Methods for the synthesis of functionalized nucleic acids

A process for the preparation of an oligonucleotide of structure IIIb, comprising the steps of:

• i) reacting an H-phosphonate of structure Ib with a silylating reagent to provide a silyloxyphosphonate;
• and
• ii) reacting the silyloxyphosphonate with a thiosulfonate reagent of structure IIb to provide an oligonucleotide of structure IIIb;
• wherein:
• the H-phosphonate of structure Ib has the following structure:
• wherein:
• at least one H-phosphonate linkage is non-stereorandom;
• W is independently O, NH, or CH2;
• R3 is —OH, —SH, —NRdRd, —N3, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y1—, alkenyl-Y1—, alkynyl-Y1—, aryl-Y1—, heteroaryl-Y1—, —P(O)(Re)2, —ORa or —SRc;
• Y1 is O, NRd, S, or Se;
• Ra is a blocking group;
• Rc is a blocking group;
• each instance of Rd is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, or tertiary silyl, or when Rd is attached to a nitrogen atom, —C(O)—O-alkyl, —C(O)—O-alkenyl, —C(O)—O-alkynyl, —C(O)—O-heteroalkyl, —C(O)—O-heteroalkenyl, —C(O)—O-heteroalkynyl, —C(O)—O-aryl, or —C(O)—O-heteroaryl,
• each instance of Re is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y2—, alkenyl-Y2—, alkynyl-Y2—, aryl-Y2—, or heteroaryl-Y2—, or a cation which is Na+1, Li+1, or K+1;
• Y2 is O, NRd, or S;
• each instance of R4 is independently hydrogen, —OH, —SH, —NRdRd, —N3, halogen, alkyl, alkenyl, alkynyl, alkyl-Y1—, alkenyl-Y1—, alkynyl-Y1—, aryl-Y1—, heteroaryl-Y1—, —ORb, or —SRc, and Rb is a blocking group;
• each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase;
• R5 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 R1—R2;
• R1 is —S-alkenylene-, —S-alkylene-, —S-alkylene-aryl-alkylene-, —S—CO-aryl-alkylene-, or —S—CO-alkylene-aryl-alkylene-;
• R2 is heterocyclo-alkylene-S—, heterocyclo-alkenylene-S—, aminoalkyl-S—, or (alkyl)4N-alkylene-S—;
• and the oligonucleotide of structure IIIb has the following structure:
• wherein:
• at least one phosphorothiotriester linkages is non-stereorandom,
• W is O;
• R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or R1—R2;
• R1 is —S-alkenylene-, —S-alkylene-, —S-alkylene-aryl-alkylene-, —S—CO-aryl-alkylene-, or —S—CO-alkylene-aryl-alkylene-;
• R2 is heterocyclo-alkylene-S—, heterocyclo-alkenylene-S—, aminoalkyl-S—, or (alkyl)4N-alkylene-S—;
• R3 is —OH, —SH, —NRdRd, —N3, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y1—, alkenyl-Y1—, alkynyl-Y1—, aryl-Y1—, heteroaryl-Y1—, —P(O)(Re)2, —ORa or —SRc;
• Y1 is O, NRd, S, or Se;
• Ra is a blocking group;
• Rc is a blocking group;
• each instance of Rd is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, or tertiary silyl, or when Rd is attached to a nitrogen atom, —C(O)—O-alkyl, —C(O)—O-alkenyl, —C(O)—O-alkynyl, —C(O)—O-heteroalkyl, —C(O)—O-heteroalkenyl, —C(O)—O-heteroalkynyl, —C(O)—O-aryl, or —C(O)—O-heteroaryl;
• each instance of Re is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y2—, alkenyl-Y2—, alkynyl-Y2—, aryl-Y2—, or heteroaryl-Y2—, or a cation which is Na+1, Li+1, or K+1;
• Y2 is O, NRd, or S;
• each instance of R4 is independently hydrogen, —OH, —SH, —NRdRd, —N3, halogen, alkyl, alkenyl, alkynyl, alkyl-Y1—, alkenyl-Y1—, alkynyl-Y1—, aryl-Y1—, heteroaryl-Y1—, —ORb, or —SRc, and Rb is a blocking group;
• each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase;
• R5 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
• wherein each heteroaryl group is a mono-, bi-, or poly-cyclic 5-14 membered ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

A process for the preparation of an oligonucleotide of structure IIIc comprising the steps of:

• i) reacting an H-phosphonate of structure Ic with a 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 oligonucleotide of structure IIIc;
• wherein:
• the H-phosphonate of structure Ic has the following structure:
• wherein:
• W is O;
• R3 is —OH, —SH, —NRdRd, —N3, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y1—, alkenyl-Y1—, alkynyl-Y1—, aryl-Y1—, heteroaryl-Y1—, —P(O)(Re)2, —ORa or —SRc;
• Y1 is O, NRd, S, or Se;
• Ra is a blocking group;
• Rc is a blocking group;
• each instance of Rd is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, or tertiary silyl, or when Rd is attached to a nitrogen atom, —C(O)—O-alkyl, —C(O)—O-alkenyl, —C(O)—O-alkynyl, —C(O)—O-heteroalkyl, —C(O)—O-heteroalkenyl, —C(O)—O-heteroalkynyl, —C(O)—O-aryl, or —C(O)—O-heteroaryl;
• each instance of Re is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y2—, alkenyl-Y2—, alkynyl-Y2—, aryl-Y2—, or heteroaryl-Y2—, or a cation which is Na+1, Li+1, or K+1;
• Y2 is O, NRd, or S;
• each instance of R4 is independently hydrogen, —OH, —SH, —NRdRd, —N3, halogen, alkyl, alkenyl, alkynyl, alkyl-Y1—, alkenyl-Y1—, alkynyl-Y1—, aryl-Y1—, heteroaryl-Y1—, —ORb, or —SRc, and Rb is a blocking group;
• each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase;
• R5 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 R6 is independently alkyl, cycloalkyl, aryl, or heteroaryl;
• the nucleophile of structure VIc has the following structure:
• R7—SH,
• wherein R7 is alkyl, alkenyl, aryl, heterocyclo, aminoalkyl, or (heterocyclo)alkyl;
• and the oligonucleotide of structure IIIc has the following structure:
• wherein:
• W is O;
• R is R7—S—S—X—;
• R7 is alkyl, alkenyl, aryl, heterocyclo, aminoalkyl, or (heterocyclo)alkyl;
• X is alkylene, alkenylene, arylene, or heteroarylene;
• R3 is —OH, —SH, —NRdRd, —N3, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y1—, alkenyl-Y1—, alkynyl-Y1—, aryl-Y1—, heteroaryl-Y1—, —P(O)(Re)2, —ORa or —SRc;
• Y1 is O, NRd, S, or Se;
• Ra is a blocking group;
• Rc is a blocking group;
• each instance of Rd is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, or tertiary silyl, or when Rd is attached to a nitrogen atom, —C(O)—O-alkyl, —C(O)—O-alkenyl, —C(O)—O-alkynyl, —C(O)—O-heteroalkyl, —C(O)—O-heteroalkenyl, —C(O)—O-heteroalkynyl, —C(O)—O-aryl, or —C(O)—O-heteroaryl;
• each instance of Re is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y2—, alkenyl-Y2—, alkynyl-Y2—, aryl-Y2—, or heteroaryl-Y2—, or a cation which is Na+1, Li+1, or K+1;
• Y2 is O, NRd, or S;
• each instance of R4 is independently hydrogen, —OH, —SH, —NRdRd, —N3, halogen, alkyl, alkenyl, alkynyl, alkyl-Y1—, alkenyl-Y1—, alkynyl-Y1—, aryl-Y1—, heteroaryl-Y1—, —ORb, or —SRc, and Rb is a blocking group;
• each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase;
• R5 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 oligonucleotide of structure IIIc may optionally comprise non-stereorandom phosphorous linkages;
• and
• wherein each heteroaryl group is a mono-, bi-, or poly-cyclic 5-14 membered ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.