US6448407B1
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Atropisomers of asymmetric xanthene fluorescent dyes and methods of DNA sequencing and fragment analysis
Assignee: Applied Biosystems LLC
•
Filing: 2000-11-01
•
Granted: 2002-09-10
•
Expires: 2020-11-01
Abstract
Abstract Substantially pure atropisomers of xanthene compounds are disclosed. A variety of molecular biology applications utilize atropisomeric xanthene fluorescent dyes as labels for substrates such as nucleotides, nucleosides, polynucleotides, polypeptides and carbohydrates. Methods include DNA sequencing, DNA fragment analysis, PCR, SNP analysis, oligonucleotide ligation, amplification, minisequencing, and primer extension.
Inventors
- 👤 Linda G. Lee
- 👤 Meng C. Taing
- 👤 Barnett B. Rosenblum
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-01 01:59
CPC Classifications
C; C07; C07H; C07H19/00; C07H19/02; C07H19/04; C07H19/06; C07H19/10; C07H19/16; C07H19/20; C07H21/00; C07H21/04; C09; C09B; C09B11/00; C09B11/04; C09B11/06; C09B11/08; C09B11/10; C09B11/24; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/531; G01N33/532; G01N33/533
Claims Analysis
Total Claims: 63
Claim 1
Independent Claim
87 words
A compound having the structure: Z 1 is OH, NH 2 , NHR, or NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; Z 2 is O, 30 NH 2 , + NHR, + NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; X is carboxylate or sulfonate; and said structure includes aryl-substituted forms thereof; wherein the compound is atropisomerically enriched.
Claim 2
Dependent Claim
87 words
A compound having the structure: Z 1 is OH, NH 2 , NHR, or NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; Z 2 is O, 30 NH 2 , + NHR, + NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; X is carboxylate or sulfonate; and said structure includes aryl-substituted forms thereof; wherein the compound is atropisomerically enriched.
Claim 3
Dependent Claim
21 words
The substantially pure atropisomer compound of claim 1 wherein Z 1 is OH, Z 2 is O, and X is carboxylate.
Claim 4
Dependent Claim
23 words
The compound of claim 1 wherein Z 1 is NR 2 , Z 2 is + NR 2 , and X is carboxylate.
Claim 5
Dependent Claim
99 words
The compound of claim 1 having the structure: wherein at least one of R 1 , R 4 , R 5 , R 11 , R 13 R 14 , R 17 , R 18 , R 19 , R 20 , Z 1 , or Z 2 is a linking moiety selected from the group consisting of azido, monosubstituted primary amine, disubstituted secondary amine, thiol, hydroxyl, halide, epoxide, N-hydroxysuccinimidyl ester, carboxyl, isothiocyanate, sulfonyl chloride, sulfonate ester, silyl halide, chlorotriazinyl, succinimidyl ester, pentafluorophenyl ester, maleimide, haloacetyl, epoxide, alkylhalide, allyl halide, aldehyde, ketone, acylazide, anhydride, iodoacetamide and an activated ester.
Claim 6
Dependent Claim
17 words
The compound of claim 4 wherein one of R 1 and R 11 is a linking moiety.
Claim 7
Dependent Claim
138 words
The compound of claim 4 comprising one or more substituents R 1 , R 4 , R 5 , R 11 , R 13 , R 14 , R 17 , R 18 , R 19 , and R 20 , each of which is independently flourine, chlorine, C 1 -C 8 alkyl, carboxylate, sulfate, sulfonate, alkylsulfonate, aminomethyl (—CH 2 NH 2 ), aminoalkyl, 4-dialkylaminopyridinium, hydroxymethyl (—CH 2 OH), methoxy (—OCH 3 ), hydroxyalkyl (—ROH), thiomethyl (—CH 2 SH), thioalkyl (—RSH), alkylsulfone (—SO 2 R), arylthio (—SAr), arylsulfone (—SO 2 Ar), sulfonamide (—SO 2 NR 2 ), alkylsulfoxide (—SOR), arylsulfoxide (—SOAr), amino (—NH 2 ), ammonium (—NH 3 + ), amido (—CONR 2 ), nitrile (—CN), C 1 -C 8 alkoxy (—OR), phenoxy, phenolic, tolyl, phenyl, aryl, benzyl, heterocycle, phosphonate, phosphate, quaternary amine, sulfate, polyethyleneoxy, and linking moiety.
Claim 8
Dependent Claim
15 words
The compound of claim 4 wherein R 4 and R 5 taken together form benzo.
Claim 9
Dependent Claim
67 words
The compound of claim 4 wherein at least one of R 1 , R 4 , R 5 , R 11 , R 13 , and R 14 is an electron-deficient heterocycle selected from 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 2-imidazole, 4-imidazole, 3-pyrazole, 4-pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline, pthalazine, quinazoline, quinoxaline, 3-(1,2,4-N)-triazolyl, 5-(1,2,4-N)-triazolyl, 5-tetrazolyl, 4-(1-O, 3-N)-oxazole, 5-(1-O, 3-N)-oxazole, 4-(1-S, 3-N)-thiazole, 5-(1-S, 3-N)-thiazole, 2-benzoxazole, 2-benzothiazole, 4-(1,2,3-N)-benzotriazole, and benzimidazole.
Claim 10
Dependent Claim
33 words
The compound of claim 4 wherein at least one of R 1 , R 4 , R 5 , R 11 , R 13 , and R 14 is phenyl or substituted phenyl.
Claim 11
Dependent Claim
33 words
The compound of claim 4 wherein at least one of R 1 , R 4 , R 5 , R 11 , R 13 , and R 14 is naphthyl or substituted naphthyl.
Claim 12
Dependent Claim
32 words
The compound of claim 4 wherein at least one of R 1 , R 4 , R 5 , R 11 , R 13 , and R 14 is fluorine or chlorine.
Claim 13
Dependent Claim
32 words
The compound of claim 4 wherein at least one of R 1 , R 4 , R 5 , R 11 , R 13 , and R 14 is 2-pyridyl or 3-pyridyl.
Claim 14
Dependent Claim
32 words
The compound of claim 4 wherein at least one of R 1 , R 4 , R 5 , R 11 , R 13 , and R 14 is 2-quinolyl or 3-quinolyl.
Claim 15
Dependent Claim
33 words
The compound of claim 4 wherein at least one of R 1 , R 4 , R 5 , R 11 , R 13 , and R 14 is methoxy (—OCH 3 ).
Claim 16
Dependent Claim
34 words
The compound of claim 4 wherein at least one of R 1 ,R 4 , R 5 , R 11 , R 13 , and R 14 is aminomethyl (—CH 2 NH 2 ).
Claim 17
Dependent Claim
24 words
The compound of claim 4 wherein at least one of R 17 , R 18 , R 19 , and R 20 is chlorine.
Claim 18
Dependent Claim
24 words
The compound of claim 4 wherein at least one of R 17 , R 18 , R 19 , and R 20 is fluorine.
Claim 19
Dependent Claim
24 words
The compound of claim 4 wherein at least one of R 17 , R 18 , R 19 , and R 20 is 4-dialkylaminopyridinium.
Claim 20
Dependent Claim
24 words
The compound of claim 4 wherein at least one of R 17 , R 18 , R 19 , and R 20 is thiophenyl.
Claim 21
Dependent Claim
24 words
The compound of claim 4 wherein at least one of R 17 , R 18 , R 19 , and R 20 is thio-4-carboxyphenyl.
Claim 22
Dependent Claim
22 words
The compound of claim 4 wherein at least one of R 18 and R 19 is carboxyl and the other is hydrogen.
Claim 23
Dependent Claim
22 words
The compound of claim 4 wherein one of R 18 and R 19 is a linking moiety and the other is hydrogen.
Claim 24
Dependent Claim
32 words
The compound of claim 4 wherein R 17 and R 20 are chlorine; one R 18 and R 19 is a linking moiety and the other is hydrogen; and X is carboxyl.
Claim 25
Dependent Claim
78 words
The compound of claim 4 comprising a first bridging group which when taken together with a Z 1 nitrogen, the Z 1 -bonded carbon, and the R 1 -bonded carbon, forms a first ring structure having from 4 to 7 members; and optionally, a second bridging group which when taken together with a Z 2 nitrogen, the Z 2 -bonded carbon, and the R 11 -bonded carbon forms a second ring structure having from 4 to 7 members.
Claim 26
Dependent Claim
19 words
The compound of claim 24 wherein one or both of the first and second ring structures has five members.
Claim 27
Dependent Claim
16 words
The compound of claim 25 wherein the five membered ring structure includes one gem disubstituted carbon.
Claim 28
Dependent Claim
15 words
The compound of claim 26 wherein the gem substituents are C 1 -C 8 alkyl.
Claim 29
Dependent Claim
11 words
The compound of claim 27 wherein the gem substituents are methyl.
Claim 30
Dependent Claim
16 words
The compound of claim 25 wherein the five membered ring is substituted with a linking moiety.
Claim 31
Dependent Claim
15 words
A compound selected from the group consisting of the structures. wherein the compound atropisomerically enriched.
Claim 32
Dependent Claim
15 words
A compound selected from the group consisting of the structures. wherein the compound atropisomerically enriched.
Claim 33
Dependent Claim
13 words
The compound of claim 1 wherein the compound is a substantially pure atropisomer.
Claim 34
Dependent Claim
151 words
A labelled nucleoside or nucleotide having the formula: wherein DYE is a substantially pure atropisomer of a xanthene compound having the structure: Z 1 is OH, NH 2 , NHR, or NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; Z 2 is O, + NH 2 , + NHR, or + NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; X is carboxylate or sulfonate; B is a nucleobase; L is a linker; R 25 is H, monophosphate, diphosphate, triphosphate, thiophosphate, or phosphate analog; R 25 and R 27 , when taken alone, are each independently H, HO, F or other moiety which blocks polymerase-mediated target-directed polymerization, or when taken together R 26 and R 27 form 2′-3′-didehydroribose; and wherein DYE is optionally aryl-substituted.
Claim 35
Dependent Claim
151 words
A labelled nucleoside or nucleotide having the formula: wherein DYE is a substantially pure atropisomer of a xanthene compound having the structure: Z 1 is OH, NH 2 , NHR, or NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; Z 2 is O, + NH 2 , + NHR, or + NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; X is carboxylate or sulfonate; B is a nucleobase; L is a linker; R 25 is H, monophosphate, diphosphate, triphosphate, thiophosphate, or phosphate analog; R 25 and R 27 , when taken alone, are each independently H, HO, F or other moiety which blocks polymerase-mediated target-directed polymerization, or when taken together R 26 and R 27 form 2′-3′-didehydroribose; and wherein DYE is optionally aryl-substituted.
Claim 36
Dependent Claim
25 words
The labelled nucleoside or nucleotide of claim 32 wherein B is selected from the group consisting of uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, and 7-deazaguanosine.
Claim 37
Dependent Claim
19 words
The labelled nucleoside or nucleotide of claim 32 in which L is: wherein n is 0, 1, or 2.
Claim 38
Dependent Claim
12 words
The labelled nucleoside or nucleotide of claim 32 which is enzymatically incorporatable.
Claim 39
Dependent Claim
12 words
The labelled nucleoside or nucleotide of claim 32 which is a terminator.
Claim 40
Dependent Claim
41 words
The labelled nucleoside or nucleotide of claim 33 which has the structure: wherein R 26 and R 27 , when taken alone, are each independently H, F or other moiety which blocks polymerase-mediated target-directed polymerization, or when taken together form 2′-3′-didehydroribose.
Claim 41
Dependent Claim
12 words
The labelled nucleoside or nucleotide of claim 33 which is enzymatically extendable.
Claim 42
Dependent Claim
104 words
A labelled polynucleotide comprising a polynucleotide covalently attached to a label, wherein the label is a substantially pure atropisomer of a xanthene compound having the structure; wherein Z 1 is OH, NH 2 , NHR, or NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; Z 2 is O, + NH 2 , + NHR, or + NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; X is carboxylate or sulfonate, and said structure includes aryl-substituted forms thereof.
Claim 43
Dependent Claim
104 words
A labelled polynucleotide comprising a polynucleotide covalently attached to a label, wherein the label is a substantially pure atropisomer of a xanthene compound having the structure; wherein Z 1 is OH, NH 2 , NHR, or NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; Z 2 is O, + NH 2 , + NHR, or + NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; X is carboxylate or sulfonate, and said structure includes aryl-substituted forms thereof.
Claim 44
Dependent Claim
84 words
The labelled polynucleotide of claim 39 comprising the formula: wherein DYE is a substantially pure atropisomer of a xanthene compound; B is a nucleobase; L is a linker; R 27 is H, OH, halide, azide, amine, alkylamine, C 1 -C 6 alkyl, allyl, C 1 -C 6 alkoxy, OCH 3 , or OCH 2 CH═CH 2 ; and R 28 and R 29 when taken alone, are each independently H, phosphate, internucleotide phosphodiester, or internucleotide analog; wherein the polynucleotide comprises 2 to 100 nucleotides.
Claim 45
Dependent Claim
23 words
The labelled polynucleotide of claim 40 wherein B is selected from the group consisting of uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, and 7-deazaguanosine.
Claim 46
Dependent Claim
80 words
The labelled polynucleotide of claim 39 comprising the formula: wherein DYE is a substantially pure atropiscmer of a xanthene compound; B is a nucleobase; X is O, NH, or S; L is a linker; R 27 is H, OH, halide, azide, amine, alkylamine, C 1 -C 6 alkyl, allyl, C 1 -C 6 alkoxy, OCH 3 , or OCH 2 CH═CH 2 ; and R 28 is internucleotide phosphodiester or internucleotide analog; wherein the polynucleotide comprises 2 to 100 nucleotides.
Claim 47
Dependent Claim
23 words
The labelled polynucleotide of claim 42 wherein B is selected from the group consisting of uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, and 7-deazaguanosine.
Claim 48
Dependent Claim
15 words
The labelled polynucleotide of claim 42 in which L is C 1 -C 12 alkyldiyl.
Claim 49
Dependent Claim
23 words
The labelled polynucleotide of claim 42 in which L comprises —(CH 2 CH 2 O) n —, where n is 1 to 100.
Claim 50
Dependent Claim
64 words
A labelled polypeptide comprising a polypeptide covalently attached to a substantially pure atropisomer of a xanthene compound having the structure: wherein Z 1 is OH, NH 2 , NHR, or NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; X is carboxylate or sulfonate, and said structure includes aryl-substituted forms thereof.
Claim 51
Dependent Claim
64 words
A labelled polypeptide comprising a polypeptide covalently attached to a substantially pure atropisomer of a xanthene compound having the structure: wherein Z 1 is OH, NH 2 , NHR, or NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; X is carboxylate or sulfonate, and said structure includes aryl-substituted forms thereof.
Claim 52
Dependent Claim
154 words
A phosphoramidite compound having the formula: wherein DYE is a substantially pure atropisomer of a xanthene compound having the structure: wherein Z 1 is OH, NH 2 , NHR, or NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; Z 2 , is O, + NH 2 , + NHR, or + NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; X is carboxylate or sulfonate, L is a linker; R 30 and R 31 taken separately are selected from the group consisting of C 1 -C 12 alkyl, C 1 -C 12 cycloalkyl, and aryl; or R 30 and R 31 taken together with the nitrogen atom form a saturated nitrogen heterocycle; and R 32 is a phosphite ester protecting group; and wherein DYE is optionally aryl-substituted.
Claim 53
Dependent Claim
154 words
A phosphoramidite compound having the formula: wherein DYE is a substantially pure atropisomer of a xanthene compound having the structure: wherein Z 1 is OH, NH 2 , NHR, or NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; Z 2 , is O, + NH 2 , + NHR, or + NR 2 , wherein each R is independently hydrogen, C 1 -C 12 alkyl, phenyl, benzyl, aryl, heterocycle, or a linking moiety; X is carboxylate or sulfonate, L is a linker; R 30 and R 31 taken separately are selected from the group consisting of C 1 -C 12 alkyl, C 1 -C 12 cycloalkyl, and aryl; or R 30 and R 31 taken together with the nitrogen atom form a saturated nitrogen heterocycle; and R 32 is a phosphite ester protecting group; and wherein DYE is optionally aryl-substituted.
Claim 54
Dependent Claim
20 words
The phosphoramidite compound of claim 47 wherein R 32 is selected from the group consisting of methyl, 2-cyanoethyl, and 2-(4-nitrophenyl)ethyl.
Claim 55
Dependent Claim
15 words
The phosphoramidite compound of claim 47 wherein R 30 and R 31 are each isopropyl.
Claim 56
Dependent Claim
16 words
The phosphoramidite compound of claim 47 wherein R 30 and R 31 taken together is morpholino.
Claim 57
Dependent Claim
14 words
The phosphoramidite compound of claim 47 wherein L is C 1 -C 12 alkyidyl.
Claim 58
Dependent Claim
21 words
The phosphoramidite compound of claim 47 wherein L is attached at R 18 or R 19 of DYE having the structure:
Claim 59
Dependent Claim
9 words
The phosphoramidite compound of claim 52 having the structure:
Claim 60
Dependent Claim
16 words
The phosphoramidite compound of claim 47 wherein L is and n ranges from 1 to 10.
Claim 61
Dependent Claim
32 words
A kit for generating a labelled primer extension product, comprising one or more enzymatically-incorporatable nucleotides and a primer, wherein at least one nucleotide is a labelled nucleotide according to claim 52 .
Claim 62
Dependent Claim
12 words
The kit of claim 55 wherein the labelled nucleotide is a terminator.
Claim 63
Dependent Claim
41 words
The kit of claim 56 which comprises four different terminators, one which terminates at a target A, one which terminates at a target G, one which terminates at a target C and one which terminates at a target T or U.
References Cited (Backward Citations)
No citation records indexed.
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