AU626305B2 - Nucleoside and polynucleotide thiophosphoramidite and phosphorodithioate compounds and processes - Google Patents

Description

4- OPI DATE 12/12/89 AOJP DATE 25/01/90 APPLN. ID 37392 89 PCT NUMBER PCT/US89/02293 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 89/11486 CO7H 17/00 Al (43) International Publication Date: 30 November 1989 (30.11.89) (21) International Application Number: PCT/US89/02293 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European pa- (22) International Filing Date: 25 May 1989 (25.05.89) tent), DK, FI, FR (European patent), GB (European patent), IT (European patent), JP, KR, LU (European patent), NL (European patent), NO, SE (European patent), Priority data: SU.

198,886 26 May 1988 (26.05.88) US 314,011 22 February 1989 (22.02.89) US Published With international search report.

(71) Applicant: UNIVERSITY PATENTS, INC. [US/US]; P.O.

Box 901, Westport, CT 06881 (US).

(72) Inventors: CARUTHERS, Marvin ;2450 Cragmoor, Boulder, CO 80303 BRILL, Wolfgang 1138 Grand View Avenue, Boulder, CO 80302 NIELSEN, John Skyttehaven 2 11B, DK-2950 Vedbaek 6 2 (74) Agent: YAHWAK, George, University Patents, Inc., P.O. Box 915, Westport, CT 06881 (US).

(54)Title: NUCLEOSIDE AND POLYNUCLEOTIDE THIOPHOSPHORAMIDITE AND PHOSPHORODITHIOATE COMPOUNDS AND PROCESSES (57) Abstract The present invention relates to new and useful nucleoside thiophosphoramidite, polynucleotide dithioate phosphoramidite and polynucleotide phosphorthioamidate phosphoramidite compounds as well as the processes whereby these compounds can be used for synthesizing new mononucleotides and polynucleotides having phosphorodithioate, phosphorothioamidate, phosphorothiotriesters, and phosphorothioate internticlo'tide linkages.

T i I SWO 89/11486 PCT/US89/02293 Nucleoside and Polynucleotide Thiophosphoramidite and Phosphorodithioate Compounds and Processes Research leading to the making of the invention described herein was supported, in part, with federal funds. Accordingly, the United Stated Government has certain statutory rights to the invention described herein.

This is a continuation-in-part application of our earlier filed United States Patent Application 198,886, filed 26 May 1988.

This invention relates to new and useful phosphorus compounds which are particularly useful in the production of polynucleotides having analogs attached to phosphorus.

The present invention relates to new and useful nucleoside thiophosphoramidite, polynucleotide dithioate phosphoramidite and polynucleotide phosphorthioamidate phosphoramidite compounds as well as the processes whereby these compounds can be used for synthesizing new mononucleotides and polynucleotides having phosphorodithioate, phosphorothioamidate, phosphorothiotriesters, and phosphorothioate internucleotide 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 WO 89/11486 PCT/US89/02293 2 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 internucleotide linkage H. Caruthers, Science 230, 281-285, 1985; M. H. Caruthers and S. L. Beaucage, U.S. Patent 4,415,732; M. H. 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 Uznanski, M. Koziolkiewicz, W.

J. Stec, G. Zon, K. Shinozuka, and L. Marzili, Chemica ScriDta 26, 221,224, 1986; M. J. Nemer and K.

K. Ogilvie, Tetrahedron Lett. 21, 4149-4152, 1980).

Other methods employing H-phosphonate internucleotide linkages can also be used to synthesize phosphoramidates 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 WO 89/1148 6 PCT/US89/02293 S3 synthesize deoxynucleoside phosphorodithioates or nucleoside phosphorodithioates useful for synthesizing polynucleotides containing the dithioate linkage.

The procedure also yields a mixture of mononucleotides having p'.osphorodithioate and phosphorothioate moieties. Additionally the yield of uridine 2',3'-cyclic phosphorodithioate is only 28% and the acidity of P2S5 and the high temperatures used in the synthesis of the cyclic phosphorodithioate would preclude the use of this procedure with protected deoxyadenosine which would undergo depurination.

Similarly adenosine cyclic can be synthesized by treating suitably protected adenosine with 4-nitrophenylphosphoranilidochloridothioate followed by cyclization with potassium t-butoxide and conversion to the dithioate in a reaction with sodium hydride/carbondisulfide Boraniak and W. Stec, J.

Chem. Soc. Trans. I, 1645,1987). Unfortunately these reaction conditions and the low synthesis yields preclude the use of this chemistry for synthesizing oligonucleotides having phosphorodithioate linkages.

The present invention provides new and useful nucleotides, dinucleotides and polynucleotides having structure modifications at phosphorus. It also describes processes which for the first time lead to the synthesis of these compounds.

In general, the compounds, according to the present invention, can be represented by general formulae I and II: 0 Y P I

OU)

WO 89/11486 PCT/US89/02293 4 where R 1 is H or a blocking group; P V, W, X, Y, Z) is a phosphorus derivative such that L, V, W, X, Y, Z are substituents where heteroatoms are linked covalently to phosphorus; A is K or KR 2 where K is OH, H, halogen, SH, NH 2 or azide and KR 2 is oxygen, sulfur or nitrogen as K and R 2 is a heteroatom substituted or unsubstituted blocking group; B is a nucleoside or deoxynucleoside base; R 3 is H or a blocking group. Substituents V,W, X and Y may also be covalently linked to heteroatom substituted or unsubstituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl, cyloalkenyl, aralkenyl, alkynyl, aralkynyl, or cycloalkynyl groups.

The compounds of general formulae I and II wherein L, V, W, X, Y and Z are substituents where heteroatoms are linked to phosphorus include those in which the heteroatoms are sulfur, nitrogen and oxygen. The substituent V is oxygen single bonded to phosphorus and to either H or R 4 where R 4 is a heteroatom substituted or unsubstituted alkyl, aryl, aralkyl, cycloalkyl, cyclcalkylalkyl, alkenyl, aralkenyl, cycloalkenyl, alkynyl, aralkynyl or cycloalkynyl group. The substituent Y is sulfur single bonded to phosphorus and to either H or R where R 5 is a heteroatom substituted or unsubstituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl, aralkenyl, cycloalkenyl, alkynyl, aralkynyl or cycloalkynyl. The substituents W and X are nitrogen heteroatoms where W is primary amino, NHR 6 and X is secondary amino, NR 6

R

7 groups. The R and

R

7 groups taken separately each represent heteroatom substituted or unsubstituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, aralkenyl, cycloalkenyl, alkenyl, aralkynyl, cycloalkynyl, or alkynyl groups.

R

6 and R 7 when taken together form an alkylene chain containing up to 5 carbon atoms in the principal chain and a total of up to 10 carbon atoms with both

II

(I

terminal valence bonds of said chain being attached to the nitrogen atom to which R6 and R 7 are attached; or R6 and R7 when taken together with the nitrogen atom to which they are attached may also form a nitrogen heterocycle and which nitrogen heterocycle optionally includes at least one additional heteroatom from the group consisting of nitrogen, oxygen or sulfur.

The new compounds of general formula I are of two classes, Ia and Ib; class Ia consists of those in which phosphorus is single bonded through the heteroatoms to each of two substituents, X and Y where Y is attached to Ry and class Ib are those in which Z is sulfur double bonded to phosphorus plus two 10 other substituents from the group V and Y where the heteroatom of each of these substituents is single bonded to phosphorus. Compounds in class Ia are useful for synthesizing polynuclectides containing phosphorodithioate and phosphorothioate internucleotide linkages. Compounds in class Ib are useful for various therapeutic and biological studies and as intermediates for synthesizing nucleotides having 15 phosphorodithioate moieties.

Compounds of general formula II are those in which all compounds have phosphorus double bonded to Z or L and single bonded to one of the substituents V, X, Y or W. Compound II is preferably phosphorus double bonded to sulfur and single bonded to Y, V, W or X. Compounds of general formula II may also be those in which L is oxygen double bonded to phosphorus plus Y which is single bonded to phosphorus. Compounds II are useful for various therapeutic, diagnostic, and biological studies and for synthesizing polynucleotides containing }4phosphorodithioate, phosphorothioamidate, phosphorothioate triester and phosphorothioate and phosphorodithioate internudleotide linkages which are also useful as therapeutic, diagnostic, or research reagents.

R T- WIO 89/11486 PCT/US89/02293 6 As used herein the symbols for nucleotides and polynucleotides are according to the IUPAC-IUB Commission of Biochemical Nomenclature Recommendations (Biochemistry 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 differently.

alkyl a non-cyclic branched or unbranched hydrocarbon radical having from 1 to (preferably 1 to 12) carbon atoms. Heteroatoms, preferably oxygen, sulfur, or nitrogen, can replace carbon atoms, preferably 1 to 4 carbon atoms (or bonded to the carbon atoms) in this non-cyclic branched or unbranched radical.

aryl an organic radical derived from an aromatic hydrocarbon by the removal of one hydrogen atom. This radical can contain one or more heteroatoms as part of the aromatic hydrocarbon ring system.

aralkyl an organic radical in which one or more aryl groups, preferably 1 to 3, are substituted for hydrogen atoms of an alkyl radical, cycloalkyl a cyclic hydrocarbon radical containing from 3 to 20 (preferably 3 to 12) carbons; heteroatoms, preferably oxygen, sulfur, and nitrogen, can replace (or be bonded to) the atoms in this cyclic hydrocarbon radical.

cycloalkylalkyl an organic radical in which one or more cycloalkyl radicals, preferably 1 to 3, are substituted for hydrogen atoms of an alkyl radical containing from 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms.

SWO 89/11486 PCT/US89/02293 7 alkenyl an aliphatic, unsaturated, branched or unbranched hydrocarbon having at least one double bond and 2 to 20 (preferably 3 to carbons. Heteroatoms, preferably sulfur, oxygen, and nitrogen, can replace saturated carbon atoms in this radical or be bonded to the saturated carbon atoms.

aralkenyl an organic radical with one or more aryl groups, preferably 1 to 3, are substituted for hydrogen atoms of an alkenyl radical.

cycloalkenyl a cyclic hydrocarbon radical having from 3 to 20 (preferably 4 to 12) carbons, and at least one double bond.

Heteroatoms, preferably oxygen, sulfur and nitrogen, can replace saturated carbons in this radical or be bonded to the saturated carbons.

alkynyl an aliphatic, unsaturated, branched or unbranched hydrocarbon radical containing at least one triple bond and 2 to 20 (preferably 3 to 10) carbons. Heteroatoms, preferably oxygen, sulfur, and nitrogen, can replace (or be bonded to) saturated carbons in this radical.

aralkynyl an organic radical in which one or more aryl groups, preferably 1 to 3, are substituted for hydrogen atoms of an alkynyl radical.

cycloalkynyl a cyclic hydrocarbon radical containing from 6 to 20 carbon atoms, preferably 7 to 12 carbon atoms, and at least one triple bond. Heteroatoms, preferably oxygen, sulfur and nitrogen, can replace saturated carbon atoms in this radical.

WO 89/11486 PCT/US89/02293 Heteroatom substituted radicals In all these radicals, including alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl, aralkenyl, cycloalkenyl, alkynyl, aralkynyl, and cycloalkynyl, heteroatoms, preferably sulfur, oxygen, nitrogen, and halogens, can replace hydrogen atoms attached to carbon. As described in the definition for each radical, heteroatoms, preferably oxygen, sulfur and nitrogen, can replace carbon atoms at saturated positions in alkyl, aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl, aralkenyl, cycloalkenyl, alkynyl, Soralkynyl, and cycloalkynyl radicals.

Heteroatoms, preferably sulfur, oxygen, and nitrogen can also replace carbon as part of the aromatic ring system in aryl radicals. Of course, heteroatoms cannot replace carbon atoms in a radical where the carbon atom to be replaced is joined to the heteroatom linked to phosphorus.

phosphorodithioate internucleotide linkage an internucleotide linkage having the general formula 5'-nucleoside-0-PS2-0-nucleoside-3' which can be illustrated with the following sfructure where B and A are as defined previously.

I 0 phosphorothioate internucleotide linkage an internucleotide linkage having the general formula 5'-nucleoside-0-POS-0-nucleoside-3' WO089/11486PC/U9/23 PCF/US89/02293 which can be illustratred with the following structure where B and A aa:e as defined previously. -3 o phosphorothioamidate internucleotide linkage an internucleotide linkage having the general formula 5 -nucleoside-O-PSNHR 6-O--nucleoside-3 and 5'-nucleoside-O-PSNR 6 R 7 -O-nucleoside-3' which can be illustrated with the following structures where B,A,R 6 and R 7are as previously defined. I L4 0' t?1

A

phosphoromidate internucleotide linkage an internucleotide linkage having the general formulae 5 '-nucleoside-O-PONHR 6-O-nucleoside-3 and 5'-nucleoside-O--PONR 6R 7-O-nucleoside-3' which can be illustrated with the following structures where B,A,R 6 and R7are as previously defined.

I,3 NA) )0=0 0 13 0 1 13 R6 0 A' WO 89/11486 PCT/US89/02293 phosphorothiotriester internucleotide linkage an internucleotide linkage having the general formulae 5 '-nucleoside-0-PSOR 4 -0-nucleoside-3' which can be illustrated with the following structure where B,A, and R4 are as previously defined. 8 8 R* o -Ps 0 3' Amines from which the substituent group W can be derived include a wide variety of primary amines such as methylamine, ethylamine, propylamine, isopropylamine, aniline, cyclohexylamine, benzylamine, polycyclic amines containing up t6 carbons, heteroatom substituted aryl or alkylamines having up to ten heteroatoms, 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 dimethylamine, diethylamine, diisopropylamine, dibutylamine, methylpropylamine, methylhexylamine, methyicyclopropylamine, ethylcyclohexylamine, methylbenzylamine, methylcyclohexylmethylamine, butylcyclohexylamine, morpholine, 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, adenine, hypoxanthine, guanine, and their derivatives, and pyrimidines, T i i 1 I WO89/11486 PCT/US89/02293 cytosine, uracil, thymine, and their derivatives.

The blocking groups represented by R 1

R

2 and R3 in the above formulae include triphenylmethyl (trityl), p-anisyldiphenylmethyl (methoxytrityl), di-p-anisylphenylmethyl (dimethoxytrityl), pivalyl, acetyl, 4-methoxytetrahydropyran-4-yl, tetrahydropyranyl, phenoxyacetyl, isobutyloxycarbonyl, t-butyldimethylsilyl, triisopropylsilyl, alkyl or aryl carbonoyl, and similar blocking groups well known in the art.

The general reaction scheme A for synthesizing compound Ia and II is shown in the- following overview: WO 89/11486 WO 9/1486PCI/US89/02293

Q

y

I

-P

I

CIL

I-

X_ '1

I-

t1 -o \C 'N 7L/%I

C

-P

13 -0 43

C

C

~jjiI WO 89/11486 W089/1486PCI7/US89/02293 The preferred reaction scheme A is represented as follows: RI HO X -I p, Cfe PiO N~x5 p-X .ZZ?7L S 14 ;-11 0/

.P-K

/1 o fo 0

LL

_R'

3 11- 0-- ~-ii~i o (t WO 89/11486 PCI/US89/02293 14 wherein R 1

R

3 B, A, X, Z, L and Y are as previously defined; and M is sulfur single bonded to phosphorus and to R 8 where R 8 is a heteroatom substituted or unsubstituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl, cycloalkenyl, aralkenyl, alkynyl, aralkynyl or cycloalkynyl. Compounds VIII and VIIla 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 VIIIa are a subset of compounds II. likewise, compounds V and Va are a subset of compounds Ia.

The process of reaction scheme A involves condensation of IIIa with IVa, which preferably is bis(dimethylamino)chlorophosphine or dipyrrolidinylchlorophosphine, to yield IXa in the presence of triethylamine. Further addition of a mercaptan, which preferably is 2,4dichlorobenzylmercaptan, in the presence -of triethylamine hydrochloride generated in the first step leads to the conversion of IXa to Va. Table 1 31 lists the P-NMR characterization data for a series of Va derivatives where the nucleoside base amine functionality and mercaptan are altered in a systematic manner. Reaction of Va with VIa and an activator tetrazole, tetrazoles and substituted triazoles, alkylammonium salts, arylalkylammonium salts, substituted and unsubstituted pyridinium salts of tetrafluoroborate, and substituted and unsubstituted pyridinium and imidazolium salts of acids, 5-substituted tetrazoles, halogenated carboxylic acids and N-hydroxybenzotriazole) yields VIIa, the dinucleoside S-(2,4-dichlorobenzyl) phosphite, which can be preferably oxidized with sulfur to yield VIIIa, the dinucleoside phosphorodithioate triester with P(Y,Z).

Of course oxidation with t-butylperoxide yields the Iiuloiepopooihot retrwt WO 89/11486 2 PCr/US89/02293 corresponding dinucleoside phosphorothioate triester P L) Table 1. 31PNR Characterization of Deoxynucleoside Phosphorothioamidites (Va) Base (B3) Amine

MX

Mercaptan

(M)

31p-NMRa Ui)

T

T

T

T

CB z C

B

C z C

B

AB z AB z AB z A Bz G iB G iB G iB G iB pyrrolidinyl pyrrolidinyl dime thy lamino dimethylamino pyrrolidinyl pyrrolidinyl dimethylamino dirnethylamino pyrrol idinyl pyrrolidinyl dimethylamino dimethylamino pyrrolidinyl pyrrolidinyl dimethylamino dimethylamino 2',4-dichlorobenzyl 4 -chlorobenzyl 4 -chlorobenzyl 2 ,4-dichlorobenzyl 2 ,4-dichlorobenzyl 4 -chlorobenzyl 4 -chlorobenzyl 2, 4-dichlorobenzyl 2 ,4-dichlorobenzyl 4 -chlorobenzyl 4-chlorobenzyl 2 ,4-dichlorobenzyl 2 ,4-dichlorobenzy.

4 -chlorobenzyl 4 -chlorobenzyl 2, 4-dichlorobenzyl 164.8; 161.8 164.2; 161.0 172.3; 170.5 172.1; 170.4 165.1; 162.6 161.8 ;159 .9 171.9 ;170 .7 172 .0;171.0 163 .8 ;162 .7 163 162 .3 171 170.9 171.7;170 .9 163.9 ;160 .9 163 .4 ;161.6 171.5 ;169 171 .9;169 .6 a 31 P-NMR were recorded in CDC1 3on a Brucker WM-250 Bz with 85% aqueous H 3P0 as external standard. T, C A ,z and G iBrefer to thymine, N-benzoylcytosine, N-benzoyladenine, and N-isobutyrylguanine respectively; R I is dimethoxytrityl; A is hydrogen.

A second general reaction scheme for synthesizing compounds la and II, scheme B, is shown in the following overview: WO 89/11486 WO 8911486PcI/US89/02293',* -0

H

13A /1 0 0 L0 81 t7r '3 7.

O 0

I

C.

-WO 89/11486 PCU/US89/02293 17 The preferred reaction scheme B is represented as follows:* Rf C) LTo_ -1- /1 73 0 (1 HO 0 I'3

R

1 0 o* R~cB -Z \1

T?

L I 0i WO 89/11486 PCT/US89/02293 18 Thus it can be seen that the processes of reaction schemes A 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 IIIa with IVb which is 4-chlorobenzylmercaptyl-bis(diisopropylamino)phosphine to yield Va with tetrazole as catalyst. Reaction of Va with VIa and an activator tetrazoles and substituted triazoles, alkylammonium salts, arylalkylammonium salts, substituted and unsubstituted pyridinium salts of tetrafluoroborate, and substituted and unsubstituted pyridinium and imidazolium salts of acids, 5-substituted tetrazoles, halogenated carboxylic acids and N-hydroxybenzotriazole) yields VIIa, the dinucleoside S-(4-chlorobenzyl)phosphite, which can be preferably oxidized with sulfur to yield VIIIa, the dinucleoside phosphorodithioate triester with P(Y, Of course oxidation with t-butylperoxide yields the corresponding dinucleoside phosphorothioate triester, P L).

Activators that are more acidic than tetrazole, such as certain 5-substituted tetrazoles -nitrophenyl)tetrazole) and pyridinium tetrafluoroborate, can be used with success to Ao IWO 89/11486

C,

PCr/US89/02293 activate Va. Certain side reactions, however, can lead to reductions in yields of the correct product.

A 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: -4- X o P -ZT 7I -O-s^O, 0 P 1 I i' 7

Z

Lp 0 1 3 -0 WO 89/11486 WO 8911486PCT/US89/02293 The preferred reaction scheme C is represented as follows: 3 4 (9 _7 0

>QJZ

xo Ii f 1 oQ j 0 RID 7 0 Al1

-P

*57a.

R, 0 01 0 o 0 SWO 89/11486 PCT/US89/02293 21 wherein R, R 3 B, A, X, W, Z, Y, and V are as previously defined and Q is H. Compounds XII and XIIa 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 XIIa 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 H2S and an activator such as tetrazole yields the dinucleoside H-phosphonothioate, XIa, which can be chemically converted by oxidation with sulfur to dinucleoside phosphorodithioates, P by oxidation with iodine in the presence of amines to phosphorothioamidates, P W or by alkylation of the corresponding dinucleoside phosphorodithioate to phosphorodithioate triesters, P by oxidation with iodine in the presence of alcohols to phosphorothioate triesters, P and by oxidation with aqueous iodine to phosphorothioates, P Compound Xa can also be reacted with a mercaptan in the presence of an activator such as tetrazole to yield the dinucleoside phosphorothioite, VIIa, which can be chemically converted to XIIa by oxidation with sulfur to dinucleoside phosphorodithioates, P and by oxidation with t-butylperoxide or aqueous iodine to phosphorothioates, P Thus it can be seen that the compounds XII and XIIa, as synthesized by process C, can be derived either from two intermediates, XIa and VIIa, or from one of these two intermediates. For example XIIa, where P can be derived from either intermediate XIa or VIIa. For i ~u WO 89/11486 PCT/US89/02293 22 XIIa 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 A, B,and C. In some cases where Z and Y are linked to phospho)ru anO, therefore, yield a dinucleoside phosphorodithioate, processes A, 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 internucleotide 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 ;1 IWO 89/11486 PCUS89/02293 23 for synthesizing the other polynucleotide linkages (although, other methods such as phosphate triester and phosphate diester and H-phosphonate 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 particularly useful in the chemical synthesis of any deoxyribonucleic acid (DNA) 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 Ib can be represented by the following general reaction scheme, scheme D: pCT/US89/O 22 93 a WO 89/11486 0 4 -c 13 0 3

II

'f-i,

V

WO 89/11486 W089/1486PC'F/US89/02293 The preferred reaction Scheme D is represented as follows: IR3 0o =U7 C%

C

0

Y

P0

-Z

wherein R 1 B, A, Q, X, 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.

WO 89/11486 PCT/US89/02293 26 The process of scheme D involves synthesis of XIV and XIVa from IIIa and XIII or XIIIa. Reaction of XIV or XIVa with H2S and an activator such as tetrazole yields a new compound, XVa, the nucleoside H-phosphonothioate, which can be chemically converted by oxidation with sulfur to nucleoside phosphorodithioates, P V, Y) and by alkylation of the nucleoside phosphorodithioate to the nucleoside phosphorodithioate triesters, P V, Y) The preferred novel compounds according to the present invention are those compounds of general formula Ia and II wherein (for Ia) Y is a substituent having sulfur single bonded to phosphorus and to R where R 5 is a heteroatom substituted or unsubstituted blocking group; A is H; R 1 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 5 where R 5 is a heteroatom substituted or unsubstituted blocking group; A is H; R 1 is a blocking group; B is a nucleoside or deoxynucleoside base having art-recognized blocking groups; and R 3 is H. These new compounds can then be used to prepare oligonucleotides having phosphorodithioate internucleotide linkages with 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'-phosphorodiamidite. The initial reaction is accomplished by dissolving the nucleoside in an organic solvent such as dioxane or tetrahydrofuran containing triethylamine to take up the liberated hydrochloric acid and adding a bis(dialkylamino)chlorophosphine. The resulting lu, S WO 89/11486 PCT/US89/02293 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 XIIa. The preferred pathway is to react Xa with a mercaptan in the presence of tetrazole to yield VIIa which is further treated with elementary sulfur to form the deoxydinucleotide phosphorodithioate, XIIa, where A second pathway 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 H-phosphonothioate with elementary sulfur in an organic solvent such as a mixture of toluene and lutidine yields the dinucleoside phosphorodithioate, XIIa where Reaction of the dinucleoside phosphorodithioate with an alkyl or aryl halide capable of alkylating thiols yields the sulfur protected dinucleoside phosphorodithioate triester, XIIa. 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 3 by conventional methods from XIIa 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.

WO 89/11486 PCT/US89/02293 28 As a further embodiment of the invention, the dinucleoside phosphorodithioates are preferably prepared by either reaction schemes A 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 A, a bis (secondaryamino) chlorophosphine, which is prepared by standard procedures, is reacted with an appropriately protected nucleoside dissolved in acetonitrile and triethylamine. 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-aralkyldinucleoside phosphite, VIIa, which, after oxidation with elementary sulfur, yields VIIIa with P(Z, the dinucleoside phosphorodithioate triester. These procedures shown in schemes A 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-aralkyldialkylaminophosphoramidite or thioamidite Va in concert with sulfur oxidation, WO 89/11486 PCT/US89/02293 29 polynucleotides having only phosphorodithioate linkages can be prepared.

The synthesis of aralkylmercaptyl-bis-(dialkylamino)phosphine, IVb, is effected in an organic solvent solution whereby the bis(dialkylamino)-chlorophosphine, IVa, is first synthesized and then further condensed with an aralkylmercaptan. 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 recrystallized 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 A 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.

WO 89/11486 PCT/US89/02293 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 internucleotide 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 internucleotide 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 tetrafluoroborate, trifluoromethylphenyltetrazole and trifluoromethyltetrazolide salts. This is especially the case where X is diisopropylamino. 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 A.

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 2,4-dichlorobenzyl, phenyl and heteroatom substituted phenyl, and heteroatom substituted or unsubstituted alkyl substituents such as 3- cyanoethyl and methyl.

t? i WO 89/11486 PC/US89/02293 31 The secondary amino moieties as part of the phosphines IVa and IVb and the nucleoside thioamidites, 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 dimethylamino, diethylamino, diisopropylamino, dipropylamino, dibutylamino, dipentylamino, 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 detritylation 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 tetrafluoroborate, para-nitrophenyl tetrazole, trifluoromethylaryl 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 i prepared, Va is activated, condensed, and oxidized WO 89/11486 PCT/US89/02293 32 with sulfur as described above, repetitively with a nucleoside preferably attached to a polymer support to yield polynucleotides having phosphorodithioate linkages. Dinucleoside phosphorodithioate triesters VIIIa or XIIa 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 phosphorodithioate linkages, the product can, if desirable, be freed of blocking groups. Thus the first step is treatment with preferably trialkylammonium 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 trichloroethyl or/ -cyanoethyl), then the deprotection protocol should be modified accordingly.

SWO 89/11486 PCT/US89/02293 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 Bis(dimethylamino)chlorophosphine was prepared by adding tris(diniethylamino)-phosphine (36.3 ml, 32.6 g, 0.2 mole) and trichlorophosphine (8.7 mld, 13.7 g, 0.1 mole) to anhydrous ether (100 mol). After stirring for 3 hours at room temperature, solvent was removed by concentration in vacuo at room temperature. The product was then distilled p. 72-75 C)Q at reduced pressure (approx. 16 mm Hg) using a water aspirator to yield 30 g of product.

31 P-NM(CHC3)6 163.06. This procedure is also used to produce dipyrrolidinylchlorophosphine. Preparation of thiophosphoramidites of the .0p formula: *DMTO

B

0 P

M

1 5 represented as Va (Reaction Scheme A) where B 1-Thyminyl; B 1-(N-4-benzoylcytosinyl); B 9-(N-6-benzoyladeninyl); B 9-(N-2-isobutyrylguariinyl); and DMT =di-p-anisylphenylmethyl (diinethoxytrityl) M =4-chlorobenzylmercaptan or 2,4-dichlorobenzylmercaptan

SRA

4 X =N,N-dimethylamino or pyrrolidinyl VT o 34a and the further use of these compounds to prepare oligonudeotides having phosphorodithioate internudleotide linkages.

The following example describes the synthesis of 5'-O-dimethoxytrity.1-N4benzoyldeoxycytidylyl-3'-S(4-chlorobenzyl) phosphorthiopyrrolidinite. The same procedure can be used for the other suitably protected deoxynudeosides. Similarly the same procedure is S WO 89/11486 PCT/US89/02293 useful for the 2.4-dichlorobenzyl and 4-chlorobenzyl protected sulfur derivatives and for the N,N-dimethylamino and pyrrolidinyl amidites. Table 1 31 summarized the P-NMR data for all these amidites.

5'-0-Dimethoxytrityl-N4-benzoyldeoxycytidine (317 mg, 0.5 mmol) was dissolved in acetonitrile (2 ml) and triethylamine (1 ml) under argon. Bispyrrolidinylchlorophosphine (124 mg, 0.6 mmol) was added which was followed by the immediate formation of a precipitate (31P-NMR of the reaction product was at 133.8 ppm). After 5 minutes stirring at room temperature, 4-chlorobenzylmercaptan (159 mg, Immol) was added to the reaction mixture and the solution, including the precipitate, was concentrated to a glass in vacuo at room temperature. The glass was resuspended in acetonitrile (2 ml). The 31

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 bispyrrolidinylchlorophosphine 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 backextracted with deacidified ethylacetate (10 ml). The organic solutions were combined, dried for 1 hour over sodium sulfate in the presence of 10% (volume) triethylamine, filtered, and the filtercake washed with 5 ml deacidified ethylacetate. The organic solution was then concentrated in 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, After filtration, the product was -I r C i~L i WO 89/11486 PCT/US89/02293 36 dried in vacuo over phosphorus pentoxide and potassium hydroxide and isolated in 83.1% yield (741 mg).

H-NMR (CDC1 3 8.76 (broad s, 1H, NH), 8.37 JHH 7.47 Hz, 0.5H, H5, cytosine), 8.31 JHH S7.48 Hz, 0.5H, H5, cytosine), 7.94 JHH 7.37 Hz, 2H, H2 and H6 of benzoyl group), 7.68-7.54 (m, 3H, H3, H4, H5 of benzoyl group), 7.44-7.14 14H, aromatic protons of 4-chlorobenzyl group, H2, H6 of anisyl (DMTr), ph-protons (DMTr), H6 cytosine)), 6.91 JHH 7.57 Hz, 4H, H3, H5 of anisyl DMTr), 6.33 1H, 4.72 1H, 4.22 1H, 4'H), 3.84 (d of singletts, 6H, methyl protons of anisyl DMTr), 3.84-3.76 2H, methylene protons of 4-chlorobenzyl group), 3.59-3.35 2H, 3.19-3.01 4H, methylene protons of pyrr-lidinyl group a to nitrogen), 2.84-2.75; 2.37-2.26 2H, 1.79-1.71 4H, methylene protons of pyrrolidinyl group b to nitrogen). 31P-NMR (CDC1) 161.79, 159.97. Fab+: 923 (M S) 907 (M 0) tic: Rf .75 (chloroform:ethylacetae:triethylamine (45:45:10, v/v/v).

Using a deoxynucleoside attached covalently to a silica based polymer support through the 3'-hydroxyl Patent 4,458,066), synthesis of deoxyoligonucleotides containing phosphorodithioate 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 C1, mole of deoxynucleoside on silica for 30 sec (step i) followed by a 400 sec oxidation with sulfur in pyridine:carbon disulfide 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

A

.I

WO 89/11486 PC/US89/02293 37 previously for synthesizing natural DNA from deoxynucleoside phosphoramidites Patent 4,415,732 and Science 230, 281-285, 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 internucleotide bonds.

Synthetic deoxyoligonucleotides were isolated free of protecting groups via a two-step protocol (thiophenol:triethylamine:dioxane, 1:1:2, v/v/v for 24 h followed by cone. ammonium hydroxide for and then purified to homogeneity by standard procedures (polyacrylamide gel electrophoresis and reverse phase hplc). 3 1 P-NMR spectra (Figure 2) of phosphorodithioate DNA indicated that this synthesis protocol yielded DNA containing exclusively phosphorodithioate internucleotide linkages. No hydrolysis of these dithioates to phosphorothioates 31 P-NMR 56) or phosphate was observed. So far a pentadecaner homopolymer containing fourteen dithioate linkages, lac and cro operators (O 1) with multiple dithioates at defined sites, and a cro operator segment (0 l) containing seventeen contiguous dithioates have been synthesized.

t v i 4- WO 89/11486 PCT/US89/02293 Figure 1. Synthesis of DNA on a Polymer Support. ,silica based polymer support.

B

HO0 0

B

HO0 0 1 P

-I

2 WO089/11486 PCr/US89/O2293 Figure 2. 31P-NMR Spectra of a Polynucleotide Derivatives. Spectra of d(C) 15containing exclusively. phosphorodithioate internucleotide linkages (113 ppm in D 2 0) 31P-NMR spectra was recorded on a Varian VXR-500S. Aqueous 85% H 3P0 was the external standard.

150 140 130 120 110 100 90 so 70 60 50 40 30 20 10 0 Ppm i: II II WO 89/11486 PCT/US89/02293 EXAMPLE II Preparation of thiophosphoramidites of the formula: 0 Sp-s

L

represented as Va (Reaction Scheme B) B 1-Thyminyl; B l-(N-4-benzoylcytosinyl); B 9-(N-6-benzoyladeninyl); B 9-(N-2-isobutyrylguaninyl); and DMT dimethoxytrityl The synthesis of compounds Va begins with the preparation of 4-chlorobenzylmercaptyl-bis- (diisopropylamino)phosphine. Phosphorus trichloride mole, 68,665 g, 43.6 ml) was dissolved in 300 ml anhydrous tetrahydrofuran (THF). The PC13 solution was cooled to -18 0 C by a NaCI ice mixture. Diisopropylamine (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 diisopropylammonium 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 1 material in the reaction mixture P-NMR 132.4 ppm). The newly formed diisopropylammonium chloride SWO 89/11486 PCT/US89102293 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. 4-Chlorobenzylmercaptan (50 mmol, 7.93 g, 6.6 ml) was dissolved in anhydrous ether (300 ml) and an amount o a sodium hydride suspension in oil (50% NaH 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 mnol, 13.34 g) was added and the reaction mixture was stirred until gas evolution stopped (4 hours at rt). 3P-NMR of the reaction mixture indicated quantitative conversion of the chlorophosphine to the desired 31 product without any side reactions (1P-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)phosphine) 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-dimethoxytrityl nucleoside (5 mmol) and 4-chlorobenzylmercaptyl-bis-(diisopropylamino)phosphine (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 (diisopropylammonium tetrazolide) precipitates. After 16 hours, the reaction was quenched with pyridine (1 ml) and diluted into acid WO 89/11486 PCT/US89/02293 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, ethylacetate and triethylamine (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 phosphorthioamidite was isolated after drying the precipitate in vacuo over P 05/KOH (3.33 g, 80.1% yield).

P NMR 161.3 and 159,97 ppm (two diastereomers) with respect to external standard of H 3 P0 4 for the thymidine derivative. H NMR 8.0 7.59 and 7.58 (2 x d, JHH 1.2 Hz), 7.42-7.19 6.83 (d, JHH 8.7 Hz), 6.37 H 1 4.65-4.58 H 3 2.05-1.83 H 6 3.80-3.61 CH 2 of p-chloro benzyl), 3.78 H 6 3.48-3.29 H 5 2.45-2.24

H

2 1.44 (CH 3 1.17-1.04 CH 3 of isopropyl).

I

IWO 89/11486 PCIT/US89/02293 43 EXAMPLE III Synthesis of Dinucleoside Phosphorodithioate Triesters of the formula: p/nTO 0 0 0 o Cq-3 represented as VIIIa (Reaction Scheme B) where B 1-Thyminyl; B 1-(N-4-benzoylcytosinyl); B 9-(N-6-benzoyladeninyl); B 9-(N-2-isobutyrylguaninyl); and DMT dimethoxytrityl 5'-O-dimethoxytritylthymidine-3'-S- (4-chlorobenzyl)diisopropylaminophosphoramidite (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 with sulfur (1 atomic equivalent, 32 mg). The reaction mixture was then diluted with ethylacetate 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 WO 89/11486 44

I

PCT/US89/02293

E

i

L

B

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 l.l.l-trichloroethane and methanol (92.5:7.5, 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% H3PO 4 as an external standard) FAB mass spectrum, 1047 (M 921 (-p-chlorobenzyl), 743 619 (-DMT and 4-chlorobenzyl), 519 (3'-O-acetylthymidine 5'-0-4-chlorobenzyl phosphorodithioate), 395 acetylthymidine phosphorodithioate).

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

These dinucleoside phosphorodithioate triesters can also be prepared by using pyridinium tetrafluoroborate as an activating agent. Pyridinium tetrafluoroborate was prepared by dissolving HBF 4 mmole, 1.9 g of a diethyletherate, Aldrich Chemical Co.) in dry dichloromethane (5 ml) and adding this solution with stirring to dry pyridine (791 mg, 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 P 2 0 5 In a typical reaction, 3'-O-acetylthymidine (142 mg. mmole) was allowed to react with tritylthymidine-3'-S(4-chlorobenzyl) diisopropylaminophosphoramidite (833 mg. 1 mmole) in the presence of pyridinium tetrafluoroborate (334 mg, 2 mmole) in dry acetonitrile (5 ml). After ten minutes the reaction mixture was quenched by addition of W0 89/1 1486

T

PCT/US89/02293 atomic equivalents of sulfur (640 mg) in pyridine (2 ml), concentrated in 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 chromatography (CH3CC13: CH3OH, 95:5, v/v), the dinucleoside phorphorodithioate in protected form was isolated by precipitation into pentane yield). The following dinucleoside phosphorodithioates in approximately 60% yield have been prepared via this procedure.

S-(4-chlorobenzyl)-3'-0-(5'-0-thymidylyl)-phosphorodithioate. FAB mass spectrum, 1005 (M 847 (M 4-chlorobenzylmercaptyl), 703 (M DMT 455 (M DMT-4-chlorobenzylmercaptyl H) FAB mass spectrum, 879 (M 4-chlorobenzyl) 779 (M 477 (thymidine-S-4-chlorobenzylphosphorodithioate), 355 (thymidine dithioate); 3P NMR (CDC1 3 96.44 UV (EtOH) max 228, 268 nm.

5'-O-Dimethoxytritylthymidine S-(4chlorobenzyl)-3'-0-(5'-O-N2-isobutyryldeoxyguanosinyl)- phosphorodithioate. FAB+ mass spectrum, 1277 (M Na) 952 (M DMTr) 3 1 -pNMR (CDC1 3 95.8, 96.14; UV (EtOH) max 262 nm.

5'-O-Dimethoxytrityl-N6-benzoyldeoxyadenosine S-(4-chlorobenzyl)-3'-O-(5'-O-N4-benzoyl- 31 deoxycytidine)-phosphorodithioate. P NMR (CDC1 3 93.89, 93.31.

Synthesis of dinucleoside phosphorodithioates, especially with strong acid catalysts such as 4-nitrophenyltetrazole or pyridinium tetrafluoroborate, 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 WO 89/11486 PCT/US89/02293 4-chlorobenzylphosphonothioamidates are formed when phosphorothioamidites 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.

c, WO 89/11486 PC1'/US89/02293 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.

i i i WO 89/11486 PCJ/US89/02293 47 EXAMPLE IV Synthesis of Dinucleoside H-Phosphonothioate of the formula: 0

O

C 3 represented as XIa (Reaction Scheme C) where B 1-Thyminyl; B 1-(N-4-benzoylcytosinyl); B 9-(N-6-benzoyladeninyl); B 9-(N-2-isobutyrylguaninyl); and DMT dimethoxytrityl The first step was condensation of oxytritylthymidine with bis(diisopropylamino)-.

chlorophosphine in dioxane containing triethylamine.

The resulting phosphorodiamidite was reacted without isolation with 3'-O-acetylthymidine to yield a homogeneous dinucleoside amidite in 62% yield after silica gel chromatography triethylamine in ethylacetate). Synthesis of the dinucleoside Hphosphonothioate proceeds by dissolving the dinucleoside phosphoroamidite (470 mg, 0.5 mmol) in acetonitrile (5 ml), bubbling H2S 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 WO 89/11486 PCT/US89/02293 48 concentrated to a gum on a rotary evaporator, redissolved in ethylacetate (50 ml) and extracted twice with 2 M triethylammonium bicarbonate (pH 7.4, 20 ml each). After concentrating in vacuo to a gum, the product was dissolved in dichloromethane (5 ml) and isolated by precipitation into pentane (400 mg, FAB+ mass spectrum, 527 (anhydro DMT dT): FAB mass spectrum, 890 623 (DMT dT-3'-PHO 2 363 (M-527, 5'-PHO 2 -dT-3'-OAc); 31 NMR 71.7 and 70.7 1 JH 673.8 Hz and 676.3 Hz; H NMR 7.81 and 7.80

HP

JHP 671.4 Hz and 676.7 Hz) 7.55 and 7.53

H

6 7.37-7.20 aromatic), 6.82 J 8.8 Hz, DMT), 6.49 and 6.26 H 1 5.49 and 5.25 (m, 31 4.35 H4,) 4.19 H5 4.07 (mi H4,) 3.76 MeO-DMT), 3.42 2.54-2.32 H 2 2.08 and 2.07 (2 x s, CH -acetyl), 1.90 CH 3 1.43 CH 3 Rf 0.35 and 0.28 (methanol/dichloromethane, 1:9, v/v).

WO089/11486 PCT/US89/02293 49 EXAMPLE V Synthesis of a Dinucleoside Phosphorodithioate of the formula:' 0 B 1-Thyminyl; B 1-(N-4-benzoylcytosinyl); 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 inmol 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 triethylamine) afforded 70% isolated yield.

FAB +mass spectrum, 303 (DMT FAB- mass spectrum, 921 395 (5-P 2 0 -dT-3'-OAc) 3P NMR 112.7; 1HNMR 8.12 NH), 7.90 and 7.60 (2 x s, H 6 7.40-724 (in, aromatic), 6.80 JH 8.8 Hz, DMT), 6.43 (in, Hl,) ,5.46-5.36 (in, H 3 4 .40 (in, H 4 4.16 (in, Hr,) 3.76 MeO-DMT) 3.52 (in, H 5 1 2.28 (in, H 21 2. 05 (Ch 3 -acetyl) 1.97 (CH 3 T) 1. 58 1 WO89/11486 PCT/US89/02293

CH

3 Rf 0.14 (methanol/dichloromethane, 1:9, v/v).

The dinucleoside phosphorodithioate was deprotected by standard procedures and isolated in 86% yield after ether extractions (3x),sephadex G10 gel filtration (H 2 and lyophilization as the ammonium 31 salt. FAB mass spectrum, 579 P NMR (D.0) 1 113.3; H NMR 7.60 and 7.46 (2 x s, H 6 6.11 and 5.99 5.17 H 3 4.85 H 3 4.15 (m, 4.03 and 3.62 H 5 2.21 1.88 m, CH R 0.25 (methanoltriethylamine/chloroform, 15:1:84, When the dinucleoside phosphorodithioate was phosphorylated with T4-polynucleotide kinase and -32P]ATP, the rate of kination was approximately one-half that of unmodified dithymidine phosphate under identical conditions.

Further testing with snake venom phosphodiesterase (Crotalus adamanteus venon, Sigma) indicated that the phosphorodithioate 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 conc. ammonium hydroxide at 55 0 C (16 h) as no degradation or isomerization was observed P NMR, thin layer chromatography).

~WO 89/11486 PCr/US89/02293 51 EXAMPLE VI Synthesis of a Dinucleoside Phosphorodithioate 3'-Phosphoramidite of the formnula:

-O

CL -P represented as XVIIa where B I-Thyminyl; B 1-(N-4-benzoylcytosinyl); B 9-(N-6-benzoyladeninyl); B =9-(N-2-isobutyrylguaninyl); and DMT dimethoxytrityl In order to introduce the phosphorodithioate linkage into oligonucleotides, a protection/deprotection scheme for the phosphorodithioate internucleotide linkage was developed. Thus the dinucleoside phosphorodithioate in protected form (XIIa) (57 mg, 0.06 nunol) was alkylated with c< ,2,4-trichlorotoluene (50 'Al, I h, 55'C) in acetonitrile to yield the dinucleoside phosphorodithioate triester quantitatively. Further testing revealed that it was completely stable to reagents used in DNA synthesis trifluoroacetic acid in dichloromethane and iodine in aqueous lutidine/THF) and that the phosphorodithioate triester was WO 89/11486 PCT/US89/02293 52 specifically S-dealkylated by treatment with thiophenolate (thiophenol:triethylamine:dioxane, 1:1:2, v/v/v, tj 3 min at rt). FAB mass spectrum, 527 (anhydro DMT dT); FAB mass spectrum, 923 (M 1-dichlorobenzyl) 813 (DMT dT-3'-PSOS-dcb) 553 31 (5'-PSOS-dcb-dT-3'OAc); 31P NMR (CH 3 CN, ext. lock) 1 94.4 and 93.7, H NMR 7.55 and 7.52 (2 x s, H 6 7.37-7.23 aromatic), 681 J 4.6 Hz, DMT), 6.34 and 6.28 H 1 5.38 and 5.01 4.24-4.08 (m,CH 2 -benzyl, H 5 1)

H

4 3.76 (s,MeO-DMT), 3.42 (m,H 5 2.39 (m,H 2 1 2.08 CH3-acetyl), 1.89 and 1.87 (2 x s CH 3 1.43 and 1.42 (2 s, CH3-T) Rf 0.74 (methanol/triethylamine/chloroform, 15:1:84, v/v/v) Conversion to a synthon useful for DNA synthesis was a two step process. The dinucleoside phosphorodithioate triester was first deacylated (the 3' acetyl group) using 0.15 M tert-butylamine in methanol (0 0 C, 10 h) and purified by silica gel chromatography to yield IIa. Less than 5% cleavage 31 of the internucleotide linkage (31P NMR, TLC) was observed. The deacylated compound was then reacted with bis (diisopropylamino)-2-cyanoethoxy phosphine 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 H 6 7.33-7.27 (in, aromatic), 6.84 3 8.5 Hz, DMT), 6.39-6.29 H 1 5.44 (m, H '1 3.79 MeO-DMT) 1.90 CH 3 1.45 (s,

CH

3 -T),1.18 J 6.6 Hz, CH 3 -iPr). R 0.29 and 0.17 (chloroform/ethylacetate/triethylamine, 45:45:10, The resulting dinucleotide phosphoramidite, XVIIa, has been used successfully in combination with unmodified mononucleoside phosphoramidites for the synthesis of 26-mer DNA fragments containing the phosphorodithioate linkage (98.2% SWO 89/11486 PCT/US89/02293 53 coupling efficiency). These syntheses were completed on silica be ed 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) i WO 89/11486 WO 8911486PCT/US89/02293 EXAMPL~E VII Synthesis of Dinucleoside Thioarnidates, Thiotriesters, and Thioates of the formulae: A) P 0 C O

CIG

0 CU3 0 11 o C NY 0

C~-

I.

X I,,I1 6 IO(ZI-4) ZW Cy where B -Thyminyl; B l-(N-4-benzoylcytosinyl) B =9-(N-6-benzoyvladeninyl); B =9-(N-2-isobutyry.guaninyl) and DMT =dimethoxytrityl The dinucleoside H-phosphonothioate was also found to be useful as a versatile synthon for preparing several analogs rapidly (5 min) in quantitative yield 31P NMR). Thus, when oxidized with iodine/nbutylamine the phosphorothioamidate, 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); 31P NMR 74.4 and 74.0; 1H NMR 8.36 and 8.34 (2 x s, NH) 7.59 and 7.56 (2 x s, H 6 7.44-7.24 (in, aromatic) 6.82 J 8.7 Hz, DMT), 6.41 and 6.28 (in, Hi t 5.28 and 5.23 (in, H3 4.21 and 4.13 (i, H 4 1 (2 x) H 5 3.77 MeO-DMT), 3.43 (mn, H)5 2.94 (mn, CH 2 2.41 (mn, H 2 2.09 and 2.07 (2 x s, AV ~W89/11486 PCI/US89/02293 CH 3 -acetyl), 1.93 and 1.88 (2 x s, CH 3 1. 42 (s, CH 3 1.39-1.23 (in, CH 2 0.90 and 0.83 (2 x t, j 7.2 Hz and 7,1 Hz, CH 3 Rf 0.56 (methanol/dichioromethane, 1:9, vlv).

The dinucleoside H-phosphonothioate was converted quantitatively to a phosphorothioate triester by oxidation with iodine and 9-anthracenyl methanol equivalents) under anhydrous conditions, XIIa, FAB+ mass spectrum, 527 (anhydro DMT dT): FAB mass spectrum, 906 (m-anthracenylmethyl) 639 (DMT dT-3'-PSO 2 379 (5'-PSO 2 dT-3'-OAc). 31 pNMR 51.7 and 51.0. R f 0.41 (methanol/dichloromethane, 1:9, v/v).

Treatment of the dinucleoside H-phosphonothioate 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, 906 603 (M-DMT) 379 2 dT-3'-OAc) 31 PNMR 60.2 and WO 89/11486 PCT/US89/02293 56 EXAMPLE VIII Synthesis of Dinucleoside Phosphorodithioate Triesters of the formula: T8 0

I

0 oAc represented as XIIa, (Reaction Scheme C) where B 1-thyminyl; B 1-(N-4-toluoylcytosinyl) 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.

(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 -e WO 89/11486 PCT/US89/02293 57 THF (1 ml) The combined filtrates containing the deoxynucleoside phosphorodiamidite were pooled, concentrated in vacuo, and redissolved in acetonitrile (5 ml). 3'-O-acetylthymidine (639 mg, 2.25 mmol) and tetrazole (142 mg, 2.0 mmol) 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 was diluted with dichloromethane ml), extracted with an aqueous sodium bicarbonate solution w/v) dried over sodium sulfate, filtered, and concentrated in vacuo to a gum. The product was 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, 31 P-NMR (CH3CN 148.5, 148.1.

The deoxydinucleoside phosphoramidite was then converted to the deoxydinucleoside phosphorodithioate triester. The deoxydinucleoside phosphoramidite (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 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, 2-12zmethanol in dichloromethane as WO 89/11486 PCT/US89/02293 58 eluant) yields the deoxydinucleoside phosphoro- 31 dithioate triester (1.59 g, 1.52 mmol, P-NMR (CHC1 3 97.9, 96.4.

Removal of the 3'-0-acetyl group (0.15 M tert-butylamine in methanol, 0 0 C, 10 h) yields a deoxydinucleoside phosphorodithioate that can be used 31 for DNA synthesis (1.26 g, 1.28 mmol, 84%) P3-NMR (CHC1 3 97.3, 96.9. The deoxydinucleoside phosp horodithioate is converted to the 3'-phosphoramidite (see example V) and then used to synthesize DNA on a polymer support.

B. Synthesis of Deoxycytidine Oligomers Containing Phosphorodithioates was prepared by minor modification of a published procedure Koster, K. Kulinowski, T. Liese, W. Heikens, and V. Kohli, Tetrahedron 37, 363, 1981). Deoxycytidine hydrochloride (10 mmol, 2.64 g) was co-evaporated twice with anhydrous pyridine and resuspended in pyridine (50 ml) Trimethylchlorosilane (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 ml) for 30 min, and the suspension removed by filtration. The resulting solution was concentrated to dryness in vacuo. The resulting solid was suspended in 40 ml dichloromethane:methanol 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 redissolved in pyridine (50 ml). After addition of 0.9 equivalents of dimethoxytrityl chloride (3.05 the reaction mixture was stirred for 30 min at 0 0 C and min at room temperature. Dimethoxytritylchloride WO 89/11486 PCT/US89/02293 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 in vacuo. The resulting oil was dissolved in dichloromethane (75 ml) and extracted sequentially with aqueous 5% sodium bicarbonate 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 g silica, dichloromethane:methanol:pyridine gradient from 0 to 3% methanol; 400 ml each). Fractions containing dimethoxytrityl-N-toluoyldeoxycytidine were pooled, concentrated in vacuo, redissolved in ethylacetate and precipitated into pentane (5.01 g, 7.7 mmol, 77%).

was prepared by minor modification of a published procedure B. Reese and J. C. M. Stewart, Tetrahedron Letters 4273, 1968). N-toluoyldeoxycytidine (1.94 g, 3 mmol) and phenoxyacetic anhydride (1.72 g, 6 mmol) was dissolved in tetrahydrofuran (50 ml). After addition of pyridine (0173 ml, 9 mmol), the solution was stirred for 14 hours at room temperature and then concentrated in vacuo. The resulting oil was dissolved in dichloromethane (75 ml), extracted twice with 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 in vacuo, redissolved in ethylacetate and precipitated in pentane. The solid corresponding to totally protected deoxycytidine was dissolved in dichloromethane:methanol v/v) and chilled in an ice bath. A solution of t WO 89/11486 PCT/US89/02293 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 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-phenoxyacetyl-Ntoluoyldeoxycytidine were pooled, concentrated to an oil, and the product isolated as a precipitate by addition of ethylacetate (1.20 g, 83%).

Deoxydicytidine phosphoroamidite in protected form was prepared using the following procedure.

(647 mg, 1 mmol) was co-evaporated three times with THF, dissolved in THF (5 ml) and triethylamine (0.21 ml, mmol) and reacted with bis(N,N-diisopropylamino) chlorosphosphine (320 mg, 1.2 mmol). After 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 redissolved in acetonitrile (2 ml). 3'-O-Phenoxyacetyl-Ntoluoyldeoxycytidine (527 mg, 1.1 mmol) and tetrazole 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 min. and then poured into ethylacetate:triethylamine (99:1, V/V, After two extractions with 2M triethylammonium bicarbonate (20 ml each) and back c* WO 89/11486 PCIF/US89/02293 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:triethylamine; 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 in vacuo, and precipitated into pentane 31P-NMR (CHC1 3 149.3, 149.1. H-NMR 8.22 and 8.19 H6), 7.54-6.80 HAr) 6.30 HI'), 5.39 4.67 CH 2 phenoxyacetyl H iPr), 4.25 3.78 (2s, Me DMT), 3.5 H5', 2.8 and 2.3 H2', 2' 2.47 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 ml) (previously flushed with helium to avoid oxygen oxidation of thiophosphite) and 4-chlorobenzylmercaptan (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 rasulting thiophosphite (completely formed in 15 minutes as shown by 31P-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 toluene:lutidine (19.1, Based on 31P-NMR analysis 94.9, 94.7), oxidation was complete after 10 minutes. The reaction mixture was diluted with ethylacetate (75 ml), extracte" twice with aqueous sodium bicarbonate (75 ml each), and the combined aqueous phases back extracted with WO 89/11486 PCI/US89/02293 62 ethylacetate (50 ml). The combined org£ 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:triethylamine, 66:33:0.03, 400 ml and dichloromethane:triethylamine, 100:0.03,200 ml). Fractions containing the completely protected product were pooled, concentrated in vacuo, redissolved in dichloromethane, and precipitated into pentane 31 P-NMR (CHC1 3 97.5, 96.7. H-NMR 8.1 (m, H6), 7.6-6.8 HAr), 6.25 5.25 H3 4.70 CH 2 phenoxyacetyl), 4.5-4.0 CH 2 benzyl, 3.79 Me DMT), 3.73-3.35 3.0-2.55 and 2.45-1.95 H2', 2.50 Me tol).

The 3'-O-phenoxyacetyl protecting group was removed using the following procedure. The completely protected deoxydicytidine phosphorodithioate triester (355 mg, .264 mmol) was dissolved in acetonitrile (3 ml) and diluted with methanol (9 ml).

After chilling the solution in an ice bath, tertbutylamine 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:triethylamine, 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, WO 89/11486 PCT/US89/02293 63 redissolved in dichloromethane, and precipitated into pentane (95% yield).

31P-NMR (CDCL 3 96.5, 96.2. 1 H-NMR 8.2-8.06 H6) 7.52-6.81 HAr) 6.25 HI') 5.24 (m, 4.5-4.0 CH 2 benzyl, H3', H4', 3.79 Me DMT) 3.6-3.3 2.95-2.55 and 2.45-2.05 H2', 2.50 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 mmol) was dissolved in acetonitrile (5 ml). Bis(diisopropylamino)- ./-cyanoethoxyphosphine (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 ethylacetate:triethylamine (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 The organic layer was dried over sodium sulfate, filtered, and concentrated to an oil in vacuo. The resulting oil was redissolved. in dry ethylacetate and precipitated into pentane (87% yield).

31 P-NMR (dichloromethane) 149.5, 149.2, 149.0, 96.5, 96.0.

Deoxycytidine pentadecamers containing phosphorodithioate internucleotide linkages at selected sites were synthesized using the deoxydicytidine phosphorodithioate synthons having a /,-cyanoethyl)-N,N-diisopropylphosphoramidite moiety a, described above and N-benzoyldeoxycytidine -cyanoethyl)-N,Ndiisopropylphosphoramidite. The standard WO 89/11486 PCT/US89/02293 64 phosphoramidite synthesis methodology was used H.

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 when analyzed by gel electrophoresis and concentrated ammonium hydroxide at 55 0 C (15 hours). Purification of the final product was by either polyacrylamide gel electrophoresis or high performance liquid chromatography. Three pentadecamers having phosphorodithioate linkages at specific positions were synthesized and have the following sequence: d(CpCxCpCpCpCpCpCpCpCpCpCpxCpC) d(CpCpCpCpCpCpCxCpCpCpCpCpCpCpC) d(CxCpCxCpCxCpCxCpCxCpCxCpCxCpC) where x represents a dithioate linkage and p represents the natural internucleotide linkage.

EXAMPLE IX Synthesis of Nucleoside 3'-Phosphorodithioate of the formula: DMTO 0 B1 0

OCH

2 CH:t 2

CN

represented as XVla: YO B =1-Thyminyl; B =1-(N-4-benzoylcytosinyl); 9-(N-6-benzoyladeninyl); B =9-(N-2-isobutyrylguaninyl); Although B may be as described above, the following description is limited to a specific Nucleoside Phosphorodithioate wherein B is I-thyminyl DMT =dimethoxytrityl 3'-O-(Diisopropylamino)-2-cyanoethylphosphino-5'-0'(di-p-nethoxytrityl) thymidine (27.7 mg, 0.04 mmol) was prepared by art form methods (M.

Caruthers and S.L. Beaucage U.S. Patent 4,415,732) and then dissolved in anhydrous acetonitrile (440 Hydrogen sulfide was bubbled through for 1 min and tetrazole (7.0 mg in 220 1 CH 3 CH, 0.2 mmol) was added. After 10 rain 3

P

NMR spectroscopy showed quantitative conversion to the two diastereomers (70.9 and 70.2 ppm, lJPH= 675 Hz) of the nucleoside H-phosphonothioate. Excess of elementary sulfur converted the H-phosphonothioate in quantitative yield within A o<4> 65A 1/2 h under stirring at rt to the nucleoside 3'-phosphorodithioate. M 1 P NMR

(CH

3 CN) 114.0 ppm. FAB- 708 182 (M-DMTdT+O). 1 H NMR (CDCI 3 7.53

H

6 7.35-6.81 (m aromatic), 6.15 Hl', J 6.4 Hz), 5.12 (in, H 3 4.20 (in,

H

5 9' 3.95 (in, H 4 3.18 MeO-DMT), 3.47 (in, CH 2

O-P),

S

-ii WO 89/11486 PCT/US89/02293 66 2.77 CH 2 CN, J 6.2 Hz), 2.56-2.44 H 2 1.91

CH

3 Protected nucleoside 3'-phosphorodithioate 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 0 C for 16 h. The mixture was reevaporated and lyophilized with water to yield the nucleoside 3'-phorphorodithioate. FAB 338 FAB+ 338 (dT-P+ S).

I, -67- EXAMPLE X Synthesis of Nucleoside 5'-Phosphorodithioate of the formula:

S

NCCH

2

CH

2 -O-P-O B S H H>OCH 3 represented as compound XVIa where B 1-Uracilyl; B 1-(N-4-benzoylcytosinyl); B 9-(N-6-benzoyladeninyl); B -N2-sb.rlgaiy) Alhog B apea ecie bvtefloigdsrpini 1. ltahyou B H1 may be as d decie above,(6g the follwin dsrion is li.me tos ade specic Nucloside 5'-osph oprothiote hrin- is 9(N345 mg, 1.1 mmol) and stirred at RT for 20 min. Precipitation of the ammonium appeared after 1/2 min. The reaction mixture was diluted with CHCI *:Ago (50 ml) and extracted with NaHCO 3 w/v, 50 ml), back-extracted with CH 2

CL

2 ml), the organic phase dried over Na 2

S

4 filtered and evaporated to dryness in vacuo. 31 P NMR analysis (CH 3 CN) showed 147.9 ppm. Crude product (0.71 g) was dissolved in anhydrous CH 3 CN (5 ml) and bubbled with hydrogen sulfide for one min. Tetrazole (175 mg, 2.5 nimol in CH CN (5ml) was added and again

SRA

4 .WO 89/11486 PCT/US89/02293 68 hydrogen sulfide was bubbled through the reaction mixture for 1 min. The reaction mixture was sealed and after 10 min a precipitate of ammonium 31 tetrazolide appeared 31 P N'"R (CH 3 CN) 72.2 and 71.8 ppdm., Jp 669 Hz). The reaction mixture was

PH

evaporated to an oil in vacuo, redissolved in ethylacetate (50ml), extracted with TEAB (1 M, pH 7.4, 50ml), and back-extracted with ethylacetate The combined organic phases were dried over Na2SO4' filtered, evaporated, and the oil was redissolved in CH 2 C12 (5ml). Excess elementary sulfur mg, 2.5 mmol, in 5 ml toluene/2,6-lutidine, 19:lm v/v) was added. Stirring at room temperature for 1 h gave the phosphorodithioate product. 31P NMR (CH 3

CN)

114.4 and 114.3. Rf (silica) 0.34 in CH2Cl (9:1, v/v).

Thus while we have illustrated and described the preferred embodiment of our invention, it is to be understood that this invention is capable of variation and modification and we therefore do not wish to be limited to the precise terms set forth, but desire to avail ourselves of such changes and alterations which may be made for adapting the invention to various usages and conditions. Accordingly, such changes and alterations are properly intended to be within the full range of equivalents, and therefore within the purview of the following claims.

Having thus described our invention and the manner and process of making and using it in such full, clear, concise, and exact terms so as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same;

Claims (2)

1. 69- The claims defining the invention are as follows: 1. A compound according to the formula: R 1 0 0 B 0 A P -M S.. *0 IAO p-X M 1 01 wherein B is a nucleoside or deoxynucleoside base; A is K or KR 2 where K is OH, H, halogen, SH, NH 2 or azide and KR 2 is oxygen, sulfur or nitrogen as K and R2 is a heteroatom substituted or unsubstituted blocking group; R 1 is a blocking group; X is a secondary amino group of the formula NR 6 R 7 wherein R 6 and R7 taken separately each represent a heteroatom substituted or unsubstituted alkyl, Saryl, aralkyl, cycloalkyl, cycloalkylalkyl, aralkenyl, cycloalkenyl, alkenyl, aralkynyl, N0 70 cycloalkynyl or alkynyl, R 6 and R 7 when taken together form an alkylene chain containing up to 5 carbon atoms in the principal chain and a total of up to carbon atoms with both terminal valence bonds of the chain being attached to the nitrogen atom to which R 6 and R 7 are attached, or when R 6 and R 7 are taken together with the nitrogen atom to which they are attached form a nitrogen heterocycle and which nitrogen heterocycle optionally includes at least one additional heteroatom from the group of nitrogen, oxygen, and sulfur; and M is sulfur single bonded to phosphorus and to Rg where R 8 is a heteroatom substituted or unsubstituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, aralkenyl, cycloalkenyl, alkenyl, alkynyl, aralkynyl or cycloalkynyl. 2. A compound according to the formula: RO B I 0 A P M x wherein B is a nucleoside or deoxynucleoside base, A is K or KR 2 where K is OH, H, halogen, SH, NH 2 or azide and KR 2 is oxygen, sulfur or nitrogen as K and R 2 is a heteroatom substituted or unsubstituted blocking group; R 1 is a blocking group; X is a secondary amino group of the formula NR 6 R 7 wherein R 6 and R 7 taken separately each represent a heteroatom substituted or unsubstituted alkyl, Saryl, aralkyl, cydoalkyl, cydoalkylalkyl, aralkenyl, cycloalkenyl, alkenyl, aralkynyl, IL-- II: -71 cycloalkynyl or alkynyl, R 6 and R 7 when taken together form an alkylene chain containing up to 5 carbon atoms in the principal chain and a total of up to carbon atoms with both terminal valence bonds of the chain being attached to the nitrogen atom to which R 6 and R7 are attached, or when R 6 and R 7 are taken together with the nitrogen atom to which they are attached form a nitrogen heterocycle and which nitrogen heterocycle optionally includes at least one additional heteroatom from the group of nitrogen, oxygen, and sulfur; and M is sulfur single bonded to phosphorus and to R 8 where R is a heteroatom substituted or unsubstituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, aralkenyl, cycloalkenyl, alkenyl, alkynyl, aralkynyl or cycloalkynyl. 3. The compound according to claim 1 or claim 2, wherein R 1 is a trityl group, a di-p-anisylphenylmethyl group or a p-anisyldiphenylmethyl group. 4. The compound according to claim 1 or claim 2, wherein R is benzyl, a j substituted benzyl, 2,4-dichlorobenzyl, a lower alkyl, a heteroatom substituted lower alkyl, or S-cyanoethyl. The compound according to claim 1 or claim 2, wherein X is selected from the class consisting of dimethylamino, diethylamino, diisopropylamino, dibutylamino, methylpropylamino, methylhexylamino, methylcyclohexylamino, ethylcyclopropylamino, methylbenzylamino, methylphenylamino, ethylchloroethylamino, methyltoluylamino, methyl-p-chlorophenylamine, methylcyclohexylmethylamino, bromobutylcyclohexylamino, methyl-p- cyanophenylamino, ethyl-g-cyanoethylamino, morpholino, thiomorpholino, RAQ r I.. 0 0 72 pyrrolidino, piperidino, 2,6-dimethylpiperidino and piperazino. 6. The compound according to claim 5, where X is diisopropylarniino, dimethylamino, diethylamino, dibutylamino, and pyrrolidinyl. 7. The compound according to claim 6, wherein X is dimethylamino. 8. The compound according to any one of claims 1 to 7, wherein B is adenine, guanine, cytosine, uracil, and thymine. 9. The compound according to any one of claims 1 to 8, where M is selected from a class consisting of ethylmercaptyl, methylmercaptyl, propylmercaptyl, 0 butylmercaptyl, 1-cyanoethylmercaptyl, benzylmercaptyl, 4-chlorophenylmercaptyl, 4-chlorobenzylmercaptyl, 2,4-dichlorobenzylmercaptyl, cyclohexylmercaptyl, and 4-nitrophenylethylmercaptyl. The compound according to claim 9, wherein M is 2,4- dichlorobenzylmercaptyl. 11. The compound according to claim 2, wherein Riis di-p-anisylphenylm-ethyl, B is thyminyl, M is 2,4-dichlorobenzylmercaptyl, A is H, and X is dimethylamino. 12. The compound according to claim 2, wherein Riis di-p-anisylphenylmethyl, B is 9-(N-6-berizoyladeninyl), M is 2,4-dichlorobenzyhnercaptyl, A is H, and X is dimethylamino. 13. The compound according to claim 2, wherein R 1 is di-p-anisylphenylmethyl, B is 1-(N-4-benzoylcytosinyl), M is 2,4-dichlorobenzylmercaptyl, A is H, and X is dimethylamino. 14. The compound according to claim 2, wherein Riis di-p-anisylphenylmethyl, -I- :~r -73- B is 9-(N-2-isobutyrylguaninyl), M is 2,4-dichlorobenzylmercaptyl, A is H, and X is dimethylamino. A compound according to claim 1 or claim 2, substantially as herein described with reference to any one of the Examples. 16. A process for production of oligonucleotides which comprises the step of condensing the 3'-OH or 5'-OH group of nucleoside or oligonucleotide by a S coupling agent through the or respectively, of said nucleoside or oligonucleotide, with a compound according to claim 1 or claim 2. 17. A process for production of oligonucleotides which comprises the step of *11 condensing the 5'-OH group of a nucleoside or oligonucleotide by a coupling agent through the respectively, of said nucleoside or oligonudeotide with a compound according to claim 2 of the following formula: R 0 B o A I P -M X wherein R 1 is a blocking group; B is a nucleoside or deoxynudeoside base; A is K or KR 2 where K is OH, H, halogen, SH, NH 2 or azide and KR, is oxygen, sulfur or nitrogen as K and R2 is a heteroatom substituted or unsubstituted blocking group; X is NR 6 R 7 and M is sulfur single bonded to phosphorus and to R 8 where R 6 R7 and R are as defined in claim 2. 74 18. The process according to claim 17, wherein B is adenine, guanine, cytosine or thymine and A is H. 19. The process according to claim 17 or claim 18, wherein X is dimethylamino. The process according to any one of claims 17 to 19, where M is 2,4- dichlorobenzylmercaptyl. 21. The process according to any one of claims 17 to 20, where the nucleoside or oligonucleotide having a free 5'-OH group is linked to a polymer support. 22. The process according to any one of claims 17 to 21, where the coupling agent is tetrazole, substituted tetrazole, tetrazolide salts, substituted tetrazolide salts and amine salts. S 23. The process according to any one of claims 17 to 22, including the further .e step of oxidizing the resulting thiophosphite triester to a phosphorodithioate triester. 24. The process according to claim 23, wherein elementary sulfur is the oxidizing agent. The process according to any one of claims 17 to 22, including the further step of oxidizing the thiophosphite triester to a phosphorothioate. 26. The process according to claim 25, wherein t-butylhydroperoxide is the oxidizing reagent. 27. A process for the production of oligonucleotides having phosphorodithioate, phosphorothioate or phosphate internucleotide linkages or a combination of such linkages which comprises the step of condensing the 3'-OH 4 T^^ T or 5'-OH group of a nucleoside or oligonucleotide by a coupling agent through the or respectively, of said nucleoside or oligonucleotide with a compound according to claim 1 or claim 2. 28. Oligonucleotides whenever prepared by the process of any one of claims 16 to 27. 29. Oligonucleotides in protected or unprotected form having at least one phosphorodithioate linkage or phosphorodithioate and phosphothioate internucleotide linkages. A repetitive process whereby the compound of claim 1 or claim 2 is used 0 in combination with nucleoside phosphoramidites, nucleoside phosphate diesters, having combinations of natural phosphate, phosphorodithioate, phosphorothioate and phosphoroamidate internucleotide linkages whereby combinations of these linkages are produced by oxidation of the appropriate thiophosphite, H- phcbphonate or phosphite with t-butylhydroperoxide, sulfur, or iodine in combination with water or amines. 31. Oligonucleotides in protected or unprotected form having at least one phosphate, phosphorothioate, phosphorodithioate and phosphoroamidate internucleotide linkages, wherein at least one linkage is a phosphorodithioate linkage. -4 32. A compound of the formula: -76- ,B 4* wherein B is a nucleoside or deoxynucleoside base; A is K or KR 2 where K is OH, O0 H, halogen, SH, NH 2 or azide and KR 2 is oxygen, sulfur or nitrogen as K and R2 is a heteroatom substituted or unsubstituted blocking group; R 1 and R 3 are blocking groups; and R 5 is a blocking group. 33. A compound according to claim 32, wherein R 1 is di-p-anisylphenylmethyl, R 3 is acetyl, levulinyl, phenoxyacetyl or other blocking group and R 5 is 2,4- dichlorobenzylmethyl or S-cyanoethyl, A is H, and B is a deoxynucleoside base. 34. A compound according to claim 32 or claim 33, substantially as herein described with reference to any one of the Examples III, VI or VIII. A compound of the formula: 77 0 A R 5 S P =S 0 0 B 0 A 1 6 or B 7 OR 4 R 5S -S B OR 3 A wherein B is a nucleoside or deoxynucleoside base; A is K or KR 2 where K is OH, H, halogen, SH, NH 2 is oxygen, sulfur or nitrogen as K and R 2 is a heteroatom substituted or unsubstituted blocking group; R 1 R 3 R 4 and R. are blocking groups, and R 6 and R 7 are heteroatom substituted or unsubstituted alkyl, aryl or aralkyl substituents. 36. A compound according to claim 35, substantially as herein described with reference to Example VI. i -78- 37. A compound of the formula: R 1 0 B 0 A 1 R5S P S 0o B O B 0 RA R *R R 0 P N wherein B is a nucleoside or deoxynucleoside base; A is K or KR 2 where K is OH, H, halogen, SH, NH 2 or azide and KR 2 is oxygen, sulfur or nitrogen as K and R 2 is a heteroatom substituted or unsubstituted blocking group; R 1 R 4 and R 5 are blocking groups, and R 6 and R 7 are heteroatom substituted or unsubstituted alkyl, aryl or aralkyl substituents. 38. A compound according to claim 37, substantially as herein described with reference to Example VI. 39. A process for production of oligonucleotides which comprises the step of condensing the 3'-OH or 5'-OH group of a nucleoside or oligonucleotide by a coupling agent through the or respectively, of said nucleoside or oligonucleotide, with a compound according to any one of claims 35 to 38 followed I by oxidation to pentavalent phosphorus. V V o 1 1 1 -79- A process for production of oligonucleotides which comprises the step of condensing the 5'-OH group of a nucleoside or oligonucleotide by a coupling agent through the respectively of said nucleoside or oligonucleotide with a compound according to claim 37 followed by oxidation to pentavalent phosphorus. 41. A process of claim 40, wherein B is adenine, guanine, cytosine or thymine, and A is H. 42. The process according to claim 40 or claim 41, wherein NR 6 R 7 is diisopropylamino, R 4 is methyl or f-cyanoethyl, R 5 is methyl or 2,4- dicholorobenzyl or -cyanoethyl, and R 1 is di-p-anisylphenylmethyl. S S S 10 43. The process according to any one of claims 40 to 42, wherein 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 S• sequence. 44. An oligonucleotide in blocked or unblocked form and containing phosphorodithioate linkages said oligonucleotide is produced from a compound of claim 1 or claim 2. An oligonucleotide in protected or unprotected form and containing a phosphorodithioate linkage said oligonucleotide is produced from a compound of claim 32, wherein B is a nucleoside or deoxynucleoside base, A is K or KR 2 where K is OH, H, halogen, SH, NH 2 or azide and KR 2 is oxygen, sulfur or nitrogen as K and R 2 is a heteroatom substituted or unsubstituted blocking group; R 5 is a blocking group, R 1 is H or a blocking group, and R 3 is H or a blocking group. in -83 wherein B is a nucleoside or deoxynudeoside base; A is K or KR 2 where K is OH, H, halogen, SH, NH 2 or azide and KR 2 is oxygen, sulfur or nitrogen as K and R 2 is a heteroatom substituted or unsubstituted blocking group; and R 1 and R 3 are blocking groups. 47. A compound according to claim 46, substantially as herein described with reference to Example IV. 48. A compound of the formula: L, -81- wherein B is a nucleoside or deoxynucleoside base; A is K or KR 2 where K is OH, H, halogen, SH, NH 2 or azide and KR 2 is oxygen, sulfur or nitrogen as K and R 2 is a heteroatom substituted or unsubstituted blocking group; R 1 and R 3 are blocking groups; and R 6 and R 7 are substituted or unsubstituted alkyl, aryl, or aralkyl substituents. 49. A compound according to claim 48, substantially as herein described with reference to Example VII. 50. A compound of the formula: B "0 A R R B 77 0 A or \R iirJ I. 82 R 6 R N- P 0 R I 7 OR 0 N P S R/ I 7 4 i5 wherein B is a nucleoside or deoxyoligonuceotide base; A is K or KR 2 where K is OH,. H, halogen, SH, NH 2 or azide and KR 2 is oxygen, sulfur or nitrogen as K and R 2 is a heteroatom substituted or unsubstituted blocking group; and R 1 R 3 and R 4 are blocking groups; and Rg and R 7 are substituted or unsubstituted alkyl, aryl, or aralkyl substituents. 51. A compound of the formula: R 1 0 R N 0 B 0 N P OR 7 A i
2. 83- wherein B is a nucleoside or deoxynucleoside base; A is K or KR 2 where K is OH, H, halogen, SH, NH 2 or azide and KR 2 is oxygen, sulfur or nitrogen as K and R 2 is a heteroatom substituted or unsubstituted blocking group; and R 1 and R 4 are blocking groups; and R 6 and R 7 are substituted or unsubstituted alkyl, aryl, or aralkyl substituents. 52. A process for production of oligonucleotides which comprises the step of condensing the 3'-OH or 5'-OH group of a nucleoside or oligonucleotide by a coupling agent through the or respectively, of said nucleoside or S oligonucleotide, with a compound according to claim 50 or claim 51 followed by i'"0 oxidation to pentavalent phosphorus. 53. A process for production of oligonucleotides which comprises the step of condensing the 5'-OH group of a nucleoside or oligonucleotide by a coupling agent through the respectively, of said nucleoside or oligonucleotide with a compound according to claim 51 followed by oxidation to pentavalent phosphorus. 54. The process of claim 53, wherein B is adenine, guanine, cytosine, or thymine and A is H. A process according to claim 53 or claim 54, wherein NR 6 R 7 attached to trivalent phosphorus is diisopropylamine, R 4 is methyl or 1-cyanoethyl, and R1 is di-p-anisylphenylmethyl. 56. The process according to any one of claims 53 to 56, wherein the nucleoside or oligonucleotide having a free 5'-OH is linked to a polymer support and the synthesis is repeated many times to form an oligonucleotide of defined sequence. RA4 .z tU i-i:~i -I -84- 57. An oligonucleotide in blocked or unblocked form and containing phosphorothioamidate linkages produced by the process of any one of claims 53 to 56. 58. An oligonucleotide in blocked or unblocked form and containing a phosphorothioamidate linkage said oligonucleotide is produced from a compound of claim 48 or claim 49, wherein B is a nucleotide or deoxynuceoside base, A is K or KR 2 where K is OH, H, halogen, SH, NH 2 or azide and KR 2 is oxygen, sulfur or nitrogen as K and R 2 is a heteroatom substituted or unsubstituted blocking 4 Sgroup; R 1 is H or a blocking group, R 3 is H or a blocking group, and R 6 and R 7 are heteroatom substituted or unsubstituted alkyl, aryl or aralkyl groups. S 59. A compound of the formula: o i 0 B RR A 0 A ORQ A wherein B is a nudeoside or deoxynuceoside base; A is K or KR 2 where K is OH, H, halogen, SH, NH 2 or azide and KR 2 is oxygen, sulfur or nitrogen as K and R 2 is a heteroatom substituted or unsubstituted blocking group; R 1 and R 3 are z 85 blocking groups; and R6is a substituted or unsubstituted alkyl, aryl, or aralkyl sub'stituent. A compound according to claim 59, substantially as herein described with reference to Example VII. 61. A compound of the formula: R I H99 0 A X@ II :a R 6 0- B OR 3 A R 00B H 09 B N -P OR 4 7 T£ -86- wherein B is a nucleoside or deoxynucleoside base; A is K or KR 2 where K is OH, H, halogen, SH, NH 2 or azide and KR 2 is oxygen, sulfur or nitrogen as K and R 2 is a heteroatom substituted or unsubstituted blocking group; R 1 R 3 and R 4 are blocking groups; and R 6 and R 7 are substituted or unsubstituted alkyl, aryl, or aralkyl substituents. 62. A compound of formula: R 1 B H. 0 0 A H H H 0 A N P OR 4 wherein B is a nudeoside or deoxynucleoside base, A is K or KR 2 where K is OH, H, halogen, SH, NH 2 or azide and KR 2 is oxygen, sulfur or nitrogen as K and R2 is a heteroatom substituted or unsubstituted blocking group; R 1 and R 4 are blocking groups; and R 6 and R 7 are substituted or unsubstituted alkyl, aryl, or aralkyl substituents. i -87 63. A process for production of oligonucleotides which comprises the step of condensing the 3'-OH or 5'-OH group of a nucleoside or oligonucleotide by a coupling agent through the or respectively, of said nucleoside or oligonucleotide, with a compound according to claim 61 or claim 62 followed by oxidation to pentavalent phosphorus. 64. A process for production of oligonucleotides which comprises the step of condensing the 5'-OH group of a nucleoside or oligonucleotide by a coupling agent through the respectively, of said nucleoside or oligonucleotide with a compound according to claim 62 following by oxidation to pentavalent phosphorus. .10 65. The process of claim 64, wherein B is guanine, adenine, cytosine or thymine Sand A is H. 66. The process according to claim 64 or claim 65, wherein NR 6 R 7 attached to *ee, trivalent phosphorus is diisopropylamine, R 4 is methyl or 1-cyanoethyl and R 1 is di-p-anisylphenylmethyl. 67. The process according to any one of claims 64 to 66, wherein the nucleoside or oligonucleotide having a free 5'-OH is linked to a polymer support and the synthesis is repeated many times to form an oligonucleotide of defined sequence. 68. An oligonucleotide in blocked or unblocked form and containing phosphorothioamidate linkages produced by the process of any one of claims 64 to 67. 69. An oligonucleotide in blocked or unblocked form and containing a phosphorothioamidate linkage said oligonucleotide is produced from a compound Cr S. r -88- of claim 59 or claim 60, wherein B is a nucleoside or deoxynucleoside base, A is K or KR 2 where K is OH, H, halogen, SH, NH 2 or azide and KR2 is oxygen, sulfur or nitrogen as K and R 2 is a heteroatom substituted or unsubstituted blocking group; R 1 is H or a blocking group, R 3 is H or a blocking group, and R6 is a heteroatom substituted or unsubstituted alkyl, aryl or aralkyl group. A process of converting a compound of claim 46 or claim 47, to a phosphorothioate triester by oxidation with R 4 0H and an oxidizing agent where R 4 is a heteroatom substituted or unsubstituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl, cycloalkenyl, aralkenyl, alkynyl, aralkynyl, or cycloalkynyl substituents. S 71. The process of claim 70, wherein the oxidizing agent is 12 and R40H and R4 is anthracenylmethyl. 72. A process of converting a compound of claim 46 or claim 47 to a phosphorothioate diester by oxidation with aqueous iodine in pyridine. 73. Oligonucleotides containing phosphorothioate diesters when produced by the process of claim 67. 74. A process for production of oligonucleotides which comprises condensing the 5'-OH group of a nucleoside or oligonucleotide by a coupling agent through the 5'-O of said nucleoside or oligonucleotide with a compound according to claim 32 wherein R 1 and R3 are H-phosphonates, a phosphate monoester, or a phosphate. 75. Oligonucleotides containing phosphorodithioates whenever produced by the T~V -~iu iiudn r 89 process of claim 74. 76. A process of producing oligonucleotides which comprises condensing the group of a nucleoside or oligonucleotide by a coupling agent through the of said nucleoside or oligonucleotide with a compound according to claim 48 wherein R 1 and R 3 are H-phosphonates, a phosphate monoester, or a phosphate. 77. Oligonucleotides containing phosphorothioamidates whenever produced by the process of claim 76. 78. A process of producing oligonucleotides which comprises condensing the 5'-OH group of a nucleoside or oligonucleotide by a coupling agent through the 10 5'-O of said nucleoside or oligonucleotide with a compound according to claim 59 Swherein R, and R 3 are H-phosphonates, a phosphate monoester, or a phosphate. 79. A compound of the formula: S HS P 0 B HO C H or R 1 0- B 0 A HO P S SH wherein B is a nucleoside or deoxynucleoside base; A is K or KR 2 where K is OH, H, halogen, SH, NH 2 or azide and KR 2 is oxygen, sulfur or nitrogen as K and R 2 is a heteroatom substituted or unsubstituted blocking group; and R 1 is H or a blocking group, C is H, OH or OR 3 where R 3 is a blocking group. Oligonucleotides in protected or unprotected form wherein the internucleotide linkages in the oligonucleotide are phosphorodithioate internucleotide linkages. DATED this 7 day of May 1992. 9* UNIVERSITY PATENTS, INC. By their Patent Attorneys: CALLINAN LAWRIE 9* I o•* *oo* _IY_ it 1 i i -i e *i L INTERNATIONAL SEARCH REPORT Internalional Application No. PCT/US8 9/02293 I. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, indicate all) 4 According to Internation Patent Classification (IPC) or to both National Classification and IPC INT. CL. CO7H 17/00 U.S. CL. 536/27, 28, 29; 538/18 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System Classification Symbols U.S. CL. 536/27, 28, 29; 538/18 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched s III. DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of Document, I' with indication, where appropriate, of the relevant passages 12 Relevant to Claim No, 12 A US, A, 3,846,402 (ECKSTEIN) 5 NOVEMBER 1984 1-77 SEE ABSTRACT. A US, 3,451,997 (FUJIMOTO) 24 JUNE 1969 1-77 SEE COLUMNS' 1-2 A US, A, 3,853,844 (SHUMAN) 12 DECEMBER 1974 1-77 SEE COLUMN 1. A US, A, 4,728,730 (FREY) 1 MARCH 1988 1-77 SEE COLUMNS 1-2 A,P US, A, 808,708 (YOSHIDA) 28 FEBRUARY 1989 1-77 SEE ABSTRACT. X JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 33,35,71 A VOLUME 106, ISSUED 1984 (WASHINGTON, DC) 1-77 WOJCIECH J. STEC ET AL., "AUTOMATED SOLID-PHASE SYNTHESIS, SEPARATION, AND STERO CHEMISTRY OF PHOSPHOROTHIOATE ANALOGUES OF OLIGODEOXY-RIBONUCLEOTIDES, PAGES 6077-6079, SEE COMPLETE DOCUMENT. SSpecial categories of cited documents: to later document published after the international filing date document defning the general state of the art which is not or priority date and not in conflict with the application but cited to understand the principle or theory underlying the considered to be of particular relevance invention earlier document but published on or after the international document of particular relevance the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establish the publication date of another document of particular relevance: the claimed Invention citation or other secia b l i °eason (as specified) document of particular relevance: the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 04 August 1989 1 SEP 1989 International Searching Authority Signature of Authorized Offir RO/US L. ?T FomPCTASAii20 (seoand she (Rev.1117) .I International Application No. PCT/US89/02293 III. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category Citation of Document, with indication, where appropriate, of the relevant passages Relevant to Claim No A TETRAHEDRON LETTERS, VOLUME 27, NO. 46, 47-77 ISSUED 1986 (GREAT BRITIAN) BRIAN C. FROEHLER, "DEOXYNUCLEOSIDE H-PHOSPHONATE DIESTER INTERMEDIATES IN THE SYNTHESIS OF INTERNUCLEOTIDE PHOSPHATE ANALOGUES," PAGES 5575 TO 5578, SEE PAGE 5575. Y JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1-46 VOLUME 92, NO. 15, ISSUED 1970 (WASHINGTO F. ECKSTEIN, "NUCLEOSIDE PHOSPHO- THIOATES," PAGES 4718 TO 4723, SEE STRUCTURES 11-13. A US, A, 4,373,071 (ITAKURA) 8 FEBRUARY 1983 1-77 SEE COLUMNS 1-3. A US, A, 4,668,777 (CARUTHERS) 26 MAY 1987 1-77 SEE COLUMNS 1-3. A US, A, 4,415,732 (CARUTHERS) 15 NOVEMBER 198 1-77 FoI PCT/SA100 (etrazhW (RPv.l -7) International Application No. PCT/US89/02293 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE' This international search report has not been established in respect of certain claims under Article 17(2) for the following reasons: Claim numbers because they relate to subject matter 12 not required to be searched by this Authority, namely: 2.E Claim numbers ,because they relate to parts of the international application that do not comply with the prescribed require- ments to such an extent that no meaningful international search can be carried out i 3 specifically: 3. Claim numbers_, because they are dependent claims not drafted in accordrnce with the second and third sentences of PCT Rule 6.4(a). VI.] OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING 2 This International Searching Authority found multiple inventions in this international application as follows: (SEE ATTACHMENT) As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims of the international application. TELEPHONE PRACTICE As only some of the required additional search fees were timel, paid by the applicant, this international search report covers only those claims of the international application for which fees were paid, specifically claims: 3.I No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claim numbers: As all searchableclaims could be searched without effort justifying an additional fee, the International Searching Authority did not invite payment of any additional fee. Remark on Protest F The additional search fees were accompanied by applicant's protest. E No protest accompanied the payment of additional search fees. Fan PCTASA1210 suppritusa h1e (Z (Rov. 11-87)