IP Library › Patent Application 19062789

Patent Application

App. No. 19/062,789

EFFICIENT METHOD FOR MAKING HIGHLY PURIFIED 5’- CAPPED OLIGONUCLEOTIDES

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Patent No.: US None
App. No.: 19/062,789

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Abstract

Described herein are highly pure, chemically synthesized, stabilized, 5′-capped oligonucleotides. Additionally, described herein are methods for making and using said oligonucleotides.

Claims (80)

1 .- 183 . (canceled)

184 . An oligonucleotide comprising 50-12000 nucleotides, whose 5′ end comprises a structure of formula

(I)

or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof;

wherein:

B 1 and B 3 are each independently a natural, modified, or unnatural nucleoside base;

each B 2 is independently a natural, modified, or unnatural nucleoside base;

Ring A is a substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

X 1 and X 2 are each independently —O—, —CH 2 —, —CX 2 —, —N(R 101 )—, —BH—, or —S—;

Y 1 , Y 2 , Y 3 , Y 4 , and Y 5 are each independently O, S, or Se;

R 1 is independently hydrogen, —C(O)R 1A , —C(O)OR 1A , —OR 1A , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R 2 is independently hydrogen, —C(O)R 2A , —C(O)OR 2A , —OR 2A , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R 1 and R 2 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocyclyl;

R 3 is hydrogen, —C(O)R 3A , —C(O)OR 3A , —OR 3A , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

each R 7 is independently hydrogen, halogen, —CCl 3 , —CBr 3 , —CF 3 , —CI 3 , —CHCl 2 , —CHBr 2 , —CHF 2 , —CHI 2 , —CH 2 Cl, —CH 2 Br, —CH 2 F, —CH 2 I, —CN, —OR 7A , —NR 7A R 7B , —COOH, —CONH 2 , —NO 2 , —SH, —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2 , —NHC(O)NH 2 , —NHSO 2 H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl 3 , —OCF 3 , —OCBr 3 , —OCI 3 , —OCHCl 2 , —OCHBr 2 , —OCHI 2 , —OCHF 2 , —OCH 2 Cl, —OCH 2 Br, —OCH 2 I, —OCH 2 F, —N 3 , —SF 5 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

each R 19 is independently hydrogen, halogen, —CCl 3 , —CBr 3 , —CF 3 , —CI 3 , —CHCl 2 , —CHBr 2 , —CHF 2 , —CHI 2 , —CH 2 Cl, —CH 2 Br, —CH 2 F, —CH 2 I, —CN, —OR 19A , —NR 19A R 19B , —COOH, —CONH 2 , —NO 2 , —SH, —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2 , —NHC(O)NH 2 , —NHSO 2 H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl 3 , —OCF 3 , —OCBr 3 , —OCI 3 , —OCHCl 2 , —OCHBr 2 , —OCHI 2 , —OCHF 2 , —OCH 2 Cl, —OCH 2 Br, —OCH 2 I, —OCH 2 F, —N 3 , —SF 5 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

or R 7 and R 19 together with the carbon atoms to which they are connected form a substituted or unsubstituted cycloalkylene or substituted or unsubstituted heterocycloalkylene;

each R 11 is independently hydrogen, halogen, —CCl 3 , —CBr 3 , —CF 3 , —CI 3 , —CHCl 2 , —CHBr 2 , —CHF 2 , —CHI 2 , —CH 2 Cl, —CH 2 Br, —CH 2 F, —CH 2 I, —CN, —OR 11A , —NR 11A R 11B , —COOH, —CONH 2 , —NO 2 , —SH, —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2 , —NHC(O)NH 2 , —NHSO 2 H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl 3 , —OCF 3 , —OCBr 3 , —OCI 3 , —OCHCl 2 , —OCHBr 2 , —OCHI 2 , —OCHF 2 , —OCH 2 Cl, —OCH 2 Br, —OCH 2 I, —OCH 2 F, —N 3 , —SF 5 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

or R 11 and R 19 together with the carbon atoms to which they are connected form a substituted or unsubstituted cycloalkylene or substituted or unsubstituted heterocycloalkylene;

each R 1A , R 2A , R 3A , R 7A , R 7B , R 11A , R 11B , R 19A , and R 19B is independently hydrogen, —CX 3 , —CHX 2 , —CH 2 X, —C(O)OH, —C(O)NH 2 , —CN, —OH, —NH 2 , —COOH, —CONH 2 , —NO 2 , —SH, —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NHNH 2 , —ONH 2 , —NHC═(O)NHNH 2 , —NHC═(O)NH 2 , —NHSO 2 H, —NHC═(O)H, —NHC(O)OH, —NHOH, —OCX 3 , —OCHX 2 , —OCH 2 X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

or R 7A and R 7B substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; R 11A and R 11B substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; R 19A and R 19B substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl;

each R 101 is independently hydrogen, oxo, halogen, —CCl 3 , —CBr 3 , —CF 3 , —CI 3 , —CHCl 2 , —CHBr 2 , —CHF 2 , —CHI 2 , —CH 2 Cl, —CH 2 Br, —CH 2 F, —CH 2 I, —CN, —OH, —NH 2 , —COOH, —CONH 2 , —NO 2 , —SH, —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2 , —NHC(O)NH 2 , —NHSO 2 H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl 3 , —OCF 3 , —OCBr 3 , —OCI 3 , —OCHCl 2 , —OCHBr 2 , —OCHI 2 , —OCHF 2 , —OCH 2 Cl, —OCH 2 Br, —OCH 2 I, —OCH 2 F, —N 3 , —SF 5 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

m is an integer from 0 to 8;

n is an integer from 0 to 3; and

each X is independently —Cl, —Br, —I or —F.

185 . An oligonucleotide comprising 50-12000 nucleotides, whose 5′ end comprises a structure of formula (II):

wherein:

B 1 and B 2 are each independently a natural, modified, or unnatural nucleoside base; Ring A is a substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

X 1 and X 2 are each independently —O—, —CH 2 —, —CX 2 —, —N(R 101 )—, —BH—, or —S—;

Y 1 , Y 2 , Y 3 , and Y 4 are each independently O, S, or Se;

R 3 is hydrogen, —C(O)R 3A , C(O)OR 3A , —OR 3A , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R 7 is independently hydrogen, halogen, —CCl 3 , —CBr 3 , —CF 3 , —CI 3 , —CHCl 2 , —CHBr 2 , —CHF 2 , —CHI 2 , —CH 2 Cl, —CH 2 Br, —CH 2 F, —CH 2 I, —CN, —OR 7A , —NR 7A R 7B , —COOH, —CONH 2 , —NO 2 , —SH, —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2 , —NHC(O)NH 2 , —NHSO 2 H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl 3 , —OCF 3 , —OCBr 3 , —OCI 3 , —OCHCl 2 , —OCHBr 2 , —OCHI 2 , —OCHF 2 , —OCH 2 Cl, —OCH 2 Br, —OCH 2 I, —OCH 2 F, —N 3 , —SF 5 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

or R 7 and R 19 together with the carbon atoms to which they are connected form a substituted or unsubstituted cycloalkylene or substituted or unsubstituted heterocycloalkylene; each R 11 is independently hydrogen, halogen, —CCl 3 , —CBr 3 , —CF 3 , —CI 3 , —CHCl 2 , —CHBr 2 , —CHF 2 , —CHI 2 , —CH 2 Cl, —CH 2 Br, —CH 2 F, —CH 2 I, —CN, —OR 11A , —NR 11A R 11B , —COOH, —CONH 2 , —NO 2 , —SH, —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2 , —NHC(O)NH 2 , —NHSO 2 H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl 3 , —OCF 3 , —OCBr 3 , —OCI 3 , —OCHCl 2 , —OCHBr 2 , —OCHI 2 , —OCHF 2 , —OCH 2 Cl, —OCH 2 Br, —OCH 2 I, —OCH 2 F, —N 3 , —SF 5 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

or R 11 and R 19 together with the carbon atoms to which they are connected form a substituted or unsubstituted cycloalkylene or substituted or unsubstituted heterocycloalkylene;

n is an integer from 0 to 3; and

each X is independently —Cl, —Br, —I or —F;

with the proviso that R 11 is not OH.

186 . The oligonucleotide of claim 185 , wherein the oligonucleotide comprises 3 or more modified nucleosides and 2 or more nucleotides that are linked together by a modified internucleotide linkage, at its 3′ end.

187 . The oligonucleotide of claim 185 , wherein the structure of formula (I) or formula (II) comprises one or more removable hydrophobic group(s).

188 . The oligonucleotide of claim 185 , wherein the structure of formula (I) or formula (II) comprises one or more non-removable hydrophobic group(s).

189 . The oligonucleotide of claim 185 , wherein Ring A is a substituted or unsubstituted heterocycloalkylene.

190 . The oligonucleotide of claim 185 , wherein R 1 is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, phenyl, benzyl,

or modified trityl.

191 . The oligonucleotide of claim 185 , wherein R 2 is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, phenyl, benzyl,

or modified trityl and/or wherein R 3 is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, phenyl, benzyl, or 4-chlorobenzyl.

192 . The oligonucleotide of claim 185 , wherein R 1 , R 2 and R 3 are independently hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl.

193 . The oligonucleotide of claim 185 , wherein R 7 is independently hydrogen, halogen, or —OR 7A .

194 . The oligonucleotide of claim 185 , wherein R 7 and R 11 are independently hydrogen, hydroxy, methoxy, ethoxy, propoxy, butoxy, or t-butoxy.

195 . The oligonucleotide of claim 185 , wherein R 19 is independently hydrogen and/or wherein R 11 is independently hydrogen, halogen, or —OR 11A .

196 . The oligonucleotide of claim 185 , wherein X 1 is —O—, —CH 2 —, or —CX 2 — and/or wherein X 2 is —O—, —CH 2 —, —CX 2 —, —N(R 101 )—, or —BH—.

197 . The oligonucleotide of claim 185 , wherein Y 1 , Y 2 , Y 3 , and Y 4 are each independently O or S.

198 . The oligonucleotide of claim 185 , wherein n is 1 or 2.

199 . An oligonucleotide whose 5′ end comprises a compound selected from the group consisting of:

200 . The oligonucleotide of claim 185 , wherein each non-removable hydrophobic group is a non-removable purification handle.

201 . A 5′-capped oligonucleotide prepared by removing the protecting group(s) of the protected 5′-capped oligonucleotide of claim 185 .

202 . A composition comprising the chemically synthesized oligonucleotide of claim 185 , wherein the composition comprises less than 1% by weight of oligonucleotide whose 5′ end does not comprise a structure of formula (I) or formula (II).

203 . A process for preparing an oligonucleotide comprising 50-12000 nucleotides, whose 5′ end comprises a structure of formula (I):

or formula (II):

comprising (a) reacting an imidazolide of formula (III)

wherein:

Ring A is a substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

X 1 is independently —O—, —CH 2 —, —CX 2 —, —N(R 101 )—, —BH—, or —S—;

Y 1 and Y 2 are each independently O, S, or Se;

each R 1A , R 2A , and R 3A is independently hydrogen, —CX 3 , —CHX 2 , —CH 2 X, —C(O)OH, —C(O)NH 2 , —CN, —OH, —NH 2 , —COOH, —CONH 2 , —NO 2 , —SH, —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NHNH 2 , —ONH 2 , —NHC═(O)NHNH 2 , —NHC═(O)NH 2 , —NHSO 2 H, —NHC═(O)H, —NHC(O)OH, —NHOH, —OCX 3 , —OCHX 2 , —OCH 2 X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

each X is independently —Cl, —Br, —I or —F;

with a 5′-phosphate-oligonucleotide

and (b) optionally removing the removable hydrophobic group(s).

Assignments (2)

SECURITY INTEREST Recorded Jun 3, 2026

From: TRILINK BIOTECHNOLOGIES, LLC; CYGNUS TECHNOLOGIES, LLC; GLEN RESEARCH, LLC

To: BSP AGENCY, LLC, AS COLLATERAL AGENT

Reel/Frame 074835/0788 →

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 24, 2025

From: ZHAO, CHANFENG; JIANG, TAO TOM; LANG, HENGYUAN; LUDFORD, PAUL THEODORE, III; XU, CHUNPING

To: TRILINK BIOTECHNOLOGIES, LLC

Reel/Frame 073022/0762 →