Water-soluble rhodamine dye conjugates

1. A labeled nucleoside/tide or nucleoside/tide analog comprising a rhodamine dye conjugated by a linker (L′) to a nucleoside/tide or nucleoside/tide analog (NUC), wherein:

the rhodamine is a rhodamine-type parent xanthene having attached to the xanthene C9 carbon a phenyl group, wherein the phenyl group is further substituted with (i) a carboxy group or a sulfonate group or a salt thereof; (ii) one to three substituted or unsubstituted aminopyridinium groups; and (iii) a substituted or unsubstituted alkylthio or arylthio group; and the nucleoside/tide or nucleoside/tide analog and linker taken together comprise the structure:

wherein:

B is a nucleobase selected from a purine, a 7-deazapurine, an 8-aza,7-deazapunne, a pyrimidine, a normal nucleobase and an analog of a normal nucleobase;

L′ is the linker comprising one to twenty carbon atoms;

R 70 and R 71 , when taken alone, are each independently selected from 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 hydroxyl, a phosphate ester having the formula:

where a is an integer from 0 to 2, and a phosphate ester analog, or a salt thereof.

2. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 comprising the formula:

wherein:

Y is a rhodamine-type parent xanthene ring attached to the illustrated phenyl group at the xanthene C9 carbon;

R 22 , R 23 , R 25 , and R 26 are independently selected from hydrogen and (C 1 -C 6 ) alkyl;

R 24 , when taken alone, is (C 1 -C 6 ) alkyl, or when taken together with R 24′ is (C 4 -C 10 ) alkyldiyl, (C 4 -C 6 ) alkyleno, (C 4 -C 6 ) heteroalkyldiyl and (C 4 -C 6 ) heteroalkyleno;

R 24′ , when taken alone, is (C 1 -C 6 ) alkyl, or when taken together with R 24 is (C 4 -C 10 ) alkyldiyl, (C 4 -C 6 ) alkyleno, (C 4 -C 6 ) heteroalkyldiyl and (C 4 -C 6 ) heteroalkyleno;

n is 1, 2, or 3;

S is sulfur;

Z 1 is selected from (C 1 -C 12 ) alkyldiyl, (C 1 -C 12 ) alkyldiyl independently substituted with one or more of the same or different W 1 groups, (C 5 -C 14 ) aryldiyl, and (C 5 -C 14 ) aryldiyl independently substituted with one or more of the same or different W 2 groups;

W 1 is selected from —X, —R, ═O, —OR, —SR, ═S, —NRR, ═NR, —CX 3 , —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO 2 , ═N 2 , —N 3 , —S(O) 2 O − , —S(O) 2 OH, —S(O) 2 R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR, —C(O)O − , —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR;

W 2 is selected from —R, —OR, —SR, —NRR, —S(O) 2 O − , —S(O) 2 OH, —S(O) 2 R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR, —C(O)O − , —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR;

L is a selected from a bond, (C 1 -C 12 ) alkyldiyl, (C 1 -C 12 ) substituted alkyldiyl, (C 6 -C 26 ) arylalkyldiyl, —O—, —S—, —NR—, —C(O)O—, —C(O)NR—, —NRS(O) 2 —, —NR—NR—, —NRC(O)O—, and —NRC(O)NR—;

R 46 is selected from —C(O)NR—, —C(O)O—, and —C(O)S—,

L′ is selected from (C 1 -C 20 ) alkyldiyl, (C 1 -C 20 ) heteroalkyldiyl, (C 1 -C 20 ) alkyleno, (C 1 -C 20 ) heteroalkyleno, (C 6 -C 26 ) arylalkyldiyl, (C 5 -C 20 ) heteroarylalkyldiyl, and substituted forms thereof; and

NUC is a nucleoside/tide or nucleoside/tide analog;

each R is independently selected from hydrogen, (C 1 -C 6 ) alkyl, (C 5 -C 20 ) aryl, (C 6 -C 26 ) arylalkyl, and (C 5 -C 20 ) arylaryl; or when two R groups on the same nitrogen atom are taken together, those two R groups are (C 4 -C 10 ) alkyldiyl or (C 4 -C 10 ) alkyleno; and

each X is independently a halogen.

3. The labeled nucleoside/tide or nucleoside/tide analog of claim 2 wherein Y comprises the rhodamine-type parent xanthene ring structures:

and a salt thereof, wherein:

R 1 and R 2 when taken alone, are independently hydrogen or (C 1 -C 6 ) alkyl; or R 1 and R 2 when taken together is benzo or naphtho;

R 3 and R 3′ when taken alone, are independently selected from hydrogen, (C 1 -C 6 ) alkyl, (C 5 -C 14 ) aryl and (C 5 -C 14 ) arylaryl, or when taken together is (C 4 -C 6 ) alkyldiyl or (C 4 -C 6 ) alkyleno, or when individually taken together with R 2 or R 4 is (C 2 -C 6 ) alkyldiyl or (C 2 -C 6 ) alkyleno;

R 4 , when taken alone, is selected from hydrogen and (C 1 -C 6 ) alkyl, or when taken together with R 3 or R 3′ is (C 2 -C 6 ) alkyldiyl or (C 2 -C 6 ) alkyleno;

R 5 , when taken alone, is selected from hydrogen and (C 1 -C 6 ) alkyl, or when taken together with R 6 or R 6′ is (C 2 -C 6 ) alkyldiyl or (C 2 -C 6 ) alkyleno;

R 6 and R 6′ when taken alone, are selected from hydrogen, (C 1 -C 6 ) alkyl, (C 5 -C 14 ) aryl and arylaryl, or when taken together are (C 4 -C 6 ) alkyldiyl or alkyleno, or when individually taken together with R 5 or R 7 is (C 2 -C 6 ) alkyldiyl or alkyleno;

R 7 , when taken alone, is selected from hydrogen and (C 1 -C 6 ) alkyl, or when taken together with R 6 or R 6′ is (C 2 -C 6 ) alkyldiyl or alkyleno;

R 8 , when taken alone, is selected from hydrogen and (C 1 -C 6 ) alkyl;

R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 and R 21 are each independently selected from hydrogen and (C 1 -C 6 ) alkyl, or

when R 10 , R 1l , R 12 , and R 13 , taken together are (C 5 -C 14 ) aryleno or (C 5 -C 14 ) aryleno substituted with one or more of the same or different (C 1 -C 6 ) alkyl, or

when R 18 , R 19 , R 20 and R 21 taken together are (C 5 -C 14 ) aryleno or aryleno substituted with one or more of the same or different (C 1 -C 6 ) alkyl; and

R 9 is the point of attachment to the xanthene C9 carbon.

4. The labeled nucleoside/tide or nucleoside/tide analog of claim 3 wherein R 2 when taken together with R 3 or R 3′ is (C 2 -C 6 ) alkyldiyl.

5. The labeled nucleoside/tide or nucleoside/tide analog of claim 3 wherein:

an alkyldiyl or alkyleno bridge formed by taking R 2 together with R 3 or R 3′ , R 7 together with R 6 or R 6′ , or R 4 together with and R 3 or R 3′ , is ethano, propano, 1,1-dimethylethano, 1,1-dimethylpropano or 1,1,3-trimethylpropano;

an aryleno bridge formed by taking R 1 together with R 2 is benzo or naphtho;

an alkyldiyl or alkyleno bridge formed by taking R 3 together with R 3′ , or R 6 together with R 6′ , is butano;

an alkyldiyl or alkyleno bridge formed by taking R 5 together with R 6 or R 6′ is ethano, propano, 1,1-dimethylethano, 1,1-dimethyipropano and 1,1,3-trimethylpropano; and

an aryleno bridge formed by taking R 10 , R 11 , R 12 and R 13 together, or R 18 , R 19 , R 20 and R 21 together, is benzo.

6. The labeled nucleoside/tide or nucleoside/tide analog of claim 2 in which Z 1 is phenyldiyl.

7. The labeled nucleoside/tide or nucleoside/tide analog of claim 2 in which L′ is selected from:

8. The labeled nucleoside/tide or nucleoside/tide analog of claim 2 in which L′ is:

wherein R 47 is hydrogen or (C 1 -C 6 ) alkyl, and R 48 is selected from:

wherein each r is independently an integer from 1 to 6; R 49 is hydrogen, (C 1 -C 6 ) alkyl, or an amino acid side chain; and φ is phenyldiyl or substituted phenyldiyl.

9. The labeled nucleoside/tide or nucleoside/tide analog of claim 2 in which R 9 comprises a phenyl ring and Y is selected from the structures:

10. The labeled nucleoside/tide or nucleoside/tide analog of claim 2 wherein R 22 , R 23 , R 25 , and R 26 are each hydrogen.

11. The labeled nucleoside/tide or nucleoside/tide analog of claim 2 which comprises the structure:

or a salt thereof.

12. The labeled nucleoside/tide or nucleoside/tide analog of claim 11 in which L′ is selected from: —C≡C—CH 2 — and —C≡C—CH 2 —O—CH 2 —.

13. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 comprising the formula:

wherein:

Y is a rhodamine-type parent xanthene ring attached to the illustrated phenyl group at the xanthene C9 carbon;

R 22 , R 23 , R 25 , and R 26 are independently selected from hydrogen and (C 1 -C 6 ) alkyl;

R 24 , when taken alone, is (C 1 -C 6 ) alkyl, or when taken together with R 24′ is (C 4 -C 10 ) alkyldiyl, (C 4 -C 6 ) alkyleno, (C 4 -C 6 ) heteroalkyldiyl or (C 4 -C 6 ) heteroalkyleno;

R 24′ , when taken alone, is (C 1 -C 6 ) alkyl, or when taken together with R 24 is (C 4 -C 10 ) alkyldiyl, (C 4 -C 6 ) alkyleno, (C 4 -C 6 ) heteroalkyldiyl or (C 4 -C 6 ) heteroalkyleno;

n is 1, 2, or 3;

S is sulfur;

Z is (C 1 -C 12 ) alkyl, (C 1 -C 12 ) alkyl substituted with one or more of the same or different W 1 groups, (C 5 -C 20 ) aryl, (C 5 -C 20 ) aryl substituted with one or more of the same or different W 2 groups, pyridyl or purinyl;

W 1 is selected from —X, —R, ═O, —OR, —SR, ═S, —NRR, ═NR, —CX 3 , —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO 2 , ═N 2 , —N 3 , —S(O) 2 O − , —S(O) 2 OH, —S(O) 2 R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR, —C(O)O − , —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR;

W 2 is selected from —R, —OR, —SR, —NRR, —S(O) 2 O − , —S(O) 2 OH, —S(O) 2 R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR, —C(O)O − , —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR;

L is a selected from a bond, (C 1 -C 12 ) alkyldiyl, (C 1 -C 12 ) substituted alkyldiyl, (C 6 -C 26 ) arylalkyldiyl, —O—, —S—, —NR—, —C(O)O—, —C(O)NR—, —NRS(O) 2 —, —NR—NR—, —NRC(O)O—, and —NRC(O)NR—;

R 46 is selected —C(O)NR—, —C(O)O—, and —C(O)S—,

L′ is selected from (C 1 -C 20 ) alkyldiyl, (C 1 -C 20 ) heteroalkyldiyl, (C 1 -C 20 ) alkyleno, (C 1 -C 20 ) heteroalkyleno, (C 6 -C 26 ) arylalkyldiyl, (C 5 -C 20 ) heteroarylalkyldiyl, and substituted forms thereof; and

NUC is a nucleoside/tide or nucleoside/tide analog;

each R is independently selected from hydrogen, (C 1 -C 6 ) alkyl, (C 5 -C 20 ) aryl, (C 6 -C 20 ) arylalkyl, and (C 6 -C 20 ) arylaryl; or when two R groups on the same nitrogen atom are taken together, those two R groups are (C 4 -C 10 ) alkyldiyl or (C 4 -C 10 ) alkyleno; and

each X is independently a halogen.

14. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 in which Y is selected from:

wherein the dashed line at the nitrogen or C4 atom indicates the point of attachment of L.

15. The labeled nucleoside/tide or nucleoside/tide analog of claim 14 wherein:

an alkyldiyl or alkyleno bridge formed by taking R 2 together with R 3 , R 4 together with R 3′ , R 5 together with R 6 , or R 7 together with R 6′ , is ethano, propano, 1,1-dimethylethano, 1,1-dimethyipropano or 1,1,3-trimethylpropano; and

an aryleno bridge formed by taking R 10 , R 11 , R 12 and R 13 together or R 18 , R 19 , R 20 and R 21 together is benzo.

16. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 in which L is selected from phenyldiyl and naphthyldiyl.

17. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 in which L is —(CH 2 ) i -φ- where i is an integer from 1 to 6 and φ is phenyldiyl or naphthyldiyl.

18. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 in which Z is selected from phenyl, benzyl, naphthyl, pyridyl and purinyl.

19. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 in which L is selected from:

20. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 in which L′ is:

wherein R 47 is hydrogen or (C 1 -C 6 ) alkyl, and R 48 is selected from:

wherein each r is independently an integer from 1 to 6; R 49 is hydrogen, (C 1 -C 6 ) alkyl, or an amino acid side chain; and φ is phenyldiyl or substituted phenyldiyl.

21. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 wherein R 22 , R 23 , R 25 , and R 26 are each hydrogen.

22. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 in which Y is selected from the group consisting of:

wherein the dash at the nitrogen or C4 atom indicates the point of attachment of L.

23. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 which has the structure:

24. The labeled nucleoside/tide or nucleoside/tide analog of claim 23 in which L′ is selected from:

25. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 used in conjunction with a donor dye or an acceptor dye whereby the rhodamine dye and the donor dye or acceptor dye form an energy-transfer dye pair.

26. The labeled nucleoside/tide or nucleoside/tide analog of claim 25 wherein the donor dye or acceptor dye is a fluorescein, rhodamine, cyanine, phthalocyanine or squaraine.

27. The labeled nucleoside/tide or nucleoside/tide analog of claim 25 wherein the donor dye or acceptor dye is 4′-aminomethyl-6-carboxyfluorescein and the 4′-aminomethyl-6-carboxyfluorescein is covalently attached to the rhodamine dye by a linker.

28. The labeled nucleoside/tide or nucleoside/tide analog of claim 27 wherein the 4′-aminomethyl-6-carboxyfluorescein is further covalently attached by the linker L′ to the nucleobase B of the nucleoside/tide or nucleoside/tide analog.

29. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 which is enzymatically incorporatable.

30. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 which is a terminator.

31. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 which is enzymatically extendable.

32. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 wherein R 71 and R 70 are hydrogen.

33. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 wherein R 71 and R 70 are hydroxyl.

34. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 wherein R 71 is hydroxyl, and R 70 is hydrogen.

35. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 in which nucleobase B is selected from adenine, 7-deazaadenine, cytosine, guanine, 7-deazaguanine, thymine and uracil.

36. A labeled polynucleotide or polynucleotide analog comprising a rhodamine dye conjugated to a nucleoside/tide or nucleoside/tide analog, wherein the rhodamine is a rhodamine-type parent xanthene having attached to the xanthene C9 carbon a phenyl group, wherein the phenyl group is further substituted with (i) a carboxy group or a sulfonate group or a salt thereof; (ii) one to three substituted or unsubstituted aminopyridinium groups; and (iii) a substituted or unsubstituted alkylthio or arylthio group.