WO1989011486A1 - Composes de thiophosphoramidite et de phosphorodithioate de nucleosides et de polynucleotides et procedes - Google Patents

Composes de thiophosphoramidite et de phosphorodithioate de nucleosides et de polynucleotides et procedes Download PDF

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WO1989011486A1
WO1989011486A1 PCT/US1989/002293 US8902293W WO8911486A1 WO 1989011486 A1 WO1989011486 A1 WO 1989011486A1 US 8902293 W US8902293 W US 8902293W WO 8911486 A1 WO8911486 A1 WO 8911486A1
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nucleoside
sulfur
oligonucleotide
compound
group
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PCT/US1989/002293
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Marvin Caruthers
Wolfgang Brill
John Nielsen
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University Patents, Inc.
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Priority to KR1019900700156A priority Critical patent/KR900701813A/ko
Publication of WO1989011486A1 publication Critical patent/WO1989011486A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

  • 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.
  • these compounds can be used to deliver metal ions, toxins, intercalating agents and other reagents that alter the biochemical reactivity of polynucleotides and proteins to specific sites in cells and tissues. These compounds can also be used for diagnostic purposes. By attaching fluorescent or other chemically reactive reagents, antigens, antibodies, proteins, and metal ions to these compounds, they can be used for diagnosing diseases and the normal and abnormal biochemistry of cells, tissues, and body fluids such as blood and urine. There are also many uses in modern biology and chemistry as well. For example, these compounds can be used to develop improved methods for sequencing and cutting DNA, for imaging in X-ray crystallography, NMR, and electron microscopy, and for studying enzymic reactions.
  • phosphite triesters can also be oxidized under anhydrous conditions with amines or ammonia and iodine to yield variable reported amounts of phosphoramidates or with sulfur to yield phosphorothioates (B. Uznanski, M. Koziolkiewicz, W. J. Stec, G. Zon, K. Shinozuka, and L. Marzili, Chemica Scripta 26, 221,224, 1986; M. J. Nemer and K. K. Ogilvie, Tetrahedron Lett. 21, 4149-4152, 1980).
  • Other methods employing H-phosphonate internucleotide linkages can also be used to synthesize phosphoramidates (B. C.
  • 3',5'-phosphorodithioate 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 (J. 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.
  • R 1 is H or a blocking group
  • 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
  • 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, cycloalkylalkyl, alkenyl, aralkenyl, cycloalkenyl, alkynyl, aralkynyl or cycloalkynyl group.
  • the substituent Y is sulfur single bonded to phosphorus and to either H or R 5 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.
  • R 6 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 terminal valence bonds of said chain being attached to the nitrogen atom to which R 6 and R 7 are attached; and R 6 and R 7 when taken together with the nitrogen atom to which they are attached may also form a nitrogen heterocycle including 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, la and lb; class la 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 R 5 ; and class lb are those in which Z is sulfur double bonded to phosphorus plus two other substituents from the group V and Y where the heteroatom of each of these substituents is single bonded to phosphorus.
  • Compounds in class la are useful for synthesizing polynucleotides containing phosphcrodithioate and phosphorothioate internucleotifie linkages.
  • Compounds in class lb are useful for various therapeutic and biological studies and as intermediates for synthesizing nucleotides having 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 phosphorodithioate, phosphorothiosmidate, phosphorothioate triester and phosphorothicete and phosphorodithioate internucleotide linkages which are also usfful as therapeutic, diagnostic, or research reagents.
  • 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: The.se definitions apply unless, in special cases, these terms are defined differently.
  • alkyl - a non-cyclic branched or unbranched hydrocarbon radical having from 1 to 20 (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.
  • 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.
  • alkenyl - an aliphatic, unsaturated, branched or unbranched hydrocarbon having at least one double bond and 2 to 20 (preferably 3 to 10) 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.
  • heteroatoms preferably oxygen, sulfur and nitrogen
  • Heteroatoms, preferably sulfur, oxygen, and nitrogen can also replace carbon as part of the aromatic ring system in aryl radicals.
  • 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-O-PS 2 -O-nucleoside-3' which can be illustrated with the following structure where B and A are as defined previously.
  • phosphoroth ioate internucleotide linkage an internucleotide linkage having the general formula 5'-nucleoside-O-POS-O-nucleoside-3' which can be illustratred with the following structure v/here B and A are as defined previously .
  • phosphorothioamidate internucleotide linkage an internucleotide linkage having the general formula 5'-nucleoside-0-PSNHR 6 -0-nucleoside-3' and 5'-nucleoside-0-PSNR 6 R 7 -0-nucleoside-3' which can be illustrated with the following structures where B,A,R 6 and R 7 are as previously defined.
  • phosphoromidate internucleotide linkage an internucleotide linkage having the general formulae 5'-nucleoside-0-PONHR 6 -0-nucleoside-3' and 5'-nucleoside-0-PONR 6 R 7 -0-nucleoside-3' which can be illustrated with the following structures where B,A,R 6 and 7 6 are as previously defined.
  • 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 R 4 are as previously defined.
  • 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 to 20 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.
  • primary amines such as methylamine, ethylamine, propylamine, isopropylamine, aniline, cyclohexylamine, benzylamine, polycyclic amines containing up to 20 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, methylcyclopropylamine, ethylcyclohexylamine, methylbenzylamine, methylcyclohexyImethylamine, 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.
  • secondary amines such as dimethylamine, diethylamine, diisopropylamine, dibutylamine, methylpropylamine, methylhexylamine, methylcyclopropylamine
  • nucleoside and deoxynucleoside bases represented by B in the above formulae are well known and include purines, e.g., adenine, hypoxanthine, guanine, and their derivatives, and pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives.
  • the blocking groups represented by R 1 , R 2 and R 3 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 preferred reaction scheme A is represented as follows: wherein R 1 , R 2 , 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 Villa are those in which phosphorus is linked through a single bond to Y and through a double bond to Z or L. Thus, it can be seen that compounds VIII and Villa are a subset of compounds II. Likewise, compounds V and Va are a subset of compounds la.
  • reaction scheme A involves condensation of IlIa with IVa, which preferably is bis(dlmethylamino)chlorophosphine or dipyrrolidinylchlorophosphine, to yield IXa in the presence of triethylamine. Further addition of a mercaptan, which preferably is 2,4- dichlorobenzylmercaptan, in the presence of triethylamine hydrochloride generated in the first step leads to the conversion of IXa to Va.
  • Table 1 lists the 31 P-NMR characterization data for a series of Va derivatives where the nucleoside base (B), amine functionality (X), and mercaptan (M) are altered in a systematic manner. Reaction of Va with
  • an activator e.g., tetrazole, 5-substituted 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
  • an activator e.g., tetrazole, 5-substituted 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 te
  • S-(2,4-dichlorobenzyl) phosphite which can be preferably oxidized with sulfur to yield Villa, the dinucleoside phosphorodithioate triester with P(Y,Z).
  • T, C Bz , A Bz , and G iB refer to thymine, N-benzoylcytosine,
  • 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
  • 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.
  • IVa generates an intermediate diamidite, IX and IXa, to which the mercaptan is added to form V and Va.
  • IVb leading directly to V and Va, is restricted to compounds IVb where the thiodiamidite can be purified by crystallization or distillation without decomposition.
  • the process of reaction scheme B involves condensation of Ilia with IVb which is
  • oxidation with t-butylperoxide yields the corresponding dinucleoside phosphorothioate triester, P (Y, L).
  • Activators that are more acidic than tetrazole such as certain 5-substituted tetrazoles (e.g. 5-(p -nitrophenyl) tetrazole) and pyridinium tetrafluoroborate, can be used with success to activate Va. Certain side reactions, however, can lead to reductions in yields of the correct product.
  • the preferred reaction scheme C is represented as follows wherein R, R 3 , B, A, X, W, Z, Y, and V are as previously defined and Q is H.
  • Compounds XII and Xlla are those in which Z is sulfur double bonded to phosphorus plus one other substituent from the group of substituents V, W X and Y which are single bonded to phosphorus. These are derived from XI or XIa.
  • Compounds XII and Xlla can also be L which is oxygen double bonded to phosphorus plus Y which is single bonded to phosphorus. These are derived from XI or XIa.
  • 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 H 2 S 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 (Z,Y); by oxidation with iodine in the presence of amines to phosphorothioamidates, P (Z, W or X); by alkylation of the corresponding dinucleoside phosphorodithioate to phosphorodithioate triesters, P (Z,Y); by oxidation with iodine in the presence of alcohols to phosphorothioate triesters, P (Z,V): and by oxidation with aqueous iodine to phosphorothi
  • Compound Xa can also be reacted with a mercaptan in the presence of an activator such as tetrazole to yield the dinucleoside phosphorothioite, Vila, which can be chemically converted to Xlla by oxidation with sulfur to dinucleoside phosphorodithioates, P (Z,Y); and by oxidation with t-butylperoxide or aqueous iodine to phosphorothioates, P (L,Y).
  • an activator such as tetrazole
  • Xlla dinucleoside phosphorothioite
  • Xlla can be chemically converted to Xlla by oxidation with sulfur to dinucleoside phosphorodithioates, P (Z,Y); and by oxidation with t-butylperoxide or aqueous iodine to phosphorothioates, P (L,Y).
  • 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.
  • processes A, B and C can all be used to prepare the same compound.
  • 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.
  • 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 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.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • 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.
  • XIV and XlVa from IlIa and XIII or Xllla. Reaction of XIV or XlVa with H 2 S 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 (Z, V, Y) and by alkylation of the nucleoside phosphorodithioate to the nucleoside phosphorodithioate triesters, P (Z, V, Y).
  • the preferred novel compounds according to the present invention are those compounds of general formula la and II wherein (for la) Y is a substituent having sulfur single bonded to phosphorus and to R 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 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 2 is H. These new compounds can then be used to prepare oligonucleotides having phosphorodithioate internucleotide linkages with P(Z,Y). These oli
  • 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 nucleoside phosphorodiamidite is reacted without isolation with a second nucleoside.
  • the isolated product of this reaction is a dinucleoside dialkylamino phosphoramidite, Xa, which can be reacted by two different pathways to form Xlla.
  • the preferred pathway is to react Xa with a mercaptan in the presence of tetrazole to yield Vila which is further treated with elementary sulfur to form the deoxydinucleotide phosphorodithioate, Xlla, where P(Z,Y).
  • 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.
  • 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.
  • 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.
  • 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, VIla, which, after oxidation with elementary sulfur, yields VIlla with P(Z, Y), the dinucleoside phosphorodithioate triester.
  • 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.
  • S-aralkyldialkylaminophosphoramidite or thioamidite Va in concert with sulfur oxidation, polynucleotides having only phosphorodithioate linkages can be prepared.
  • 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.
  • 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. 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.
  • 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.
  • 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.
  • 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 ⁇ -cyanoethyl and methyl.
  • 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.
  • the present new compounds When used to form polynucleotides, they are employed in combination with art recognized nucleoside phosphoramidites or in the absence of nucleoside phosphoramidites.
  • 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.
  • 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.
  • Va is activated, condensed, and oxidized with sulfur as described above, repetitively with a nucleoside preferably attached to a polymer support to yield polynucleotides having phosphorodithioate linkages.
  • Dinucleoside phosphorodithioate triesters VIlla or Xlla where P(Z,Y) can also be used as synthons for polynucleotide synthesis.
  • dinucleoside phosphorodithioate 3'-phosphoramidites 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.
  • the product can, if desirable, be freed of blocking groups.
  • 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.
  • Bis(dimethylamino)chlorophosphine was prepared by adding tris(dimethylamino)-phosphine (36.3 ml,
  • M 4-chlorobenzyl or 2,4-chlorobenzyl
  • X N,N-dimethylamino or pyrrolidinyl and. the further use of these compounds to prepare oligonucleotides having phosphorodithioate internucleotide linkages.
  • step iii Acylation of unreactive deoxynucleoside (step iii) , detritylation (step iv) and various washes were the same as those described previously for synthesizing natural DNA from deoxynucleoside phosphoramidites (U.S. 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 conc. ammonium hydroxide for 15h) and then purified to homogeneity by standard procedures (polyacrylamide gel electrophoresis and reverse phase hplc).
  • 31 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.
  • VXR-500S Aqueous 85% H 3 PO 4 was the external standard .
  • reaction mixture 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 material in the reaction mixture ( 31 P-NMR 132.4 ppm). The newly formed diisopropylammonium chloride was removed by filtration and washed with anhydrous ether.
  • the product was dissolved in toluene and precipitated into n-pentane.
  • the nucleoside phosphorthioamidite was isolated after drying the precipitate in vacuo over P 2 O 5 /KOH (3.33 g, 80.1% yield).
  • 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.
  • the reaction to the dinucleoside thiophosphite was quenched with sulfur (1 atomic equivalent, 32 mg).
  • the reaction mixture was then diluted with ethylacetate (50 ml) and the sulfur removed by filtration through a cotton plug. After removal of the solvents in high vacuo, the desired product was dissolved in ethylacetate (10 ml) and extracted twice with an aqueous saturated solution of sodium bicarbonate and once with brine successively. The organic layer was dried over sodium sulfate. After removal of the salt, the product was chromatographed on silica with a mixture of 1.1.1-trichloroethane and methanol (92.5:7.5, v/v).
  • the 4-chlorobenzyl group was removed from the phosphorodithioate triester with a mixture of dioxane:triethylamine:thiophenol (2:2:1, v/v/v) within 1.5 hours at room temperature.
  • dinucleoside phosphorodithioate triesters can also be prepared by using pyridinium tetrafluoroborate as an activating agent.
  • Pyridinium tetrafluoroborate was prepared by dissolving HBF 4 (10 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, 10 mmole) in dry ethyl ether (50 ml). After 2 h the salt was removed by filtration, washed with dry ethyl ether, and dried in a dessicator over P 2 O 5 .
  • DMT dimethoxytrityl
  • Dithymidine phosphorodithioate was synthesized by stirring the dinucleoside H-phosphonothioate (104 mg. 0.1 mmol in 1 ml dichloromethane) with elementary sulfur (1 mmol in 2 ml toluene: 2 ,6-lutidine, 19:1, v/v) for 0.5 h. Purification via silica gel column chromatography (0-12% methanol in dichloromethane and 0.5% triethylamine) afforded 70% isolated yield.
  • the deacylated compound was then reacted with bis (diisopropylamino) -2-cyanoethoxy phosphine (1.5 eq) in the presence of tetrazole (1 eq, 1 h at rt) to yield the dinucleoside phosphorodithioate triester as the 3'-phorphoramidite in 76% yield.
  • 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(ApApTpTpGpTpTpApTpCpCpGpCpTpCpApApTxTpCpCpApAp-CpA)
  • the dinucleoside H-phosphonothioate was also found to be useful as a versatile synthon for preparing several analogs rapidly (5 min) in quantitative yield ( 31 P NMR).
  • the phosphorothioamidate, XIIa,P(Z,W) was isolated in 92% yield.
  • FAB mass spectrum 961 (M-)
  • the dinucleoside H-phosphonothioate was converted quantitatively to a phosphorothioate triester by oxidation with iodine and 9-anthracenyl methanol (10 equivalents) under anhydrous conditions, Xlla, P(Z,V).
  • FAB + mass spectrum 527 (anhydro DMT dT): FAB- mass spectrum, 906 (m ⁇ anthracenylmethyl), 639 (DMT dT-3'-PSO 2 -), 379 (5'-PSO 2 --dT-3'-OAc).
  • R f 0.41 (methanol/dichloromethane, 1:9, v/v).
  • Ac acetyl and the further conversion of the deoxydicytidine derivative to deoxycytidine oligodeoxynucleotides having phosphorodithioate internucleotide linkages at various positions.
  • 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.
  • the reaction mixture was diluted with ethylacetate (75 ml), extracted with an aqueous sodium bicarbonate solution (5%, w/v), dried over sodium sulfate, filtered and concentrated in vacuo to an oil.
  • the oil was dissolved in ethylacetate (40 ml) and triturated with hexanes (200 ml) to give a crude product as a white powder.
  • Purification by silica column chromatography 100 ml silica, 2-12%methanol in dichloromethane as % eluant yields the deoxydinucleoside phosphorodithioate triester (1.59 g, 1.52 mmol, 91%).
  • 31 P-NMR (CHCl 3 ) 97.9, 96.4.
  • 5'-O-Dimethoxytrityl-N-toluoyldeoxycytidine was prepared by minor modification of a published procedure (H. 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
  • o-Toluoyl chloride (1.44 ml, 11 mmol) was added and the reaction stirred for two additional hours.
  • the reaction mixture was chilled in an ice bath, treated with methanol (10 ml) and 25% ammonium hydroxide (20 ml) for 30 min, and the suspension removed by filtration.
  • the resulting solution was concentrated to dryness in vacuo.
  • the resulting solid was suspended in 40 ml dichloromethane:methanol (8:2) and the insoluble salts removed by filtration.
  • the filtrate was concentrated in vacuo to an oil, reconcentrated twice in vacuo after addition of pyridine and redissolved in pyridine (50 ml).
  • 3'-O-Phenoxyacetyl-N-toluoyldeoxycytidine was prepared by minor modification of a published procedure (C. B. Reese and J. C. M. Stewart, Tetrahedron Letters 4273, 1968).
  • 5'-O-Dimethoxytrityl-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.
  • Deoxydicytidine phosphoroamidite in protected form was prepared using the following procedure. 5'-O-Dimethoxytrityl-N-toluoyldeoxycytidme (647 mg, 1 mmol) was co-evaporated three times with THF, dissolved in THF (5 ml) and triethylamine (0.21 ml, 1.5 mmol) and reacted with bis (N,N-diisopropylammo) chlorosphosphine (320 mg, 1.2 mmol). After 90 minutes under argon, the reaction mixture was filtered under argon pressure to remove insoluble salts. The salts were washed with THF (2 ml).
  • Deoxydicytidine phosphorodithioate was prepared using the following procedure.
  • the deoxydicytidine phosphoramidite as prepared in the previous procedure (1.40 g, 1.12 mmol) was dissolved in acetonitrile (5 ml) (previously flushed with helium to avoid oxygen oxidation of 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 resulting thiophosphite (completely formed in 15 minutes as shown by 31 P-NMR,
  • 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 (60%).
  • 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.
  • 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, redissolved in dichloromethane, and precipitated into pentane (95% yield).
  • 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.
  • Deoxycytidine pentadecamers containing phosphorodithioate internucleotide linkages at selected sites were synthesized using the deoxydicytidine phosphorodithioate synthons having a 3'-O-( ⁇ -cyanoethyl)-N,N-diisopropylphosphoramidite moiety as described above and 5'-O-dimethoxytrityl-N-benzoyldeoxycytidine -3'-O-( ⁇ -cyanoethyl)-N,N-diisopropylphosphoramidite.
  • the standard phosphoramidite synthesis methodology was used (M. H.
  • 3'-O-(Diisopropylamino)-2-cyanoethylphosphino--5'-O'(di-p-methoxytrityl) thymidine (27.7 mg, 0.04 mmol) was prepared by art form methods (M. H. Caruthers and S. L. Beaucage U.S. Patent 4,415,732) and then dissolved in anhydrous acetonitrile (440 1). Hydrogen sulfide was bubbled through for 1 min and tetrazole (7.0 mg in 220 1 CH 3 CN, 0.2 mmol) was added.
  • 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°C for 16 h. The mixture was reevaporated and lyophilized with water to yield the nucleoside 3'-phorphorodithioate.
  • FAB+ 338 (dT-P + SH S).

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Abstract

Cette invention concerne des composés nouveaux et utiles de thiophosphoramidite de nucléosides, de phosphoramidite de dithioate de polynucléotides et de phosphoramidite de phosphorthioamidate de polynucléotides, ainsi que les procédés selon lesquels on peut utiliser ces composés pour synthétiser de nouveaux mononucléotides et polynucléotides ayant du phosphorodithioate, du phosphorothioamidate, des phosphorothiotriesters, et des liaisons internucléotides de phosphorothioate.
PCT/US1989/002293 1988-05-26 1989-05-25 Composes de thiophosphoramidite et de phosphorodithioate de nucleosides et de polynucleotides et procedes WO1989011486A1 (fr)

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EP0463712A2 (fr) 1990-06-27 1992-01-02 University Patents, Inc. Phosphorothioates des polynucléotides comme agents actifs contre les infections retrovirales
WO1992005186A1 (fr) * 1990-09-20 1992-04-02 Gilead Sciences Liaisons internucleosides modifiees
US5194599A (en) * 1988-09-23 1993-03-16 Gilead Sciences, Inc. Hydrogen phosphonodithioate compositions
EP0549107A1 (fr) 1991-10-11 1993-06-30 BEHRINGWERKE Aktiengesellschaft Méthode pour produire un polynucléotide qui peut être utilisé pour des amplifications à amorce unique et utilisation d'oligonucléotides contenant le phosphorotioate comme amorces pour l'amplification d'acides nucléiques
US5378825A (en) * 1990-07-27 1995-01-03 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs
US5470967A (en) * 1990-04-10 1995-11-28 The Dupont Merck Pharmaceutical Company Oligonucleotide analogs with sulfamate linkages
EP0689433A1 (fr) * 1993-03-16 1996-01-03 Board Of Regents Of The University Of Nebraska Nouveaux agents de liaison des metaux, procedes et compositions permettant de les utiliser pour traiter la toxicite des metaux
US5602240A (en) * 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5610289A (en) * 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
EP0766688A1 (fr) * 1994-05-26 1997-04-09 Isis Pharmaceuticals, Inc. Synthese d'oligonucleotides
US5623070A (en) * 1990-07-27 1997-04-22 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5965722A (en) * 1991-05-21 1999-10-12 Isis Pharmaceuticals, Inc. Antisense inhibition of ras gene with chimeric and alternating oligonucleotides
US6001982A (en) * 1993-07-29 1999-12-14 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
US6080727A (en) * 1996-03-26 2000-06-27 Istituto Regina Elena Oligonucleotide treatments and compositions for human melanoma
US6756496B1 (en) 1988-09-23 2004-06-29 Isis Pharmaceuticals, Inc. Nucleoside hydrogen phosphonodithioate diesters and activated phosphonodithioate analogues
US7255874B1 (en) 2001-12-21 2007-08-14 Closure Medical Corporation Biocompatible polymers and adhesives: compositions, methods of making and uses related thereto
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WO2018100558A2 (fr) 2016-12-01 2018-06-07 Takeda Pharmaceutical Company Limited Dinucléotide cyclique
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WO2022122613A1 (fr) * 2020-12-08 2022-06-16 F. Hoffmann-La Roche Ag Nouvelle synthèse d'oligonucléotides phosphorodithioate

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US5194599A (en) * 1988-09-23 1993-03-16 Gilead Sciences, Inc. Hydrogen phosphonodithioate compositions
US5565555A (en) * 1988-09-23 1996-10-15 Gilead Sciences, Inc. Nucleoside hydrogen phosphonodithioate diesters and activated phosphonodithioate analogues
US6756496B1 (en) 1988-09-23 2004-06-29 Isis Pharmaceuticals, Inc. Nucleoside hydrogen phosphonodithioate diesters and activated phosphonodithioate analogues
US5470967A (en) * 1990-04-10 1995-11-28 The Dupont Merck Pharmaceutical Company Oligonucleotide analogs with sulfamate linkages
US5151510A (en) * 1990-04-20 1992-09-29 Applied Biosystems, Inc. Method of synethesizing sulfurized oligonucleotide analogs
WO1991016331A1 (fr) * 1990-04-20 1991-10-31 Applied Biosystems, Inc. Procede de synthese des analogues oligonucleotidiques sulfures
EP0463712A3 (en) * 1990-06-27 1992-04-08 University Patents, Inc. Polynucleotide phosphorodithioates as therapeutic agents for retroviral infections
EP0463712A2 (fr) 1990-06-27 1992-01-02 University Patents, Inc. Phosphorothioates des polynucléotides comme agents actifs contre les infections retrovirales
US6900301B2 (en) 1990-07-27 2005-05-31 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5378825A (en) * 1990-07-27 1995-01-03 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs
US5602240A (en) * 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5610289A (en) * 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5965721A (en) * 1990-07-27 1999-10-12 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5623070A (en) * 1990-07-27 1997-04-22 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5777092A (en) * 1990-07-27 1998-07-07 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
WO1992005186A1 (fr) * 1990-09-20 1992-04-02 Gilead Sciences Liaisons internucleosides modifiees
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JPH03501128A (ja) 1991-03-14
EP0378615A4 (en) 1992-04-08
ES2015665A6 (es) 1990-09-01
IL90359A0 (en) 1989-12-15
KR900701813A (ko) 1990-12-04
AU626305B2 (en) 1992-07-30
EP0378615A1 (fr) 1990-07-25
AU3739289A (en) 1989-12-12

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