WO2010021897A1

WO2010021897A1 - Nucleosidic phosphoramidite crystalline material and a process for purifying the same

Info

Publication number: WO2010021897A1
Application number: PCT/US2009/053640
Authority: WO
Inventors: Raymond Cvetovich; Arlene E. Mckeown; Michael J. Mcnevin
Original assignee: Merck Sharp & Dohme Corp.
Priority date: 2008-08-18
Filing date: 2009-08-13
Publication date: 2010-02-25

Abstract

The instant invention provides for novel nucleosidic phosphoramidite crystalline material and crystallization processes for purifying the same, useful for the synthesis of oligonucleotides.

Description

TITLE OF THE INVENTION

NUCLEOSIDIC PHOSPHORAMIDITE CRYSTALLINE MATERIAL AND A PROCESS FOR

PURIFYING THE SAME

BACKGROUND OF THE INVENTION

Oligonucleotides, in particular antisense and siRNA, are now accepted as therapeutic agents or potential therapeutic agents holding great promise for human health.

Synthesis of oligonucleotides, including the synthesis of DNA and RNA oligonucleotides with or without chemical modifications, is well known in the art. Known methods include: 1) the phosphotriester method, as described by Reese, Tetrahedron 1978, 34, 3143; 2) the phosphoramidite method, as described by Beaucage, in Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs; Agrawal, ea.; Humana Press: Totowa, 1993, Vol. 20, 33-61; and 3) the H-phosphonate method, as described by Froehler in Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs; Agrawal, ea.; Humana Press: Totowa, 1993, Vol. 20, 63-80.

Using the phosphoramidite method, oligonucleotides are incorporated with nucleosidic phosphoramidate linkages derived from nucleosidic phosphoramidites. Nucleosidic phosphoramidites, with or without chemical modifications, are well known in the art and can be purchased from many vendors. Typically, nucleosidic phosphoramidites are purified by standard chromatography methods. Unfortunately, the current chromatographic methods have significant drawbacks. Nucleosidic phosphoramidites purified via chromatography provide low product yield, high impurity levels, and may be unstable to reactive degradation. Further, the synthesis of oligonucleotides, incorporating nucleosidic phosphoramidites purified via chromatographic methods, would be expected to provide oligonucleotides in low product yield and with high impurity levels.

Thus, there exists a need to provide a better purification process that generates nucleosidic phosphoramidites with fewer impurities (for example, a process that generates nucleosidic phosphoramidite crystalline material). Some benefits of generating nucleosidic phosphoramidite crystalline material may also include greater stability, high overall yield, and conservation of resources. Further, the synthesis of oligonucleotides, incorporating nucleosidic phosphoramidite crystalline material, would be expected to provide oligonucleotides in greater product yield and with lower impurity levels. SUMMARY OF THE INVENTION

The instant invention provides for novel nucleosidic phosphoramidite crystalline material and crystallization processes for purifying the same, useful for the synthesis of oligonucleotides . DETAILED DESCRIPTION OF THE INVENTION The instant invention provides for novel nucleosidic phosphoramidite crystalline material and crystallization processes for purifying the same, useful for the synthesis of oligonucleotides.

In a first embodiment, the invention provides for the production of crystalline material containing a nucleosidic phosphoramidite comprising:

(1) exposure of the nucleosidic phosphoramidite to processing conditions under which crystallization of material containing the nucleosidic phosphoramidite occurs; and

(2) isolation of crystalline material containing the nucleosidic phosphoramidite. In another aspect of the first embodiment, the invention provides for the production of crystalline material containing a nucleosidic phosphoramidite of Formula A,

wherein:

W is substituted or unsubstituted C, N, O, Si, P₅ S, B, and Se; X is a base;

Y is a protecting group;

Z is a 2'-chemical modification;

R¹ is a protecting group; and

R² is NR³R⁴, wherein each of R³ and R⁴ is independently substituted or unsubstituted alkyl or alternatively R³ and R⁴ are joined to form a substituted or unsubstituted 4 to 7 membered heteroaryl or heterocycle including the nitrogen to which R³ and R⁴ are attached; comprising the steps of:

(1) exposure of the nucleosidic phosphoramidite to processing conditions under which crystallization of material containing the nucleosidic phosphoramidite occurs; and (2) isolation of crystalline material containing the nucleosidic phosphoramidite.

In another aspect of the first embodiment, the invention provides for the production of crystalline material containing a nucleosidic phosphoramidite of Formula A wherein:

W is O, N, S, Si, P or C wherein C is optionally substituted with alkyl; X is selected from: guanine, N2-isobutyrylguanine, N2~t~ butylphenoxyacetylguanine, N2-(N,N~dimethylformamidine)guanosine, adenine, Nό- benzoyladenine, N4-phenoxyacetyladenine, thymine, uracil, cytosine, N4-benzoylcytosine, N4- acetylcytosine, N4-phenoxyacetylcytosine, 2,6-diaminopurine, 6-phenyllumazine, 7-(4 biphenyl)lumazine, 5-methylcytosine, 5-propynyluracil, 5-propynylcytosine, 5-(thiazol 2- yl)υracil, 5-(5-methyl-2-yl)uracil, 7-deazaguanine, tubercine (7-deazaadenine), 7-deaza-7- methylguanine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoguanine, 7-(ρropyn-l-yl)-7- deazaguanine, 7-(hex-l-ynyl)-7~deazaguanine_; 7-iodo-7-deaza-2-aminoadenine, 7-(proρ-l-ynyl)- 7-deaza-2-aminoadenine, 7-cyano-7-deaza-2-aminoadenine, 7-(proρ-l-ynyl)-7~deazadenine, 7- ethynyl-7-deazadenine, 7-bromo-7-deazadenine, 7-chloro-7-deazadenine, 7-methyl-7 deazadenine, 7-deaza-8-azadenine, 7-deaza-8-azaguanine, spermine-conjugated guanine, 5-(N- aminohexyl)carbamoyluracilj triaminoalkylamidouracil, 7~(3-aminopropyn- 1 -yl)-7-deazadenine, 3 -aminopropyn- 1 -yluracil, 2,7 -dioxopyridopyrimidine, phenoxazinopyrimidine, phenothiazinopyrimidine, tetracyclic deazadenine, thiothynine, 2-thiouracil, hypoxanthine, xanthine, pyrrolopyriraidinone, N-chloroethylcytosine, haloacetylcytosine, N4,N4- ethanocytosme, imidazolylpropylguanine, N2-imidazolylpropyl-2-aminoadenine, 5-methyl-N4-(l pyrenylmethyl)cytosine, N4-diphenylether-5-methylcytidine, and aminoethoxyphenoxazinopyrimidine-2-one;

Y is 4_!4'-dimethoxytriphenylmethyl (DMT), 4~monomethoxytrityl(MMT), or 9- phenylxanthen-9-yl (pixyl);

Z is selected from: H (i.e. deoxy),-OH, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkoxy, substituted alkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy, substituted alkynyloxy, aminoalkoxy, alkoxyalkoxy, alkylaminoalkoxy, imidazolylalkoxy, alkenylthiO_j alkynylthio, alkenylamino, alkynylamino, aryloxy, arylthio, aralkyloxy, aralkylthio, aralkylamino, N-ρhthalimido, halogen (e.g. fluoro),-C(=^:O)-R (wherein R is an organic radical), carboxyl, nitro, nitroso, cyano, trifluoromethyl, trifluoromethoxy, imidazolyl, azido, hydrazino, aminooxy, isocyanato, isothiocyanato, O~tert-butyldimethyl silyl, sulfoxide (~S(^~O)-R), sulfone (~S(=O)2-R (wherein R is an organic radical)), disulfide (-S-S-R (wherein R is an organic radical)), silyl, O- silyl, methoxyethoxy, thioalkyl, a heterocycle, a carbocycle, a substituted or unsubstituted ether, and a LNA (locked nucleic acid) wherein the LNA forms a bridge between Z and the 4 carbon position of the ribose in Formula A;

R¹ is cyanoethyl, methyl, tetrazole, triazole, or benzyl; and R² is NR³R⁴, wherein each of R³ and R⁴ is independently alkyl, or wherein R³ and R⁴ are joined to form a substituted or unsubstituted triazole, tetrazole, or benzotriazole; comprising the steps of:

(2) isolation of crystalline material containing the nucleosidic phosphoramidite. In another aspect of the first embodiment, the invention provides for the production of crystalline material containing a nucleosidic phosphoramidite of Formula A wherein:

W is O;

X is selected from adenine, guanine, thymine, cytosine, and uracil; Y is 4,4'-dimethoxytriphenylmethyl (DMT);

Z is selected from: H₅ OMe, F, O-tert-butyldimethylsilyl, and methoxyethoxy; R¹ is cyanoethyl or Me; and

R² is NR³R⁴, wherein each of R³ and R⁴ is independently alkyl, or wherein R³ and R⁴ are joined to form a triazole, tetrazole, or benzotriazole; comprising the steps of:

(2) isolation of crystalline material containing the nucleosidic phosphoramidite. In a second embodiment, the invention provides a process for crystallization of nucleosidic phosphoramidite comprising:

(1) combination of the nucleosidic phosphoramidite with a liquid or gas;

(2) crystallization of material containing nucleosidic phosphoramidite; and

(3) isolation of nucleosidic phosphoramidite containing crystalline material. In another aspect of the second embodiment, the invention provides a process for crystallization of nucleosidic phosphoramidites of Formula A,

wherein: W is substituted or unsubstituted C, N, O, Si, P, S, B, and Se;

X is a base;

Y is a protecting group; Z is a 2'-chemical modification; R¹ is a protecting group; and R² is NR³R⁴, wherein each of R³ and R⁴ is independently substituted or unsubstituted alkyl or alternatively R³ and R⁴ are joined to form a substituted or unsubstituted 4 to 7 membered heteroaryl or heterocycle including the nitrogen to which R³ and R⁴ are attached; comprising the steps of:

(1) combination of the nucleosidic phosphoramidite with a liquid or gas; (2) crystallization of material containing the nucleosidic phosphoramidite; and

(3) isolation of nucleosidic phosphoramidite containing crystalline material. In another aspect of the second embodiment, the invention provides a process for crystallization of nucleosidic phosphoramidites of Formula A wherein,

W is O, N, S, Si, P or C wherein C is optionally substituted with alkyl; X is selected from: guanine, N2-isobutyrylguanine, N2-t- butylphenoxyacetylguanine, N2-(N,N-dimethylformamidine)guanosine, adenine, N6- benzoyladenine, N4-phenoxyacetyladenine, thymine, uracil, cytosϊne, N4-benzoylcytosine, N4- acetylcytosine, N4-phenoxyacetylcytosine, 2,6-diaminopurine, 6-phenyllumazine, 7-(4 biphenyl)lumazine, 5-methylcytosine, 5-propynyluracil, 5-propynylcytosine, 5-(thiazol 2- yl)uracil, 5-(5-methyl-2-yl)uracil, 7-deazaguanine, tubercine (7-deazaadenine), 7~deaza-7- methylguanine, 7-deaza-7-iodoguanine_f 7-deaza-7-bromoguara^'ne, 7-(propyn-l-yl)-7~ deazaguanine, 7-(hex-l-ynyl)-7-deazaguanine_J 7-iodo~7-deaza-2-aminoadenine, 7-(prop-l-ynyl)- 7-deaza-2-aminoadenine, 7-cyano-7-deaza-2-aminoadem^'ne, 7-(prop-l-ynyl)-7-deazadenine, 7- ethynyl-7-deazadenine_f 7-bromo-7-deazadenine, 7-chloro-7-deazadenine, 7-methyl-7 deazadenine, 7-deaza-8-azadenine, 7-deaza-8-a2aguanine, spermine-conjugated guanine, 5-(N- aminohexyl)carbamoyluracil_s triaminoalkylamidouracil, 7-(3-aminopropyn-l ~yi)-7-deazadenine, 3 -aminopropyn- 1 -yluracil, 2,7-dioxopyridopyrimidine , phenoxazinopyrimidine, phenothiazinopyrimidine, tetracyclic deazadenine, thiothynine, 2-thiouracil, hypoxanthine, xanthine, pyrrolopyrimidinone, N-chloroethylcytosine, haloacetylcytosine, N4,N4- ethanocytosine, imidazolylpropylguanine, N2-iraidazolylpropyl-2-aminoadenine, 5-methyl-N4-(l pyτenylmethyl)cytosine, N4-diphenylether-5-methylcytidine, and aminoethoxyphenoxazinopyrimidine-2-one;

Y is 4,4'-dimethoxytriphenylmethyI (DMT), 4-monomethoxytrityl(MMT), or 9- phenylxanthen-9-yl (pixyl);

Z is selected from; H (i.e. deoxy),-OH, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkoxy, substituted alkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy, substituted alkynyloxy, aminoalkoxy, alkoxyalkoxy, alkylaminoalkoxy, ϊmidazolylalkoxy, alkenylthio, alkynylthio, alkenylamino, alkynylamino, aryloxy, arylthϊo, aralkyloxy, aralkylthio, aralkylamino, N-phthalimido, halogen (e.g. fluoro),-C(=O)-R (wherein R is an organic radical), carboxyl, nitro, nitroso, cyano, trifluoromethyl, trifluoromethoxy, imidazolyl, azido, hydrazino, aminooxy, isocyanato, isothiocyanato, O-tert-butyldimethyl silyl, sulfoxide (-S(=O)-R), sulfone (-S(=O)2-R (wherein R is an organic radical)), disulfide (-S-S-R (wherein R is an organic radical)), silyl, O- silyl, methoxyethoxy, thioalkyl, a heterocycle, a carbocycle, a substituted or unsubstituted ether, and a LNA (locked nucleic acid) wherein the LNA forms a bridge between Z and the 4 carbon position of the ribose in Formula A;

(1) combination of the nucleosidic phosphoramidite with a liquid or gas;

(2) crystallization of material containing the nucleosidic phosphoramidite; and

(3) isolation of the nucleosidic phosphoramidite containing crystalline material. In another aspect of the second embodiment, the invention provides for the production of crystalline material containing a nucleosidic phosphoramidite of Formula A wherein:

W is O; X is selected from adenine, guanine, thymine, cytosine, and uracil;

Y is 4,4'-dimethoxytriphenylmethyl (DMT);

Z is selected from: H, OMe, F, O-tert-butyldimethylsilyl, and methoxoyethoxy;

R¹ is cyanoethyl or Me; and

(1) combination of the nucleosidic phosphoramidite with a liquid or gas;

(2) crystallization of material containing the nucleosidic phosphoramidite; and

(3) isolation of nucleosidic phosphoramidite containing crystalline material, In a third embodiment, the invention provides a process for crystallization of nucleosidic phosphoramidite comprising:

(1) combination of the nucleosidic phosphoramidite with a solvent, creating a solution;

(2) crystallization of material containing the nucleosidic phosphoramidite from the solution; and

(3) isolation of nucleosidic phosphoramidite containing crystalline material from the solution.

In another aspect of the third embodiment, the invention provides a process for crystallization of nucleosidic phosphoramidites of Formula A,

Formula A wherein:

W is substituted or unsubstituted C₅ N, O, Si, P, S, B, and Se; X is a base; Y is a protecting group;

Z is a 2'-chemical modification; R^! is a protecting group; and R² is NR³R⁴, wherein each of R³ and R⁴ is independently substituted or unsubstituted alkyl or alternatively R³ and R⁴ are joined to form a substituted or unsubstituted 4 to 7 membered heteroaryl or heterocycle including the nitrogen to which R³ and R⁴ are attached; comprising the steps of: ( 1 ) combination of the nucleosidic phosphoramidite with a solvent, creating a solution;

In another aspect of the third embodiment, the invention provides a process for crystallization of nucleosidic phosphoramidites of Formula A wherein,

W is O_j N, S, Si, P or C wherein C is optionally substituted with alkyl; X is selected from: guanine, N2-isobutyrylguanine, N2-t- butylphenoxyacetylguanine, N2-(N,N-dimethylformamidine)guanosme, adenine, N6- benzoyladenine, N4-phenoxyacetylademne, thymine, uracil, cytosine, N4-benzoylcytosine, N4- acetylcytosine, N4-phenoxyacetylcytosine, 2,6-diaminopurme, 6-phenyllumazine, 7-(4 biphenyl)lumazine, 5-methylcytosine, 5-propynyluracil, 5-propynylcytosine₅ 5-(thiazol 2- yl)uracil, 5-(5-methyl-2-yl)uracil, 7-deazaguanine, tubercine (7-deazaadenine), 7-deaza-7- methylguanine, 7-deaza-7-iodoguanine, 7~deaza-7-bromoguanine, 7-(propyn-l-yl)-7- deazaguanine, 7-(hex-l-ynyl)-7-deazaguanine, 7-iodo-7-deaza-2-aminoadenine, 7-(prop-l-ynyl)- 7-deaza-2-aminoadenine, 7-cyano-7-deaza-2-aminoadenine, 7-(prop-l-ynyl)-7-deazadenine_J 7- ethynyl-7-deazadenine, 7-bromo-7-deazadenine, 7-chloro-7-deazadenine, 7-methyl-7 deazadenine, 7-deaza-S-azadenine, 7-deaza~8-azaguanme, spermine-conjugated guanine, 5-(N- aminohexyl)carbamoyluracil, triaminoalkylamidouracil, 7-(3-aminopropyn-l-yl)-7-deazadenine, 3-aminopropyn-l -yluracil, 2,7-dioxopyridopyrimidine, phenoxazinopyrirmdine, phenothiazinopyrimidine, tetracyclic deazadenine, thiothynine, 2-thiouracil, hypoxanthine, xanthine, pyrrolopyrimidinone, N-chloroethylcytosine_; haloacetylcytosine, N4,N4- ethanocytosine, imidazolylpropylguanine, N2-imidazolylpropyl-2-aminoadenine, 5-methyl-N4-(l ρyrenylmethyl)cytosine, N4-diphenylether-5-methylcytidine, and aminoethoxyphenoxazinopyrimidine-2-one;

Y is 4,4'-dimethoxytriphenylmethyl (DMT), 4-monomethoxytrityl(MMT), or 9- phenylxanthen-9-yl (pixyl);

Z is selected from: H (i.e. deoxy)_f-OH, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkoxy, substituted alkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy, substituted alkynyloxy, aminoalkoxy, alkoxyalkoxy, alkylaminoalkoxy, imidazolylalkoxy, alkenylthio, alkynylthio, alkenylamino, alkynylamino, aryloxy, arylthio, aralkyloxy, aralkylthio, aralkylamino, N-phthalimido, halogen (e.g. fluoro),-C(⁼=O)-R (wherein R is an organic radical), carboxyl, nitro, nitroso, cyano, trifluoromethyl, trifluoromethoxy, imidazolyl, azido, hydrazino, aminooxy, isocyanato, isothiocyanato, O-tert-butyldimethyl silyl, sulfoxide (-S(=O)-R), sulfone (-S(=O)2-R (wherein R is an organic radical)), disulfide (-S-S-R (wherein R is an organic radical)), silyl, O- silyl, methoxyethoxy, thioalkyl, a heterocycle, a carbocycle, a substituted or unsubstituted ether, and a LNA (locked nucleic acid) wherein the LNA forms a bridge between Z and the 4 carbon position of the ribose in Formula A;

R¹ is cyanoethyl, methyl, tetrazole, triazole, or benzyl; and R² is NR³R⁴ _J wherein each of R³ and R⁴ is independently alkyl, or wherein R³ and R are joined to form a substituted or unsubstituted triazole, tetrazole, or benzotriazole; comprising the steps of:

(2) crystallization of material containing the nucleosidic phosphoramidite from the solution; and (3) isolation of nucleosidic phosphoramidite containing crystalline material from the solution.

In another aspect of the third embodiment, the invention provides for the production of crystalline material containing a nucleosidic phosphoramidite of Formula A wherein: W is O;

X is selected from adenine, guanine, thymine, cytosine, and uracil; Y is 4,4'-dimethoxytriρhenylmethyl (DMT);

Z is selected from: H, OMe, F, O-tert-butyldimethylsilyl₅ and methoxoyethoxy; R¹ is cyanoethyl or Me; and R² is NR³R⁴, wherein each of R³ and R⁴ is independently alkyl, or wherein R³ and

R⁴ are joined to form a triazole, tetrazole, or benzotriazole; comprising the steps of:

(1) combination of the nucleosidic phosphoramidite with a solvent, creating a solution; (2) crystallization of material containing the nucleosidic phosphoramidite from the solution; and

In a fourth embodiment, the invention provides a process for crystallization of nucleosidic phosphoramidite comprising:

(1) combination of the nucleosidic phosphoramidite with an anhydrous solvent, in a water-free environment, creating a solution; (2) crystallization of material containing the nucleoside phosphoramidite from the solution; and

(3) isolation of nucleosidic phosphoramidite containing crystalline material from the solution. In another aspect of the fourth embodiment, the invention provides a process for crystallization of nucleosidic phosphoramidites of Formula A₅

Formula A wherein: W is substituted or unsubstituted C, N, O, Si, P, S, B, and Se;

X is a base;

Y is a protecting group;

Z is a 2'-chemical modification;

R¹ is a protecting group; and R² is NR³R⁴, wherein each of R³ and R⁴ is independently substituted or unsubstituted alkyl or alternatively R³ and R⁴ are joined to form a substituted or unsubstituted 4 to 7 membered heteroaryl or heterocycle including the nitrogen to which R³ and R⁴ are attached; comprising the steps of:

(1) combination of the nucleosidic phosphoramidite with an anhydrous solvent, in a water-free environment, creating a solution;

(3) isolation of nucleosidic phosphoramidite containing crystalline material from the solution. In another aspect of the fourth embodiment, the invention provides a process for crystallization of nucleosidic phosphoramidites of Formula A wherein,

W is O, N, S, Si, P or C wherein C is optionally substituted with alkyl; X is selected from: guanine, N2-isobutyrylguanine, N2-t- butylphenoxyacetylguanine, N2-(N,N-dimethylformamidine)guanosine, adenine, Nό- benzoyladenine, N4-phenoxyacetyladenine, thymine, uracil, cytosine, N4-benzoylcytosme, N4- acetylcytosine, N4-phenoxyacetylcytosine, 2,6-diaminopurine, ό-phenyllumazine, 7-(4 biphenyl)lumazine, 5-methylcytosine, 5-proρynyluracil, 5-propynylcytosine, 5-(thiazol 2- yl)uracil, 5-(5-methyl-2-yl)uracil, 7-deazaguanine_? tubercϊne (7-deazaadenine), 7-deaza-7- methylguanine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoguanme, 7-(propyn-l-yl)-7- deazaguanine, 7-(hex-l-ynyl)-7-deazaguanine, 7-iodo-7-deaza-2-aminoadenine, 7-(prop-l-ynyl)- 7-deaza-2-aminoadenine, 7-cyano-7-deaza-2-aminoadenine, 7-(prop-l-ynyl)-7~deazadenine, 7- ethynyl-7-deazadenine, 7-bromo-7-deazadenine, 7-chloro-7-deazadenine, 7-methyl-7 deazadenine, 7-deaza-8-azadenine, 7-deaza-8-azaguanine, spermine-conjugated guanine, 5-(N- aminohexy^carbamoyluracil, triaminoalkylamidouracil, 7-(3-aminopropyn~l-yl)-7-deazadenine_J 3 -aminopropyn- 1 -yluracil, 2 ,7-dioxopyridopyrimidine, phenoxazinopyrimidine, phenothiazinopyrimidine, tetracyclic deazadenine, thiothynine, 2-thiouracil, hypoxanthine, xanthine, pyrrolopyrimidinone, N-chloroethylcytosine, haloacetylcytosine, N4,N4- ethanocytosine, imidazolylpropylguanine, N2-imidazolylpropyl-2-aminoadenine, 5-methyl-N4-(l ρyrenylmethyl)cytosine, N4-diphenylether-5-methylcytidine, and aminoethoxyphenoxazinopyrimidine-2-one;

Y is 4,4'-dimethoxytriρhenylmethyl (DMT), 4-monomethoxytrityl(MMT), or 9- phenylxanthen-9-yl (pixyl);

Z is selected from: H (i.e. deoxy),-OH, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkoxy, substituted alkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy, substituted alkynyloxy, aminoalkoxy, alkoxyalkoxy, alkylaminoalkoxy, imidazolylalkoxy, alkenylthio, alkynylthio, alkenylamino, alkynylamino, aryloxy, arylthio, aralkyloxy, aralkylthio, aralkylamino, N-phthalimido, halogen (e.g. fluoro),-C(=0)-R (wherein R is an organic radical), carboxyl, nitro, nitroso, cyano, trifluoromethyl_> trifluoromethoxy, imidazolyl, azϊdo, hydrazino, aminooxy, isocyanato, isothiocyanato, O-tert-butyldimethyl silyl, sulfoxide (-S(==O)-R), sulfone (-S(=O)2-R (wherein R is an organic radical)), disulfide (-S-S-R (wherein R is an organic radical)), silyl, O- silyl, methoxyethoxy, thioalkyl, a heterocycle, a carbocycle, a substituted or unsubstituted ether, and a LNA (locked nucleic acid) wherein the LNA forms a bridge between Z and the 4 carbon position of the ribose in Formula A; R¹ is cyanoethyl, methyl, tetrazole, triazole, or benzyl; and

R² is NR³R⁴, wherein each of R³ and R⁴ is independently alkyl, or wherein R³ and R are joined to form a substituted or unsubstituted triazole, tetrazole, or benzotriazole; comprising the steps of:

(3) isolation of nucleosidic phosphoramidite containing crystalline material from the solution. In another aspect of the fourth embodiment, the invention provides for the production of crystalline material containing a nucleosidic phosphoramidite of Formula A wherein:

W is O; X is selected from adenine, guanine, thymine, cytosine, and uracil;

Y is 4,4'-dimethoxytriphenyImethyl (DMT);

Z is selected from: H, OMe, F, O-tert-butyldimethylsilyl, and methoxoyethoxy;

R¹ is cyanoethyl or Me; and R² is NR³R⁴, wherein each of R³ and R⁴ is independently alky I₅ or wherein R³ and

(1) combination of the nucleosidic phosphoramidite with an anhydrous solvent, in a water-free environment, creating a solution; (2) crystallization of material containing the nucleosidic phosphoramidite from the solution; and

In a fifth embodiment, the invention provides for the production of crystalline material containing a nucleosidic phosphoramidite comprising:

(1) exposure of a nucleosidic phosphoramidite precursor to processing conditions under which the precursor is transformed into the nucleosidic phosphoramidite;

(2) exposure of the resulting nucleosidic phosphoramdite to processing conditions under which the crystallization of material containing the nucleosidic phosphoramidite occurs; and

(3) isolation of the crystalline material containing the nucleosidic phosphoramidite.

In another aspect of the fifth embodiment, the invention provides for the production of crystalline material containing a nucleosidic phosphoramidite of Formula A,

Formula A wherein:

W is substituted or unsubstituted C, N, O, Si, P, S, B, and Se; X is a base; Y is a protecting group;

Z is a 2' -chemical modification; R¹ is a protecting group; and R is NR R j wherein each of R and R is independently substituted or unsubstituted alkyl or alternatively R³ and R⁴ are joined to form a substituted or unsubstituted 4 to 7 membered heteroaryl or heterocycle including the nitrogen to which R and R⁴ are attached; comprising the steps of: (1) exposure of a nucleosidic phosphoramidite precursor to processing conditions under which the precursor is transformed into the nucleosidic phosphoramidite;

(2) exposure of the resulting nucleosidic phosphoramdite to processing conditions under which the crystallization of material containing the nucleosidic phosphoramidite occurs; and (3) isolation of the crystalline material containing the nucleosidic phosphoramidite.

In another aspect of the fifth embodiment, the invention provides for the production of crystalline material containing a nucleosidic phosphoramidite of Formula A wherein: W is O₅ N_> S, Si, P or C wherein C is optionally substituted with alkyl;

X is selected from: guanine, N2-isobutyrylguam^'ne, N2-t- butylphenoxyacetylguanme, N2-(N,N-dimethylformamidine)guanosine, adenine, N6~ benzoyladenine, N4-phenoxyacetyladenine, thymine, uracil, cytosme, N4-benzoylcytosine, N4- acetylcytosine, N4~phenoxyacetylcytosine, 2,6-diaminoρurine, 6-phenyllumazine, 7-(4 biphenyl)lumazme_j 5-methylcytosine, 5-propynyluracil, 5-propynylcytosine, 5-(thiazol 2- yl)uracil, 5-(5-methyl-2-yl)uracil, 7-deazaguanine, tubercine (7-deazaadenine), 7~deaza-7- methylguanine, 7-deaza-7-iodoguanine_} 7-deaza-7-bromoguanine_; 7-(propyn-l-yl)~7- deazaguanine, 7-(hex-l-ynyl)-7-deazaguanine, 7-iodo-7-deaza-2-aminoadenine, 7-(ρrop-l-ynyl)- 7"deaza-2-aminoadenine_s 7-cyano-7-deaza-2-aminoadenine, 7-(prop-l-ynyl)-7-deazadenine, 7- ethynyl-7-deazadenine, 7-bromo-7-deazadenine, 7-chloro-7-deazadenine, 7-methyl-7 deazadenine, 7-deaza-8-azadenine, 7-deaza-8-azaguanine, spermine-conjugated guanine, 5-(N- aminohexyi)carbamoyluracil, triaminoalkylamidouracil, 7-(3-aminoproρyn-l -yl)-7-deazadenine, 3-aminoρropyn- 1 -yluracil, 2,7-dioxopyridopyrimidine, phenoxazinopyrimidine, phenothiazinopyrimidine, tetracyclic deazadenine, thiothynine, 2-thiouracil, hypoxanthine, xanthine, pyrrolopyrimidinone, N-chloroethylcytosine_? haloacetylcytosine, N4,N4~ ethanocytosine, imidazolylpropylguanine, N2-imidazolyIpropyl-2-aminoadenine, 5-methyl-N4-(l pyrenylmethyl)cytosine, N4-diphenylether-5-methylcytidine, and aminoethoxyphenoxazinopyrimidine-2-one;

Z is selected from: H (i.e. deoxy),-OH_> alkyl, alkenyl, alkynyl, aryl, aralkyl, alkoxy, substituted alkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy, substituted alkynyl oxy, aminoalkoxy, alkoxyalkoxy, alkylaminoalkoxy, imidazolylalkoxy, alkenylthio, alkynylthio, alkenylamino, alkynylamino, aryloxy, arylthio, aralkyloxy, aralkylthio, aralkylamino, N-ρhthalimido, halogen (e.g. fluoro),-C(=O)-R (wherein R is an organic radical), carboxyl, nitro, nitroso, cyano, trifluoromethyl, trifluoromethoxy, imidazolyl, azido, hydrazino, aminooxy, isocyanato, isothiocyanato, O~tert-butyldimethyl silyl, sulfoxide (-S(=O)-R), sulfone (-S(=O)2-R (wherein R is an organic radical)), disulfide (-S-S-R (wherein R is an organic radical)), sϋyl, O- silyl, methoxyethoxy, Ihioalkyl, a heterocycle, a carbocycle, a substituted or unsubstituted ether, and a LNA (locked nucleic acid) wherein the LNA forms a bridge between Z and the 4 carbon position of the ribose in Formula A;

R¹ is cyanoethyl, methyl, tetrazole, triazole, or benzyl; and R² is NR³R⁴, wherein each of R³ and R⁴ is independently alkyl, or wherein R³ and

R⁴ are joined to form a substituted or unsubstituted triazole, tetrazole, or benzotriazole; comprising the steps of:

(1) exposure of a nucleosidic phosphoramidite precursor to processing conditions under which the precursor is transformed into the nucleosidic phosphoramidite; (2) exposure of the resulting nucleosidic phosphoramdite to processing conditions under which the crystallization of material containing the nucleosidic phosphoramidite occurs; and

(3) isolation of the crystalline material containing the nucleosidic phosphoramidite. In another aspect of the fifth embodiment, the invention provides for the production of crystalline material containing a nucleosidic phosphoramidite of Formula A wherein:

W is O;

Z is selected from: H, OMe, F, O-tert-butyldimethylsilyl, and methoxoyethoxy;

R¹ is cyanoethyl or Me; and

(3) isolation of the crystalline material containing the nucleosidic phosphoramidite. In another embodiment, the invention provides a process for crystallization of 2'- OMeU β-cyanoethyl phosphoramidite comprising the steps of:

(1) combination of solid or liquid 2'-0MeU β-cyanoethyl phosphoramidite in a solvent, creating a solution; (2) crystallization of material containing 2'-0MeU β-cyanoethyl phosphoramidite from the solution; and

(3) isolation of the crystalline material containing 2'-0MeU β-cyanoethyl phosphoramidite from the solution.

In another embodiment, the invention provides a process for crystallization of 2'- OMeU β-cyanoethyl phosphoramidite comprising the steps of:

(1) combination of solid or liquid 2'-0MeU β-cyanoethyl phosphoramidite in acetonitrile, creating a solution;

(2) crystallization of material containing 2'-0MeU β-cyanoethyl phosphoramidite (Compound A) from the solution; and (3) isolation of Compound A from the solution:

Compound A

In another embodiment, the instant invention includes nucleosidic phosphoramidite crystalline material made by the crystallization processes of the instant invention and any stereoisomers thereof.

In another embodiment, the instant invention includes a method for the synthesis of oligonucleotides, wherein the method comprises the use of at least one nucleosidic phosphoramidite crystalline material.

A compound of the instant invention is the isolated acetonitrile solvate of T- OMeU β-cyanoethyl phosphoramidite (Compound A) and any tautomers or stereoisomers thereof:

Compound A

In another embodiment, the acetonitrile solvate of Compound A exhibits one or more of: (i) the X-ray powder diffractogram shown in Figure 1, as measured using CuKa radiation; (ii) reflections in the X-ray powder diffractogram as measured using CuKa radiation at the 2-theta angles: 8.2, 12.9, 15.7, 16.5, and 18.8; (iii) the chromatography data with a starting material purity of 99.11% and a crystallization material purity of 99.75%; (iv) the solution ¹H NMR data indicating the presence of 1.1 equivalent of acetonitrile in the sample; (v) the solid state ⁱ³C NMR data indicating that 2'0-MeU β-cyanoethyl phosphoramidite and acetonitrile molecules are present in the crystal in comparable numbers; and (vi) chemical shifts in the 13- carbon solid-state NMR at: 55.6, 159.9, and 131.2 ppm.

In another embodiment, the acetonitrile solvate of Compound A exhibits reflections in the X-ray powder diffractogram as measured using CuKa radiation at the 2-theta angles: 8.2, 12.9, 15.7, 16.5, and 18.8.

In another embodiment, the acetonitrile solvate of Compound A exhibits reflections in the X-ray powder diffractogram as measured using CuKa radiation at the 2-theta angles: 5.7, 9.5, 21.0, 21.8, 23.6, and 35.0.

In another embodiment, the acetonitrile solvate of Compound A exhibits reflections in the X-ray powder diffractogram as measured using CuKa radiation at the 2-theta angles: 5.7, 8.2, 9.5, 12.9, 15.7, 16.5, 18.8, 21.0, 21.8, 23.6, and 35.0. In another embodiment, the acetonitrile solvate of Compound A exhibits reflections in the X-ray powder diffractogram as measured using CuKa radiation at d-spacing: 15.5, 10.7, 9.3, 6.9, and 5.7.

In another embodiment, the acetonitrile solvate of Compound A exhibits reflections in the X-ray powder diffractogram as measured using CuKa radiation at d-spacing: 5.4, 4.7, 4.2, 4.1, 3.8, and 2.6.

In another embodiment, the acetonitrile solvate of Compound A exhibits reflections in the X-ray powder diffractogram as measured using CuKa radiation at the 2-theta angles: 5.7, 8.2, 9.5, 12.9, 15.7, 16.5, 18.8, 21.0, 21.8, 23.6, 35.0, and d-spacing: 15.5, 10.7, 9.3, 6.9, 5.7, 5.4, 4.7, 4.2, 4.1, 3.8, and 2.6.

In another embodiment, the acetonitrile solvate of Compound A exhibits reflections in the X-ray powder diffractogram as measured using CuKa radiation at the 2-theta angles: 5.7, 8.2, and 16.5. It is further characterized by reflections in the X-ray powder diffractogram as measured using CuKa radiation at the 2-theta angles: 9.5, 12.9, and 15.7. It is further characterized by reflections in the X-ray powder diffractogram as measured using CuKa radiation at the 2-theta angles: 18.8, 21.0, and 23.6. It is further characterized by reflections in the X-ray powder diffractogram as measured using CuKa radiation at the 2-theta angles: 21.8, and 35.0.

In another embodiment, the acetonitrile solvate of Compound A exhibits chemical shifts in the 13-carbon solid-state NMR at: 55.6, 159.9, and 131.2 ppm. It is further characterized by chemical shifts in the 13-carbon solid-state NMR at: 86.7, 43.2, and 115.6 ppm. It is further characterized by chemical shifts in the 13-carbon solid-state NMR at: 22.0, 140.6, and 79.7 ppm. It may also be further characetized by a chemical shift in the 13-carbon solid-state NMR at: 118.2 ppm.

In another embodiment, the acetonitrile solvate of Compound A exhibits chemical shifts in the 13-carbon solid-state NMR at: 55.6, 159.9, 131.2, and 86.7 ppm.

In another embodiment, the acetonitrile solvate of Compound A exhibits chemical shifts in the 13-carbon solid-state NMR at: 43.2, 115.6, 22.0, 140.6, and 79.7 ppm.

In another embodiment, the acetonitrile solvate of Compound A exhibits chemical shifts in the 13-carbon solid-state NMR at: 118.2 ppm.

A compound of the instant invention is 2'-OMeU β-cyanoethyl phosphoramidite (Compound A) containing crystalline material and any tautomers or stereoisomers thereof:

In an aspect of the first, second, third, fourth and fifth embodiments, the anhydrous solvent is selected from: 1,1 J-trichloroethane, 1,1,2,2,-tetrachloroethane, l-butanol_s 1-chlorobutane, l-methyl-2-pyrrolidinone, l~nitropropane, 1-octanol, 1-pentanol, 1-propanol, 1 ,2-dichlorobenzene, 1 ,2 dichloroethane, 1 ,2-dichloroethene, 1 ,2-diethoxyethane, 1 ,2- dimethoxyethane, 1 ,4-dioxane, 2,2,4-trimethylpentane, 2-butanol, 2-butanone, 2-ethoxyethanol, 2-methoxyethanol, 2-methyl- 1-propanol, 2-methyl-tetrahydrofuran, 2-methylbutane, 2- nitropropane, 2-ρentanone, 2-propanol, 3-methyl-l-butanol, 3-pentanone, acetone, acetonitrile, anisole, benzene, benzonitrile, butyl acetate, butyl amine, butyl ether, butyronitrile, carbon tetrachloride, chlorobenzene, chloroform, cumene, cyclohexane, cyclohexanone, cyclopentanone, decane, decalin, dichloromethane, diethyl ether, diethylene glycol, diisobutyl ethyl amine, diisopropyl amine, diisopropyl ether, diglyme, dimethylsulfoxide, dimethoxymethane, ethanol, ethyl acetate, ethyl alcohol, ethyl formate, ethylene glycol, fluorobenzene, formamide, glycerine, glycerol, heptane, hexane, hexafluorobenzene, isobutyl acetate, isooctane, isopropyl acetate, isopropyl ether, mesitylene, m-xylene, methanol, methyl acetate, methyl formate, methyl cyclohexane, methylene chloride, methyl isopropyl ether, methyl isopropyl ketone, N₉N- dimethylacetamide, N,N-dimethylformamide, morpholine, nitrobenzene, nitroethane, nitromethane, o-xylene, octafluorotoluene, octane, p-xylene, pentane, perfluoroheptane, perfluorohexane, perfluorodecalin, perfluorooctane, petroleum ether, pyridine, propionitrile, propyl acetate, tert-butanol, tert-butyl alcohol, tert-butyl methyl ether, terpineol, tetralintetrahydrofuran, tetrachloroethylene, toluene, trichloroethylene, triethyl amine, and trifluorotoluene,

In another aspect of the first, second, third, fourth and fifth embodiments, the anhydrous solvent is acetonitrile.

In another aspect of the first, second, third, fourth and fifth embodiments, W is substituted or unsubstituted C, N, O, Si, P, S, B₅ or Se.

In another aspect of the first, second, third, fourth and fifth embodiments, W is O, N, S, Si, P or C wherein C is optionally substituted with alkyl.

In another aspect of the first, second, third, fourth and fifth embodiments, W is oxygen. In another aspect of the first, second, third, fourth and fifth embodiments, X is selected from: guanine, N2-isobutyrylguanine, N2-t-butylphenoxyacetylguanine, N2-(N,N- dimethylformamidine)guanosine, adenine, N6-benzoyladenine, N4-phenoxyacetyladenine, thymine, uracil, cytosine, N4-benzoylcytosine, N4-acetylcytosine, N4-phenoxyacetylcytosine, 2,6-diaminopurine, 6-phenyllumazine, 7-(4-biphenyl)lumazine, 5-methylcytosine, 5- propynyluracil, 5-ρropynylcytosine, 5-(thiazol-2-yl)uracil, 5-(5-methyl-2-yl)uracil, 7- deazaguanine, tubercine (7-deazaadenine), 7-deaza-7-methylguanine, 7-deaza-7 iodoguanine, 7- deaza-7-bromoguanine_f 7-(propyn-l-yl)-7-deazaguanine, 7-(hex-l-ynyl) 7-deazaguanine, 7-iodo- 7-deaza-2-aminoadenine, 7-(prop- 1 -ynyl)-7-deaza-2-aminoadenine, 7-cyano-7-deaza-2- aminoadenine, 7-(prop4-ynyl)-7-deazadenine, 7 ethynyl-7-deazadenine, 7-bromo-7-deazadenine, 7-chloro-7-deazadenine, 7-methyl-7-deazadenme, 7-deaza-8-azadenhie_} 7-deaza-8-azaguanme, spermine-conjugated guanine, 5-(N-ammohexyl)carbamoyluracil, triaminoalkylamidouracil, 7- (3-aminopropyn-l-yl)-7 deazadenine, 3-aminoρropyn-l-yluxacil, 2,7-dioxopyridopyrimidine, phenoxazinopyrimidine, phenothiazinopyrimidine, tetracyclic deazadenine, thiothynine, 2- thiouracil, hypoxanthine, xanthine, pyrrolopyrimidinone, N-choloroethylcytosine, haloacetylcytosine, N4,N4~ethanocytosine, imidazolylpropylguanine, N2-imidazolylpropyl-2- aminoadenine, 5~methyl-N4~(l pyrenylmethyl)cytosine, N4~diphenylether-5-methylcytidine, and aminoethoxyphenoxazinopyrimidine-2-one. In another aspect of the first, second, third, fourth and fifth embodiments, X is selected from: adenine, guanine, thymine, cytosine, and uracil.

In another aspect of the first, second, third, fourth and fifth embodiments, Y is selected from: 4,4'-dimethoxytriρhenylmethyl (DMT), 4-monomethoxytrityl(MMT), and 9- phenylxanthen-9-yl (pixyl). In another aspect of the first, second, third, fourth and fifth embodiments, Y is

4 ,4'-dimethoxytriphenylmethyl (DMT) .

In another aspect of the first, second, third, fourth and fifth embodiments, Z is selected from: H (i.e. deoxy),-OH, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkoxy, substituted alkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy, substituted alkynyloxy, aminoalkoxy, alkoxyalkoxy, alkylaminoalkoxy, imidazolylalkoxy, alkenylthio, alkynylthio, alkenylamino, alkynylamino, aryloxy, arylthio, aralkyloxy, aralkylthio, aralkylamino, N-phthalimido, halogen (e.g. fluoro),-C(-O)-R (wherein R is an organic radical), carboxyl, nitro, nitroso, cyano, trifiuoromethyl, trifluoromethoxy, imidazolyl, azido, hydrazino, aminooxy, isocyanato, isothiocyanato, O-tert-butyldimethyl silyl, sulfoxide (-S(=O)-R), sulfone (-S(=O)2-R (wherein R is an organic radical)), disulfide (-S-S-R (wherein R is an organic radical)), silyl, O-silyl, methoxyethoxy, thioallcyl, a heterocycle, a carbocycle, a substituted or unsubstituted ether, a LNA (locked nucleic acid), an intercalator, a reporter group, conjugate, polyamine, polyamide, polyalkylene glycol, and polyethers of the formula (O-alkyl)m, where m is 1 to 10.

In another aspect of the first, second, third, fourth and fifth embodiments, Z is selected from: H (i.e. deoxy), -OH, alkyl, alkoxy, substituted alkoxy, alkoxyalkoxy, halogen (e.g. fluoro), 2'-C-α-(hydroxyalkyl), 2'-C-α-(alkyl), -trifluoromethyl, trifluoromethoxy, silyl, O-silyl, methoxyethoxy, O-tert-butyldimethylsilyl, and thioalkyl.

In another aspect of the first, second, third, fourth and fifth embodiments, Z is selected from: H, OMe, F, O-tert-butyldimethyl silyl, and methoxyethoxy. In another aspect of the first, second, third, fourth and fifth embodiments, Z is selected from: H, OMe, and F. In another aspect of the first, second, third, fourth and fifth embodiment, R¹ is selected from: substituted or unsubstituted 4-[N-Methyl-N(2,2,2-trifluoroacetyl)amino]butyl, (2- acetyoxyphenoxy)ethyl, and aryloxycarbonyl.

In another aspect of the first, second, third, fourth and fifth embodiments, R¹ is selected from: cyanoethyl, methyl, tetrazole, triazole, and benzyl.

In another aspect of the first, second, third, fourth and fifth embodiments, R¹ is selected from: cyanoethyl and Me.

In another aspect of the first, second, third, fourth and fifth embodiments, R² is NR³R⁴, wherein each of R³ and R⁴ is independently substituted or unsubstituted alkyl or alternatively R³ and R⁴ are joined to form a substituted or unsubstituted 4 to 7 membered heteroaryl or heterocycle including the nitrogen to which R³ and R⁴ are attached.

In another aspect of the first, second, third, fourth and fifth embodiments, R² is NR³R⁴, wherein each of R³ and R⁴ is independently alkyl, or wherein R³ and R⁴ are joined to form a substituted or unsubstituted triazole, tetrazole, or benzotriazole. In another aspect of the first, second, third, fourth and fifth embodiments, R² is

NR³R⁴, wherein each of R³ and R⁴ is independently alkyl, or wherein R³ and R⁴ are joined to form a triazole, tetrazole, or benzotriazole.

In another aspect of the first, second, third, fourth and fifth embodiments, R is N(J-Pr)₂. DEFINITIONS

As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, Ci-CiO, as in "Cl-Cio alkyl" is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons in a linear or branched arrangement. For example, "Cj-Cio alkyl" specifically includes methyl, ethyl, ^-propyl, /-propyl, rz-butyl, r-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and so on.

When used in the phrases "alkylaryl", "alkylcycloalkyl" and "alkylheterocyclyl" the term "alkyl" refers to the alkyl portion of the moiety and does not describe the number of atoms in, for example, the heterocyclyl portion of the moiety. In an embodiment, if the number of carbon atoms is not specified, the "alkyi" of "alkylaryl", "alkylcycloalkyl" and

"alkylheterocyclyl" refers to Cj -C 12 alkyl and in a further embodiment, refers to Ci-Cg alkyl.

In an embodiment, if the number of carbon atoms is not specified, "alkyl" refers to C1-C12 alkyl and in a further embodiment, "alkyl" refers to C1-C6 alkyl. In an embodiment, if the number of carbon atoms is not specified, "cycloalkyl" refers to C3-C10 cycloalkyl and in a further embodiment, "cycloalkyl" refers to C3-C7 cycloalkyl. In an embodiment, examples of "alkyl" include methyl, ethyl, ^-propyl, /-propyl, n-butyl, Λ-butyl and /-butyl.

The term "alkylene" means a hydrocarbon diradical group having the specified number of carbon atoms. For example, "alkylene" includes -CH2-, -CH2CH2- and the like. In an embodiment, if the number of carbon atoms is not specified, "alkylene" refers to Cl -C 12 alkylene and in a further embodiment, "alkylene" refers to Ci-Cg alkylene.

If no number of carbon atoms is specified, the term "alkenyl" refers to a non- aromatic hydrocarbon radical, straight, branched or cyclic, containing from 2 to 10 carbon atoms and at least one carbon to carbon double bond. Preferably one carbon to carbon double bond is present, and up to four non-aromatic carbon-carbon double bonds may be present. Thus, "C2-C6 alkenyl" means an alkenyl radical having from 2 to 6 carbon atoms. Alkenyl groups include ethenyl, propenyl, butenyl, 2-methylbutenyl and cyclohexenyl. The straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated.

The term "alkynyl" refers to a hydrocarbon radical straight, branched or cyclic, containing from 2 to 10 carbon atoms and at least one carbon to carbon triple bond. Up to three carbon-carbon triple bonds may be present. Thus, "C2-C0 alkynyl" means an alkynyl radical having from 2 to 6 carbon atoms. Alkynyl groups include ethynyl, propynyl, butynyl, 3- methylbutynyl and so on. The straight, branched or cyclic portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated.

In certain instances, substituents may be defined with a range of carbons that includes zero, such as (Co-C6)alkylene-aryl. If aryl is taken to be phenyl, this definition would include phenyl itself as well as -CJfePh, -CH2CH2Ph, CH(CH3)CH2CH(CH3)Ph, and so on. "Aryl" is intended to mean any stable monocyclic, bicyclic or tricyclic carbon ring of up to 7 atoms in each ring, wherein at least one ring is aromatic. Examples of such aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl. In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring. In one embodiment, "aryl" is an aromatic ring of 6 to 14 carbons atoms, and includes a carbocyclic aromatic group fused with a 5 -or 6-membered cycloalkyl group such as indan. Examples of carbocyclic aromatic groups include, but are not limited to, phenyl, naphthyl, e.g. 1-naphthyl and 2-naphthyl; anthracenyl, e.g. 1-anthracenyl, 2-anthracenyl; phenanthrenyl; fluorenonyl, e.g. 9-fluorenonyl, indanyl and the like. The term heteroaryl, as used herein, represents a stable monocyclic, bicyclic or tricyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains carbon and from 1 to 4 heteroatoms selected from the group consisting of O, N and S. In another embodiment, the term heteroaryl refers to a monocyclic, bicyclic or tricyclic aromatic ring of 5- to 14-ring atoms of carbon and from one to four heteroatoms selected from O, N, or S. As with the definition of heterocycle below, "heteroaryl" is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively. Beteroaryl groups within the scope of this definition include but are not limited to acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indoryl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline. Additional examples of heteroaryl include, but are not limited to pyridyl, e.g., 2-pyridyl (also referred to as α-pyridyl), 3-pyridyl (also referred to as β-pyridyl) and 4-pyridyl (also referred to as (γ-pyridyl); thienyl, e.g., 2-thienyl and 3-thienyl; furanyl, e.g., 2-furanyl and 3-furanyl; pyrimidyl, e.g., 2- pyrimidyl and 4-pyrimidyl; imidazolyl, e.g., 2-imidazolyl; pyranyl, e.g., 2-pyranyl and 3-pyranyl; pyrazolyl, e.g., 4-ρyrazolyl and 5-pyrazolyl; thiazolyl, e.g., 2-thiazolyl, 4-thiazolyl and 5- thiazolyl; thiadiazolyl; isothiazolyl; oxazolyl, e.g., 2-oxazoyl, 4-oxazoyl and 5-oxazoyl; isoxazoyl; pyrrolyl; pyridazinyl; pyrazinyl and the like.

In an embodiment, "heteroaryl" may also include a "fused polycyclic aromatic", which is a heteroaryl fused with one or more other heteroaryl or nonaromatic heterocyclic ring. Examples include, quinolinyl and isoquinolinyl, e.g. 2~quinolinyl, 3-quinolinyl, 4-quinolinyl, 5- quinolinyl, 6-quinolinyl, 7-quinolinyl and 8-quinolinyl, 1 -isoquinolinyl, 3-quinolinyl, 4- isoquinolinyl, 5-isoquϊnolinyl, 6-isoquinolinyl, 7-isoquinolinyl and 8-isoquinolinyl; benzofuranyl, e.g. 2-benzofuranyl and 3 -benzofuranyl; dibenzofuranyl, e.g. 2,3- dihydrobenzofuranyl; dibenzothiophenyl; benzothienyl, e.g. 2-benzothienyl and 3-benzothienyl; indolyl, e.g. 2-indolyl and 3-indolyl; benzothiazolyl, e.g., 2-benzothiazolyl; benzooxazolyl, e.g., 2-benzooxazolyl; benzimidazolyl, e.g. 2-benzoimidazolyl; isoindolyl, e.g, 1-isoindolyl and 3- isoindolyl; benzotriazolyl; purinyl; thianaphthenyl, pyrazinyland the like.

The term "heterocycle" or "heterocyclyl" as used herein is intended to mean monocyclic, spirocyclic, bicyclic or tricyclic ring of up to 7 atoms in each ring, wherein each ring is aromatic or non-aromatic and contains carbon and from 1 to 4 heteroatoms selected from the group consisting of O, N, P and S. A nonaromatic heterocycle may be fused with an aromatic aryl group such as phenyl or aromatic heterocycle.

"Heterocyclyl" or "heterocycle" therefore includes the above mentioned heteroaryls, as well as dihydro and tetrahydro analogs thereof. Further examples of "heterocyclyl" include, but are not limited to the following: azetidinyl, benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, 1 ,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrotbiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof. Attachment of a heterocyclyl substituent can occur via a carbon atom or via a heteroatom.

In an embodiment, "heterocycle" (also referred to herein as "heterocyclyl"), is a monocyclic, spirocyclic, bicyclic or tricyclic saturated or unsaturated ring of 5- to 14-ring atoms of carbon and from one to four heteroatoms selected from O, N₅ S or P. Examples of heterocyclic rings include, but are not limited to: pyrrolidinyl, piperidinyl, morpholinyl, thiamorpholinyl, piperazinyl, dihydrofuranyl, tetrahydrofuranyl, dihydropyranyl, tetrahydrodropyranyl, dihydroquinolinyl, tetrahydroquinolinyl, dihydroisoquinolinyl, tetrahydroisoquinolinyl, dihydropyrazinyl, tetrahydropyrazinyl, dihydropyridyl, tetrahydropyridyl and the like. An "alkoxy group" (alkyloxy) as used herein, is a straight chain or branched Ci-

Ci₂ or cyclic C₃-C₁₂ alkyl group that is connected to a compound via an oxygen atom. Examples of alkoxy groups include but are not limited to methoxy, ethoxy and propoxy.

An "aralkyl group" as used herein refers to an aryl-substituted alkyl group. Preferable araklyl groups are "lower aralkyl" groups having aryl groups attached to alkyl groups having one to six carbon atoms. Even more preferred are lower aralkyl radicals phenyl attached to alkyl portions having one to three carbon atoms. Examples of such radicals include benzyl, diphenylmethyl and phenylethyl. The aryl in said aralkyl may be additionally substituted with halo, alkyl, alkoxy, haloalkyl, and haloalkoxy.

An "alkenyloxy group" as used herein refers to a straight-chain or branched saturated alkenyl group, as defined above, which is attached to a compound via an oxygen atom. Examples include vinyloxy, allyloxy, methaUyloxy, and buten-4-yloxy.

An "alkynyloxy group" as used herein refers to a straight-chain or branched saturated alkynyl group, as defined above, which is attached to a compound via an oxygen atom. Examples include propargyloxy, butin-3-ylsufanyl and butin-4-ylsulfanyl. An "aminoalkoxy group" as used herein embraces alkoxy groups substituted with an amino group. More preferred aminoalkoxy groups are "lower aminoalkoxy" groups having alkoxy groups of one to six carbon atoms. Suitable aminoalkoxy groups may be aminoethoxy, aminomethoxy, aminopropoxy and the like.

An "alkoxyalkoxy group" as used herein means an alkoxy group, as defined herein, appended to the parent molecular moiety through another alkoxy group. Representative examples of alkoxyalkoxy include, but are not limited to, tert-butoxymethoxy, 2-ethoxyethoxy, 2-methoxyethoxy, and methoxymethoxy. An "alkylamino group" as used herein denotes amino groups which have been substituted with one alkyl group and with two alkyl groups, including the terms "N-alkylamino" and "N,N-dialkylamino". More preferred alkylamino groups are "lower alkylamino" groups having one or two alkyl groups of one to six carbon atoms, attached to a nitrogen atom. Even more preferred are lower alkylamino groups having one to three carbon atoms. Suitable alkylamino may be mono or dialkylamino such as N-methylamino, N-ethylamino, N₅N- dimethylamino and the like.

An "alkylaminoalkoxy group" as used herein embraces alkoxy groups substituted with alkylamino groups. More preferred alkylaminoalkoxy groups are "lower alkylaminoalkoxy" groups having alkoxy radicals of one to six carbon atoms. Even more preferred are lower alkylaminoalkoxy groups having alkyl groups of one to three carbon atoms.

An "imidazolylalkoxy group" as used herein embraces alkoxy groups, as defined above, that is substituted with an imidazole group.

An "alkenylthio group" as used herein refers to a straight-chain or branched saturated alkenyl group, as defined above, which is attached to a compound via a sulfur atom. Examples include vinylsulfanyl, allylsulfanyl, methallylsufanyl, and buten-4-ylsulfanyl.

An "alkynylthio group" as used herein refers to a straight-chain or branched saturated alkynyl group, as defined above, which is attached to a compound via a sulfur atom. Examples include propargylsulfanyl, butin-3-ylsufanyl and butin-4-ylsulfanyl. An "alkenylamino group" as used herein refers to a straight-chain or branched saturated alkenyl group, as defined above, which is attached to a compound via a nitrogen atom. Examples include vinylamino, allylamino, methallylamino, and buten-4-ylamino.

An "alkynylamino group" as used herein refers to a straight-chain or branched saturated alkynyl group, as defined above, which is attached to a compound via a nitrogen atom. Examples include propargylamino, butin-3 -amino, and butin-4-yIamino.

An "aryloxy group" as used herein embraces optionally substituted aryl group, as defined above, attached to an oxygen atom (e.g., phenoxy).

An "arylthio group" as used herein is an aryl group, as defined above, which is attached to a compound via a sulfur atom. An example of arylthio is phenylthio. An "aralkyloxy group" (arylalkyloxy) as used herein embraces aralkyl groups, as defined above, attached to an oxygen atom. More preferred are phenyl-Ct to C₃ alkyloxy groups.

An "aralkylthio group" as used herein embraces aralkyl groups as defined above, attached to a divalent sulfur atom. More preferred are phenyl-Cj to C₃-alkylthio groups. An example of aralkylthio is benzylthio. An "aralkylamino group" as used herein denotes amino groups which have been substituted with one or two aralkyl groups. More preferred are phenyl-Ci to C₃-alkylamino groups, such as N-benzylamino. The aralkylamino group may be further substituted on the aryl ring portion of the radical. A "halogen group" as used herein means halogens such as fluorine, chlorine, bromine or iodine atoms.

An "organic radical" as used herein is an unsubstiluted or substituted aliphatic, aromatic, or araliphatic radical having from 1 to 30 carbon atoms. This radical can contain one or more heteroatoms such as oxygen, nitrogen, sulfur, or phosphorus and/or be substituted by one or more functional groups containing, for example, oxygen, nitrogen, sulfur and/or halogen, for example by fluorine, chlorine, bromine, iodine and/or a cyano group. If the carbon-containing organic radical contains one or more heteroatoms, it can also be bound via a heteroatom. Thus, for example, ether, thioether and tertiary amino groups are also included. The carbon-containing organic radical can be a monovalent or polyvalent, for example divalent, radical.

An "amϊnooxy group" as used herein is an amino group that is attached to a compound via an oxygen atom.

A "carbocycle" as used herein means a stable aliphatic 3- to 15-membered monocyclic or polycyclic monovalent or divalent radical consisting solely of carbon and hydrogen atoms which may comprise one or more fused or bridged rings, preferably a 5- to 7~ membered monocyclic or 7- to 10-membered bicyclic ring. Unless otherwise specified, the carbocycle may be attached at any carbon atom which results in a stable structure and, if substitute, may be substituted at any suitable carbon atom which results in a stable structure. The term "cycloalkyl" means a monocyclic saturated or unsaturated aliphatic hydrocarbon group having the specified number of carbon atoms. The cycloalkyl is optionally bridged (i.e., forming a bicyclic moiety), for example with a methylene, ethylene or propylene bridge. The cycloalkyl may be fused with an aryl group such as phenyl, and it is understood that the cycloalkyl substituent is attached via the cycloalkyl group. For example, "cycloalkyl" includes cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, cyclopentenyl, cyclobutenyl and so on.

An "ether" as used herein means a class of organic compounds which contain an ether group (an oxygen atom connected to two alkyl or aryl groups). Polyethers are compounds with more than one ether group.

When a moiety is referred to as "unsubstituted" or not referred to as "substituted" or "optionally substituted", it means that the moiety does not have any substituents. When a moiety is referred to as substituted, it denotes that any portion of the moiety that is known to one skilled in the art as being available for substitution can be substituted. The phrase "optionally substituted with one or more substituents" means, in one embodiment, one substituent, two substituents, three substituents, four substituents or five substituents. For example, the substitutable group can be a hydrogen atom that is replaced with a group other than hydrogen (i.e., a substituent group). Multiple substituent groups can be present. When multiple substituents are present, the substituents can be the same or different and substitution can be at any of the substitutable sites. Such means for substitution are well known in the art. For purposes of exemplification, which should not be construed as limiting the scope of this invention, some examples of groups that are substituents are: alkyl, alkenyl or alkynyl groups (which can also be substituted, with one or more substituents), alkoxy groups (which can be substituted), a halogen or halo group (F, Cl, Br, I), hydroxy, nitro, oxo, -CN, -COH, -COOH, amino, azido, N-alkylamino or N,N-dialkylamino (in which the alkyl groups can also be substituted), N-arylamino or N,N~diarylamino (in which the aryl groups can also be substituted), esters (-C(O)-OR, where R can be a group such as alkyl, aryl, etc., which can be substituted), ureas (-NHC(O)-NHR, where R can be a group such as alkyl, aryl, etc., which can be substituted), carbamates (-NHC(O)-OR, where R can be a group such as alkyl, aryl, etc., which can be substituted), sulfonamides (-NHS(O)2R, where R can be a group such as alkyl, aryl, etc., which can be substituted), alkylsulfonyl (which can be substituted), aryl (which can be substituted), cycloalkyl (which can be substituted) alkylaryl (which can be substituted), alkylheterocyclyl (which can be substituted), alkylcycloalkyl (which can be substituted), and aryloxy. As used herein, "amorphous nucleosidic phosphoramidite" means nucleosidic phorphoramidite which is not crystalline material or which does not have a degree of structural order in its solid state.

As used herein, "anhydrous solvent" means any solvent system which minimizes the amount of water present in solution. Examples of anhydrous solvents include but are not limited to: 1,1,1-trichloroethane, 1,1,2,2,-tetrachloroethane, 1-butanol, 1-chlorobutane, 1-methyl- 2-pyrrolidinone, 1-nitropropane, 1-octanol, 1-pentanol, 1-propanol, 1,2-dichlorobenzene, 1,2 dichloroethane, 1,2-dichloroethene, 1 ,2-diethoxyethane, 1,2-dimethoxyethane, 1,4-dioxane, 2,2,4-trimethylρentane, 2-butanol, 2-butanone, 2-ethoxyethanoI, 2-methoxyethanol, 2-methyl-l- propanol, 2-methyl-tetrahydrofuran, 2-methylbutane, 2-nitropropane, 2-pentanone, 2-propanol, 3- methyl- 1-butanol, 3-pentanone, acetone, acetonitrile, anisole, benzene, benzonitrile, butyl acetate, butyl amine, butyl ether, butyronitrile, carbon tetrachloride, chlorobenzene, chloroform, cumene, cyclohexane, cyclohexanone, cyclopentanone, decane, decalin, dichloromethane, diethyl ether, diethylene glycol, diisopropyl amine, diisopropyl ether, diglyme, dimethylsulfoxide, dimethoxymethane, ethanol,, ethyl acetate, ethyl alcohol, ethyl formate, ethylene glycol, fluorobenzene, formamide, glycerine, glycerol, heptane, hexane, hexafluorobenzene, isobutyl acetate, isooctane, isopropyl acetate, isopropyl ether, mesitylene, m-xylene, methanol, methyl acetate, methyl formate, methyl cyclohexane, methylene chloride, methyl isopropyl ketone, N,N- dimethylacetamide, N,N-dimethylformamide, morpholine, nitrobenzene, nitroethane, nitromethane, o-xylene, octafluorotoluene, octane, p-xylene, pentane, perfluoroheptane, perfluorohexane, perfluorodecalin, perfluorooctane, petroleum ether, pyridine, propionitrile, propyl acetate, tert-butanol, tert-butyl alcohol, tert-butyl methyl ether, terpineol, tetralintetrahydrofuran, tetrachloroethylene, toluene, trichloroethylene, triethyl amine, and trifluorotoluene. As used herein, "base" means a group capable of binding, whether via Watson Crick binding, Hoogstein binding, clamp-type binding, or non-specific binding to a complementary base of an oligonucleotide. Included within the meaning of "base" is a heterocyclic base moiety. Heterocyclic bases useful in the present invention include both naturally and non-naturally occurring bases. The heterocyclic base moiety further may be protected wherein one or more functionalities of the base bears a protecting group. As used herein, "unmodified" or "natural" bases include the purine bases adenine and guanine, and the pyrimidine bases thymine, cytosine and uracil. Modified and unmodified bases include other synthetic and natural bases such as bases of the purine and pyrimidine family (protected or unprotected) or a derivative of such base. Preferred base moieties include, but are not limited to : guanine, N2-ϊsobutyrylguanine, N2-t-butylphenoxyacetylguanine_J N2-(N,N- dimethylformamidine)guanosine, adenine, N6-benzoyladenine, N4-phenoxyacetyladenine, thymine, uracil, cytosine, N4-benzoylcytosine, N4-acetylcytosine, N4-phenoxyacetylcytosine, 2,6-diaminopurine, 6-phenyllumazine, 7-(4 biphenyl)lumazine, 5-methylcytosine, 5- propynyluracil, 5-propynylcytosine, 5-(thiazol 2-yl)uracil; 5-(5-methyl-2-yl)uracil; 7 deazaguanine; tubercine (7-deazaadenine); 7-deaza-7-methylguanine; 7-deaza-7 iodoguanine; 7- deaza-7-bromoguanine; 7-(proρyn-l-yl)-7-deazaguanine; 7-(hex-l-ynyl) 7-deazaguanine; 7-iodo- 7-deaza-2-aminoadenine; 7-(prop-l-ynyl)-7-deaza-2 aminoadenine; 7-cyano-7-deaza-2- aminoadenine; 7-(prop-l-ynyl)-7-deazadenine; 7 ethynyl-7-deazadenine; 7-bromo-7- deazadenine; 7-chloro-7-deazadenine; 7-methyl-7 deazadenine; 7-deaza-8-azadenine; 7-deaza-8- azaguanine; spermine-conjugated guanine; 5-(N-aminohexyl)carbamoyluracil; triaminoalkylamidouracil; 7-(3-amϊnopropyn-l-yl)-7 deazadenine; 3-aminopropyn-l-yluracil; 2,7-dioxopyridopyrimidine; phenoxazinopyrimidϊne; phenothiazinopyrimidine; tetracyclic deazadenine; thiothynine; 2-thiouracil; hypoxanthine; xanthine; pyrrolopyrimidinone; N choloroethylcytosine; haloacetylcytosine; N4,N4-ethanocytosine; imidazolylpropylguanine; N2- imidazolylpropyl-2-aminoadem^'ne; 5-methyl-N4-(l pyrenylmethyl)cytosine; N4-diphenylether-5- methylcytidine; Aminoethoxyphenoxazinopyrimidine-2-one, Further bases 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.L, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al, Angewandte Chemie, 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., ea., CRC Press, 1993.

As used herein, "crystalline material" means any material possessing crystallinity (crystallinity is defined as the degree of structural order in a solid. In a crystal, the atoms or molecules are arranged in a regular, periodic manner).

As used herein, "crystallization" means the natural or artificial process of formation of solid crystalline material from a uniform solution or melt, or more rarely directly from a gas. It is also a chemical solid-liquid separation technique in which mass transfer of a solute from the liquid solution to a pure solid crystalline phase occurs. This can also be achieved by exposing a non-crystalline or partly crystalline solid to processing conditions which cause it to crystallize or to crystallize further. In some processes it is also a phase reaction in which mass transfer of a solute from a liquid solution to a pure solid crystalline phase occurs. It may also be a process in which a chemical reaction occurs, generating a compound in solution which may then crystallize from the solution.

As used herein, "nucleosidic phosphoramidite(s)" means a phosphoramidite with a bound nucleoside. Nucleosidic phosphoramidites are well known to those skilled in the art. Examples of nucleosidic phosphoramidites include:

dA Q-methyl-A

rG rC

ΓU fiuoro-C dT

As used herein, "nucleosidic phosphoramidite precursor" means a chemical compound, raw material, or synthetic intermediate, whether isolated or not, which when exposed to the appropriate processing conditions may undergo a chemical transformation to form a desired compound such as a desired nucleosidic phosphoramidite.

As used herein, "oligonucleotide(s)" means an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mirnetics. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Oligonucleotides can be 8 to 50 nucleobases (i.e. from about less than 8 to above 50 linked nucleotides). Oligonucleotides include antisense or siRNA comprising from about 12 to about 30 nucleobases. As used herein, "precursor" means a chemical compound, raw material, or synthetic intermediate, whether isolated or not, which when exposed to the appropriate processing conditions may undergo a chemical transformation to form a desired compound such as a desired nucleosidic phosphoramidite.

As used herein, "processing conditions" means exposure to solvents, gases, co- crystal forming reagents, or other reagents in the solid, liquid, or gaseous state, fluids, or mixtures or solutions of these, exposure to conditions of temperature, humidity, and pressure, and also include manipulation of temperature, pressure, humidity, and the mixing of gases, liquids, slurries, or powders in the dry state, and any combination of these. This also includes common unit operations, such as distillation, evaporation, use of antisolvent, filtration, centrifugation, and other procedures well known to those skilled in the art.

As used herein, "protecting group" means a chemical group that is labile under selected conditions and protects a functional group from participating in, or interfering with, a reaction.

As used herein, "siRNA" shall mean a short (15-30 mer) double stranded RNA (blunt ended or with overhangs), including micro-RNA, which can be chemically modified or unmodified, wherein the chemical modification(s) may include, but are not limited to, one or more chemical modifications to any base and/or sugar moiety (2'-OH) and/or chemical modifications to the phosphate backbone, which can be used to silence genes expression in a cell. As used herein, "solvent" means a liquid, gas, or any combination thereof, that dissolves a solid, liquid, or gaseous solute, resulting in a solution. A solution is a mixture composed of two or more substances. In such a mixture a solute is dissolved (whole or in part) in another substance, known as a solvent. Examples of anhydrous solvents include but are not limited to solvents deemed or determined to be anhydrous of the type: 1,1,1-trichloroethane, 1,1,2,2,-tetrachloroethane, 1-butanol, 1-chlorobutane, 1 -methyl-2-pyrrolidinone, 1-nitropropane, 1-octanol, 1-pentanol, 1-ρropanol, 1 ,2-dichlorobenzene, 1,2 dichloroethane, 1 ,2-dichloroethene, 1 ,2-diethoxyethane, 1 ,2-dimethoxyethane, 1,4-dioxane, 2,2,4-trirnethylρentane, 2-butanol, 2~ butanone, 2-ethoxyethanol, 2-methoxyethanol₅ 2-methyM-propanol, 2-methyl-tetrahydrofuran, 2-methylbutane, 2-nitropropane, 2-pentanone, 2-propanol, 3-methyl- 1-butanol, 3~pentanone, acetone, acetonitrile, anisole, benzene, benzonitrile, butyl acetate, butyl amine, butyl ether, butyronitrile, carbon tetrachloride, chlorobenzene, chloroform, cumene, cyclohexane, cyclohexanone, cyclopentanone, decane, decalin, dichloromethane, diethyl ether, diethylene glycol diisobutyl ethyl amine, diisopropyl amine, diisopropyl ether, diglyme, dimethylsulfoxide, dimethoxymethane, ethanol, ethyl acetate, ethyl alcohol, ethyl formate, ethylene glycol, fluorobenzene, formamide, glycerine, glycerol, heptane, hexane, hexafluorobenzene, isobutyl acetate, isooctane, isopropyl acetate, isopropyl ether, mesitylene, m-xylene, methanol, methyl acetate, methyl formate, methyl cyclohexane, methylene chloride, methyl isopropyl ether, methyl isopropyl ketone, N,N-dimethylacetamide, N,N-dimethylformamide, morpholiπe, nitrobenzene, nitroethane, nitromethane, o-xylene, octafluorotoluene, octane, p-xylene, pentane, perfluoroheptane, perfluorohexane, perfluorodecalin, perfiuorooctane, petroleum ether, pyridine, propionitrile, propyl acetate, tert-butanol, tert-butyl alcohol, tert-butyl methyl ether, terpineol, tetralintetrahydrofuran, tetrachloroethylene, toluene, trichloroethylene, triethyl amine, trifluorotoluene and water. As used herein, "water-free environment" means an environment designed to negate the presence of water within itself. Examples of water-free environments include glove and dry boxes, dessication chamber, flow cell, sealed ampoule, inerted flask, and other techniques, apparatuses, and conditions known to those skilled in the art.

As used herein, ^M2'-chemical modification" means any chemical modification to the 2' position of a ribose (designated "Z" in Formula A). Examples of chemical modifications are disclosed in US 2006/0240554 and US 2008/0020058. Additional examples of chemical modifications are found in Reese, Colin B.; Current Protocols in Nucleic Acid Chemistry (2000) 2.2.1-2.2.24; Piccirilli, J. A.; Li, N. J.; Org. Chem. 2007, 72, 1198-1210; Oretskaya, T. S.; et. al.; Russian Chemical Reviews 2004, 73, 701-733. UTILITY

The present invention provides a purification process which produces crystalline material containing the nucleosidic phosphoramidites that are useful in the synthesis of oligonucleotides. Such oligonucleotides include antisense and siRNA, which are useful for therapeutic purposes. The synthesis of oligonucleotides, in particular antisense and siRNA, is well known in the art (Beaucage et al., 2008 Curr. Opin. Drug Discov. DeveL, 1 1:203-16; U.S. Pat. No. 6,989,442). The utility of antisense and siRNA for therapeutic purposes is well known in the art (Karagiannis, T. and El-Osta, A., 2004 Cancer Biol. Ther., 3:1069-74; Karagiannis, T. and El-Osta, A., 2005 Cancer Gene Ther,, 12:787-95; Dallas, A. and Vlassov A., 2006 Med. Sci. Monit., 12:67-74; Spurgers et al., 2008 Antiviral Research, 78:26-36; Fuchs et al., 2004 Curr. MoI. Med., 4:507-17; Eckstein, F., 2007 Expert Opin. Biol. Ther., 7:1021-34).

GENERAL SYNTHESIS OF NUCLEOSIDIC PHOSPHORAMIPITES

The general synthesis of nucleosidic phosphoramidites is well known in the art. Nucleosidic Phosphoramidites may be purchased from commercial sources such as Thermo Fisher Scientific. Further, nucleosidic phosphoramidites may be prepared as described in U.S. Pat. No. 6,426,220 and PCT WO 02/36743.

GENERAL OLIGONUCLEOTIDE SYNTHESIS Oligonucleotides, which may include novel crystalline nucleosidic phosphoramidites of the instant invention (made by the purification process of the instant invention), may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis on scales from small to large is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.), GE Healthcare (US and UK). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides on all scales. Further, synthesis of oligonucleotides is described in the following references (Ohkubo et al., 2006 Curr. Protoc, Nucleic Acid Chem., Chapter 3:Unit 3.15; PCT WO 1996/040708; U.S. Pat Nos. 4,458,066 and 4,973,679; Beaucage et al., 1992 Tetrahedron Lett. 22:1859-69; U.S. Pat. No. 4,415,732). GENERAL RNA SYNTHESIS

Methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J Am. Chem. Soc, 1998, 120, 11820-11821 ; Matteucci, M. D. and Carathers, M. H. J Am. Chem. Soc, 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers. M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett, 1994, 25, 4311- 4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffm, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).

Once synthesized, complementary RNA oligonucleotides can then be annealed by methods known in the art to form double stranded (duplexed) oligonucleotide compounds. For example, duplexes can be formed by combining 30 μl of each of the complementary strands of RNA oligonucleotides (50 μM RNA oligonucleotide solution) and 15 μl of 5X annealing buffer (100 mM potassium acetate, 30 raM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90° C, then 1 hour at 37° C. The resulting duplexed RNA oligonucleotides can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid, or for diagnostic or therapeutic purposes.

GENERAL siRNA SYNTHESIS

RNA oligonucleotides, which may be derived from novel crystalline nucleosidic phosphoramidites of the instant invention (made by the purification processes of the instant invention) can be synthesized in a stepwise fashion comprising at least one nucleosidic phosphoramidate linkage derived from nucleosidic phosphoramidites. Each nucleotide can be added sequentially (3'- to 5 '-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3 '-end of the chain can be covalently attached to a solid support. The 5'-O- dimethoxy trityl group of the nucleoside bound to the solid support is removed by treatment with an acid such dicloroacetic acid. The nucleotide precursor, a nucleosidic phosphoramidite, and activator can be added, coupling the second base onto the 5 '-end of the first nucleoside. The linkage may be then oxidized to the more stable and ultimately desired P(V) linkage. The support is washed and any unreacted 5'-hydroxyl groups can be capped with acetic anhydride to yield 5'- acetyl moieties. The cycle can be repeated for each subsequent nucleotide. This cycle is repeated until the desired oligonulcoetide sequence has been completed.

Following synthesis, the support bound oligonucleotide can be treated with a base such a diethylamine to remove the cyanoethyl protecting groups of the phosphate backbone. The support may then be treated with a base such as aqueous methylamine. This releases the oligonucleotides into solution, deprotects the exocyclic amines. Any 2' silyl protecting groups can be removed by treatment with fluoride ion. The oligonucleotide can be analyzed by anion exchange HPLC at this stage.

The oligonucleotides synthesized by this method can be purified by HPLC. Once purified complementary RNA oligonucleotides can then be annealed by methods known in the art to form double stranded (duplexed) oligonucleotide compounds.

SPECIFIC siRNA SYNTHESIS Solid Phase Synthesis

The single-strand oligonucleotides are synthesized using phosphoramidite chemistry on an automated solid-phase synthesizer. An adjustable synthesis column is packed with solid support derivatized with the first nucleoside residue. Synthesis is initiated by detritylation of the acid labile 5'-O-dimethoxytrϊtyl group to release the 5'-hydroxyl. Phosphoramidite and a suitable activator (in acetonitrile) are delivered simultaneously to the synthesis column resulting in coupling of the amidite to the 5'-hydroxyl (the column is then washed with acetonitrile). Oxidizers such as I₂ are pumped through the column to oxidize the phosphite triester linkage P(III) to its phosphotriester P(V) analog. Alternately, sulfurizing reagent (in acetonitrile) replaces the iodine solution when a phosphorothioate triester linkage is required by the sequence. Unreacted 5'-hydroxyl groups are capped using reagents such as acetic anhydride in the presence of 2,6-lutidine and N-methylimidazole. The elongation cycle resumes with the detritylation step for the next phosphoramidite incorporation. This process is repeated until the desired sequence has been synthesized. The synthesis concludes with the removal of the terminal dimethoxytrityl group.

Cleavage and Deprotection

On completion of the synthesis, the solid support and associated oligonucleotide is filtered, dried under vacuum and transferred to a reaction vessel. Aqueous base is added and the mixture is heated to effect cleavage of the succinyl linkage, removal of the cyanoethyl phosphate protecting group and the exocyclic amine protecting groups. The mixture is filtered under vacuum to remove the solid support. The solid support is rinsed with DMSO which is combined with the filtrate. The mixture is cooled, fluoride reagent such as triethylamine trihydrofluoride is added and the solution is heated. The reaction is quenched with suitable buffer to provide a solution of crude single strand product.

Anion Exchange Purification

The oligonucleotide strand is purified using chromatographic purification. The product is eluted using a suitable buffer gradient. Fractions are collected in closed sanitized containers, analyzed by HPLC and the appropriate fractions are combined to provide a pool of product which is analyzed for purity (HPLC), identity (HPLC and LCMS) and concentration (UV A₂₆₀).

Annealing

Based on the analysis of the pools of product, equal molar amounts (calculated using the theoretical extinction coefficient) of the sense and antisense oligonucleotide strands are transferred to a reaction vessel. The solution is mixed and analyzed for purity of duplex by chromatographic methods. If the analysis indicates an excess of either strand, then additional non-excess strand is titrated until duplexing is complete. When analysis indicates that the target product purity has been achieved, the material is transferred to the Tangential Flow Filtration (TFF) system for concentration and desalting.

Ultrafiltration

The annealed product solution is concentrated using a TFF system containing an appropriate molecular weight cut-off membrane. Following concentration, the product solution is desalted via diafiltration using WFI quality water until the conductivity of the filtrate is that of water.

Lvophilization

The concentrated solution is transferred to sanitized trays or containers in a shelf lyophilizer. The product is then freeze-dried to a powder. The trays are removed from the lyophilizer.

FORMULATIONS Oligonucleotides, which may include novel crystalline material incorporated with nucleosidic phosphoramidate linkages derived from nucleosidic phosphoramidites of the instant invention and made by a purification process of the instant invention, may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Formulations for oligonucleotides are well known in the art (U.S. Pat. Nos. 6,559,129, 6,042,846, 5,855,911, 5,976,567, 6,815,432, and 6,858,225 and US 2006/0240554, US 2008/0020058 and PCT/US08/002006).

Pharmaceutical compositions of oligonucleotides may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including but not limited to ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer (intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Sites of administration are known to those skilled in the art.

One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

DOSING The formulation of therapeutic compositions and their subsequent administration

(dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on ECsoS found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, from 0.1 μg to 10 g per kg of body weight, from 1.0 μg to 1 g per kg of body weight, from 10.0 μg to 100 mg per kg of body weight, from 100 μg to 10 mg per kg of body weight, or from 1 mg to 5 mg per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

INDICATIONS It is well known in the art that oligonucleotides (including antisense and siRNA) are useful therapeutically in mammals, in particular humans (Karagiannis, T. and El-Osta, A., 2004 Cancer Biol. Ther,, 3:1069-74; Karagiannis, T. and El-Osta, A., 2005 Cancer Gene Ther., 12:787-95; Dallas, A. and Vlassov A., 2006 Med. Sci. Monit., 12:67-74; Spurgers et al., 2008 Antiviral Research, 78:26-36; Fuchs et al., 2004 Curr. MoI. Med, 4:507-17; Eckstein, F., 2007 Expert Opin. Biol Ther. , 7:1021-34). While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. Each of the references, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.

EXAMPLES

PURIFICATION OF COMPOUND A General Considerations. Standard glovebox techniques were used unless stated otherwise. 2'O- MeU β-cyanoethyl phosphoramidite was purchased from commercial sources (Thermo Fisher Scientific Lot IE 102, Bulk Lot HG0075, lOOg container) in an argon filled sealed container and was used as received. Anhydrous acetonitrile (99.8%, CAS 75-05-8, 1 L bottle) was purchased from commercial sources (Sigma- Aldrich) and was sealed with a Sure- Seal cap. Experimental. 2'O-MeU β-cyanoethyl phosphoramidite is taken into the glovebox along with the bottle of acetonitrile. A 530.75 mg/mL solution of the 2'0-MeU β-cyanoethyl phosphoramidite in acetonitrile is made by adding 2123 mg 2'0-MeU β-cyanoethyl phosphoramidite to a vial containing 4.00 mL acetonitrile solution. The vial is swirled until all material dissolves and it is capped. The vial is then cooled to -42 ⁰C for 18 hours. The vial is seeded as soon as low temperature is reached. If no solid has formed, the vial is again seeded with crystalline material. Results'. 2'0-MeU β-cyanoethyl phosphoramidite acetonitrile solvate forms a crystalline solid in the vial. The crystalline solid is isolated via vacuum filtration and is gently heated in a vacuum oven (25 in. Hg ) at 40 ⁰C for 5 minutes in order to remove residual solvent.

ANALYTICAL DATA FOR COMPOUND A A. X-ray Powder Diffraction:

The X-ray powder diffraction (XRPD) pattern for the solid phase of 2'0-MeU β-cyanoethyl phosphoramidite acetonitrile solvate were generated on a Philips Analytical X'Pert PRO X-ray Diffraction System with PW3040/60 console. The diffraction peak positions were referenced by silicon which has a 2 theta value of 28.443 degree. A PW3373/00 ceramic Cu LEF X-ray tube K- alpha radiation was used as the source. The experiments were run at ambient condition unless noted otherwise.

Figure 1 is the X-ray powder diffraction (XRPD) pattern for the 2'0-MeU β-cyanoethyl phosphoramidite acetonitrile solvate with selected d-spacings listed in Table 1.

Figure 1 : XRPD 2'0-MeU β-cyanoethyl phosphoramidite acetonitrile solvate

Table 1 : XRPD 2'0-MeU β-cyanoethyl phosphoramidite acetonitrile solvate

2Θ°(2 thetaYdeεrees) d-spacing (A)

5.7 15.5

8.2 10.7

9.5 9.3

12.9 6.9

15.7 5.7

16.5 5.4

18.8 4.7

21.0 4.2

21.8 4.1

23.6 3.8

35.0 2.6

B. High Performance Liquid Chromatography:

Chromatographic Method:

Instrument: Agilent 1100 with UV detector

Column: Agilent Zorbax SB-C 18, 100mm x 4.6mm, 1.8μm

Column Temperature: 22⁰C

Sample Tray Temperature: 5⁰C

Sample Concentration: 0.65 mg/mL

Flow Rate: 1.2 mL/min.

UV Detection of Sample: 260 nm Injection Volume: 6 μL

Run Time: lO min.

Equilibration Time: 5 min.

Mobile Phase: A - IOOmM TEAA

B - ACN

Mobile Phase Program: Time (min) %B

0.0 60

4.0 74

7.0 80

10.0 80

Analysis Results:

Figure 2: 2'0-MeU β-cyanoethyl phosphoramidite starting material; Thermo Fischer Scientific

Figure 3: 2'0-MeU β-cyanoethyl phosphoramidite starting material; Thermo Fischer Scientific

Table 2: 2O-MeU β-cyanoethyl phosphoramidite starting material retention time and area percent

Component Retention Time (min.) Area Percent

4.271 0.04 4.402 0.14 4.534 0.02 4.767 0.04 5.175 0.01 6.161 0.14

6.273 0.12

2'-OMeTJ Diastereomer 6.443 56.82

6.681 0.03

2'-O-MeU Diastereomer 6.978 42.29

7.449 0.03 7.609 0.01 8.168 0.31

Total 2'0-MeU β-cyanoethyl phosphoramidite purity = 99.11%

Figure 4: 2'0-MeU β-cyanoethyl phosphoramidite

Figure 5: 2'0-MeU β-cyanoethyl phosphoramidite

Table 3: 2'0-MeU β-cyanoethyl phosphoramidite acetonitrile solvate retention time and area percent

Component Retention Time (min.) Area Percent

2.250 0.01 4.271 0.02

Conclusion:

The crystalline material produced from the Thermo Scientific 2'0-MeU β-cyanoethyl phosphoramidite starting material was confirmed to be 2'0-MeU β-cyanoethyl phosphoramidite based on a retention time match with the starting material.

The crystallization procedure developed for 2'0-MeU β-cyanoethyl phosphoramidite acetonitrile solvate produced an upgrade in 2'0-MeU β-cyanoethyl phosphoramidite purity. Specifically, the purity was increased by 0.64 area % by crystallizing the 99.11% pure 2'0-MeU β-cyanoethyl phosphoramidite starting material (Table 2) from Thermo Scientific and obtaining the 99.75% pure 2'0-MeU β-cyanoethyl phosphoramidite acetonitrile solvate crystalline material (Table 3).

C. Proton Solution Nuclear Magnetic Resonance:

The ¹H spectrum was recorded in CD₃CN on a Bruker DRX 600 at a frequency of 600.13 MHz using a Bruker 5mm broadband inverse probe with z-gradient. The chemical shifts are reported in ppm relative to residual CHD₂CN for proton (δ=l .94).

2'0-MeU β-cyanoethyl phosphoramidite acetonitrile solvate: ¹H NMR (600.13 MHz, CD₃CN; compound has a 7/3 ratio of diastereomers) 5 9.03 (br s, IH), 7.82 (d, J=8.2 Hz, .7H), 7.72 (d, J=8.2 Hz, .3H), 7.47-7.42 (m, 2H)₅ 7.36-7.30 (m, 6H), 7.28-7.24 (m, IH), 6.90-6.86 (m, 4H), 5.87 (d, J=3.4 Hz, .3H), 5.85 (d, J=3.0 Hz, .7H), 5.24 (d, J=8.2 Hz, .3H), 5.22 (d, J=8.2 Hz, .7H), 4.53-4.47 (m, .7H), 4.45-4.41 (m, .3H), 4.16-4.12 (m, IH)₅ 3.95 (dd, J=4.9, 3.0 Hz, .7H), 3.93 (dd, J-4.9, 3.4 Hz, .3H), 3.89-3.83 (m, .3H), 3.80-3.70 (m, IH), 3.78 (s, 4.2H), 3.77 (s, 1.8H), 3.67-3.56 (m, 2.7H), 3.50 (s, .9H), 3.48 (s, 2.1H), 3.46-3.35 (m, 2H), 2.71-2.63 (m, .6H), 2.56- 2.48 (m, 1.4H), 1.96 (s, 3H-CH₃CN), 1.18 (d, J=6.8, 4.2H), 1.16 (d, J=6.8, 4.2H), 1.15 (d, J=6.8, 1.8H), 1.05 (d, J=6.8, 1.8H). Conclusion:

This method determined that there is 1.1 equivalents acetonitrile (singlet at 1.96 ppm), in the

2'0-MeU β-cyanoethyl phosphoramidite containing crystalline material.

D. Carbon- 13 Solid-State Nuclear Magnetic Resonance:

The 2'0-MeU β-cyanoethyl phosphoramidite acetonitrile solvate was characterized by carbon- 13 solid-state nuclear magnetic resonance (ssNMR). The carbon- 13 NMR spectrum was collected of a dry powdered sample on a Bruker AV500 NMR spectrometer using a Bruker 4 mm triple resonance CPMAS probe. The spectrum was collected utilizing proton/carbon- 13 variable- amplitude cross-polarization (VACP) with a contact time of 5 ms, a pulse delay of 2 s, and a magic-angle spinning (MAS) rate of 12 kHz. Line broadening of 50 Hz was applied to the spectrum before Fourier Transformation. Chemical shifts are reported on the TMS scale using the carbonyl carbon of glycine (176.7 ppm) as a secondary reference. The spectrum of the 2'O- MeU β-cyanoethyl phosphoramidite acetonitrile solvate and a list with the ten most intense peaks in the spectrum, as well as their relative intensities are displayed in Figure 6 and Table 4.

250 200 150 50 - 50 ppnr δ(Carbon-13)

Figure 6: Carbon- 13 VACP MAS spectrum of the 2'0-MeU β-cyanoethyl phosphoramidite acetonitrile solvate. Table 4: 2'0-MeU β-cyanoethyl phosphoramidite acetonitrile solvate chemical shift and relative intensity

Chemical Shift [ppmj Relative Intensity [%]

55.6 100.0

159.9 80.5

131.2 79.3

86.7 49.8

43.2 49.3

118.2 45.3

115.6 44.8

22.0 44.4

140.6 41.8

79.7 41.0

The carbon-13 ssNMR spectrum of the 2'0-MeU β-cyanoethyl phosphoramidite acetonitrile solvate exhibits two peaks (1.8 ppm and 118.2 ppm) at positions very close to the respective positions of the carbon-13 NMR signals of acetonitrile in solution (1.3 ppm and 118.2 ppm relative to TMS) as shown in Figure 7. This observation shows that acetonitrile is present in the crystal lattice of the 2¹O-MeU β-cyanoethyl phosphoramidite acetonitrile solvate. The signal at 1.8 ppm, appearing in a spectral region where typically no carbon-13 signals are observed, is very indicative for the acetonitrile solvate.

Figure 7: Expansion of the carbon-13 VACP MAS spectrum of the 2'0-MeU β-cyanoethyl phosphoramidite acetonitrile solvate. The arrows indicate the carbon-13 solution NMR chemical shifts of acetonitrile (ppm relative to TMS).

The similar peak intensities of the acetonitrile and 2'0-MeU β-cyanoethyl phosphoramidite peaks indicate that both 2'0-MeU β-cyanoethyl phosphoramidite and acetonitrile molecules are present in the crystal in comparable numbers.

FURTHER ANALYTICAL DATA FOR COMPOUND A Smgie-Crystaϊ X-ray Crystallography

The structure of Compound A, C₄₂Hs₂N₅θ₉P, was determined by single-crystal X-ray crystallography. The crystal selected was representative of a bulk sample. Crystal data at 100 K:

The crystal selected was representative of a bulk sample. Crystal data at 100 K: a = 9Λ6 λ α = 90.00 ° F= 2066.20(5) A³ b - 14.68 A β = 93.17 ° Space group = Pl₁ , U c = 15.40 A γ = 90.00 ° Z= 2

Data were collected on a Oxford Diffraction CCD diffractometer using molybdenum Ka radiation and integrated to a resolution of 0.69 A^"' which yielded 10121 unique reflections from 27932 measured reflections.

The structure was solved using direct methods. The refined model has all non-H atoms refined anisotropically, and H atoms at their calculated positions, with agreement statistics of: Ri = 5.9%, for 515 variables and 8843 reflections and wR₂ = 16.2% using all 10121 reflections. The stereogenic P atom is observed to occupy two positions (labeled as Pl and P2) with occupancies of approximately 3: 1 for the major: minor positions. These two positions correspond to an inversion of the stereochemistry at the P atom thereby resulting in an approximately 3:1 mixture of diastereomers of the compound.

The structure model is shown below

and a perspective view calculated from the crystallographic coordinates is presented in Figure 8. Table 5 contains additional experimental details while crystallographic atom parameters are given in Table 6 along with derived geometric parameters in Tables 7 and 8.

Table 5. Table of Experimental Details

Formula C₄₂H₅₂N₅O₉P

Fw 801.864

Crystal colour colorless

Crystal dimen. (mm) 0.11 x 0.20 x 0.38

Lattice symmetry monoclinic

Space group Pl₁

Z 2

Dcalc (Mg πr³) 1.289

Radiation (K{) Mo

Wavelength (A) 0.7107

Temperature (K) 100 μ (mnr') 0.127

Diffractometer Oxford Diffraction Gemini Ruby CCD

Reflections measured 27932

Resolution (A) 0.69

Unique reflections 10121

Rint 0.030

Absorp. corr. multi-scan

Trans, (max, min) 0.9860, 0.9190

Reflections used 10121

Refl. obsd. criterion > 2σ(7)

Variables 515

Refined on p.

R 0.059

R_w 0.167

S 1.02 residual peak (eA^~3) 1.46(8)

Computer programs:

Solution SHELXS-97

Refinement SHELXL-97 Table 6. Atomic Parameters for Compound A Fractional atomic coordinates and equivalent isotropic displacement parameters (A²)

x y Z Ueq

Cl 1.0263(3) 0.31799(16) 1.09920(15) 0.0130(4)

C2 0.8812(3) 0.26849(16) 1.08154(15) 0.0132(4)

C3 0.9245(3) 0.19573(16) 1.01649(15) 0.0123(4)

C4 1.0756(3) 0.16826(16) 1.05710(15) 0.0122(4)

C5 0.5236(4) 0.4662(2) 1.0612(2) 0.0346(7)

C6 0.6022(4) 0.5400(3) 1.0096(3) 0.0451(10)

C7 0.3745(4) 0.5008(3) 1.0848(3) 0.0384(8)

C8 0.4386(4) 0.3815(3) 0.9232(2) 0.0386(8)

C9 0.5433(4) 0.3672(4) 0.8500(2) 0.0496(11)

ClO 0.3114(3) 0.3138(2) 0.91465(18) 0.0273(6)

CI l 0.4697(3) 0.2328(2) 1.1784(2) 0.0328(7)

C12 0.5201(4) 0.1612(3) 1.2406(2) 0.0397(9)

C13 0.6380(3) 0.1039(2) 1.21016(19) 0.0260(6)

C14 0.9549(4) 0.17469(19) 0.86457(17) 0.0232(6)

C15 1.0448(3) 0.00595(17) 1.07858(16) 0.0150(4)

C16 1.0903(3) -0.06610(19) 1.22300(16) 0.0192(5)

C17 1.1057(3) 0.02473(19) 1.25855(16) 0.0197(5)

C18 1.0959(3) 0.09783(18) 1.20647(15) 0.0153(5)

C19 1.0437(3) 0.37531(18) 1.17991(15) 0.0176(5)

C20 1.0039(3) 0.37301(17) 1.33481(14) 0.0133(4)

C21 0.9341(3) 0.46782(17) 1.32979(15) 0.0146(4)

C22 0.9861(3) 0.54302(18) 1.37766(16) 0.0167(5)

C23 0.9157(3) 0.62783(18) 1.37189(17) 0.0180(5)

C24 0.7911(3) 0.63750(19) 1.31707(16) 0.0174(5)

C25 0.7360(3) 0.5626(2) 1.26997(17) 0.0195(5)

C26 0.8047(3) 0.47911(18) 1.27718(15) 0.0162(5)

C27 0.7746(4) 0.7976(2) 1.3457(2) 0.0298(6)

C28 0.9173(3) 0.31493(18) 1.39706(15) 0.0151(4)

C29 0.8969(3) 0.34722(18) 1.48066(16) 0.0171(5)

C30 0.8162(3) 0.2981(2) 1.53964(15) 0.0191(5)

C31 0.7565(3) 0.21494(18) 1.51396(16) 0.0171(5)

C32 0.7789(3) 0.17993(19) 1.43202(17) 0.0193(5)

C33 0.8584(3) 0.23028(19) 1.37340(16) 0.0187(5)

C34 0,6479(4) 0.1974(2) 1.65033(18) 0.0303(7) C35 1.1696(3) 0.37117(19) 1.36126(16) 0.0171(5)

C36 1.2642(3) 0.43979(19) 1.33518(19) 0.0230(5)

C37 1.4150(3) 0.4321(2) 1.3516(2) 0.0320(7)

C38 1.4751(3) 0.3555(2) 1.3946(2) 0.0315(7)

C39 1.3820(3) 0.2876(2) 1.4207(2) 0.0294(6)

C40 1.2313(3) 0.2953(2) 1.40355(17) 0.0209(5)

Nl 0.5103(2) 0.38082(16) 1.01026(14) 0.0160(4)

N2 0.7333(3) 0.0631(2) 1.1851(2) 0.0397(7)

N3 1.0691(2) 0.08945(14) 1.11819(13) 0.0126(4)

N4 1.0601(2) -0.06769(15) 1.13299(14) 0.0163(4)

Ol 1.13136(18) 0.24374(12) 1.10458(11) 0.0151(3)

02 0.9852(2) 0.32780(12) 1.25124(11) 0.0156(3)

03 0.7708(2) 0.32968(14) 1.04939(13) 0.0233(4)

04 0.94047(19) 0.23877(12) 0.93490(10) 0.0150(3)

05 0.5839(2) 0.2839(2) 1.14618(16) 0.0373(6)

06 1.0154(2) -0.00051(13) 1.00069(12) 0.0207(4)

07 1.1019(3) -0.13722(15) 1.26397(13) 0.0289(5)

08 0.7156(2) 0.71729(14) 1.30421(14) 0.0254(4)

09 0.6742(2) 0.16176(15) 1.56646(12) 0.0237(4)

Pl 0.60640(8) 0.28885(6) 1.04159(5) 0.0129(2)

P2 0.6202(3) 0.34995(19) 1.09047(17) 0.0158(8)

C41 0.5675(10) 0.5056(6) 0.5705(5) 0.103(3)

C42 0.6442(8) 0.5480(6) 0.5021(5) 0.100(2)

N5 0.5078(11) 0.4691(7) 0.6254(5) 0.145(3)

Hl 1.0461 0.3567 1.0477 0.016

H2 0.8504 0.2388 1.1361 0.016

H3 0.8542 0.1435 1.0126 0.015

H4 1.1423 0.1532 1.0099 0.015

H5 0.5834 0.4536 1.1162 0.042

H6A 0.6980 0.5171 0.9941 0.068

H6B 0.6154 0.5949 1.0454 0.068

H6C 0.5431 0.5547 0.9564 0.068

H7A 0,3251 0.4538 1.1176 0.058

H7B 0.3154 0.5151 1.0315 0.058

H7C 0.3867 0.5559 1.1205 0.058

H8 0.3952 0.4435 0.9144 0.046

H9A 0.6235 0.4113 0.8563 0.074

H9B 0.4902 0.3760 0.7936 0.074 H9C 0.5831 0.3053 0.8534 0.074

HlOA 0.2452 0.3243 0.9615 0.041

HlOB 0.3496 0.2515 0.9187 0.041

HlOC 0.2580 0.3223 0.8583 0.041

HI lA 0.4135 0.2039 1.1290 0.039

HUB 0.4028 0.2744 1.2076 0.039

Hl 2A 0.5544 0.1906 1.2959 0.048

H12B 0.4360 0.1219 1.2529 0.048

H14A 0.9661 0.2080 0.8102 0.035

H14B 0.8673 0.1363 0.8588 0.035

H14C 1.0410 0.1363 0.8770 0.035

H17 1.1228 0.0327 1.3195 0.024

H18 1.1077 0.1568 1.2312 0.018

H19A 1.1485 0.3886 1.1932 0.021

Hl 9B 0.9916 0.4339 1.1705 0.021

H22 1.0714 0.5366 1.4151 0.020

H23 0.9529 0.6781 1.4051 0.022

H25 0.6505 0.5692 1.2327 0.023

H26 0.7640 0.4284 1.2461 0.019

H27A 0.7103 0.8495 1.3316 0.045

H27B 0.8721 0.8098 1.3252 0.045

H27C 0.7818 0.7885 1.4089 0.045

H29 0.9387 0.4040 1.4981 0.021

H30 0.8027 0.3214 1.5962 0.023

H32 0.7403 0.1219 1.4157 0.023

H33 0.8722 0.2066 1.3170 0.022

H34A 0.5890 0.1540 1.6817 0.045

H34B 0.5953 0.2553 1.6439 0.045

H34C 0.7415 0.2073 1.6830 0.045

H36 1.2252 0.4921 1.3060 0.028

H37 Ϊ .4776 0.4792 1.3336 0.038

H38 1.5778 0.3503 1.4056 0.038

H39 1.4211 0.2355 1.4503 0.035

H40 1.1692 0.2476 1.4211 0.025

H4A 1.0498 -0.1217 1.1087 0.020

H42A 0.7182 0.5899 0.5275 0.150

H42B 0.5747 0.5819 0.4638 0.150

H42C 0.6920 0.5010 0.4685 0.150

Table 8. Selected Interatomic Angles (deg.)

Claims

WHAT IS CLAIMED IS:

1. An isolated acetonitrile solvate of 2'-OMeU β-cyanoethyl phosphoramidite (Compound A) and any tautomers or stereoisomers thereof:

^ Compound A

2. The acetonitrile solvate of Claim 1 which exhibits one or more of: (i) the X-ray powder diffractogram shown in Figure 1 , as measured using CuKa radiation; (ii) reflections in the X-ray powder diffractogram as measured using CuKa radiation at the 2-theta angles: 8.2, 12.9, 15.7, 16.5, and 18.8; (iii) the solution ¹H NMR data indicating the presence of0 1.1 equivalent of acetonitrile in the sample; and (iv) chemical shifts in the 13-carbon solid-state NMR at: 55.6, 159.9, and 131.2 ppm.

3. The acetonitrile solvate of Claim 2 which exhibits reflections in the X-ray powder diffractogram as measured using CuKa radiation at the 2-theta angles: 8.2, 12.9, 15.7,5 16.5, and 18.8.

4. The acetonitrile solvate of Claim 2 which exhibits reflections in the X-ray powder diffractogram as measured using CuKa radiation at the 2-theta angles: 5.7, 9.5, 21.0, 21.8, 23.6, and 35.0. 0

5. The acetonitrile solvate of Claim 2 which exhibits reflections in the X-ray powder diffractogram as measured using CuKa radiation at the 2-theta angles: 5.7, 8.2, 9.5, 12.9, 15.7, 16.5, 18.8, 21.0, 21.8, 23.6, and 35.0. 5 6. The acetonitrile solvate of Claim 2 which exhibits reflections in the X-ray powder diffractogram as measured using CuKa radiation at the 2-theta angles: 5.7, 8.2, 9.5, 12.9, 15.7, 16.5, 18.8, 21.0, 21.8, 23.

6 and 35.0.

7. The acetonitrile solvate of Claim 2 which exhibits reflections in the X-ray powder cliffractogram as measured using CuKa radiation at the 2-theta angles: 5.7, 8.2, and 16.5.

8. The acetonitrile solvate of Claim 2 which exhibits chemical shifts in the 13-carbon solid-state NMR at: 55.6, 159.9, and 131.2 ppm.

9. An isolated acetonitrile solvate of 2'-0MeU β-cyanoethyl phosphoramidite (Compound A) with unit cell parameters of: a = 9.15603(14) k', b = 14.6794(2) A; c = 15.3965(2) A; α - 90.00 °; β = 93.1697(14) °; γ = 90.00 °; and Space group = P2_h

10. An isolated acetonitrile solvate of 2'-OMeU β-cyanoethyl phosphoramidite (Compound A) with unit cell parameters of: a = 9.16 A; b = 14.68 A; c = 15.40 A; α = 90.00 °; β = 93.17 °; γ = 90.00 °; and Space group = P2_h