# US 9,751,909 B2 · Synthesis and uses of nucleic acid compounds with conformationally restricted monomers

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- Number: US 9,751,909 B2
- Document ID: US9751909B2
- Status: Granted
- Kind: B2
- Application number: 14/343,607
- Filed: 2012-09-07
- Published: 2015-03-19
- Granted: 2017-09-05
- Earliest claimed: 2011-09-07
- Assignees: Marina Biotech, Inc.
- Applicants: Tracy J. Matray; Iwona M. Maciagiewicz; Michael E. Houston, Jr.
- Inventors: Tracy J. Matray; Iwona M. Maciagiewicz; Michael E. Houston, Jr.
- Web page: https://patentgrep.com/patent/US9751909B2

## CPC

- C07H 19/06: Pyrimidine radicals
- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 19/16: Purine radicals
- C07H 23/00: Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12 (esters with inorganic acids C07H11/00)
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Processes for synthesis of conformationally restricted nucleomonomers (CRN) by ozonolysis or oxidation of a 2′-vinyl substituted nucleoside precursor with protected 3′-hydroxyl, 4′-hydroxymethyl and 5′-hydroxyl positions. The CRN nucleomonomers can be used to prepare nucleic acid compounds. CRN nucleomonomers for nucleic acid compounds can be prepared in high yields and in multi-gram scale. The nucleic acid compounds can be of various regulatory classes such as antisense RNA for therapeutic modalities useful for treating or preventing diseases or disorders by up- or down-regulating the expression of genes and other nucleic acid based regulatory systems in a cell.

## Claims

### Claim 1

A method for making a compound having Formula V
wherein R1 is a substituent protecting group, R2 is a substituent protecting group, one or both of R1 and R2 are bulky substituent protecting groups, R1 and R2 are optionally linked, R3 is a protecting group, and B is a nucleobase or nucleobase analog, the method comprising providing a compound having Formula II,
transforming the compound having Formula II by ozonolysis or oxidation, followed by contacting with a reducing agent, and isolating the compound having Formula V, wherein the bulky substituent protecting groups are selected from trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM) and dimethoxytrityl (DMT).

### Claim 2 (depends on 1)

The method of claim 1, wherein B is 1-uracilyl or 5-methyl-1-uracilyl.

### Claim 3 (depends on 1)

The method of claim 1, wherein R1 and R2, optionally linked, is a 1,1,3,3-tetraalkyldisiloxane-1,3-diyl group.

### Claim 4 (depends on 1)

The method of claim 1, wherein R1 and R2, optionally linked, is a 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl group.

### Claim 5 (depends on 1)

The method of claim 1, wherein R3 is benzyl.

### Claim 6 (depends on 1)

The method of claim 1, wherein the yield of the compound having Formula V by ozonolysis is at least 50% at a scale of at least 5 grams of the compound having Formula V.

### Claim 7 (depends on 1)

The method of claim 1, wherein the yield of the compound having Formula V by ozonolysis is at least 50% at a scale of at least 10 grams of the compound having Formula V.

### Claim 8 (depends on 1)

The method of claim 1, wherein the yield of the compound having Formula V ozonolysis is at least 70% at a scale of at least 10 grams of the compound having Formula V.

### Claim 9 (depends on 1)

The method of claim 1, wherein the yield of the compound having Formula V ozonolysis is at least 80% at a scale of at least 10 grams of the compound having Formula V.

### Claim 10 (depends on 1)

The method of claim 1, further comprising transforming the compound having Formula V to a conformationally restricted nucleomonomer CRN having Formula VI,
by the steps of:
deprotecting the 4′-hydroxymethyl groups of Formula V; and
forming a 2′-4′ —CH2OCH2— bridge.

### Claim 11 (depends on 10)

The method of claim 10, wherein the conformationally restricted nucleomonomer is a pyrimidine CRN.

### Claim 12

A compound having the structure
wherein R is methyl, R1 is independently H, alkyl, or a protecting group, and R2 is independently H, alkyl, a protecting group, or a phosphoramidityl group.

### Claim 13 (depends on 12)

A nucleic acid compound comprising a residue of the compound of claim 12.

### Claim 14 (depends on 13)

The nucleic acid compound of claim 13, wherein the nucleic acid compound is a single stranded oligonucleotide or a double-stranded oligonucleotide.

### Claim 15 (depends on 13)

The nucleic acid compound of claim 13, wherein the nucleic acid compound is a microRNA, an antimir, an antagomir, a microRNA mimetic, a microRNA precursor, an RNA, a short interfering RNA or siRNA, a DNA, an RNA and DNA, a meroduplex RNA or mdRNA, a short hairpin RNA or shRNA, or an antisense oligonucleotide.

### Claim 16

A compound having the structure

### Claim 17 (depends on 16)

A nucleic acid compound comprising a residue of the compound of claim 16.

### Claim 18 (depends on 17)

The nucleic acid compound of claim 17, wherein the nucleic acid compound is a single stranded oligonucleotide or a double-stranded oligonucleotide.

### Claim 19 (depends on 17)

The nucleic acid compound of claim 17, wherein the nucleic acid compound is a microRNA, an antimir, an antagomir, a microRNA mimetic, a microRNA precursor, an RNA, a short interfering RNA or siRNA, a DNA, an RNA and DNA, a meroduplex RNA or mdRNA, a short hairpin RNA or shRNA, or an antisense oligonucleotide.