WO1991004983A1 - Composes thiophosphoramidite et phosphorodithioate de nucleoside et polynucleotide, ainsi que leur procede - Google Patents

Composes thiophosphoramidite et phosphorodithioate de nucleoside et polynucleotide, ainsi que leur procede Download PDF

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Publication number
WO1991004983A1
WO1991004983A1 PCT/US1990/005653 US9005653W WO9104983A1 WO 1991004983 A1 WO1991004983 A1 WO 1991004983A1 US 9005653 W US9005653 W US 9005653W WO 9104983 A1 WO9104983 A1 WO 9104983A1
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compound according
sulfur
nucleoside
phosphorodithioate
diisopropylamino
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PCT/US1990/005653
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English (en)
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Marvin H. Caruthers
Wolfgang Brill
John Nielsen
Eric Yau
Yun-Xi Ma
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University Patents, Inc.
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Publication of WO1991004983A1 publication Critical patent/WO1991004983A1/fr

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    • 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
    • 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
    • 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

Definitions

  • This invention described and claimed herein relates to novel and useful phosphorous compounds which are particularly useful in the production of polynucleotides having analogs attached to phosphorous.
  • the present invention relates to novel and useful nucleoside thiophosphoramidite, polynucleotide dithioate phosphoramidite, polynucleotide phosphoramidite, nucleoside 3'- hydrogenphosphonodithioates, nucleosid-3'-yl-S- aralkylphosphorodithioate, nucleoside 3'- hydrogenphosphonothioate, nucleoside 3'-methylphosphonothioate, dinucleoside H-phosphonothioate, dinucleoside phosphorodithioate and nucleoside 3'-amidophosphorodithioate compounds as well as the processes whereby these compounds can be used for
  • These novel mononucleotides and polynucleotides 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 to specific sites in cells and tissues such reagents as metal ions, toxins, intercalating agents and other reagents that alter the biochemical reactivity of polynucleotides and proteins. These compounds can also be joined to sugars, steroids, proteins, peptides and lipids so as to deliver these oligonucleotides to specific cells and thus to target certain cells for various biological and therapeutic applications with these oligonucieotide analogs. These compounds can also be used for various diagnostic purposes.
  • fluorescent or other chemically reactive reagents, antigens antibodies, proteins, and metal ions 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 enzyme reactions.
  • phosphite triesters can also be oxidized under anhydrous conditions with amines or ammonia and iodine to yield variable reported amounts of oligonucieotide phosphoramidates or with sulfur to yield oligonucieotide
  • oligonucieotide phosphoramidates and oligonucieotide phosphorothioates (Froehler, B. C, Tetrahedron Letters 27, 5575- 5578, 1986).
  • a process has also been developed for synthesizing methylphosphonothioate internucleotide linkages (Brill, W. K.-D. and Caruthers, M. H., Tetrahedron Letters 28, 3205-3208, 1987). Unfortunately, none of these procedures can be used to synthesize polynucleotides containing the phosphorodithioate or the
  • 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 synthesize deoxynucleoside phosphorodithioates or nucleoside phosphorodithioates useful for synthesizing polynucleotides containing the dithioate linkage, the procedure also yields a mixture of mononucleotides having phosphorodithioate and phosphorothioate moieties. Additionally the yield or uridine 2',3'- cyclic phosphorodithioate is only 28 % and the acidity of P 2 S 5 and the high temperatures used in the synthesis of the cyclic
  • adenosine cyclic 3',5'-phosphorodithioate can be synthesized by treating suitably protected adenosine with 4- nitrophenylphosphoranilidochloridothioate followed by
  • R 1 is H or a blocking group
  • A is D or DR 2 where D is OH, H, halogen, SH, NH 2 or azide and DR 2 is oxygen, sulfur or nitrogen as D 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
  • T, G, X and M are substituents where heteroatoms are linked covalently to phosphorous.
  • Substituents T, G, X and M may also be covalently linked to heteroatom substituted or unsubstituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl, cycloalkenyl,
  • alkynyl, aralkynyl or cycloalkynyl groups alkynyl, aralkynyl or cycloalkynyl groups.
  • the compounds of general formulae I and II wherein T, G, X and M are substituents where heteroatoms are linked to phosphorus include those in which the heteroatoms are sulfur, nitrogen and oxygen.
  • novel compounds of general formula I are of two
  • class la consists of those in which phosphorus is single bonded to each of two substituents, X and M, through the heteroatoms; and class lb are those in which phosphorous is single and double bonded to sulfur and also to one other
  • R 4 is a heteroatom substituted or unsubstituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl, cycloalkenyl, aralkenyl, alkynyl, aralkynyl or cycloalkynyl group.
  • substituent M is sulfur single bonded to phosphorous and to R 5 where R 5 is a heteroatom substituted or unsubstituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl, cycloalkenyl, aralkenyl, alkynyl, aralkynyl or cycloalkynyl.
  • the substituents G and X are nitrogen single bonded to phosphorous where G is amino or primary amino, NHR 6 , and X is secondary amino NR 6 R 7 .
  • 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 where R 6 and R 7 taken separately each represent hetroatom substituted or unsubstituted alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkenyl, cycloalkenyl, aralkenyl, alkynyl, aralkynyl, or cycloalkynyl groups; 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; and R 6 and R 7 when taken together with the nitrogen atom to which they are attached may also form a ring nitrogen
  • heterocycle compound which contains unsaturated bonds in the ring structure and may also contain at least one additional heteroatom from the group consisting of nitrogen, oxygen or sulfur.
  • Compounds of general formula II my also be those in which oxygen is double bonded to phosphorous plus M which is single bonded to phosphorous.
  • Compounds II are useful for various biological uses and for synthesizing polynucleotides containing phosphorodithioate, phophorothioamidate, phosphorothioate triester and phosphorothioate internucleotide linkages which are also useful for biological studies.
  • Amines from which the substituent group G can be derived include a wide variety of primary amines such as methylamine, ethylamine, propylamine, isopropylamine, aniline,
  • cyclohexylamine benzylamine, polycyclic amines, heteroatom substituted aryl or alkylamines, and similar primary amines.
  • Amines from which the substituent group X can be derived include a wide variety of secondary amines such as dimethyJamine, diethylamine, diisopropylamine, dibutylamine,
  • nucleoside and deoxynucleoside bases represented by B in the above formulae are well known and include purines, e.g.
  • 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 trityl, methoxytrityl, dimethoxytrityl, pivalyl, acetyl, tetrahydropyranyl, methoxytetrehydropyranyl, phenoxyacetyl, isobutyloxycarbonyl, t-butyldimethylsilyl, triisopropylsilyl, alkyl or aryl carbonoyl, and similar blocking groups well known in the art. Common blocking groups
  • R 4 and R 5 include 4-chlorobenzyl, 2,4- dichlorobenzyl, and ⁇ -cyanoethyl.
  • R 1 -9 can represent blocking groups and in many cases these blocking groups are removed at some point during synthesis, these radicals may also remain covalently attached to nucleosides, nucleotides, and polynucleotides following synthesis and correspond to
  • fluorescent probes antigens, steroids, sugars, peptides, proteins, lipids or other groups that are useful for a large number of therapeutic, diagnostic, biological or biochemical applications.
  • polynucleotides are according to the IUPAC-IUB Commission of Biochemical Nomenclature recommendations (Biochemistry 9, 4022, 1970).
  • 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:
  • Heteroatoms, preferably oxygen, sulfur, or nitrogen can replace or be bonded to the carbon atoms, preferably 1 to 4 carbon atoms in this non-cyclic branched or unbranched radical.
  • heteroatoms such as halogens can be bonded to the carbon atoms in this radical.
  • aryl - an organic radical derived from an aromatic
  • This radical can contain one or more heteroatoms, preferably sulfur, nitrogen, or oxygen, as part of the aromatic ring system. Heteroatoms, preferably halogen, sulfur, oxygen, or nitrogen, can also replace hydrogen attached to carbon that is part of the ring system.
  • aralkyl - an organic radical in which one or more aryl radicals, preferably 1 to 3, are substituted for hydrogen atoms of an alkyl radical.
  • Heteroatoms preferably oxygen, sulfur, and nitrogen, can replace or be bonded to the carbon atoms in this cyclic hydrocarbon radical.
  • Certain heteroatoms such as halogens can be bonded to the carbon atoms in this cyclic 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 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
  • Heteroatoms preferably sulfur, oxygen, and nitrogen, can replace saturated carbon atoms in this radical or be bonded to the saturated carbon atoms. Heteroatoms such as halogens can be bonded to the saturated carbon atom.
  • Heteroatoms such as oxygen, sulfur, and nitrogen can also replace carbon at an unsaturated position to generate ketone, thioketone, or imine, respectively. carbon at an unsaturated position to generate ketone, thioketone, or imine, respectively.
  • aralkenyl - an organic radical with one or more aryl radicals, 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 the cyclic part of this radical would be preferable 50 to 10 carbon atoms with the remainder attached to the cycle, the cyclic part of this radical would be preferably 5 to 10 carbon atoms with the remainder attached to the cycle, heteroatoms, preferably oxygen, sulfur and nitrogen, can replace saturated carbons in this radical or be bonded to the saturated carbons. Heteroatoms such as halogens can be bonded to the carbon atoms in this radical.
  • 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. Heteroatoms such as nitrogen can be replaced carbon at an unsaturated position to generate a nitrile.
  • aralkynyl - an organic radical in which one or more aryl groups, preferably 1 to 3, are substituted for the hydrogen atoms of an alkynyl radical.
  • cycloalkynyl - a cyclic hydrocarbon radical containing from 6 to 20 carbon atoms, preferably 7 or 12 carbon atoms, and at least one triple bond in the cycle with the remaining carbon atoms attached to the cycle, Heteroatoms, preferably oxygen, sulfur, and nitrogen, can replace saturated carbon atoms in this radical. Heteroatoms such as halogens can be bonded to the saturated carbon atoms.
  • Heteroatom substituted radicals include 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 carbons.
  • 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 can also replace carbon atoms as part of unsaturated systems such as where oxygen replaces carbon in an alkene to generate a ketone or aldehyde and nitrogen replaces carbon in an alkyne to generate a nitrile.
  • Examples of common heteroatoms substituted radicals used in nucleotide chemistry are ⁇ -cyanoethyl, 4-chlorobenzyl, 2,4-dichlorobenzyl, 4- chlorophenyl, 2,4-dichlorophenyl, acetyl, tetrahydropyranyl, di-p- methoxytrityl, and benzoyl radicals.
  • internucleotide iinkage having the general formula 5'-nucleoside-
  • internuleotide linkage an internuleotide linkage having the general formula 5'-nuleoside-OPOS-O- nucleoside 3' which can be illustrated with the following
  • internucleotide linkage having the general formula 5'-nucleoside- O-PSNHR6-O-nucleoside-3' and 5'-nucleoside-O-PSNR6R7-O- nucleoside-3' which can be illustrated with the following structures where B and A are as previously defined:
  • O-alkyl or arylphosphorothiotriester internucleotide linkage an internucleotide linkage having the general formula 5'- nucleoside-O-PSOR4-O-nucleoside-3" which can be illustrated with the following structure where B, A and R4 are previously defined:
  • internucleotide linkage having the general formula 5'-nucleoside- O-PSH-O-nucleoside-3' which can be illustrated with the
  • the preferred reaction scheme A for synthesizing compounds la, IIa, and IIc is represented as follows: wherein R 1 , R 3 , B, A, X, and M are as previously defined, compounds Vila and lia are those in which phosphorous is linked through single bonds to nucleosides and to sulfur and through a double bond to sulfur, compounds Vllb and IIc are those in which phosphorous is linked through single bonds to nucleosides and to sulfur and through a double bond to oxygen.
  • reaction scheme A involves condensation of IlIa with IVa which can be 2,4-dichlorobenzylmercaptyl-bis (diisopropylamino)phosphine or 4-chlorobenzylmercaptyl-bis (diisopropylamino)phosphine to yield la.
  • Reaction of la with Va and an activator e.g. 5-substituted tetrazoles and substituted triazoles, alkylammonium salts, aralkylammonium salts, substituted and unsubstituted pyridinium salts of
  • a second reaction scheme B was also discovered for the purpose of synthesizing compounds IIa and additionally lIb, lId, IIe, and llf.
  • the general reaction scheme B for synthesizing compounds IIa, lIb, IId, IIe and llf is as follows:
  • the preferred reaction scheme B is represented as follows:
  • R 1 , R 3 , B, A, X, M, G and T are as previously defined, compounds IIa, lla-1 , lIb, llb-1 , lId, IIe, and llf are those in which phosphorous is double bonded to sulfur and single bonded to nucleosides and one other substituent from the group of
  • reaction scheme B involves synthesis of Villa and condensation with Va to yield IXa.
  • Reaction of IXa with H 2 S and an activator such as tetrazole yields the dinucleoside H- phosphonothioate, lId, which can be chemically converted by oxidation with sulfur to lla-1 , the dinucleoside
  • the present novel compounds of general structure II having different heteroatoms containing substituents covalently linked to phosphorous can thus be prepared by processes A and B.
  • processes A and B can both be used to prepare the same compound IIa.
  • IIc where phosphorous is double bonded to oxygen and single bonded to nucleosides and to M
  • only process A can be used to produce this compound.
  • compounds lIb, llb-1 , IIe, and llf having phosphorous double bonded to sulfur and single bonded to
  • Process A also illustrates how compound la can be used to synthesize polynucleotides having
  • Process A when used to synthesize polynucleotides can be completed either on art form polymer support or in the absence of these supports.
  • nucleoside moiety of the present invention can include more than one nucleoside and may include a number of nucleosides condensed as having one or more phosphorous
  • moieties in combination with additional internucleotide phosphatediester linkages, these polynucleotides having a mixture of internucleotide linkages, and the presently described linkages as in lla-f, are prepared using the novel 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, 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
  • RNA ribonucleic acid
  • G, T, X or M sulfur double bonded to phosphorus at one or more internucleotide phosphorus containing linkages as found in DNA and RNA.
  • the synthesis of compounds according to the general formula lb can be represented by the following general reaction scheme C:
  • the preferred reaction scheme C is represented as follows:
  • compounds lb are those in which all compounds have phosphorus double bonded to sulfur and single bonded to a nucleoside, sulfur and T.
  • scheme C involves synthesis of XIII and Xllla from Ilia and Xll or Xlla. Reaction of Xlll or Xllla with H 2 S and an activator such as tetrazole yields a novel compound, XIV, the nucleoside H-phosphonothioate, which can be chemically
  • the preferred novel compounds according to the present invention are those compounds of general formula la and IIa wherein for la, M is a substituent having sulfur bonded to
  • R 5 is a heteroatom substituted or unsubstituted blocking group
  • A is H
  • R 1 is a trityl group
  • B is a nucleoside or deoxynucleoside base having art form blocking groups
  • X is a secondary amino group
  • Z is sulfur double bonded to phosphorous
  • M 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 trityl group
  • B is a nucleoside or deoxynucleoside base having art- recognized blocking groups
  • R 3 is H.
  • M is the sulfhydryl group
  • the novel compound lla-f of the present invention can be prepared as shown in scheme B from art-recognized starting materials such as Villa, a nucleoside 3'-phosphorodiamidite.
  • the initial reaction is accomplished by dissolving the nucleoside in an organic solvent such as dioxane or tetrahydrofuran containing triethyiamine 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 which can be reacted with hydrogen sulfide and tetrazole in an organic solvent such as acetonitrile to yield the dinucleoside H-phosphonothioate, lId.
  • the dinucleoside phosphorodithioates, lia are preferably synthesized as shown in scheme A by forming the aralkylmercaptyl-bis(dialkylamino- phosphine, IVa, and thereafter condensing this compound with the selected nucleoside using tetrazole as an activator in order to form a nucleoside S-(aralkyl)dialkylaminophosphoramidite.
  • the nucleoside S-(aralkyl)dialkylaminophosphoramidite, la can then be condensed with a second nucleoside using an activator in order to form an S-(aralkyl)dinucleoside phosphite, VIa, which after oxidation with elementary sulfur, yields IIa, the dinucleoside phosphorodithioate triester.
  • This procedure obviates the
  • nucleoside S-(aralkyl)dialkylaminophosphoramidite and the 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 a selected combination of
  • aralkylm ⁇ rcaptyl-bis-dialkylamino phosphine is effected in an organic solvent solution whereby the bis(dialkylamino)-chlorophosphine 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 be recrystallized from chemically inert solvents such as pentane, hexane and heptane. Distillation of the
  • synthesis 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 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
  • 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 the aralkylmercaptyl- bis(dialkylamino)phosphine requires acidic species, preferably mildly acidic, and includes tetrazole and 3-nitrotriazole.
  • the resulting nucleoside aralkylmercaptyl-phosphoramidite is difficult to activate and requires more acidic species such as aromatic amine salts of strong acids, para-nitrophenyltetrazole, trifluoromethylphenytetrazole and trifluoromethyltetrazolide salts.
  • the mercaptyl moiety as part of the bis(dialkyiamino) phosphine can vary considerably in structure. The criteria are that it facilitates activation of the mercaptyl-bis (dialkylamino) phosphine by acids, and that it can be easily removed after termination of the polynucleotide synthesis.
  • the preferred mercaptans include benzyl and heteroatom substituted benzyl moieties, phenyl and heteroatom substituted phenyl moieties, and heteroatom substituted alkyl substituents such as ⁇ -cyanoethyl.
  • aralkylmercaptyl-bis(dialkylamino) phosphine are preferable substituents that stabilize both the phosphine and the nucleoside aralkylmercaptylphosphoramidite toward storage and synthesis.
  • dialkylamino groups should also preferably facilitate activation of the phosphine during the reactions leading to the formation of internucleotide bonds.
  • substituents such as dimethylamino, diethyiamino, diisopropylamino, dipropylamino, dibutylamino, dipentylamino, various isomeric alkyl groups, aralkyl groups, and heteroatom substituted cycloalkyl groups such as pyrrolidino and piperidino.
  • the present novel compounds are used to form polynucleotides, they are preferably employed in combination with art recognized 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.
  • phosphorodithioate linkages are to be
  • a nucleoside, aralkylmercaptyl phosphoramidite is activated with aromatic amine salts, tetrazole, 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 the aralkylmercaptyl nucleoside phosphoramidite in the polynucleotide synthesis are the same as with art recognized nucleoside phosphoramidites.
  • Dinucleoside phosphorodithioate triesters can also be used as synthons for polynucleotide synthesis.
  • phosphorodithioate 3'-phosphoramidites they can be activated with tetrazole and used directly as dinucieotide synthons via the normal art-recognized polynucleotide synthesis procedure, 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 and, if methyl groups are used to protect normal internucleotide linkages, the methyl group from these phosphate 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 (e.g. trichloroethyl or ⁇ - cyanoethyl), then the de protection protocol should be modified accordingly.
  • 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 diisopropylamino dichlorophosphine was complete, the reaction mixture was refluxed for 12 hours to afford the desired product. After 12 hours the reaction mixture was cooled to room temperature 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 delta 132.4 ppm).
  • 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.
  • the reaction was quenched with pyridine (1 ml) and diluted into and free ethyl-acetate (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
  • nucleoside phosphorothioamidate was isolated after drying the precipitate in vacuo over P 2 O 5 /KOH (3.33 g, 80.1 % yield).
  • the reaction to the dinucleoside thiophosphite was quenched with sulfur (1 mmole, 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 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
  • the first step was condensation of 5'-O- dimethoxytritylthymidine with bis(diisopropylamino)- chlorophosphine in dioxane containing triethylamino.
  • the resulting phosphorodiamidite was reacted without isolation with 3'-O-acetylthymidine to yield a homogeneous dinucleoside amidite in 62 % yield after silica gel chromatography (5% triethylamine in ethylacetate).
  • Synthesis of the dinucleoside H- phosphonothioate processed by dissolving the dinucleoside phosphoroamidite (470 mg.
  • 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 hours. Purification via silica gel column chromatography (0-12% methanol in dichloromethane and 0.5 % triethylamine) afforded 70 % isolated yield. FAB+ mass spectrum, 303 (DMT+); FAB- mass spectrum, 921 (M-), 395 (5'- PSO 2 --dt-3'-OAc); 31 P-NMR delta 112.7; 1 H NMR delta 8.12
  • the dinucleoside H-phosphonoth ate was a o found to be useful as a versatile ynthon for prep e ing sever analogs rapidly (5 min) in quantitative yield ( 31 P-NMR).
  • the phosphorothioamidate (llf) was isolated in 92 % yield.
  • the dinucleoside H-phosphonothioate was converted quantitatively to a phosphorothioate triester by oxidation with iodine and 9-anthracenyl methanol (10 equivalents) under anhydrous conditions (lIb).
  • 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).
  • Rf 0.41 (methanol/dichloromethane, 1 :9, v/v).
  • 3'-O-(Diisopropylamino)-2-cyanoethyphosphino-5'-O-(di-p- methoxytrityl) thymidine (27.7 mg, 0.04 mmol) was prepared by art form methods (M.H. Caruthers and S.L. Beacage U. S. Patent 4,415,732) and then dissolved in anhydrous acteonitrile (440 ⁇ l). Hydrogen sulfide was bubbled through for 1 min and tetrazole (7.0 mg in 200 ⁇ l CH 3 CN, 0.1 mmol) was added. After 10 min 31 P-NMH spectroscopy showed quantitative conversion to the
  • nucleoside 3'-phosphorodithioate was dissolved in 80 % aqueous acetic acid (4 ml) and left for 30 min at room temperature. The reaction mixture was then diluted with water (4 ml) and extracted 3 timed 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 in 25 % aqueous ammonia and incubated at 55°C for 16 h, The mixture was re-evaporated and lyophilized with water to yield the nucleoside 3'- phosphorodithioate.
  • FAB+ mass spectrum, 338 (dt-P+ SH S).
  • the invention describes procedures for synthesizing polynucleotide phosphorodithioate, H-phosphonothioate, phosphorothioate and phosphorothioamidate compounds from nucleosid-3'-yl
  • the invention therefore provides procedures for preparing polynucleotide phosphorodithioate, H- phosphonothioate, phosphorothioate, alkylphosophonothioate and phosphorothioamidate compounds from nucleosid-3'-yl hydrogen phosphonodithioate, nucleosid-3'-yl-S-aralkylphosphorodithioate and nucleosid 3'-methylphosphonothioate synthons.
  • polynucleotide phosphorodithioate compounds synthesized with the nucleosid-3'- yl hydrogenphosphonodithioate and nucleosid-3'-yl-S-aralkyl phosphorodithioate synthons also appear to have less
  • the compounds according to this second aspect of the present invention may be represented specifically than previously described (for example, compound XXI is more specific than compound la described earlier) by the following general formulae XXI to XXIX:
  • the compounds of general formula XXI, XXII, XXIII and XXIV are useful for the synthesis of polynucleotides containing phosphorodithioate, phosphorothioamidate, alkyl or aryl phosphonothioate and phosphorothioate internucleotide linkages which are useful for various biological applications. These compounds are also useful for various biological applications.
  • the process of the generalized reaction scheme involves first the synthesis of XXIa and the conversion of this novel compound to various mononucleotides and oligonucleotides having modified chemical structures.
  • the synthesis of XXIa proceeds by reacting XXXa with preferably bis(triazoyl)chlorophosphine, compound XXXIa, followed by a treatment with H 2 S for five minutes.
  • Various other bis-aminophosphines such as tetrazoyl, imidazoyl, diisopropylamino, dimethylamino, diethylamino, morpholino, piperidino and pyrrolidono derivatives are additional examples of amino groups that can be used.
  • compound XXIa After purging with an inert gas to remove H 2 S, compound XXIa can be isolated by purification and precipitation, compound XXIa can then be converted via novel processes to XXIIa.
  • compound XXIA when compound XXIA is treated with one equivalent each of water and dicyclohexylcarbodiimide or N-methyl-2-chloropyridinium iodide in pyridine for 30 minutes, the nucleoside 3'- hydrogenphosphonothioate forms in essentially quantitative yield.
  • Formation of compound XXIXa via a similar reaction was possible by treatment of compound XXIa with compound XXXIIIa and N- methyl-2-chloropyridinium iodide. After 15 minutes reaction time, compound XXIXa can be isolated by purification and
  • compound XXIa can be used to prepare dinucleoside hydrogenphosphdnothioates.
  • novel compounds XXIa are not as reactive as the nucleoside diamidites and not as unstable, but reacts readily with unblocked 3'-OH or 5'- OH of nucleosides under normal reaction conditions.
  • the novel nucleoside hydrogenphosphonodithioates are stable under normal laboratory conditions to hydolysis and air oxidation and may be stored as dry, stable powders. Therefore, the novel compounds are more easily employed in the process of forming
  • novel compound XXIa may be used to form novel
  • compound XXVIa may be isolated by purification and precipitation from n-pentane.
  • compound XXIa can be used to prepare
  • condensation of XXIa with XXXIIIa may be monitored by
  • the resulting novel dinucleoside phosphorodithioate can then be reacted with various alkylating agents to yield XXVI la, and this compound may then be incorporated into polynucleotides.
  • R 1 , R 4 , and R 7 may be removable as blocking groups under different chemical conditions so that each can be selectively eliminated in the presence of the other.
  • One such preferable combination of conditions would be R 1 removed with acid (as in the case of di-p-methoxytrityl), R 7 removed by a base (as in the case of ⁇ -cyanoethyl), and R 4 removed by thiophenol (as in the case of 2,4-dichlorobenzyl).
  • all other "blocking groups" according to the invention may also be selected so that each can be selectively eliminated in the presence of the others.
  • XXVII can be extended to form polynucleotides simply by removing either R 1 or R 1 preferentially followed by the chemistry outlined in the scheme immediately above.
  • Reaction of XXXVIa without isolation with sulfur yields XXXVIIa which can them be converted to XXXVI IIa with triethylamine under anhydrous conditions.
  • the triethylammonium salts of XXXVIIIa may then be stored as a solid.
  • bases that preferentially remove the R 7 protecting group in the presence of R 4 may also be used.
  • Reaction of XXXVIIIa with XXXIIIa in the presence of triisopropylbenzenesulfonyl chloride then yields XXVIIa, the completely protected dinucleoside phosphorodithioate.
  • activating agents such as mesitylenesulfonyl chloride and tetrazolide can be used to synthesize XXVIIa.
  • Compound XXVIIa may then be further extended to synthesize larger polynucleotides by removing R 1 from XXVIIa with acid and condensing the resulting compound with XXXVIIIa using
  • XXVIIa may be treated with a base to remove R 3 and then converted to the dinucleoside 3'-phosphoramidite analogous to XXXIVa, using the known conditions in United States Patent 4,415,732, which can
  • triisopropylbenzenesulfonyl chloride to yield a tetranucleotide having three phosphorodithioate linkages.
  • These polynucleotides may then be further extended in a similar manner to form longer polynucleotides having phosphorodithioate linkages or by using nucleoside 3'-phosphate diesters to polynucleotides having both phosphorodithioate and phosphate internucleotide linkages.
  • R 8 is a heteroatom substituted or unsubstituted alkyl, aryl, aralkly, cycloalkyl, cycloalkylalkyl, alkenyl, cycloalkenyl, aralkenyl, alkynyl, aralkynyl, or cycloalkynyl group.
  • the preferred novel compounds of this aspect of the invention are those compounds of general formula XXI, XXIII, XXIV, and XXVII. These novel compounds may be used to prepare XXIX, the dinucleoside H-phosphonothioates. Compound XXIX may then be converted to preferably dinucleoside phosphorodithioates (XXVI), dinucleoside phosphorothioamidates and dinucleoside phosphorothioates.
  • Compound XXI may also be condensed with an appropriate nucleoside, XXXIII, with iodine to form XXVI, the dinucleoside phosphorodithioate which can be converted to XXVII via a conventional alkylating agent.
  • Preferred compound XXlll can react with an appropriate nucleoside, XXXIII, and a condensing agent such as triisopropylsulfonyl chloride, to form XXVII.
  • R 9 2-anthracenyl
  • R 4 2,4-dichlorobenzyl
  • R 3 acetyl
  • the deacylated compound was then reacted with bis(diisopropylamino)-2-cyanoethoxy phosphine (1 ,5 eq) in the presence of tetrazole (1 eq) for 1 hour at room temperature to produce the dinucieotide phosphorodithioate triester as the 3'-phosphoramidite in 76% yield.
  • the resulting dinucieotide phosphoramidite has been used successfully in combination with modified mononucleoside phosphoramidites for the synthesis of a 26-mer DNA fragment containing the
  • reaction mixture was subjected to column chromatography using CH 3 CCI 3 /CH 3 OH (4:1. v:v) containing 0.5% of triethylamine to yield the desired product.
  • R 3 acetyl
  • R 4 4-chlorobenzyl
  • deoxygenated CH 3 CN was added 0.22 ml (0.262 g, 1.65 mmol) of 4- chlorobenzylmercaptan and a solution of 84,8 mg (1.2 mmol) of tetrazole in 2 ml of CH 3 CN.
  • the reaction mixture was stirred at room temperature under argon for 40 minutes, at which time a saturated solution of sulfur (2.25 ml) in toluene/2,6-lutidine (19/1) was added.
  • the resulting mixture was allowed to continue to stir at room temperature for 1 hour.
  • the mixture was then diluted with EtOAc and the organic layer was washed with 5% aqueous NaHCO 3 , water and saturated NaCL, dried over MgSO 4 , filtered, and evaporated.
  • the crude residue obtained was
  • R 4 4-chlorobenzyl
  • R 3 acetyl
  • the 31 P-NMR spectrum of the reaction mixture indicated two peaks, one of the desired product at 98.06 and 97.18 ppm, and several side-products at 87.05 and 86.58 ppm (30%).
  • the reaction mixture was subjected to column chromatography using CH 3 CCI 3 /CH 3 OH (9:1 , v:v). The product fractions were combined and evaporated to dryness. Precipitation from CHCI 3 into n-pentane followed. The product was obtained as a white solid in 47% (47 mg) yield. If the reaction was carried out in CH 2 CI 2 , almost no formation of dimer was observed by 31 P-NMR. Instead, several products giving NMR-signals from 85.3-93.4 ppm were formed.

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Abstract

Nouveaux composés utiles de thiophosphoramidite de nucléoside, phosphoramidite de thioate de polynucléotide et phosphoramidite de phosphorothioamidate de polynucléotide, et procédés selon lesquels ces composés peuvent être utilisés afin de synthétiser de nouveaux mononucléotides et polynucléotides comportant des liaisons internucléotides phosphorodithioate, phosphorothioamidate, phosphorothiotriesters et phosphorothioate.
PCT/US1990/005653 1989-10-05 1990-10-04 Composes thiophosphoramidite et phosphorodithioate de nucleoside et polynucleotide, ainsi que leur procede WO1991004983A1 (fr)

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US5194599A (en) * 1988-09-23 1993-03-16 Gilead Sciences, Inc. Hydrogen phosphonodithioate compositions
EP0538194A1 (fr) * 1991-10-17 1993-04-21 Ciba-Geigy Ag Nucléosides et oligonucléosides bicycliques, leur procédé de préparation et leurs intermédiaires
WO1994004548A1 (fr) * 1992-08-19 1994-03-03 Gilead Sciences, Inc. Aptameres chimiquement reversibles
FR2705099A1 (fr) * 1993-05-12 1994-11-18 Centre Nat Rech Scient Oligonucléotides phosphorothioates triesters et procédé de préparation.
WO1996029337A1 (fr) * 1995-03-23 1996-09-26 Hybridon, Inc. Phosphorothioates d'oligodesoxynucleotides anti-sens modifies de thiono-triester
US5571902A (en) * 1993-07-29 1996-11-05 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
WO1997009340A1 (fr) * 1995-09-01 1997-03-13 Polska Akademia Nauk Centrum Badan Molekularnych I Makromolekularnych Compositions et procedes de synthese de derives organo-phosphoreux
US5614621A (en) * 1993-07-29 1997-03-25 Isis Pharmaceuticals, Inc. Process for preparing oligonucleotides using silyl-containing diamino phosphorous reagents
WO1997019092A1 (fr) * 1995-11-17 1997-05-29 Isis Pharmaceuticals, Inc. Procede ameliore de synthese de composes oligomeres
US5955591A (en) * 1993-05-12 1999-09-21 Imbach; Jean-Louis Phosphotriester oligonucleotides, amidites and method of preparation
US6001982A (en) * 1993-07-29 1999-12-14 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
US6294664B1 (en) 1993-07-29 2001-09-25 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides

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IL90359A0 (en) * 1988-05-26 1989-12-15 University Patents Inc Nucleoside and polynucleotide thiophosphoramidite and phosphorodithioate compounds and their production
WO1990012802A1 (fr) * 1989-04-18 1990-11-01 The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce Nouveaux oligodeoxynucleotides dotes de groupes chimiques lies en 5', procede de preparation, et leur utilisation
FI102072B1 (fi) * 1990-09-14 1998-10-15 Hoechst Ag 2-dansyylietoksiklooriformiaattihydrokloridin käyttö hydroksiryhmän suojaamiseen oligonukleotidisynteesissä, näin suojattuja välituotteita, menetelmiä niiden valmistamiseksi ja menetelmä oligonukleotidien syntetisoimiseksi

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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
EP0538194A1 (fr) * 1991-10-17 1993-04-21 Ciba-Geigy Ag Nucléosides et oligonucléosides bicycliques, leur procédé de préparation et leurs intermédiaires
US5319080A (en) * 1991-10-17 1994-06-07 Ciba-Geigy Corporation Bicyclic nucleosides, oligonucleotides, process for their preparation and intermediates
US5393878A (en) * 1991-10-17 1995-02-28 Ciba-Geigy Corporation Bicyclic nucleosides, oligonucleotides, process for their preparation and intermediates
WO1994004548A1 (fr) * 1992-08-19 1994-03-03 Gilead Sciences, Inc. Aptameres chimiquement reversibles
US5770713A (en) * 1993-05-12 1998-06-23 Centre National De La Recherche Scientifique Phosphorothioate triester oligonucleotides and method of preparation
FR2705099A1 (fr) * 1993-05-12 1994-11-18 Centre Nat Rech Scient Oligonucléotides phosphorothioates triesters et procédé de préparation.
WO1994026764A1 (fr) * 1993-05-12 1994-11-24 Centre National De La Recherche Scientifique (Cnrs) Oligonucleotides phosphorothioates triesters et procede de preparation
US5955591A (en) * 1993-05-12 1999-09-21 Imbach; Jean-Louis Phosphotriester oligonucleotides, amidites and method of preparation
US6001982A (en) * 1993-07-29 1999-12-14 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
US6294664B1 (en) 1993-07-29 2001-09-25 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
US6870039B2 (en) 1993-07-29 2005-03-22 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
US6646114B2 (en) 1993-07-29 2003-11-11 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
US6486312B2 (en) 1993-07-29 2002-11-26 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
US5847106A (en) * 1993-07-29 1998-12-08 Isis Pharmaceuticals Inc. Monomeric and dimeric nucleosides with silyl-containing diamino phosphorous linkages
US5614621A (en) * 1993-07-29 1997-03-25 Isis Pharmaceuticals, Inc. Process for preparing oligonucleotides using silyl-containing diamino phosphorous reagents
US5571902A (en) * 1993-07-29 1996-11-05 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
US6211350B1 (en) 1993-07-29 2001-04-03 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
US6124450A (en) * 1993-07-29 2000-09-26 Isis Pharmaceuticals, Inc. Processes and intermediates for phosphorous-containing covalent linkages
US6124445A (en) * 1994-11-23 2000-09-26 Isis Pharmaceuticals, Inc. Phosphotriester oligonucleotides, amidities and method of preparation
WO1996029337A1 (fr) * 1995-03-23 1996-09-26 Hybridon, Inc. Phosphorothioates d'oligodesoxynucleotides anti-sens modifies de thiono-triester
WO1997009340A1 (fr) * 1995-09-01 1997-03-13 Polska Akademia Nauk Centrum Badan Molekularnych I Makromolekularnych Compositions et procedes de synthese de derives organo-phosphoreux
US6051699A (en) * 1995-11-17 2000-04-18 Isis Pharmaceuticals, Inc. Process for the synthesis of oligomeric compounds
US5859232A (en) * 1995-11-17 1999-01-12 Isis Pharmaceuticals, Inc. Process for the synthesis of oligomeric phosphite, phosphodiester, phosphorothioate and phosphorodithioate compounds
US5705621A (en) * 1995-11-17 1998-01-06 Isis Pharmaceuticals, Inc. Oligomeric phosphite, phosphodiester, Phosphorothioate and phosphorodithioate compounds and intermediates for preparing same
WO1997019092A1 (fr) * 1995-11-17 1997-05-29 Isis Pharmaceuticals, Inc. Procede ameliore de synthese de composes oligomeres

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