# US 7,465,544 B2 · Synthetic cofactor analogs of S-adenosylmethionine as ligatable probes of biological methylation and methods for their use

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- Number: US 7,465,544 B2
- Document ID: US7465544B2
- Status: Granted
- Kind: B2
- Application number: 11/330,370
- Filed: 2006-01-11
- Published: 2007-07-12
- Granted: 2008-12-16
- Earliest claimed: 2006-01-11
- Assignees: Wisconsin Alumni Research Foundation
- Inventors: Scott R. Rajski; Lindsay R. Comstock; Rachel L. Weller
- Web page: https://patentgrep.com/patent/US7465544B2

## CPC

- C07H 19/16: Purine radicals
- C07H 19/20: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 21/04: with deoxyribosyl as saccharide radical
- 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

The present invention discloses compounds and methods used to specifically target substrates of methylation by S-adenosyl-L-methionine (SAM)-dependent methyltransferases. The substrates can be peptides, single stranded nucleic acids or double stranded nucleic acids, including RNA, DNA and PNA or phospholipids. The compounds disclosed are SAM analogs that are ligated to a methylation site by the methyltransferase. Also disclosed, are reacting groups that are ligatable to the cofactor analogs and can also be used as detectable labels. The reacting group can be used to cleave the substrate providing a methylation footprint. The invention can be used clinically to determine methylation state of a gene or gene promoter such as those involved in imprinting and transcription. In some preferred embodiments, the invention includes a kit, which can include one or more suitable SAM analogs and may include one or more detectable labels. In other preferred embodiments, the invention includes a pharmaceutical composition.

## Claims

### Claim 1

A method of derivatizing a methyltransferase substrate comprising:
providing (i) a methyltransferase substrate selected from the group consisting of a nucleic acid and a polynucleotide, (ii) a methyltransferase, and (iii) a cofactor analog, wherein the cofactor analog is modified at its 5′ carbon atom to result in site-specific covalent ligation of the cofactor analog to the methyltransferase substrate by the methyltransferase and wherein the cofactor analog further includes at least one reacting group allowing for ligation of the cofactor analog to a detectable label; and
reacting the methyltransferase substrate, the methyltransferase, and the cofactor analog under conditions wherein the co-factor analog ligates to the methyltransferase substrate in a reaction catalyzed by the methyltransferase.

### Claim 2

A cofactor analog of the Formula I:
wherein R1 is attached through a nitrogen atom and is selected from the group consisting of
X is a leaving group selected from the group consisting of I, Br, Cl, F, tosylate (OTs), mesylate (OMs) or triflate (OTf);
R3 is selected from the group consisting of:
R2 is independently selected from the group consisting of H, —N3, —NH(CH2)n2N3, —NH(CH2CH2O)n1N3, —NH(CH2)n2C≡CH, —NH(CH2O)n1C≡CH,
R4 is selected from the group consisting of H, CH3, and triarylphosphine;
n1 is an integer from 1-10 inclusive and n2 is an integer from 2-10 inclusive; and zwitterionic forms and salts thereof;
provided that when R1 is
R2 is not N3 or H.

### Claim 3 (depends on 2)

A method of determining the methylation state of substrate comprising:
(a) providing a substrate selected from the group consisting of a nucleic acid and a polynucleotide;
(b) contacting the substrate with the cofactor analog of claim 2, in the presence of a methyltransferase to form a detectable complex; and then
(c) detecting the detectable complex, wherein the detected complex identifies an unmethylated methylation site on the substrate, thereby determining the methylation state of the substrate.

### Claim 4 (depends on 3)

The method of claim 3, wherein detecting in step (c) is accomplished by gel electrophoresis, phosphorimaging, spectroscopy or photoluminescence.

### Claim 5 (depends on 3)

The method of claim 3, further comprising, prior to step (c), contacting the detectable complex with Cu(I) to cleave the substrate.

### Claim 6 (depends on 5)

The method of claim 5, further comprising electrophoretically separating the cleaved substrate thereby resulting in a methylation footprint of the substrate.

### Claim 7 (depends on 2)

A kit for determining methylation state of a methyltransferase substrate, the kit comprising a cofactor analog according to claim 2 disposed in a first container, and instructions for application of the kit.

### Claim 8 (depends on 7)

The kit of claim 7, further comprising a methyltransferase disposed in a second container.

### Claim 9 (depends on 2)

The cofactor analog of claim 2, wherein R1 is selected from the group consisting of:

### Claim 10 (depends on 2)

The cofactor analog of claim 2, wherein R1 is selected from the group consisting of:

### Claim 11 (depends on 2)

The cofactor analog of claim 2, wherein R1 is selected from the group consisting of:

### Claim 12 (depends on 1, 2)

The method of claim 1, wherein the cofactor analog is selected from the analogs recited in claim 2.

### Claim 13

A cofactor analog having the formula:
wherein X is a leaving group selected from the group consisting of F, Cl, Br, I, tosylate (OTs), mesylate (OMs) or triflate (OTf), zwitterionic forms and salts thereof.

### Claim 14 (depends on 13)

The cofactor analog of claim 13, wherein the cofactor analog is selected from the group consisting of 8-azido-5′-aziridino-5′-deoxy adenosine, 8-(4″-azido-butylamino)-5′-aziridino-5′-deoxy adenosine, 5′-(diaminobutyric acid)-N-iodoethyl-5′deoxyadenosine ammonium hydrochloride, 5′-(Nα-Boc diaminobutyric acid-O-tert-Butyl ester)-N-hydroxyethyl-5′-deoxy-2′,3′-bis-(O-triethylsilyl)adenosine, 5′-(diaminobutyric acid)-N-iodoethyl-5′-deoxy-8-aminobutylazido adenosine (bis)hydrochloride and 5′-(diaminobutyric acid)-N-iodoethyl-5′-deoxy-8-azidoadenosine bis(hydrochloride).

### Claim 15 (depends on 1, 13)

The method of claim 1, wherein the cofactor analog is selected from the analogs recited in claim 13.

### Claim 16

A cofactor analog of the Formula I:
wherein R1 is selected from the group consisting of
R2 is selected from the group consisting of H, —N3, —NH(CH2)n1N3, —NH(CH2CH2O)n1N3, —NH(CH2)n2C≡CH, —NH(CH2O)n1C≡CH,
Su is a substrate selected from the group consisting of a nucleic acid, a polynucleotide, a phospholipid, a peptide, a polypeptide, a peptide nucleic acid and a protein;
n1 is an integer from 1-10 inclusive and n2 is an integer from 2-10 inclusive, and zwitterionic forms and salts thereof
provided that when R1 is
R2 is not N3 or H.

### Claim 17 (depends on 16)

The cofactor analog of claim 16, wherein the analog is selected from the group consisting of:
R4 is selected from the group consisting of H, CH3 and
and zwitterionic forms and salts thereof.