 ) ; B = 9 - ( N-6 -benzoy laden iny 1 ) ; B = 9 - (M-2-isobu y ry lguaniny 1 ) and DMT = dime thoxy r i ty 1 Although B may be as described above, the following description is limited to a specific Nucleoside 5 ' -Phosphorodi thioa te wherein B is 1-thyminyl. 3'-0-(Diisopropylamino) -2 -cy anoe thy Iphosph i no- -5 ' -0' (di-p-methoxytrityl) thymidine (27.7 mg , 0.04 lumol) was prepared by art form methods (M. II. Cairuthers and S. L. Beaucage U.S. Patent 4,415,732) and then dissolved in anhydrous acetonitrile (Ή0 1) . Hydrogen sulfide was bubbled through for 1 mi. n and tetrazole (7.0 mg in 220 1 CH CN , 0.2 mrnol) was added. After 10 min P MM spectroscopy showed quantitative conversion to the two d ias tereomer s ( 70.9 and 70.2 ppm, 1 J = 675 Hz) of the nucleoside H-phosphonothioa te . Excess of elementary sulfur converted the H-phosphono thioa e in quantitative yield within 1/2 h under stirring at rt to the nucleoside 3 ' -phosphorodi thioa te . 3''P NMK (CI^CN) - - I 114.0 ppm. FAB 708 (M ) , 182 (M-DMTdT+0) · M NMK (CDC1-) 7.53 (s, Hc) , 7.35-6.01 (m aromatic) , 6.15 3 6 (t, II ' r J = 6.4 Hz) , 5.12 (m, H3 ' ) , 4.20 (rn, !I^) , 3.95 (ni, Π4') , 3.18 (s, MeO-DMT) , 3.47 (m, CI^O-P) , 66 2.77 (t, CH2CN, J = 6.2 Hz) , 2.56-2.44 (in, II2 ' ) , 1.91 (s, CH3-T) .

Protected nucleoside 3 ' -phosphorodithioa te was dissolved in 80% aqueous acetic acid (4 ml) and left for 30 min at rt. The reaction mixture was then diluted with water (4 ml) and extracted 3 times with ether (5 ml) . The water phase was evaporated to an oil followed by a co-evaporation with water (5 ml) . The oil was redissolved 25% aqueous ammnia and incubated ag 55°C for 16 h. The mixture was re-evaporated and lyophilized with water to yield the nucleoside 3 ' -phorphorodithioa te . FAB 338 (M ). FAB+ 338 (dT-P+_.It = S) . 67 EXAMPLE X Syntliesis of Muceloside 5 ' -Fhosphorodi thioa te of. the formula: Γ n represented as compound XVIa where B = 1-Uracilyl; D = 1 - ( Μ-Ί -benzoy Icy tos i ny 1 ) ; B = 9- (N-6-benzoyladeninyl) ; B = 9- (N-2-isobu yrylguaninyl) .

Although B may be as described above, the following description is limited to a specific Nucleoside 5 ' -Phosphoro-dithioate wherein B is 9- (N-6-benzoyladeninyl) .

A solution of t^-benzoyl-2-3-metho:

Claims (6)

- 70 - CLAIMS:

1. An oligonucleotide comprising at least two nucleotide moieties having an intcrnuclcolidc linkage between said moieties wherein at least one intcrnuclcotidc linkage is a p osphorodithioatc linkage of the structure RS - P = S I wherein R is H or a blocking group (as herein defined).

2. An oligonucleotide according to Claim 1 wherein the phosphorodithioate linkage of the structure is l HS - P = S. i

3. A compound according to Claim 1 of the formula: wherein B is a nucleoside or deoxynucleoside base (as herein defined); A is H, halo, OR-,, SR2) N(R2)2 or azido where R2 is independently a blocking group (as herein defined); R( and R3 arc blocking groups (as herein defined) or one of R[ and R3 may also represent a group -P(OR4)NR'6R'7 where R.( is a blocking group (as herein defined) and R'0 and R'7 are the same or different substituted (as herein exemplified) or unsubstituted aryl, alkyl or aralkyl moieties; R5 is a blocking group (as herein defined).

4. A compound according to Claim 3 wherein R, is di-p-anisylphenyl- mcthyl, R3 is acetyl, lcvulinyl, phenoxyacctyl or another blocking group (as herein defined); R5 is 2,4-dichlorobcuzyImcthyl or β- cyanoethyl; A is H, and B is a deoxynucleoside base (as herein defined). 110600/ 3

5. . A compound according to Claim 3 of the formulae: wherein A, B, R1; R3, R4, R5, R'6 and R' 7 are as defined in Claim 3 ·

6. - A compound according to Claim 5 of the formula: wherein A, B, R R4, R5, R'6 and R'7 are as defined in Claim 3. . A process for production of oligonucleotides which comprises the step of condensing the 3'-OH or 5'-OH group of a nucleoside or oligonucleotide compound according to Claim 3 which has been activated by a coupling agent, followed by oxidation to pentavalent phosphorus. - 71 - 1 10600/ A process of Claim 7·, wherein in the compound of Claim ; β ine, guanine, cytosine or thymine, and A is H. 9.. A process according to Claim 7, wherein in the compound of Claim 5 -NR'6R'7 is diisopropylamino, R4 is methyl or β-cyanoethyl, R5 is methyl or 2,4-dichlorobenzyl or β-cyanoethyl, and R, is di— p— anisyl-phenylmethyl. 0 · A process according to Claim 7 where the nucleoside or oligonucleotide having a free 5' -OH group is linked to a polymer support and the synthesis is repeated many times to form an oligonucleotide of defined sequence. 1 1 - An oligonucleotide in protected or unprotected form and containing a phosphorodithioate linkage as shown in Claim 3 wherein A and B are as defined in Claim 3, R5 is a blocking group (as herein defined); and each of Rj and R3 is H or a blocking group (as herein defined). 1 2. A process of producing oligonucleotides from a compound of Claim 3 wherein R3 or Rj represents H-phosphonates, a phosphate monoester, or phosphate. For the Applicants, - 72 -