Orthoester compositions for affinity purification of oligonucleotides

Patent metadata

Field	Value
Publication number	US-11548876-B2
Application number	US-202016793808-A
Country	US
Kind code	B2
Filing date	Feb 18, 2020
Priority date	Aug 18, 2017
Publication date	Jan 10, 2023
Grant date	Jan 10, 2023

Abstract

Compounds and methods for purifying oligonucleotides such as RNA and DNA. A target oligonucleotide is reacted with an orthoester linker comprising an affinity tag to form an orthoester oligonucleotide-orthoester linker conjugate which is subjected to a purification technique to separate the target oligonucleotide from impurities such as truncated oligonucleotides. The orthoester linker can be then removed under mild conditions to generate the target oligonucleotide in high purity.

First claim

We claim:

1. An oligonucleotide orthoester linker conjugate comprising an oligonucleotide which comprises at its 5′ or 3′end a moiety of formula (IV),

wherein the oxygen linked to the squiggly line is the oxygen of the 5′end or the 3′end of the oligonucleotide;
wherein at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 is C 1 -C 24 alkyl, C 1 -C 24 heteroalkyl, C 2 -C 24 alkenyl, C 2 -C 24 heteroalkenyl, C 2 -C 24 alkynyl, C 2 -C 24 heteroalkynyl, halogen, aryl, heteroaryl, heterocyclyl, carbocyclyl, any substituted equivalent or a combination thereof and the remaining R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are H, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 6 -C 12 arylalkyl, carbocyclyl, heterocyclyl, any substituted equivalent or combination thereof, and n is 0, 1, or 2, wherein at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 or R 7 comprises an affinity tag; and with the proviso that said moiety is not

2. The oligonucleotide orthoester linker conjugate of claim 1 , wherein n=0 and the affinity tag is a fluorous or a hydrophobic tag with a cLog P value of at least 3, wherein the cLog P value is calculated from solubility in octanol and water.

3. The oligonucleotide orthoester linker conjugate of claim 1 , wherein the oligonucleotide comprises an oligoribonucleic acid (RNA).

4. The oligonucleotide orthoester linker conjugate of claim 1 , wherein at least one of R 1 , R 2 , R 3 , R 4 , R 5 or R 6 is a fluorosubstituted alkyl, a fluorosubstituted alkenyl, a fluorosubstituted alkynyl, a fluorosubstituted aryl, a fluorosubstituted heteroalkyl, a fluorosubstituted heteroalkenyl, a fluorosubstituted heteroalkynyl, or a fluorosubstituted heterocyclyl.

5. The oligonucleotide orthoester linker conjugate of claim 1 , wherein at least one of R 1 , R 2 , R 3 , R 4 , R 5 or R 6 is a fluorous tag and the remaining R groups including R 7 are H.

6. The oligonucleotide of claim 1 , wherein at least two of R 1 , R 2 , R 3 , R 4 , R 5 or R 6 are a fluorous tag.

7. The oligonucleotide orthoester linker conjugate of claim 1 , wherein said moiety is selected from the group consisting of:

wherein R 11 and R 12 are each independently H, C 1 -C 23 alkyl, C 1 -C 23 heteroalkyl, C 1 -C 23 substituted alkyl, C 2 -C 23 alkenyl, C 2 -C 23 heteroalkenyl, C 2 -C 23 substituted alkenyl, C 2 -C 23 alkynyl, C 2 -C 23 heteroalkynyl, C 2 -C 23 substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl or substituted heterocyclyl provided that at least one of R 11 and R 12 comprises an affinity tag; R 7 is H, methyl, ethyl, n-propyl, phenyl or benzyl; Z are each independently CR a R b , O, S or NR a wherein R a and R b are each independently H, C 1 -C 6 alkyl, or R a and R 11 or R a and R 12 together form a heterocycle with N; n is 0, 1, or 2.

8. The oligonucleotide orthoester linker conjugate of claim 1 , wherein said moiety is:

wherein X 1 is H, F, an azido, a protected sulfhydryl, a protected poly-sulfhydryl, a poly-histidine, a protected amino group, a protected hydrazide group, a protected oxyamine group, a maleimide, a cyclooctyne, a conjugated diene, a C 2 alkenyl group, a C 2 substituted alkenyl group, a C 2 alkynyl group or a C 2 substituted alkynyl group; R 18 and R 19 are each independently H, F, C 1-3 heteroalkyl or C 1-3 substituted alkyl; n is 1 or 2; and m is an integer ranging from 0 to 12.

9. The oligonucleotide orthoester linker conjugate of claim 1 , wherein said moiety is selected from the group consisting of:

Assignees

• Agilent Technologies Inc

Inventors

• Dellinger Douglas J
• Myerson Joel
• Smart Brian

Classifications

• C07H21/02

  with ribosyl as saccharide radical · CPC title

• C07D317/34

  Oxygen atoms · CPC title

• C07H1/06 Primary

  Separation; Purification · CPC title

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• C07D403/06

  linked by a carbon chain containing only aliphatic carbon atoms · CPC title