# US 11,299,483 B2 · Orthoester compositions for affinity purification of oligonucleotides

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- Number: US 11,299,483 B2
- Document ID: US11299483B2
- Status: Granted
- Kind: B2
- Application number: 16/640,005
- Filed: 2018-08-17
- Published: 2020-06-18
- Granted: 2022-04-12
- Earliest claimed: 2017-08-18
- Assignees: Agilent Technologies, Inc.
- Applicants: Agilent Technologies, Inc.
- Inventors: Douglas J. Dellinger; Joel Myerson; Brian Smart
- Web page: https://patentgrep.com/patent/US11299483B2

## CPC

- C07D 405/12: linked by a chain containing hetero atoms as chain links
- C07D 317/34: Oxygen atoms
- C07D 403/06: linked by a carbon chain containing only aliphatic carbon atoms
- C07H 21/02: with ribosyl as saccharide radical
- C07H 21/04: with deoxyribosyl as saccharide radical
- C07H 1/06: Separation; Purification
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- 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)

## 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.

## Claims

### 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 7)

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.

### Claim 9 (depends on 7)

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

### Claim 10 (depends on 9)

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

### Claim 11 (depends on 9)

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.

### Claim 12 (depends on 7)

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.

### Claim 13 (depends on 7)

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

### Claim 14 (depends on 1)

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 C1-C24 alkyl, a C2-C24 alkenyl, a C2-C24 alkynyl, a halosubstituted C1-C24 alkyl, a halosubstituted C2-C24 alkenyl, a halosubstituted C2-C24 alkynyl, a carbocyclyl, a heteroalkyl, an aryl, a heteroaryl, a heterocycle or any substituted equivalents, with a proviso that R may be a hydrogen.

### Claim 15 (depends on 14)

The method of claim 14, wherein R is H or C1-C3 alkyl and R′, R″, R′″ are each independently an aryl or a substituted aryl.

### Claim 16 (depends on 14)

The method of claim 14, wherein R is H or CH3 and R′, R″, R′″ are phenyl or a substituted phenyl.

### Claim 17 (depends on 14)

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.

### Claim 18 (depends on 1)

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 C1-C24 alkyl, a C2-C24 alkenyl, a C2-C24 alkynyl, a halosubstituted C1-C24 alkyl, a halosubstituted C2-C24 alkenyl, a halosubstituted C2-C24 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 C1-C12 hydrocarbon chain that is optionally substituted with F, Cl, Br, I or C1-C3 alkyls and optionally interspersed with heteroatoms independently selected from: O, S, N, or with groups independently selected from S—S, NRa, NRa—CO, —CO—NRa—CO—NRb, CO wherein Ra, Rb are each independently H or C1-C6 alkyl.

### Claim 19 (depends on 1)

The method of claim 1, wherein the orthoester linker is a compound of formula (I)
wherein each of R1, R2, R3, R4, R5, R6 and R7 is independently H, C1-C24 alkyl, C1-C24 heteroalkyl, C2-C24 alkenyl, C2-C24 heteroalkenyl, C2-C24 alkynyl, C2-C24 heteroalkynyl, halogen, aryl, heteroaryl, heterocyclyl, carbocyclyl, or any substituted equivalents or combination thereof provided that the total number of carbons doesn't exceed 24; R8 and R9 are H; X is H, methyl or an electron withdrawing group; and n is 0, 1, or 2, wherein at least one of R1, R2, R3, R4, R5, R6 or R7 comprises an affinity tag.

### Claim 20 (depends on 19)

The method of claim 19, wherein at least one of R1, R2, R3, R4, R5, R6 or R7 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.

### Claim 21 (depends on 1)

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.

### Claim 22 (depends on 1)

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

### Claim 23 (depends on 1)

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