Methods for the synthesis of functionalized nucleic acids

Abstract

Described herein are methods for the synthesis of derivatives of thiosulfonate reagents. Said reagents have utility for the synthesis of phosphorothiotriesters from H-phosphonates in a stereospecific fashion.

1 . A process for the preparation of phosphorothiotriesters of structure IIIa comprising the steps of:
 i) reacting an H-phosphonate of structure Ia with an silylating reagent to provide a silyloxyphosphonate; and   ii) reacting the silyloxyphosphonate with a thiosulfonate reagent of structure IIa to provide a phosphorothiotriester of structure IIIa;   
       wherein, 
       the H-phosphonate of structure Ia has the following structure: 
       
         
           
           
               
               
           
         
       
       wherein,
 W is independently selected from O, S, NH, or CH 2 ; 
 R 3  is —OH, —SH, —NR d R d , —N 3 , halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —P(O)(R e ) 2 , —HP(O)(R e ), —OR a  or —SR c ; 
 Y 1  is O, NR d , S, or Se; 
 R a  is a blocking group; 
 R c  is a blocking group; 
 each instance of R d  is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, —P(O)(R e ) 2 , or —HP(O)(R e ); 
 each instance of R e  is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y 2 —, alkenyl-Y 2 —, alkynyl-Y 2 —, aryl-Y 2 —, or heteroaryl-Y 2 —, or a cation which is Na +1 , Li +1 , or K +1 ; 
 Y 2  is O, NR d , or S; 
 each instance of R 4  is independently hydrogen, —OH, —SH, —NR d R d , —N 3 , halogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —OR b , or —SR c , and R b  is a blocking group; 
 each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase; 
 R 5  is hydrogen, a blocking group, a linking moiety connected to a solid support or a linking moiety connected to a nucleic acid; and 
 n is between 1 and about 200; and 
 
       the thiosulfonate reagent of structure IIa has the following structure: 
       
         
           
           
               
               
           
         
       
       wherein,
 X is alkyl, cycloalkyl, or heteroaryl; 
 R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or R 1 -R 2 ; 
 R 1  is selected from —S-alkenylene-, —S-alkylene-, —S-alkylene-aryl-alkylene-, —S—CO-aryl-alkylene-, or —S—CO-alkylene-aryl-alkylene-; 
 R 2  is selected from heterocyclo-alkylene-S—, heterocyclo-alkenylene-S—, aminoalkyl-S—, or (alkyl) 4 N-alkylene-S—; 
 
       and the phosphorothiotriester of structure IIIa has the following structure: 
       
         
           
           
               
               
           
         
       
       wherein,
 W is independently selected from O, S, NH, or CH 2 ; 
 R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or R 1 -R 2 ; 
 R 1  is selected from —S-alkenylene-, —S-alkylene-, —S-alkylene-aryl-alkylene-, —S—CO-aryl-alkylene-, or —S—CO-alkylene-aryl-alkylene-; 
 R 2  is selected from heterocyclo-alkylene-S—, heterocyclo-alkenylene-S—, aminoalkyl-S—, or (alkyl) 4 N-alkylene-S—; 
 R 3  is —OH, —SH, —NR d R d , —N 3 , halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —P(O)(R e ) 2 , —HP(O)(R e ), —OR a  or —SR c ; 
 Y 1  is O, NR d , S, or Se; 
 R a  is a blocking group; 
 R c  is a blocking group; 
 each instance of R d  is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, —P(O)(R e ) 2 , or —HP(O)(R e ); 
 each instance of R e  is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y 2 —, alkenyl-Y 2 —, alkynyl-Y 2 —, aryl-Y 2 —, or heteroaryl-Y 2 —, or a cation which is Na +1 , Li +1 , or K +1 ; 
 Y 2  is O, NR d , or S; 
 each instance of R 4  is independently hydrogen, —OH, —SH, —NR d R d , —N 3 , halogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —OR b , or —SR c , and R b  is a blocking group; 
 each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase; 
 R 5  is hydrogen, a blocking group, a linking moiety connected to a solid support or a linking moiety connected to a nucleic acid; and 
 n is between 1 and about 200. 
 
     
     
         2 . A process for the preparation of phosphorothiotriesters comprising non-stereorandom phosphorous linkages of structure IIIb comprising the steps of:
 i) reacting a H-phosphonate comprising non-stereorandom phosphorous linkages of structure Ib with an silylating reagent to provide a silyloxyphosphonate; and   ii) reacting the silyloxyphosphonate with a thiosulfonate reagent of structure IIb to provide a phosphorothiotriester comprising non-stereorandom phosphorous linkages of structure IIIb;   
       wherein, 
       the H-phosphonate comprising non-stereorandom phosphorous linkages of structure Ib has the following structure: 
       
         
           
           
               
               
           
         
       
       wherein,
 W is independently selected from O, NH, or CH 2 ; 
 R 3  is —OH, —SH, —NR d R d , —N 3 , halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —P(O)(R e ) 2 , —HP(O)(R e ), —OR a  or —SR c ; 
 Y 1  is O, NR d , S, or Se; 
 R a  is a blocking group; 
 R c  is a blocking group; 
 each instance of R d  is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, —P(O)(R e ) 2 , or —HP(O)(R e ); 
 each instance of R e  is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y 2 —, alkenyl-Y 2 —, alkynyl-Y 2 —, aryl-Y 2 —, or heteroaryl-Y 2 —, or a cation which is Na +1 , Li +1 , or K +1 ; 
 Y 2  is O, NR d , or S; 
 each instance of R 4  is independently hydrogen, —OH, —SH, —NR d R d , —N 3 , halogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —OR b , or —SR c , and R b  is a blocking group; 
 each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase; 
 R 5  is hydrogen, a blocking group, a linking moiety connected to a solid support or a linking moiety connected to a nucleic acid; and 
 n is between 1 and about 200; and 
 
       the thiosulfonate reagent of structure IIb has the following structure: 
       
         
           
           
               
               
           
         
       
       wherein,
 X is alkyl, cycloalkyl, aryl, or heteroaryl; 
 R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or R 1 -R 2 ; 
 R 1  is selected from —S-alkenylene-, —S-alkylene-, —S-alkylene-aryl-alkylene-, —S—CO-aryl-alkylene-, or —S—CO-alkylene-aryl-alkylene-; 
 R 2  is selected from heterocyclo-alkylene-S—, heterocyclo-alkenylene-S—, aminoalkyl-S—, or (alkyl) 4 N-alkylene-S—; 
 and the chiral phosphorothiotriester comprising non-stereorandom phosphorous linkages of structure IIIb has the following structure: 
 
       
         
           
           
               
               
           
         
       
       wherein,
 W is independently selected from O, NH, or CH 2 ; 
 R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or R 1 -R 2 ; 
 R 1  is selected from —S-alkenylene-, —S-alkylene-, —S-alkylene-aryl-alkylene-, —S—CO-aryl-alkylene-, or —S—CO-alkylene-aryl-alkylene-; 
 R 2  is selected from heterocyclo-alkylene-S—, heterocyclo-alkenylene-S—, aminoalkyl-S—, or (alkyl) 4 N-alkylene-S—; 
 R 3  is —OH, —SH, —NR d R d , —N 3 , halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —P(O)(R e ) 2 , —HP(O)(R e ), —OR a  or —SR c ; 
 Y 1  is O, NR d , S, or Se; 
 R a  is a blocking group; 
 R c  is a blocking group; 
 each instance of R d  is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, —P(O)(R e ) 2 , or —HP(O)(R e ); 
 each instance of R e  is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y 2 —, alkenyl-Y 2 —, alkynyl-Y 2 —, aryl-Y 2 —, or heteroaryl-Y 2 —, or a cation which is Na +1 , Li +1 , or K +1 ; 
 Y 2  is O, NR d , or S; 
 each instance of R 4  is independently hydrogen, —OH, —SH, —NR d R d , —N 3 , halogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —OR b , or —SR c , and R b  is a blocking group; 
 each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase; 
 R 5  is hydrogen, a blocking group, a linking moiety connected to a solid support or a linking moiety connected to a nucleic acid; and 
 n is between 1 and about 200. 
 
     
     
         3 . The process of  claim 1 , wherein W is O. 
     
     
         4 . The process of  claim 1 , wherein R 1  is selected from: 
       
         
           
           
               
               
           
         
       
       and
 R 2  is selected from: 
 
       
         
           
           
               
               
           
         
       
     
     
         5 . The process of  claim 1 , wherein the silylating reagent is selected from
 1,1,3,3-tetramethyl-1,3-diphenyldisilazane;   1,3-dimethyl-1,1,3,3-tetraphenyldisilazane;   1-(trimethylsilyl)imidazole;   N-trimethylsilyl-N-methyl trifluoroacetamide;   bis(dimethylamino)dimethylsilane;   bromotrimethylsilane;   chlorodimethyl(pentafluorophenyl)silane;   chlorotriethyl silane;   chlorotriisopropylsilane;   chlorotrimethylsilane;   dichlorodimethylsilane;   hexamethyldisilazane;   N,N′-bis(trimethylsilyl)urea;   N,N-bis(trimethylsilyl)methylamine;   N,N-dimethyltrimethylsilylamine;   N,O-bis(trimethylsilyl)acetamide;   N,O-bis(trimethylsilyl)carbamate;   N,O-bis(trimethylsilyl)trifluoroacetamide;   N-methyl-N-(trimethylsilyl)trifluoroacetamide;   N-methyl-N-trimethylsilylacetamide;   N-methyl-N-trimethylsilylheptafluorobutyramide;   N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide;   N-methyl-N-trimethylsilylheptafluorobutyramide;   trimethylsilyltriflate;   triethylsilyltriflate;   triisopropylsilyltriflate; or   tert-butyldimethylsilyltriflate.   
     
     
         6 . The process of  claim 5 , wherein the silylating reagent is selected from N,O-bis(trimethylsilyl)trifluoroacetamide, trimethylsilyltriflate, chlorotrimethylsilane, or 1-(trimethyl silyl)imidazole. 
     
     
         7 . The process of  claim 6 , wherein the silylating reagent is selected from N,O-bis(trimethylsilyl)trifluoroacetamide. 
     
     
         8 . The process of  claim 1 , wherein the H-phosphonate is covalently linked to a solid phase. 
     
     
         9 . A process for the preparation of phosphorothiotriesters of structure IIIc comprising the steps of:
 i) reacting a H-phosphonate of structure Ic with an silylating reagent to provide a silyloxyphosphonate;   ii) reacting the silyloxyphosphonate with a bis(thiosulfonate) reagent of structure IVc to provide a phosphorothiotriester comprising a thiosulfonate group of structure Vc;   iii) reacting the phosphorothiotriester comprising a thiosulfonate group of structure Vc with a nucleophile of structure VIc to provide the phosphorothiotriesters of structure IIIc;   
       wherein, 
       the H-phosphonate of structure Ic has the following structure: 
       
         
           
           
               
               
           
         
       
       wherein,
 W is independently selected from O, S, NH, or CH 2 ; 
 R 3  is —OH, —SH, —NR d R d , —N 3 , halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, P(O)(R e ) 2 , —HP(O)(R e ), —OR a  or —SR c ; 
 Y 1  is O, NR d , S, or Se; 
 R a  is a blocking group; 
 R c  is a blocking group; 
 each instance of R d  is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, —P(O)(R e ) 2 , or —HP(O)(R e ); 
 each instance of R e  is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y 2 —, alkenyl-Y 2 —, alkynyl-Y 2 —, aryl-Y 2 —, or heteroaryl-Y 2 —, or a cation which is Na +1 , Li +1 , or K +1 ; 
 Y 2  is O, NR d , or S; 
 each instance of R 4  is independently hydrogen, —OH, —SH, —NR d R d , —N 3 , halogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —OR b , or —SR c , and R b  is a blocking group; 
 each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase; 
 R 5  is hydrogen, a blocking group, a linking moiety connected to a solid support or a linking moiety connected to a nucleic acid; and 
 n is between 1 and about 200; and 
 
       the bis(thiosulfonate) reagent of structure IVc has the following structure: 
       
         
           
           
               
               
           
         
       
       wherein,
 X is alkylene, alkenylene, arylene, or heteroarylene; 
 each R 6  is independently alkyl, cycloalkyl, aryl, or heteroaryl; 
 the nucleophile of structure VIc has the following structure: 
 R 7 —SH, wherein R 7  is selected from alkyl, alkenyl, aryl, heterocyclo, aminoalkyl, or (heterocyclo)alkyl; 
 and phosphorothiotriesters of structure IIIc has the following structure: 
 
       
         
           
           
               
               
           
         
       
       wherein,
 W is independently selected from O, S, NH, or CH 2 ; 
 R is R 7 —S—S—X— 
 R 7  is alkyl, alkenyl, aryl, heterocyclo, aminoalkyl, or (heterocyclo)alkyl; 
 X is alkylene, alkenylene, arylene, or heteroarylene; 
 R 3  is —OH, —SH, —NR d R d , —N 3 , halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —P(O)(R e ) 2 , —HP(O)(R e ), —OR a  or —SR c ; 
 Y 1  is O, NR d , S, or Se; 
 R a  is a blocking group; 
 R c  is a blocking group; 
 each instance of R d  is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, —P(O)(R e ) 2 , or —HP(O)(R e ); 
 each instance of R e  is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y 2 —, alkenyl-Y 2 —, alkynyl-Y 2 —, aryl-Y 2 —, or heteroaryl-Y 2 —, or a cation which is Na +1 , Li +1 , or K +1 ; 
 Y 2  is O, NR d , or S; 
 each instance of R 4  is independently hydrogen, —OH, —SH, —NR d R d , —N 3 , halogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —OR b , or —SR c , and R b  is a blocking group; 
 each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase; 
 R 5  is hydrogen, a blocking group, a linking moiety connected to a solid support or a linking moiety connected to a nucleic acid; 
 n is between 1 and about 200; and 
 wherein the phosphorous linkages of the H-phosphonate of structure Ic, the phosphorothiotriester comprising a thiosulfonate group of structure Vc, and the phosphorothiotriesters of structure IIIc may optionally comprise non-stereorandom phosphorous linkages. 
 
     
     
         10 . The process of  claim 8 , wherein the phosphorothiotriesters of structure IIIb comprise non-stereorandom phosphorous linkages and the H-phosphonate of structure Ic comprise non-stereorandom phosphorous linkages; and W is independently selected from O, NH, or CH 2 . 
     
     
         11 . The process of  claim 9 , wherein W is O. 
     
     
         12 . The process of  claim 9 , wherein R 6  is methyl. 
     
     
         13 . The process of  claim 9 , wherein bis(thiosulfonate) reagent of structure IVc is selected from: 
       
         
           
           
               
               
           
         
       
     
     
         14 . The process of  claim 9 , wherein the nucleophile of structure VIc has the following structure: 
       
         
           
           
               
               
           
         
       
     
     
         15 . The process of  claim 9 , wherein the silylating reagent is selected from
 1,1,3,3-tetramethyl-1,3-diphenyldisilazane;   1,3-dimethyl-1,1,3,3-tetraphenyldisilazane;   1-(trimethylsilyl)imidazole;   N-trimethylsilyl-N-methyl trifluoroacetamide;   bis(dimethylamino)dimethylsilane;   bromotrimethylsilane;   chlorodimethyl(pentafluorophenyl)silane;   chlorotriethyl silane;   chlorotriisopropylsilane;   chlorotrimethylsilane;   dichlorodimethylsilane;   hexamethyldisilazane;   N,N′-bis(trimethylsilyl)urea;   N,N-bis(trimethylsilyl)methylamine;   N,N-dimethyltrimethylsilylamine;   N,O-bis(trimethylsilyl)acetamide;   N,O-bis(trimethylsilyl)carbamate;   N,O-bis(trimethylsilyl)trifluoroacetamide;   N-methyl-N-(trimethylsilyl)trifluoroacetamide;   N-methyl-N-trimethylsilylacetamide;   N-methyl-N-trimethylsilylheptafluorobutyramide;   N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide;   N-methyl-N-trimethylsilylheptafluorobutyramide;   trimethylsilyltriflate;   triethylsilyltriflate;   triisopropylsilyltriflate; or   tert-butyldimethylsilyltriflate.   
     
     
         16 . The process of  claim 15 , wherein the silylating reagent is selected from N,O-bis(trimethylsilyl)trifluoroacetamide, trimethylsilyltriflate, chlorotrimethylsilane, or 1-(trimethyl silyl)imidazole. 
     
     
         17 . The process of  claim 16 , wherein the silylating reagent is selected from N,O-bis(trimethylsilyl)trifluoroacetamide. 
     
     
         18 . The process of  claim 17 , wherein the H-phosphonate is covalently linked to a solid phase.