Mobility-modifying cyanine dyes

Abstract

The present invention provides a novel class of fluorescent cyanine dye compounds that are modified at one of the hetercyclic ring nitrogen atoms with a mobility-modifying moiety that permits the electrophoretic mobilities of polynucleotides labeled with the mobility-modifying cyanine dyes to be adjusted or tuned in a predictable fashion while retaining enzymatic activity. The ability to predictably tune the relative electrophoretic mobilities of the dyes permits the creation of sets of mobility-matched fluorescent dyes of a variety of structures for a variety of applications, including fluorescence-based 4-color nucleic acid sequencing reactions.

1 . A mobility-modifying cyanine dye comprising: (i) a first substituted or unsubstituted parent heteroaromatic benzazole/benzazolium ring system having a linking moiety of the formula -L-LG, where L is a linker and LG is a linking group, attached to the heteroaromatic ring nitrogen; (ii) a second substituted or unsubstituted parent heteroaromatic benzazole/benzazolium ring system having a mobility-modifying moiety attached to the heteroaromatic ring nitrogen; and an electron delocalizing bridge connecting the first and second parent benzazole/benzazolium rings via their respective C2 carbons, wherein when said mobility-modifying moiety has a net charge of −2 or less or +1 or greater.  
     
     
         2 . The mobility-modifying cyanine dye of  claim 1  in which the bridge is a compound selected from the group consisting of:  
         —(CR 1 ═CR 2 ) k (CR 3 ═CR 4 ) l —(CR 5 —CR 6 ) m —CR 7 ═  (B.1)  and                    wherein: 
 k, l, and m are each independently integers from 0 to 1;  
 R 1 , R 2 , R 3 , R 4 , R 5 , R 6  and R 7  are each independently selected from the group consisting of hydrogen, halogen, —F, —Cl, —CN, —CF 3 , (C 1 -C 6 ) alkyl, (C 5 -C 14 ) aryl or 5-14 membered heteroaryl;  
 R 10  and R 10′  are each independently selected from the group consisting of hydrogen, oxygen, halogen, —F, —Cl, —CN, —CF 3 , —OR, —SR, —NRR, (C 1 -C 6 ) alkyl, (C 5 -C 14 ) aryl or 5-14 membered heteroaryl, where each R is independently hydrogen or (C 1 -C 6 ) alkyl; and  
   p is an integer from 0 to 2, where in structural formula (B.2), the dotted lines at substituents R 10′  represent bonds that may be independently either single bonds or a double bonds, depending upon the identities of the substituents.    
     
     
         3 . The mobility-modifying cyanine dye of  claim 2  in which the bridge is a compound of structural formula (B. 1).  
     
     
         4 . The mobility-modifying cyanine dye of  claim 3  in which the sum of k, l and m is 2.  
     
     
         5 . The mobility-modifying cyanine dye of  claim 2  in which the bridge is a compound of structural formula (B. 1) wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6  and R 7  are each hydrogen.  
     
     
         6 . The mobility-modifying cyanine dye of  claim 1  in which the bridge is —CH═CH—CH═CH—CH═.  
     
     
         7 . The mobility-modifying cyanine dye of  claim 1  in which the mobility-modifying moiety has a net positive charge.  
     
     
         8 . The mobility-modifying cyanine dye of  claim 1  in which the mobility-modifying moiety has a net negative charge.  
     
     
         9 . The mobility-modifying cyanine dye of  claim 1  in which the mobility-modifying moiety has the structure:  
       
         
           
           
               
               
           
         
         or a salt thereof, wherein: 
 n is an integer from 1 to 6 (preferably 1 to 3);  
 R 24 , when taken alone, is hydrogen, a strong anionic substituent, —S(O) 2 O − , or —O—S(O) 2 O − , or when taken together with R 25  is a benzo group or a benzo group independently substituted with one or more strong anionic substituents, —S(O) 2 O − , or —O—S(O) 2 O −  groups;  
 R 25 , when taken alone, is hydrogen, a strong anionic substituent, —S(O) 2 O − , or —O—S(O) 2 O − , or when taken together with R 24  or R 26  is a benzo group or a benzo group independently substituted with one or more strong anionic substituents, —S(O) 2 O − , or —O—S(O) 2 O −  groups;  
 R 26 , when taken alone, is hydrogen, a strong anionic substituent, —S(O) 2 O − , or —O—S(O) 2 O − , or when taken together with R 25  or R 27  is a benzo group or a benzo group independently substituted with one or more strong anionic substituents, —S(O) 2 O − , or —O—S(O) 2 O −  groups; and  
 R 27 , when taken alone, is hydrogen, a strong anionic substituent, —S(O) 2 O − , or —O—S(O) 2 O − , or when taken together with R 26  or R 28  is a benzo group or a benzo group independently substituted with one or more strong anionic substituents, —S(O) 2 O − , or —O—S(O) 2 O −  groups; and  
 R 28 , when taken alone, is hydrogen, a strong anionic substituent, —S(O) 2 O − , or —O—S(O) 2 O − , or when taken together with R 27  is a benzo group or a benzo group independently substituted with one or more strong anionic substituents, —S(O) 2 O − , or OS(O) 2 O −  groups.  
 
       
     
     
         10 . The mobility-modifying cyanine dye of  claim 1  in which the mobility-modifying moiety has the structure:  
       
         
           
           
               
               
           
         
         or a salt thereof, wherein: 
 o is an integer from 1 to 3;  
 q is an integer from 1 to 3;  
 R 29  is a strong anionic substituent, —S(O) 2 O −  or —O—S(O) 2 O − ,  
 each R 30  is independently selected from the group consisting of hydrogen a strong anionic substituent, —S(O) 2 O − and —O—S(O) 2 O − ; and  
 R 31  is selected from the group consisting of hydrogen, a strong anionic substitutent, —S(O) 2 O − , —O—S(O) 2 O −  and —CH 3 ,  
 with the proviso that MM has a net charge of at least −2 at a pH in the range of about pH 6 to pH 10.  
 
       
     
     
         11 . The mobility-modifying cyanine dye of  claim 1  in which the first and second heteroaromatic benzazole/benzazolium ring systems are the same.  
     
     
         12 . The mobility-modifying cyanine dye of  claim 1  in which the first parent heteroaromatic benzazole/benzazolium ring system has the structure:  
       
         
           
           
               
               
           
         
         or a salt thereof, wherein: 
 Z is selected from the group consisting of —S—, —O—, —Se— and —CR1R 9 —, where R 8  and R 9  when taken alone, are each independently (C 1 -C 6 ) alkyl, or when taken together are (C 4 -C 5 ) alkyleno or (C 4 -C 5 ) alkano;  
 R 11 , R 12 , R 13  and R 14 , when taken alone, are each independently selected from the group consisting of hydrogen, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkyl independently substituted with one or more W, 2-6 membered heteroalkyl, 2-6 membered heteroalkyl independently substituted with one or more W, (C 5 -C 10 ) aryl, (C 5 -C 10 ) aryl independently substituted with one or more W, (C 5 -C 6 ) arylaryl, (C 5 -C 6 ) arylaryl independently substituted with one or more W, (C 6 -C 16 ) arylalkyl, (C 6 -C 16 ) independently substituted with one or more W, 6-16 membered arylheteroalkyl, 6-16 membered arylheteroalkyl independently substituted with one or more W, 5-10 membered heteroaryl, 5-10 membered heteroaryl independently substituted with one or more W, 5-6 membered heteroaryl-heteroaryl, 5-6 membered heteroaryl independently substituted with one or more W, 6-16 membered heteroarylalkyl, 6-16 membered heteroarylalkyl independently substituted with one or more W, 6-16 membered heteroaryl-heteroalkyl and 6-16 membered heteroaryl-heteroalkyl independently substituted with one or more W,  
 or when taken together with an adjacent R n  are each independently selected from the group consisting of (C 6 -C 10 ) aryleno, (C 6 -C 10 ) aryleno independently substituted with one or more W, 6-10 membered heteroaryleno and 6-10 membered heteroaryleno independently substituted with one or more W; and  
 each W is independently —R, —X, ═O, —OR, ═S, —SR, —NRR, ═NR, (C 1 -C 6 ) perhaloalkyl, —CX 3 , —CN, —OCN, —SCN, —NO, —NO 2 , =N 2 , —N 3 , —NHOH, —S(O) 2 R, —C(O)R, —C(S)R, —C(O)OR′, —C(S)OR′, —C(O)SR′, —C(S)SR′, and —C(NR)NRR, wherein:  
 each X is independently a halogen;  
 each R is independently —H, —NR″R″, —C(O)R″, —S(O 2 )R″, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 5 -C 10 ) aryl, (C 6 -C 16 ) arylalkyl, 5-10 membered heteroaryl or 6-16 membered heteroarylalkyl; and  
 each R′ is independently (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkenyl and (C 2 -C 6 ) alkynyl, (C 5 -C 10 ) aryl, (C 6 -C 16 ) arylalkyl, 5-10 membered heteroaryl or 6-16 membered heteroarylalkyl; and each R″ is independently —H, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkynyl, (C 5 -C 10 ) aryl, (C 6 -C 16 ) arlyalkyl, 5-10 membered heteroaryl or 6-16 membered heteroarylalkyl.  
 
       
     
     
         13 . The mobility-modifying cyanine dye of  claim 1  in which the second parent heteroaromatic benzazole/benzazolium ring system has the structure:  
       
         
           
           
               
               
           
         
         or a salt thereof, wherein: 
 Z′ is selected from the group consisting of —S—, —O—, —Se— and —CR 8 R 9 , where R 8′  and R 9′ , when taken alone, are each independently (C 1 -C 6 ) alkyl, or when taken together are (C 4 -C 5 ) alkyleno or (C 4 -C 5 ) alkano;  
 R 19 , R 20 , R 21  and R 22 , when taken alone, are each independently selected from the group consisting of hydrogen, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkyl independently substituted with one or more W, 2-6 membered heteroalkyl, 2-6 membered heteroalkyl independently substituted with one or more W, (C 5 -C 10 ) aryl, (C 5 -C 10 ) aryl independently substituted with one or more W, (C 5 -C 6 ) arylaryl, (C 5 -C 6 ) arylaryl independently substituted with one or more W, (C 6 -C 16 ) arylalkyl, (C 6 -C 16 ) independently substituted with one or more W, 6-16 membered arylheteroalkyl, 6-16 membered arylheteroalkyl independently substituted with one or more W, 5-10 membered heteroaryl, 5-10 membered heteroaryl independently substituted with one or more W, 5-6 membered heteroaryl-heteroaryl, 5-6 membered heteroaryl independently substituted with one or more W, 6-16 membered heteroarylalkyl, 6-16 membered heteroarylalkyl independently substituted with one or more W, 6-16 membered heteroaryl-heteroalkyl and 6-16 membered heteroaryl-heteroalkyl independently substituted with one or more W,  
 or when taken together with an adjacent R n  are each independently selected from the group consisting of (C 6 -C 10 ) aryleno, (C 6 -C 10 ) aryleno independently substituted with one or more W, 6-10 membered heteroaryleno and 6-10 membered heteroaryleno independently substituted with one or more W; and  
 each W is independently —R, —X, ═O, —OR, ═S, —SR, —NRR, ═NR, (C 1 -C 6 ) perhaloalkyl, —CX 3 , —CN, —OCN, —SCN, —NO, —NO 2 , =N 2 , —N 3 , —NHOH, —S(O) 2 R, —C(O)R, —C(S)R, —C(O)OR′, —C(S)OR′, —C(O)SR′, —C(S)SR′, and —C(NR)NRR, wherein:  
 each X is independently a halogen;  
 each R is independently —H, —NR″R″, —C(O)R″, —S(O 2 )R″, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 5 -C 10 ) aryl, (C 6 -C 16 ) arylalkyl, 5-10 membered heteroaryl or 6-16 membered heteroarylalkyl; and  
 each R′ is independently (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkenyl and (C 2 -C 6 ) alkynyl, (C 5 -C 10 ) aryl, (C 6 -C 16 ) arylalkyl, 5-10 membered heteroaryl or 6-16 membered heteroarylalkyl; and each R″ is independently —H, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkynyl, (C 5 -C 10 ) aryl, (C 6 -C 16 ) arlyalkyl, 5-10 membered heteroaryl or 6-16 membered heteroarylalkyl.  
 
       
     
     
         14 . A mobility-modifying cyanine dye according to  claim 1  in which the first and second heteroaromatic benzazole/benzazolium rings are each the same or different substituted or unsubstituted indoline/indolinium ring.  
     
     
         15 . The mobility-modifying cyanine dye of  claim 1  which has the structure:  
       
         
           
           
               
               
           
         
         or a salt thereof, wherein: 
 k, l, and m are each independently integers from 0 to 1;  
 R 1 , R 2 , R 3 , R 4 , R 5 , R 6  and R 7  are each independently selected from the group consisting of hydrogen, halogen, —F, —Cl, —CN, —CF 3 , (C 1 -C 6 ) alkyl, (C 5 -C 14 ) aryl and 5-14 membered heteroaryl;  
 MM is a mobility-modifying moiety;  
 L is a linker;  
 R x  is a reactive functional group;  
 Z is selected from the group consisting of —S—, —O—, —Se—and —CR 8 R 9′ , where R 8  and R 9  when taken alone, are each independently (C 1 -C 6 ) alkyl, or when taken together are (C 4 -C 5 ) alkyleno or (C 4 -C 5 ) alkano;  
 Z′ is selected from the group consisting of —S—, —O—, —Se— and —CR 8′ R 9′ , where R 8′  and R 9′ , when taken alone, are each independently (C 1 -C 6 ) alkyl, or when taken together are (C 4 -C 5 ) alkyleno or (C 4 -C 5 ) alkano;  
 R 11 , R 12 , R 13  and R 14 , when taken alone, are each independently selected from the group consisting of hydrogen, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkyl independently substituted with one or more W, 2-6 membered heteroalkyl, 2-6 membered heteroalkyl independently substituted with one or more W, (C 5 -C 10 ) aryl, (C 5 -C 10 ) aryl independently substituted with one or more W, (C 5 -C 6 ) arylaryl, (C 5 -C 6 ) arylaryl independently substituted with one or more W, (C 6 -C 16 ) arylalkyl, (C 6 -C 16 ) independently substituted with one or more W, 6-16 membered arylheteroalkyl, 6-16 membered arylheteroalkyl independently substituted with one or more W, 5-10 membered heteroaryl, 5-10 membered heteroaryl independently substituted with one or more W, 5-6 membered heteroaryl-heteroaryl, 5-6 membered heteroaryl independently substituted with one or more W, 6-16 membered heteroarylalkyl, 6-16 membered heteroarylalkyl independently substituted with one or more W, 6-16 membered heteroaryl-heteroalkyl and 6-16 membered heteroaryl-heteroalkyl independently substituted with one or more W,  
 or when taken together with an adjacent R n  are each independently selected from the group consisting of (C 6 -C 10 ) aryleno, (C 6 -C 10 ) aryleno independently substituted with one or more W, 6-10 membered heteroaryleno and 6-10 membered heteroaryleno independently substituted with one or more W;  
 R 19 , R 20 , R 21  and R 22 , when taken alone, are each independently selected from the group consisting of hydrogen, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkyl independently substituted with one or more W, 2-6 membered heteroalkyl, 2-6 membered heteroalkyl independently substituted with one or more W, (C 5 -C 10 ) aryl, (C 5 -C 10 ) aryl independently substituted with one or more W, (C 5 -C 6 ) arylaryl, (C 5 -C 6 ) arylaryl independently substituted with one or more W, (C 6 -C 16 ) arylalkyl, (C 6 -C 16 ) independently substituted with one or more W, 6-16 membered arylheteroalkyl, 6-16 membered arylheteroalkyl independently substituted with one or more W, 5-10 membered heteroaryl, 5-10 membered heteroaryl independently substituted with one or more W, 5-6 membered heteroaryl-heteroaryl, 5-6 membered heteroaryl independently substituted with one or more W, 6-16 membered heteroarylalkyl, 6-16 membered heteroarylalkyl independently substituted with one or more W, 6-16 membered heteroaryl-heteroalkyl and 6-16 membered heteroaryl-heteroalkyl independently substituted with one or more W,  
 or when taken together with an adjacent R n  are each independently selected from the group consisting of (C 6 -C 10 ) aryleno, (C 6 -C 10 ) aryleno independently substituted with one or more W, 6-10 membered heteroaryleno and 6-10 membered heteroaryleno independently substituted with one or more W;  
 each W is independently —R, —X, ═O, —OR, ═S, —SR, —NRR, =NR, (C 1 -C 6 ) perhaloalkyl, —CX 3 , —CN, —OCN, —SCN, —NO, —NO 2 , =N 2 , —N 3 , —NHOH, —S(O) 2 R, —C(O)R, —C(S)R, —C(O)OR′, —C(S)OR′, —C(O)SR′, —C(S)SR′, and —C(NR)NRR, wherein:  
 each X is independently a halogen;  
 each R is independently —H, —NR″R″, —C(O)R″, —S(O 2 )R″, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 5 -C 10 ) aryl, (C 6 -C 16 ) arylalkyl, 5-10 membered heteroaryl or 6-16 membered heteroarylalkyl; and  
 each R′ is independently (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkenyl and (C 2 -C 6 ) alkynyl, (C 5 -C 10 ) aryl, (C 6 -C 16 ) arylalkyl, 5-10 membered heteroaryl or 6-16 membered heteroarylalkyl; and each R″ is independently —H, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkynyl, (C 5 -C 10 ) aryl, (C 6 -C 16 ) arlyalkyl, 5-10 membered heteroaryl or 6-16 membered heteroarylalkyl.  
 
       
     
     
         16 . The mobility-modifying cyanine dye of  claim 15  in which Z is —NR 8 R 9 —, where R 8  and R 9  are each independently (C 1 -C 6 ) alkano; and Z′ is —NR 8′ R 9′ , where R 8′  and R 9′  are each independently (C 1 -C 6 ) alkano.  
     
     
         17 . The mobility-modifying cyanine dye of  claim 15  which is selected from the group consisting of:  
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         or a salt thereof, wherein: 
 R 11 , R 12 , R 13 , R 14 , R 19 , R 20 , R 21 , R 22 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36  and R 37  are each independently selected from the group consisting of hydrogen, —S(O) 2 O −  and —O—S(O) 2 O − .  
 
       
     
     
         18 . A labeled nucleoside/tide or nucleoside/tide analog having the structure:  
         NUC-L′-R 41 -L-D  or a salt thereof, wherein: 
 D is a mobility-modifying cyanine dye chromophore;  
 L is a first linker which is attached to D at a heteroaromatic ring nitrogen;  
 R 41  is a covalent linkage;  
 NUC is a nucleoside/tide or nucleoside/tide analog; and  
   L′ is a second linker which is attached to the nucleobase or sugar moiety of NUC.    
     
     
         19 . The labeled nucleoside/tide or nucleoside/tide analog of  claim 18  which is enzymatically incorporable.  
     
     
         20 . The labeled nucleoside/tide or nucleoside/tide analog of  claim 18  which is a terminator.  
     
     
         21 . The labeled nucleoside/tide or nucleoside/tide analog of  claim 18  which is enzymatically extendable.  
     
     
         22 . The labeled nucleoside/tide or nucleoside/tide analog of  claim 18  in which -L′-NUC taken together has the structure:  
       
         
           
           
               
               
           
         
         or a salt thereof, wherein: 
 B is a nucleobase;  
 L′ is (C 1 -C 20 ) alkyldiyl, (C 1 -C 20 ) alkyleno, (C 2 -C 20 ) alkyno, (C 2 -C 20 ) alkeno 2-20 membered heteroalkyldiyl, 2-20 membered heteroalkyleno, 2-20 membered heteroalkyno or 2-20 membered heteroalkeno;  
 R 70  and R 71 , when taken alone, are each independently selected from the group consisting of hydrogen, hydroxyl and a moiety which blocks polymerase-mediated template-directed polymerization, or when taken together form a bond such that the illustrated sugar is 2′,3′-didehydroribose; and  
 R 72  is selected from the group consisting of hydroxyl, a phosphate ester having the formula  
                     
 where α is an integer from 0 to 2 and a phosphate ester analog.  
 
       
     
     
         23 . The labeled nucleoside/tide or nucleoside/tide analog of  claim 22  in which L′ is selected from the group consisting of: 
 —C≡C—CH 2 —, where the terminal sp carbon is covalently attached to nucleobase B and the terminal methylene (sp 3 ) carbon is covalently attached to R 41 ; and    —C≡C—CH 2 —OCH 2 —CH 2 —NR 47 —R 48 —, where R 47  is hydrogen or (C 1 -C 6 ) alkyl and R 48  is —C(O)—(CH 2 ) r —, —C(O)—CHR 49 —, —C(O)C≡C—CH 2 — or —C(O)-φ-CH 2 ) r —, where each r is independently an integer from 1 to 5 and φ is C 6  aryldiyl or 6-membered heteroaryldiyl and R 49  is hydrogen, (C 1 -C 6 ) alkyl or a side chain of an encoding or non-encoding amino acid, and where the terminal sp carbon is covalently attached to nucleobase B and the other terminal group is covalently attached to R 41 .    
     
     
         24 . The labeled nucleoside/tide or nucleoside/tide analog of  claim 22  in which nucleobase B is a purine, a 7-deazapurine, a pyrimidine, a normal nucleobase or a common analog of a normal nucleobase.  
     
     
         25 . The labeled nucleoside/tide or nucleoside/tide analog of  claim 18  in which D has the structure:  
       
         
           
           
               
               
           
         
         or a salt thereof, wherein: 
 k, l, and m are each independently integers from 0 to 1;  
 R 1 , R 2 , R 3 , R 4 , R 5 , R 6  and R 7  are each independently selected from the group consisting of hydrogen, halogen, —F, —Cl, —CN, —CF 3 , (C 1 -C 6 ) alkyl, (C 5 -C 14 ) aryl or 5-14 membered heteroaryl;  
 MM is a mobility-modifying moiety;  
 Z is selected from the group consisting of —S—, —O − , —Se— and —CR 8 R 9 —, where R 8  and R 9  when taken alone, are each independently (C 1 -C 6 ) alkyl, or when taken together are (C 4 -C 5 ) alkyleno or (C 4 -C 5 ) alkano;  
 Z′ is selected from the group consisting of —S—, —O—, —Se— and —CR 8′ R 9′ , where R 8′  and R 9′ , when taken alone, are each independently (C 1 -C 6 ) alkyl, or when taken together are (C 4 -C 5 ) alkyleno or (C 4 -C 5 ) alkano;  
 R 11 , R 12 , R 13  and R 14 , when taken alone, are each independently selected from the group consisting of hydrogen, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkyl independently substituted with one or more W, 2-6 membered heteroalkyl, 2-6 membered heteroalkyl independently substituted with one or more W, (C 5 -C 10 ) aryl, (C 5 -C 10 ) aryl independently substituted with one or more W, (C 5 -C 6 ) arylaryl, (C 5 -C 6 ) arylaryl independently substituted with one or more W, (C 6 -C 16 ) arylalkyl, (C 6 -C 16 ) independently substituted with one or more W, 6-16 membered arylheteroalkyl, 6-16 membered arylheteroalkyl independently substituted with one or more W, 5-10 membered heteroaryl, 5-10 membered heteroaryl independently substituted with one or more W, 5-6 membered heteroaryl-heteroaryl, 5-6 membered heteroaryl independently substituted with one or more W, 6-16 membered heteroarylalkyl, 6-16 membered heteroarylalkyl independently substituted with one or more W, 6-16 membered heteroaryl-heteroalkyl and 6-16 membered heteroaryl-heteroalkyl independently substituted with one or more W,  
 or when taken together with an adjacent R n  are each independently selected from the group consisting of (C 6 -C 10 ) aryleno, (C 6 -C 10 ) aryleno independently substituted with one or more W, 6-10 membered heteroaryleno and 6-10 membered heteroaryleno independently substituted with one or more W;  
 R 19 , R 20 , R 21  and R 22 , when taken alone, are each independently selected from the group consisting of hydrogen, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkyl independently substituted with one or more W, 2-6 membered heteroalkyl, 2-6 membered heteroalkyl independently substituted with one or more W, (C 5 -C 10 ) aryl, (C 5 -C 10 ) aryl independently substituted with one or more W, (C 5 -C 6 ) arylaryl, (C 5 -C 6 ) arylaryl independently substituted with one or more W, (C 6 -C 16 ) arylalkyl, (C 6 -C 16 ) independently substituted with one or more W, 6-16 membered arylheteroalkyl, 6-16 membered arylheteroalkyl independently substituted with one or more W, 5-10 membered heteroaryl, 5-10 membered heteroaryl independently substituted with one or more W, 5-6 membered heteroaryl-heteroaryl, 5-6 membered heteroaryl independently substituted with one or more W, 6-16 membered heteroarylalkyl, 6-16 membered heteroarylalkyl independently substituted with one or more W, 6-16 membered heteroaryl-heteroalkyl and 6-16 membered heteroaryl-heteroalkyl independently substituted with one or more W,  
 or when taken together with an adjacent R n  are each independently selected from the group consisting of (C 6 -C 10 ) aryleno, (C 6 -C 10 ) aryleno independently substituted with one or more W, 6-10 membered heteroaryleno and 6-10 membered heteroaryleno independently substituted with one or more W;  
 each W is independently —R, —X, ═O, —OR, ═S, —SR, —NRR, ═NR, (C 1 -C 6 ) perhaloalkyl, —CX 3 , —CN, —OCN, —SCN, —NO, —NO 2 , =N 2 , —N 3 , —NHOH, —S(O) 2 R, —C(O)R, —C(S)R, —C(O)OR′, —C(S)OR′, —C(O)SR′, —C(S)SR′, and —C(NR)NRR, wherein:  
 each X is independently a halogen;  
 each R is independently —H, —NR″R″, —C(O)R″, —S(O 2 )R″, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 5 -C 10 ) aryl, (C 6 -C 16 ) arylalkyl, 5-10 membered heteroaryl or 6-16 membered heteroarylalkyl;  
 each R′ is independently (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkenyl and (C 2 -C 6 ) alkynyl, (C 5 -C 10 ) aryl, (C 6 -C 16 ) arylalkyl, 5-10 membered heteroaryl or 6-16 membered heteroarylalkyl; and each R″ is independently —H, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkynyl, (C 5 -C 10 ) aryl, (C 6 -C 16 ) arlyalkyl, 5-10 membered heteroaryl or 6-16 membered heteroarylalkyl; and  
 the dotted line at the heteroaromatic ring nitrogen indicates the point of attachment of linker L.  
 
       
     
     
         26 . A mobility-modifying phosphoramidite reagent having the structure:  
       
         
           
           
               
               
           
         
         or a salt thereof, wherein: 
 N, O and P are nitrogen, oxygen and phosphorous, respectively;  
 D is a mobility-modifying dye chromophore or a protected derivative thereof;  
 L is a first linker;  
 R 41  is a bond or a covalent linkage;  
 
         L″ is a bond or a second linker; 
 R 60  is a phosphite ester protecting group;  
 
         R 61 , when taken alone, is selected from the group consisting of (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 3 -C 10 ) cycloalkyl, (C 5 -C 20 ) aryl and (C 6 -C 26 ) arylalkyl, or when taken together with R 62  forms a straight-chain or branched (C 2 -C 10 ) alkyleno or a straight-chain or branched 2-10 membered heteroalkyleno; and 
 R 62 , when taken alone, is selected from the group consisting of (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 3 -C 10 ) cycloalkyl, (C 5 -C 20 ) aryl and (C 6 -C 26 ) arylalkyl, or when taken together with R 62  forms a straight-chain or branched (C 2 -C 10 ) alkyleno or a straight-chain or branched 2-10 membered heteroalkyleno.  
 
       
     
     
         27 . The mobility-modifying phosphoramidite reagent according to  claim 26  which has the structure:  
       
         
           
           
               
               
           
         
         wherein: 
 N, P and O are nitrogen, phosphorous and oxygen, respectively;  
 R 41 , L, D, R 60 , R 61  and R 62  are as previously defined;  
 R 63  is hydrogen or an acid-labile hydroxylprotecting group; and  
 v is an integer from 1 to 30.  
 
       
     
     
         28 . A mobility-modifying phosphoramidite reagent having the structure:  
       
         
           
           
               
               
           
         
         or a salt thereof, wherein: 
 O, P and N are oxygen, phosphorous and nitrogen, respectively;  
 B is a nucleobase or a protected derivative thereof;  
 D is a mobility-modifying dye chromophore or a protected derivative thereof;  
 L is a first linker which is attached to D at a heteroaromatic ring nitrogen;  
 R 41  is a bond or a covalent linkage;  
 L′ is a bond or a second linker;  
 R 60  is a phosphite ester protecting group;  
 R 61 , when taken alone, is selected from the group consisting of (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 3 -C 10 ) cycloalkyl, (C 5 -C 20 ) aryl and (C 6 -C 26 ) arylalkyl, or when taken together with R 62  forms a straight-chain or branched (C 2 -C 10 ) alkyleno or a straight-chain or branched 2-10 membered heteroalkyleno; and  
 R 62 , when taken alone, is selected from the group consisting of (C 6 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 3 -C 10 ) cycloalkyl, (C 5 -C 20 ) aryl and (C 6 -C 26 ) arylalkyl, or when taken together with R 61  forms a straight-chain or branched (C 2 -C 10 ) alkyleno or a straight-chain or branched 2-10 membered heteroalkyleno; and  
 R 63  is hydrogen or an acid-labile hydroxyl protecting group  
 
       
     
     
         29 . The mobility-modifying phosphoramidite reagent according to  claim 26 ,  27  or  28  in which D has the structure:  
       
         
           
           
               
               
           
         
         or a salt thereof, wherein: 
 k, l, and m are each independently integers from 0 to 1;  
 R 1 , R 2 , R 3 , R 4 , R 5 , R 6  and R 7  are each independently selected from the group consisting of hydrogen, halogen, —F, —Cl, —CN, —CF 3 , (C 1 -C 6 ) alkyl, (C 5 -C 14 ) aryl or 5-14 membered heteroaryl;  
 MM is a mobility-modifying moiety;  
 Z is selected from the group consisting of —S—, —O—, —Se— and —CR 8 R 9 —, where R 8  and R 9  when taken alone, are each independently (C 1 -C 6 ) alkyl, or when taken together are (C 4 -C 5 ) alkyleno or (C 4 -C 5 ) alkano;  
 Z′ is selected from the group consisting of —S—, —O—, —Se— and —CR 8′ R 9′ , where R 8  and R 9  when taken alone, are each independently (C 1 -C 6 ) alkyl, or when taken together are (C 4 -C 5 ) alkyleno or (C 4 -C 5 ) alkano;  
 R 11 , R 12 , R 13  and R 14 , when taken alone, are each independently selected from the group consisting of hydrogen, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkyl independently substituted with one or more W, 2-6 membered heteroalkyl, 2-6 membered heteroalkyl independently substituted with one or more W, (C 5 -C 10 ) aryl, (C 5 -C 10 ) aryl independently substituted with one or more W, (C 5 -C 6 ) arylaryl, (C 5 -C 6 ) arylaryl independently substituted with one or more W, (C 6 -C 16 ) arylalkyl, (C 6 -C 16 ) independently substituted with one or more W, 6-16 membered arylheteroalkyl, 6-16 membered arylheteroalkyl independently substituted with one or more W, 5-10 membered heteroaryl, 5-10 membered heteroaryl independently substituted with one or more W, 5-6 membered heteroaryl-heteroaryl, 5-6 membered heteroaryl independently substituted with one or more W, 6-16 membered heteroarylalkyl, 6-16 membered heteroarylalkyl independently substituted with one or more W, 6-16 membered heteroaryl-heteroalkyl and 6-16 membered heteroaryl-heteroalkyl independently substituted with one or more W,  
 or when taken together with an adjacent R n  are each independently selected from the group consisting of (C 6 -C 10 ) aryleno, (C 6 -C 10 ) aryleno independently substituted with one or more W, 6-10 membered heteroaryleno and 6-10 membered heteroaryleno independently substituted with one or more W;  
 R 19 , R 20 , R 21  and R 22 , when taken alone, are each independently selected from the group consisting of hydrogen, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkyl independently substituted with one or more W, 2-6 membered heteroalkyl, 2-6 membered heteroalkyl independently substituted with one or more W, (C 5 -C 10 ) aryl, (C 5 -C 10 ) aryl independently substituted with one or more W, (C 5 -C 6 ) arylaryl, (C 5 -C 6 ) arylaryl independently substituted with one or more W, (C 6 -C 16 ) arylalkyl, (C 6 -C 16 ) independently substituted with one or more W, 6-16 membered arylheteroalkyl, 6-16 membered arylheteroalkyl independently substituted with one or more W, 5-10 membered heteroaryl, 5-10 membered heteroaryl independently substituted with one or more W, 5-6 membered heteroaryl-heteroaryl, 5-6 membered heteroaryl independently substituted with one or more W, 6-16 membered heteroarylalkyl, 6-16 membered heteroarylalkyl independently substituted with one or more W, 6-16 membered heteroaryl-heteroalkyl and 6-16 membered heteroaryl-heteroalkyl independently substituted with one or more W,  
 or when taken together with an adjacent R n  are each independently selected from the group consisting of (C 6 -C 10 ) aryleno, (C 6 -C 10 ) aryleno independently substituted with one or more W, 6-10 membered heteroaryleno and 6-10 membered heteroaryleno independently substituted with one or more W;  
 each W is independently —R, —X, ═O, —OR, ═S, —SR, —NRR, ═NR, (C 1 -C 6 ) perhaloalkyl, —CX 3 , —CN, —OCN, —SCN, —NO, —NO 2 , =N 2 , —N 3 , —NHOH, —S(O) 2 R, —C(O)R, —C(S)R, —C(O)OR′, —C(S)OR′, —C(O)SR′, —C(S)SR′, and —C(NR)NRR, wherein:  
 each X is independently a halogen;  
 each R is independently —H, —NR″R″, —C(O)R″, —S(O 2 )R″, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 5 -C 10 ) aryl, (C 6 -C 16 ) arylalkyl, 5-10 membered heteroaryl or 6-16 membered heteroarylalkyl;  
 each R′ is independently (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkenyl and (C 2 -C 6 ) alkynyl, (C 5 -C 10 ) aryl, (C 6 -C 16 ) arylalkyl, 5-10 membered heteroaryl or 6-16 membered heteroarylalkyl; and each R″ is independently —H, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkanyl, (C 2 -C 6 ) alkynyl, (C 5 -C 10 ) aryl, (C 6 -C 16 ) arlyalkyl, 5-10 membered heteroaryl or 6-16 membered heteroarylalkyl; and  
 the dotted line at the heteroaromatic ring nitrogen indicates the point of attachment of linker L.  
 
       
     
     
         30 . The mobility-modifying phosphoramidite reagent of  claim 29  in which MM has the structure:  
       
         
           
           
               
               
           
         
         or a salt thereof, wherein: 
 n is an integer from 1 to 6 (preferably 1 to 3);  
 R 24 , when taken alone, is hydrogen, a strong anionic substituent, —S(O) 2 O − , or —O—S(O) 2 O − , or when taken together with R 25  is a benzo group or a benzo group independently substituted with one or more strong anionic substituents, —S(O) 2 O − , or —O—S(O) 2 O −  groups;  
 R 25 , when taken alone, is hydrogen, a strong anionic substituent, —S(O) 2 O − , or —O—S(O) 2 O − , or when taken together with R 24  or R 26  is a benzo group or a benzo group independently substituted with one or more strong anionic substituents, —S(O) 2 O − , or —O—S(O) 2 O −  groups;  
 R 26 , when taken alone, is hydrogen, a strong anionic substituent, —S(O) 2 O − , or —O—S(O) 2 O − , or when taken together with R 25  or R 27  is a benzo group or a benzo group independently substituted with one or more strong anionic substituents, —S(O) 2 O − , or —O—S(O) 2 O −  groups; and  
 R 27 , when taken alone, is hydrogen, a strong anionic substituent, —S(O) 2 O − , or —O—S(O) 2 O − , or when taken together with R 26  or R 28  is a benzo group or a benzo group independently substituted with one or more strong anionic substituents, —S(O) 2 O − , or —O—S(O) 2 O −  groups; and  
 R 28 , when taken alone, is hydrogen, a strong anionic substituent, —S(O) 2 O − , or —O—S(O) 2 O − , or when taken together with R 27  is a benzo group or a benzo group independently substituted with one or more strong anionic substituents, —S(O) 2 O − , or —O—S(O) 2 O −  groups.  
 
       
     
     
         31 . The mobility-modifying phosphoramidite reagent according to  claim 26 ,  27  or  28  in which L is —CH 2 —CH 2 —CH 2 —CH 2 —CH 2 — (pentano).  
     
     
         32 . The mobility-modifying phosphoramidite reagent according to  claim 26 ,  27  or  28  in which R 41  is a covalent linkage formed upon the reaction between an electrophile and a nucleophile.  
     
     
         33 . The mobility-modifying phosphoramidite reagent according to  claim 31  in which R 41  has the structure —(O)NR 56 —, where R 56  is hydrogen or (C 1 -C 6 ) alkyl.  
     
     
         34 . A polynucleotide labeled with a mobility-modifying dye according to  claim 1 .  
     
     
         35 . A method of generating a labeled primer extension product, comprising the step of enzymatically extending a primer-target hybrid in the presence of a mixture of enzymatically-extendable nucleotides capable of supporting continuous primer extension and a terminator, wherein said primer or said terminator is labeled with a mobility-modifying dye according to  claim 1 .  
     
     
         36 . The method of  claim 35  in which the terminator is a mixture of four different terminators, one which terminates at a template A, one which terminates at a template G, one which terminates at a template C and one which terminates at a template T or U and wherein at least one of the terminators is labeled with a mobility-modifying dye according to  claim 1 .  
     
     
         37 . The method of  claim 36  in which each of the four different terminators is labeled with a different, spectrally-resolvable fluorophore, and one of the terminators is selected from the group consisting of Compound 29 and Compound 32.  
     
     
         38 . A kit for generating a labeled primer extension product, comprising enzymatically-extendable nucleotides capable of supporting continuous primer extension and a terminator, wherein said primer or said terminator is labeled with a mobility-modifying cyanine dye according to  claim 1 .  
     
     
         39 . The kit of  claim 38  in which the terminator is a set of four different mobility-modified terminators, one which terminates at a template A, one which terminates at a template G, one which terminates at a template C and one which terminates at a template T or U.  
     
     
         40 . The kit of  claim 39  in which the set of four different terminators is a set of mobility-matched terminators.  
     
     
         41 . The kit of  claim 39  in which the set of mobility-matched terminators comprises Compounds 31, 32, 33 and 34.