Compositions and methods for quadricyclane modification of biomolecules

Abstract

The present disclosure features a strain-promoted [2+2+2] reaction that can be carried out under physiological conditions. In general, the reaction involves reacting a pi-electrophile with a low lying LUMO with a quadricyclane on a biomolecule, generating a covalently modified biomolecule. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo and in vitro. The reaction is compatible with modification of living cells. In certain embodiments, the pi-electrophile can comprise a molecule of interest that is desired for delivery to a quadricyclane-containing biomolecule via [2+2+2] reaction.

1 .- 14 . (canceled) 
     
     
         15 . A compound selected from:
 (a) an azo compound of the formula (IIa) or (III):
                     Y 1 —R 1 —N═N—R 2 —Y 2   (III),
 
   wherein   Ar 1  is an optional aryl or substituted aryl group;   R 1  and R 2  are optional and are independently selected from hydrogen, alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, acylamino, aminoacyl, aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl ester, sulfonyl, sulfonyloxy, and thioalkoxy; and   Y 1  and Y 2  are independently selected from hydrogen, halogen, a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest; and a molecule of interests;   wherein at least one of Y 1  and Y 2  is a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest or a molecule of interest:   
       
         
           
           
               
               
           
         
         wherein 
         R 1  and R 2  are optional and are independently selected from hydrogen, alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, acylamino, aminoacyl, aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl ester, sulfonyl, sulfonyloxy, and thioalkoxy; and 
         Y 1  and Y 2  are independently selected from hydrogen; halogen; a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest; and a molecule of interest; 
         wherein at least one of Y 1  and Y 2  is a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest or a molecule of interest; and 
         (c) a ketone compound of formula (VIa) or formula (VIb): 
       
       
         
           
           
               
               
           
         
         wherein 
         R 1  is optional and is selected from alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, acylamino, aminoacyl, aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl ester, sulfonyl, sulfonyloxy, and thioalkoxy; and 
         R 2 , R 3 , and R 4  are independently selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, aminocarbonyl, carboxyl, carboxyl ester, cyano, halogen, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, trihalomethyl, aryl, and substituted aryl; 
         each R 5  is independently selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, aminocarbonyl, carboxyl, carboxyl ester, cyano, halogen, nitro, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and trihalomethyl; and 
         n is number from one to five; and 
         Y 1  is selected from a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest; and a molecule of interest. 
       
     
     
         16 . The compound of  claim 15 , wherein the compound is an azo compound of the formula: 
       
         
           
           
               
               
           
         
         wherein 
         R 1  is optional and is selected from alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, acylamino, aminoacyl, aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl ester, sulfonyl, sulfonyloxy, and thioalkoxy; 
         Y 1  is selected from a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest; and a molecule of interest; 
         R 2  is selected from alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, aminocarbonyl, carboxyl, carboxyl ester, cyano, halogen, nitro, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and trihalomethyl; and 
         n is a number from zero to four. 
       
     
     
         17 . The compound of  claim 15 , wherein the compound is an azo compound of the formula:
   Y 1 —R 1 —N═N—R 2 Y 2   (III),
   wherein   R 1  and R 2  are optional and are independently selected from hydrogen, alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, acylamino, aminoacyl, aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl ester, sulfonyl, sulfonyloxy, and thioalkoxy; and   Y 1  and Y 2  are independently selected from hydrogen; halogen; a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest; and a molecule of interest;   wherein at least one of Y 1  and Y 2  is a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest or a molecule of interest.   
     
     
         18 . The compound of  claim 15 , wherein the compound is an alkynyl compound of the formula: 
       
         
           
           
               
               
           
         
         wherein 
         R 1  and R 2  are optional and are independently selected from hydrogen, alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, acylamino, aminoacyl, aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl ester, sulfonyl, sulfonyloxy, and thioalkoxy; and 
         Y 1  and Y 2  are independently selected from hydrogen; halogen; a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest; and a molecule of interest; 
         wherein at least one of Y 1  and Y 2  is a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest or a molecule of interest. 
       
     
     
         19 . The compound of  claim 15 , wherein the compound is an alkenyl compound of the formula: 
       
         
           
           
               
               
           
         
         wherein 
         R 1  and R 2  are optional and are independently selected from hydrogen, alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, acylamino, aminoacyl, aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl ester, sulfonyl, sulfonyloxy, and thioalkoxy; and 
         Y 1  and Y 2  are independently selected from hydrogen; halogen; a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest; and a molecule of interest; 
         wherein at least one of Y 1  and Y 2  is a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest or a molecule of interest. 
       
     
     
         20 . The compound of  claim 15 , wherein the compound is a ketone compound of the formula: 
       
         
           
           
               
               
           
         
         wherein 
         R 1  is optional and is selected from alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, acylamino, aminoacyl, aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl ester, sulfonyl, sulfonyloxy, and thioalkoxy; and 
         R 2 , R 3 , and R 4  are independently selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, aminocarbonyl, carboxyl, carboxyl ester, cyano, halogen, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, trihalomethyl, aryl, and substituted aryl; and 
         Y 1  is selected from a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest; and a molecule of interest. 
       
     
     
         21 . The compound of  claim 15 , wherein the compound is a ketone compound of the formula: 
       
         
           
           
               
               
           
         
         wherein 
         R 1  is optional and is selected from alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, alkoxy, substituted alkoxy, aryl, substituted aryl, acyl, acylamino, aminoacyl, aminocarbonylamino, acyloxy, aminosulfonyl, sulfonylamino, amino, substituted amino, carboxyl ester, sulfonyl, sulfonyloxy, and thioalkoxy; and 
         R 2  and R 3  are independently selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, aminocarbonyl, carboxyl, carboxyl ester, cyano, halogen, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and trihalomethyl; 
         each R 5  is independently selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, aminocarbonyl, carboxyl, carboxyl ester, cyano, halogen, nitro, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and trihalomethyl; and 
         n is number from one to five; 
         Y 1  is selected from a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest; and a molecule of interest. 
       
     
     
         22 . The compound of  claim 15 , wherein the molecule of interest is selected from a detectable label; a toxin, a peptide, a drug; a member of a specific binding pair, and an epitope tag. 
     
     
         23 .- 25 . (canceled) 
     
     
         26 . A method for chemoselective modification of a biomolecule comprising a quadricyclane, the method comprising reacting a quadracyclane in a quadricyclane-containing biomolecule with a compound of  claim 15 , wherein said reacting produces a conjugate between the quadricyclane of the quadracyclane-containing biomolecule and a pi electrophile of the compound of  claim 15 . 
     
     
         27 . A method for synthetically modifying a cellular component, the method comprising:
 introducing a quadricyclane moiety into a cellular component, thereby generating a quadricyclane-modified cellular component; and   contacting the cell comprising the quadricyclane-modified cellular component with a reaction partner comprising a pi-electrophile selected from a metal bis(dithiolene) compound, an azo compound, an alkynyl compound, an alkenyl compound and a ketone compound, said contacting being under physiological conditions;   wherein said contacting with said reaction partner results in reaction between the quadricyclane group of quadricyclane-modified cellular component and the pi-electrophile of the reaction partner, thereby synthetically and covalently modifying the cellular component.   
     
     
         28 . The method of  claim 27 , wherein said contacting is in vitro. 
     
     
         29 . The method of  claim 27 , wherein said contacting is ex vivo. 
     
     
         30 . The method of  claim 27 , wherein said contacting is in vivo. 
     
     
         31 . The compound of  claim 15 , wherein the azo compound is selected from: 
       
         
           
           
               
               
           
         
       
     
     
         32 . The compound of  claim 18 , wherein the alkynyl compound is: 
       
         
           
           
               
               
           
         
       
     
     
         33 . The compound of  claim 21 , wherein the ketone compound is 
       
         
           
           
               
               
           
         
       
     
     
         34 . The compound of  claim 15 , wherein each Y 1  independently comprises a reactive group selected from carboxyl, amine, ester, thioester, sulfonyl halide, alcohol, thiol, succinimidyl ester, isothiocyanate, iodoacetamide, maleimide, hydrazine, aminooxy, aldehyde, ketone, nitrile oxide, nitrone, tetrazine, azirine, tetrazole, alkene, alkyne, cyclooctyne, trans-cyclooctene, norbornene, and azide.