Dithioamine reducing agents

Abstract

Dithioamine reducing agents useful for the reduction of disulfide bonds. The reducing agents of this invention are useful, for example, to reduce disulfide bonds, particularly in proteins, or to prevent the formation of disulfide bonds, particularly in proteins and other biological molecules. Reducing agents of this invention can be employed to regulate protein function in proteins in which a sulfhydryl group is associated with biological activity. Reducing agents of this invention can prevent inactivation of a given protein or enhance activation of a given protein or other biological molecule in vitro and/or in vivo. Reducing agents of this invention can prevent or reduce oxidation of cysteine residues in proteins and prevent the formation of reduced activity protein dimers (or other oligomers). Reducing agents of this invention are useful and suitable for application in a variety of biological applications, particularly as research and synthetic reagents. The invention provides S-acylated dithioamines which can be selectively activated reducing agents by removal of the S-acyl groups enzymatically or chemically. The invention further provides dithiane precursors of thioamino reducing agents. The invention provides dithioamine reducing agents, S-acylated dithioamines and dithianes which are immobilized on surfaces, including among others, glass, quartz, microparticles, nanoparticles and resins.

We claim: 
     
         1 . A compound having formula I: 
       
         
           
           
               
               
           
         
         or salts thereof where: 
         R 1  is hydrogen, or an unsubstituted alkyl group having 1 to 3 carbon atoms; 
         each R 2  and R 3  is independently hydrogen, an alkyl group having 1-3 carbon atoms, a phenyl or a benzyl group each of which is optionally substituted with one or more of substituents W 2 ; 
         each R 4  and R 5  is independently hydrogen, a halogen, a cyano group, a nitro group, a hydroxyl, an alkyl group having 1-6 carbon atoms, a phenyl, a benzyl group, an N(R 9 ) 2 , or a —COR 10  group, wherein each alkyl, phenyl, or benzyl group is optionally substituted with one or more of substituents W 2 ; 
         each R 6  and R 7 , is independently hydrogen, a 1-12 carbon alkyl group, an aryl group, a heterocyclic group, a heteroaryl group, a —COR 11  group, a —CO—NHR 11  group, a —CO—NHR 11  group, a —SO 2 —R 11  group, or a —(CH 2 ) n —R 12  group, wherein each alkyl, aryl heterocylic or heteroaryl group is optionally substituted with one or more of substituents W 3 ; and 
         each R 8  is independently hydrogen or —CO—R 13 ,
 wherein: 
 each R 9  is independently hydrogen, a 1-12 carbon alkyl group, an aryl group, a heterocyclic group, a heteroaryl group, a —COR 11  group, a —COOR 11  group, a —CO—NHR 11  group, a —CO—NHR 11  group, a —SO 2 —R 11  group, or a —(CH 2 )n-R 12  group where n is an integer ranging from 1-12, wherein each alkyl, aryl heterocyclic or heteroaryl group is optionally substituted with one or more of substituents W 3 ; 
 each R 10  is independently hydrogen, a 1-12 alkyl group, a phenyl or benzyl group which are optionally substituted with one or more of substituents W 2 ; 
 each R 11  and R 12  is independently hydrogen, a 1-12 carbon alkyl group, an aryl group, a heterocyclic group, a heteroaryl group, a -L-T group or a -M group, wherein each alkyl, aryl heterocylic or heteroaryl group is optionally substituted with one or more of substituents W 3 , -L- is a divalent linker group and T is a biological species or a surface to which the reducing agent is linked and -M is a reactive group or a spacer moiety carrying a reactive group; and 
 
         each R 13  is independently hydrogen, a 1-12 carbon alkyl group, an aryl group, a heterocyclic group, or a heteroaryl group, wherein each alkyl, aryl heterocyclic or heteroaryl group is optionally substituted with one or more of substituents W 3 ; 
         wherein: 
         W 2  is one or more substituents selected from halogen, an oxo group (═O), cyano group, nitro group, hydroxyl, unsubstituted alkyl group having 1-3 carbon atoms, halogen-substituted alkyl group having 1-3 carbon atoms, or unsubstituted alkoxy group having 1-3 carbon atoms; and 
         W 3  is one or more substituents selected from halogen, an oxo group (═O), cyano group, nitro group, hydroxyl, optionally substituted alkyl group having 1-6 carbon atoms, unsubstituted alkyl group having 1-6 carbon atoms, hydroxyl-substituted alkyl group having 1-6 carbon atoms, halogen-substituted alkyl group having 1-6 carbon atoms, unsubstituted alkoxy group having 1-6 carbon atoms, alkenyl group having 2-6 carbon atoms; alkynyl group having 2-6 carbon atoms, a 3-6-member alicyclic ring, wherein one or two ring carbons are optionally replaced with —CO— and which may contain one or two double bonds, an aryl group having 6-14 carbon ring atoms, a phenyl group, a benzyl group, a 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein one or two ring carbons are optionally replaced with —CO— and which may contain one or two double bonds, or a heteroaryl group having 1-3 heteroatoms (N, O or S), —CO 2 R 14  group, —CON(R 15 ) 2  group, —OCON(R 15 ) 2  group, —N(R 15 ) 2  group, a —SO 2 —OR 15  group, —(CH 2 ) m —OR 14  group, —(CH 2 ) m —N(R 15 ) 2 , 
         where m is 1-8, and 
         each R 14  and R 15  is independently hydrogen, an unsubstituted alkyl group having 1-6 carbon atoms; an unsubstituted aryl group having 6-14 carbon atoms, an unsubstituted phenyl group; an unsubstituted benzyl group, an unsubstituted 5- or 6-member ring heterocyclic group, having 1-3 heteroatoms and wherein one or two ring carbons are optionally replaced with —CO— and which may contain one or two double bonds, or a unsubstituted heteroaryl group having 1-3 heteroatoms, with the exception that no R 14  is hydrogen. 
       
     
     
         2 . The compound of  claim 1  wherein neither R 4  or R 5  is an —NH 2  group. 
     
     
         3 . The compound of  claim 1  wherein one of R 6  or R 7  is a —COR 11  group. 
     
     
         4 . The compound of  claim 3  wherein R 1 , R 2  and R 3  are all hydrogens. 
     
     
         5 . The compound of  claim 3  wherein R 1 , R 2 , R 3 , R 4  and R 5  are all hydrogens. 
     
     
         6 . The compound of  claim 3  wherein both R 8  are hydrogens. 
     
     
         7 . The compound of  claim 3  wherein both of R 8  are acyl groups. 
     
     
         8 . The compound of  claim 3  wherein R 11  is independently hydrogen, a 1-12 carbon alkyl group, an aryl group, a heterocyclic group, or a heteroaryl group. 
     
     
         9 . The compound of  claim 3  wherein R 1 -R 5  are all hydrogens and each R 8  is a —CO—R 13  wherein R 13  is an alkyl group having 1-6 carbon atoms, a halogen-substituted alkyl group having 1-6 carbon atoms, a hydroxy-substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted phenyl group or an optionally substituted benzyl group. 
     
     
         10 . The compound of  claim 3  wherein R 1 , R 2 , R 3  and one of R 4  or R 5  are all hydrogens and the other of R 4  or R 5  is an optionally substituted alkyl group having 1-3 carbons atoms, a halogen, a hydroxyl, or an acyl group. 
     
     
         11 . The compound of  claim 1  which is non-racemic having formula: 
       
         
           
           
               
               
           
         
       
     
     
         12 . The compound of  claim 3  which is non-racemic having formula: 
       
         
           
           
               
               
           
         
       
     
     
         13 . The compound of  claim 3  which is covalently conjugated to a surface. 
     
     
         14 . The compound of  claim 1  which is covalently conjugated to a peptide, protein, carbohydrate or a nucleic acid. 
     
     
         15 . A method for reducing or preventing disulfide bond formation in one or more molecules having one or more sulfhydryl groups which comprises the step of contacting the one or more molecules with one or more compounds of formula I of  claim 1 . 
     
     
         16 . A method for reducing or preventing disulfide bond formation in one or more molecules having one or more sulfhydryl groups which comprises the step of contacting the one or more molecules with one or more compounds of formula I of  claim 3 . 
     
     
         17 . The method of  claim 16  wherein the one or more compounds are covalently attached to a surface. 
     
     
         18 . The method of  claim 16  wherein both R 8  of the one or more compounds are acyl groups and the acyl groups of R 8  are removed prior to or at the same time as the contacting. 
     
     
         19 . The method of  claim 16  wherein reducing or preventing disulfide bond formation reduces or prevents the formation of dimers or other oligomers of the one or more molecules having sulfhydryl groups. 
     
     
         20 . A kit comprising one or more of the compounds of  claim 3  in combination with one or more components selected from one or more molecules having one or more sulfhydryl groups, or one or more solvents or buffers for the one or more molecules or one or more compounds. 
     
     
         21 . A compound having formula II: 
       
         
           
           
               
               
           
         
         or salts thereof where: 
         R 1  is hydrogen, or an unsubstituted alkyl group having 1 to 3 carbon atoms; 
         each R 2  and R 3  is independently hydrogen, an alkyl group having 1-3 carbon atoms, a phenyl or a benzyl group each of which is optionally substituted with one or more of substituents W 2 ; 
         each R 4  and R 5  is independently hydrogen, a halogen, a cyano group, a nitro group, a hydroxyl, an alkyl group having 1-6 carbon atoms, a phenyl, a benzyl group, an N(R 9 ) 2 , or a —COR 10  group, wherein each alkyl, phenyl, or benzyl group is optionally substituted with one or more of substituents W 2 ; 
         each R 6  and R 7 , is independently hydrogen, a 1-12 carbon alkyl group, an aryl group, a heterocyclic group, a heteroaryl group, a —COR— group, a —CO—NHR 11  group, a —CO—NHR 11  group, a —SO 2 —R 11  group, or a —(CH 2 ) n —R 12  group, wherein each alkyl, aryl heterocylic or heteroaryl group is optionally substituted with one or more of substituents W 3 ; and 
         each R 8  is independently hydrogen or —CO—R 13 ,
 wherein: 
 each R 9  is independently hydrogen, a 1-12 carbon alkyl group, an aryl group, a heterocyclic group, a heteroaryl group, a —COR 11  group, a —COOR 11  group, a —CO—NHR 11  group, a —CO—NHR 11  group, a —SO 2 —R 11  group, or a —(CH 2 )n-R 12  group where n is an integer ranging from 1-12, wherein each alkyl, aryl heterocyclic or heteroaryl group is optionally substituted with one or more of substituents W 3 ; 
 each R 10  is independently hydrogen, a 1-12 alkyl group, a phenyl or benzyl group which are optionally substituted with one or more of substituents W 2 ; 
 each R 11  and R 12  is independently hydrogen, a 1-12 carbon alkyl group, an aryl group, a heterocyclic group, a heteroaryl group, a -L-T group or a -M group, wherein each alkyl, aryl heterocylic or heteroaryl group is optionally substituted with one or more of substituents W 3 , -L- is a divalent linker group and T is a biological species or a surface to which the reducing agent is linked and -M is a reactive group or a spacer moiety carrying a reactive group; and 
 each R 13  is independently hydrogen, a 1-12 carbon alkyl group, an aryl group, a heterocyclic group, or a heteroaryl group, wherein each alkyl, aryl heterocyclic or heteroaryl group is optionally substituted with one or more of substituents W 3 ; 
 
         wherein: 
         W 2  is one or more substituents selected from halogen, an oxo group (═O), cyano group, nitro group, hydroxyl, unsubstituted alkyl group having 1-3 carbon atoms, halogen-substituted alkyl group having 1-3 carbon atoms, or unsubstituted alkoxy group having 1-3 carbon atoms; and 
         W 3  is one or more substituents selected from halogen, an oxo group (═O), cyano group, nitro group, hydroxyl, optionally substituted alkyl group having 1-6 carbon atoms, unsubstituted alkyl group having 1-6 carbon atoms, hydroxyl-substituted alkyl group having 1-6 carbon atoms, halogen-substituted alkyl group having 1-6 carbon atoms, unsubstituted alkoxy group having 1-6 carbon atoms, alkenyl group having 2-6 carbon atoms; alkynyl group having 2-6 carbon atoms, a 3-6-member alicyclic ring, wherein one or two ring carbons are optionally replaced with —CO— and which may contain one or two double bonds, an aryl group having 6-14 carbon ring atoms, a phenyl group, a benzyl group, a 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein one or two ring carbons are optionally replaced with —CO— and which may contain one or two double bonds, or a heteroaryl group having 1-3 heteroatoms (N, O or S), —CO 2 R 14  group, —CON(R 15 ) 2  group, —OCON(R 15 ) 2  group, —N(R 15 ) 2  group, a —SO 2 —OR 15  group, —(CH 2 ) m —OR 14  group, —(CH 2 ) m —N(R 15 ) 2 , 
         where m is 1-8, 
         each R 14  and R 15  is independently hydrogen, an unsubstituted alkyl group having 1-6 carbon atoms; an unsubstituted aryl group having 6-14 carbon atoms, an unsubstituted phenyl group; an unsubstituted benzyl group, an unsubstituted 5- or 6-member ring heterocyclic group, having 1-3 heteroatoms and wherein one or two ring carbons are optionally replaced with —CO— and which may contain one or two double bonds, or a unsubstituted heteroaryl group having 1-3 heteroatoms, with the exception that no R 14  is hydrogen; and 
         wherein not all of R 1 -R 7  is hydrogen.