Methods and compositions for labeling polypeptides

Abstract

Synthesis of many proteins is tightly controlled at the level of translation and plays an essential role in fundamental processes such as cell growth and proliferation, signaling, differentiation or death. Methods that allow imaging and identification of nascent proteins allow for dissecting regulation of translation, both spatially and temporally, including in whole organisms. Described herein are robust chemical methods for imaging and affinity-purifying nascent polypeptides in cells and in animals, based on puromycin analogs. Puromycin analogs of the present invention form covalent conjugates with nascent polypeptide chains, which are rapidly turned over by the proteasome and can be visualized and specifically captured by a bioorthogonal reaction (e.g., [3+2] cycloaddition). The methods of the present invention have broad applicability for imaging protein synthesis and for identifying proteins synthesized under various physiological and pathological conditions in vivo.

1 - 161 . (canceled) 
     
     
         162 . A labeled polypeptide comprising a puromycin analog conjugate of the following formula: 
       
         
           
           
               
               
           
         
       
       wherein:
 R A  is a bond, or an optionally substituted aliphatic group, heteroaliphatic group, aryl group, and heteroaryl group, or a combination thereof; 
 R B  is a bond or a C 1-6  aliphatic moiety; 
 R 1  is hydrogen or a reactive group capable of undergoing a bioorthogonal reaction; 
 R 1′  is hydrogen or a reactive group capable of undergoing a bioorthogonal reaction; wherein R1 and R1′ are not simultaneously hydrogen; 
 R 2  is hydrogen or C 1-6  aliphatic group; 
 R 3  and R 4  are each independently hydrogen or a protecting group; and 
 R 6  is hydrogen or C 1-6  aliphatic group; 
 
       or a salt thereof. 
     
     
         163 . The labeled polypeptide according to  claim 162 , wherein the puromycin analog conjugate is of the following formula: 
       
         
           
           
               
               
           
         
       
       or a salt thereof. 
     
     
         164 . The labeled polypeptide according to  claim 162 , wherein R A  is: 
       
         
           
           
               
               
           
         
       
     
     
         165 . The labeled polypeptide according to  claim 162 , wherein the —R A —R 1  group is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         166 . The labeled polypeptide according to  claim 162 , wherein R A  is an -alkylaryl group, a tyrosine side chain, or an alkyl group (e.g. a C 1-3  alkyl group). 
     
     
         167 . The labeled polypeptide according to  claim 162 , wherein R 1  comprises a dipolarophile. 
     
     
         168 . The labeled polypeptide according to  claim 162 , wherein R 1  comprises an ethynyl or a propargyl group. 
     
     
         169 . The labeled polypeptide according to  claim 162 , wherein R 1  comprises a 1,3-dipole. 
     
     
         170 . The labeled polypeptide according to  claim 162 , wherein R 1  comprises an azido or an azidoethyl group. 
     
     
         171 . The labeled polypeptide according to  claim 162 , wherein R 1  is an aldehyde or a quadricyclane. 
     
     
         172 . The labeled polypeptide according to  claim 162 , wherein R 1  comprises a tetrazine or a trans-cyclooctene. 
     
     
         173 . The labeled polypeptide according to  claim 162 , wherein the bioorthogonal reaction is a Staudinger ligation, a tetrazine ligation, an oxime ligation, a hydrazone ligation, and a quadricyclane ligation. 
     
     
         174 . A labeled polypeptide prepared by the method comprising:
 providing a polypeptide-puromycin analog conjugate comprising a first reactive group; and   contacting the polypeptide-puromycin analog conjugate with a compound comprising a second reactive group and a label, such that a bioorthogonal reaction occurs between the first and second reactive groups.   
     
     
         175 . The labeled polypeptide according to  claim 174 , wherein the first reactive unsaturated group comprises a 1,3-dipole and the second reactive unsaturated group comprises a dipolarophile or wherein the first reactive unsaturated group comprises a dipolarophile and the second reactive unsaturated group comprises a 1,3-dipole. 
     
     
         176 . The labeled polypeptide according to  claim 174 , wherein the first and second reactive groups are capable of undergoing a [3+2] cycloaddition, a Staudinger ligation, an inverse electron demand Diels-Alder reaction (e.g., tetrazine ligation), an oxime addition, a hydrazone addition, or a [2+2+2] cycloaddition (e.g., quadricyclane ligation). 
     
     
         177 . The labeled polypeptide according to  claim 175 , wherein the a 1,3-dipole is a nitrile oxide, an azide, a diazomethane, a nitrone, or a nitrile imine. 
     
     
         178 . The labeled polypeptide according to  claim 175 , wherein the dipolarophile is an alkene or an alkyne. 
     
     
         179 . The labeled polypeptide according to  claim 174 , wherein the first reactive group is an ethynyl or propargyl group and the second reactive group is an azido group. 
     
     
         180 . The labeled polypeptide according to  claim 174 , wherein the first reactive group is an azido group and the second reactive group is an ethynyl group or propargyl group. 
     
     
         181 . The labeled polypeptide according to  claim 174 , comprising a cycloadduct resulting from a [3+2] cycloaddition between an ethynyl group and an azido group. 
     
     
         182 . The labeled polypeptide according to  claim 174 , comprising a label that is directly detectable. 
     
     
         183 . The polypeptide according to  claim 182 , wherein the label is a fluorescent agent. 
     
     
         184 . The polypeptide according to  claim 183 , wherein the label is biotin. 
     
     
         185 . A cell comprising a labeled polypeptide according to  claim 162 . 
     
     
         186 . An organism comprising a labeled polypeptide according to  claim 162 . 
     
     
         187 . The organism according to  claim 186 , wherein the organism is a non-human whole animal. 
     
     
         188 . The labeled polypeptide according to  claim 162 , wherein the puromycin analog is of the formula: 
       
         
           
           
               
               
           
         
       
       or a salt thereof. 
     
     
         189 . A compound of the following formula: 
       
         
           
           
               
               
           
         
       
       or a salt thereof.