Compositions and methods for modification of biomolecules
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).
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, sulfonic acid, or a halogen; 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.
10. The method of claim 9 , wherein the modified cycloalkyne of formula Y—R 1 —X has the structure:
11. 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 has the structure of one of Formulas XI-XVI:
wherein R, R 1 , R 2 , and R 3 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—(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, R 1 , R 2 , and R 3 is linked to Y; 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.
12. The method of claim 1 , wherein Y is a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest.
13. The method of claim 12 , 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.
14. The method of claim 1 , 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.
15. The method of claim 1 , wherein the modified cycloalkyne is of the structure:
16. The method of claim 1 , wherein the modified cycloalkyne is of one of the following structures:
17. The method of claim 1 , wherein the chemoselective modification is carried out in the absence of a catalyst.
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 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, 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 the cellular component is a polypeptide, selected from a lipoprotein or a glycoprotein.
20. The method of claim 18 , wherein said contacting is in vivo.