Methods and kits using extended rhodamine dyes

Patent metadata

Field	Value
Publication number	US-9309561-B2
Application number	US-201314098519-A
Country	US
Kind code	B2
Filing date	Dec 5, 2013
Priority date	Jun 3, 1999
Publication date	Apr 12, 2016
Grant date	Apr 12, 2016

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.

First claim

We claim:

1. A method of polynucleotide sequencing comprising the steps of:
forming a mixture of a first, a second, a third, and a fourth class of polynucleotides such that:
each polynucleotide in the first class includes a 3′-terminal dideoxyadenosine labeled with a first dye;
each polynucleotide in the second class includes a 3′-terminal dideoxycytidine and is labeled with a second dye;
each polynucleotide in the third class includes a 3′-terminal dideoxyguanosine and is labeled with a third dye; and
each polynucleotide in the fourth class includes a 3′-terminal dideoxythymidine and is labeled with a fourth dye;
wherein one of the first, second, third or fourth dyes is an extended rhodamine dye having a structure of one of the following formulae:

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; 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 ;
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 linkage to the polynucleotide; and
R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and a linkage to the labeled polynucleotide wherein the linkage is attached to the extended rhodamine compound at R 1 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , Y 1 , Y 2 , Y 3 , or Y 4 ; and
wherein each of the other of the four dyes is spectrally resolvable from the extended rhodamine dye and from each other;
electrophoretically separating the labeled polynucleotides thereby forming bands of similarly sized polynucleotides;
illuminating the bands with an illumination beam capable of causing the dyes to fluoresce; and
identifying the classes of the labeled polynucleotides in the bands by the fluorescence spectrum of the dyes.

2. The method of claim 1 , wherein the extended rhodamine dye is configured to be excited by an excitation light source having a wavelength greater than about 630 nm.

3. The method of claim 1 , wherein the one or more labeled polynucleotides is labeled at a nucleobase of the polynucleotide, and further wherein:
(a) when the nucleobase comprises a purine base, the linking group connecting the extended rhodamine dye to the nucleobase is attached to the 8-position of the purine base;
(b) when the nucleobase comprises a 7-deazapurine base, the linking group connecting the extended rhodamine dye to the nucleobase is attached to the 7-position of the 7-deazapurine base; and
(c) when the nucleobase comprises a pyrimidine base, the linking group connecting the extended rhodamine dye to the nucleobase is attached to the 5-position of the pyrimidine base.

4. The method of claim 1 , wherein the linking group connecting the extended rhodamine dye to a nucleobase of the one or more labeled polynucleotide is attached to R 8 of the extended rhodamine dye.

5. The method of claim 1 , wherein the linking group connecting the extended rhodamine dye to the labeled polynucleotide is an alkynylamino- derivatized or alkenyl-derivatized linkage.

6. The method of claim 1 , wherein the one or more labeled polynucleotides has a structure of one of the following formulae:

wherein NUC is a nucleobase of the one or more labeled polynucleotides;
D is the extended rhodamine dye;
R 3 is selected from the group consisting of —H and lower alkyl;
X is selected from one of the following formulae:

n is an integer of 1 to 5; and
R 1 is —H, lower alkyl, or protecting group.

7. A method for sequencing a nucleic acid sequence comprising:
forming a mixture of extended labeled primers by hybridizing a nucleic acid sequence with a fluorescently labeled oligonucleotide primer in the presence of deoxynucleotide triphosphates, at least one dideoxynucleotide triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxynucleotide triphosphates until a dideoxynucleotide triphosphate is incorporated which terminates extension of the primer;
separating the mixture of extended primers; and
determining the sequence of the nucleic acid sequence by fluorescently measuring the mixture of extended primers formed;
wherein the fluorescently labeled oligonucleotide primer comprises an extended rhodamine dye having a structure of the formula:

wherein
R 1 , R 2 , R 3 , R 4 , R 5 , R 6 ,

Assignees

• Applied Biosystems Llc

Inventors

• Lam Joe Y L
• Benson Scott C
• Menchen Steven M

Classifications

• C07D487/06

  Peri-condensed systems · CPC title

• Y10S436/80

  Fluorescent dyes, e.g. rhodamine · CPC title

• C09B11/24

  Phthaleins containing amino groups {; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes} · CPC title

• C07H19/04

  Heterocyclic radicals containing only nitrogen atoms as ring hetero atom · CPC title

• C07D221/10

  Aza-phenanthrenes · CPC title