US 9,551,029 B2Granted

Methods and kits using extended rhodamine dyes

AssigneeApplied Biosystems, LLC

InventorsJoe Y. L. Lam, Scott C. Benson, Steven M. Menchen

Life of this patent

Estimating the term…

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.

Claims3

Dependent claims

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 labelled nucleic acid compound comprising the structure:

wherein B is a nucleobase;
W1 and W2 taken separately are selected from —H, —OH, and —F;
and W3 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 the structure:
wherein
R1, R2, R3, R4, R5, R6, R7, R9, R10, R11, and R13 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z1, halogen, —OS(O)2OR, —S(O)2OR, —S(O)2R, —S(O)2NR, —S(O)R, —OP(O)O2RR, —P(O)O2RR, —C(O)OR, —NR2, —NR3, —NC(O)R, —C(O)R, —C(O)NR2, —CN, and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group;
or
R1 taken together with R2, Y1, or Y2;
or
R4 taken together with R3, Y3, or Y4;
or
R5 taken together with R6, Y3, or Y4;
or
R6 taken together with R7, Y3, or Y4;
or
R10 taken together with R9 or R11;
or
R11 taken together with Y1, or Y2;
or
R13 taken together with Y3 or Y4 are selected from alkyleno, alkyleno independently substituted with one or more Z1, heteroalkyleno, heteroalkyleno independently substituted with one or more Z1, aryleno, aryleno independently substituted with one or more Z1, heteroaryleno, and heteroaryleno independently substituted with one or more Z1;
R5 is selected from —H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z1;
Y1, Y2, Y3, Y4 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z1;
or
Y1 taken together with R1, R11 or Y2;
or
Y2 taken together with R1, R11 or Y1;
or
Y3 taken together with R4, R5, R6, R13 or Y4;
or
Y4 taken together with R4, R5, R6, R13 or Y3 are selected from alkyleno, alkyleno independently substituted with one or more Z1, heteroalkyleno, heteroalkyleno independently substituted with one or more Z1, aryleno, aryleno independently substituted with one or more Z1, heteroaryleno, and heteroaryleno independently substituted with one or more Z1;
Z1 is selected from —R, halogen, —OS(O)2OR, —SO2OR, —SO2R, —SO2NR, —S(O)R, —OP(O)O2RR, —P(O)O2RR, —CO2R, —NR2, —NR3, —NC(O)R, —C(O)R, —C(O)NR2, —CN, —O and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group;
and
wherein the linkage is attached to R8 of the extended rhodamine dye.

33 more elements

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 labelled 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 the structure:
wherein
R1, R2, R3, R4, R5, R6, R7, R9, R10, R11, and R13 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z1, halogen, —OS(O)2OR, —S(O)2OR, —S(O)2R, —S(O)2NR, —S(O)R, —OP(O)O2RR, —P(O)O2RR, —C(O)OR, —NR2, —NR3, —NC(O)R, —C(O)R, —C(O)NR2, —CN, and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group;
or
R1 taken together with R2, Y1, or Y2;
or
R4 taken together with R3, Y3, or Y4;
or
R5 taken together with R6, Y3, Or Y4;
or
R6 taken together with R7, Y3, Or Y4;
or
R10 taken together with R9 or R11;
or
R11 taken together with Y1, or Y2;
or
R13 taken together with Y3 or Y4 are selected from alkyleno, alkyleno independently substituted with one or more Z1, heteroalkyleno, heteroalkyleno independently substituted with one or more Z1, aryleno, aryleno independently substituted with one or more Z1, heteroaryleno, and heteroaryleno independently substituted with one or more Z1;
R5 is selected from —H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z1;
Y1, Y2, Y3, Y4 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z1;
or
Y1 taken together with R1, R11 or Y2;
or
Y2 taken together with R1, R11 or Y1;
or
Y3 taken together with R4, R5, R6, R13 or Y4;
or
Y4 taken together with R4, R5, R6, R13 or Y3 are selected from alkyleno, alkyleno independently substituted with one or more Z1, heteroalkyleno, heteroalkyleno independently substituted with one or more Z1, aryleno, aryleno independently substituted with one or more Z1, heteroaryleno, and heteroaryleno independently substituted with one or more Z1;
Z1 is selected from —R, halogen, —OS(O)2OR, —SO2OR, —SO2R, —SO2NR, —S(O)R, —OP(O)O2RR, —P(O)O2RR, —CO2R, —NR2, —NR3, —NC(O)R, —C(O)R, —C(O)NR2, —CN, —O and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group;
and
wherein the linkage is attached to R5 of the extended rhodamine dye.

31 more elements

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 labelled 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 the structure:
wherein
R1, R2, R3, R4, R5, R6, R7, R9, R10, R11, and R13 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z1, halogen, —OS(O)2OR, —S(O)2OR, —S(O)2R, —S(O)2NR, —S(O)R, —OP(O)O2RR, —P(O)O2RR, —C(O)OR, —NR2, —NR3, —NC(O)R, —C(O)R, —C(O)NR2, —CN, and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group;
or
R1 taken together with R2, Y1, or Y2;
or
R4 taken together with R3, Y3, or Y4;
or
R5 taken together with R6, Y3, or Y4;
or
R6 taken together with R7, Y3, Or Y4;
or
R10 taken together with R9 or R11;
or
R11 taken together with Y1, or Y2;
or
R13 taken together with Y3 or Y4 are selected from alkyleno, alkyleno independently substituted with one or more Z1, heteroalkyleno, heteroalkyleno independently substituted with one or more Z1, aryleno, aryleno independently substituted with one or more Z1, heteroaryleno, and heteroaryleno independently substituted with one or more Z1;
R5 is selected from —H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z1;
Y1, Y2, Y3, Y4 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z1;
or
Y1 taken together with R1, R11 or Y2;
or
Y2 taken together with R1, R11 or Y1;
or
Y3 taken together with R4, R5, R6, R13 or Y4;
or
Y4 taken together with R4, R5, R6, R13 or Y3 are selected from alkyleno, alkyleno independently substituted with one or more Z1, heteroalkyleno, heteroalkyleno independently substituted with one or more Z1, aryleno, aryleno independently substituted with one or more Z1, heteroaryleno, and heteroaryleno independently substituted with one or more Z1;
Z1 is selected from —R, halogen, —OS(O)2OR, —SO2OR, —SO2R, —SO2NR, —S(O)R, —OP(O)O2RR, —P(O)O2RR, —CO2R, —NR2, —NR3, —NC(O)R, —C(O)R, —C(O)NR2, —CN, —O and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group;
and
wherein the linkage is attached to R8 of the extended rhodamine dye.

31 more elements

Dependencies are inferred from the claim text.

Facts

Application: 14/927,908Filed 30 Oct 2015 · continuation
Priority: 3 Jun 1999
Published: US 2016/0122813 A15 May 2016
Granted: 24 Jan 2017
Term ends: Not estimatedThe source records don’t support an estimate.

Classification22 codes

C12Q 1/6869
Involving nucleic acids: methods for sequencing
C07D 209/60
[b]- or [c]-condensed: naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
C07D 221/08
Ring systems of three rings: aza-anthracenes
C07D 221/10
Ring systems of three rings: aza-phenanthrenes

Family

Application09/325,243Parent of this division · filed 3 Jun 1999
Application09/780,600Parent of this division · filed 9 Feb 2001
US 6,248,884 B1Parent of this division · filed 19 Jun 2001
Application10/925,411Parent of this division · filed 24 Aug 2004
US 6,781,001 B2Parent of this division · filed 24 Aug 2004
Application12/846,773Parent of this continuation · filed 29 Jul 2010
US 7,776,568 B2Parent of this division · filed 17 Aug 2010
Application13/365,554Parent of this continuation · filed 3 Feb 2012
US 8,124,746 B2Parent of this continuation · filed 28 Feb 2012
Application14/098,519Parent of this continuation · filed 5 Dec 2013
US 8,628,975 B2Parent of this continuation · filed 14 Jan 2014
US 2016/0122813 A1Pre-grant publication · published 5 May 2016
US 9,309,561 B2Parent of this continuation
Application12/846,773Parent of this division
US 2001/0007905 A1Published application
US 2005/0027130 A1Published application
US 2011/0053282 A1Published application
US 2012/0135885 A1Published application
US 2014/0171336 A1Published application
US 9,551,029 B2This patent

From the US filing history. Foreign family members aren’t listed.

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Abstract

wherein B is a nucleobase;
W1 and W2 taken separately are selected from —H, —OH, and —F;
and W3 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 the structure:
wherein
R1, R2, R3, R4, R5, R6, R7, R9, R10, R11, and R13 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z1, halogen, —OS(O)2OR, —S(O)2OR, —S(O)2R, —S(O)2NR, —S(O)R, —OP(O)O2RR, —P(O)O2RR, —C(O)OR, —NR2, —NR3, —NC(O)R, —C(O)R, —C(O)NR2, —CN, and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group;
or
R1 taken together with R2, Y1, or Y2;
or
R4 taken together with R3, Y3, or Y4;
or
R5 taken together with R6, Y3, or Y4;
or
R6 taken together with R7, Y3, or Y4;
or
R10 taken together with R9 or R11;
or
R11 taken together with Y1, or Y2;
or
R13 taken together with Y3 or Y4 are selected from alkyleno, alkyleno independently substituted with one or more Z1, heteroalkyleno, heteroalkyleno independently substituted with one or more Z1, aryleno, aryleno independently substituted with one or more Z1, heteroaryleno, and heteroaryleno independently substituted with one or more Z1;
R5 is selected from —H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z1;
Y1, Y2, Y3, Y4 when taken alone are selected from —H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z1;
or
Y1 taken together with R1, R11 or Y2;
or
Y2 taken together with R1, R11 or Y1;
or
Y3 taken together with R4, R5, R6, R13 or Y4;
or
Y4 taken together with R4, R5, R6, R13 or Y3 are selected from alkyleno, alkyleno independently substituted with one or more Z1, heteroalkyleno, heteroalkyleno independently substituted with one or more Z1, aryleno, aryleno independently substituted with one or more Z1, heteroaryleno, and heteroaryleno independently substituted with one or more Z1;
Z1 is selected from —R, halogen, —OS(O)2OR, —SO2OR, —SO2R, —SO2NR, —S(O)R, —OP(O)O2RR, —P(O)O2RR, —CO2R, —NR2, —NR3, —NC(O)R, —C(O)R, —C(O)NR2, —CN, —O and —OR, wherein R is independently selected from —H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and linking group;
and
wherein the linkage is attached to R8 of the extended rhodamine dye.

33 more elements

Facts

Application

14/927,908Filed 30 Oct 2015 · continuation

Priority

3 Jun 1999

Published

US 2016/0122813 A15 May 2016

Granted

24 Jan 2017

Term ends

Not estimatedThe source records don’t support an estimate.

Classification22 codes

C12Q 1/6869
Involving nucleic acids: methods for sequencing
C07D 209/60
[b]- or [c]-condensed: naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
C07D 221/08
Ring systems of three rings: aza-anthracenes
C07D 221/10
Ring systems of three rings: aza-phenanthrenes

Family

Application09/325,243Parent of this division · filed 3 Jun 1999

Application09/780,600Parent of this division · filed 9 Feb 2001

US 6,248,884 B1Parent of this division · filed 19 Jun 2001

Application10/925,411Parent of this division · filed 24 Aug 2004

US 6,781,001 B2Parent of this division · filed 24 Aug 2004

Application12/846,773Parent of this continuation · filed 29 Jul 2010

US 7,776,568 B2Parent of this division · filed 17 Aug 2010

Application13/365,554Parent of this continuation · filed 3 Feb 2012

US 8,124,746 B2Parent of this continuation · filed 28 Feb 2012

Application14/098,519Parent of this continuation · filed 5 Dec 2013

US 8,628,975 B2Parent of this continuation · filed 14 Jan 2014

US 2016/0122813 A1Pre-grant publication · published 5 May 2016

US 9,309,561 B2Parent of this continuation

Application12/846,773Parent of this division

US 2001/0007905 A1Published application

US 2005/0027130 A1Published application

US 2011/0053282 A1Published application

US 2012/0135885 A1Published application

US 2014/0171336 A1Published application

US 9,551,029 B2This patent

From the US filing history. Foreign family members aren’t listed.