Orthoester compositions for affinity purification of oligonucleotides

Patent metadata

Field	Value
Publication number	US-11299483-B2
Application number	US-201816640005-A
Country	US
Kind code	B2
Filing date	Aug 17, 2018
Priority date	Aug 18, 2017
Publication date	Apr 12, 2022
Grant date	Apr 12, 2022

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. A method of purifying a target oligonucleotide, the method comprising:
synthesizing the target oligonucleotide on a solid support and obtaining a mixture comprising the target oligonucleotide and truncated oligonucleotides;
reacting the target oligonucleotide with an orthoester linker, and thereby forming an oligonucleotide-orthoester linker conjugate, wherein the orthoester linker either comprises an affinity tag at the time of conjugation or the affinity tag is reacted with the oligonucleotide-orthoester linker conjugate in a second reaction after the conjugation reaction;
cleaving the oligonucleotide-orthoester linker conjugate and the truncated oligonucleotides from the solid support;
loading the oligonucleotide-orthoester linker conjugate and the truncated oligonucleotides onto a chromatographic column or an affinity capture support and binding the oligonucleotide-orthoester linker conjugate by the affinity tag to the affinity capture support;
washing off the truncated oligonucleotides form the column or the affinity capture support; and
cleaving the orthoester linker from the target oligonucleotide and eluting the target oligonucleotide from the chromatographic column or the affinity capture support, and thereby releasing the purified target oligonucleotide.

2. The method of claim 1 , wherein the affinity tag is one or more groups in the orthoester linker or the affinity tag is linked with a linker to the orthoester linker.

3. The method of claim 1 , wherein the second reaction is conducted before or after the oligonucleotide-orthoester linker conjugate is cleaved from the solid support.

4. The method of claim 1 , wherein the orthoester linker is attached at the 5′-hydroxyl of the target oligonucleotide.

5. The method of claim 1 , wherein the orthoester linker is attached at the 3′-hydroxyl of the target oligonucleotide.

6. The method of claim 1 , wherein the affinity tag comprises one or more of the following: a fluorous tag, a hydrophobic tag, a biotin tag, a glutathione tag, a maltose tag, an arylboronic acid tag, a poly-histidine peptide tag, a poly-sulfhydryl tag, a cyclodextrin tag, an adamantane tag, a polyamine tag, a maleimide tag, an alkyne tag, an azido tag, a hydrazide tag, an amino tag, a diol tag, a thiol tag, or any combination thereof.

7. The method of claim 1 , wherein the target oligonucleotide comprises an oligoribonucleotide (RNA).

8. The method of claim 7 , wherein the RNA comprises a 2′-hydroxyl protecting group selected from a thionocarbamate (TC) protecting group, bis(2-acetoxyethoxy)methyl (ACE) protecting group, t-butyldimethylsilyl (TBDMS) protecting group, triisopropylsilyloxymethyl (TOM) protecting group, pivaloyloxymethyl (PivOM) protecting group and 2-cyanoethoxymethyl (CEM) protecting group.

9. The method of claim 7 , wherein the RNA further comprises a phosphorous protecting group, a nucleobase protecting group, or a combination thereof.

10. The method of claim 9 , wherein the phosphorous protecting group is deprotected before reacting the oligonucleotide with the orthoester linker.

11. The method of claim 9 , wherein the nucleobase protecting group and optionally the phosphorous protecting group is deprotected after reacting the target oligonucleotide with the orthoester linker.

12. The method of claim 7 , wherein the cleavage of the oligonucleotide-orthoester linker conjugate from the solid support and the deprotection of the nucleobase protecting group and optionally of the phosphorus protecting group is performed in a single reaction.

13. The method of claim 7 , wherein the RNA comprises at least 70 nucleotides.

14. The method of claim 1 , wherein the orthoester linker is a compound of formula (Ia):

wherein each of R, R′, R″, and R′″ is independently a C 1 -C 24 alkyl, a C 2 -C 24 alkenyl, a C 2 -C 24 alkynyl, a halosubstituted C 1 -C 24 alkyl, a halosubstituted C 2 -C 24 alkenyl, a halosubstituted C 2 -C 24 alkynyl, a carbocyclyl, a heteroalkyl, an aryl, a heteroaryl, a heterocycle or any substituted equivalents, with a proviso that R may be a hydrogen.

15. The method of claim 14 , wherein R is H or C 1 -C 3 alkyl and R′, R″, R′″ are each independently an aryl or a substituted aryl.

16. The method of claim 14 , wherein R is H or CH 3 and R′, R″, R′″ are phenyl or a substituted phenyl.

17. The method of claim 14 , wherein the orthoester linker is a compound of formula (Ia) and at least one of R, R′, R″, and R′″ is a hydrophobic tag, partial hydrophobic tag, fluorous tag, or partial fluorous tag.

18. The method of claim 1 , wherein the orthoester linker is a compound of formula (Ib):

wherein AfTg is an affinity tag and R, R′, R″ are each independently AfTg-L or a C 1 -C 24 alkyl, a C 2 -C 24 alkenyl, a C 2 -C 24 alkynyl, a halosubstituted C 1 -C 24 alkyl, a halosubstituted C 2 -C 24 alkenyl, a halosubstituted C 2 -C 24 alkynyl, a carbocyclyl, a heteroalkyl, an aryl, a heteroaryl, a heterocycle, any substituted equivalents or a combination thereof provided that the total number of carbons doesn't exceed 24; and
wherein L is a covalent bond or a straight, branched, mono- or poly-cyclic, saturated, partially unsaturated or unsaturated C 1 -C 12 hydrocarbon chain that is optionally substituted with F, Cl, Br, I or C 1 -C 3 alkyls and optionally interspersed with heteroatoms independently selected from: O, S, N, or with groups independently selected from S—S, NR a , NR a —CO, —CO—NR a —CO—NR b , CO wherein R a , R b are each independently H or C 1 -C 6 alkyl.

19. The method of claim 1 , wherein the orthoester linker is a compound of formula (I)

wherein each of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 is independently H, 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, or any substituted equivalents or combination thereof provided that the total number of carbons doesn't exceed 24; R 8 and R 9 are H; X is H, methyl or an electron withdrawing group; 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.

20. The method of claim 19 , wherein at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 or R 7 is a fluorosubstituted alkyl, a fluorosubstituted alkenyl, a fluorosubstituted alkynyl, a fluorosubstituted carbocyclyl, a fluorosubstituted heteroalkyl, a fluorosubstituted heteroalkenyl, a fluorosubstituted heteroalkynyl, or a fluorosubstituted heterocyclyl.

21. The method of claim 1 , wherein the affinity tag is a fluorous or a hydrophobic tag with a cLogP value of at least 3, wherein the cLogP value is the calculated ratio of solubility of the affinity tag in octanol and water.

22. The method of claim 1 , wherein the orthoester linker is:

23. The method of claim 1 , wherein the affinity tag is a fluorosubstituted alkyl, a fluorosubstituted alkenyl, a fluorosubstituted alkynyl, or a fluorosubstituted carbocyclyl.

Assignees

• Agilent Technologies Inc

Inventors

• Dellinger Douglas J
• Myerson Joel
• Smart Brian

Classifications

• C07H21/02

  with ribosyl as saccharide radical · CPC title

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• C07H1/06 Primary

  Separation; Purification · CPC title

• C07D317/34

  Oxygen atoms · CPC title

• C07D403/06

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