Compositions and Methods for Modification of Biomolecules

Patent metadata

Field	Value
Publication number	US-2016243135-A1
Application number	US-201615043181-A
Country	US
Kind code	A1
Filing date	Feb 12, 2016
Priority date	Nov 1, 2004
Publication date	Aug 25, 2016
Grant date	—

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).

First claim

1 .- 9 . (canceled)

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.

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

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

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

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

15 . (canceled)

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

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

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.

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

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

21 . The method of claim 10 , wherein the modified cycloalkyne has the formula
Y—R 1 —X
wherein: X is a cycloalkyne group substituted with R 1 ; 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 R 1 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.

22 . The method of claim 21 , wherein the modified cycloalkyne is selected from:
a) a compound of the formula:

wherein each of R 1 -R 6 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—(CH 2 ) 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); —(CH 2 ) n —W—(CH 2 ) 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 —(CH 2 ) n —Z (where: n is an integer from 1-4, and wherein Z is nitro, cyano, sulfonic acid, or a halogen);
wherein R 3 is optionally linked to R 4 through Y 1 thus forming a substituted or unsubstituted cycloalkyl or substituted or unsubstituted heterocycloalkyl substituient on the cycloalkyne ring, wherein Y 1 , if present, is C, O, N, or S; and

wherein each of R 1 -R 6 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 X 1 -X 6 are carbon atoms;
wherein one of X 1 -X 6 is N, O, P or S;
wherein each of R 1 -R 6 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—(CH 2 ) 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); —(CH 2 ) n —W—(CH 2 ) 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 —(CH 2 ) 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 R 1 -R 6 is optionally independently linked to Y;
f) a compound of the formula:

wherein each of R 1 -R 6 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—(CH 2 ) 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); —(CH 2 ) n —W—(CH 2 ) 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 —(CH 2 ) 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 R 1 -R 6 is optionally independently linked to Y;
g) a compound of the formula:

where R 1 and R 2 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 R 1 and R 2 is optionally independently linked to Y; and
h) a compound of the formula:

wherein Z is —CH 2 —CH 2 —, —CH═CH—, —CH≡CH—, —Se(O)O—, —C(O)O—, —C(R 3 )(R 4 )O—, —N(R 5 )N(R 6 )—, —CH(OR 7 )CH 2 —, or —S(O)O—;
wherein R 1 and R 2 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 k

Assignees

• Univ California

Inventors

• Bertozzi Carolyn Ruth
• Agard Nicholas J
• Prescher Jennifer A
• Baskin Jeremy Michael
• Sletten Ellen May

Classifications

• C07C59/72

  containing six-membered aromatic rings and other rings · CPC title

• C07D207/416

  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 · CPC title

• C07C49/457

  containing halogen · CPC title

• C07C57/26

  containing rings other than six-membered aromatic rings · CPC title

• C07D495/04

  Ortho-condensed systems · CPC title