# US 7,612,197 B2 · Thermolabile hydroxyl protecting groups and methods of use

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- Number: US 7,612,197 B2
- Document ID: US7612197B2
- Status: Granted
- Kind: B2
- Application number: 10/556,219
- Filed: 2004-05-07
- Published: 2006-12-14
- Granted: 2009-11-03
- Earliest claimed: 2003-05-09
- Assignees: United States of America, repesented by the Secretary, Department of Health and Human Services
- Inventors: Serge L Beaucage; Marcin K Chmielewski
- Web page: https://patentgrep.com/patent/US7612197B2

## CPC

- C07H 19/20: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 19/04: Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H 19/06: Pyrimidine radicals
- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 19/16: Purine radicals
- C07H 21/02: with ribosyl as saccharide radical
- C12C 11/02: Pitching yeast
- Y02P 20/55: Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
- 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)
- C12C: BEER; PREPARATION OF BEER BY FERMENTATION (ageing or ripening by storing C12H1/22; methods for reducing the alcohol content after fermentation C12H3/00; methods for increasing the alcohol content after fermentation C12H6/00; venting devices for casks, barrels or the like C12L9/00); PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

Provided is a hydroxyl-protected alcohol comprising a thermolabile hydroxyl-protecting group comprising a 2-pyridyl substituent and a precursor of the thermolabile hydroxyl-protected alcohol. An exemplary thermolabile hydroxyl-protected alcohol is represented by the formula Pg-O—R, wherein Pg is a protecting group of the formula: (Formula) wherein: A is a 2-pyridyl; Z is CH2 or NR1; R1, R2, R2′, R3 and R3′ are the same or different and each can be, e.g., H, alkyl, or alkyl comprising an aryl substituent; W is CO, CS, or SO; and R is the organic residue of the hydroxyl-protected alcohol. Also provided is a method of producing an alcohol, which method comprises heating the hydroxyl-protected alcohol, which optionally may be obtained from a precursor, at a temperature effective to cleave the hydroxyl-protecting group. The method can be used to produce oligonucleotides.

## Claims

### Claim 1

A hydroxyl-protected alcohol of the formula Pg-O—R, wherein Pg is a protecting group of the formula:
wherein:
A is a 2-pyridyl group of the formula:
Z is CH2 or NR1;
R1, R2, R2′, R3 and R3′ are the same or different and each is H, a saturated alkyl, an unsaturated alkyl, an aryl, a saturated alkyl comprising an aryl substituent, or an unsaturated alkyl comprising an aryl substituent, wherein the saturated alkyl, unsaturated alkyl and aryl portions are optionally substituted;
R4, R5, R6, and R7 are the same or different and each is H, an amino, a monoalkylamino, a dialkylamino, a hydroxy, an alkoxy, a halogen, an amide, a saturated alkyl, an unsaturated alkyl, an aryl, a saturated alkyl comprising an aryl, or an unsaturated alkyl comprising an aryl;
W is CO; and
R is a nucleoside, an oligonucleotide, or an oligomer comprising a nucleoside,
wherein the protecting group is thermolabile.

### Claim 2 (depends on 1)

The hydroxyl-protected alcohol of claim 1, wherein Z is NR1.

### Claim 3 (depends on 2)

The hydroxyl-protected alcohol of claim 2, wherein R1 is H or a saturated alkyl.

### Claim 4 (depends on 1)

The hydroxyl-protected alcohol of claim 1, wherein, when R1, R2, R2′, R3 or R3′is a saturated alkyl, an unsaturated alkyl, a saturated alkyl comprising an aryl substituent, or an unsaturated alkyl comprising an aryl substituent, the unsaturated alkyl or saturated alkyl is unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of a halogen, a saturated alkyl, an unsaturated alkyl, a hydroxyl, an alkoxy, an aryloxy, an aralkoxy, an ester, an amide, a sulfhydryl, an alkyl sulfide, an aryl sulfide, an alkyl sulfoxide, an aryl sulfoxide, an alkyl sulfonyl, an aryl sulfonyl, a keto, a thioketo, a cyano, and a nitro.

### Claim 5 (depends on 1)

The hydroxyl-protected alcohol of claim 1, wherein Pg is selected from the group consisting of:

### Claim 6

A hydroxyl-protected alcohol of the formula Pg-O—R, wherein Pg is a protecting group of the formula:
wherein:
A is a 2-pyridyl group of the formula:
Z is CH2 or NR1;
R1, R2, R2′, R3 and R3′ are the same or different and each is H, a saturated alkyl, an unsaturated alkyl, an aryl, a saturated alkyl comprising an aryl substituent, or an unsaturated alkyl comprising an aryl substituent, wherein the saturated alkyl, unsaturated alkyl and aryl portions are optionally substituted;
R4, R5, R6, and R7 are the same or different and each is H, an amino, a monoalkylamino, a dialkylamino, a hydroxy, an alkoxy, a halogen, an amide, a saturated alkyl, an unsaturated alkyl, an aryl, a saturated alkyl comprising an aryl, or an unsaturated alkyl comprising an aryl;
W is CO; and
R is the organic residue of the hydroxyl-protected alcohol of the formula:
wherein:
Q is a nucleoside, an oligonucleotide, or an oligomer comprising a nucleoside;
X is O, S or Se;
R8 is H, a protecting group or a solid support;
R9 is H or a protecting group;
B is a labeling group, a saturated alkyl, an unsaturated alkyl, a cycloalkyl, an aryl, a heteroaryl comprising from 5 to about 10 atoms in the ring skeleton thereof, a heterocycloalkyl comprising from 3 to about 10 atoms in the ring skeleton thereof, a saturated alkyl comprising an aryl substituent, an unsaturated alkyl comprising an aryl substituent, an amino, an alkylamino, a dialkylamino, a purine, a pyrimidine, adenine, guanine, cytosine, uracil, or thymine, wherein B is unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of a nucleobase protecting group, R10, OR10, NHR10, NR10R11, N═CH—NR10aR11a, N═C(alkyl)-NR10aR11a, CN, NO2, N3, and a halogen, wherein R10 and R11 are the same or different and each is H, a saturated alkyl, an unsaturated alkyl, a keto, or a thioketo, and R10a and R11 are the same or different and each is a saturated alkyl or an unsaturated alkyl, or, R10a and R11a, together with the nitrogen atom to which they are bonded, form a heterocycle containing 3 to about 7 atoms in the ring skeleton thereof; and
E is H, a halogen, OR12, NHR12, or NR12R13, wherein R12 and R13 are the same or different and each is H, a protecting group, a saturated alkyl, an unsaturated alkyl, a keto or a thioketo;
wherein the protecting group is thermolabile.

### Claim 7 (depends on 1)

A method of producing an alcohol, the method comprising heating the hydroxyl-protected alcohol of claim 1, so as to deprotect the hydroxyl-protected alcohol, whereupon an alcohol is produced.

### Claim 8 (depends on 7)

The method of claim 7, wherein A is 2-pyridyl or 2-pyridyl having an amino substituent.

### Claim 9 (depends on 8)

The method of claim 8, wherein the hydroxyl-protected alcohol is provided by reducing a precursor of the formula:
wherein
A′ is a 2-pyridyl comprising a nitro-substituent, a 2-pyridyl-1-oxide, or a 2- pyridyl-1-oxide comprising a nitro substituent;
Z is CH2 or NR1,
R1, R2, R2′, R3 and R3′ are the same or different and each is H, a saturated alkyl, an unsaturated alkyl, an aryl, a saturated alkyl comprising an aryl substituent, or an unsaturated alkyl comprising an aryl substituent, wherein the saturated alkyl, unsaturated alkyl and aryl portions are optionally substituted;
W is CO; and
R is the organic residue of the hydroxyl-protected alcohol,
so as to produce the hydroxyl-protected alcohol.

### Claim 10 (depends on 9)

The method of claim 9, wherein A′ is selected from the group consisting of

### Claim 11 (depends on 9)

The method of claim 9, wherein, when R1, R2, R2′, R3 or R3′is a saturated alkyl, an unsaturated alkyl, a saturated alkyl comprising an aryl substituent, or an unsaturated alkyl comprising an aryl substituent, the unsaturated alkyl or saturated alkyl is unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of a halogen, a saturated alkyl, an unsaturated alkyl, a hydroxy, an alkoxy, an aryloxy, an aralkoxy, an ester, an amide, a sulfhydryl, an alkyl sulfide, an aryl sulfide, an alkyl sulfoxide, an aryl sulfoxide, an alkyl sulfonyl, an aryl sulfonyl, a keto, a thioketo, a cyano, and a nitro.

### Claim 12 (depends on 9)

The method of claim 9, wherein the precursor is of the formula:

### Claim 13 (depends on 7)

The method of claim 7, wherein the hydroxyl-protected alcohol is a 2-(2-pyridyl)aminoethoxycarbonyl protected alcohol.

### Claim 14 (depends on 13)

The method of claim 13, wherein the protecting group is selected from the group consisting of:

### Claim 15 (depends on 1)

A method of producing an oligonucleotide, the method comprising:
(a) reacting a nucleophile with an electrophile to produce an adduct of the formula:
(b) reacting the adduct obtained in step (a) with a reagent selected from the group consisting of oxidizing agents, sulfurizing agents, and selenizing agents to produce a hydroxyl-protected oligonucleotide of the formula:
(c) thermally cleaving Pg to produce a nucleophile of the formula;
(d) optionally repeating steps (a)-(c) to produce an oligomer of the formula:
(e) optionally removing R14; and
(f) optionally removing R15;
wherein:
Pg is a thermolabile hydroxyl-protecting group as defined in claim 1;
n is an integer from 2 to about 300;
R14 is a protecting group or a solid support;
R15 R is a protecting group;
Q and Q′ are the same or different and each is a nucleoside, an oligonucleotide, or an oligomer comprising a nucleoside; and
X is O, S or Se.

### Claim 16

An alcohol protected with a hydroxyl-protecting group selected from the group consisting of a 2-(2-pyridyl)aminoethoxycarbonyl and a 3-(2-pyridyl) propoxycarbonyl, wherein the alcohol is a nucleoside, an oligonucleotide, or an oligomer comprising a nucleoside.

### Claim 17

An alcohol protected with a hydroxyl-protecting group selected from the group consisting of a 2-(substituted-2-pyridyl)aminoethoxycarbonyl and a 3-(substituted-2-pyridyl) propoxycarbonyl, wherein the 2-pyridyl ring is substituted with a nitro substituent, a 1-oxide substituent, or a nitro substituent and a 1-oxide substituent.

### Claim 18 (depends on 2)

The hydroxyl-protected alcohol of claim 2, wherein R1 is methyl.

### Claim 19 (depends on 1)

The hydroxyl-protected alcohol of claim 1, wherein, when R1, R2, R2′, R3 or R3′ is an aryl, a saturated alkyl comprising an aryl substituent, or an unsaturated alkyl comprising an aryl substituent, the aryl or aryl substituent is unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of a halogen, a saturated alkyl, an unsaturated alkyl, a hydroxy, an alkoxy, an aryloxy, an aralkoxy, an ester, an amide, a sulfhydryl, an alkyl sulfide, an aryl sulfide, an alkyl sufoxide, an aryl sulfoxide, an alkylsulfonyl, an arylsulfonyl, a keto, a thioketo, a cyano, a nitro, an amino, a monoalkylamino, and a dialkylamino.

### Claim 20 (depends on 1)

The hydroxyl-protected alcohol of claim 1, wherein, when R1, R2, R2′, R3 or R3′ is a saturated alkyl, an unsaturated alkyl, a saturated alkyl comprising an aryl substituent, or an unsaturated alkyl comprising an aryl substituent, the unsaturated alkyl or saturated alkyl is unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of a halogen, a saturated alkyl, an unsaturated alkyl, a hydroxyl, an alkoxy, an aryloxy, an aralkoxy, an ester, an amide, a sulfhydryl, an alkyl sulfide, an aryl sulfide, an alkyl sulfoxide, an aryl sulfoxide, an alkyl sulfonyl, an aryl sulfonyl, a cyano, and a nitro.

### Claim 21 (depends on 1)

The hydroxyl-protected alcohol of claim 1, wherein, when R1, R2, R2′, R3 or R3′ is an aryl, a saturated alkyl comprising an aryl substituent, or an unsaturated alkyl comprising an aryl substituent, the aryl or aryl substituent is unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of a halogen, a saturated alkyl, an unsaturated alkyl, a hydroxy, an alkoxy, an aryloxy, an aralkoxy, an ester, an amide, a sulfhydryl, an alkyl sulfide, an aryl sulfide, an alkyl sufoxide, an aryl sulfoxide, an alkylsulfonyl, an arylsulfonyl, a cyano, a nitro, an amino, a monoalkylamino, and a dialkylamino.

### Claim 22 (depends on 1)

The hydroxyl-protected alcohol of claim 1, wherein R2 and R2′ are H.

### Claim 23 (depends on 1)

The hydroxyl-protected alcohol of claim 1, wherein R3 and R3′ are H.

### Claim 24 (depends on 1)

The hydroxyl-protected alcohol of claim 1, wherein one of R3 and R3′ is H and the other is an aryl.

### Claim 25 (depends on 1)

The hydroxyl-protected alcohol of claim 1, wherein, when R1, R2, R2′, R3 or R3′ is an unsaturated alkyl or an unsaturated alkyl comprising an aryl substituent, the unsaturated alkyl is an alkenyl or an alkynyl.

### Claim 26

A hydroxyl-protected alcohol of the formula Pg-O—R, wherein Pg is a protecting group of the formula:
wherein:
A is a 2-pyridyl group of the formula:
Z is CH2 or NR1;
R1, R2, R2′, R3 and R3′ are the same or different and each is H, a saturated alkyl, an unsaturated alkyl, an aryl, a saturated alkyl comprising an aryl substituent, or an unsaturated alkyl comprising an aryl substituent, wherein the saturated alkyl, unsaturated alkyl and aryl portions are optionally substituted;
R4, R5, R6, and R7 are the same or different and each is H, an amino, a monoalkylamino, a dialkylamino, a hydroxy, an alkoxy, a halogen, an amide, a saturated alkyl, an unsaturated alkyl, an aryl, a saturated alkyl comprising an aryl, or an unsaturated alkyl comprising an aryl;
W is CO; and
R is the organic residue of the hydroxyl-protected alcohol,
wherein when Z is CH2, at least one of R2, R2′, R3, R3′, R4, R5, R6, and R7 is not H, and
wherein the protecting group is thermolabile.

### Claim 27

A hydroxyl-protected alcohol of the formula Pg-O—R, wherein Pg is a protecting group of the formula:
wherein:
A is a 2-pyridyl group of the formula:
Z is CH2 or NR1;
R1, R2, R2′, R3 and R3′ are the same or different and each is H, a saturated alkyl, an unsaturated alkyl, an aryl, a saturated alkyl comprising an aryl substituent, or an unsaturated alkyl comprising an aryl substituent, wherein the saturated alkyl, unsaturated alkyl and aryl portions are optionally substituted;
R4, R5, R6, and R7 are the same or different and each is H, an amino, a monoalkylamino, a dialkylamino, a hydroxy, an alkoxy, a halogen, an amide, a saturated alkyl, an unsaturated alkyl, an aryl, a saturated alkyl comprising an aryl, or an unsaturated alkyl comprising an aryl;
W is CO; and
R is the organic residue of the hydroxyl-protected alcohol,
wherein R is not a low molecular weight aromatic alcohol, and
wherein the protecting group is thermolabile.