Field of the Invention The invention relates generally to the synthesis of oligonucleotides, and more particularly, to a method for sulfurizing oligonucleotides with thiophosphorus compounds to form oligodeoxyribonucleoside phosphorothioates and/or phosphorodithioates.

BACKGRQUND With the development of efficient methods of synthesis, interest has arisen in the use of anti-sense oligonucleotides to treat a variety of diseases, particularly viral infections, e.g. Matsukura et al, Proc. Natl. Acad. Sci., Vol. 86, pgs. 4244-4448 (1989). An antisense oligonucleotide is a synthetic oligonucleotide of varying length, usually in the range of about 12 to 30 nucleotides, or nucleotide analogs, whose sequence is complementary to a predetermined segment of the RNA, either freshly transcribed or the messenger (mRNA), critical to some viral function. It is believed that when an antisense oligonucleotide hybridizes to its target RNA, it either blocks translation or processing of the RNA or makes it susceptible to enzymatic degradation. One problem with this approach has been the difficulty of getting the antisense oligonucleotide to its target RNA in sufficient concentration and for sufficient duration to be effective in shutting do n che synthesis of undesired proteins, e.g. viral enzymes, coat proteins, and the like. The susceptibility of the phosphodiester

linkage of the oligonucleotides to nuclease digestion is believed to be an important cause of this difficulty, and has prompted the development of a variety of nucleoside oligomerε linked by nuclease-resistant analogs of the natural phosphodiester bond, e.g. Miller et al, U.S. patent 4,511,713 and Ts'o U.S. patent 4,469,863 (methyl- and arylphosphonates); Miro et al, Nucleic Acids Research, Vol. 17, pgs. 8207-8219 (1989) (phosphoroselenoates); Brill et ^' al, J. Am. Chem. Soc, Vol. Ill, pg. 2321 (1989) (phosphorodithioates); and Matsukura et al, Proc. Natl.

Acad. Sci., Vol. 84, pgs. 7706-7710 (1987), and Gene, Vol. 72, pgs. 343-347 (1988) (phosphorothioates).

The phosphorothioate and phosphorodithioate analogs are especially promising because they are highly nuclease- resistant, have the same charge as natural oligonucleotides, and are taken up by cells in effective amounts.

Phosphorothioates are conveniently synthesized by automated DNA synthesizers using hydrogen phosphonate chemistry, which permits the phosphonate backbone to be sulfurized in a single step off of the automated synthesizer after synthesis. This is advantageous because the phosphonate moieties are sulfurized by exposure to elemental sulfur dissolved in an organic solvent. Since the sulfur readily precipitates out of solution, the off- column sulfurization avoids costly blockages of valves and tubing of the synthesizer by sulfur precipitates. A drawback of of this route of phosphorothioate synthesis is that coupling yields during chain elongation are typically lower than those obtained using phosphoramidite chemistry, Gaffney and Jones, Tett. Lett., Vol. 29, pgs. 2619-2622 (1988). The practical importance of high coupling yields is demonstrated by the synthesis of a 28-mer where a 99% coupling yield per step results in an overall yield of 76%

(.99 ), whereas a 96% yield per step results in an overall yield of only 33% (.96 ²⁷ ).

Phosphoramidite chemistry, with coupling yields typically greater than 99%, would be a highly desirable approach to phosphorothioate and phosphorodithioate synthesis. However, the phosphite intermediates, which would be sulfurized, are unstable under the conditions of the detritylation step of the reaction cycle. This requires that the phosphite linkage be sulfurized after each coupling step. For practical purposes, such sulfurizations would have to be carried out on an automated synthesizer, but the sulfur precipitation problem discussed above precludes the use of any of the commercially available machines. Moreover, the sulfurization rate of the phosphites is relatively slow and suffers from side reactions that lead to increased contamination of the final product.

In view of the desire to employ phosphorothioate and phosphorodithioate analogs of oligonucleotides aε pharmaceutical compounds, it would be advantageous to have available a method for sulfurizing that achieved the highest possible yields of completely sulfurized analogs and that was amenable for use with automated synthesizers, particularly with phosphoramidite and/or phosphorthioamidite chemistries.

SPMMARY QF THE INVFNTJPN The invention relates to a method of synthesizing sulfur-containing analogs of oligonucleotides, particularly but not exclusively, phosphorothioate and phosphorodithioate analogs. The method of the invention comprises the step of treating phosphorous(III) linkages of the intermediates of the desired analog with a thiophosphorus compound selected from the group defined by

Formula I and Formula II to obtain the desired analog. In particular, when phosphoramidite chemistry is employed the phosphorous(III) linkage is a phosphite and the end product is a phosphorothioate, when phosphorothioamidite chemistry is employed the phosphorous(III) linkage is a thiophosphite and the end product is a phosphorodithioate, and when hydrogen phosphonate chemistry is employed the phosphorous(III) linkage is a hydrogen phosphonate diester and the end product is a phosphorothioate. Preferably, the thiophosphorus compounds used in the invention are selected from the group consisting of thiophosphoric, dithiophosphoric, thiophosphinic, and dithiophosphinic acid polysulfides. More particularly, the thiophosphorus compounds defined by the formulas:

x y

f VW^ - I

x c

R, o - p «),- P- o - _:

/ o o \ /

FpπttμlH £E

-5-

wherein:

X is oxygen or sulfur; and most preferably, X is sulfur.

Y is oxygen or sulfur; and most preferably, Y is sulfur. n is within the range of 2-10, inclusive, and most preferably, n is 2.

Rπ, R2, R3, and R4 are inert side chains that can vary greatly in composition. Generally, they should not contain reactive groups that could lead to side reactions and inefficient sulfurization, and when taken together, they should permit the thiophosphorus compound to be soluble to an effective concentration. Preferably, R^, R2, R3, and R ₄ separately are alkyl, alkenyl, aryl, acyl, aralkyl, cycloalkyl, or cycloalkylalkyl containing up to 10 carbon atoms; halo-, nitro-, sulfo-, cyano-, lower alkoxy-substituted alkyl, alkenyl, aryl, acyl, aralkyl, cycloalkyl, or cycloalkylalkyl containing up to 10 carbon atoms; a heterocycle containing from 1 to 3 heteroatoms of nitrogen, oxygen, or sulfur, and from 2 to 8 carbon atoms; or a lower alkyl-, halo-, nitro-, sulfo-, cyano-, lower alkoxy-substituted heterocycle containing from 1 to 3 heteroatoms of nitrogen, oxygen, or sulfur, and from 2 to 8 carbon atoms. More preferably, R^, R2, R3, and R ₄ are separately lower alkyl; lower alkenyl; or cycloalkylalkyl, aryl or aralkyl containing up to 8 carbon atoms; halo-, nitro-, sulfo-, cyano-, lower alkoxy-substituted lower alkyl, lower alkenyl; or lower alkyl-, halo-, nitro-, sulfo-, cyano-, lower alkoxy-substituted aryl or aralkyl containing up to 8 carbon atoms; morpholinyl; thiomorpholinyl; piperidinyl; piperazinyl; or lower alkyl- , halo-, nitro-, sulfo-, cyano-, lower alkoxy-cubεtituted morpholinyl; thiomorpholinyl; piperidinyl; piperazinyl. In further preference, R-^, R2, R3, and R ₄ are separately

methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, cyclopentyl, cyclopentylmethyl, isopentyl, neopentyl, n-hexyl, neohexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 2-ethylhexyl, beta- cyclopentylethyl; methyl-, ethyl-, ethoxy-, nitro-, or halo-substituted phenyl; or methyl-, ethyl-, methoxy-, nitro- or halo-substituted benzyl. Preferably, the halo- substituents of the substituted phenyl or benzyl are chloro or bro o. Most preferably, R^, R ₂ , R3, and R ₄ are separately methyl, ethyl, n-propyl, isopropyl, or 2- ethylhexyl.

The term "lower alkyl" as used herein denotes straight-chain, branched-chain, and cyclic alkyl groups containing from 1-8 carbon atoms, e.g. methyl, ethyl, propyl, isopropyl, tert-butyl, isobutyl, sec-butyl, neopentyl, tert-pentyl, cyclohexyl, and the like. Likewise, the term "lower alkenyl" as used herein denotes straight-chain, branched-chain, and cyclic alkenyl groups containing from 2 to 8 carbon atoms. The term "oligonucleotide" as used herein includes linear oligomers of natural or modified nucleosides or of non-nucleosidic analogs linked by phosphodiester bonds or analogs thereof ranging in size from a few monomeric units, e.g. 2-3, to several hundred monomeric units. In particular, the term includes non-natural oligomers having phosphorus-containing linkages whose phosphorous(III) precursors are amenable to sulfurization, e.g. Takeshita et al, J. Biol. Chem., Vo. 282, pgs. 10171-10179 (1987); and Eapienis et al, pgs. 225-230 in, Bruzik and Stec, eds., Bipphpsphates and Thejyr Analogs—Synthesis.? gtructuye., Metafrpljspi., ^nd Acfrjyjty (Elsevier, Amsterdam, 19861 ,

PETA1LEP PEgCRXPPJPN ∑ THE INVgNTIPN The invention includes a method of synthesizing phosphorothioates and phosphorodithioateε. An important feature of the invention is the step of reacting phosphorous Ill-containing moieties of oligonucleotide intermediates with a thiophosphorus compound selected from Formula I or Formula II to bring about sulfurization. Because the thiophosphorus compounds of Formulas I and II are efficient sulfurizing agents that do not precipitate out of solution, the invention is particularly useful in the automated synthesis of phosphorothioate and phosphorodithioate analogs of oligonucleotides by all the commercially viable approaches, including hydrogen phosphonate, phosphoramidite, or phosphorothioamidite chemistries.

Petailed procedures for the phosphoramidite, phosphorthioa idite, and hydrogen phosphonate methods of oligonucleotide synthesis are described in the following references, which are incorporated by reference: Caruthers et al, U.S. Patents 4,458,066 and 4,500,707;

Koester et al, U.S. patent 4,725,677; Matteucci et al, J,. Apiey.. Chem. Sop.., Vol. 103, pgs. 3185-3191 (1981); Caruthers et al. Ge e c Engineering, Vol. 4, pgs. 1-17 (1981); Jones, chapter 2, and Atkinson et al, chapter 3, in Gait, ed., piigpnμpileptifle Synthesis.; A pra cal pprp _f rC (IRL Press, Waεhingtpn, P.C., 1984); Froehler et al, e ra edr n e ers, Vol. 27, Pgs. 469-472 (1986); Garegg et al, Tetra edrpfl etters, VPI. 27, pgs. 4051-4054 and 4055-4058 (1986); Andrus et al, U.S. patent 4,816,571; Brill et al, J. Am. Chem. Soc, Vol. Ill, pgs. 2321-

(1989); and Froehler et al, NμpJ-eΪP Acjds Vol. 14, pgs, 5399-5407 (1986).

Thiophoshoric, dithiophosphoric, thiophosphinic, and dithiophosphinic acid disulfides of the invention are

readily prepared by oxidation of the salts, e.g. triethylammonium, sodium, and the like, of the corresponding thiophosphoric, dithiophosphoric, thiophosphinic, or dithiophpsphinic apids with pxidizing agents suph as iodine, bromine, and the like. Methods of synthesis and properties of these disulfides are described in the following references which are incorporated by reference: Shishkov et al, Nauch.« r. Plpydjyεki Uμiy, S iP P l Vol. lO, pgs. 117-122 (1972); Almasi et al, Monat h.. Chepi., Vol. 100, pgs. 798-805

(1969); Krawczyk et al, phogpftprpμg Sμ fiay, Vol. 9, pgs. 189-192 (1980); Ko ber et al, PhPSPhPrpμs Sulfur, Vol. 35, pgs. 335-343 (1988); and Haegele et al, £__ N&tμrfprPPfr.r Teil B, Vol. 29, pgs. 349-357 (1974). Many of the aoid prepursors to the above disulfides are commercially available materials which are used industrially as solvent extraction and floatation agents. Examples include bis(2,4,4-trimethylpentyl)-dithiophosphinic acid and bis(2,4 ,4-trimethylpenty1)-monothiophosphinic acid (American Cyanamide) and bis(n-propyl)-dithiophosphoric acid and bis(2-ethylhexyl)-dithiophosphoric acid disulfide (ELCO). The synthesis of the dithiophosphoric acid compounds involves the reaction of the appropriate alcohol with phosphorous pentasulfide. The reaction product mixture generally contains varying amounts of the bis(alkyl)-dithiophosphoric acid thioanhydride (monosulfide), bis(alkyl)-dithiophosphoric acid disulfide, and bis(alkyl)-dithiophosphoric acid polysulfides, as well as the bis(alkyl)-dithiophosphoric acid product. Although the bis(alkyl)-dithiophosphoric acid may be purified and then oxidized to the disulfide, the entire reaction product mixture may be oxidized to produce a mixture of monosulfides, disulfides, and polysulfides which comprises a useful εulfurizing reagent.

When employed as a sulfurizing agent in the hydrogen phosphonate approach, a thiophosprous compound of the invention is delivered to the completed oligonucleotide chain in a εuitable organic εolvent, εuch aε acetonitrile, pyridine, tetrahydrofuran, dichloromethane, or the like, in a conεentration of between about .01 M to about 2.0 M. Preferably, the sulfurization is accomplished on an automated PNA synthesizer, e.g. an Applied Biosyεtemε model 380B, or like machine. Most preferably, the compounds of the invention are employed as a sulfurizing agents in the phoεphoramidite or phosphorthioamidite approaches. A thiophosphoruε compound of the invention iε delivered to the growing oligomer as a separate step within each addition cycle. Generally, the addition cycles of theεe methodε of synthesis involve the following steps: (1) deblocking a blocked functionality (usually a 5'-tritylated hydroxyl) pn the grpwing correct- sequence chain, cr pn the initial monomer attached to a solid phase support, to form a reactive functionality (e.g. a 5'-hydroxyl), (2) reacting an appropriately blocked and protected nucleoside phosphoramidite or phosphorthioamidite monomer or analog thereof (uεually in the preεence of an activator, e.g. tetrazole) with the reactive functionality of the growing correct- εequence chain, (3) capping unreacted reactive functionalities, and (4) oxidizing the newly formed phosphorous(III) linkage to form the naturally occurring pentacoordinate state. The sequence of above εtepε (3) and (4) can be reversed. The term "prptected" in reference tc monomer, partioularly nuplepside phosphoramiditeε or phoεphorthioamidites, means that moietieε εuoh as exυcyclie nitrogens, 2'- hydroxylε, oxygenε bonded to the phoεphorouε, or the like, have proteotipn groupε (usually base-labile)

attached which are removed after synthesis is completed, e.g. as those described in Koester et al (cited above), or in Molko et al, European patent publication no. 241,363 dated 14 Cctober 1987. The term is also meant to include monomers which may not have moieties requiring protective groups, e.g. some nucleoεide analogs, abaεic nucleosides, and the like. In the method of the invention, the thiophosphorus compounds _, defined by Formulas I and II are employed as sulfurizing agents in place of the oxidation step.

Preferably, such a thiophoεphoruε compound iε delivered to the growing oligomer in a εuitable organic εolvent, εuoh aε apetpnitrile, tetrahydrpfuran, dichloromethane, or the like, in a concentration of between about .01 M to about 2.0 M. Preferably, the εtep of sulfurizing with a thiophosphoruε compound of Formula I or II iε accomplished on an automated DNA synthesizer. In both approaches a wide variety of reaction temperatures may be uεed. Preferably, the εulfurization iε carried out at a temperature in the range of 0°C to 100°C, and more preferably, in the range of 15°C to 60°C.

Synthesis of Bis.(diispprppp?cyphPSPhinpthipylJ disulfide .(cp-djisppropylPhPSPhprpdithiPiP aci djgμ fjdeJ

PART A: Into 50 ml of absolute iεopropanol waε added at room temperature with stirring 20 g of phosphorous pentasulfide (Fluka) in small portions. Hydrogen sulfide iε releaεe and needε to be trapped. The reaction mixture waε εtirred at room temperature for about 3 hours until a clear, transparent liquid was obtained. Thiε εolution waε concentrated by rotary evaporation and the residue waε diεtilled with a 30 cm Vigroux column, collecting the fraction at 57-58°C under 0.02 mm Hg to give 34 g of C,C-

diiεopropylphoεphorodithioic acid.

PART B: To a εtirred εolution of 19.4 g of 0,0- diiεopropylphoεphorothioic acid in 30 ml of methylene chloride, cooled in an ice bath, waε added dropwiεe 9.3 g (12.6 ml) of triethylamine. Thiε solution waε cooled below 5°C and 12.5 g of iodine was added in small portionε, maintaining the temperature below 10°C. After 0.5 hours of stirring, the reaction mixture waε extracted three times with water, dried over anhydrous magnesium sulfate, and filtered. Ethanol (100 ml) was added to the solution which was then concentrated by rotary evaporation, during which pale-yellow crystalline bis(diisopropoxyphosphinothioyl) disulfide was formed. This, material was collected by filtration and washed with cold ethanol and dried to give 16.7 g pf pure prpduct.

The ^lp NMR εpectra was a single peak at 82.6 ppm (H P0 ₄ , external reference). M.P. 92-93°C.

sypfl pj.g o£ a ^7τPΨ- phpsphpyothip^e Pligpnucleotide fis aa Pr ?pprppy p]p?ψ p pdj.t j. c ftpj.d Pisulfide

£§. g ^μ lf ^μ risiftg Agent A 27-baεe phcεphorothioate oligonucleotide, 5*- TCGTCTTGTCCCGTCATCGTTGCCCCT-3" waε εyntheεized by the phosphoramidite method on an automated syntheεizer (model 380B Applied Biosystem, Foster City, CA). The standard syntheεiε protocol waε followed, except that in place of the oxidation step a sulfurization step waε εubεtituted, and thiε εtep preceded the capping εtep. In other words, the syntheεiε consisted of repeated cycles of detritylation, coupling, sulfurization, and capping. Separation of the final product from the synthesis column and purification were accomplished by standard means. The sulfurization step waε accomplished by exposing the

growing chain to a 0.2 M solution of 0,0- diiεopropylphoεphorodithioic acid diεulfide in pyridine for 1 minute at room temperature.

The yield of trityl cation releaεed during the detritylation εteps averaged 99%. The trityl yield iε a both a measure of coupling efficiency and a measure of the extent of sulfurization, since non-sulfurized (or oxidized) trivalent phosphorous linkages in the oligonucleotide are labile to cleavage during detritylation.

The 27-mer was cleaved from the support and deprotected with concentrated ammonium hydroxide at 55°C for 6 hours. The trityl-on oligonucleotide waε isolated by HPLC, detritylated, and precipitated as the sodium salt. The ³¹ P-NMR spectra (JEOL, 36.5 MHz, ppm vs H ₃ PP ₄ external reference) of the product showed greater than 98.5% sulfur incorporation (55.1 ppm) with less than 1.5% oxygen incorporation (-1.1 ppm).

I£££££I 1 pyptfte J- S PPJ-yrft P PSP orPt P&te oligonucleotide using P _^ prdiisppropy .-Phpshprpdithipip £id dj-^μ^ ^ __n sμi uriging ~~ _, A poly-A 19-mer phosphorothioate oligonucleotide waε synthesized fol lowing the same protocol as used in example 2. The yield of trityl cation averaged 98.5% per detritylation step. ³¹ P-NMR of the prpdupt indicated 99 % sulfur incorporation and 1% oxygen incorporation.

EXAMPLE £

Synthesis pf p _^ p.-diiscprppy rphpsphprpthipic

?P ft disulfide A sclutipn of 25.6 g of Sg and 117 ml of triethyla ine in 750 ml of oarbon disulfide waε added to

132.8 g cf neat diisppropyl phoεphite. After several hourε of εtirring at ambient temperature the reaction mixture waε concentrated by rotary evaporation. The P,P- diiscprppyl-phpsphprpthipic acid prcdupt was dissplved in 750 ml of methylene ohlpride and stirred while the splutipn was ccpled in an ipe bath. To this solution waε added 20.5 ml of bromine dropwise. The solution was allowed to stir an additional hour at ambient temperature, and then extraoted three times with deionized water. The organic phase waε then dried with anhydrouε εodiu εulfate and filtered. The filtrate was concentrated by rotary evaporation, and dried overnight under reduced pressure, to yield 137 g of 0,P-diisoprppyl-phosphprothipic acid disulfide (87% yield). The ³¹ P-NMR spectra cf the prpduct dissclved in methylene chlpride consisted of a single peak at 18.1 ppm vs. and external εtandard of phoεphoric acid.

EXAMPLE H Synthesis pf 1.4.-ba pfrpsphpypthipate pligpnupleptide M- i- . P-rP.-di.ispprppy -PhPSPhPFPttiiPiP ___-___[ ftLpμj.fj.fle

&£ £3& ?-fμ ?J-P9 WPfc A 14-base phosphorothioate oligonucleotide, 5 ¹ - CGCTTCTTCCTGCC, was synthesized as in example 2, with the exception that the sulfurization step was carried out using a 0.2 M solution of C,P-diisopropyl-phosphorothioic acid diεulfide in 2:1 pyridine:acetonitrile for 10 minutes at ambient temperature. The yield of trityl cation released during the detritylation εtep averaged 99%. The ³¹ P-NMR spectra of the 14-mer showed 92% sulfur incorporation and 8% oxygen incorporation.

EXAMPLE H Synthesis pf p.,p.-dir2rethylhe?ylrpftpsphprpdithipic

A solution of 177 g of P,P-di-2-ethylhexyl- phosphorodithioic acid (Elco L-21612) in 400 ml of methylene chloride waε cooled in an ice bath and 73.5 ml of triethylamine waε added dropwiεe with εtirring. While continuing to cool the εtirred solution in an ice bath 61.5 g of. iodine was added in small portions. The reaction mixture was allowed to stir at ambient temperature an additional 30 minutes and then was extrapted once with deicnized water and then twipe with aqueous brine. The organic phase was dried with anhydrous sodium sulfate, filtered, and then concentrated by rotary evaporation. The product was dried overnight under reduced pressure to yield 171 g (97% yield) of P,P-di-2- ethylhexyl-phoεphorodithioic acid disulfide as a yellow oil. The ³¹ P-NMR spectra of the product consisted of a resonance at 86.0 ppm corresponding to the desired product and another resonance at 79.0 ppm (4%) corresponding to bis(P,P-di-2-ethylhexyl-phosphorodithioic acid thioanhydride (the monsulfide), which was present as an impurity in the starting material.

EXAMPLE 7

Synthesis f 27rme:F spfr ^p & e pligphusleotide -ΨΪ -. P,Prdir2retfrylhe?ylrPhPSPhPrpditfriPig .-^ disulfide £§. __i_§. fμrwfig ?9 fi A 27-baεe phphprpthioate oligonuoleotide, 5 ¹ - TCGTCGCTTCTCTGCTTCCGTCTGCC-3 ¹ , was synthesized following the same protoopl as used in example 2, with the following exoeption. The sulfurization step was carried out using C,P-di-2-ethylhexylphosphorodithioic acid disulfide which waε 0.2 M in a mixture of 20 parts by volume pyridine and

31.5 parts acetonitrile. The sulfurization step was carried out at room temperature for 15 minutes. The yield of trityl cation averaged 98% per detritylation εtep. ³¹ P-NMR of the product indicated 99% sulfur incorporation and 1% oxygen incorporation.

EXAMPLE I Synthesis ££ bis.( Z _* *■ ■4rtrimethylPentylJ.-dithiophosphinic

A splutipn pf 161 g pf bis(2,4,4-trimethylpentyl)- dithiophoεphinic acid (Cyanex 301, American Cyanamide, 77% pure) in 400 ml of methylene chloride waε cooled in an ice bath and 73.2 ml of triethylamine was added dropwise with stirring. While continuing to cool the stirred solution in an ice bat 50.7 g of iodine was added in small portions. The reaction mixture was allowed to stir an additional 30 minutes at ambient temperature after which it was extracted once with deionized water, then twice with aqueous brine. The organic phase waε dried with anhydrouε εodiu sulfate, filtered, and then concentrated by rotary evaporation. The product was dried overnight under reduced presεure to yield 158.7 g of crude product. The ^P-NMR εpectra of the crude product indicated the preεence of a complex mixture. The desired bis(2,4,4- trimethylpentyl)-dithiophosphinic acid diεulfide comprised about half of the mixture (a multiplet at ca. 80 ppm vs. phoεphoric acid external standard). Impurities included the monothiophosphinic acid disulfide and the mixed disulfide of the monothiophoεphinic acid and the dithiophoεphinic acid. The monothiophosphinic acid waε preεent aε an impurity in the εtarting material.

EXAMPLE £

Synthesis g£. __& Wry? pft ppftpr flij-pjiEs ing bis a _^ .^i.- riϋi hylP n ylJrd tti Ph SP nic as d

££ §. sulfu z n ^g ^ggft An 18-mer phosphorothioate oligonucleotide, 5'- TCTCTGCTTCCGTCTGCC-3', was synthesized using the same protocol as used in example 2, with the following exception. The sulfurization εtep waε carried out uεing a solution of 137.9 g pf prude biε(2,4,4-trimethylpentyl)- dithiophosphinio acid disulfide in a mixture of 360 ml of acetonitrile and 100 ml of pyridine. The sulfurization step was carried out for 15 minutes at ambient temperature. The yield of trityl cation averaged 97.3% per detritylation step. The ³¹ P-NMR spectra of the 18-mer showed 98% sulfur incorporation and 2% oxygen incorporation.

The foregoing disclosure of preferred embodiments of the invention haε been presented for purposes of illustration and description. It iε not intended to be exhaustive or to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The embodiments were choεen and described in order to best explain the principles of the invention and its practical application, to thereby enable others skilled in the art to best utilize the invention in various embodiments and with variouε modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto.