# US 2016/0243135 A1 · Compositions and Methods for Modification of Biomolecules

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2016/0243135 A1
- Document ID: US20160243135A1
- Status: Published application
- Kind: A1
- Application number: 15/043,181
- Filed: 2016-02-12
- Published: 2016-08-25
- Earliest claimed: 2004-11-01
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Carolyn Ruth Bertozzi; Nicholas J. Agard; Jennifer A. Prescher; Jeremy Michael Baskin; Ellen May Sletten
- Web page: https://patentgrep.com/patent/US20160243135A1

## CPC

- A61K 31/655: Azo (—N=N—), diazo (=N2), azoxy (>N—O—N< or N(=O)—N<), azido (—N3) or diazoamino (—N=N—N<) compounds
- C07C 45/292: {with chromium derivatives}
- C07C 45/511: {involving transformation of singly bound oxygen functional groups to >C = O groups (involving two hydroxy groups C07C45/52; hydroperoxides C07C45/53)}
- C07C 45/63: by introduction of halogen; by substitution of halogen atoms by other halogen atoms
- C07C 49/457: containing halogen
- C07C 49/753: containing ether groups, [IMAGE cpc-sch-C07C-0958.gif] groups,[IMAGE cpc-sch-C07C-0959.gif] groups, or[IMAGE cpc-sch-C07C-0960.gif] groups
- C07C 57/26: containing rings other than six-membered aromatic rings
- C07C 59/72: containing six-membered aromatic rings and other rings
- C07C 63/66: Polycyclic acids with unsaturation outside the aromatic rings
- C07C 63/74: having unsaturation outside the aromatic rings
- C07C 69/708: Ethers
- C07C 69/76: Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring
- C07D 207/416: with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to other ring carbon atoms
- C07D 207/452: with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom
- C07D 225/02: not condensed with other rings
- C07D 249/16: condensed with carbocyclic rings or ring systems
- C07D 495/04: Ortho-condensed systems
- G01N 33/531: Production of immunochemical test materials
- G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins}
- G01N 33/6842: {Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins}
- C07C 2601/18: with a ring being at least seven-membered
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention provides modified cycloalkyne compounds; and method of use of such compounds in modifying biomolecules. The present invention features a cycloaddition reaction that can be carried out under physiological conditions. In general, the invention involves reacting a modified cycloalkyne with an azide moiety on a target biomolecule, generating a covalently modified biomolecule. The selectivity of the reaction and its compatibility with aqueous environments provide for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

## Claims

### Claim at position 1

-9. (canceled)

### Claim 10

A method for chemoselective modification of a target molecule comprising an azide, the method comprising:
reacting an azide of a target molecule with a modified cycloalkyne linked to a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest, or a molecule of interest, wherein said reacting produces a conjugate between the azide of the target molecule and the modified cycloalkyne.

### Claim 11 (depends on 10)

The method of claim 10, wherein the target molecule is a sugar.

### Claim 12 (depends on 11)

The method of claim 11, wherein the sugar is a substrate of sialic acid biosynthesis.

### Claim 13 (depends on 11)

The method of claim 11, wherein the sugar is mannosamine or acetylated mannosamine.

### Claim 14 (depends on 10)

The method of claim 10, wherein the target molecule is an amino acid or a polypeptide.

### Claim 15

(canceled)

### Claim 16 (depends on 10)

The method of claim 10, wherein said reacting is performed under physiological conditions.

### Claim 17 (depends on 10)

The method of claim 10, wherein the target molecule comprising the azide is expressed on a cell surface.

### Claim 18

A method for synthetically modifying a cellular component, the method comprising:
introducing an azide moiety into a cellular component, thereby generating an azide-modified cellular component; and
contacting the cell comprising the azide-modified cellular component with a reactive partner comprising a modified cycloalkyne linked to a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest, or a molecule of interest, said contacting being under physiological conditions;
wherein said contacting with said reactive partner results in reaction between the azide group of azide-modified cellular component and the cycloalkyne of the reactive partner, thereby synthetically and covalently modifying the cellular component.

### Claim 19 (depends on 18)

The method of claim 18, wherein cellular component is a polypeptide.

### Claim 20 (depends on 18)

The method of claim 18, wherein said contacting is in vivo.

### Claim 21 (depends on 10)

The method of claim 10, wherein the modified cycloalkyne has the formula
Y—R1—X
wherein:
X is a cycloalkyne group substituted with R1;
Y is H; a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest; or a molecule of interest; and
R1 is selected from a carboxylic acid, an alkyl ester, an aryl ester, a substituted aryl ester, an aldehyde, an amide, an aryl amide, an alkyl halide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone, a substituted aryl ketone, a halosulfonyl, a nitrile and a nitro.

### Claim 22 (depends on 21)

The method of claim 21, wherein the modified cycloalkyne is selected from:
a) a compound of the formula:
wherein each of R1-R6 is independently H; one or two halogen atoms; a carboxylic acid; an alkyl ester; an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol; an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester; an alkyl ketone; an aryl ketone; a substituted aryl ketone; a halosulfonyl; a nitrile; a nitro; —W—(CH2)n—Z (where: n is an integer from 1-4, wherein W, if present, is O, N, or S; and Z is nitro, cyano, sulfonic acid, or a halogen); —(CH2)n—W—(CH2)n—Z (where: n and m are each independently 1 or 2; W is O, N, S, or sulfonyl, wherein if W is O, N, or S, then Z is nitro, cyano, or halogen, and wherein and if W is sulfonyl, then Z is H); or —(CH2)n—Z (where: n is an integer from 1-4, and wherein Z is nitro, cyano, sulfonic acid, or a halogen);
wherein R3 is optionally linked to R4 through Y1 thus forming a substituted or unsubstituted cycloalkyl or substituted or unsubstituted heterocycloalkyl substituient on the cycloalkyne ring, wherein Y1, if present, is C, O, N, or S; and
wherein each of R1-R6 is optionally independently linked to Y;
b) a compound of the formula:
c) a compound of the formula:
d) a compound of the formula:
e) a compound of the formula:
wherein five of X1-X6 are carbon atoms;
wherein one of X1-X6 is N, O, P or S;
wherein each of R1-R6 is independently H; one or two halogen atoms; a carboxylic acid; an alkyl ester; an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol; an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester; an alkyl ketone; an aryl ketone; a substituted aryl ketone; a halosulfonyl; a nitrile; a nitro; —W—(CH2)n—Z (where: n is an integer from 1-4, wherein W, if present, is O, N, or S; and Z is nitro, cyano, sulfonic acid, or a halogen); —(CH2)n—W—(CH2)n—Z (where: n and m are each independently 1 or 2; W is O, N, S, or sulfonyl, wherein if W is O, N, or S, then Z is nitro, cyano, or halogen, and wherein and if W is sulfonyl, then Z is H); or —(CH2)n—Z (where: n is an integer from 1-4, and wherein Z is nitro, cyano, sulfonic acid, or a halogen); and
wherein each of R1-R6 is optionally independently linked to Y;
f) a compound of the formula:
wherein each of R1-R6 is independently H; one or two halogen atoms; a carboxylic acid; a methoxy group; an alkyl ester; an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol; an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester; an alkyl ketone; an aryl ketone; a substituted aryl ketone; a halosulfonyl; a nitrile; a nitro; —W—(CH2)n—Z (where: n is an integer from 1-4, wherein W, if present, is O, N, or S; and Z is nitro, cyano, sulfonic acid, or a halogen); —(CH2)n—W—(CH2)n—Z (where: n and m are each independently 1 or 2; W is O, N, S, or sulfonyl, wherein if W is O, N, or S, then Z is nitro, cyano, or halogen, and wherein and if W is sulfonyl, then Z is H); or —(CH2)n—Z (where: n is an integer from 1-4, and wherein Z is nitro, cyano, sulfonic acid, or a halogen); and
wherein each of R1-R6 is optionally independently linked to Y;
g) a compound of the formula:
where R1 and R2 are each independently H; one or two halogen atoms; a carboxylic acid; an alkyl ester; an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol; an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester; an alkyl ketone; an aryl ketone; a substituted aryl ketone; a halosulfonyl; a nitrile; or a nitro; and
wherein each of R1 and R2 is optionally independently linked to Y; and
h) a compound of the formula:
wherein Z is —CH2—CH2—, —CH═CH—, —CH≡CH—, —Se(O)O—, —C(O)O—, —C(R3)(R4)O—, —N(R5)N(R6)—, —CH(OR7)CH2—, or —S(O)O—;
wherein R1 and R2 are each independently H; one or two halogen atoms; a carboxylic acid; an alkyl ester; an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol; an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester; an alkyl ketone; an aryl ketone; a substituted aryl ketone; a halosulfonyl; a nitrile; or a nitro;
wherein R3 to R7 is each independently selected from H; a halogen atom; an aliphatic group, a substituted or unsubstituted alkyl group; an alkenyl group; an alkynyl group; a carboxylic acid, an alkyl ester; an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol; an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester; an alkyl ketone; an aryl ketone; a substituted aryl ketone; a halosulfonyl; a nitrile; and a nitro; and
wherein each of R1 and R2 is optionally independently linked to Y.

### Claim 23 (depends on 22)

The method of claim 22, wherein the modified cycloalkyne has the structure:
wherein each of X and X′ is independently:
(a) H;
(b) one or two halogen atoms;
(c) —W—(CH2)n—Z, wherein n is an integer from 1-4; wherein W, if present, is O, N, or S; and wherein Z is nitro, cyano, sulfonic acid, or a halogen;
(d) —(CH2)n—W—(CH2)m—Z, wherein n and m are each independently 1 or 2; wherein W is O, N, S, or sulfonyl; with the proviso that if W is O, N, or S, then Z is nitro, cyano, or halogen; and with the proviso that if W is sulfonyl, then Z is H; or
(e) —(CH2)n—Z, wherein n is an integer from 1-4; and wherein Z is nitro, cyano, sulfonic acid, or a halogen; and
R1 is selected from a carboxylic acid, an alkyl ester, an aryl ester, a substituted aryl ester, an aldehyde, an amide, an aryl amide, an alkyl halide, a thioester, a sulfonyl ester, an alkyl ketone, an aryl ketone, a substituted aryl ketone, a halosulfonyl, a nitrile, and a nitro.

### Claim 24 (depends on 22)

The method of claim 22, wherein the modified cycloalkyne has the structure:

### Claim 25 (depends on 22)

The method of claim 22, wherein the modified cycloalkyne has the structure of one of Formulas XI-XVI:
wherein R, R1, R2, and R3 are each independently H; a carboxylic acid; a methoxy; an alkyl ester; an aryl ester; a substituted aryl ester; an aldehyde; an amine; a thiol; an amide; an aryl amide; an alkyl halide; a thioester; a sulfonyl ester; an alkyl ketone; an aryl ketone; a substituted aryl ketone; a halosulfonyl; —W—(CH2)n—Z (wherein n is an integer from 1-4, wherein W, if present, is O, N, or S; and Z is nitro, cyano, sulfonic acid, or a halogen); —(CH2)n—W—(CH2)mZ (wherein n and m are each independently 1 or 2; W is O, N, S, or sulfonyl, wherein if W is O, N, or S, then Z is nitro, cyano, or halogen, and wherein and if W is sulfonyl, then Z is H); or —(CH2)n—Z (wherein n is an integer from 1-4, and wherein Z is nitro, cyano, sulfonic acid, or a halogen); and;
wherein each R, R1, R2, and R3 is optionally independently linked to Y.

### Claim 26 (depends on 21)

The method of claim 21, wherein Y is a reactive group that facilitates covalent attachment of a molecule.

### Claim 27 (depends on 26)

The method of claim 26, wherein the reactive group is selected from a carboxyl, an amine, an ester, a thioester, a sulfonyl halide, an alcohol, a thiol, a succinimidyl ester, an isothiocyanate, an iodoacetamide, a maleimide, and a hydrazine.

### Claim 28 (depends on 21)

The method of claim 21, wherein Y is a molecule of interest selected from a detectable label, a toxin, a linker, a peptide, a drug, a member of a specific binding pair, and an epitope tag.