Phosphoroamidate esters, and use and synthesis thereof

Abstract

Phosphoramidate esters and related nucleotide analogs useful in polynucleotide sequencing techniques, and synthetic methods for preparing those compounds, are disclosed. These compounds include nucleotide phosphoramidates analogs that are modified on the alpha-phosphate to enable attachment of a variety of application-specific substituents such as tether molecules.

What is claimed is: 
     
         1 . A compound of the formula 
       
         
           
           
               
               
           
         
         wherein 
         R 1  is selected from
 a) an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen; and 
 b) an alkyl group and an oxyalkyl group, either of which terminates in a linker group (LG1), the LG1 bonded to a tether (T); 
 
         R 2  is selected from hydrogen and C 1 -C 4 alkyl; 
         R 3  is selected from R 5  and -[Pn-O] m —R 5 , where Pn is independently selected from P(OR 5 ) and P(═O)(OR 5 ) at each occurrence, and m is selected from 1, 2, 3, 4, 5 and 6; 
         R 4  is selected from 
       
       
         
           
           
               
               
           
         
         R 5  is selected from H and G 1 ; 
         R 6  is a heterocycle, the heterocycle optionally comprising a substituent R 13 , where R 13  is selected from
 a) an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen; and 
 b) an alkyl group and an oxyalkyl group, either of which terminates in a linker group (LG2), the LG2 bonded to the tether (T); 
 
         R 7  is selected from hydrogen, —CH 2 -halogen, C 1 -C 4 alkyl, hydroxyl and —CH 2 —OR 10 ; 
         R 8  is —OR 11  or —O-L-SS where L-SS represents a solid support optionally bound to a linker; 
         R 9  is hydrogen or, when R 7  is —CH 2 —OR 10  then R 9  may be —CH 2 —R 12  where R 10  and R 12  form a direct bond; 
         R 11  is selected from H and G 3 ; 
         G 1  is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom; 
         G 2  is selected from oxygen, sulfur and CH 2 ; and 
         G 3  is a protecting group for a hydroxyl group that is bonded to a carbon atom. 
       
     
     
         2 . The compound of  claim 1  wherein each of R 1  and R 13  is selected from an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen. 
     
     
         3 . The compound of  claim 1  wherein each of R 1  and R 13  is selected from an alkyl group and an oxyalkyl group, either of which terminates in a linker group (LG1), the LG1 bonded to a tether (T). 
     
     
         4 . The compound of  claim 3  wherein LG1 and LG2 are triazole groups. 
     
     
         5 . The compound of  claim 1  wherein R 1  is —(CH 2 ) q —C≡CH and q is an integer selected from 2-10. 
     
     
         6 . The compound of  claim 1  wherein R 3  is selected from 
       
         
           
           
               
               
           
         
       
     
     
         7 . The compound of  claim 1  wherein R 4  is 
       
         
           
           
               
               
           
         
       
     
     
         8 . The compound of  claim 1  wherein R 6  is selected from:
 an adenosine analog of formula 
 
       
         
           
           
               
               
           
         
         a guanosine analog of formula 
       
       
         
           
           
               
               
           
         
         a uridine analog of formula 
       
       
         
           
           
               
               
           
         
          and 
         a cytidine analog of formula 
       
       
         
           
           
               
               
           
         
          and wherein R 13  is selected from
 a) an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen; and 
 b) an alkyl group and an oxyalkyl group, either of which terminates in a linker group (LG2), the LG2 bonded to the tether (T). 
 
       
     
     
         9 . The compound of  claim 8  wherein R 13  is —C≡C—(CH 2 ) 4 —C≡CH. 
     
     
         10 . The compound of  claim 1  having the formula 
       
         
           
           
               
               
           
         
         wherein:
 G 1  is H or a protecting group; 
 R 6  is a heterocycle comprising a substituent R 13 ; 
 R 8  is selected from OR 11  and O-L-SS where SS represents a solid support and L represents a linking group between 0 and the SS; 
 R 11  is selected from H and G 3 ; and 
 G 3  is a protecting group for a hydroxyl group that is bonded to a carbon atom. 
 
       
     
     
         11 . The compound of  claim 1  having the formula 
       
         
           
           
               
               
           
         
       
       wherein:
 R 6  is a heterocycle comprising a substituent R 13 ; 
 R 8  is —OR 11  or —O-L-SS where L-SS represents a solid support bound to a linker; 
 R 11  is selected from H and G 3 ; and 
 G 3  is a protecting group for a hydroxyl group that is bonded to a carbon atom. 
 
     
     
         12 . The compound of  claim 1  having a formula selected from the group 
       
         
           
           
               
               
           
         
         wherein:
 R 8  is —OR 11  or —O-L-SS where L-SS represents a solid support bound to a linker; 
 R 11  is selected from H and G 3 ; and 
 G 3  is a protecting group for a hydroxyl group that is bonded to a carbon atom. 
 
       
     
     
         13 . The compound of  claim 1  wherein each of LG1 and LG2 is a triazole group. 
     
     
         14 . A process of forming a phosphoromonoamidate diester 110 from a phosphite triester compound (100) and an azide compound (105), 
       
         
           
           
               
               
           
         
         the process comprising combining (100) with (105) in the presence of a halide anion, wherein: 
         R 1  is selected from an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen; 
         R 2  is selected from hydrogen and C 1 -C 4 alkyl; 
         R 4  is selected from 
       
       
         
           
           
               
               
           
         
         R 6  is a heterocycle, the heterocycle optionally comprising a substituent R 13 , where R 13  is selected from
 a) an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen; and 
 b) an alkyl group and an oxyalkyl group, either of which terminates in a linker group (LG2), the LG2 bonded to the tether (T); 
 
         R 7  is selected from hydrogen, —CH 2 -halogen, C 1 -C 4 alkyl, hydroxyl and —CH 2 —OR 10 ; 
         R 8  is —OR 11  or —O-L-SS where L-SS represents a solid support bound to a linker (L); 
         R 9  is hydrogen or, when R 7  is —CH 2 —OR 10  then R 9  may be —CH 2 —R 12  where R 10  and R 12  form a direct bond; 
         R 11  is selected from H and G 3 ; 
         G 1  is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom; 
         G 2  is selected from oxygen, sulfur and CH 2 ; and 
         G 3  is a protecting group for a hydroxyl group that is bonded to a carbon atom. 
       
     
     
         15 . A process for forming a phosphate protected N-phosphoroamidate-monoester disphosphate (120) from a phosphoroamidate diester compound (110) and a phosphorylating phosphoramidite compound 115, 
       
         
           
           
               
               
           
         
         the process comprising combining (110) with a base and a silylating agent to provide a first intermediate, combining the first intermediate with (115) and an activator to provide a second intermediate, and combining the second intermediate with an oxidizing agent to form the phosphate protected N-phosphoroamidate-monoester diiphosphate (120), wherein: 
         R 1  is selected from an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen; 
         R 2  is selected from hydrogen and C 1 -C 4 alkyl; 
         R 4  is selected from 
       
       
         
           
           
               
               
           
         
         R 6  is a heterocycle, the heterocycle optionally comprising a substituent R 13 , where R 13  is selected from
 a) an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen; and 
 b) an alkyl group and an oxyalkyl group, either of which terminates in a linker group (LG2), the LG2 bonded to the tether (T); 
 
         R 7  is selected from hydrogen, —CH 2 -halogen, C 1 -C 4 alkyl, hydroxyl and —CH 2 —OR 10 ; 
         R 8  is —OR 11  or —O-L-SS where L-SS represents a solid support optionally bound to a linker (L); 
         R 9  is hydrogen or, when R 7  is —CH 2 —OR 10  then R 9  may be —CH 2 —R 12  where R 10  and R 12  form a direct bond; 
         R 11  is selected from H and G 3 ; 
         G 1  is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom; 
         G 2  is selected from oxygen, sulfur and CH 2 ; and 
         G 3  is a protecting group for a hydroxyl group that is bonded to a carbon atom. 
       
     
     
         16 . A process for forming a phosphate protected N-phosphoroamidate-monoester triphosphate (125) from a phosphate protected N-phosphoroamidate-monoester diphosphate compound (120) and a phosphorylating phosphoramidite compound (115), 
       
         
           
           
               
               
           
         
         the process comprising combining (120) with a base and a silylating agent to provide a first intermediate, combining the first intermediate with (115) and an activator to provide a second intermediate, and combining the second intermediate with an oxidizing agent to form the phosphate protected N-phosphoroamidate-monoester triphosphate (125), wherein: 
         R 1  is selected from an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen; 
         R 2  is selected from hydrogen and C 1 -C 4 alkyl; 
         R 4  is selected from 
       
       
         
           
           
               
               
           
         
         R 6  is a heterocycle, the heterocycle optionally comprising a substituent R 13 , where R 13  is selected from
 a) an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen; and 
 b) an alkyl group and an oxyalkyl group, either of which terminates in a linker group (LG2), the LG2 bonded to the tether (T); 
 
         R 7  is selected from hydrogen, —CH 2 -halogen, C 1 -C 4 alkyl, hydroxyl and —CH 2 —OR 10 ; 
         R 8  is —OR 11  or —O-L-SS where L-SS represents a solid support optionally bound to a linker (L); 
         R 9  is hydrogen or, when R 7  is —CH 2 —OR 10  then R 9  may be —CH 2 —R 12  where R 10  and R 12  form a direct bond; 
         R 11  is selected from H and G 3 ; 
         G 1  is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom; 
         G 2  is selected from oxygen, sulfur and CH 2 ; and 
         G 3  is a protecting group for a hydroxyl group that is bonded to a carbon atom. 
       
     
     
         17 . A process for forming a N-phosphoroamidate-monoester triphosphate (160) from a cyclotriphosphite (155) and an azide (105) 
       
         
           
           
               
               
           
         
         the process comprising combining (155) and (105) in the presence of solvent so as to form (160), wherein: 
         R 1  is selected from an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen; 
         R 4  is selected from 
       
       
         
           
           
               
               
           
         
         R 6  is a heterocycle, the heterocycle optionally comprising a substituent R 13 , where R 13  is selected from
 a) an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen; and 
 
         b) an alkyl group and an oxyalkyl group, either of which terminates in a linker group (LG2), the LG2 bonded to the tether (T); 
         R 7  is selected from hydrogen, —CH 2 -halogen, C 1 -C 4 alkyl, hydroxyl and —CH 2 —OR 10 ; 
         R 8  is —OR 11  or —O-L-SS where L-SS represents a solid support optionally bound to a linker; 
         R 9  is hydrogen or, when R 7  is —CH 2 —OR 10  then R 9  may be —CH 2 —R 12  where R 10  and R 12  form a direct bond; 
         R 11  is selected from H and G 3 ; 
         G 2  is selected from oxygen, sulfur and CH 2 ; and 
         G 3  is a protecting group for a hydroxyl group that is bonded to a carbon atom. 
       
     
     
         18 . The process of  claim 17  further comprising reacting the N-phosphoroamidate-monoester triphosphate (160) with a tether precursor of the formula X-T-X where X represents a reactive functional group that is reactive with the terminating functional group of R 1  and R 13 , so as to form linker groups LG1 and LG2. 
     
     
         19 . The process of  claim 18  wherein X is an azide group and the terminating functional groups of R 1  and R 13  are alkyne groups. 
     
     
         20 . A cyclic phosphite of the formula 
       
         
           
           
               
               
           
         
         wherein R 1  is selected from an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen.