Synthesis of 2', 3'-dideoxynucleosides for automated dna synthesis and pyrophosphorolysis activated polymerization

Abstract

Methods for preparation of 2′,3′-dideoxynucleotides support structures, such as 2′,3′-dideoxyguanosine, 2′,3′-dideoxyadenosine, and 3′-deoxythymidine support structures are disclosed. Various methods of using such structures are also provided, such as their use for automated DNA synthesis and pyrophosphorolysis activated polymerization.

1 - 11 . (canceled) 
     
     
         12 . A method for detecting a nucleic acid, the method comprising:
 (a) preparing a 2′,3′-dideoxynucleoside selected from the group consisting of 2′,3′-dideoxyguanosine, 2′,3′-dideoxyadenosine, and 3′-deoxythymidine support structures.   (b) preparing a 2′,3′-dideoxynucleotide from the 2′,3′-dideoxynucleoside, wherein the 2′,3′-dideoxynucleotide has a non-extendable 3′ end, which is removable by pyrophosphorolysis;   (c) annealing the 2′,3′-dideoxynucleotide to a nucleic acid;   (d) removing the 3′ non-extendable terminus of the 2′,3′-dideoxynucleotide by pyrophosphorolysis to produce an unblocked oligonucleotide;   (e) extending the unblocked oligonucleotide; and   (f) detecting the presence of the nucleic acid by detecting the extended oligonucleotide to thereby detect the nucleic acid.   
     
     
         13 . The method of  claim 12 , wherein pyrophosphorolysis is performed in the presence of a nucleic acid polymerase. 
     
     
         14 . The method of  claim 12 , wherein the extending of the unblocked oligonucleotide is performed with a nucleic acid polymerase. 
     
     
         15 . The method of  claim 12 , wherein the unblocked oligonucleotide present in the extension step contains a label and the detecting step comprises detecting the presence of the label. 
     
     
         16 . A method of synthesizing a nucleic acid, the method comprising:
 (a) preparing a 2′,3′-dideoxynucleoside selected from the group consisting of 2′,3′-dideoxyguanosine, 2′,3′-dideoxyadenosine, and 3′-deoxythymidine support structures;   (b) preparing a 2′,3′-dideoxynucleotide from the 2′,3′-dideoxynucleoside, wherein the 2′,3′-dideoxynucleotide has a non-extendable 3′ end, which is removable by pyrophosphorolysis;   (c) annealing the 2′,3′-dideoxynucleotide to a nucleic acid;   (d) removing the 3′ non-extendable terminus of the 2′,3′-dideoxynucleotide by pyrophosphorolysis to produce an unblocked oligonucleotide; and   (e) extending the unblocked oligonucleotide using a nucleic acid polymerase to thereby synthesize the nucleic acid.   
     
     
         17 . The method of  claim 16 , wherein pyrophosphorolysis is performed in the presence of a nucleic acid polymerase. 
     
     
       claims  18 - 20 . (canceled) 
     
     
         21 . The method of  claim 17 , wherein at least one of the unblocked oligonucleotides present in the extending step contains a label and the detecting step comprises detecting the presence of the label. 
     
     
         22 - 26 . (canceled) 
     
     
         27 . A pyrophosphorolysis activated polymerization method of synthesizing a desired nucleic acid strand on a nucleic acid template strand, comprising serially:
 (a) preparing a 2′,3′-dideoxynucleoside selected from the group consisting of 2′,3′-dideoxyguanosine, 2′,3′-dideoxyadenosine, and 3′-deoxythymidine support structures;   (b) preparing an activatable 2′,3′-dideoxynucleotide from the 2′,3′-dideoxynucleoside, wherein the activatable 2′,3′-dideoxynucleotide has a non-extendable 3′-deoxynucleotide end, which is removable by pyrophosphorolysis and has a mismatch with respect to the corresponding nucleotide on the template strand;   (c) annealing to the template strand the activatable 2′,3′-dideoxynucleotide;   (d) pyrophosphorolyzing the resulting duplex with pyrophosphate and an enzyme that has pyrophosphorolysis activity and activates the 2′,3′-dideoxynucleotide by removal of the terminal 3′-deoxynucleotide; and   (e) polymerizing by extending the activated 2′,3′-dideoxynucleotide on the template strand in presence of four nucleoside triphosphates and a nucleic acid polymerase to synthesize the desired nucleic acid strand.   
     
     
         28 . The method of  claim 12 , wherein the 2′,3′-dideoxynucleoside is a 2′,3′-dideoxyguanosine support structure prepared by a method comprising:
 (a) reacting 2′,3′-dideoxyguanosine with N,N-dimethylformamide dimethylacetal, to obtain a first intermediate; 
 (b) reacting the first intermediate with 4,4-dimethoxytriphenylmethyl chloride, to obtain a second intermediate comprising a dimethylformamide protecting group; 
 (c) cleaving the dimethylformamide protecting group off the second intermediate, to obtain a third intermediate; and 
 (d) reacting the third intermediate with adipic anhydride, to thereby obtain a precursor for the 2′,3′-dideoxyguanosine support structure, wherein the precursor is a derivative of 2′,3′-dideoxyguanosine. 
 
     
     
         29 . The method of  claim 12 , wherein the 2′3′-dideoxynucleoside is a 2′,3′-dideoxyguanosine support structure prepared by a method comprising:
 (a) reacting 2′,3′-dideoxyguanosine with isobutyric anhydride, to obtain a first intermediate; 
 (b) reacting the first intermediate with 4,4-dimethoxytriphenylmethyl chloride, to obtain a second intermediate comprising an isobutyramide protecting group; 
 (c) cleaving the isobutyramide protecting group off the second intermediate, to obtain a third intermediate; and 
 (d) reacting the third intermediate with adipic anhydride, 
 
       to thereby obtain a precursor for the 2′,3′-dideoxyguanosine support structure, wherein the precursor is a derivative of 2′,3′-dideoxyguanosine. 
     
     
         30 . The method of  claim 12 , wherein the 2′,3′-dideoxynucleoside is a 2′,3′-dideoxyadenosine support structure prepared by a method comprising:
 (a) reacting 2′,3′-dideoxyadenosine with 4,4-dimethoxytriphenylmethyl chloride, to obtain a first intermediate; 
 (b) reacting pentane-1,3,5-tricarboxylic acid with oxalyl chloride, to obtain a second intermediate; and 
 (c) reacting the first intermediate with the second intermediate, 
 
       to thereby obtain a precursor for the 2′,3′-dideoxyadenosine support structure, wherein the precursor is a derivative of 2′,3′-dideoxyadenosine. 
     
     
         31 . The method of  claim 12 , wherein the 2′,3′-dideoxynucleoside is a 3′-deoxythymidine support structure, prepared by a method comprising:
 (a) esterifying 3-fluoro-6-nitrobenzoic acid, to obtain an ester intermediate; 
 (b) reacting the ester intermediate with 2-mercaptoethanol, to obtain a thiol intermediate; 
 (c) reacting 1-(5-((bis(4-methoxyphenyl) (phenyl)methoxy)methyl) tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4-dione with the thiol intermediate under Mitsunobu conditions to obtain a third intermediate; and 
 (d) subjecting the third intermediate to saponification, 
 
       to thereby obtain a precursor for the 3′-deoxythymidine support structure, wherein the precursor is a derivative of 3′-deoxythymidine. 
     
     
         32 . A 2′,3′-dideoxyguanosine support structure, wherein the 2′,3′-dideoxyguanosine support structure is prepared by the method of  claim 28 . 
     
     
         33 . A 2′,3′-dideoxyguanosine support structure, wherein the 2′,3′-dideoxyguanosine support structure is prepared by the process of  claim 29 . 
     
     
         34 . A 2′,3′-dideoxyadenosine support structure, wherein the 2′,3′-dideoxyadenosine support structure prepared by the process of  claim 30 . 
     
     
         35 . A 3′-deoxythymidine support structure, wherein the 3′-deoxythymidine support structure is prepared by the process of  claim 31 .