Compositions and methods for modification of biomolecules
Provided are modified cycloalkyne compounds; and methods of use of such compounds in modifying biomolecules. Embodiments include a cycloaddition reaction that can be carried out under physiological conditions. The cycloaddition reaction 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 and in vitro.
1. A compound of the formula:
wherein
X 1 -X 8 are each independently selected from carbon and nitrogen;
each L is a divalent moiety independently selected from alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, arylene, substituted arylene, cycloalkylene, substituted cycloalkylene, heteroarylene, substituted heteroarylene, heterocyclene, substituted heterocyclene, carboxamido, C 1 to C 7 acyloxy, urethanylene, sulfonyl, sulfonamido, —O—, —S—, —NH—, and substituted amino; wherein substituents of the substituted divalent moieties are selected from carboxy, amino, halo, hydroxy, nitro, cyano, trifluoromethyl, C 1 to C 7 alkyl, C 1 to C 7 alkoxy, C 1 to C 7 acyloxy, carboxamide, carboxymethyl, and hydroxymethyl; and wherein the substituted and unsubstituted heteroarylene and heterocyclene divalent moieties are five or six-membered rings having 1 to 4 heteroatoms independently selected from oxygen, nitrogen and sulfur ring atoms;
each n is a number independently selected from zero to 40; and
Y 1 -Y 3 are independently selected from H; a group selected from a carboxyl, an amino, an alkoxycarbonyl, a RSC(O)—, a sulfonyl halide, a hydroxyl, an alkoxy, an —SH, an N-succinimidyl ester, an isothiocyanate, an iodoacetamide, a maleimidyl, a hydrazinyl, a hydrazide, a halogen, a cyano, a diazo, an azide, a guanidinyl, a sulfone, an epoxide, a diazirine, an alkenyl, an alkynyl, a phosphine, a silane and an alkylsulfonic acid; and a molecule of interest selected from a detectable label, a toxin, a peptide, a drug, a member of a specific binding pair, an epitope tag and a strained azacycloalkynone group.
2. The compound of claim 1 , wherein the compound is of formula III:
wherein
each L is a divalent moiety independently selected from alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, arylene, substituted arylene, cycloalkylene, substituted cycloalkylene, heteroarylene, substituted heteroarylene, heterocyclene, substituted heterocyclene, carboxamido, C 1 to C 7 acyloxy, urethanylene, sulfonyl, sulfonamido, —O—, —S—, —NH—, and substituted amino; wherein substituents of the substituted divalent moieties are selected from carboxy, amino, halo, hydroxy, nitro, cyano, trifluoromethyl, C 1 to C 7 alkyl, C 1 to C 7 alkoxy, C 1 to C 7 acyloxy, carboxamide, carboxymethyl, and hydroxymethyl; and wherein the substituted and unsubstituted heteroarylene and heterocyclene divalent moieties are five or six-membered rings having 1 to 4 heteroatoms independently selected from oxygen, nitrogen and sulfur ring atoms;
n is a number selected from zero to 40;
each R is independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, —SH, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO 2 -alkyl, —SO 2 -substituted alkyl, —SO 2 -aryl and —SO 2 -heteroaryl; wherein substituents are selected from carboxy, amino, halo, hydroxy, nitro, cyano, trifluoromethyl, C 1 to C 7 alkyl, C 1 to C 7 alkoxy, C 1 to C 7 acyloxy, carboxamide, carboxymethyl, and hydroxymethyl;
each a is a number selected from zero to four; and
Y is H; a group selected from a carboxyl, an amino, an alkoxycarbonyl, a RSC(O)—, a sulfonyl halide, a hydroxyl, an alkoxyl, an —SH, an N-succinimidyl ester, an isothiocyanate, an iodoacetamide, a maleimidyl, a hydrazinyl, a hydrazide, an aldehyde, a haloalkyl, a halogen, a cyano, a diazo, an azide, a guanidinyl, a sulfone, an epoxide, a diazirine, an alkenyl, an alkynyl, a phosphine, a silane, and an alkylsulfonic acid; or a molecule of interest selected from a detectable label, a toxin, a peptide, a drug, a member of a specific binding pair, an epitope tag and a strained azacycloalkynone group.
3. The compound of claim 2 , wherein the compound is of the formula:
wherein R is selected from hydrogen, alkyl, sulfonate, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl, sulfonyl, sulfonamide, amino and substituted amino; wherein the substituted and unsubstituted heteroaryl and heterocyclyl are five or six-membered rings having 1 to 4 heteroatoms independently selected from oxygen, nitrogen and sulfur ring atoms; and wherein substituents are selected from carboxy, amino, halo, hydroxy, nitro, cyano, trifluoromethyl, C 1 to C 7 alkyl, C 1 to C 7 alkoxy, C 1 to C 7 acyloxy, carboxamide, carboxymethyl, and hydroxymethyl.
4. The compound of claim 2 , wherein the compound is of formula V:
wherein each L is a divalent moiety independently selected from alkylene, alkenylene, alkynylene, arylene, cycloalkylene, heteroarylene, heterocyclene, acylamino, C 1 to C 7 acyloxy, urethanylene, sulfonyl, sulfonamido, —O—, —S—, and —NH—, and wherein the heteroarylene and heterocyclene divalent moieties are five or six-membered rings having 1 to 4 heteroatoms independently selected from oxygen, nitrogen and sulfur ring atoms;
n is a number selected from zero to 40; and
Y is H; a group selected from a carboxyl, an amino, an alkoxycarbonyl, a RSC(O)—, a sulfonyl halide, a hydroxyl, an —SH, an N-succinimidyl ester, an isothiocyanate, an iodoacetamide, a maleimidyl, a hydrazinyl, a hydrazide, an aldehyde, a haloalkyl, a halogen, a cyano, a diazo, an azide, a guanidinyl, a sulfone, an epoxide, a diazirine, an alkenyl, an alkynyl, a phosphine, a silane, and an alkylsulfonic acid; or a molecule of interest selected from a detectable label, a peptide, a member of a specific binding pair and an epitope tag.
5. The compound of claim 2 , wherein the compound is of formula VI:
wherein:
each L is a divalent moiety independently selected from alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, arylene, substituted arylene, cycloalkylene, substituted cycloalkylene, heteroarylene, substituted heteroarylene, heterocyclene, substituted heterocyclene, carboxamido, C 1 to C 7 acyloxy, urethanylene, sulfonyl, sulfonamido, —O—, —S—, —NH—, and substituted amino; wherein substituents of the substituted divalent moieties are selected from carboxy, amino, halo, hydroxy, nitro, cyano, trifluoromethyl, C 1 to C 7 alkyl, C 1 to C 7 alkoxy, C 1 to C 7 acyloxy, carboxamide, carboxymethyl, and hydroxymethyl; and wherein the substituted and unsubstituted heteroarylene and heterocyclene divalent moieties are five or six-membered rings having 1 to 4 heteroatoms independently selected from oxygen, nitrogen and sulfur ring atoms;
n is a number selected from zero to 40;
each R is independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, —SH, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO 2 -alkyl, —SO 2 -substituted alkyl, —SO 2 -aryl, —SO 2 -heteroaryl; wherein substituents are selected from carboxy, amino, halo, hydroxy, nitro, cyano, trifluoromethyl, C 1 to C 7 alkyl, C 1 to C 7 alkoxy, C 1 to C 7 acyloxy, carboxamide, carboxymethyl, and hydroxymethyl; and
each a is a number selected from zero to four.
6. The compound of claim 4 , wherein the compound is of one of the following structures:
7. The compound of claim 2 , wherein the compound is of one of the following structures:
8. The compound of claim 4 , wherein:
each L is a divalent moiety independently selected from alkylene, arylene, cycloalkylene, heteroarylene, heterocyclene, C 1 to C 7 acyloxy, acylamino, urethanylene, sulfonyl, sulfonamido, —O—, —S—, and —NH—, and wherein the heteroarylene and heterocyclene divalent moieties are five or six-membered rings having 1 to 4 heteroatoms independently selected from oxygen, nitrogen and sulfur ring atoms;
n is a number selected from zero to 40; and
Y is selected from H, a carboxyl, an amino, a hydroxyl, an alkoxycarbonyl, an N-succinimidyl ester, a RSC(O)—, an isothiocyanate, an iodoacetamide, a maleimidyl, a hydrazinyl, a hydrazide, a halogen, an epoxide, a fluorophore, an epitope tag, and a biotin.