Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
  • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• • 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

Definitions

• the present invention relates to modified triphosphate nucleoside compounds and methods for their preparation.
• the present methods provide modified nucleoside triphosphate compounds having at least one modification at the ⁇ -position of the triphosphate.
• Nucleoside triphosphate compounds may be polymerized to provide ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Apart from the traditional role in normal cells these nucleic acids form the genetic materials of a variety of pathogenic viruses such as HTV, herpes, measles, mumps and many others. [0003] Simoncsits, A. and Tomasz, J., Tetrahedron Letters 1976, 44, 3995-
• Chem., 1989, 54, 631-635 disclose a method of making nucleoside 5 '-triphosphates and ⁇ -thio analogues that requires the protection of the ribose 2' and 3' hydroxyl groups and produces hard to remove by products.
• R.K. Gaur, B.S. Sproat, G. Krupp, Tetrahedron Letters 1992, 33, 3301- 3304 disclose a method of making a 5 '-triphosphate that requires the attachment of a functionalized solid support that serves as an anchor and protecting group throughout all of the chemical manipulations. This synthesis method also results in modest yield of ⁇ -thiotriphosphates that may be the result of an incomplete sulphurisation step.
• nucleoside triphosphate analogues are useful in elucidation of the enzymic functions and mechanisms.
• Nucleoside 5'-( ⁇ -P-(N- alkyl)-nucleoside triphosphates are modified nucleotides in which one of the ⁇ - phosphate oxygens is replaced by a primary amine. Exchange of one of the - phosphate oxygens for sulfur, borane or methyl has also proven to be useful in investigations of the stereochemical course of enzymic reactions. (See Eckstein, F. Ann. Rev. Biochem. 1985, 54, 367-402; Kaizhang, H., et al. J. Org. Chem.
• nucleotide triphosphate compounds can be used as intermediates in the biosynthesis of nucleosides, proteins and hormones.
• nucleotide triphosphates are difficult because of the hydrolytic lability of the triphosphate and related intermediates and also due to the short half life related to these compounds. In short they are widely useful per se.
• a wider variety of modified nucleoside triphosphate compounds is needed including those that mimic natural analogs that have desired downstream pharmacologic effects not elicited by unmodified compounds. Other properties desirable in such modified nucleoside triphosphate compounds include increased hydrolytic stability in cellular systems and low toxicity.
• nucleoside triphosphate compounds of formula I there are methods providing nucleoside triphosphate compounds of formula I:
• Y is Se and Y 2 is OH or SH; or Y, is O and Y 2 is NHR, or BH 3 ; or Y, is S and Y 2 is NHR l5 SH or BH 3 ; and Y 3 is OH; NHR ⁇ alkyl or alkoxy; R, is hydrogen, substituted or unsubstituted -C 12 alkyl, substituted or unsubstitiited C 2 -C 12 alkenyl, substituted or unsubstituted C 2 -C 12 alkynyl, substituted or unsubstituted C 5 to C 7 cyclic alkyl, substituted or unsubstituted C 5 -C 20 alkyaryl, substituted or unsubstituted heterocyclyl, or a nitrogen protecting group, wherein said sub- stituent is substituted or unsubstituted C $ -C 20 aryl, O-alkyl, O-alkylalkoxy, O- alkylaminoal
• Bx is an optionally protected heterocyclic base moiety; one of X, and X 2 is H, hydroxyl, a protected hydroxyl, or a sugar substituent group and the other of X, and X 2 is hydroxyl, a protected hydroxyl or -O-J-SM; J is a bifunctional linking moiety; and SM is a support medium; comprising the steps of: a) providing a nucleosidyl-5'-cyclotriphosphite having the formula
• one of X 2 and X t is H, hydroxyl, a protected hydroxyl, or a sugar substituent group and the other of X 2 and X ! is -O-J-SM; b) reacting said nucleosidyl-5'-cyclotriphosphite with an oxidizing reagent for a time and under conditions effective to give an oxidized compound having the formula III:
• Z is Se, S, BH 3 , orNHR,; e) treating said oxidized compound with a ring opening reagent effective to give compound I.
• Y is Se and Y 2 is OH or SH;
• Y is O and Y 2 is NHR, or BH,; or
• Y is S and Y 2 is NHR réelle SH or BH 3 ; and Y 3 is OH; NHR,; alkyl or alkoxy;
• R is hydrogen, substituted or unsubstituted -C 12 alkyl, substituted or unsubstituted C 2 -C 12 alkenyl, substituted or unsubstituted C 2 -C 12 alkynyl, substituted or unsubstituted C 5 to C 7 cyclic alkyl, substituted or unsubstituted C 5 -C 20 alkyaryl, substituted or unsubstituted heterocyclyl, or a nitrogen protecting group, wherein said substituent is substituted or unsubstituted C 5 -C 20 aryl, O-alkyl, O-alkylalkoxy, O- alkylaminoalkyl, O-alkyl imidazole, S-alkyl, NH-alkyl, N-dialkyl, aryl, heterocycle, substituted heterocycle, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, or NH-aralkyl
• Bx is an optionally protected heterocyclic base moiety; one of X, and X 2 is H, hydroxyl, a protected hydroxyl, or a sugar substituent group and the other of X ! and X 2 is hydroxyl, a protected hydroxyl or -O-J-SM;
• J is a bifunctional linking moiety
• SM is a support medium; with the proviso that when Y 2 is BH 3 or NH 2 then Y 3 is other than OH.
• the oxidizing agent is mixture of I 2 and NH 3 in a suitable solvent or I 2 and NH 2 R, in a suitable solvent.
• the solvent can also be 1,4-dioxane, THF, (MeO) 3 PO, acetonitrile, DMF or formamide.
• Rj is methyl, pro- pyl, butyl, methoxypropyl, methoxyethyl, methylthioethyl, methylthiopropyl, cyclopentyl, benzyl, 2,4-methoxybenzyl, phenylbenzyl, crown ether or 7- deazaguanine.
• oxidizing agent that is a solution of 3H- l,2-benzothiaselenol-3-one dissolved in a suitable solvent.
• the solvent of some embodiments is a mixture of acetonitrile and triethylamine. That mixture may comprise from about 1% to about 30% triethyl amine in acetonitrile by volume.
• General embodiments can have oxidizing agents that are solutions of S in a mixture of suitable solvents.
• the mixture can comprise carbon disulfide, pyridine, and tryiethlyamine.
• Another embodiment can have a borane-N,N-diisopropylethyl complex as oxidizing agent.
• the nucleosidyl-5'-cyclotriphosphite can be reacted with said oxidizing agent for up to about 3 hours in some embodiments or up to about 2 hours in others. In yet other embodiments, the reaction can take place for up to about 40 minutes.
• the ring opening reagent is H 2 O, NHR, or Li 2 S.
• the invention also encompasses those versions further comprising treating the nucleoside triphosphate compound with a cleaving reagent to cleave the compound from the support medium.
• Other versions can exist wherein the treatment with the cleaving reagent removes protecting groups present on the heteiocyclic base moiety.
• Y is Se and
• Y 2 is OH; wherein Y, is O and Y 2 is NHR, wherein said R, is other than H; wherein Y, is O and Y 2 is NHR,; wherein Y, is O and Y 2 is BH 3 or wherein Y,is S and Y 2 is SH.
• Bx is an optionally protected heterocyclic base moiety; one of X, and X 2 is H, hydroxyl, a protected hydroxyl, or a sugar substituent group and the other of X, and X 2 is -O-J-SM;
• J is a bifunctional linking moiety
• SM is a support medium.
• Z is Se, S, BH 3 , or NHR,; Bx is an optionally protected heterocyclic base moiety; one of X, and X 2 is H, hydroxyl, a protected hydroxyl, or a sugar substituent group and the other of X, and X 2 is -O-J-SM;
• J is a bifunctional linking moiety
• SM is a support medium.
• the support bound cyclic triphosphite nucleoside is oxidized with an amino, seleno, borano or thio agent followed by treatment with a reagent effective to open the cyclic functionality and give the support bound ⁇ -modif ⁇ ed triphosphate nucleoside.
• Oxidizing a support bound cyclic triphosphite nucleoside which is further treated with a reagent effective to give the corresponding support bound nucleoside triphosphate which is cleaved and purified to give the free nucleoside triphosphate.
• ⁇ -modified triphosphate nucleosides are provided. Further modified ⁇ -modified triphosphate nucleosides are provided comprising modifications on the nucleoside and/or the ⁇ -position of the triphosphate.
• ⁇ -Modified triphosphate nucleosides of the present invention include those having the formula:
• Y is Se and Y 2 is OH or SH; or Y, is O and Y 2 is NHR, or BH 3 ; or Y, is S and Y 2 is NHR réelle SH or BH 3 ;
• Y 3 is OH; NHR,; alkyl or alkoxy; R, is hydrogen, substituted or unsubstituted C,-C, 2 alkyl, substituted or unsubstituted C 2 -C, 2 alkenyl, substituted or unsubstituted C 2 -C, 2 alkynyl, substituted or un- substituted C 5 to C 7 cyclic alkyl, substituted or unsubstituted C 5 -C 20 alkyaryl, substituted or unsubstituted heterocyclyl, or a nitrogen protecting group, wherein said substituent is substituted or unsubstituted C C 20 aryl, O-alkyl, O-alkylalkoxy, 0- alkylaminoalkyl, O-alkyl imidazole, S-alkyl, NH-alkyl, N-dialkyl, aryl, heterocycle, substituted heterocycle, O-aryl, S-aryl, NH-aryl, O-a
• Bx is an optionally protected heterocyclic base moiety; one of X, and X 2 is H, hydroxyl, a protected hydroxyl, or a sugar substituent group and the other of X, and X 2 is hydroxyl, a protected hydroxyl or -O-J-SM;
• J is a bifunctional linking moiety
• SM is a support medium; with the proviso that when Y 2 is BH 3 or NH 2 then Y 3 is other than OH.
• Some of the ⁇ -modified nucleoside triphosphates provided by this invention have one of the ⁇ -phosphate oxygens exchanged for sulfur, borane, selenium, amine or alkyl.
• Many embodiments can be used as model compounds for research on enzyme functions and mechanisms of action. Such research and mechanisms may include stereochemical course of a variety of NTP based enzymic reaction and metal ion dependance for diastereomeric forms of ⁇ -P substituted anologues of nucleoside 5 '-triphosphates.
• the modified triphosphates can be used in direct sequencing by incorporation of 2'-deoxynucleoside 5'-O-( ⁇ -P-boranothio) triphosphates into PCR products followed by chemical degradation in order to reveal their positions.
• amino functional groups are covalently attached to the ⁇ -position of modified triphosphate nucleosides.
• the amino functional group is covalently attached to the ⁇ -position of the ⁇ -modified triphosphate nucleoside.
• An amino functional group according to the present invention includes compounds having the formula -NHR,.
• R includes hydrogen, substituted or unsubstituted C,-C, 2 alkyl, substituted or unsubsi- tuted C 2 -C, 2 alkenyl, substituted or unsubstituted C 2 -C 12 alkynyl, substituted or unsub- stituted C 5 to C 7 cyclic alkyl, substituted or unsubstituted Cj-C,,, alkyaryl, substituted or unsubstituted heterocyclyl, or a nitrogen protecting group, wherein said substituent is substituted or unsubstituted -C 20 aryl, O-alkyl, O-alkylalkoxy, O-alkylamino- alkyl, O-alkyl imidazole, S-alkyl, NH-alkyl, N-dialkyl, aryl, heterocycle, substituted heterocycle, O-aryl, S-aryl
• a heterocyclic base moiety (often referred to in the art simply as a
• base or a “nucleobase” amenable to the present invention includes both naturally and non-naturally occurring nucleobases.
• the heterocyclic base moiety further may be protected wherein one or more functionalities of the base bears a protecting group.
• unmodified or “natural” nucleobases include the purine bases a - nine and guanine, and the pyrimidine bases thymine, cytosine and uracil.
• Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6- methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- halouracil and cytosine, 5-propynyl uracil and cytosine, 6-aza uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8- hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-sub
• nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al, Angewandte Cheniie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., ed., CRC Press, 1993. [0028] Certain heterocyclic base moieties are particularly useful for increasing the binding affinity of the oligomeric compounds that incorporate them to complementary targets.
• 5-substituted pyrimidines include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
• 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Id., pages 276-278) and are presently preferred base substitutions, even more particularly when combined with selected 2'- sugar modifications such as 2'-methoxyethyl groups.
• Representative United States patents that teach the preparation of heterocyclic base moieties include, but are not limited to, U.S. Patents 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, certain of which are commonly owned, and each of which is herein incorporated by reference, and commonly owned United States patent application 08/762,587, filed on December 10, 1996, also herein incorporated by reference.
• a representative list of sugar substituent groups amenable to the present invention include C,-C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, C 5 -C 20 aryl, O- alkyl, O-alkenyl, O-alkynyl, O-alkylamino, O-alkylalkoxy, O-alkylaminoalkyL O- alkyl imidazole, S-alkenyl, S-alkynyl, NH-alkyl, NH-alkenyl, NH-alkynyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, halogen (particularly fluoro), keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoro- methoxy, imid
• polyethers linear and cyclic polyethylene glycols (PEGs), and (PEG)- containing groups, such as crown ethers and those which are disclosed by Ouchi ⁇ t al. (Drug Design and Discovery 1992, 9, 93), Ravasio et al. (J. Org. Chem. 1991, 56, 4329) and Delgardo et. al. (Critical Reviews in Therapeutic Drag Carrier Systems 1992, 9, 249), each of which is herein incorporated by reference in its entirety. Further sugar modifications are disclosed in Cook, P .D., Anti-Cancer Drug Design, 1991, 6, 585-607.
• Additional sugar substituent groups amenable to the present invention include -SR and -NR 2 groups, wherein each R is, independently, hydrogen, a protecting group or substituted or unsubstituted alkyl, alkenyl, or alkynyl.
• 2'-SR nucleosides are disclosed in United States Patent No. 5,670,633, issued September 23, 1997, hereby incorporated by reference in its entirety. The incorporation of 2'-SR monomer synthons are disclosed by Hamm et al, J. Org. Chem., 1997, 62, 3415-3420.
• 2'-NR 2 nucleosides are disclosed by Goettingen, M, J. Org. Chem., 1996, 61, 73-6281; and Polushin et al, Tetrahedron Lett, 1996,37, 3227-3230.
• Further sugar substituent groups have one of formula IN or V:
• each R 6 , R 7 , Rg, R,, and R, 0 is, independently, hydrogen, C(O)R, attorney substituted or unsubstituted C,-C, 0 alkyl, substituted or unsubstituted C 2 -C, 0 alkenyl, substituted or unsubstituted C 2 -C, 0 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the sugar substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl; or optionally, R 7 and R 8 , together form a phthalimido moiety with the nitrogen atom to which they are attached; or optionally, Rg and R, 0 , together form a phthalimi
• R 5 is T-L
• T is a bond or a linking moiety
• L is a chemical functional group, a conjugate group or a solid support material; each R, and R 2 is, independently, H, a nitrogen protecting group, substituted or unsubstituted C,-C, 0 alkyl, substituted or unsubstituted C 2 -C, 0 alkenyl, substituted or unsubstituted C 2 -C, 0 alkynyl, wherein said substitution is OR ⁇ SR 3 , NH 3 + , N(R 3 )(R 4 ), guanidino or acyl where said acyl is an acid amide or an ester; or R, and R 2 , together, are a nitrogen protecting group or are joined in a ring structure that optionally includes an additional heteroatom selected from N and O; or R perpetrat T and L, together, are a chemical functional group; each R 3 and R 4 is, independently, H, C,-C, 0 alkyl, a nitrogen protecting group, or R 3 and R 4 , together, are a nitrogen
• R 5 is H or C,-C 8 alkyl
• Zache Z 2 and Z 3 comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsatu- rated heterocyclic;
• Z 5 is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R,)(R 2 ) OR,, halo, SR, or CN; each q, is, independently, an integer from 1 to 10; each q 2 is, independently, 0 or 1; q 3 is 0 or an integer from 1 to 10; q 4 is an integer from 1 to 10; q 5 is from 0, 1 or 2; and provided that when q 3 is 0, q 4 is greater than 1.
• sugar substituent groups include
• Another particularly preferred modification includes 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 or 2'-MOE, Martin et al, Helv. Chim. Ada, 1995, 78, 486).
• a further preferred sugar substituent group is 2'-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE.
• Representative aminooxy sugar substituent groups are described in co-owned United States Patent Application serial number 09/344,260, filed June 25, 1999, entitled “Aminooxy-Functionalized Oligomers”; and United States Patent Application serial number 09/370,541, filed August 9, 1999, also identified by attorney docket number ISIS-3993, entitled Aminooxy-Functionalized Oligomers and Methods for Making Same; hereby incorporated by reference in their entirety.
• Other preferred modifications include 2'-methoxy (2'-O-CH 3 ), 2'- aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F).
• the configuration of the sugar substituent group is also variable such as at the 2'-position.
• arabinose configuration is also amenable to the present invention.
• Arabinose modifications are known to those skilled in the art and include more recent procedures described in for example, Damha et. al., J.A.C.S., 1998, 120, 12976-12977; Bioconjugate Chem., 1999, 10, 299-305; Nucleic Acids Res. (2000), 28(18), 3625-3635; Biochemistry (2000), 39(24), 7050-7062.
• nucleosides and oligomers may also be made at other positions on nucleosides and oligomers, particularly the 3' position of the sugar on the 3' terminal nucl ⁇ - side or at a 3 '-position of a nucleoside that has a linkage from the 2-position such as a 2 '-5' linked oligomer and at the 5 '-position at a 5 '-terminus.
• Oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
• Representative United States patents that teach the preparation of such modified sugars structures include, but are not limited to, U.S.
• a " bifunctional linking moiety” refers to a hydrocarbyl chain which connects the monomers and oligomers of the invention to a support ms- dium.
• a preferred linking moiety is a succinyl group.
• linking moieties include, but are not limited to, substituted or unsubstituted C,-C, 0 alkyl, substituted or unsubstituted C 2 -C, 0 alkenyl or substituted or unsubstituted C 2 -C, 0 alkynyl, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
• Other linking moE- ties are known in the art and are equally amenable to the present invention.
• modified triphosphate nu- cleotides are prepared with the nucleoside portion linked to a support media.
• Preferred attachments are via the 2' or 3 '-position of the sugar portion of the nucleoside but other points of attachment such as via the heterocyclic base moiety are know in the art and are amenable to the present invention.
• Preferred support media is insoluble and allows all of the reactants to be easily removed by washing with an appropriate solvent.
• soluble or variable soluble support media is also known in the art. Such support permits variable solubility of the nucleoside bound synthon in different solvents thereby allowing variable solubility.
• Traditional solid supports are insoluble and are routinely placed in a reaction vessel while reagents and solvents react and or wash the growing chain until cleavage frees the final oligomer. More recent approaches have introduced soluble supports including soluble polymer supports to allow precipitating and dissolving of the synthon at desired points in the synthesis (Gravert et al, Chem. Rev., 1997, 97, 489-510).
• Representative support media that are amenable to the methods of the present invention include without limitation: controlled pore glass (CPG); oxalyl- controlled pore glass (see, e.g., Alul, et al, Nucleic Acids Research 1991, 19, 1527); TENTAGEL Support, (see, e.g., Wright, et al, Tetrahedron Letters 1993, 34, 3373); or POROS, a copolymer of polystyrene/divinylbenzene available from Perceptive Biosystems.
• CPG controlled pore glass
• oxalyl- controlled pore glass see, e.g., Alul, et al, Nucleic Acids Research 1991, 19, 1527
• TENTAGEL Support see, e.g., Wright, et al, Tetrahedron Letters 1993, 34, 3373
• POROS a copolymer of polystyrene/divinylbenzene available from Perceptive Biosystems.
• a soluble support media poly(ethylene glycol), with molecular weights between 5 and 20 kDa, for large-scale synthesis of oligonucleotides is described in, Bonora et al, Organic Process Research & Devdopment, 2000, 4, 225- 231.
• hydroxyl protecting groups known in the art are amenable to use in the present invention.
• a further list of hydroxyl protecting groups amenable to the present invention may be represented by the group having the formula: -C(R,)(R 2 )(R 3 ) wherein each of R perpetrat R 2 and R 3 is an unsubstituted or mono-substituted aryl or het- eroaryl group selected from phenyl, naphthyl, anthracyl, and five or six membered heterocylic rings with a single heteroatom selected from N, O and S, or two N het- eroatoms, including quinolyl, furyl, and thienyl; where the substituent is selected from halo (i.e., F, CI, Br, and I), nitro, or alkoxy, and aryl, aralkyl and cycloal- kyl containing up to 10 carbon atoms; and wherein R 2 and R 3 may each also be C r C 4 - alkyl or aralkyl or
• Y is Se and Y 2 is OH or SH; or Y, is O and Y 2 is NHR, or BH 3 ; or Y, is S and Y 2 is NHR whatsoever SH or BH 3 ; Y 3 is OH; NHR,; alkyl or alkoxy; R, is hydrogen, substituted or unsubstituted -C, 2 alkyl, substituted or unsubstituted C 2 -C, 2 alkenyl, substituted or unsubstituted C 2 -C 12 alkynyl, substituted or unsubstituted C 5 to C 7 cyclic alkyl, substituted or unsubstituted C 5 -C 20 alkyaryl, substituted or unsubstituted heterocyclyl, or a nitrogen protecting group, wherein said substituent is substituted or unsubstituted C ⁇ -C 20 aryl, O-alkyl, O-alkylalkoxy, O- alkylaminoalkyl, O-al
• Bx is an optionally protected heterocyclic base moiety; one of X, and X 2 is H, hydroxyl, a protected hydroxyl, or a sugar substituent group and the other of X, and X 2 is hydroxyl, a protected hydroxyl or -O-J-SM; J is a bifunctional linking moiety; and SM is a support medium; comprising the steps of: a) providing a nucleosidyl-5'-cyclotriphosphite having the formula
• one of X 2 and X is H, hydroxyl, a protected hydroxyl, or a sugar substituent group and the other of X 2 and X, is -O-J-SM; b) reacting said nucleosidyl-5'-cyclotriphosphite with an oxidizing reagent for a time and under conditions effective to give an oxidized compound having the formula III:
• Z is Se, S, BH 3 , or NHR,; and e) treating said oxidized compound with a ring opening reagent to give compound I.
• Another embodiment includes a process wherein the oxidizing agent is a mixture of I 2 and ⁇ 3 in a suitable solvent.
• the oxidizing agent is a mixture of I 2 and NH 2 R, in a suitable solvent.
• the oxidizing agent is a solution of sulfur in a mixture of suitable solvents.
• the oxidizing agent is a borane-N,N-diisopropylethyl complex.
• Suitable solvents useful in connection with this invention can be 1,4- dioxane, THF, (MeO) 3 PO, acetonitrile, DMF, and formamide. In many forms, the solvent is 1,4-dioxane, whereas other embodiments have THF as the suitable solvent. Mixtures of carbon disulfide, pyridine, and tryiethylamine can also be used in some embodiments.
• This invention can be conducted wherein said oxidizing agent is a solution of 3H-l,2-benzothiaselenol-3-one dissolved in a suitable solvent.
• suitable solvent can also be a mixture of acetonitrile and triethylamine. Such mixture comprises from about 1% to about 30% triethyl amine in acetonitrile by volume.
• the oxidation step can be conducted for up to about 3 hours or up to about 2 hours. In other embodiments, the step can be conducted for up to about 40 minutes.
• Oxidized cyclic intermediates of the present invention may be opened up in a number of ways.
• the cyclic intermediates may be opened using a solution of Li 2 S and 18-crown-6ether in dimethyl-formamide.
• the ring may be opened by hydrolysis or action of different amines.
• the cyclic intermediates can be hydrolized with water.
• the processes of the present invention can be augmented to include treating the nucleoside triphosphate compound with a cleaving reagent to cleave said compound from the support medium.
• the treatment with said cleaving reagent removes protecting groups present on the heterocyclic base moiety.
• Protecting groups can also be removed by treating the compound with a deprotecting reagent in other embodiments.
• Certain embodiments of the invention may exist in wherein Y, is Se and Y 2 is OH, Y, is O and Y 2 is NHR, wherein said R, is other than H, Y, is O and Y 2 is NHR
• Y is O and Y 2 is NHR
• Y is O and Y 2 is BH 3 and Y, is S and Y 2 is SH.
• nucleosidyl-5'- cyclotriphosphite compound having the formula II:
• Bx is an optionally protected heterocyclic base moiety; one of X, and X 2 is H, hydroxyl, a protected hydroxyl, or a sugar substituent group and the other of X, and X 2 is -O-J-SM; J is a bifunctional linking moiety; and SM is a support medium.
• Other forms of the invention are compounds having the formula III:
• Z is Se, S, BH 3 , or NHR,
• Bx is an optionally protected heterocyclic base moiety; one of X, and X j is H, hydroxyl, a protected hydroxyl, or a sugar substituent group and the other of X, and X 2 is -O-J-SM;
• J is a bifunctional linking moiety
• SM is a support medium.
• the flow rate was 10 mL/min.
• the mass spectra of the purified triphosphates was determined using on-line HPLC mass spectrometry on a Hewlett-Packard (Palo Alto, CA) MSD 1100.
• RP HPLC was performed on a Phenomenex Luna (C-18(2)), 150 x 2 mm, plus 30 2 mm guard column, 3- ⁇ m particle size.
• a 0 to 50% linear gradient of acetonitrile in 20 mM TEAA (pH 7) was performed in series with mass spectra detection in the negative ionization mode. Nitrogen gas and a pneumatic nebulizer were used to generate the electrospray.
• the mass range of 150-900 was sampled. Molecular masses were determined using the HP Chemstation analysis package.
• Nucleosides were protected if necessary and attached to support media using standard procedures. A fully protected nucleoside is selected having a free hydroxyl group at the 2' or 3 '-position. A typical selection is a 5'-DMT-nucleoside having any exocyclic amino groups protected as the N-acyl group. The free hydroxyl group is attached to an amino functionalized solid support (e.g. CPG or polystyrene using a succinate linking group).
• an amino functionalized solid support e.g. CPG or polystyrene using a succinate linking group.
• the cyclic triphosphite (4, Example 2) was oxidized using iodine and a primary amine such as an alkylamine.
• the selected primary amine (4 mL) was suspended in a solution of iodine in tetrahydrofuran (224 mg in 5 mL).
• the solid support was treated with the oxidation mixture containing the primary amine and agitated for 40 minutes at 25°C. Excess oxidation mixture was removed by extensive washing with dry tetrahydrofuran (10 mL) and dry acetonitrile (10 mL).
• Oxidized intermediate (5) was hydrolized with water (5 mL at 25°C for 90 minutes).
• the ⁇ -amino modified nucleoside triphosphate was cleaved and deprotected by treating the solid support with 25%o aqueous ammonia in a sealed glass vial (15 hours at 25 ⁇ and 2 hours at 55°C).
• the oxidized material was hydrolyzed with a mixture of water and acetonitrile (9/1, v/v, 8 mL) for 90 minutes, followed by washing with acetonitrile, methanol and di- chloromethane (15 mL of each).
• the longer hydrolysis time proved to be necessary as the ⁇ -seleno compound was less reactive compared with the ⁇ -Sulfur analogue ( ⁇ -S- analogue was hydrolyzed completely in 30 minutes).
• the solid support was dried using a stream of dry nitrogen and removed from the reactor. The product was cleaved from the solid support using concentrated ammonia in a closed glass vial (for 12 hours at 25°C and 3 hours at 55°C).
• the ⁇ -Se-nucleoside triphosphate was obtained as essentially equal amounds of Rp and Sp stereoisomers. These stereoisomers were separated using ion-exchange and reverse-phase ⁇ PLC. The ion-exchange ⁇ PLC purification was preformed using MonoQ column (Pharmacia), 100 X 30 mm, and the following gradient: 0-0.72M NaCl at constant Tris (20 mM, p ⁇ 7), flow rate 6.5 mL/min. R,. for the stereoisomers was 17.7 minutes.
• the RP- ⁇ PLC separations were performed using a Luna (C-18(2)) column, 250 X 21.2 mm (Phenomenex, CA), 5- ⁇ m particle size column using linear gradient of MeO ⁇ (l%-25 %>) in 60 minutes, at constant TEAA (p ⁇ 7, 50 mM), flow rate 10 mL/minutes. Theretention times were 45.6 and 50 minutes for the Sp and Rp isomers respectively.
• Solid support bound cyclic triphosphite nucleoside (4, Scheme 1) was prepared as illustrated in Example 2 above.
• the oxidation of the cyclic triphosphife nucleoside was carried out using borane-N,N-diisopropylethylamine complex.
• the reaction mixture was agitated for 180 minutes at 25°C and the oxidation reagent was removed by repeated washing with dry acetonitrile (20 mL) and DMF (10 mL).
• the oxidized intermediate was hydrolized with water (5 mL at 25*C for 90 minutes).
• the ⁇ -borano modified nucleoside triphosphate was cleaved and deprotected by treating the solid support with 25% aqueous ammonia in a sealed glass vial (15 hours at 25°C and 2 hours at 55°C).
• the cooled ammonia solution of crude ⁇ -amino modified nucleoside triphosphate was evaporated to dryness.
• Solid support bound cyclic triphosphite nucleoside (4, Scheme 1) was prepared as illustrated in Example 2 above.
• the cyclic triphosphite nucleoside was oxidized with borane-N,N-diisopropylethylamine complex.
• the reaction mixture was agitated for 180 minutes at 25°C and excess oxidation reagent was removed by extensive washing with dry acetonitrile (20 mL) and DMF (10 mL).
• the resulting cyclic triphosphate was treated with a solution of Li 2 S in dimethyl-formamide in the presence of crown ether (18-crown-6) (8 mL at 25°C for 120 minutes) to give the solid support bound triphosphate.
• the solid support was treated with 25% aqueous ammonia in a sealed glass vial (15 hours at 25°C and 2 hrs at 55C).
• Solid support bound cyclic triphosphite nucleoside (4, Scheme 1) was prepared as illustrated in Example 2 above.
• the cyclic triphosphite nucleoside was oxidized with a 5% solution of elemental sulfur in carbon disulfide, pyridine and tri- etylamine (95/95/5, v/v/v) with agitation for 120 minutes at 25O.
• the solid support was repeatedly washed with dry acetonitrile (20 mL).
• the resulting cyclic triphosphate nucleoside was treated with a 0.2M solution of Li ⁇ and 18-crown-6 ether in dimethyl-formamide (8 L at 25°C for 120 minutes).
• the solid support was treated with 25% aqueous ammonia in a sealed glass vial (15 hours at 25°C and 2 hrs at 55°C).
• Solid support bound cyclic triphosphite nucleoside (4, Scheme 1) is prepared as illustrated in Example 2 above.
• the cyclic triphosphite nucleoside was oxidized with a 0.2M solution of 3H-l,2-benzothiaselenol-3-one in acetonitrile and triethylamine (95/5, v/v).
• the reaction mixture is agitated for 120 minutes at 25°C.
• the support is washed repeatedly with dry acetonitrile (20 mL).
• the oxidized cyclic intermediate is treated with a 0.2M solution of Li ⁇ in dimethylformamide (8 mL) in the presence of a crown ether (18-crown-6, 500 mg) at 25°C for 120 minutes).
• the support is treated with 25% aqueous ammonia in a sealed glass vial (15 hours at 25°C and 2 hrs at 55°C).
• the resulting ammonia solution of ⁇ -seleno- ⁇ -thio triphosphate nucleoside is evaporated to dryness.
• Solid support bound cyclic triphosphite nucleoside (4, Scheme 1) is prepared as illustrated in Example 2 above.
• the cyclic triphosphite nucleoside is oxidized with iodine in the presence of the corresponding amine in an organic solvent.
• Ammonia is used to attach an amino group and a primary amine having a single substituent defined by R, in example 3 above is used to attach a substituted amino group at the ⁇ -position.
• Reaction is performed by addition of ammonia (0.5M in dioxane) or a desired primary amine (XX mmoles) (4 mL) to the solid support suspended in iodine solution in an organic solvent such as tetrahydrofuran (224 mg in 5 mL). Next the reaction mixture is agitated for 40 minutes at 25°C. The solid support is repeatedly washed with dry tetrahydrofuran (10 mL) and dry acetonitrile (10 mL). The solid support is treated with a 0.2M solution of Li,S in dimethylformamide (8 mL) in the presence of a crown ether (18-crown-6, 500 mg) at 25O for 120 minutes.
• ammonia 0.5M in dioxane
• XX mmoles a desired primary amine
• the support is treated with 25% aqueous ammonia in a sealed glass vial (15 hours at 25°C and 2 hrs at 55°C).
• the resulting ammonia solution of ⁇ -alkylamino- ⁇ -thio triphos- phate nucleoside is evaporated to dryness.
• Solid support bound cyclic triphosphite nucleoside (4, Scheme 1) is prepared as illustrated in Example 2 above.
• the oxidation of the cyclic triphosphite nucleoside is carried out using borane-N,N-diisopropylethylamine complex.
• the reaction mixture is agitated for 180 minutes at 25°C and the oxidation reagent is removed by repeated washing with dry acetonitrile (20 mL) and DMF (10 mL).
• the oxidized intermediate is procedure for treatment of the oxidized cyclic triphosphate with an amine to prepare the ⁇ -amino derivative.
• the ⁇ -borano modified nucleoside triphosphate is cleaved and deprotected by treating the solid support with 25% aqueous ammonia in a sealed glass vial (15 hours at 25°C and 2 hours at 55°C).
• the cooled ammonia solution of crude ⁇ - amino modified nucleoside triphosphate is evaporated to dryness.

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