# US 2017/0283453 A1 · Cofactor analogues for methyltransferases

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- Number: US 2017/0283453 A1
- Document ID: US20170283453A1
- Status: Published application
- Kind: A1
- Application number: 15/507,628
- Filed: 2015-08-31
- Published: 2017-10-05
- Earliest claimed: 2014-08-29
- Assignees: Katholieke Universiteit Leuven
- Applicants: Katholieke Universiteit Leuven
- Inventors: Robert NEELY; Volker LEEN; Johan HOFKENS
- Web page: https://patentgrep.com/patent/US20170283453A1

## CPC

- C07H 19/16: Purine radicals
- C07H 19/167: with ribosyl as the saccharide radical
- C09B 11/22: containing OH groups bound to an aryl nucleus {and their ethers and esters}
- C09B 23/04: one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
- C12N 9/1007: {Methyltransferases (general) (2.1.1.)}
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Y 201/01072: Site-specific DNA-methyltransferase (adenine-specific) (2.1.1.72)
- C12Q 2600/154: Methylation markers
- 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)
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Y: ENZYMES

## Abstract

Cofactor analogues for methyltransferases are disclosed. The compounds are represented by formula (I) wherein R1 is COOH or COO—; X is an organic or inorganic anion carrying one or more negative charges; Y and Y′ are H, or an alkyl; R2 is NH2, NHBoc, or H; and Z is S or Se. R comprises a carbon-carbon double bond, carbon-oxygen double bond, carbon-sulfur double bond, carbon-nitrogen double bond, a carbon-carbon triple bond, carbon-nitrogen triple bond, an aromatic carbocyclic or heterocyclic system in β-position to the sulfonium center, unsaturated c-c bond, or c-heteroatom bond where the heteroatom is O, N, S.

## Claims

### Claim at position 1

1-23. (canceled)

### Claim 24

A compound represented by formula (I)
wherein
R1 is COOH or COO—;
X is an organic or inorganic anion carrying one or more negative charges;
Y and Y′ are H, or an alkyl;
R2 is NH2, NHBoc, or H; and
Z is S or Se
R comprises a carbon-carbon double bond, carbon-oxygen double bond, carbon-sulfur double bond, carbon-nitrogen double bond, a carbon-carbon triple bond, carbon-nitrogen triple bond, an aromatic carbocyclic or heterocyclic system in β-position to the sulfonium center, unsaturated c-c bond, or c-heteroatom bond where the heteroatom is O, N, S.

### Claim 25 (depends on 24)

A compound according to claim 24, wherein
R, R2, Y, and Y′ are selected from the following:
Y
Y′
R2
R
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
NH2
H
H

### Claim 26 (depends on 25)

A compound according to claim 25, selected from the group comprising:
Compound
Name
(Benzyl)(5′-Deoxyadenosyl)-(N- Boc-L-Cysteine)sulfonium salt
(Allyl)(5′-Deoxyadenosyl)-(N- Boc-L-Cysteine)sulfonium salt
(8-Azido-oct-2-ene)(5′- Deoxyadenosyl)-(N-Boc-L- Cysteine)sulfonium salt
(6-Azido-hex-2-yne)(5′- Deoxyadenosyl)-(N-Boc-L- Cysteine)sulfonium salt
(6-Amino-hex-2-yne)(5′- Deoxyadenosyl)-(N-Boc-L- Cysteine)sulfonium salt
(Ethyl Carboxymethyl)(5′- Deoxyadenosyl)-(N-Boc-L- Cysteine)sulfonium salt
(Benzyl)(5′-Deoxyadenosyl)-(3- Propionate)sulfonium salt
(Benzyl)(5′-Deoxy-N6,N6- dimethyladenosyl)-(N-Boc-L- Cysteine) sulfonium salt
(6-Azido-hex-2-yne)(5′- Deoxyadenosyl)-(L- Cysteine)sulfonium salt
(6-Amino-hex-2-yne)(5′- Deoxyadenosyl)-(L- Cysteine)sulfonium salt
Fluorescent sulfonium salt
Fluorescent sulfonium salt II
Fluorescent sulfonium salt III
Dethiobiotin sulfonium cofactor.

### Claim 27 (depends on 24)

A compound according to claim 24, wherein said organic or inorganic anion is selected from trifluoroacetate, formate, halide and sulfonate.

### Claim 28 (depends on 24)

A compound according to claim 24, wherein R additionally comprises at least one member selected from functional groups, heavy atoms or heavy atom clusters suitable for phasing of X-ray diffraction data, radioactive or stable rare isotopes, and a residue of a member selected from fluorophores, fluorescence quenchers, affinity tags, crosslinking agents, nucleic acid cleaving reagents, spin labels, chromophores, proteins, peptides or amino acids which may optionally be modified, nucleotides, nucleosides, nucleic acids which may optionally be modified, carbohydrates, lipids, transfection reagents, intercalating agents, nanoparticles and beads.

### Claim 29 (depends on 28)

A compound according to claim 28, wherein said functional group is selected from an amino group, a thiol group, a 1,2-diol group, a hydrazino group, a hydroxyamino group, a haloacetamide group, a maleimide group, an aldehyde group, a ketone group, an 1,2-aminothiol group, an azido group, an alkyne group, a 1,3-diene function, a dienophilic function (e.g. activated carbon-carbon double bond), an arylhalide group, a terminal alkyne group, an arylboronic acid group, a terminal haloalkyne group, a terminal silylalkyne group and a protected amino, thiol, 1,2-diol, hydrazino, hydroxyamino, aldehyde, ketone and 1,2-aminothiol group.

### Claim 30 (depends on 28)

A compound according to claim 28, wherein said fluorophore is selected from Alexa, BODIPY, bimane, coumarin, Cascade blue, dansyl, dapoxyl, fluorescein, mansyl, MANT, Oregon green, pyrene, rhodamine, Texas red, TNS, fluorescent nanocrystals (quantom dots), a cyanine fluorophore and derivatives thereof and/or wherein said fluorescence quencher is selected from dabcyl, QSY and BHQ.

### Claim 31 (depends on 28)

A compound according to claim 28, wherein said affinity tag is selected from peptide tags, metal-chelating groups, isotope coded affinity tags, biotin, maltose, mannose, glucose, Λ/-acetylglucosamine, Λ/-acetylneuraminic acid, galactose, Λ/-acetylgalactosamine, digoxygenin and dinitrophenol.

### Claim 32 (depends on 31)

A compound according to claim 31 wherein said peptide tag is selected from his-tags, tags with metal chelating properties, strep-tags, flag-tags, c-myc-tags, HA-tags, epitopes and glutathione or wherein said metal-chelating group is selected from nitrilotriacetic acid, ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline, a crown ether and a HiS4-8 peptide.

### Claim 33 (depends on 28)

A compound according to claim 28, wherein said crosslinking agent is selected from mono- or bifunctional platinum(ll) complexes, maleimides, iodacetamides, aldehydes and photocrosslinking agents like arylazide, a diazo compound, a 2-nitrophenyl compound, psoralen and a benzophenone compound and/or wherein said heavy atom or heavy atom cluster is selected from copper, zinc, selenium, bromine, iodine, ruthenium, palladium, cadmium, tungsten, platinum, gold, mercury, bismuth, samarium, europium, terbium, uranium, Ta6Br14, Fe4S4 and P2W-|8O62 suitable for phasing X-ray diffraction data and/or wherein R comprises a nucleic acid cleaving reagent selected from the group consisting of iron-EDTA, copper-1,10-phenanthroline, acridine or a derivative thereof, an enediyne compound and a rhodium complex, more preferably selected from iron-EDTA, copper-1,10-phenanthroline and a rhodium complex.

### Claim 34 (depends on 24)

A complex of a compound according to claim 24 and a methyltransferase capable of using S-adenosyl-L-methionine (SAM or AdoMet) as a cofactor.

### Claim 35 (depends on 34)

A complex according to claim 34, wherein said methyltransferase is capable of transferring the methyl residue of S-adenosyl-L-methionine (SAM or AdoMet) onto a nucleic acid molecule, a polypeptide, a carbohydrate or a small molecule and/or wherein said methyltransferase is an orphan DNA methyltransferase or part of a bacterial restriction modification system.

### Claim 36 (depends on 34)

A complex according to claim 34, wherein said methyltransferase is selected from the group consisting of the DNA methyltransferases M.Taql, M.Hhal, M.XbahI, M.PvuII, M.BsahI, M.FokI, M.BcnlB (M2.Bcnl), M.Sssl, M.MpeI, M.PstI, M.XhoI, M.BseCI, M.M.Rsrl, M.EcoRI, or a derivative thereof.

### Claim 37 (depends on 24)

A kit comprising a compound (I) according to claim 24 packed in a container.

### Claim 38 (depends on 37)

The kit according to claim 37, said kit comprising the compound (I) and furthermore comprising a methyltransferase capable of using S-adenosyl-L-methionine (SAM or AdoMet) as a cofactor.

### Claim 39 (depends on 38)

A kit according to claim 38, wherein said compound and said methyltransferase are packed in one or more containers and/or wherein said compound and said methyltransferase are dissolved in a buffer.

### Claim 40 (depends on 24)

A pharmaceutical or diagnostic composition comprising a compound according to claim 24.

### Claim 41 (depends on 24)

A method for the preparation of a modified target molecule, the method comprising the incubation of the target molecule with a compound (I) according to claim 24 in the presence of a methyltransferase which is capable of using the compound (I) as a cofactor and under conditions which allow for the transfer of part of the compounds onto the target molecule.

### Claim 42 (depends on 24)

A method for detecting sequence-specific methylation in a biomolecule, comprising:
(a) contacting a biomolecule with an S-adenosyl-L-methionine-dependent methyltransferase in the presence of a detectable cofactor of said methyltransferase; and
(b) detecting whether the recognition site of said methyltransferase has been modified with the cofactor or a derivative thereof, wherein modification of the recognition site of said methyltransferase is indicative of an absence of methylation at said recognition site, wherein said cofactor is a compound of formula (I) according to claim 24.

### Claim 43 (depends on 24)

A method for synthesizing a compound according to claim 24, the method comprising coupling a thioether with a lactone.