IP Library › Granted Patent US 8,124,746
Granted Patent B2
US 8,124,746 · App. 12/846,773 · Granted Feb 28, 2012

Methods and kits using extended rhodamine dyes

Assignee: Applied Biosystems, LLC
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Quick Facts
Patent No.
US 8,124,746
App. No.
12/846,773
Granted
Feb 28, 2012
Kind
B2
Abstract

Extended rhodamine compounds exhibiting favorable fluorescence characteristics having the structure are disclosed. In addition, novel intermediates for synthesis of these dyes are disclosed, such intermediates having the structure In addition, methods of making and using the dyes as fluorescent labels are disclosed.

Claims (81)

1. A kit for fragment analysis comprising one or more nucleoside or nucleotide, a chain-terminating nucleotide analog and a primer, wherein one or more of the nucleoside or nucleotide is a labeled nucleic acid compound comprising the structure:

wherein B is a nucleobase;

W 1 and W 2 taken separately are selected from —H, —OH, and —F; and W 3 is selected from —OH, monophosphate, diphosphate, triphosphate and phosphate analog

L is a linkage; wherein if B comprises a purine base, the linkage is attached to the 8-position of the purine, if B comprises a 7-deazapurine base, the linkage is attached to the 7-position of the 7-deazapurine, and if B comprises a pyrimidine base, the linkage is attached to the 5-position of the pyrimidine; and

D is an extended rhodamine dye comprising a structure selected from the group consisting of:

wherein

R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 9 , R 10 , R 11 , and R 13 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z 1 , halogen, —OS(O) 2 OR, —S(O) 2 OR, —S(O) 2 R, —S(O) 2 NR, —S(O)R, —OP(O)O 2 RR, —P(O)O 2 RR, —C(O)OR, —NR 2 , —NR 3 , —NC(O)R, —C(O)R, —C(O)NR 2 , —CN, and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group; or

R 1 taken together with R 2 , Y 1 , or Y 2 ; or

R 4 taken together with R 3 , Y 3 , or Y 4 ; or

R 5 taken together with R 6 , Y 3 , or Y 4 ; or

R 6 taken together with R 7 , Y 3 , or Y 4 ; or

R 10 taken together with R 9 or R 11 ; or

R 11 taken together with Y 1 , or Y 2 ; or

R 13 taken together with Y 3 or Y 4 are selected from alkyleno, alkyleno independently substituted with one or more Z 1 , heteroalkyleno, heteroalkyleno independently substituted with one or more Z 1 , aryleno, aryleno independently substituted with one or more Z 1 , heteroaryleno, and heteroaryleno independently substituted with one or more Z 1 ;

R 8 is selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z 1 ;

Y 1 , Y 2 , Y 3 , Y 4 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z 1 ; or

Y 1 taken together with R 1 , R 11 or Y 2 ; or

Y 2 taken together with R 1 , R 11 or Y 1 ; or

Y 3 taken together with R 4 , R 5 , R 6 , R 13 or Y 4 ; or

Y 4 taken together with R 4 , R 5 , R 6 , R 13 or Y 3 are selected from alkyleno, alkyleno independently substituted with one or more Z 1 , heteroalkyleno, heteroalkyleno independently substituted with one or more Z 1 , aryleno, aryleno independently substituted with one or more Z 1 , heteroaryleno, and heteroaryleno independently substituted with one or more Z 1 ; and

Z 1 is selected from —R, halogen, —OS(O) 2 OR, —SO 2 OR, —SO 2 R, —SO 2 NR, —S(O)R, —OP(O)O 2 RR, —P(O)O 2 RR, —CO 2 R, —NR 2 , —NR 3 , —NC(O)R, —C(O)R, —C(O)NR 2 , —CN, —O and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group.

2. A kit for fragment analysis comprising one or more nucleotide triphosphates, a chain-terminating nucleotide analog and a primer, wherein one or more of the nucleotide triphosphates is a labeled nucleic acid compound comprising the structure:

wherein B is a nucleobase;

L is a linkage; wherein if B comprises a purine base, the linkage is attached to the 8-position of the purine, if B comprises a 7-deazapurine base, the linkage is attached to the 7-position of the 7-deazapurine, and if B comprises a pyrimidine base, the linkage is attached to the 5-position of the pyrimidine; and

D is an extended rhodamine dye comprising a structure selected from the group consisting of:

wherein

R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 9 , R 10 , R 11 , and R 13 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z 1 , halogen, —OS(O) 2 OR, —S(O) 2 OR, —S(O) 2 R, —S(O) 2 NR, —S(O)R, —OP(O)O 2 RR, —P(O)O 2 RR, —C(O)OR, —NR 2 , —NR 3 , —NC(O)R, —C(O)R, —C(O)NR 2 , —CN, and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group; or

R 1 taken together with R 2 , Y 1 , or Y 2 ; or

R 4 taken together with R 3 , Y 3 , or Y 4 ; or

R 5 taken together with R 6 , Y 3 , or Y 4 ; or

R 6 taken together with R 7 , Y 3 , or Y 4 ; or

R 10 taken together with R 9 or R 11 ; or

R 11 taken together with Y 1 , or Y 2 ; or

R 13 taken together with Y 3 or Y 4 are selected from alkyleno, alkyleno independently substituted with one or more Z 1 , heteroalkyleno, heteroalkyleno independently substituted with one or more Z 1 , aryleno, aryleno independently substituted with one or more Z 1 , heteroaryleno, and heteroaryleno independently substituted with one or more Z 1 ;

R 8 is selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z 1 ;

Y 1 , Y 2 , Y 3 , Y 4 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z 1 ; or

Y 1 taken together with R 1 , R 11 or Y 2 ; or

Y 2 taken together with R 1 , R 11 or Y 1 ; or

Y 3 taken together with R 4 , R 5 , R 6 , R 13 or Y 4 ; or

Y 4 taken together with R 4 , R 5 , R 6 , R 13 or Y 3 are selected from alkyleno, alkyleno independently substituted with one or more Z 1 , heteroalkyleno, heteroalkyleno independently substituted with one or more Z 1 , aryleno, aryleno independently substituted with one or more Z 1 , heteroaryleno, and heteroaryleno independently substituted with one or more Z 1 ; and

Z 1 is selected from —R, halogen, —OS(O) 2 OR, —SO 2 OR, —SO 2 R, —SO 2 NR, —S(O)R, —OP(O)O 2 RR, —P(O)O 2 RR, —CO 2 R, —NR 2 , —NR 3 , —NC(O)R, —C(O)R, —C(O)NR 2 , —CN, —O and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group.

3. A kit for fragment analysis comprising one or more nucleotide triphosphates, a chain-terminating nucleotide analog and a primer, wherein said chain-terminating nucleotide analog is a labeled nucleic acid compound comprising the structure:

wherein B is a nucleobase;

L is a linkage; wherein if B comprises a purine base, the linkage is attached to the 8-position of the purine, if B comprises a 7-deazapurine base, the linkage is attached to the 7-position of the 7-deazapurine, and if B comprises a pyrimidine base, the linkage is attached to the 5-position of the pyrimidine; and

D is an extended rhodamine dye comprising a structure selected from the group consisting of:

wherein

R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 9 , R 10 , R 11 , and R 13 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z 1 , halogen, —OS(O) 2 OR, —S(O) 2 OR, —S(O) 2 R, —S(O) 2 NR, —S(O)R, —OP(O)O 2 RR, —P(O)O 2 RR, —C(O)OR, —NR 2 , —NR 3 , —NC(O)R, —C(O)R, —C(O)NR 2 , —CN, and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group; or

R 1 taken together with R 2 , Y 1 , or Y 2 ; or

R 4 taken together with R 3 , Y 3 , or Y 4 ; or

R 5 taken together with R 6 , Y 3 , or Y 4 ; or

R 6 taken together with R 7 , Y 3 , or Y 4 ; or

R 10 taken together with R 9 or R 11 ; or

R 11 taken together with Y 1 , or Y 2 ; or

R 13 taken together with Y 3 or Y 4 are selected from alkyleno, alkyleno independently substituted with one or more Z 1 , heteroalkyleno, heteroalkyleno independently substituted with one or more Z 1 , aryleno, aryleno independently substituted with one or more Z 1 , heteroaryleno, and heteroaryleno independently substituted with one or more Z 1 ;

R 8 is selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z 1 ;

Y 1 , Y 2 , Y 3 , Y 4 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z 1 ; or

Y 1 taken together with R 1 , R 11 or Y 2 ; or

Y 2 taken together with R 1 , R 11 or Y 1 ; or

Y 3 taken together with R 4 , R 5 , R 6 , R 13 or Y 4 ; or

Y 4 taken together with R 4 , R 5 , R 6 , R 13 or Y 3 are selected from alkyleno, alkyleno independently substituted with one or more Z 1 , heteroalkyleno, heteroalkyleno independently substituted with one or more Z 1 , aryleno, aryleno independently substituted with one or more Z 1 , heteroaryleno, and heteroaryleno independently substituted with one or more Z 1 ; and

Z 1 is selected from —R, halogen, —OS(O) 2 OR, —SO 2 OR, —SO 2 R, —SO 2 NR, —S(O)R, —OP(O)O 2 RR, —P(O)O 2 RR, —CO 2 R, —NR 2 , —NR 3 , —NC(O)R, —C(O)R, —C(O)NR 2 , —CN, —O and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group.

4. A kit for fragment analysis comprising one or more nucleotide triphosphates, a chain-terminating nucleotide analog and a primer, wherein said chain-terminating nucleotide analog is a labeled nucleic acid compound comprising the structure:

wherein B is a nucleobase;

L is a linkage; wherein if B comprises a purine base, the linkage is attached to the 8-position of the purine, if B comprises a 7-deazapurine base, the linkage is attached to the 7-position of the 7-deazapurine, and if B comprises a pyrimidine base, the linkage is attached to the 5-position of the pyrimidine; and

D is an extended rhodamine dye comprising a structure selected from the group consisting of:

wherein

R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 9 , R 10 , R 11 , and R 13 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z 1 , halogen, —OS(O) 2 OR, —S(O) 2 OR, —S(O) 2 R, —S(O) 2 NR, —S(O)R, —OP(O)O 2 RR, —P(O)O 2 RR, —C(O)OR, —NR 2 , —NR 3 , —NC(O)R, —C(O)R, —C(O)NR 2 , —CN, and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group; or

R 1 taken together with R 2 , Y 1 , or Y 2 ; or

R 4 taken together with R 3 , Y 3 , or Y 4 ; or

R 5 taken together with R 6 , Y 3 , or Y 4 ; or

R 6 taken together with R 7 , Y 3 , or Y 4 ; or

R 10 taken together with R 9 or R 11 ; or

R 11 taken together with Y 1 , or Y 2 ; or

R 13 taken together with Y 3 or Y 4 are selected from alkyleno, alkyleno independently substituted with one or more Z 1 , heteroalkyleno, heteroalkyleno independently substituted with one or more Z 1 , aryleno, aryleno independently substituted with one or more Z 1 , heteroaryleno, and heteroaryleno independently substituted with one or more Z 1 ;

R 8 is selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z 1 ;

Y 1 , Y 2 , Y 3 , Y 4 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z 1 , heteroalkyl, heteroalkyl independently substituted with one or more Z 1 , aryl, aryl independently substituted with one or more Z 1 , heteroaryl, heteroaryl independently substituted with one or more Z 1 , arylalkyl, arylalkyl independently substituted with one or more Z 1 , heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z 1 ; or

Y 1 taken together with R 1 , R 11 or Y 2 ; or

Y 2 taken together with R 1 , R 11 or Y 1 ; or

Y 3 taken together with R 4 , R 5 , R 6 , R 13 or Y 4 ; or

Y 4 taken together with R 4 , R 5 , R 6 , R 13 or Y 3 are selected from alkyleno, alkyleno independently substituted with one or more Z 1 , heteroalkyleno, heteroalkyleno independently substituted with one or more Z 1 , aryleno, aryleno independently substituted with one or more Z 1 , heteroaryleno, and heteroaryleno independently substituted with one or more Z 1 ; and

Z 1 is selected from —R, halogen, —OS(O) 2 OR, —SO 2 OR, —SO 2 R, —SO 2 NR, —S(O)R, —OP(O)O 2 RR, —P(O)O 2 RR, —CO 2 R, —NR 2 , —NR 3 , —NC(O)R, —C(O)R, —C(O)NR 2 , —CN, —O and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group.

Continuity (4)
Division 10925411 · Aug 24, 2004
Division 09780600 · Feb 9, 2001
Division 09325243 · Jun 3, 1999
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