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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, 1997Also published asCA2311476A1, CA2311476C, EP1034221A1, 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 (49), Legal Events (5) External Links: USPTO, USPTO Assignment, EspacenetMethods of labelling polynucleotides with dibenzorhodamine dyesUS 7833756 B2Abstract Dibenzorhodamine compounds 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.
Z1 is selected from the group consisting of �NH, sulfur and oxygen;
Z2 is selected from the group consisting of �NH, sulfur and oxygen; and
1. CROSS REFERENCE TO RELATED APPLICATIONS 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.
2. FIELD OF THE INVENTION This invention relates generally to fluorescent dye compounds. More specifically, this invention relates to modified rhodamine dyes useful as fluorescent labeling reagents.
3. BACKGROUND The non-radioactive detection of biological analytes utilizing fluorescent labels is an important technology in modern molecular biology. By eliminating the need for radioactive labels, safety is enhanced and the environmental impact and costs associated with reagent disposal is greatly reduced. Examples of methods utilizing such non-radioactive fluorescent detection include 4-color automated DNA sequencing, oligonucleotide hybridization methods, detection of polymerase-chain-reaction products, immunoassays, and the like.
4. SUMMARY The present invention is directed towards our discovery of a class of dibenzorhodamine dye compounds suitable for the creation of sets of spectrally-resolvable fluorescent labels useful for multi-color fluorescent detection. The subject dye compounds are particularly well suited for use in automated 4-color DNA sequencing systems using an excitation light source having a wavelength greater than about 630 nm, e.g., a helium-neon gas laser or a solid state diode laser.
5. BRIEF DESCRIPTION OF THE FIGURES FIGS. 1-3 show exemplary synthetic pathways for the synthesis of the 1-amino-3-hydroxynapthalene intermediates of the invention.
6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Reference will now be made in detail to the preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the preferred embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims.
1.1. DEFINITIONS Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:
�Rhodamine dye� refers to dyes including the general polycyclic structure
1.2. 1-Amino-3-Hydroxynapthalene Intermediates 1.2.1. Structure
1.3. Dibenzorhodamine Dye Compounds 1.3.1. 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.
1.4. Energy Transfer Dyes Incorporating the Dibenzorhodamine Dyes In another aspect, the present invention comprises energy transfer dye compounds incorporating the dibenzorhodamine dye compounds of Formula I. Generally, the energy transfer dyes of the present invention include a donor dye which absorbs light at a first wavelength and emits excitation energy in response, an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, and a linker which attaches the donor dye to the acceptor dye, the linker being effective to facilitate efficient energy transfer between the donor and acceptor dyes. A through discussion of the structure, synthesis and use of such energy transfer dyes is provided by Lee et al., U.S. patent application Ser. No. 08/726,462, and Mathies et al., U.S. Pat. No. 5,654,419.
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.
NUC�C≡C�CH2OCH2CH2NR3X-D
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.
1.6. Methods Utilizing the Dibenzorhodamine Dyes The dyes and reagents of the present invention are well suited to any method utilizing fluorescent detection, particularly methods requiring the simultaneous detection of multiple spatially-overlapping analytes. Dyes and reagents of the invention are particularly well suited for identifying classes of polynucleotides that have been subjected to a biochemical separation procedure, such as electrophoresis, or that have been distributed among locations in a spatially-addressable hybridization array.
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 The invention will be further clarified by a consideration of the following examples, which are intended to be purely exemplary of the invention and not to in any way limit its scope.
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.
Example 4 Synthesis of 3-(5-Hydroxybenzoquinolin-1-yl) propanesulfonic acid 17 (FIG. 2) 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.
Example 5 Synthesis of N-Methyl-2,2,4-Trimethyl-5-Hydroxy-(Tetrahydro)benzoquinoline 22 (FIG. 3) 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.
Example 6 Synthesis of N-Methyl-3,3-Dimethyl-4-Hydroxy-Benzoindoline 27 (FIG. 3) 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.
Example 7 Synthesis of N-Ethyl-3,3-Dimethyl-4-Hydroxy-Benzoindoline 29 (FIG. 3) 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.
Example 8 Synthesis of Selected Dibenzorhodamine Dye Compounds 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.
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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 ClassificationC09B11/24, C07D221/10, C07D311/78, C07D491/147, C07H19/10, C09B11/02, C07H21/04, C07H21/00, C07D491/14, C12Q1/6869, C07H19/20, C07D209/60European ClassificationC07D221/10, C07D491/14, C07D311/78, C09B11/02, C07D209/60, C09B11/24, C07H19/20, C07H21/00, C07H19/10, C12Q1/68E, C07H21/04, C07D491/147Legal EventsDateCodeEventDescriptionApr 9, 2013ASAssignmentEffective date: 20100528Owner name: APPLIED BIOSYSTEMS, INC., CALIFORNIAFree format text: LIEN RELEASE;ASSIGNOR:BANK OF AMERICA, N.A.;REEL/FRAME:030182/0677Feb 26, 2010ASAssignmentOwner name: APPLIED BIOSYSTEMS, LLC,CALIFORNIAFree format text: MERGER;ASSIGNOR:APPLIED BIOSYSTEMS INC.;US-ASSIGNMENT DATABASE UPDATED:20100226;REEL/FRAME:23985/801Effective date: 20081121Free format 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