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Matched Legal Cases: ['Application No. 2002219896', 'Application No. 14182', 'Application No. 2008200036', 'Application No. 2', 'Application No. 2', 'Application No. 98', 'Application No. 98', 'Application No. 98', 'Application No. 01', 'Application No. 98', 'Application No. 98', 'Application No. 01', 'Application No. 98', 'Application No. 98', 'Application No. 2003', 'Application No. 2053069', 'Application No. 2000']

Patent US7833756 - Methods of labelling polynucleotides with dibenzorhodamine dyes - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsDibenzorhodamine compounds having the structure are disclosed, including nitrogen- and aryl-substituted forms thereof. In addition, two intermediates useful for synthesizing such compounds are disclosed, a first intermediate having the structure including nitrogen- and aryl-substituted forms thereof,...http://www.google.com/patents/US7833756?utm_source=gb-gplus-sharePatent US7833756 - Methods of labelling polynucleotides with dibenzorhodamine dyesAdvanced Patent SearchPublication numberUS7833756 B2Publication typeGrantApplication numberUS 12/169,624Publication dateNov 16, 2010Filing dateJul 8, 2008Priority dateNov 25, 1997Fee statusPaidAlso published asCA2311476A1, CA2311476C, EP1034221A1, EP1034221B1, EP1408090A1, EP2186801A2, EP2186801A3, EP2295503A1, US5936087, US6051719, US6111116, US6221606, US6326153, US6566071, US6919445, US20010011139, US20020034761, US20040072209, US20060051791, US20070099210, US20090068751, US20110124510, WO1999027020A1Publication number12169624, 169624, US 7833756 B2, US 7833756B2, US-B2-7833756, US7833756 B2, US7833756B2InventorsScott C. Benson, Joe Y. L. Lam, Steven Michael MenchenOriginal AssigneeApplied Biosystems, LlcExport CitationBiBTeX, EndNote, RefManPatent Citations (44), Non-Patent Citations (37), Classifications (50), Legal Events (6) External Links: USPTO, USPTO Assignment, EspacenetMethods of labelling polynucleotides with dibenzorhodamine dyes
US 7833756 B2Abstract
Dibenzorhodamine compounds having the structure
are disclosed, including nitrogen- and aryl-substituted forms thereof. In addition, two intermediates useful for synthesizing such compounds are disclosed, a first intermediate having the structure
including nitrogen- and aryl-substituted forms thereof, and a second intermediate having the structure
including nitrogen- and aryl-substituted forms thereof, wherein substituents at positions C-14 to C18 taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alkyl, carboxylic acid, sulfonic acid, —CH2OH, alkoxy, phenoxy, linking group, and substituted forms thereof. The invention further includes energy transfer dyes comprising the dibenzorhodamine compounds, nucleosides labeled with the dibenzorhodamine compounds, and nucleic acid analysis methods employing the dibenzorhodamine compounds.
1. A method of identifying polynucleotide classes comprising the steps of:
providing multiple classes of polynucleotides distributed among locations in a spatially addressable hybridization array;
wherein a first class of polynucleotide comprises a dibenzorhodamine dye having a structure of the formula:
including nitrogen- and aryl-substituted forms thereof;
and further wherein other classes of polynucleotides are labeled with dyes configured to be spectrally resolvable from the dibenzorhodamine dye of the first class of polynucleotide and from each other;
illuminating the spatially addressable hybridization array with an illumination beam configured to cause the dyes to fluoresce; and
identifying the classes of the polynucleotides in the spatially addressable hybridization array by the fluorescence spectrum of the dyes.
2. The method of claim 1 wherein the dibenzorhodamine dye comprises a first bridging group which when taken together with the C-12-bonded nitrogen and the C-12 and C-13 carbons forms a first ring structure having from 4 to 7 members; and/or
a second bridging group which when taken together with the C-2-bonded-nitrogen and the C-1 and C-2 carbons forms a second ring structure having from 4 to 7 members.
3. The method of claim 2 wherein one or both of the first and second ring structures has five members.
4. The method of claim 3 wherein the five membered ring structure includes one gem disubstituted carbon.
5. The method of claim 4 wherein the gem substituents are lower alkyl.
6. The method of claim 5 wherein the gem substituents are methyl.
7. The method of claim 3 wherein the five membered ring is not aromatic.
8. The method of claim 2 wherein the first and second ring structures are the same.
9. The method of claim 2 wherein the first and second ring structures are different.
10. The method of claim 1 wherein the dibenzorhodamine dye comprising one or more nitrogen substituents selected from the group consisting of lower alkyl, lower alkene, lower alkyne, phenyl, aromatic, electron-rich heterocycle, polycyclic aromatic, water-solubilizing group, linking group, including substituted forms thereof.
11. The method of claim 10 wherein the nitrogen substituents are selected from the group consisting of lower alkyl, phenyl, and substituted forms thereof.
12. The method of claim 10 wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is linking group.
13. The method of claim 10 wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is sulfonate.
14. The method of claim 10 wherein the linking group is isothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinyl, succinimidyl ester, maleimide, haloacetyl, or iodoacetamide.
15. The method of claim 10 wherein the linking group is carboxylate.
16. The method of claim 10 wherein the nitrogen substituents are water-solubilizing group.
17. The method of claim 10 wherein the water-solubilizing group is selected from the group consisting of sulfonate, phosphate, quaternary amine, sulfate, polyhydroxyl, and water-soluble polymer.
18. The method of claim 1 wherein the dibenzorhodamine dye and the polynucleotide are connected by a linkage at a position on the polynucleotide selected from the 8-position of a purine nucleobase, the 7- or 8-position of a deazapurine nucleobase, the 5-position of a pyrimidine nucleus, the 5′ terminus, the 3′ terminus, and the phosphodiester backbone.
19. The method of claim 18 wherein the linkage is attached to the linking group substituent of the dibenzorhodamine dye.
20. The method of claim 18 wherein the linkage is an acetylenic amido or alkenic amido linkage.
21. The method of claim 20 wherein the linkage has a structure of one of the following formulae:
wherein NUC is a nucleobase of the polynucleotide; D is the dibenzorhodamine dye; R3 is H or C1-C8 alkyl; and
X is a moiety having a structure of one of the following formulae:
wherein R1 is H or (C1-C8)alkyl, and n is an integer of 1 to 5.
22. The method of claim 1 wherein the dibenzorhodamine dye has a structure of the following formula:
including aryl-substituted forms thereof;
wherein R1 and R2 are independently selected from lower alkyl, lower alkene, lower alkyne, phenyl, aromatic, electron-rich heterocycle, polycyclic aromatic, water-solubilizing group, linking group, including substituted forms thereof; and
the dibenzorhodamine is covalently attached to the polynucleotide by a linkage connected to a linking group substituent of R1 or R2.
23. A method of identifying polynucleotide classes comprising the steps of:
wherein a first class of polynucleotide comprises an energy transfer dye comprising:
a donor dye configured to absorb light at a first wavelength and emit excitation energy in response;
an acceptor dye configured to absorb the excitation energy emitted by the donor dye and fluoresce at a second wavelength in response; and
wherein at least one of the donor dye and acceptor dye is a dibenzorhodamine dye having a structure of the formula:
and further wherein other classes of polynucleotides are labeled with dyes configured to be spectrally resolvable from the energy transfer dye of the first class of polynucleotide and from each other;
24. The method of claim 23 wherein the first wavelength is greater than about 600 nm.
25. The method of claim 23 wherein the dibenzorhodamine dye comprises a first bridging group which when taken together with the C-12-bonded nitrogen and the C-12 and C-13 carbons forms a first ring structure having from 4 to 7 members; and/or
26. The method of claim 25 wherein one or both of the first and second ring structures has five members.
27. The method of claim 26 wherein the five membered ring structure includes one gem disubstituted carbon.
28. The method of claim 27 wherein the gem substituents are lower alkyl.
29. The method of claim 28 wherein the gem substituents are methyl.
30. The method of claim 26 wherein the five membered ring is not aromatic.
31. The method of claim 25 wherein the first and second ring structures are the same.
32. The method of claim 25 wherein the first and second ring structures are different.
33. The method of claim 23 wherein the dibenzorhodamine dye comprises one or more nitrogen substituents selected from the group consisting of lower alkyl, lower alkene, lower alkyne, phenyl, aromatic, electron-rich heterocycle, polycyclic aromatic, water-solubilizing group, and linking group, including substituted forms thereof.
34. The method of claim 33 wherein the nitrogen substituents are selected from the group consisting of lower alkyl, phenyl, and substituted forms thereof.
35. The method of claim 33 wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is linking group.
36. The method of claim 35 wherein the linking group is isothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinyl, succinimidyl ester, maleimide, haloacetyl or iodoacetamide.
37. The method of claim 35 wherein the linking group is carboxylate.
38. The method of claim 33 wherein one nitrogen substituent is water-solubilizing group.
39. The method of claim 33 wherein the water-solubilizing group is selected from the group consisting of sulfonate, phosphate, quaternary amine, sulfate, polyhydroxyl, and water-soluble polymer.
40. The method of claim 33 wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is sulfonate.
41. The method of claim 23 wherein the linker has the structure
Z1 is selected from the group consisting of —NH, sulfur and oxygen;
R21 is a lower alkyl attached to the donor dye;
R22 is a substituent selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond and a fused ring structure which is attached to the carbonyl carbon; and
R28 includes a functional group which attaches the linker to the acceptor dye.
42. The method of claim 41 wherein R22 is a five or six membered ring selected from the group consisting of cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, triazine, pyrazine, oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.
43. The method of claim 23 wherein the linker has the structure
Z2 is selected from the group consisting of —NH, sulfur and oxygen; and
R29 is a lower alkyl.
44. The method of claim 23 wherein the linker has the structure
45. The method of claim 23 wherein the energy transfer dye and the polynucleotide are connected by a linkage at a position on the polynucleotide selected from the 8-position of a purine nucleobase, the 7- or 8-position of a deazapurine nucleobase, the 5-position of a pyrimidine nucleus, the 5′ terminus, the 3′ terminus, and the phosphodiester backbone.
46. The method of claim 45 wherein the linkage is attached to a linking group substituent of the dibenzorhodamine dye.
47. The method of claim 45 wherein the linkage is an acetylenic amido or alkenic amido linkage.
48. The method of claim 47 wherein the linkage has a structure of one of the following formulae:
wherein NUC is a nucleobase of the polynucleotide; D is the energy transfer dye; R3 is H or C1-C8 alkyl; and
49. The method of claim 23 wherein the dibenzorhodamine dye has a structure of the following formula:
wherein R1 and R2 are independently selected from lower alkyl, lower alkene, lower alkyne, phenyl, aromatic, electron-rich heterocycle, polycyclic aromatic, water-solubilizing group, linking group, including substituted forms thereof.
50. The method of claim 23 wherein the donor or the acceptor dye is a dibenzorhodamine dye and the donor or acceptor dye is a fluorescein dye.
51. A method of identifying polynucleotide classes comprising the steps of:
wherein R1 and R2 are independently selected from lower alkyl, lower alkene, lower alkyne, phenyl, aromatic, electron-rich heterocycle, polycyclic aromatic, water-solubilizing group, linking group, including substituted forms thereof;
52. The method of claim 51 wherein the water-solubilizing group is selected from the group consisting of sulfonate, phosphate, quaternary amine, sulfate, polyhydroxyl, and water-soluble polymer.
53. The method of claim 51 wherein the linking group is isothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinyl, succinimidyl ester, maleimide, haloacetyl or iodoacetamide.
54. The method of claim 51 wherein the linking group is carboxylate.
55. The method of claim 51 wherein the water-solubilizing group is selected from the group consisting of sulfonate, phosphate, quaternary amine, sulfate, polyhydroxyl, and water-soluble polymer.
This application is a continuation of application Ser. No. 11/466,085, file Aug. 21, 2006, now abandoned, which is a continuation of application Ser. No. 11/177,233, filed Jul. 7, 2005, now abandoned, which is a continuation of application Ser. No. 10/441,950, filed May 20, 2003, now U.S. Pat. No. 6,919,445, which is a continuation of application Ser. No. 09/969,430 filed Oct. 2, 2001,now U.S. Pat. No. 6,556,071, which is a division of application Ser. No. 09/784,943, filed Feb. 14, 2001, now U.S. Pat. No. 6,326,153 which is a continuation of application Ser. No. 09/556,040, filed Apr. 20, 2000, now U.S. Pat. No. 6,221,606, which is a division of application Ser. No. 09/199,402, filed Nov. 24, 1998, now U.S. Pat. No. 6,111,116, which is a division of application Ser. No. 08/978,775, filed Nov. 25, 1997, now U.S. Pat. No. 5,936,087, which are all incorporated herein by reference.
In a first aspect, the invention comprises dibenzorhodamine dye compounds having the structure
including nitrogen- and aryl-substituted forms thereof.
In a second aspect, the invention comprises intermediates useful for the synthesis of dibenzorhodamine compounds having the structure
In a third aspect, the invention comprises intermediates useful for the synthesis of dibenzorhodamine compounds having the structure
including nitrogen- and aryl-substituted forms thereof, wherein R1 taken together with the C-12-bonded nitrogen and the C-12 and C-13 carbons forms a first ring structure having from 4 to 7 members; and/or R1 taken together with the C-12-bonded nitrogen and the C-11 and C-12 carbons forms a second ring structure having from 5 to 7 members.
In a fourth aspect, the invention includes energy transfer dye compounds comprising a donor dye, an acceptor dye, and a linker linking the donor and acceptor dyes. The donor dye is capable of absorbing light at a first wavelength and emitting excitation energy in response, and the acceptor dye is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response. The linker serves to facilitate the efficient transfer of energy between the donor dye and the acceptor dye. According to the present invention, at least one of the donor and acceptor dyes is a dibenzorhodamine dye having the structure set forth above.
NUC-D
“Lower alkene” denotes a hydrocarbon containing from 1 to 8 carbon atoms wherein one or more of the carbon-carbon bonds are double bonds.
“Lower alkyne” denotes a hydrocarbon containing from 1 to 8 carbon atoms wherein one or more of the carbons are bonded with a triple bond.
“Nucleoside” refers to a compound consisting of a purine, deazapurine, or pyrimidine nucleoside base, e.g., adenine, guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, and the like, linked to a pentose at the 1′ position. When the nucleoside base is purine or 7-deazapurine, the sugar moiety is attached at the 9-position of the purine or deazapurine, and when the nucleoside base is pyrimidine, the sugar moiety is attached at the 1-position of the pyrimidine, e.g., Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). The term “nucleotide” as used herein refers to a phosphate ester of a nucleoside, e.g., triphosphate esters, wherein the most common site of esterification is the hydroxyl group attached to the C-5 position of the pentose. The term “nucleoside/tide” as used herein refers to a set of compounds including both nucleosides and nucleotides. “Analogs” in reference to nucleosides/tides include synthetic analogs having modified base moieties, modified sugar moieties and/or modified phosphate moieties, e.g. described generally by Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Phosphate analogs comprise analogs of phosphate wherein the phosphorous atom is in the +5 oxidation state and one or more of the oxygen atoms is replaced with a non-oxygen moiety. Exemplary analogs include but are not limited to phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, boronophosphates, including associated counterions, e.g., H+, NH4 +, Na+, if such counterions are present. Exemplary base analogs include but are not limited to 2,6-diaminopurine, hypoxanthine, pseudouridine, C-5-propyne, isocytosine, isoguanine, 2-thiopyrimidine, and other like analogs. Exemplary sugar analogs include but are not limited to 2′- or 3′-modifications where the 2′- or 3′-position is hydrogen, hydroxy, alkoxy, e.g., methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy and phenoxy, amino or alkylamino, fluoro, chloro and bromo. The term “labeled nucleoside/tide” refers to nucleosides/tides which are covalently attached to the dye compounds of Formula I through a linkage.
“Rhodamine dye” refers to dyes including the general polycyclic structure
including any and all substituted versions thereof.
1.2. 1-Amino-3-Hydroxynapthalene Intermediates
1.2.1. Structure
In a first aspect, the present invention comprises a novel class of 1-amino-3-hydroxynapthalene compounds useful as intermediates in the synthesis of dibenzorhodamine dyes. These compounds have the general structure shown in Formula I immediately below, including substituted forms thereof, where R1 taken together with the C-12-bonded nitrogen and the C-12 and C-13 carbons forms a first ring structure having from 4 to 7 members; and/or R1 taken together with the C-12-bonded nitrogen and the C-11 and C-12 carbons forms a second ring structure having from 5 to 7 members. (Note that all molecular structures provided herein are intended to encompass not only the exact electronic structures presented, but also include all resonant structures, protonation states and associated counterions thereof.)
In the compound of Formula I, preferably the first ring structure has five members. More preferably, the five membered first ring structure includes one gem disubstituted carbon, e.g., wherein the gem substituents are lower alkyl, e.g., methyl. In an alternative preferred embodiment, the five membered ring is substituted with linking group or water-solubilizing group.
Preferably, the compound of Formula I includes one or more nitrogen substituents. Exemplary nitrogen substituents include but are not limited to lower alkyl, lower alkene, lower alkyne, phenyl, aromatic, electron-rich heterocycle, polycyclic aromatic, water-solubilizing group, and linking group, including substituted forms thereof. In a particularly preferred embodiment, the nitrogen substituents are lower alkyl and/or phenyl, including substituted forms thereof. More preferably, the nitrogen substituents are substituted lower alkyl or substituted phenyl, wherein the substituent is linking group, or water-solubilizing group.
In an additional preferred embodiment, one or more of carbons at positions C-8 to C-11 are substituted. Exemplary substituents include but are not limited to fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfate, sulfonate, sulfone, sulfonamide, sulfoxide, amino, ammonium, amido, nitrile, lower alkoxy, phenoxy, aromatic, phenyl, polycyclic aromatic, electron-rich heterocycle, water-solubilizing group, and linking group, including substituted forms thereof. Preferably, one or more of the substituents is sulfonate.
1.2.2. Synthetic Methods
A third preferred synthesis method suitable for the synthesis of N-substituted-5-hydroxy-(tetrahydro)benzoquinoline compounds, e.g., N-methyl-5-hydroxy-(tetrahydro)benzoquinoline 15, is shown in FIG. 2. In this method, compound 10 is synthesized from methoxy-napthaldehyde by condensation with malonic acid using a piperidine catalyst in pyridine. Compound 10 is then reduced with hydrogen, followed by LAH reduction, and reacted with trifluoromethanesulfonic anhydride to give the triflate 11. The triflate 11 is reacted with NaN3 to give compound 12. Compound 12 is complexed with a Lewis acid, e.g., AlCl3, and refluxed yielding the cyclized benzoquinoline derivative 13. Next, a nitrogen substituent is added, e.g., the nitrogen is alkylated using a conventional alkylation procedure, e.g., the benzoquinoline derivative 13 is reacted with n-butyl lithium and an alkylating agent, e.g., MeI to give compound 14 or propane sultone to give compound 16. The methoxy group is then removed by a boron tribromide procedure giving a N-alkylbenzoquinoline derivative, e.g., compound 15 or 17. An example of this synthesis is provided in Example 3 below.
A fourth preferred synthesis method suitable for the synthesis of N-substituted-2,2,4-trimethyl-5-hydroxy-benzoquinoline compounds, e.g. N-methyl-2,2,4-trimethyl-5-hydroxy-(tetrahydro)benzoquinoline 22, is shown in FIG. 3. In this method, following the procedure of A. Rosowsky and E. J. Modest (J.O.C. 30 1832 1965, and references therein), 1-amino-3-methoxynapthalene 18 is reacted with acetone catalyzed by iodine and then quenched with saturated Na2S2O3 to give the benzoquinoline compound 19. Compound 19 is then alkylated with an alkylating agent, e.g., MeI, according to a general alkylation procedure to give compound 20. The alkylated compound 20 is reduced with H2 catalyzed by Pd/C to give a N-methyl-methoxyquinoline intermediate 21, and subsequent methoxy group deprotection by a general boron tribromide procedure yields the N-substituted-2,2,4-trimethyl-5-hydroxy-benzoquinoline compound, e.g., N-methyl-2,2,4-trimethyl-5-hydroxy-(tetrahydro)benzoquinoline 22. An example of this synthesis is provided in Example 4 below.
1.3. Dibenzorhodamine Dye Compounds
In a second aspect, the present invention comprises a novel class of dibenzorhodamine dye compounds useful as molecular labels having the general structure shown in Formula II immediately below, including aryl- and nitrogen-substituted forms thereof.
In one preferred embodiment of the compound of Formula II, the compound includes a first bridging group which when taken together with the C-12-bonded nitrogen and the C-12 and C-13 carbons forms a first ring structure having from 4 to 7 members, and/or a second bridging group which when taken together with the C-2-bonded-nitrogen and the C-1 and C-2 carbons forms a second ring structure having from 4 to 7 members. More preferably, one or both of the first and second ring structures has five members. In yet a more preferred embodiment, the five membered ring structure includes one gem disubstituted carbon, wherein the gem substituents are lower alkyl, e.g., methyl. In an alternative preferred embodiment, the five membered ring is substituted with linking group.
In another preferred embodiment, the compound of Formula II includes a C-7 substituent selected from the group consisting of acetylene, lower alkyl, lower alkene, cyano, phenyl, heterocyclic aromatic, electron-rich heterocycle, and substituted forms thereof. In a more preferred embodiment, the C-7 substituent is a phenyl or substituted phenyl having the structure
wherein aryl substituents at positions C-14 to C-18 taken separately may be selected from the group consisting of hydrogen, chlorine, fluorine, lower alkyl, carboxylic acid, sulfonic acid, —CH2OH, alkoxy, phenoxy, linking group, and substituted forms thereof. Preferably, the phenyl substituent at C-18 is selected from the group consisting of carboxylic acid and sulfonate, and is most preferably carboxylic acid. In another preferred embodiment, substituents at positions C-14 and C-17 are chlorine. In yet another preferred embodiment, substituents at positions C-14 to C-17 are all chlorine or all fluorine. In a particularly preferred embodiment, substituents at one of positions C-15 and C-16 is linking group and the other is hydrogen, substituents at positions C-14 and C-17 are chlorine, and a substituent at position C-18 is carboxy.
In yet another preferred embodiment of the invention, the compound of Formula II includes one or more nitrogen substituents. Preferably, such substituents are selected from the group consisting of lower alkyl, lower alkene, lower alkyne, phenyl, aromatic, electron-rich heterocycle, polycyclic aromatic, water-solubilizing group, linking group, and substituted forms thereof. More preferably, the nitrogen substituents are selected from the group consisting of lower alkyl, phenyl, and substituted forms thereof, where exemplary substituents include linking group, and water-solubilizing group.
In another preferred embodiment of the invention, the compound of Formula II includes aryl substituents at one or more of carbons C-1, C-3 through C-6, C-8 through C-11, and C-13. Exemplary aryl substituents include but are not limited to fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfate, sulfonate, sulfone, sulfonamide, sulfoxide, amino, ammonium, amido, nitrile, lower alkoxy, phenoxy, aromatic, phenyl, polycyclic aromatic, water-solubilizing group, electron-rich heterocycle, and linking group, including substituted forms thereof. In a particularly preferred embodiment, at least one substituent is sulfonate.
1.3.2. Synthetic Methods
Generally, the dibenzorhodamine dyes of the present invention are synthesized as follows. See FIG. 4. An anhydride derivative 30, e.g., a phthalic anhydride, is mixed with 1-amino-3-methoxy intermediates 31 and 32, and Lewis acid, e.g., ZnCl2, where the R-substituents in compound 30 may be the same or different, but are preferably the same. Exemplary R-substituents include but are not limited to acetylene, lower alkyl, lower alkene, phenyl, heterocyclic aromatic, electron-rich heterocycle, and substituted forms thereof. The mixture is heated briefly until melting is observed. A solvent, e.g., 1,2-dichlorobenzene, is added to the reaction mixture, and the heterogeneous mixture is heated to about 130� C. to about 180� C. The crude reaction mixture is cooled and purified by normal phase flash chromatography to yield dye compound 33. When the anhydride is part of a substituted phthalic anhydride, e.g., compound 34, two isomers are formed. See FIG. 5. The isomers 35 and 36 are separated by PTLC. The isomerically pure dyes are identified by single spots on normal and reverse phase TLC and by their UV/Visible absorption spectra and their long wavelength fluorescent excitation and emission spectra.
1.4. Energy Transfer Dyes Incorporating the Dibenzorhodamine Dyes
One linker according to the present invention for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure
where R21 is a lower alkyl attached to the donor dye, Z1 is either NH, sulfur or oxygen, R22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R28 includes a functional group which attaches the linker to the acceptor dye.
In one embodiment of this linker, illustrated below, the linker has the general structure
where R21, and R22 are as detailed above, Z1 and Z2 are each independently either NH, sulfur or oxygen, R29 is a lower alkyl, and the terminal carbonyl group is attached to a ring structure of the acceptor dye. In the variation where Z2 is nitrogen, the C(O)R22R29Z2 subunit forms an amino acid subunit. Particular examples of five or six membered rings which may be used as R22 in the linker include, but are not limited to cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, triazine, pyrazine and oxazine. Examples of fused ring structures include, but are not limited to indene, benzofuran, thionaphthene, indole and naphthalene. A preferred embodiment of this linker is where R21 and R29 are methylene, Z1 and Z2 are NH, and R22 is benzene, as shown below.
1.5. Reagents Incorporating the Dibenzorhodamine Dyes
In another aspect, the present invention comprises reagents labeled with the dibenzorhodamine dye compounds of Formula I. Reagents of the invention can be virtually anything to which the dyes of the invention can be attached. Preferably, the dye is covalently attached to the reagent. Reagents may include but are not limited to proteins, polypeptides, polysaccharides, nucleotides, nucleosides, polynucleotides, lipids, solid supports, organic and inorganic polymers, and combinations and assemblages thereof, such as chromosomes, nuclei, living cells, such as bacteria or other microorganisms, mammalian cells, tissues, and the like.
1.5.1. Nucleoside/Tide Reagents
NUC-D FORMULA III
Preferably, the linkages are acetylenic amido or alkenic amido linkages, the linkage between the dye and the nucleoside/tide base being formed by reacting an activated N-hydroxysuccinimide (NHS) ester of the dye with an alkynylamino- or alkenylamino-derivatized base of a nucleoside/tide. More preferably, the resulting linkage is 3-(carboxy)amino-1-propyn-1-yl having the structure
NUC—C≡C—CH2OCH2CH2NR3X-D
where n ranges from 1 to 5,
R1 is selected from the group consisting of —H, lower alkyl and protecting group; and R3 is selected from the group consisting of —H and lower alkyl. See Khan et al., U.S. patent application Ser. No. 08/833,854 filed Apr. 10, 1997.
Particularly preferred nucleosides/tides of the present invention are shown below in Formula IV wherein
B is a nucleoside/tide base, e.g., uracil, cytosine, deazaadenine, or deazaguanosine; W1 and W2 taken separately are OH or a group capable of blocking polymerase-mediated template-directed polymerization, e.g., H, fluorine and the like; W3 is OH, or mono-, di- or triphosphate or phosphate analog; and D is a dye compound of Formula I. In one particularly preferred embodiment, the nucleotides of the present invention are dideoxynucleotide triphosphates having the structure shown in Formula IV.1 below, including associated counterions if present.
Labeled dideoxy nucleotides such as that shown in Formula IV.1 find particular application as chain terminating agents in Sanger-type DNA sequencing methods utilizing fluorescent detection.
In a second particularly preferred embodiment, the nucleotides of the present invention are deoxynucleotide triphosphates having the structure shown in Formula IV.2 below.
Labeled deoxynucleotides such as that shown in Formula IV.2 find particular application as reagents for labeling polymerase extension products, e.g., in the polymerase chain reaction or nick-translation.
1.5.2. Polynucleotide Reagents
Generally, if the labeled polynucleotide is made using enzymatic synthesis, the following procedure may be used. A template DNA is denatured and an oligonucleotide primer is annealed to the template DNA. A mixture of deoxynucleotide triphosphates is added to the mixture including dGTP, dATP, dCTP, and dTTP where at least a fraction of the deoxynucleotides is labeled with a dye compound of the invention as described above. Next, a polymerase enzyme is added under conditions where the polymerase enzyme is active. A labeled polynucleotide is formed by the incorporation of the labeled deoxynucleotides during polymerase-mediated strand synthesis. In an alternative enzymatic synthesis method, two primers are used instead of one, one primer complementary to the + strand and the other complementary to the − strand of the target, the polymerase is a thermostable polymerase, and the reaction temperature is cycled between a denaturation temperature and an extension temperature, thereby exponentially synthesizing a labeled complement to the target sequence by PCR, e.g., PCR Protocols, Innis et al. eds., Academic Press (1990).
The following briefly describes the steps of a typical polynucleotide synthesis cycle using the phosphoramidite method. First, a solid support including a protected nucleotide monomer is treated with acid, e.g., trichloroacetic acid, to remove a 5′-hydroxyl protecting group, freeing the hydroxyl for a subsequent coupling reaction. An activated intermediate is then formed by simultaneously adding a protected phosphoramidite nucleoside monomer and a weak acid, e.g., tetrazole, to the reaction. The weak acid protonates the nitrogen of the phosphoramidite forming a reactive intermediate. Nucleoside addition is complete within 30 s. Next, a capping step is performed which terminates any polynucleotide chains that did not undergo nucleoside addition. Capping is preferably done with acetic anhydride and 1-methylimidazole. The internucleotide linkage is then converted from the phosphite to the more stable phosphotriester by oxidation using iodine as the preferred oxidizing agent and water as the oxygen donor. After oxidation, the hydroxyl protecting group is removed with a protic acid, e.g., trichloroacetic acid or dichloroacetic acid, and the cycle is repeated until chain elongation is complete. After synthesis, the polynucleotide chain is cleaved from the support using a base, e.g., ammonium hydroxide or t-butyl amine. The cleavage reaction also removes any phosphate protecting groups, e.g., cyanoethyl. Finally, the protecting groups on the exocyclic amines of the bases and the hydroxyl protecting groups on the dyes are removed by treating the polynucleotide solution in base at an elevated temperature, e.g., 55� C.
Subsequent to synthesis, the polynucleotide may be labeled at a number of positions including the 5′-terminus, e.g., Oligonucleotides and Analogs, Eckstein ed., Chapter 8, IRL Press (1991) and Orgel et al., Nucleic Acids Research 11 (18): 6513 (1983); U.S. Pat. No. 5,118,800; the phosphodiester backbone, e.g., ibid., Chapter 9; or at the 3′-terminus, e.g., Nelson, Nucleic Acids Research 20(23): 6253-6259, and U.S. Pat. Nos. 5,401,837 and 5,141,813. For a through review of oligonucleotide labeling procedures see R. Haugland in Excited States of Biopolymers, Steiner ed., Plenum Press, NY (1983).
1.6. Methods Utilizing the Dibenzorhodamine Dyes
In another such fragment analysis method known as nick translation, a reaction is used to replace unlabeled nucleoside triphosphates in a double-stranded DNA molecule with labeled ones. Free 3′-hydroxyl groups are created within the unlabeled DNA by “nicks” caused by deoxyribonuclease I (DNAase I) treatment. DNA polymerase I then catalyzes the addition of a labeled nucleotide to the 3′-hydroxyl terminus of the nick. At the same time, the 5′ to 3′-exonuclease activity of this enzyme eliminates the nucleotide unit from the 5′-phosphoryl terminus of the nick. A new nucleotide with a free 3′-OH group is incorporated at the position of the original excised nucleotide, and the nick is shifted along by one nucleotide unit in the 3′ direction. This 3′ shift will result in the sequential addition of new labeled nucleotides to the DNA with the removal of existing unlabeled nucleotides. The nick-translated polynucleotide is then analyzed using a separation process, e.g., electrophoresis.
Sanger-type sequencing involves the synthesis of a DNA strand by a DNA polymerase in vitro using a single-stranded or double-stranded DNA template whose sequence is to be determined. Synthesis is initiated at a defined site based on where an oligonucleotide primer anneals to the template. The synthesis reaction is terminated by incorporation of a nucleotide analog that will not support continued DNA elongation. Exemplary chain-terminating nucleotide analogs include the 2′,3′-dideoxynucleoside 5′-triphosphates (ddNTPs) which lack the 3′-OH group necessary for 3′ to 5′ DNA chain elongation. When proper proportions of dNTPs (2′-deoxynucleoside 5′-triphosphates) and one of the four ddNTPs are used, enzyme-catalyzed polymerization will be terminated in a fraction of the population of chains at each site where the ddNTP is incorporated. If labeled primers or labeled ddNTPs are used for each reaction, the sequence information can be detected by fluorescence after separation by high-resolution electrophoresis. In the chain termination method, dyes of the invention can be attached to either sequencing primers or dideoxynucleotides. Dyes can be linked to a complementary functionality on the 5′-end of the primer, e.g. following the teaching in Fung et al, U.S. Pat. No. 4,757,141; on the base of a primer; or on the base of a dideoxynucleotide, e.g. via the alkynylamino linking groups disclosed by Hobbs et al, supra.
In each of the above fragment analysis methods labeled polynucleotides are preferably separated by electrophoretic procedures, e.g. Gould and Matthews, cited above; Rickwood and Hames, Eds., Gel Electrophoresis of Nucleic Acids: A Practical Approach, IRL Press Limited, London, 1981; Osterman, Methods of Protein and Nucleic Acid Research, Vol. 1 Springer-Verlag, Berlin, 1984; or U.S. Pat. Nos. 5,374,527, 5,624,800 and/or 5,552,028. Preferably the type of electrophoretic matrix is crosslinked or uncrosslinked polyacrylamide having a concentration (weight to volume) of between about 2-20 weight percent. More preferably, the polyacrylamide concentration is between about 4-8 percent. Preferably in the context of DNA sequencing in particular, the electrophoresis matrix includes a denaturing agent, e.g., urea, formamide, and the like. Detailed procedures for constructing such matrices are given by Maniatis et al., “Fractionation of Low Molecular Weight DNA and RNA in Polyacrylamide Gels Containing 98% Formamide or 7 M Urea,” in Methods in Enzymology, 65: 299-305 (1980); Maniatis et al., “Chain Length Determination of Small Double- and Single-Stranded DNA Molecules by Polyacrylamide Gel Electrophoresis,” Biochemistry, 14: 3787-3794 (1975); Maniatis et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, New York, pgs. 179-185 (1982); and ABI PRISM™ 377 DNA Sequencer User's Manual, Rev. A, January 1995, Chapter 2 (p/n 903433, The Perkin-Elmer Corporation, Foster City, Calif.). The optimal electrophoresis conditions, e.g., polymer concentration, pH, temperature, concentration of denaturing agent, employed in a particular separation depends on many factors, including the size range of the nucleic acids to be separated, their base compositions, whether they are single stranded or double stranded, and the nature of the classes for which information is sought by electrophoresis. Accordingly application of the invention may require standard preliminary testing to optimize conditions for particular separations.
1.7. Examples
1.8. Materials and Methods
All chemicals were purchased from Aldrich Chemical Company unless otherwise noted. Martius yellow was purchased from Fluka. Acetone was dried over CaSO4 and distilled. Dichloromethane (CH2Cl2) and nitrobenzene were dried over CaH2 and distilled. Tetrahydrofuran (THF) was dried over lithium aluminum hydride (LAH) and distilled as needed. Triethylamine (Et3N) was dried over sodium and distilled. DMSO (99.9%) and N,N-diisopropylethylamine (99.5%) were dried and stored over activated molecular sieves. Silica gel (220-400 mesh) from VWR was used for normal phase flash chromatography. Reverse phase chromatography was performed on octadecyl functionalized silica gel from Aldrich. Preparative thin layer chromatography (PTLC) was performed on 1 and 2 mm pre-made silica gel plates from EM science (VWR). TLC was performed on aluminum back silica gel 60 plates from EM science (VWR). Developed spots were visualized with both long and short wavelength UV irradiation.
Absorption spectroscopy was performed on a Hewlett Packard model 8451A UV/Vis diode array spectrophotometer. Fluorescence measurements were made on a Perkin-Elmer LS-50B luminescence spectrophotometer. NMR spectra were determined on a Varian 300 MHz NMR referenced relative to a solvent peak at 7.26 ppm in CD3Cl or 3.31 ppm in CD3OD. HPLC purification of oligomer labeled dye fragments was performed on a Perkin-Elmer Series 200 pump, employing a reverse phase C-18 column, with both UV and fluorescence emission detection. Fluorescence detection was performed by a Perkin-Elmer LC 2-40 fluorescence detector equipped with a red sensitive PMT, and UV detection was performed with a Model LC 295 UV/Vis detector. Pump and detectors were all interfaced with a Perkin-Elmer Model 1022 computer run in two-channel mode. Buffers were made up fresh from the following concentrated stock: 10�TBE (0.89 M tris-(hydroxymethyl)aminomethane, 0.89 M borate, 0.02 M ethylenediaminetetraacetic acid disodium salt), and 0.1 M TEAA (triethylamine acetate).
3-methoxy-1-hydroxy napthalene 1 (1 gm) was suspended in dry CH2Cl2 (30 mL). Dry triethylamine (1.2 equivalents) was added and the reaction was cooled to −5� C. Trifluoromethanesulfonic anhydride (1.1 equivalents) suspended in CH2Cl2 (15 mL) was added dropwise with vigorous stirring over a period of 2 hours. The reaction was allowed to come to room temperature and subjected to aqueous work up using 5% HCl and CH2Cl2. The resulting crude 3-methoxynapthalene-1-triflate 2 was purified by normal phase flash chromatography employing an EtOAc/Hexane (1:10) mobile phase.
The purified 3-methoxynapthalene-1-triflate 2 was converted to the 1-diethylamino-3-methoxynapthalene 3 using the palladium-catalyzed triflate/amine coupling procedure of Wolfe as follows (J. P. Wolfe and S. L. Buchwald, JOC, 61: 1133 (1996)). The 3-methoxy-napthalene-1-triflate 2 (1 gram) was suspended in 100 mL of dry toluene with 0.015 equivalents of (S)-(−)-2,2′-bis(diphenylphosphino)-1,1′-binapthyl (BINAP), 0.005 equivalents of tris(dibenzylideneacetone)dipalladium (Pd2(dba)3), and 3 equivalents of dry diethyl amine. The reaction was purged with argon, and 3.3 equivalents of solid sodium t-butoxide was added with stirring. The reaction was then heated, and stirred for 16 hours at 80� C. in an oil bath. The reaction was allowed to come to room temperature and subjected to aqueous work up using 5% HCl and CH2Cl2 resulting in a crude 1-diethylamino-3-methoxynapthalene 3, which was purified by normal phase flash chromatography employing EtOAc:hexane (1:49) as the mobile phase (1HNMR: CD3Cl d 8.20 (broad d, 1H, J=9 Hz), 7.72 (broad d, 1H, J=7.8 Hz), 7.43 (dt, 1H, J=7.2, 1.2 Hz), 7.34 (dt, 1H, J=7.7, 1.2 Hz), 6.88 (d, 1H, J=2.4 Hz), 6.82 (d, 1H, J=2.4 Hz), 3.93 (s, 3H), 3.21 (q, 4H, J=7.2 Hz), 1.08 (t, 6H, J=7.2 Hz)).
Next, the methyl group of the 1-diethyl-amino-3-methoxy-napthalene 3 was removed by boron tribromide deprotection as follows. The 1-amino-3-methoxy-napthalene (100 mg) was suspended in dry CH2Cl2 (5 mL) and the mixture was cooled to −70� C. in a dry ice/acetone bath. Boron tribromide (10 equivalents) was added dropwise and the reaction was stirred for 30 minutes, then placed in a refrigerator (0� C.) overnight. The reaction was quenched at −70� C. by careful addition of MeOH (10 mL). Solid NaHCO3 (30 equivalents) was added and the reaction was warmed to room temperature, then briefly heated to reflux. The mixture was cooled and filtered, the filtrate was acidified with AcOH, and the solvent was removed in vacuo to give the crude 1-diethylamino-3-hydroxynapthalene 4, which was purified by normal phase flash chromatography employing EtOAc:hexane (1:4) as the mobile phase.
The 1-anilino-3-methoxynapthalene 5 was acetylated by an amino group acetylation procedure as follows. The 1-amino-3-methoxynapthalene 5 (500 mg) and 1.2 equivalents of dry Et3N were suspended in 10 mL of dry CH2Cl2 and cooled to −5� C. using an ice/NaCl bath. 1.1 equivalent of 2-bromo-2-methylpropionylchloride was added dropwise and the reaction was stirred for 1 hour at −5� C. and stirred at room temperature for an additional 1 hour. The reaction was allowed to come to room temperature and subjected to aqueous work up using 5% HCl and EtOAc resulting in the crude intermediate 1-(bromoalkyl)amido-3-methoxy-napthalene 6, which was purified by normal phase flash chromatography employing EtOAc:hexane (1:9) as the mobile phase.
The 1-(bromoalkyl)amido-3-methoxy-napthalene 6 was cyclized using an AlCl3 catalyzed Friedel-Crafts cyclization procedure as follows. 1 to 3 equivalents of AlCl3 in nitrobenzene was added to the 1-(bromoalkyl)amido-3-hydroxy-napthalene 6. The reaction was heated to 130� C. and reacted for 1 hour. Aqueous work-up using NH4Cl and EtOAc gave the crude N-phenyl-benzoindolinone intermediate 7, which was purified by normal phase flash chromatography employing EtOAc:hexane (1:4) as the mobile phase. The amide carbonyl group of the N-phenyl-benzoindoline intermediate 7 was then reduced with LAH to give compound 8 (1HNMR: CD3Cl d 7.71 (d, 1H, J=7.8 Hz), 7.32 (m, 2H), 7.24 (m, 2H), 7.07 (bt, 1H, J=6.6 Hz), 6.96 (m, 3H), 6.84 (s, 1H), 3.97 (s, 3H), 3.92 (s, 2H), 1.44 (s, 6H).
Compound 10 was synthesized by condensation of methoxy-napthaldehyde and malonic acid employing piperidine catalysis in pyridine. Compound 10 was reduced with hydrogen over 10% Pd/carbon, followed by LAH reduction, and reacted as outlined for the synthesis of compound 2 above with trifluoromethanesulfonic anhydride to give the triflate 11. Triflate 11 was then reacted with NaN3 (3 equiv.) in DMF at 100� C. for 6 hours. Then, the reaction was allowed to come to room temperature and subjected to aqueous work up using pure water and EtOAc resulting in pure compound 12. Compound 12 was suspended in dry CH2Cl2, complexed with 3 to 5 equivalents of solid AlCl3, and refluxed for 2 hours yielding compound 13.
Compound 13 was alkylated with MeI according to a general amino group alkylation procedure as follows. The 3-methoxybenzoquinoline derivative (100 mg) 13 was suspended in 5 mL of dry THF and cooled to −5� C. (ice/NaCl). 1.1 equivalents of n-butyl lithium (1 M) was added dropwise, and the reaction was stirred for 1 hour. 3 equivalents of the MeI alkylating agent was added slowly and the reaction was allowed to stir at room temperature for 2 hours. Aqueous work-up using NH4Cl and EtOAc gave a crude alkylated 3-methoxybenzoquinoline intermediate 14. Intermediate 14 was then purified by normal phase flash chromatography employing EtOAc:hexane (1:19) as the mobile phase (1HNMR: CD3Cl d 8.1 (broad d, 1H, J=8.1 Hz), 7.68 (dd, 1 H, J=8.1, 1.8 Hz), 7.34 (m, 2H), 6.8 (s, 1H), 3.92 (s, 3H), 3.21 (m, 2H), 2.94 (s, 3H), 2.77 (t, 2H, J=6.6 Hz), 1.92 (m, 2H)). Subsequent methoxy group deprotection by the general boron tribromide procedure described above in Example 1 resulted in the N-methyl-hydroxybenzoquinoline derivative 15.
Compound 13 was synthesized according to the procedure outlined above in Example 3 for the synthesis of the N-methyl-hydroxybenzoquinoline derivative 15. Compound 13 was then alkylated according to the general amino group alkylation procedure described above in Example 3, this time using 1,3-propane sultone as the alkylating agent rather than MeI, to give a 5-methoxybenzoquinoline-N-propanesulfonic acid intermediate 16 (1HNMR: CD3OD d 7.94 (d, 1 H, J=8.7 Hz), 7.65 (d, 1H, J=8.4 Hz), 7.32 (t, 1H), 7.27 (t, 1H), 6.85 (s, 1H), 4.89 (s, 3H), 3.20 (m, 2H), 3.08 (bt, 2H, J=6 Hz), 2.91 (m, 2H), 2.72 (t, 2H, J=6.6 Hz), 2.33 (m, 2H), 1.89 (m, 2H). Subsequent methoxy group deprotection of compound 16 by the general boron tribromide procedure described above in Example 1 resulted in the 3-(5-hydroxybenzoquinolin-1-yl) propanesulfonic acid 17.
Following the procedure of A. Rosowsky and E. J. Modest (J.O.C., 30: 1832 (1965)), 1-amino-3-methoxynapthalene 18 (1 gm) was dissolved in dry acetone (50 mL), and 0.01 equivalent of iodine was added to the solution. The reaction was heated and stirred for 16 hours, cooled, and then quenched with saturated Na2S2O3. The reaction mixture was then subjected to aqueous work up using saturated Na2S2O3 and EtOAc resulting in the crude methoxybenzoquinoline 19. The methoxybenzoquinoline 19 was purified by flash chromatography using an EtOAc/hexane 1:9 mobile phase. Compound 19 was then alkylated with MeI according to the general amino group alkylation procedure described above in Example 3 to give compound 20. Compound 20 was reduced with H2 in a Parr hydrogenator at 70 psi and 10% Pd/C catalysis to give a N-methyl-2,2,4-trimethyl-5-methoxybenzoquinoline intermediate 21 (1HNMR: CD3Cl d 8.20 (bd, 1H, J=7.5 Hz), 7.65 (bd, 1H, J=7.5 Hz), 7.33 (m, 2H), 6.89 (s, 1 H), 3.94 (s, 3H), 3.14 (b sextet, 1H, J=6.6 Hz), 2.80 (3, 3H), 1.89 (d, 2H, J=8.7), 1.42 (d, 3 H, J=6.9 Hz), 1.34 (s, 3H), 1.05 (s, 3H). Subsequent methoxy group deprotection of compound 21 by the general boron tribromide procedure described above in Example 1 gave the N-methyl-5-hydroxy-(tetrahydro)benzoquinoline 22.
1-Amino-3-methoxynapthalene 18 was acetylated with 2-bromo-2-methylpropionyl chloride according to the general amino group acylation procedure described above in Example 2 to give compound 23. Compound 23 was cyclized by the Friedel-Crafts cyclization procedure described above in Example 2 to give compound 24. Next, compound 24 was reduced with 3 equivalents LAH in THF to give the 4-methoxybenzoindoline 25. Compound 25 was alkylated using the general amino group alkylation procedure described above in Example 3 using methyl iodide as the alkylating agent to give a N-methyl-3,3-dimethyl-4-methoxybenzoindoline intermediate 26 (1HNMR: CD3Cl d 8.07 (bd, 1H, J=8.4 Hz), 7.69 (bd, 1H, J=8.1 Hz), 7.33 (bt, 1H, J=7.8 Hz), 7.22 (bt, 1H, J=8.1 Hz), 6.70 (s, 1H), 3.92 (s, 3H), 3.32 (s, 2H), 3.32 (s, 3H), 1.44 (s, 6H). Subsequent methoxy group deprotection of compound 26 by the general boron tribromide procedure described in Example 1 resulted in the N-methyl-3,3-dimethyl-4-hydroxy-benzoindoline 27.
The 4-methoxybenzoindoline 25 was synthesized as described above in Example 6. Compound 25 was alkylated by the general amino group alkylation procedure described in Example 3 employing ethyl iodide as the alkylating agent to give the N-ethyl-3,3-dimethyl-4-methoxybenzoindoline intermediate 28 (1HNMR: CD3Cl d 7.90 (d, 1H, J=8.7 Hz), 7.68 (d, 1H, J=8.1 Hz), 7.32 (bt, 1H, J=7.5 Hz), 7.22 (bt, 1H, J=6.9 Hz), 6.69 (s, 1H), 3.83 (s, 3H), 3.52 (q, 2H J=7.5 Hz), 3.38 (s, 2H), 1.46 (s, 6H), 1.27 (t, 3H, J=7.5 Hz). Subsequent methoxy group deprotection of compound 28 by the general boron tribromide procedure described in Example 1 yielded the N-ethyl-3,3-dimethyl-4-hydroxy-benzoindoline 29.
General Procedure A (FIG. 5). A solid phthalic anhydride derivative 34 was mixed with 1.4 equivalents of an aminohydroxy intermediate 31 and 2.8 equivalents of ZnCl2. The oven dried reaction vessel was capped with a rubber septa and purged with Argon. The solid mixture was heated briefly at 130� C. until melting was observed, e.g., after approximately 15 minutes. 1,2-Dichlorobenzene (approximately 10 equivalents) was added by syringe to the reaction mixture, and the heterogeneous mixture was heated to 130� C. to 170� C. for 4 hours. The crude reaction mixture was cooled, suspended in a minimal amount of MeOH: CH2Cl2 (1:19), loaded directly onto a normal phase flash chromatography column, and the crude dye was eluted with an MeOH: CH2Cl2 (1:19) mobile phase. When necessary, the dye was purified and separated into distinct isomers 35 and 36 by PTLC developed with MeOH: CH2Cl2 (1:9). The isomerically pure dye, which migrated as a single spot on silica TLC eluting with 1:9 MeOH:CH2Cl2, was identified by its UV/Visible absorption spectra and its long wavelength fluorescent excitation and emission spectra.
Synthesis of Dibenzorhodamine Dye 41 (FIG. 7). General procedure A was followed employing dichlorotrimellitic anhydride as the phthalic anhydride derivative, i.e., compound 34 where the substituents at C-14 and C-17 are Cl and the substituent at C-15 is CO2H, and 1-diethylamino-3-hydroxynapthalene 4 as the aminohydroxy intermediate 31.
Synthesis of Dibenzorhodamine Dye 45 (FIG. 7). General procedure A was followed employing dichlorotrimellitic anhydride as the phthalic anhydride derivative, i.e., compound 34 where the substituents at C-14 and C-17 are Cl and the substituent at C-15 is CO2H, and N-methyl-3,3-dimethyl-4-hydroxy-benzoindoline 27 as the aminohydroxy intermediate 31.
The following table presents important spectral properties of several representative dibenzorhodamine dye compounds of the invention. All spectra were recorded at room temperature, in 1�TBE buffer and 8 M urea, for the free dye having 0.05 absorption at the dye's λmax, abs. Dye concentration was approximately 10−6 M.
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ClassificationC07H19/20, C09B11/28, C09K11/06, C12N15/09, C07H21/00, C07H19/14, C07D487/00, C07H19/16, C07D491/14, C07D221/10, C07C215/86, C07H19/10, C07D311/78, C12Q1/68, C09B11/02, C07D491/147, G01N21/78, C07D209/60, C09B11/24, C07H19/06Cooperative ClassificationC07D311/78, C12Q1/6869, C07H21/04, C07D209/60, C09B11/24, C09B11/02, C07H19/20, C07H19/10, Y10T436/143333, C07H21/00, C07D491/14, C07D221/10, C07D491/147European ClassificationC07D221/10, C07D491/14, C07D311/78, C09B11/02, C07D209/60, C09B11/24, C07H19/20, C07H21/00, C07H19/10, C12Q1/68E, C07H21/04, C07D491/147Legal EventsDateCodeEventDescriptionMay 16, 2014FPAYFee paymentYear of fee payment: 4Apr 9, 2013ASAssignmentOwner name: APPLIED BIOSYSTEMS, INC., CALIFORNIAFree format text: LIEN RELEASE;ASSIGNOR:BANK OF AMERICA, N.A.;REEL/FRAME:030182/0677Effective date: 20100528Feb 26, 2010ASAssignmentFree format text: MERGER;ASSIGNOR:APPLIED BIOSYSTEMS INC.;US-ASSIGNMENT DATABASE UPDATED:20100316;REEL/FRAME:23985/801Free format text: 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