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WO2003045969A1 - Reactif de couplage en chimie des h-phosphonates - Google Patents

Reactif de couplage en chimie des h-phosphonates Download PDF

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Publication number
WO2003045969A1
WO2003045969A1 PCT/GB2002/005177 GB0205177W WO03045969A1 WO 2003045969 A1 WO2003045969 A1 WO 2003045969A1 GB 0205177 W GB0205177 W GB 0205177W WO 03045969 A1 WO03045969 A1 WO 03045969A1
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group
oligonucleotide
substituted
phosphonate
unsubstituted
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PCT/GB2002/005177
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English (en)
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Nanda Dulal Sinha
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Avecia Biotechnology Inc.
Avecia Limited
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Priority to AU2002339175A priority Critical patent/AU2002339175A1/en
Publication of WO2003045969A1 publication Critical patent/WO2003045969A1/fr

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    • 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

  • the H-phosphonate method for synthesizing oligonucleotides involves condensation of a nucleoside H-phosphonate and a nucleoside or nascent oligonucleotide. This methodology does not require a capping step or oxidation step after each condensation reaction. Generally, the H-phosphonate linkages formed as a result of the condensation reaction can be oxidized once the entire oligonucleotide chain is synthesized.
  • the coupling step is performed in the presence of a sulfur transfer reagent which reacts with the H-phosphonate diester formed in the coupling reaction to form, in situ, a phosphorothioate triester (see WO99/09041).
  • Z is a halogen, preferably -Cl or -Br
  • R 2 is -O- or -S-
  • R 3 is -H or a substituent, preferably a halogen, a nitro group, or an alkyl group
  • R 1 is O
  • R 2 -O-
  • R 3 -H
  • n 1
  • X 7 is O.
  • Z is Cl.
  • the method of the invention can be used to form oligonucleotides in solution or on a solid support.
  • the coupling can be done in the presence of a sulfur transfer reagent to form a phosphorothioate triester.
  • the coupling reagent represented by Formula I is suitable for use in the methods taught in PCT publication WO 99/09041 , the entire teachings of which are hereby incorporated by reference.
  • the present invention features a method of preparing an H- phosphonate diester, involving the coupling of an H-phosphonate with a substrate comprising a free hydroxy group in the presence of a compound represented by Structural Formula I.
  • the H-phosphonate diester formed can be protected or deprotected.
  • the H- phosphate linkages formed in the coupling step can be derivatized using a sulfur transfer agent or other oxidizing agent upon completion of the oligonucleotide synthesis.
  • phosphorothioate triester linkages can be formed in situ by reacting the H- phosphonate with a sulfur transfer agent.
  • the phosphorothioate triester oligonucleotide is treated with a base upon completion of the oligonucleotide synthesis to form a phosphorothioate diester oligonucleotide or with an oximate and a base to form a phosphodiester oligonucleotide. Any of these methods may be carried out in solution or on a solid support.
  • a compound represented by Structural Formula I can also be used in coupling reactions to generate oligonucleotides, which can be protected or deprotected, and which can also be derivatized using a sulfur transfer agent or other oxidizing agent.
  • Synthesis of phosphodiester and phosphorothioate triester oligonucleotides can be achieved in situ by reacting the H-phosphonate diester with a sulfur transfer reagent.
  • preparation of oligonucleotides using a compound represented by Structural Formula I can be carried out in solution or on a solid support.
  • the synthetic oligonucleotide preferably has from 2 to about 100 nucleobases.
  • the synthetic oligonucleotide has 2 to about 75 nucleobases.
  • Many synthetic oligonucleotides of current therapeutic interest comprise from about 8 to about 40 nucleobases. Oligonucleotides of such lengths can be generated using the methods described herein.
  • the present invention has several advantages.
  • the methods described herein provide a new coupling procedure for the synthesis of phosphodiesters and oligonucleotides that in many embodiments (a) is extremely efficient and does not lead to side-reactions, (b) proceeds relatively rapidly, and (c) is equally suitable for the preparation of oligonucleotides, their phosphodiester or phosphorothioate analogs, and chimeric oligonucleotides containing both phosphodiester and phosphorothioate diester internucleotide linkages.
  • a compound represented by Structural Formula I in the promotion of H-phosphonate condensation reactions has the advantage of minimizing modification of the O6 group of guanosine and the O4 group of thymidine, which are susceptible to modification with other condensation promoters such as pivaloyl chloride, adamantyl chloride, and diphenyl phosphoryl chloride.
  • a compound represented by Structural Formula I is less expensive than other condensation promoters, such as adamantyl chloride, and is easily removed after the condensation reaction.
  • Another advantage of the present invention is that the coupling methods described herein can be used for either solution synthesis or solid support synthesis of oligonucleotides.
  • the advantages of solid support synthesis over solution synthesis are (i) that it is much faster; (ii) that coupling yields are generally higher; (iii) that it is easily automated; and (iv) that it is completely flexible with respect to sequence.
  • solid phase synthesis is particularly useful if relatively small quantities of a large number of oligonucleotides sequences are required for combinatorial purposes. However, if kilogram quantities of a particular sequence of moderate size are required, speed and flexibility become relatively unimportant, and synthesis in solution is likely to be highly advantageous.
  • Solution synthesis also has the advantage over solid phase synthesis in that block coupling (i.e., the addition of two or more nucleotide residues at a time) is more feasible and scaling-up is unlikely to present a problem.
  • block coupling i.e., the addition of two or more nucleotide residues at a time
  • the use of a coupling method involving a compound represented by Structural Formula I provides the flexibility to synthesize oligonucleotides based on the desired criteria.
  • FIG. 1 represents a preferred coupling reagent N,N-bis-( 2 -oxo- 3 -oxazolidinyl)- phosphorodiamidic chloride (also referred to as BOP-CI).
  • FIG. 2 is a reaction scheme for the activation of a nucleoside-H-phosphonate monomer in the formation of an H-phosphonate diester linkage.
  • FIG. 3A represents a protected dGC dimer phosphorothioate synthesized using methods described herein.
  • FIG. 3B represents a protected dGA dimer phosphorothioate synthesized using methods described herein.
  • FIG. 3C represents a protected dTG dimer phosphorothioate synthesized using methods described herein.
  • the H-phosphonate employed in the methods of the present invention is advantageously a protected nucleoside or oligonucleotide H-phosphonate, or an analog thereof, preferably comprising a 5' or a 3' H-phosphonate function, and more preferably, a 3" H-phosphonate function.
  • Preferred nucleosides are 2'-deoxyribonucleosides and ribonucleosides, for example, 2'-O-(alkyl, alkoxyalkyl, or alkenyl)-ribonucleosides.
  • oligonucleotides are oligo(2'-deoxyribonucleotides) and oligoribonucleotides, for example, 2'-O-(alkyl, alkyoxyalkyl, or alkenyl)-oligoribonucleotides.
  • nucleoside means a molecule that is made of a purine or pyrimidine base linked to sugar.
  • Nucleoside bases include, but are not limited to, naturally occurring bases, such as adenine, guanine, cytosine, thymine, and uracil and modified bases such as 7-deazaguanine, 7-deaza-8-azaguanine, 5-propynylcytosine, 5- propynyluracil, 7-deazaadenine, 7-deaza-8-azaadenine, 7-deaza-6-oxopurine, 6- oxopurine, 3-deazaadenosine, 2-oxo-5-methylpyrimidine, 2-oxo-4-methylthio-5- methylpyrimidine, 2-thiocarbonyl-4-oxo-5-methylpyrimidine, 4 ⁇ oxo-5-methylpyrimidine, 2- amino-purine, 5-fluorouracil, 2,6-diaminopurine, 8-
  • oligonucleotide includes naturally occurring oligonucleotides, for example, 2'-deoxyribonucleic acids (hereinafter “DNA”) and ribonucleic acids (hereinafter “RNA”) and nucleic acids containing modified sugar moieties, modified phosphate moieties, or modified nucleobases. Modification to the sugar moiety includes replacing the ribose ring with a hexose, cyclopentyl, or cyclohexyl ring.
  • DNA 2'-deoxyribonucleic acids
  • RNA ribonucleic acids
  • the D-ribose ring of a naturally occurring nucleic acid can be replaced with an L-ribose ring, or the b-anomer of a naturally occurring nucleic acid can be replaced with the a-anomer.
  • the oligonucleotide may also comprise one or more abasic moieties. Modified phosphate moieties include phosphorothioates, phosphorodithioates, methyl phosphonates, methyl phosphates, and phosphoramidates. Such nucleic acid analogs are known to those of skill in the art.
  • the H-phosphonate is a protected deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide, or oligoribonucleotide derivative comprising a 3' H-phosphonate function
  • the 5' hydroxy function is advantageously protected by a suitable protecting group.
  • suitable protecting groups include acid labile protecting groups, particularly trityl and substituted trityl groups, such as dimethoxytrityl and 9- phenylxanthen-9-yl groups; and base labile-protecting groups, such as FMOC.
  • an "acid labile protecting group” is a protecting group that can be removed by contacting the group with a Bronsted or a Lewis acid. Acid labile protecting groups are known to those skilled in the art. Examples of common acid labile protecting groups include substituted or unsubstituted trityl groups (Greene et al., Protective Groups in Organic Synthesis (1991), John Wiley & Sons, Inc., pages 60-62), substituted or unsubstituted tetrahydropyranyl groups (Id., pages 31-34), substituted or unsubstituted tetrahydrofuranyl groups (Id., pages 36-37), or pixyl groups (Id., page 65).
  • a preferred acid labile protecting group is a substituted or unsubstituted trityl, for example, 4,4'- dimethoxytrityl (hereinafter "DMT").
  • Trityl groups are preferably, substituted by electron donating substituents such as alkoxy groups.
  • the 3' hydroxy function is advantageously protected by a suitable protecting group.
  • suitable protecting groups include those disclosed above for the protection of the 5' hydroxy functions of 3' H-phosphonates and acyl groups, such as levulinoyl and substituted levulinoyl groups.
  • the 2'-hydroxy function is advantageously protected by a suitable protecting group, for example, an acid-labile acetal protecting group, particularly a 1-(aryl)- 4-alkoxypiperidin-4-yl group such as 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp) or 1-(2-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep); and trialkylsilyl groups, often tri(C 1-4 - alkyl)silyl groups such as a tertiary butyl dimethylsilyl group.
  • a suitable protecting group for example, an acid-labile acetal protecting group, particularly a 1-(aryl)- 4-alkoxypiperidin-4-yl group such as 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp) or 1-(2-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep); and trial
  • the ribonucleoside or oligoribonucleotide may be a 2'-O-alkyl, 2'-O-alkoxyalkyl, or 2'-O-alkenyl derivative, commonly a C M alkyl, C 1-4 alkoxy, C 1- alkyl, or alkenyl derivative, in which case, the 2' position does not need further protection.
  • H-phosphonates of nucleoside and oligonucleotide analogs that may be employed in the process of the present invention include 2'-fluoro, 2'-amino, 2'-C-alkyl, and 2'-C-alkenyl substituted nucleoside and oligonucleotide derivatives.
  • H-phosphonates that may be employed in the methods of the present invention are derived from other polyfunctional alcohols, especially alkyl alcohols, and preferably, diols or triols.
  • alkyl diols include ethane-1 ,2-diol, and low molecular weight poly(ethylene glycols), such as those having a molecular weight of up to 400.
  • alkyl triols include glycerol and butane triols.
  • suitable protecting groups such as those disclosed hereinabove for protection at the 5' or 2' positions of ribonucleosides.
  • H-phosphonate multimers for example, dimers or trimers are another example of H-phosphonates that can be used in the invention as described herein.
  • the multimers may contain an H-phosphonate backbone, or may be derivatized to form, for example, a phosphodiester backbone, or a phosphorothioate diester backbone, using methods described herein.
  • the H-phosphonate is coupled with a substrate comprising a free hydroxy group, often a nucleoside or an oligonucleotide, which may be protected or unprotected, preferably comprising a free 5' or 3' hydroxy function, and more preferably comprising a free 5' hydroxy function.
  • preferred nucleosides are 2'- deoxyribonucleosides and ribonucleosides, for example, 2'-O-(alkyl, alkyloxyl, or alkenyl)- ribonucleosides and preferred oligonucleotides are oligo(2'-deoxyribonucleotides) and oligoribonucleotides, for example, 2'-O-(alkyl, alkyloxyl, or alkenyl)-oligoribonucleotides.
  • the 3'-hydroxy function may be protected by a suitable protecting group.
  • protecting groups include acyl groups, commonly comprising up to 16 carbon atoms, such as those derived from gamma keto acids, such as levulinoyl groups and substituted levulinoyl groups.
  • Substituted levulinoyl groups include, for example, 5-halo- levulinoyl, such as 5,5,5-trifluorolevulinoyl and benzoylpropionyl groups.
  • Other such protecting groups include fatty alkanoyl groups, for example, linear or branched C 6-1 6 alkanoyl groups, such as lauroyl groups; benzoyl and substituted benzoyl groups, such as alkyl, commonly C 1-4 alkyl-, and halo, commonly chloro or fluoro, substituted benzoyl groups; and silyl ethers, such as alkyl, commonly C 1-4 alkyl, and aryl, commonly phenyl, silyl ethers, particularly tertiary butyl dimethyl silyl and tertiary butyl diphenyl silyl groups.
  • the substrate comprising a free hydroxy group is a deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide, or oligoribonucleotide derivative comprising a free 5'-hydroxy group
  • the substrate comprising a free hydroxy group is preferably bonded to the solid support via the 3'-hydroxy function.
  • the substrate comprising a free hydroxy group is a deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide, or oligoribonucleotide derivative comprising a free 3'-hydroxy group
  • the substrate comprising a free hydroxy group is preferably bonded to the solid support via the 5'- hydroxy function.
  • the substrate comprising a free hydroxy group is a protected deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide, or oligoribonucleotide comprising a free 3'-hydroxy group
  • the ⁇ '-hydroxy function is advantageously protected by a suitable protecting group.
  • Suitable protecting groups are those disclosed above for the protection of the 5' hydroxy group of deoxyribonucleosides, ribonucleosides, oligodeoxyribonucleotides, and oligoribonucleotide 3' H-phosphonates.
  • the 2'-hydroxy function is advantageously protected by a suitable protecting group, such as an acetal, particularly 1-(2-fluorophenyl)-4-methoxypiperidine-4- yl (Fpmp); and trialkylsilyl groups, often tri(C 1-4 -alkyl) silyl groups such as a tertiary butyl dimethyl silyl group.
  • a suitable protecting group such as an acetal, particularly 1-(2-fluorophenyl)-4-methoxypiperidine-4- yl (Fpmp); and trialkylsilyl groups, often tri(C 1-4 -alkyl) silyl groups such as a tertiary butyl dimethyl silyl group.
  • the ribonucleoside or oligoribonucleotide may be a 2'- O-alkyl, 2'-O-alkoxyalkyl, or 2'-O-alkenyl derivative, commonly a C 1-4 alkyl, C 1-4 alkoxy, or C 1-4 alkyl or alkenyl derivative, in which case, the 2' position does not need further protection.
  • substrates comprising a free hydroxy group that may be employed in the process according to the present invention are non-saccharide polyols, especially alkyl polyols, and preferably, diols or triols.
  • alkyl diols include ethane-1,2-diol, and low molecular weight poly(ethylene glycols), such as those having a molecular weight of up to 400.
  • alkyl triols include glycerol and butane triols.
  • suitable protecting groups such as those disclosed hereinabove for the protection at the 5' or 2' positions of ribonucleosides.
  • more than one free hydroxy group may be present if it is desired to perform identical couplings on more than one hydroxy group.
  • bases present in nucleosides/nucleotides employed in the present invention are also preferably protected where necessary by suitable protecting groups.
  • Protecting groups employed are those known in the art for protecting such bases.
  • a and/or C can be protected by benzoyl, including substituted benzoyl, for example, alkyl- or alkoxy-, often C 1-4 alkyl- or C 1-4 alkoxy-; benzoyl; pivaloyl; and amidine, particularly dialkylaminomethylene, preferably, di(C 1- -alkyl) aminomethylene, such as dimethyl or dibutyl aminomethylene.
  • O6 of G may be protected by a phenyl group, including a substituted phenyl, for example, 2,5-dichlorophenyl and diphenyl carbamoyl, and also by an isobutyryl group.
  • T and U generally do not require protection, but in certain embodiments may advantageously be protected, for example, at O4 by a phenyl group, including a substituted phenyl, for example, 2,4-dimethylphenyl or at N3 by a pivaloyloxymethyl, benzoyl, alkyl, or alkoxy substituted benzoyl, such as C 1-4 alkyl- or C 1-4 alkoxybenzoyl.
  • the substrate comprising a free hydroxy group and/or an H-phosphonate is a protected nucleoside or oligonucleotide having protected hydroxy groups
  • one of these protecting groups may be removed after carrying out the coupling reaction described above. Commonly, the protecting group removed is that on the 3'-hydroxy function.
  • the oligonucleotide thus formed may be converted into an H-phosphonate and may then proceed through one or more additional coupling reactions and one or more derivatizations, as described herein. The method may then proceed with steps to remove the protecting groups from the internucleotide linkages, the 3' and the 5'-hydroxy groups, and from the bases, if so desired. Similar methodology may be applied to coupling 5' H-phosphonates, wherein the protecting group removed is that on the 5' hydroxy function.
  • Protecting groups can be removed using methods known in the art for the particular protecting group and function.
  • transient protecting groups commonly employed on 3'-hydroxy groups, particularly gamma keto acids such as levulinoyl-type protecting groups, can be removed by treatment with hydrazine, for example, buffered hydrazine, such as the treatment with hydrazine under very mild conditions disclosed by van Boom. J.H.; Burgers, P.M.J. Tetrahedron Lett., 1976, 4875- 4878.
  • oligonucleotides with free 3'-hydroxy functions may then be converted into the corresponding H-phosphonates, which are intermediates that can be employed for the block synthesis of oligonucleotides and their phosphorothioate analogues.
  • such depurination which perhaps is difficult completely to avoid in solid phase synthesis, can be totally suppressed by effecting 'detritylation' with a dilute solution of hydrogen chloride at low temperature, particularly about 0.45 M hydrogen chloride in dioxane - dichloromethane (1 :8 v/v) solution at -50°C. Under these reaction conditions, 'detritylation' can be completed rapidly, and in certain cases after 5 minutes or less.
  • Silyl protecting groups may be removed by fluoride treatment, for example, with a solution of a tetraalkyl ammonium fluoride salt such as tetrabutyl ammonium fluoride.
  • Fpmp protecting groups may be removed by acidic hydrolysis under mild conditions.
  • the coupling of the H-phosphonate to a substrate comprising a free hydroxy group occurs in the presence of a compound represented by Structural Formula I.
  • the coupling reaction is conveniently carried out at a temperature in the range of approximately 15°C to about 30°C, and this range has been used for solution and solid phase.
  • the newly formed H-phosphonate diesters or oligonucleotides generated as described herein can be derivatized to form, for example, phosphodiesters or phosphorothioate triesters. Such derivatizations are carried out in situ or once the oligonucleotide is completely synthesized. As used herein, by "in situ” is meant that the product of the coupling reaction is derivatized without separation and purification of the intermediate produced by the coupling reaction.
  • protected nucleosides or oligonucleotides with a 3'-terminal H-phosphonate function and protected nucleosides or oligonucleotides with a 5'-terminal hydroxy function are coupled in the presence of a compound represented by Structural Formula I to form an oligonucleoside or oligonucleotide H-phosphonate intermediate, where the intermediates undergo sulfur- transfer in the presence of a suitable sulfur-transfer agent.
  • In situ derivatization of the H-phosphonate occurs through the use of a sulfur transfer agent.
  • the nature of the sulfur-transfer agent will depend on whether an oligonucleotide having phosphodiester linkages, a phosphorothioate analog or a mixed oligonucleotide/oligonucleotide phosphorothioate (chimeric oligonucleotide) is desired.
  • Sulfur transfer agents employed in the process of the present invention often have the general chemical formula:
  • L represents a leaving group
  • A represents an aryl group, a methyl group, a substituted alkyl group or an alkenyl group.
  • the leaving group is selected so as to comprise a nitrogen-sulfur bond.
  • suitable leaving groups include morpholines such as morpholine-3,5-dione; imides such as phthalimides, succinimides and maleimides; indazoles, particularly indazoles with electron-withdrawing substituents such as 4-nitroindazoles; and triazoles.
  • the moiety A represents a methyl, substituted alkyl, or alkenyl group.
  • suitable substituted alkyl groups include substituted methyl groups, particularly benzyl and substituted benzyl groups, such as alkyl-, commonly C 1- alkyl- and halo-, commonly chloro-, substituted benzyl groups, and substituted ethyl groups, especially ethyl groups substituted at the 2-position with an electron-withdrawing substituent such as 2-(4- nitrophenyl)ethyl and 2-cyanoethyl groups.
  • suitable alkenyl groups are allyl and crotyl.
  • Examples of a suitable class of phosphorothioate-directing sulfur-transfer agents are, for example, (2-cyanoethyl)sulfanyl derivatives such as 4-[(2-cyanoethyl)- sulfanyl]morpholine-3,5-dione, or a corresponding reagent such as 3- (phthalimidosulfanyl)propanonitrile.
  • a phosphorothioate triester can be formed by reacting a nucleoside H-phosphonate with a substrate comprising a free hydroxy group in the presence of a coupling reagent represented by Structural Formula I, in the presence of a compound represented by one of Structural Formulae II, III, or IV:
  • a cyanoethyl group can be removed by treatment with a strongly basic amine such as DABCO, 1 ,5-diazabicylo[4.3.0]non-5-ene (DBN), 1 ,8- diazabicyclo[5.4.0]undec-7-ene (DBU) or triethylamine.
  • DABCO 1 ,5-diazabicylo[4.3.0]non-5-ene
  • DBU 1 ,8- diazabicyclo[5.4.0]undec-7-ene
  • the moiety A represents an aryl group, such as a phenyl or naphthyl group.
  • Suitable aryl groups include substituted and unsubstituted phenyl groups, particularly halophenyl and alkylphenyl groups, especially 4-halophenyl and 4- alkylphenyl, commonly 4-(C 1-4 alkyl)phenyl groups, most preferably, 4-chlorophenyl and p- tolyl groups.
  • An example of a suitable class of standard phosphodiester-directing sulfur- transfer agent is an ⁇ /-(arylsulfanyl)phthalimide (succinimide or other imide may also be used).
  • phenyl and substituted phenyl groups on the phosphorothioate internucleotide linkages and on the base residues can be removed by oximate treatment, for example, with the conjugate base of an aldoxime, preferably that of E-2-nitrobenzaldoxime or pyridine-2-carboxaldoxime (Reese et. al., Nucleic Acids Res. 1981) to form a phosphotriester.
  • an aldoxime preferably that of E-2-nitrobenzaldoxime or pyridine-2-carboxaldoxime
  • a phosphotriester can be formed by reacting a nucleoside H- phosphonate with a substrate comprising a free hydroxy group in the presence of a coupling reagent represented by Structural Formula I, in the presence of a compound represented by Structural Formula V:
  • L is a leaving group as described above, and R is -H, an alkyl group, or a halogen.
  • oligonucleotide Once the entire oligonucleotide has been synthesized, it is reacted with a base and an oximate to form a phosphodiester.
  • Chimeric oligonucleotides containing both phosphodiester and phosphorothioate diester linkages can also be generated using the above reagents.
  • a chimeric oligonucleotide can be formed by reacting a nucleoside H-phosphonate with a substrate comprising a free hydroxy group in the presence of a coupling reagent represented by Structural Formula I, in the presence of a compound represented by Structural Formula V. After one or more coupling steps, the oligonucleotide is contacted with any of the compounds represented by Structural Formulae II, III, or IV. The oligonucleotide is then reacted with a base and an oximate to form a chimeric oligonucleotide.
  • the method of the invention can be used in the synthesis of
  • RNA 2'-O-alkyl-RNA, 2'-O-alkoxyalkyl-RNA and 2'-O-alkenyl-RNA sequences.
  • 2'-O- (Fpmp)-5'-O-(4,4-dimethoxytrityl)-ribonucleoside 3'-H-phosphonates an example of which is shown in Structural Formula VI:
  • nucleoside building blocks ammonium p-cresyl H-phosphonate and a compound represented by Structural Formula I.
  • a suitable temperature for carrying out the coupling reaction and in situ derivatization step is in the range of about -55°C to about 35°C.
  • the temperature is in the rage of about 0°C to about 30°C.
  • room temperature commonly in the range of from about 10°C to about 25°C, for example, from about 20°C to about 25°C is used.
  • a suitable temperature for carrying out the coupling reaction and in situ derivatization step is in the range of about -55°C to about 40°C.
  • the temperature is in the rage of about 0°C to about 30°C.
  • room temperature commonly in the range of from about 10°C to about 25°C, for example, from about 20°C to about 25°C is used.
  • organic solvents which can be employed in the process of the present invention include haloalkanes, particularly dichloromethane, esters, particularly alkyl esters such as ethyl acetate, and methyl or ethyl propionate, and basic, nucleophilic solvents such as pyridine.
  • Preferred solvents for the coupling and in situ sulfur transfer steps are pyridine, dichloromethane, and mixtures thereof.
  • Other preferred solvents include dimethylformamide, N-methylpyrollidinone, and mixtures thereof.
  • Solid supports that are employed in the methods according to the present invention are substantially insoluble in the solvent employed, and include those supports well known in the art for the solid phase synthesis of oligonucleotides. Examples include silica, controlled pore glass, polystyrene, copolymers comprising polystyrene such as polystyrene-poly(ethylene glycol) copolymers, and polymers such as polyvinylacetate. Additionally, poly(acrylamide) supports, especially microporous or soft gel supports, such as those more commonly employed for the solid phase synthesis of peptides may be employed if desired.
  • Preferred poly(acrylamide) supports are amine-functionalized supports, especially those derived from supports prepared by copolymerisation of acryloyl-sarcosine methyl ester, N,N-dimethylacrylamide and bis-acryloylethylenediamine, such as the commercially available (Polymer Laboratories) support sold under the catalogue name PL-DMA.
  • the procedure for preparation of the supports has been described by Atherton and Sheppard in Solid Phase Synthesis: A Practical Approach, Publ., IRL Press at Oxford University Press (1984).
  • the functional group on such supports is a methyl ester and this is initially converted to a primary amine functionality by reaction with an alkyl diamine, such as ethylene diamine.
  • the substrate is commonly bound to the solid support via a cleavable linker.
  • linkers that may be employed include those well known in the art for the solid phase synthesis of oligonucleotides, such as urethane, oxalyl, succinyl, and amino- derived linkers.
  • the substrate when the substrate is bound to a poly(acrylamide) support via a cleavable linker and comprises a nucleoside, the substrate is attached to the support by a process comprising either: a) reacting a 5'-protected nuceloside having a free 3'-hydroxy group with a linker, preferably succinic anhydride, to form a linker-derivatized nucleoside; and b) reacting the linker-derivatized nucleoside with an amine-functionalized poly(acrylamide) support in the presence of a coupling agent used for amide bond formation and optionally a catalyst, such as a base, for example, diisopropylethylamine (DIPEA) or N-methylmorpholine (NMM), or hydroxybenzotriazole; or c) reacting an amine-functionalized poly(acrylamide) support with a linker, preferably succinic anhydride, to form a linker-derivatized support; and
  • the 5'-protecting group in which case its removal may be omitted.
  • the 5'- protecting group can be removed when desired prior to use of the supported substrate in the process for the synthesis of phosphorothioate triesters according to the present invention.
  • Coupling agents used for amide bond formation that can be employed in the process for attaching the substrate to an amine-functionalized poly(acrylamide) support include those known in the art of peptide synthesis, see, for example, those coupling reagents disclosed by Wellings, D.A.; Atherton, E.; in Methods in Enzymology, Publ., Academic Press, New York (1997) incorporated herein by reference, such as those comprising carbodiimides, especially dialkyl carbodiimides such as N,N'- diisopropylcarbodiimide (DIC), and reagents that form active esters, particularly benzotriazole active esters in situ, such as 2-(1 H-benzotriazole-1-yl)-1 , 1 ,3,3- tetramethyluronium tetrafluoroborate (TBTU) or benzotriazole-1-yloxy-tris- (dimethylamino)phosphonium hexafluorophosphate (BOP).
  • An organic solvent such as N,N-dimethylformamide (DMF) or N- methylpyrrolidinone (NMP) is suitably employed for attaching the substrate to an amine- functionalized poly(acrylamide) support.
  • DMF N,N-dimethylformamide
  • NMP N- methylpyrrolidinone
  • the process for the synthesis of phosphodiesters or phosphorothioate triesters according to the present invention can be carried out by stirring a slurry of the substrate bonded to the solid in a solution of the H-phosphonate and coupling agent or sulfur transfer agent.
  • the solid support can be packed into a column, and solutions of H-phosphonate and coupling agent, followed by sulfur transfer agent can be passed sequentially through the column.
  • the mole ratio of H-phosphonate to substrate comprising a free hydroxy group in the process of the present invention is often selected to be in the range of from about 0.9:1 to 3:1 , commonly from about 1 :1 to about 2:1 , and preferably, from about 1.1 :1 to about 1.5:1 , such as about 1.2:1.
  • the mole ratio of a compound represented by Structural Formula I to substrate comprising a free hydroxy group is often selected to be in the range of from about 1 :1 to about 10:1 , commonly from about 1.5:1 to about 5:1 and preferably, from about 1.5:1 to about 3:1.
  • the mole ratio of oxidizing agent or sulfur transfer agent to substrate comprising a free hydroxy group is often selected to be in the range of from about 1 :1 to about 10:1 , commonly from about 2:1 to about 5:1 and preferably, from about 2:1 to about 3:1.
  • H-phosphonates formed using the coupling agent of the present invention can also be derivatized to phosphodiesters or phosphorothioate triesters through oxidation or oxidative sulfurization techniques once the H-phosphonate oligonucleotide has been completely synthesized.
  • the oxidation reaction is often carried out by treating the H-phosphonate with an oxidizing agent, such as l 2 , in the presence of water and a tertiary amine.
  • an oxidizing agent such as l 2
  • l 2 is added to the reaction mixture so that it is present in about a 0.5 M to about 3.0 M concentration.
  • Water is also added to the reaction mixture so that it is present in about 1 % (V V) to about 10% (V/V).
  • a tertiary amine is not already present in the reaction mixture, it is also added so that it is present in about 5% to about 20%.
  • Other oxidizing agents can be used in place of l 2 , for example, N-chlorosuccinimide, N-bromosuccinimide, salts of periodic acid, or a salt of meta chloroperbenzoic acid.
  • derivatizing the H-phosphonate oligonucleotide backbone can be accomplished by contacting the newly formed H-phosphonate diester with an oxidizing agent, a compound represented by R 12 -X 6 -H, and optionally, a base tertiary amine, where R 12 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group; and X 6 is -O-, -S-, or -NR 10 -, where R 10 is H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group; or X 6 is -NR 10 -, and R 12 and R 10 taken together with the nitrogen to which they are attached form a heterocycloalkyl.
  • the phosphodiester linkages can undergo oxidative sulfurization to form a phosphorothioate triester, using a sulfur transfer reagent, such as 3H-benzodithiol- 3-one 1 ,1-dioxide (also called “Beaucage reagent”), dibenzoyl tetrasulfide, phenylacetyl disulfide, N,N,N',N'-tetraethylthiuram disulfide, 3-amino-1,2,4-dithiazole-5-thione, or elemental sulfur.
  • a sulfur transfer reagent such as 3H-benzodithiol- 3-one 1 ,1-dioxide (also called "Beaucage reagent"), dibenzoyl tetrasulfide, phenylacetyl disulfide, N,N,N',N'-tetraethylthiuram disulfide, 3-amino-1,2,
  • a phosphorothioate triester is formed by derivatizing the newly formed H-phosphonate diester with elemental sulfur and a tertiary amine.
  • chimeric oligonucleotides containing both phosphodiester and phosphorothioate diester internucleotide linkages in the same molecule by selection of appropriate sulfur transfer agents, particularly when the process is carried out in a stepwise manner. For example, one or more reaction cycles are performed, ending with a condensation step to form a nascent H-phosphonate oligonucleotide. The nascent H-phosphonate oligonucleotide is then contacted with an oxidizing agent such as l 2 or CCI 4 , and water in a first derivatization step.
  • an oxidizing agent such as l 2 or CCI 4
  • the oxidizing agent is typically dissolved in a polar organic solvent such as pyridine, acetonitrile, dimethylformamide, an ether (e.g., tetrahydrofuran and dioxane), or combinations thereof.
  • a tertiary amine such as N-methylimidazole, a trialkylamine (e.g., triethylamine, trimethylamine) diisopropylethylamine and the like, must also be present, often in an amount of from about 5% (V/V) to about 40% (V/V) in the derivatization reaction mixture.
  • the oxidizing agent is present in at least 1 equivalent in relationship to the H- phosphonate groups to be derivatized. However, it can be present in a large excess which is only limited by its solubility in a particular solvent in which the derivatization reaction is preformed. Typically, the oxidizing agent is present in the reaction mixture in about 2 equivalents to about 10 equivalents in relationship to H-phosphonate groups to be derivatized.
  • the H-phosphonate oligonucleotide is contacted with the derivatizing reaction mixture for about 5 minutes to about 1 hour to form a nascent oligonucleotide having a phosphodiester backbone.
  • the nascent derivatized oligonucleotide is then subjected to one or more additional reaction cycles ending with a condensation step to form an oligonucleotide in which part of the backbone is a phosphodiester backbone and the other part is an H- phosphonate backbone.
  • the oligonucleotide is then contacted with a suitable sulfur transfer agent, or elemental sulfur, for example, those described herein.
  • the second derivatization step is performed in the same manner as the first derivatization step described above, and the result is chimeric oligonucleotide having the desired phosphorothioate diester and phosphodiester linkages. The order in which these two steps occur may be reversed.
  • the oligonucleotide can be derivatized first by sulfurization, followed by oxidation.
  • the first and second derivatization steps can be repeated one or more times.
  • the protecting groups can be removed by standard methods, such as treatment with an ammonium hydroxide solution.
  • the formation of the H-phosphonate linkage can be carried out in 10% N-methylimidazole solution, preferably in dry pyridine.
  • Other solvents such as acetonitrile and methylene chloride can also be used for the coupling and in situ sulfur transfer reaction.
  • the final step of the reaction cycle is a coupling step (or a derivatization step if a phosphodiester or phosphorothioate is desired as the final product). If a 5'-deprotected product is desired, the reaction cycle can end with the deprotection step.
  • a 5'- protected H-phosphonate or oligonucleotide is the desired product if it is to be purified by reverse phase high performance liquid chromatography (HPLC) or ion-exchange chromatography.
  • the H-phosphonate or oligonucleotide is to be purified by ion- exchange chromatography or electrophoresis, a 5'-deprotected oligonucleotide is preferred even for ion-exchange chromatography.
  • the 5'-protecting group provides additional separation between failure/shorter sequences from the desired full-length desired oligonucleotide.
  • the present invention features a method for producing an oligonucleotide H-phosphonate, comprising reacting an oligonucleotide comprising a free hydroxy function, with a substituted or unsubstituted alkyl H-phosphonate salt or a substituted or unsubstituted aryl H-phosphonate salt in the presence of a coupling agent represented by Structural Formula I.
  • the oligonucleotide comprising a free hydroxy function has a free 3' or 5' hydroxy function.
  • the oligonucleotide is a protected oligodeoxyribonucleotide.
  • the H-phosphate salt is an ammonium salt of a phenyl, alkylphenyl, or halophenyl H-phosphonate.
  • the coupling reagent represented by Structural Formula I is that shown in FIG. 1.
  • the coupling reagent represented by Structural Formula I is that shown in FIG. 1.
  • X 1 for each occurrence is, independently, -O- or -S-.
  • X 1 is -O- at every occurrence.
  • X 2 for each occurrence is, independently, -O-, -S-, -CH 2 -, or -NR 11 .
  • X 2 is -O- at every occurrence.
  • X 3 for each occurrence is, independently, -O-, -S-, -CH 2 -, or -(CH 2 ) 2 -.
  • X 3 is -O- at every occurrence.
  • X 1 , X 2 , and X 3 are all -O- at every occurrence.
  • X 4 is -OH or -SH.
  • X 4 is -OH.
  • R 2 is -H, -F, -OR 6 , -NR 7 R 8 , or -SR 9 .
  • R 6 is -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, or a hydroxy protecting group.
  • R 7 and R 8 are each, independently, -H, a substituted or substituted aliphatic group, or an amine protecting group.
  • R 9 is -H, a substituted or unsubstituted aliphatic group, or a thio protecting group.
  • R 11 is -H, an alkyl group, an aryl group, or an aralkyl group.
  • R 16 is a hydroxy protecting group, a thio protecting group, an amino protecting group, a solid support, or a cleavable linker attached to a solid support, such as a group of the formula -Y 2 -L-Y 2 -R 15 .
  • Amine, hydroxy, and thiol protecting groups are known to those skilled in the art.
  • amine protecting groups see Greene et al. (Protective Groups in Organic Synthesis (1991 ), John Wiley & Sons, Inc., pages 309-405), the teachings of which are incorporated herein by reference in their entirety.
  • amines are protected as amides or carbamates.
  • Y 2 for each occurrence is, independently, a single bond, -C(O)-, -C(O)NR 17 -, -C(O)O-, -NR 17 -, or -O-.
  • L is a linker that is preferably, a substituted or unsubstituted aliphatic group or a substituted or unsubstituted aromatic group. More preferably, L is an ethylene group.
  • R 17 is -H, a substituted or unsubstituted aliphatic group or a substituted or unsubstituted aromatic group.
  • R 15 is a solid support, such as controlled-pore glass or polystyrene.
  • B for each occurrence is, independently, H or a protected or unprotected nucleoside base, n is zero or a positive integer.
  • Aliphatic groups include straight chained or branched C r C 18 hydrocarbons that are completely saturated or that contain one or more unconjugated double bonds, or cyclic C 3 -C 18 hydrocarbons that are completely saturated or that contain one or more unconjugated double bonds.
  • Alkyl groups are straight chained or branched d-C 8 hydrocarbons or C 3 -C 8 cyclic hydrocarbons that are completely saturated.
  • Aliphatic groups are preferably alkyl groups.
  • Aryl groups include carbocyclic aromatic ring systems (e.g., phenyl) and carbocyclic aromatic ring systems fused to one or more carbocyclic aromatics (e.g., naphthyl and anthracenyl) or an aromatic ring system fused to one or more non-aromatic rings (e.g., 1 ,2,3,4-tetrahydronaphthyl).
  • carbocyclic aromatic ring systems e.g., phenyl
  • carbocyclic aromatic ring systems fused to one or more carbocyclic aromatics e.g., naphthyl and anthracenyl
  • an aromatic ring system fused to one or more non-aromatic rings e.g., 1 ,2,3,4-tetrahydronaphthyl
  • Heteroaryl groups include aromatic ring systems that have one or more heteroatoms selected from sulfur, nitrogen, or oxygen in the aromatic ring (e.g., thienyl, pyridyl, pyrazole, isoxazolyl, thiadiazolyl, oxadiazolyl, indazolyl, furans, pyrroles, imidazoles, pyrazoles, triazoles, pyrimidines, pyrazines, thiazoles, isoxazoles, isothiazoles, tetrazoles, oxadiazoles, benzo(b)thienyl, benzimidazole, indole, tetrahydroindole, azaindole, indazole, quinoline, imidazopyridine, purine, pyrrolo[2,3- djpyrimidine, and pyrazolo[3,4-d]pyrimidine).
  • heteroaryl groups include aromatic ring systems that have
  • Azaheteroaryl compounds include heteroaryl groups which have one or more nitrogen atoms in the aromatic ring.
  • azaheteroaryl compounds have five or six membered aromatic rings with from one to three nitrogens in the aromatic ring.
  • Preferred azaheteroaryl compounds are organic bases.
  • azaheteroaryl compounds that are organic bases include pyrimidine, 1-alkylpyrazole, pyrazine, N- alkylpurine, N-alkylpyrrole, pyridine, N-alkylimidazole, quinoline, isoquinoline, quinoxaline, quinazoline, N-alkylindole, N-alkylbenzimidazole, triazine, thiazole, N-alkylindole, and 1- alkyl-7-azaindole.
  • An aralkyl group as used herein, is an aromatic substituent that is linked to a moiety by an alkyl group.
  • a heterocycloalkyl group is a non-aromatic ring system that preferably has five to six atoms and includes at least one heteroatom selected from nitrogen, oxygen, and sulfur.
  • a heterocycloalkyl group is a non-aromatic ring system that preferably has five to six atoms and includes at least one heteroatom selected from nitrogen, oxygen, and sulfur.
  • heterocycloalkyl groups include morpholinyl, piperidinyl, piperazinyl, thiomorpholinyl, pyrrolidinyl, thiazolidinyl, tetrahydrothienyl, azetidinyl, tetrahydrofuryl, dioxanyl, and dioxepanyl.
  • Azaheterocycloalkyl groups are heterocycloalkyl groups that have at least one nitrogen atom in the non-aromatic ring system.
  • Examples of azaheterocycloalkyl groups include morpholines, piperidines, and piperazines.
  • Heterocyclic groups include heteroaryl groups and heterocycloalkyl groups.
  • Suitable substituents for aliphatic groups, aryl groups, aralkyl groups, heteroaryl groups, azaheteroaryl groups and heterocycloalkyl groups include aryl groups, halogenated aryl groups, alkyl groups, halogenated alkyl (e.g., trifluoromethyl and trichloromethyl), aliphatic ethers, aromatic ethers, benzyl, substituted benzyl, halogens, cyano, nitro, -S-(aliphatic or substituted aliphatic group), and -S-(aromatic or substituted aromatic).
  • the monomer to which the nucleoside or nascent oligonucleotide is coupled can be represented by Structural Formula IX:
  • R is an alcohol protecting group or a thio protecting group.
  • R 1 is an acid labile protecting group.
  • nascent oligonucleotide is contacted with the monomer and a compound represented by Structural Formula I and forms a nascent H-phosphonate (n+1) oligonucleotide represented by Structural Formula X:
  • nascent H-phosphonate (n+1) oligonucleotide can be deprotected by removing the protecting group represented by R 1 . If R 1 is an acid labile protecting group, the nascent H-phosphonate (n+1) oligonucleotide is treated with an acid to remove R 1 .
  • R 1 is a trialkylsilyl group, such as .-butyldimethylsilyl group or a triisopropylsilyl group
  • the nascent H-phosphonate (n+1) oligonucleotide can be treated with fluoride ions to remove R 1 .
  • fluoride ions typically, f-butyldimethylsilyl and a triisopropylsilyl are removed by treatment with a solution of tetrabutylammonium fluoride in THF.
  • Methods for removing .-butyldimethylsilyl can be found in Greene et al. (Protective Groups in Organic Synthesis (1991), John Wiley & Sons, Inc., pages 77-83), the teachings of which are incorporated herein by reference in their entirety.
  • reaction cycle The condensation and deprotection reaction steps, referred to herein as the reaction cycle, can be repeated one or more times to form an H-phosphonate oligonucleotide of the desired length that can be represented by Structural Formula XI:
  • the H-phosphonate oligonucleotide represented by Structural Formula XI can then be treated with a sulfur transfer agent, as defined above, to form a phosphodiester or a phosphorothioate diester, using methods described above.
  • oligonucleotide can be contacted with an oxidizing agent, a compound represented by R 12 -X 6 -H, as defined above, and optionally, a base tertiary amine, thereby forming an oligonucleotide represented by the Structural Formula XII:
  • the oligonucleotide backbone is derivatized by contacting the H-phosphonate oligonucleotide with an oxidizing agent, a tertiary amine, and water, thereby forming a phosphodiester oligonucleotide represented by Structural Formula XIII:
  • X 1 , X 2 , X 3 , R 1 , R 2 , R 16 , and B are as defined above; and m is a positive integer.
  • the H-phosphonate oligonucleotide can be derivatized to form a phosphorothioate.
  • the H-phosphonate oligonucleotide can be contacted with elemental sulfur and a tertiary amine, whereby the oligonucleotide formed is a phosphorothioate represented by Structural Formula XIV:
  • a second method for forming an oligonucleotide can be carried out as follows.
  • the oligonucleotide is formed by condensing a nucleoside or a nascent oligonucleotide represented by Structural Formula XV:
  • Structural Formula XV X 1 , X 2 , X 3 , X 4 , R 1 , R 2 , R 16 , and B are as defined above; n is zero or a positive integer; and A is a substituted or unsubstituted aryl group, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted alkenyl group.
  • the salt of a monomer to which the nucleoside or nascent oligonucleotide is coupled is represented by Structural Formula IX.
  • the sulfur transfer agent used in the reaction is represented by the following general chemical formula: L S A
  • L represents a leaving group as defined above
  • A represents a substituted or unsubstituted aryl group, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted alkenyl group.
  • the nascent H-phosphonate (n+1) oligonucleotide formed is represented by Structural Formula XVI:
  • the nascent H-phosphonate (n+1) oligonucleotide can be deprotected as described above, to remove R 1 . If desired, the coupling step and deprotection step can be repeated one or more times to form a phosphorothioate oligonucleotide represented by Structural Formula XVII:
  • X 1 , X 2 , X 3 , R 1 , R 2 , R 16 , A, and B are as defined above; and m is a positive integer.
  • the leaving group of the sulfur transfer agent is commonly selected so as to comprise a nitrogen-sulfur bond.
  • suitable leaving groups include morpholines such as morpholine-3,5-dione; imides such as phthalimides, succinimides and maleimides; indazoles, particularly indazoles with electron-withdrawing substituents such as 4-nitroindazoles; and triazoles.
  • the newly formed H-phosphonate oligonucleotide is contacted with a compound having one of Structural Formulae II, III, or IV, thereby forming an oligonucleotide represented by Structural Formula XVIII:
  • the sulfur transfer reagent is a compound represented by Structural Formula V, which is used to form a phosphorothioate triester represented by Structural Formula XIX:
  • the backbone of the newly formed oligonucleotide is deprotected by contacting the oligonucleotide with an oximate and a base, thereby forming a phosphodiester oligonucleotide.
  • the above described second method of oligonucleotide synthesis can be use to generate a chimeric oligonucleotide containing both phosphodiester linkages.
  • This method is carried out as described above, where the H-phosphonate oligonucleotide is contacted with a compound represented by Structural Formula V, and after one or more condensation steps is contacted with one of the compounds represented by Structural Formulae II, II, or IV after one or more different coupling steps, forming a phosphorothioate triester.
  • the phosphorothioate triester backbone of the oligonucleotide is deprotected by contacting the phosphorothioate triester with an oximate and a base, thereby forming a chimeric oligonucleotide.
  • the coupling step or derivatization step, if derivatization of the oligonucleotide is desired
  • the deprotection step can be the last step, and the oligonucleotide generated is a deprotected oligonucleotide.
  • synthesis of the oligonucleotide can be done in solution or on a solid support.
  • R 16 of each of Structural Formulae VIII and XV is an alcohol, amine, or thiol protecting group.
  • Solution synthesis of the desired oligonucleotide can occur, for example, when the H-phosphonate and the substrate comprising a free hydroxy group are pre-mixed in solution, and a compound represented by Structural Formula I is then added to this mixture.
  • the H-phosphonate and a compound represented by Structural Formula I can be pre-mixed, often in solution and then added to a solution of the substrate comprising a free hydroxy group, or the substrate comprising a free hydroxy group and a compound represented by Structural Formula I may be mixed, commonly in solution, and then added to a solution of the H-phosphonate.
  • the H- phosphonate optionally in the form of a solution, can be added to a solution comprising a mixture of the substrate comprising a free hydroxy group and a compound represented by Structural Formula I. Reagent additions commonly take place continuously or incrementally over an addition period.
  • each R 2 is -OH or -OR 6 and the oligonucleotide prepared is a ribonucleotide.
  • each R 2 is -H and the oligonucleotide prepared is a deoxyribonucleotide.
  • a compound represented by Structural Formula I (as shown in FIG. 1), N-ibu-5'-O- DMT-deoxyguanosine-H-phosphonate, and 5'-HO-thymidine-3'-Olev were mixed together in pyridine and CD 3 CN, as shown in FIG. 2.
  • the reaction was followed by 31 P NMR for the formation of new phosphorus peaks, indicating formation of H-phosphonate diester linkages. When two peaks were observed indicating the formation of H-phosphonate diester linkages had formed, elemental sulfur powder was added to the reaction tube. After one hour, the product was again evaluated by NMR spectra measurement. The result of this analysis showed formation of phosphorothioated diester.
  • oligonucleotide dimers were synthesized utilizing a compound represented by Structural Formula I (as shown in FIG. 1) as the coupling reagent, as shown in the reaction scheme of FIG. 2.
  • Two of the dimers were made by combining N- ibu-5'-O-DMT-deoxyguanosine-H-phosphonate (0.25M) in dry pyridine containing 10% N- methylimidazole (by volume) with either 5'-HO-deoxycytidine-3'-O-Lev or 5'-HO- deoxyadenosine-3'-O-Lev (1.1 equivalents relative to H-phosphonate).
  • the third dimer was generated by combining N-ibu-5'-O-DMT-thymidime-H-phosphonate with 5'-HO- deoxyguanosine-3'-O-Lev (1.1 equivalents relative to H-phosphonate). These condensation reactions were carried out by adding the coupling reagent a compound represented by Structural Formula I (1.5 equivalents with respect to H-phosphonate) in pyridine (4.0 ml/mmol), and was catalyzed by N-methyllmidazole (10% of the solvent) at ambient temperature. The coupling reaction was carried out for approximately 20 to 30 minutes. The reaction product was then assessed by TLC and 31 P-NMR to determine when the H-phosphonate diester linkage had formed.
  • the sulfur transfer agent 3- (1 ,3-dioxo-1,3-dihydro-isoindol-2-ylsulfanyl)-propionitrile (hereinafter "CESP") (2 equivalents with respect to H-phosphonate) was added to the reaction mixture, and the reaction was carried out for 15 to 20 minutes at ambient temperature.

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Abstract

L'invention concerne des procédés d'utilisation de composés de formule (I), dans laquelle X7 représente =O ou =S, Z représente un halogène, R1 représente =O ou =S, R2 représente -O- ou -S-, R3 représente un hydrogène, ou un substituant, de préférence un halogène, un groupe nitro, ou un groupe alkyle, et n prend la valeur 1 ou 2, en tant qu'agent de couplage afin de former des diesters et des oligonucléotides sous forme de H-phosphonate.
PCT/GB2002/005177 2001-11-21 2002-11-15 Reactif de couplage en chimie des h-phosphonates WO2003045969A1 (fr)

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EP0723973A1 (fr) * 1995-01-25 1996-07-31 King's College London Dérivés de nucléosides phosphorothioatés, leur synthèse et leur utilisation
WO1999009041A2 (fr) * 1997-08-13 1999-02-25 Avecia Limited Synthese en phase de solution d'oligonucleotides
US6207819B1 (en) * 1999-02-12 2001-03-27 Isis Pharmaceuticals, Inc. Compounds, processes and intermediates for synthesis of mixed backbone oligomeric compounds
WO2001064702A1 (fr) * 2000-03-01 2001-09-07 Avecia Limited Procede de preparation de triesters de phosphorothioate

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EP0723973A1 (fr) * 1995-01-25 1996-07-31 King's College London Dérivés de nucléosides phosphorothioatés, leur synthèse et leur utilisation
WO1999009041A2 (fr) * 1997-08-13 1999-02-25 Avecia Limited Synthese en phase de solution d'oligonucleotides
US6207819B1 (en) * 1999-02-12 2001-03-27 Isis Pharmaceuticals, Inc. Compounds, processes and intermediates for synthesis of mixed backbone oligomeric compounds
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7476709B2 (en) 2002-04-26 2009-01-13 Avecia Biotechnology Inc. Process for preparing oligonucleotides

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