METHODS FOR THE SYNTHESIS OF FUNCTIONALIZED NUCLEIC ACIDS
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.