Propargyl substituted nucleoside compounds and methods

Abstract

The present teachings relate to nucleobase, nucleoside and nucleotide compounds, methods of synthesis, and uses thereof. The present teachings provide compounds, such as nucleobase, nucleoside and/or nucleotide compounds including a propargyl linker, and methods for making or using such compounds.

1 . A nucleotide comprising a nucleobase disposed between a sugar or sugar analog and a propargylic moiety, wherein the nucleobase comprises a pyrimidine nucleobase or pyrimidine nucleobase analog, the propargylic moiety is covalently attached to the nucleobase at the C-5 position of the nucleobase, the sugar or sugar analog is covalently attached to the nucleobase at the N−1 position of the nucleobase, the sugar or sugar analog comprises a 5′-monophosphate group, 5′-diphosphate group, or 5′-triphosphate group, wherein the propargylic moiety comprises
 (i) a propargyl group covalently attached by its methylene carbon to the nucleobase,   (ii) a detectable label, and   (iii) a linker covalently linking the acetylene carbon of the propargyl group to the detectable label.   
     
     
         2 . The nucleotide of  claim 1  wherein the nucleobase comprises a pyrimidine base. 
     
     
         3 . The nucleotide of  claim 2  wherein the pyrimidine base is uridine. 
     
     
         4 . The nucleotide of  claim 3  wherein the sugar comprises ribose, 2′-deoxyribose or 2′,3′-dideoxyribose. 
     
     
         5 . The nucleotide of  claim 4  wherein the sugar is 2′,3′-dideoxyribose. 
     
     
         6 . The nucleotide of  claim 1  wherein the sugar or sugar analog comprises a compound of the structure: 
       
         
           
           
               
               
           
         
       
       wherein,
 W 1  when taken alone is —H or —OH, 
 W 2  is OH or a nonextendable moiety, 
 W 3  when taken alone is —H or when taken together with W 1  is —CH 2 —O—, and 
 W 4  is monophosphate, diphosphate, or triphosphate. 
 
     
     
         7 . The nucleotide of  claim 1  wherein the linker comprises from 1-50 atoms selected from C, H, N, O, S, P and Si. 
     
     
         8 . The nucleotide of  claim 1  wherein the linker comprises at least one anionic moiety. 
     
     
         9 . The nucleotide of  claim 1  wherein the linker comprises at least one cationic moiety. 
     
     
         10 . The nucleotide of  claim 1  wherein the linker comprises at least one amide linkage. 
     
     
         11 . The nucleotide of  claim 10  wherein the linker comprises at least one diradical selected from 
       
         
           
           
               
               
           
         
       
     
     
         12 . The nucleotide of  claim 10  wherein the linker comprises at least one diradical selected from 
       
         
           
           
               
               
           
         
       
       wherein Z is selected from oxygen, sulfur, substituted amine and unsubstituted amine, and the carbonyl carbon is covalently attached to X. 
     
     
         13 . The nucleotide of  claim 12  wherein Z is unsubstituted amine. 
     
     
         14 . The nucleotide of  claim 12  wherein Z is substituted amine. 
     
     
         15 . The nucleotide of  claim 1  wherein the detectable label comprises at least one of a fluorescent dye, an energy transfer dye, a quencher and biotin. 
     
     
         16 . The nucleotide of  claim 1  wherein the detectable label comprises a fluorescent dye comprising a rhodamine dye, a fluorescein dye, a rhodol dye, an energy transfer dye, a cyanine dye, a phthalocyanine or a squaraine. 
     
     
         17 . The nucleotide of  claim 15  wherein the fluorescent dye comprises a fluorescein dye, a rhodamine dye or an energy transfer dye. 
     
     
         18 . The nucleotide of  claim 1  wherein the detectable label comprises an energy transfer dye comprising a donor dye covalently attached to an acceptor dye, wherein either the donor dye or the acceptor dye is covalently attached to the linker, and the acceptor dye is capable of absorbing light emitted by the donor dye. 
     
     
         19 . The nucleotide of  claim 18  wherein the donor dye is covalently attached to the linker. 
     
     
         20 . The nucleotide of  claim 18  wherein the donor dye comprises a fluorescein dye or a rhodamine dye. 
     
     
         21 . The nucleotide of  claim 20  wherein the acceptor dye comprises one of a rhodamine dye, a fluorescein dye, a cyanine dye or a non-fluorescent quencher. 
     
     
         22 . The nucleotide of  claim 18  wherein the donor dye is covalently linked to the acceptor dye through a linker-moiety that is different from the first said linker. 
     
     
         23 . The nucleotide of  claim 18  wherein the donor dye comprises a fluorescein or a rhodamine dye and the acceptor dye is a non-fluorescent quencher. 
     
     
         24 . A labeled nucleoside comprising a nucleobase disposed between a sugar or a sugar analog and a propargylic moiety, wherein the nucleobase is selected from a pyrimidine base or a pyrimidine base analog, the propargylic moiety is covalently attached to the nucleobase at the C-5 position of the nucleobase, the sugar or sugar analog is covalently attached to the nucleobase at the N−1 position of the nucleobase, and wherein the propargylic moiety comprises
 (i) a propargyl group covalently attached by its methylene carbon to the nucleobase,   (ii) a detectable label, and   (iii) a linker covalently linking the acetylene carbon of the propargyl group to the detectable label.   
     
     
         25 . The nucleoside of  claim 24  wherein the nucleobase comprises a pyrimidine base. 
     
     
         26 . The nucleoside of  claim 24  wherein the pyrimidine base is uridine. 
     
     
         27 . The nucleoside of  claim 24  wherein the sugar comprises ribose, 2′-deoxyribose or 2′,3′-dideoxyribose. 
     
     
         28 . The nucleoside of  claim 27  wherein the sugar is 2′,3′-dideoxyribose. 
     
     
         29 . The nucleoside of  claim 24  wherein the sugar or sugar analog comprises a compound of the structure: 
       
         
           
           
               
               
           
         
         W 1  when taken alone is —H or —OH, 
         W 2  is —OH or a nonextendable moiety, 
         W 3  when taken alone is —H or when taken together with W 1  is —CH 2 —O—, and 
         W 4  is monophosphate, diphosphate, or triphosphate. 
       
     
     
         30 . The nucleoside of  claim 24  wherein the linker comprises from 1-50 atoms selected from C, H, N, O, S, P and Si. 
     
     
         31 . The nucleoside of  claim 24  wherein the linker comprises at least one anionic moiety. 
     
     
         32 . The nucleoside of  claim 24  wherein the linker comprises at least one cationic moiety. 
     
     
         33 . The nucleoside of  claim 24  wherein the linker comprises at least one amide linkage. 
     
     
         34 . The nucleoside of  claim 33  wherein the linker comprises at least one diradical selected from 
       
         
           
           
               
               
           
         
       
     
     
         35 . The nucleoside of  claim 33  wherein the linker comprises at least one diradical selected from 
       
         
           
           
               
               
           
         
       
       wherein Z is selected from oxygen, sulfur, substituted amine and unsubstituted amine, and the carbonyl carbon is covalently attached to X. 
     
     
         36 . The nucleoside of  claim 35  wherein Z is unsubstituted amine. 
     
     
         37 . The nucleoside of  claim 35  wherein Z is substituted amine. 
     
     
         38 . The nucleoside of  claim 24  wherein the detectable label comprises at least one of a fluorescent dye, an energy transfer dye, a quencher or biotin. 
     
     
         39 . The nucleoside of  claim 24  wherein the detectable label comprises a rhodamine dye, a fluorescein dye, a rhodol dye, an energy transfer dye, a cyanine dye, a phthalocyanine and a squaraine. 
     
     
         40 . The nucleoside of  claim 24  wherein the detectable label comprises a fluorescein dye, a rhodamine dye or an energy transfer dye. 
     
     
         41 . The nucleoside of  claim 24  wherein the detectable label comprises an energy transfer dye comprising a donor dye covalently attached to an acceptor dye, wherein either the donor dye or the acceptor dye is covalently attached to the linker, and the acceptor dye is capable of absorbing the light emitted by the donor dye. 
     
     
         42 . The nucleoside of  claim 41  wherein the donor dye is covalently attached to the linker. 
     
     
         43 . The nucleoside of  claim 41  wherein the donor dye comprises a fluorescein dye or a rhodamine dye. 
     
     
         44 . The nucleoside of  claim 43  wherein the acceptor dye comprises one of a rhodamine dye, a fluorescein dye, a cyanine dye or a non-fluorescent quencher. 
     
     
         45 . The nucleoside of  claim 44  wherein the donor dye is covalently linked to the acceptor dye through a linker-moiety. 
     
     
         46 . The nucleoside of  claim 41  wherein the donor dye comprises a fluorescein or a rhodamine dye and the acceptor dye is a non-fluorescent quencher. 
     
     
         47 . A polynucleotide comprising at least one nucleobase comprising the structure
   NUC—CH 2 —≡—X-LABEL   
       wherein NUC comprises a pyrimidine nucleobase or pyrimidine nucleobase analog, X comprises a bond or a linker having from 1-50 chain atoms selected from C, N, O, S, P and Si, and LABEL comprises at least one detectable label. 
     
     
         48 . The polynucleotide of  claim 47  wherein NUC comprises a pyrimidine nucleobase. 
     
     
         49 . The polynucleotide of  claim 47  wherein the pyrimidine nucleobase is uridine. 
     
     
         50 . The polynucleotide of  claim 47  wherein the sugar comprises ribose, 2′-deoxyribose or 2′,3′-dideoxyribose. 
     
     
         51 . The polynucleotide of  claim 50  wherein the sugar is 2′,3′-dideoxyribose. 
     
     
         52 . The polynucleotide of  claim 47  wherein the sugar or sugar analog comprises a compound of the structure: 
       
         
           
           
               
               
           
         
         wherein W 1  when taken alone is —H or —OH, 
         W 2  is —OH or a nonextendable moiety, 
         W 3  when taken alone is —H or when taken together with W 1  is —CH 2 —O—, and 
         W 4  is monophosphate, diphosphate, or triphosphate. 
       
     
     
         53 . The polynucleotide of  claim 47  wherein the linker comprises from 1-50 atoms selected from C, H, N, O, S, P and Si. 
     
     
         54 . The polynucleotide of  claim 47  wherein the linker comprises at least one anionic moiety. 
     
     
         55 . The polynucleotide of  claim 47  wherein the linker comprises at least one cationic moiety. 
     
     
         56 . The polynucleotide of  claim 47  wherein the linker comprises at least one amide linkage. 
     
     
         57 . The polynucleotide of  claim 56  wherein the linker comprises at least one diradical selected from 
       
         
           
           
               
               
           
         
       
     
     
         58 . The polynucleotide of  claim 56  wherein the linker comprises at least one diradical selected from 
       
         
           
           
               
               
           
         
       
       wherein Z is selected from oxygen, sulfur, substituted amine and unsubstituted amine, and the carbonyl carbon is covalently attached to X. 
     
     
         59 . The polynucleotide of  claim 58  wherein Z is unsubstituted amine. 
     
     
         60 . The polynucleotide of  claim 58  wherein Z is substituted amine. 
     
     
         61 . The polynucleotide of  claim 47  wherein the detectable label comprises at least one of a fluorescent dye, an energy transfer dye, a quencher or biotin. 
     
     
         62 . The polynucleotide of  claim 47  wherein the detectable label comprises a fluorescent dye selected from a rhodamine dye, a fluorescein dye, a rhodol dye, an energy transfer dye, a cyanine dye, a phthalocyanine and a squaraine. 
     
     
         63 . The polynucleotide of  claim 61  wherein the fluorescent dye comprises a fluorescein dye, a rhodamine dye or an energy transfer dye. 
     
     
         64 . The polynucleotide of  claim 47  wherein the detectable label comprises an energy transfer dye comprising a donor dye covalently attached to an acceptor dye, wherein either the donor dye or the acceptor dye is covalently attached to the linker, and the acceptor dye is capable of absorbing the light emitted by the donor dye. 
     
     
         65 . The polynucleotide of  claim 64  wherein the donor dye is covalently attached to the linker. 
     
     
         66 . The polynucleotide of  claim 64  wherein the donor dye comprises a fluorescein dye or a rhodamine dye. 
     
     
         67 . The polynucleotide of  claim 66  wherein the acceptor dye comprises one of a rhodamine dye, a fluorescein dye, a cyanine dye or a non-fluorescent quencher. 
     
     
         68 . The polynucleotide of  claim 67  wherein the donor dye is covalently linked to the acceptor dye through a linker-moiety that is different from the first said linker. 
     
     
         69 . The polynucleotide of  claim 64  wherein the donor dye comprises a fluorescein or a rhodamine dye and the acceptor dye is a non-fluorescent quencher. 
     
     
         70 . A nucleoside comprising a nucleobase disposed between a sugar or a sugar analog and a propargylic moiety, wherein the nucleobase is selected from a pyrimidine base or a pyrimidine base analog, the propargylic moiety is covalently attached to the nucleobase at the C-5 position of the nucleobase, the sugar or sugar analog is covalently attached to the nucleobase at the N−1 position of the nucleobase, and wherein the propargylic moiety comprises a propargyl group covalently attached to the nucleobase at the methylene carbon of the propargyl group. 
     
     
         71 . The nucleoside of  claim 70  wherein the nucleobase comprises a pyrimidine base. 
     
     
         72 . The nucleoside of  claim 70  wherein the pyrimidine base is uridine. 
     
     
         73 . The nucleoside of  claim 70  wherein the sugar comprises ribose, 2′-deoxyribose or 2′,3′-dideoxyribose. 
     
     
         74 . The nucleoside of  claim 70  wherein the sugar is 2′,3′-dideoxyribose. 
     
     
         75 . The nucleoside of  claim 70  wherein the sugar or sugar analog comprises a compound of the structure: 
       
         
           
           
               
               
           
         
         W 1  when taken alone is —H or —OH, 
         W 2  is —OH or a nonextendable moiety, 
         W 3  when taken alone is —H or when taken together with W 1  is —CH 2 —O—, and 
         W 4  is monophosphate, diphosphate, or triphosphate. 
       
     
     
         76 . The nucleoside of  claim 70  wherein the linker comprises from 1-50 atoms selected from C, H, N, O, S, P and Si. 
     
     
         77 . The nucleoside of  claim 70  wherein the linker comprises at least one anionic moiety. 
     
     
         78 . The nucleoside of  claim 70  wherein the linker comprises at least one cationic moiety. 
     
     
         79 . The nucleoside of  claim 70  wherein the linker comprises at least one amide linkage. 
     
     
         80 . The nucleoside of  claim 79  wherein the linker comprises at least one diradical selected from 
       
         
           
           
               
               
           
         
       
     
     
         81 . The nucleoside of  claim 79  wherein the linker comprises at least one diradical selected from 
       
         
           
           
               
               
           
         
       
       wherein Z is selected from oxygen, sulfur, substituted amine and unsubstituted amine, and the carbonyl carbon is covalently attached to X. 
     
     
         82 . The nucleoside of  claim 81  wherein Z is unsubstituted amine. 
     
     
         83 . The nucleoside of  claim 81  wherein Z is substituted amine. 
     
     
         84 . A nucleotide comprising a nucleobase disposed between a sugar or a sugar analog and a propargylic moiety, wherein the nucleobase is selected from a pyrimidine base or a pyrimidine base analog, the propargylic moiety is covalently attached to the nucleobase at the C-5 position of the nucleobase, the sugar or sugar analog is covalently attached to the nucleobase at the N−1 position of the nucleobase, the sugar or sugar analog comprises a 5′-monophosphate group, 5′-diphosphate group or 5′-triphosphate group and wherein the propargylic moiety comprises a propargyl group covalently attached to the nucleobase at the methylene carbon of the propargyl group. 
     
     
         85 . The nucleotide of  claim 84  wherein the nucleobase comprises a pyrimidine base. 
     
     
         86 . The nucleotide of  claim 85  wherein the pyrimidine base is uridine. 
     
     
         87 . The nucleotide of  claim 84  wherein the sugar comprises ribose, 2′-deoxyribose or 2′,3′-dideoxyribose. 
     
     
         88 . The nucleotide of  claim 84  wherein the sugar is 2′,3′-dideoxyribose. 
     
     
         89 . The nucleotide of  claim 84  wherein the sugar or sugar analog comprises a compound of the structure: 
       
         
           
           
               
               
           
         
         W 1  when taken alone is —H or —OH, 
         W 2  is —OH or a nonextendable moiety, 
         W 3  when taken alone is —H or when taken together with W 1  is —CH 2 —O—, and 
         W 4  is monophosphate, diphosphate, or triphosphate. 
       
     
     
         90 . The nucleotide of  claim 84  wherein the linker comprises from 1-50 atoms selected from C, H, N, O, S, P and Si. 
     
     
         91 . The nucleotide of  claim 84  wherein the linker comprises at least one anionic moiety. 
     
     
         92 . The nucleotide of  claim 84  wherein the linker comprises at least one cationic moiety. 
     
     
         93 . The nucleotide of  claim 84  wherein the linker comprises at least one amide linkage. 
     
     
         94 . The nucleotide of  claim 92  wherein the diradical comprises at least one diradical selected from 
       
         
           
           
               
               
           
         
       
     
     
         95 . The nucleotide of  claim 93  comprising at least one diradical selected from 
       
         
           
           
               
               
           
         
       
       wherein Z is selected from oxygen, sulfur, substituted amine and unsubstituted amine, and the carbonyl carbon is covalently attached to X. 
     
     
         96 . The nucleotide of  claim 95  wherein Z is unsubstituted amine. 
     
     
         97 . A method of extending a polynucleotide comprising
 forming a hybridization complex between a 3′-extendable polynucleotide and a complementary sequence in a polynucleotide, and   extending the 3′-extendable polynucleotide in the presence of a nucleotide 5′-triphosphate and a template-dependent nucleic acid polymerase under conditions effective to append the nucleotide 5′ triphosphate to the 3′-end of the extendable polynucleotide, wherein the nucleotide 5′-triphosphate comprises nucleobase disposed between a sugar or sugar analog and a propargylic moiety, the nucleobase comprises a pyrimidine nucleobase or pyrimidine nucleobase analog, the propargylic moiety is covalently attached to the nucleobase at the C-5 position of the nucleobase, the sugar or sugar analog is covalently attached to the nucleobase at the N−1 position of the nucleobase, the sugar or sugar analog comprises a 5′-monophosphate group, a 5′-diphosphate group, or a 5′-triphosphate group, and the propargylic moiety comprises (i) a propargyl group covalently attached by its methylene carbon to the nucleobase, (ii) a detectable label, and (iii) a linker covalently linking the acetylene carbon of the propargyl group to the detectable label.   
     
     
         98 . The method of  claim 97  wherein the extendable polynucleotide comprises an extended primer 10-500 nucleotides in length. 
     
     
         99 . The method of  claim 97  wherein the nucleotide 5′ triphosphate is non-extendable. 
     
     
         100 . The method of  claim 97  wherein the nucleotide 5′ triphosphate comprises a dideoxyribose moiety. 
     
     
         101 . The method of  claim 97  wherein the nucleic acid polymerase comprises a DNA polymerase. 
     
     
         102 . The method of  claim 97  wherein said extending is performed in the presence of four different nucleotide triphosphates and a compound of  claim 1 . 
     
     
         103 . A method for making a thymidine analog comprising:
 contacting a protected or unprotected 5-halomethyl uridine nucleotide with a copper acetylide to form an adduct comprising a 5-propargyl uridine.   
     
     
         104 . The method of  claim 103  wherein the copper acetylide comprises a mixture comprising a copper reagent, a base and an alkyne. 
     
     
         105 . A compound having the formula:
   NUC—CH 2 —C≡C—X-LABEL   
       wherein
 NUC comprises a nucleoside comprising a sugar moiety and a nucleobase selected from a pyrimidine nucleobase or a pyrimidine nucleobase analog, 
 the CH 2  moiety is covalently attached to the 5-atom of the nucleobase, 
 X is bond or a linker having from 1 to 50 chain atoms each selected from C, H, N, O, S, P and Si, and 
 the sugar moiety is covalently attached to the N−1 position of the nucleobase. 
 
     
     
         106 . The compound of  claim 105  wherein NUC comprises a 5′-triphosphate group. 
     
     
         107 . The compound of  claim 105  wherein X comprises a linker having from 1 to 25 chain atoms. 
     
     
         108 . A compound comprising the structure 
       
         
           
           
               
               
           
         
       
       wherein
 X comprises a bond or a linker, 
 LABEL comprises at least one detectable label, 
 W 1  taken alone is —H or —OH, 
 W 2  is —OH or a nonextendable moiety, 
 W 3  when taken alone is —H or when taken together with W 1  is —CH 2 —O—, and 
 W 4  is OH, monophosphate, diphosphate, or triphosphate.