# US 8,541,569 B2 · Phosphoramidites for synthetic RNA in the reverse direction, efficient RNA synthesis and convenient introduction of 3'-end ligands, chromophores and modifications of synthetic RNA

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- Number: US 8,541,569 B2
- Document ID: US8541569B2
- Status: Granted
- Kind: B2
- Application number: 12/708,827
- Filed: 2010-02-19
- Published: 2011-06-09
- Granted: 2013-09-24
- Earliest claimed: 2008-09-06
- Assignees: ChemGenes Corporation
- Applicants: Suresh C. Srivastava; Naveen P. Srivastava
- Inventors: Suresh C. Srivastava; Naveen P. Srivastava
- Web page: https://patentgrep.com/patent/US8541569B2

## CPC

- C07H 19/167: with ribosyl as the saccharide radical
- C07H 19/067: with ribosyl as the saccharide radical
- C07H 21/02: with ribosyl as saccharide radical
- Y02P 20/55: Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

The present invention provides building blocks and methods for synthesizing very pure RNA in a form that can efficiently be modified at the 3′ end. Reverse RNA monomer phosphoramidites have been developed for RNA synthesis in 5′→3′ direction, leading to very clean oligo synthesis that allows for the introduction of various modifications at the 3′-end cleanly and efficiently. Higher coupling efficiency per step have been observed during automated oligo synthesis with the reverse RNA amidites disclosed herein, resulting in a greater ability to achieve higher purity and produce very long oligonucleotides. The use of the reverse RNA phosphoramidites in the synthetic process of this invention leads to oligonucleotides free of N+1 species.

## Claims

### Claim 1

A compound of Formula Ia or Ib:
or a salt thereof, wherein:
J is H,
wherein indicates where J is attached to the O atom;
Q is a support comprised of: a) a linking group and a spacer that can be cleaved to form a hydroxy group; or b) an aliphatic chain, aromatic group, substituted or unsubstituted aromatic, a substituted or unsubstituted phenoxy, or levulinyl;
R1 is a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C3-C20)cycloalkyl group, or a substituted or unsubstituted (C3-C20)cycloalkyl(C1-C12)alkyl group, wherein the alkyl or cycloalkyl groups optionally include intervening heteroatoms independently selected from NH, NR7, O and S;
R2 is a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C3-C20)cycloalkyl group, or a substituted or unsubstituted (C3-C20)cycloalkyl(C1-C12)alkyl group, wherein the alkyl or cycloalkyl groups optionally include intervening heteroatoms independently selected from NH, NR7, O and S;
or R1 and R2 taken together with the nitrogen atom to which they are bound form a 4-7 membered non-aromatic heterocyclyl, wherein the heterocyclyl formed may optionally include intervening heteroatoms independently selected from NH, NR7, O and S;
R3 is a phosphate protecting group;
R4 is a -halo, —R5, —NR7R8, —OR9, —SR10, or 2′-blocking group; or when Structural Formula Ia or Ib is:
wherein R4 is further selected from O—Si(R11)3 or O—CH2—Si(R11)3; or when Structural Formula Ia or Ib is:
R4 is further selected from —OC(═O)R12;
each R5 is independently a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C2-C12)alkenyl group, a substituted or unsubstituted (C2-C12)alkynyl group, or a substituted or unsubstituted phenyl, wherein the alkyl, alkenyl, alkynyl and the phenyl groups optionally includes intervening heteroatoms independently selected from NH, NR5, O and S; and may optionally terminate with —NR7R8, (C1-C4)alkylamino, di(C1-C4)alkylamino, —OR9, (C1-C6)alkoxy, benzyl or substituted benzyl, —SR10; or —S—(C1-C6)alkyl group;
R6 is —H or —O—Z;
each R7 is independently a fluorenylmethyloxycarbonyl; —C(═O)—(CH2)1-16NR8C(═O)CF3; —C(═O)—(CH2)1-16NR8C(═O)-phthalimide; —C(═O)—(CH2)1-16-phthalimide; NR8C(═O)-phthalimide; a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group;
each R8 is H or a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group;
each R9 is independently —C(═O)—(CH2)1-16CH3; a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group;
each R10 is independently —S(C1-C6)alkyl, —C(═O)—(CH2)1-16CH3; a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group;
each R11 is independently a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group;
each R12 is independently a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group, or a substituted or unsubstituted aryl group;
Z is an unsubstituted or substituted aryl group, an unsubstituted or substituted triarylmethyl group, an unsubstituted or substituted trityl group, an unsubstituted or substituted tetrahydropyranyl group, or an unsubstituted or substituted 9-phenylxanthyl;
Bn is hydrogen or an optionally substituted nucleobase optionally functionalized at each exocyclic amine with an amine protecting group, wherein the nucleobase is selected from:
N6,N6-dimethyl adenine, N-1 methyladenine, 7-deazaadenine, 7-deaza-8-azaadenine, 3-deazaadenine, ethenoadenine, isoguanine, N1-methylguanine, 7-iodo-7-deazaguanine, 7-deaza-7-iodoadenine, 7-deaza-7-iodo-6-oxopurine, 5-iodo-5-methyl-7-deazaguanine, 7-deazaguanine substituted with —C≡C(CH2)1-8-pthlamide, 7-deaza-8-azaguanine, 8-methylguanine, 8-bromoguanine, 8-aminoguanine, hypoxanthine, 6-methoxypurine, 7-deaza-6-oxopurine, 6-oxopurine, 2-aminopurine, 2,6-diaminopurine, 8-bromopurine, 8-aminopurine, 8-alkylaminopurine, 8-alkylaminopurine, thymine, N-3 methyl thymine, 5-acetoxymethylcytosine, 5-azacytosine, isocytosine, N-4(C1-C6)alkylcytosine, N-3(C1-C6)alkylcytidine, 5-propynylcytosine, 5-iodo-cytosine, 5-(C1-C6)alkylcytosine, 5-aryl(C1-C6)alkylcytosine, 5-trifluoromethylcytosine, 5-methylcytosine, ethenocytosine, cytosine and uracil substituted with —CH═CH—C(═O)NH(C1-C6)alkyl, cytosine and uracil substituted with —C≡C—CH2-phthalimide, NH(C1-C6)alkyl, 4-thiouracil, 2-thiouracil, N3-thiobenzoylethyluracil, 5-propynyluracil, 5-acetoxymethyluracil, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, 5-iodouracil, thiouracil, N-3-(C1-C6)alkyluracil, 5-(3-aminoallyl)-uracil, 5-(C1-C6)alkyluracil, 5-aryl(C1-C6)alkyluracil, 5-trifluoromethyluracil, 4-triazolyl-5-methyluracil, 2-pyridone, 2-oxo-5-methylpyrimidine, 2-oxo-4-methylthio-5-methylpyrimidine, 2-thiocarbonyl-4-oxo-5-methylpyrimidine, and 4-oxo-5-methylpyrimidine;
wherein any substitutable nitrogen atom within the nucleobase or on the exocyclic amine is optionally substituted with fluorenylmethyloxycarbonyl; —C(═O)OPh; —C(═O)(C1-C16)alkyl; —C(═O)CH2CH2CH═CH2—; —C(═O)(C1-C16)alkylene-C(═O)OH; —C(═O)(C1-C16)alkylene-C(═O)O(C1-C6)alkyl; ═CR8N(C1-C6)alkyl)2; —C(═O)—NR8—(CH2)1-16NR8C(═O)CF3; —C(═O)—(CH2)1-16NR8C(═O)CF3; —C(═O)—NR8(CH2)1-16NR8C(═O)-phthalimide; —C(═O)—(CH2)1-16-phthalimide; and
wherein any substitutable oxygen atom within the nucleobase is optionally substituted with —C(═O)N(C1-C6alkyl)2 or —C(═O)N(phenyl)2.

### Claim 2

A compound of Formula Ia or Ib:
or a salt thereof, wherein:
J is H or
R1 is a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C3-C20)cycloalkyl group, or a substituted or unsubstituted (C3-C20)cycloalkyl(C1-C12)alkyl group, wherein the alkyl or cycloalkyl groups optionally include intervening heteroatoms independently selected from NH, NR7, O and S;
R2 is a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C3-C20)cycloalkyl group, or a substituted or unsubstituted (C3-C20)cycloalkyl(C1-C12)alkyl group, wherein the alkyl or cycloalkyl groups optionally include intervening heteroatoms independently selected from NH, NR7, O and S;
or R1 and R2 taken together with the nitrogen atom to which they are bound form a 4-7 membered non-aromatic heterocyclyl, wherein the heterocyclyl formed may optionally include intervening heteroatoms independently selected from NH, NR7, O and S;
R3 is a phosphate protecting group;
R4 is a -halo, —R5, —NR7R8, —OR9, —SR10, or 2′-blocking group; or when Structural Formula Ia or Ib is:
wherein R4 is further selected from O—Si(R11)3 or O—CH2—Si(R11)3; or when Structural Formula Ia or Ib is:
R4 is further selected from —OC(═O)R12;
each R5 is independently a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C2-C12)alkenyl group, a substituted or unsubstituted (C2-C12)alkynyl group, or a substituted or unsubstituted phenyl, wherein the alkyl, alkenyl, alkynyl and the phenyl groups optionally includes intervening heteroatoms independently selected from NH, NR5, O and S; and may optionally terminate with —NR7R8, (C1-C4)alkylamino, di(C1-C4)alkylamino, —OR9, (C1-C6)alkoxy, benzyl or substituted benzyl, —SR10; or —S—(C1-C6)alkyl group;
R6 is —H or —O—Z;
each R7 is independently a fluorenylmethyloxycarbonyl; —C(═O)—(CH2)1-16NR8C(═O)CF3; —C(═O)—(CH2)1-16NR8C(═O)-phthalimide; —C(═O)—(CH2)1-16-phthalimide; NR8C(═O)-phthalimide; a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group;
each R8 is H or a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group;
each R9 is independently —C(═O)—(CH2)1-16CH3; a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group;
each R10 is independently —S(C1-C6)alkyl, —C(═O)—(CH2)1-16CH3; a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group;
each R11 is independently a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group; provided that if R4 is —O—Si(R11)3, and two R11 groups are both methyl, the other is not t-butyl or if R4 is —O—CH2—Si(R11)3, then the three R11 groups cannot all be isopropyl;
each R12 is independently a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group, or a substituted or unsubstituted aryl group;
Z is an unsubstituted or substituted aryl group, an unsubstituted or substituted triarylmethyl group, an unsubstituted or substituted trityl group, an unsubstituted or substituted tetrahydropyranyl group, or an unsubstituted or substituted 9-phenylxanthyl;
Bn is hydrogen or an optionally substituted nucleobase optionally functionalized at each exocyclic amine with an amine protecting group,
provided that the compound is not represented by one of the following structural formulas:
wherein X is tert-butyldimethylsilyl or triisopropylsiloloxymethyl.

### Claim 3 (depends on 1)

The compound of claim 1, wherein J is

### Claim 4 (depends on 1)

The compound of claim 1, wherein Z is di-p-anisylphenyl methyl, p-fluorophenyl-1-naphthylphenyl methyl, p-anisyl-1-naphthylphenyl methyl, di-o-anisyl-1-naphthyl methyl, di-o-anisylphenyl methyl, p-tolyldiphenylmethyl, di-p-anisylphenylmethyl, di-o-anisyl-1-naphthylmethyl, di-p-anisylphenyl methyl, di-o-anisyl phenyl methyl, di-p-anisylphenyl methyl, or p-tolyldiphenylmethyl.

### Claim 5 (depends on 1)

The compound of claim 1, wherein Z is represented by the following structural formula:
wherein indicates attachment to the 3′ oxygen atom and Ra, Rb, and Rc are independently selected from the following structural formulas

### Claim 6 (depends on 1)

The compound of claim 1, wherein Z is 4-methoxytrityl, 4,4′-dimethoxytrityl, or 4,4′,4″-trimethoxytrityl.

### Claim 7 (depends on 1)

The compound of claim 1, wherein the substitutable nitrogen atom within the nucleobase or on the exocyclic amine is optionally substituted with ═CHN(CH3)2; C(═O)CH(CH3)2; —C(═O)CH3, ═C(CH3)N(CH3)2; —C(═O)OPh; —C(═O)CH2CH2CH═CH2; —C(═O)CH2CH2—C(═O)O(C1-C6)alkyl; —C(═O)—NR8—(CH2)1-16NR8C(═O)CF3; —C(═O)—(CH2)1-16NR8C(═O)CF3; —C(═O)—NR8(CH2)1-16NR8C(═O)-phthalimide; —C(═O)—(CH2)1-16-phthalimide and

### Claim 8 (depends on 1)

The compound of claim 1, wherein R3 is —CH2CH2CN, —CH2CH2—Si(CH3)2C6H5, —CH2CH2—S(O)2—CH2CH3, —CH2CH2—C6H4—NO2, —CH2CH2—NH—C(O)—C6H5, or —CH2CH2—O—C6H4—C(O)CH3, and R4 is —O—Si(R11)3 or —O—CH2—Si(R11)3.

### Claim 9 (depends on 8)

The compound of claim 8, wherein R4 is —O—Si(CH3)2(C(CH3)3) or —O—CH2—Si(CH(CH3)2)3.

### Claim 10 (depends on 1)

The compound of claim 1, wherein the compound is represented by one of the following structural formulas:
or a salt thereof.

### Claim 11 (depends on 10)

The compound of claim 10, wherein the compound is represented by one of the following structural formulas:
or a salt thereof.

### Claim 12 (depends on 11)

The compound of claim 11, wherein R3 is —CH2CH2CN.

### Claim 13 (depends on 12)

The compound of claim 12, wherein the compound is represented by one of the following structural formulas:
or a salt thereof.

### Claim 14 (depends on 1)

The compound of claim 1, wherein the compound is represented by one of the following structural formulas:
or a salt thereof.

### Claim 15 (depends on 14)

The compound of claim 14, wherein the compound is represented by one of the following structural formula:
or a salt thereof.

### Claim 16 (depends on 15)

The compound of claim 15, wherein R3 is —CH2CH2CN.

### Claim 17 (depends on 16)

The compound of claim 16, wherein the compound is represented by one of the following structural formulas:
or a salt thereof.

### Claim 18 (depends on 1)

The compound of claim 1, wherein the compound is represented by one of the following structural formulas:
or a salt thereof.

### Claim 19 (depends on 18)

The compound of claim 18, wherein the compound is represented by one of the following structural formulas:
or a salt thereof.

### Claim 20 (depends on 1)

The compound of claim 1, wherein the compound is represented by one of the following structural formulas:
or a salt thereof.

### Claim 21 (depends on 20)

The compound of claim 20, wherein the compound is represented by one of the following structural formulas:
or a salt thereof.

### Claim 22

A method of preparing an oligonucleotide by bond formations in the 5′- to 3′-direction for the synthesis of RNA oligomers and enantiomers thereof, said method comprises the following steps
a) cleavage of Z from a compound represented by the following structural formula:
thereby forming a compound represented by structural formula (XX):
b) reacting the compound represented by structural formula XX with a compound of structural formula XXI:
thereby forming 2-mer compound represented by the following structural formula:
c) reacting the 2-mer compound with a capping agent;
d) oxidizing or sulfurizing the trivalent phosphorous group of the 2-mer compound to form a compound of structural formula XXII:
e) cleavage of Z from the compound represented by structural formula XXII, thereby forming a compound of structural formula XXIII:
f) reacting the compound represented by structural formula XXIII with a compound represented by the following structural formula:
thereby forming a 3-mer represented by the following structural formula XXIV:
g) cleavage of Z from the compound represented by structural formula XXIV;
h) repeating steps a) through g) n times, thereby forming a oligonucleotide represented by the following structural formula:
or a salt thereof, wherein:
Q is a support comprised of: a) a linking group and a spacer that can be cleaved to form a hydroxy group; or b) an aliphatic chain, aromatic group, substituted or unsubstituted aromatic, a substituted or unsubstituted phenoxy, or levulinyl;
each R1 is independently a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C3-C20)cycloalkyl group, or a substituted or unsubstituted (C3-C20)cycloalkyl(C1-C12)alkyl group, wherein the alkyl or cycloalkyl groups optionally include intervening heteroatoms independently selected from NH, NR7, O and S;
each R2 is independently a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C3-C20)cycloalkyl group, or a substituted or unsubstituted (C3-C20)cycloalkyl(C1-C12)alkyl group, wherein the alkyl or cycloalkyl groups optionally include intervening heteroatoms independently selected from NH, NR7, O and S;
or each occurrence of R1 and R2 taken together with the nitrogen atom to which they are bound independently forms a 4-7 membered non-aromatic heterocyclyl, wherein the heterocyclyl formed may optionally include intervening heteroatoms independently selected from NH, NR7, O and S;
each R3 is independently a phosphate protecting group;
each R4 is independently a -halo, —R5, —NR7R8, —OR9, —SR10, or 2′-blocking group; or when a 1-mer is in the β-D-ribose, β-L-ribose, α-D-ribose or α-L-ribose configuration, wherein R4 is further selected from O—Si(R11)3 or O—CH2—Si(R11)3; or when a 1-mer is in the β-D-arabinose, β-L-arabinose, α-D-arabinose or α-L-arabinose configuration,
R4 is further selected from —OC(═O)R12;
each R5 is independently a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C2-C12)alkenyl group, a substituted or unsubstituted (C2-C12)alkynyl group, or a substituted or unsubstituted phenyl, wherein the alkyl, alkenyl, alkynyl and the phenyl groups optionally includes intervening heteroatoms independently selected from NH, NRS, O and S; and may optionally terminate with —NR7R8, (C1-C4)alkylamino, di(C1-C4)alkylamino, —OR9, (C1-C6)alkoxy, benzyl or substituted benzyl, —SR10; or —S—(C1-C6)alkyl group;
each R7 is independently a fluorenylmethyloxycarbonyl;
—C(═O)—(CH2)1-16NR8C(═O)CF3; —C(═O)—(CH2)1-16NR8C(═O)-phthalimide; —C(═O)—(CH2)1-16-phthalimide; NR8C(═O)-phthalimide; a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group;
each R8 is independently H or a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group;
each R9 is independently —C(═O)—(CH2)1-16CH3; a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group;
each R10 is independently —S(C1-C6)alkyl, —C(═O)—(CH2)1-16CH3; a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group;
each R11 is independently a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group;
each R12 is independently a substituted or unsubstituted (C1-C12)alkyl group, a substituted or unsubstituted (C2-C12)alkenyl group, or a substituted or unsubstituted (C2-C12)alkynyl group, or a substituted or unsubstituted aryl group;
each Z is independently an unsubstituted or substituted aryl group, an unsubstituted or substituted triarylmethyl group, an unsubstituted or substituted trityl group, an unsubstituted or substituted tetrahydropyranyl group, or an unsubstituted or substituted 9-phenylxanthyl;
each Bn is independently hydrogen or an optionally substituted nucleobase optionally functionalized at each exocyclic amine with an amine protecting group, wherein the nucleobase is independently selected from:
N6,N6-dimethyl adenine, N1 methyladenine, 7-deazaadenine, 7-deaza-8-azaadenine, 3-deazaadenine, ethenoadenine, isoguanine, N1-methylguanine, 7-iodo-7-deazaguanine, 7-deaza-7-iodoadenine, 7-deaza-7-iodo-6-oxopurine, 5-iodo-5-methyl-7-deazaguanine, 7-deazaguanine substituted with —C≡C(CH2)1-8-pthlamide, 7-deaza-8-azaguanine, 8-methylguanine, 8-bromoguanine, 8-aminoguanine, hypoxanthine, 6-methoxypurine, 7-deaza-6-oxopurine, 6-oxopurine, 2-aminopurine, 2,6-diaminopurine, 8-bromopurine, 8-aminopurine, 8-alkylaminopurine, 8-alkylaminopurine, thymine, N-3 methyl thymine, 5-acetoxymethylcytosine, 5-azacytosine, isocytosine, N-4(C1-C6)alkylcytosine, N-3(C1-C6)alkylcytidine, 5-propynylcytosine, 5-iodo-cytosine, 5-(C1-C6)alkylcytosine, 5-aryl(C1-C6)alkylcytosine, 5-trifluoromethylcytosine, 5-methylcytosine, ethenocytosine, cytosine and uracil substituted with —CH═CH—C(═O)NH(C1-C6)alkyl, cytosine and uracil substituted with —C≡C—CH2-phthalimide, NH(C1-C6)alkyl, 4-thiouracil, 2-thiouracil, N3-thiobenzoylethyluracil, 5-propynyluracil, 5-acetoxymethyluracil, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, 5-iodouracil, N-3-(C1-C6)alkyluracil, 5-(3-aminoallyl)-uracil, 5-(C1-C6)alkyluracil, 5-aryl(C1-C6)alkyluracil, 5-trifluoromethyluracil, 4-triazolyl-5-methyluracil, 2-pyridone, 2-oxo-5-methylpyrimidine, 2-oxo-4-methylthio-5-methylpyrimidine, 2-thiocarbonyl-4-oxo-5-methylpyrimidine, and 4-oxo-5-methylpyrimidine;
wherein any substitutable nitrogen atom within the nucleobase or on the exocyclic amine is optionally and independently substituted with fluorenylmethyloxycarbonyl; —C(═O)OPh; —C(═O)(C1-C16)alkyl; —C(═O)CH2CH2CH═CH2I; —C(═O)(C1-C16)alkylene-C(═O)OH; —C(═O)(C1-C16)alkylene-C(═O)O(C1-C6)alkyl; ═CR8N(C1-C6)alkyl)2; —C(═O)—NR8—(CH2)1-16NR8C(═O)CF3; —C(═O)—(CH2)1-16NR8C(═O)CF3; —C(═O)—NR8(CH2)1-16NR8C(═O)-phthalimide; —C(═O)—(CH2)1-16-phthalimide; and
wherein any substitutable oxygen atom within the nucleobase is optionally and independently substituted with —C(═O)N(C1-C6alkyl)2-C(═O)N(phenyl)2; and
n is 1 to 150.

### Claim 23 (depends on 22)

The method of claim 22, wherein each Y group is independently introduced via sulfurization to form a phosphorothioate linkage or via oxidation to form a phosphate linkage.

### Claim 24 (depends on 23)

The method of claim 23, wherein the R3 group is eliminated after oxidation to form a phosphate linkage.

### Claim 25 (depends on 23)

The method of claim 23, wherein the R3 group is eliminated after sulfurization to form a phosphorothioate linkage.

### Claim 26 (depends on 25)

The method of claim 25, wherein the nucleobase protecting groups are deprotected with a base.

### Claim 27 (depends on 26)

The method of claim 26, wherein the base is selected from aqueous ammonia, methanolic ammonia, or ethanolic ammonia.

### Claim 28 (depends on 26)

The method of claim 26, wherein R4 is —OSi(R11)3.

### Claim 29 (depends on 28)

The method of claim 28, wherein the method further comprises the step of cleaving —Si(R11)3 from —OSi(R11)3 with a fluoride salt.

### Claim 30 (depends on 29)

The method of claim 29, wherein the fluoride salt is tetraethylammonium fluoride.

### Claim 31 (depends on 22)

The method of claim 22, further comprising the step of reacting the compound represented by the following structural formula:
with a 3′ functional group, thereby forming a compound represented by the following structural formula:
wherein R21 is the 3′ functional group or a salt thereof.

### Claim 32 (depends on 31)

The method of claim 31, wherein R21 is
a) cyanoethyl phosphate-polyethylene glycols', where v is 2-100 and is number of glycol units;
b) cyanoethyl phosphate-linker attached with cholesterol, biotin, fluorescein, cyanine dye, psoralen, tetramethylrhodamine dye, dabcyl dye, C-3 disulfide, C-6 disulfide, symmetrical and asymmetrical hydrocarbon chain (C2-C50), symmetrical and asymmetrical hydrocarbon chain (C2-C50) with a terminal amino group protected with CF3C(═O) or phthalamido or FMOC, (C1-C16)alkylene-amine protected with a amine protecting group, (C1-C5)alkylene-amine protected with an azide group; (C1-C5)alkylene-amine protected with a C≡CH group;
c) cyanoethyl phosphate-ethane-2-ol-protected with DMT group or other acid labile group, cyanoethyl phosphate-propane-3-ol-protected with DMT group or other acid labile group;
d) (C1-C50)alkylene with a terminal hydroxy;
e) lipid, carboxyl group, or peptide; or
f) a branched phosphoramidite.