Compositions and methods for modification of biomolecules

Patent metadata

Field	Value
Publication number	US-10434111-B2
Application number	US-201615043181-A
Country	US
Kind code	B2
Filing date	Feb 12, 2016
Priority date	Nov 1, 2004
Publication date	Oct 8, 2019
Grant date	Oct 8, 2019

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

What is claimed is:

1. 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 of the formula:
Y—R 1 —X

wherein:
X is a cyclooctyne group or a heterocyclooctyne group, substituted with R 1 , and optionally substituted with one or more additional groups;
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; and
Y is a linked 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.

2. The method of claim 1 , wherein the target molecule is a sugar.

3. The method of claim 2 , wherein the sugar is a substrate of sialic acid biosynthesis.

4. The method of claim 2 , wherein the sugar is mannosamine or acetylated mannosamine.

5. The method of claim 1 , wherein the target molecule is an amino acid or a polypeptide.

6. The method of claim 1 , wherein said reacting is performed under physiological conditions.

7. The method of claim 1 , wherein the target molecule comprising the azide is expressed on a cell surface.

8. The method of claim 1 , 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 (wherein n is an integer from 1-4, is O, N, or S; and Z is nitro, cyano, sulfonic acid, or a halogen); —(CH 2 ) n —W—(CH 2 ) m —Z (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 if W is sulfonyl, then Z is H); or —(CH 2 ) n —Z (wherein n is an integer from 1-4, and 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 substituent on the cycloalkyne ring, wherein Y 1 , is C, O, N, or S; and

wherein at least one of R 1 -R 6 is linked to Y;
b) a compound of the formula:

wherein five of X 1 -X 6 are carbon atoms;
one of X 1 -X 6 is N, O, P or S;
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 (wherein n is an integer from 1-4, W is O, N, or S; and Z is nitro, cyano, sulfonic acid, or a halogen); —(CH 2 ) n —W—(CH 2 ) m —Z (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 if W is sulfonyl, then Z is H); or —(CH 2 ) n —Z (wherein n is an integer from 1-4, and Z is nitro, cyano, sulfonic acid, or a halogen); and
wherein at least one of R 1 -R 6 is linked to Y;
c) 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 (wherein n is an integer from 1-4, W is O, N, or S; and Z is nitro, cyano, sulfonic acid, or a halogen); —(CH 2 ) n —W—(CH 2 ) m —Z (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 if W is sulfonyl, then Z is H); or —(CH 2 ) n —Z (wherein n is an integer from 1-4, and wherein Z is nitro, cyano, sulfonic acid, or a halogen); and
wherein at least one of R 1 -R 6 is linked to Y;
d) a compound of the formula:

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 ketone; an aryl ketone; a substituted aryl ketone; a halosulfonyl; a nitrile; or a nitro;
wherein one of R 1 and R 2 is linked to Y; and
e) 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—;
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;
wherein R 3 to R 7 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 one of R 1 and R 2 is linked to Y.

9. The method of claim 8 , wherein the modified cycloalkyne of formula Y—R 1 —X is selected from:
i)

ii) a compound of the formula:

iii) a compound of the formula:

 and
iv) a compound of the formula:

wherein each of X and X′ is independently:
(a) H;
(b) one or two halogen atoms;
(c) —W—(CH 2 ) n —Z, wherein n is an integer from 1-4; W is O, N, or S; and Z is nitro, cyano, sulfonic acid, or a halogen;
(d) —(CH 2 ) n —W—(CH 2 ) m —Z, wherein n and m are each independently 1 or 2; 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 if W is sulfonyl, then Z is H; or
(e) —(CH 2 ) n —Z, wherein n is an integer from 1-4; and Z is nitro, cyano,

Assignees

• Univ California

Inventors

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

Classifications

• 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

• C07D207/452

  with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom · CPC title

• C07D249/16

  condensed with carbocyclic rings or ring systems · CPC title

• C07C49/457

  containing halogen · CPC title

• C07C69/708

  Ethers · CPC title