Propargylethoxyamino nucleotides

Abstract

Propargylethoxyamino nucleosides are disclosed having the structure wherein R 1 and R 2 are —H lower alkyl, or label; B is a 7-deazapurine, purine, or pyrimidine nucleoside base; W 1 is —H or —H; W 2 is —H or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′-position; and W 3 is —PO 4 , —P 2 O 7 , —P 3 O 10 , phosphate analog, or —OH. Additionaly, a primer extension method is provided employing the above propargylethoxyamino nucleosides.

1 . A nucleoside compound having the structure:  
     
       
         
         
             
             
         
       
       wherein:  
       R 1  and R 2  taken separately are selected from the group consisting of —H, lower alkyl protecting group, and label;  
       B is a 7-deazapurine, purine, or pyrimidine nucleoside base;  
       wherein when B is purine or 7-deazapurine, the sugar moiety is attached at the N 9 -position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N 1 -position of the pyrimidine; and  
       wherein when B is a purine, the adjacent triple-bonded carbon is attached to the 8-position of the purine, when B is 7-deazapurine, the adjacent triple-bonded carbon is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, the adjacent triple-bonded carbon is attached to the 5-position of the pyrimidine;  
       W 1  is selected from the group consisting of —H and —OH;  
       W 2  is —OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′-position; and  
       W 3  is selected from the group consisting of —PO 4 , —PO 2 O 7 , —P 3 O 10 , phosphate analog, and —OH.  
     
   
   
       2 . The nucleoside compound of  claim 1  wherein one of R 1  and R 2  is label.  
   
   
       3 . The nucleoside compound of  claim 2  wherein the label is a fluorescein-type dye.  
   
   
       4 . The nucleoside compound of  claim 2  wherein the label is a rhodamine-type dye.  
   
   
       5 . The nucleoside compound of  claim 1  wherein W 1  is —H and W 2  is —OH or a moiety, and W 3  is —P 3 O 10 .  
   
   
       6 . The nucleoside compound of  claim 1  wherein W 1  is —H, W 2  is —OH or a moiety, and W 3  is —P 3 O 10 .  
   
   
       7 . The nucleoside compound of  claim 1  wherein W 2  is —H or a moiety selected from the group consisting of —H, azido, amino, fluro, and methoxy.  
   
   
       8 . The nucleoside of  claim 1  wherein B is selected from the group consisting of uracil, cytosine, 7-deazaadenine, and 7-deazaguanosine.  
   
   
       9 . A method for performing a primer extension reaction comprising the steps of: 
 providing a template nucleic acid;    annealing an oligonucleotide primer to a portion of the template nucleic acid; and    adding primer-extension reagents to the primer-template hybrid for extending the primer, the primer extension reagents including a nucleoside compound having the structure:                          wherein:    R 1  and R 2  taken separately are selected from the group consisting of —H, lower alkyl, protecting group, and label;    B is a 7-deazapurine, purine, or pyrimidine nucleoside base;    wherein when B is purine or 7-deazapurine, the sugar moiety is attached at the N 9 -position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N 1 -position of the pyrimidine; and    wherein if B is a purine, the adjacent triple-bonded carbon is attached to the 8-position of the purine, if B is 7-deazapurine, the adjacent triple-bonded carbon is attached to the 7-position of the 7-deazapurine, and if B is pyrimidine, the adjacent triple-bonded carbon is attached to the 5-position of the pyrimidine;    W 1  is selected from the group consisting of —H and —OH;    W 2  is —OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′-position; and    W 3  is selected from the group consisting of —PO 4 , —P 2 O 7 , —P 3 O 10 , phosphate analog, and —H.    
   
   
       9 . The method of  claim 9  wherein one of R 1  and R 2  is label and the other is —H.