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US 8,703,936 B2Granted
Compositions and methods for modification of biomolecules
AssigneeThe Regents of the University of California
InventorsJohn C. Jewett, Carolyn R. Bertozzi, Ellen May Sletten, Chelsea G. Gordon
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Abstract
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.
Claims21
A method for labeling a target molecule comprising an azide, the method comprising:
- reacting an azide of a target molecule with a modified cycloalkyne compound of the formula:
- wherein
- X1-X8 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, C1 to C7 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, C1 to C7 alkyl, C1 to C7 alkoxy, C1 to C7 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
- Y1-Y3 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,
- wherein said reacting produces a conjugate between the azide of the target molecule and the modified cycloalkyne.
9 more elements
A method for labeling a cellular component, the method comprising:
- introducing an azide moiety into a cellular component, thereby generating an azide-modified cellular component;
- and
- contacting a cell comprising the azide-modified cellular component with a reactive partner comprising a modified cycloalkyne
- compound of the formula:
- wherein
- X1-X8 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, C1 to C7 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, C1 to C7 alkyl, C1 to C7 alkoxy, C1 to C7 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
- Y1-Y3 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,
- said contacting being under physiological conditions,
- wherein said contacting with said reactive partner results in a reaction between the azide group of the azide-modified cellular component and the cycloalkyne of the reactive partner, thereby synthetically and covalently modifying the cellular component to produce a covalently modified cellular component.
13 more elements
Dependencies are inferred from the claim text.