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WO2002055512A1 - Derives de xanthene - Google Patents

Derives de xanthene Download PDF

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Publication number
WO2002055512A1
WO2002055512A1 PCT/US2002/000801 US0200801W WO02055512A1 WO 2002055512 A1 WO2002055512 A1 WO 2002055512A1 US 0200801 W US0200801 W US 0200801W WO 02055512 A1 WO02055512 A1 WO 02055512A1
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Prior art keywords
xanthene compounds
amine
dyes
fluorescein
ester
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Ceased
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PCT/US2002/000801
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English (en)
Inventor
Jianxin Gao
Roger W. Giese
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Northeastern University China
Northeastern University Boston
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Northeastern University China
Northeastern University Boston
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Priority to US10/250,975 priority Critical patent/US20040054195A1/en
Publication of WO2002055512A1 publication Critical patent/WO2002055512A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/10—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/10—Amino derivatives of triarylmethanes
    • C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes

Definitions

  • the field of the invention is organic synthesis of xanthene derivatives for the purpose of yielding improved fluorescent dyes or dye precursors for use in biological and chemical analysis, and in other areas.
  • Fluorescent xanthene derivatives such as fluorescein are widely employed in biological and chemical analysis as labels and staining .reagents to enhance sensitivity and specificity of detection.
  • their usefulness is often limited by their photoinstability: degradation takes ' place rapidly upon exposure to light, resulting in loss of fluorescence.
  • Such photobleaching has been, reduced in some cases by adding an antioxidant to a sample, but the usefulness of antioxidants can depend on the sample and assay conditions and can lead to other complications .
  • xanthene derivatives are employed as fluorescent dyes.
  • General and specific examples are fluoresceins, rhodamines, Ale ' xa dyes, naphthofluoresceins, Oregon Greens, eosins, erythrosins, 6-carboxyfluorescein, 5-carboxyhexachlorofluorescein, tetramethyl-6-carboxyrhodamine, and Rose Bengal.
  • xanthamides Secondary amide xanthene derivatives, termed “xanthamides,” are disclosed, along with “3-bulky- xanthenes” and, in their fluorescent dye forms, they have or can have much higher photostability than related dyes such as fluorescein and BODIPY. Examples are presented in which the synthesis begins with 5- carboxyfluorescein, an inexpensive reagent. Related carboxyxanthines employed in other dyes such as rhodamines can also be used as starting materials. Simple synthetic steps yield compounds which exemplify this invention. Other notable properties of the xanthamides that are fluorescent dyes include the option of pH-independent fluorescence, and the ease of controlling their functional and other groups.
  • xanthamide dyes can be prepared with a broad variation of physicochemical properties to enhance the usefulness of fluorescence in biological and chemical analysis, and in other areas. Also disclosed are precursors to xanthamide dyes which are useful for detecting or quenching reactive oxygen or free radical species .
  • Curve A BODIPY FL in pH 10 buffer, 50 nM, excited at 505 rim, excitation and emission slit 2.5 nm
  • Curve B fluorescein in pH 10 borate buffer, 50 nM, excited at 488 nm, excitation and emission slit 2.5 nm
  • Curve C compound 8 in pH 10 borate buffer, 50 nM, excited at 459 nm, excitation slit 2.5 nm, emission slit 8 nm
  • Curve D compound 8 in methanol, 50 nM, excited at 459 nm, excitation slit 2.5 nm, emission slit 8 nm
  • Inset is UV spectrum of 8 in pH 10 borate buffer, 10 ⁇ M, peak absorption wavelength: 459 nm and 483 nm.
  • Figure 7 A. Core structural component for starting materials.
  • R13 is a C-attached secondary amide, including piperidinamide, substituted piperidinamide, piperazinaird.de, substituted piperazinamide, which may be substituted with a diversity of chemical groups such as amino, hydroxy, amide, alkenyl, alkynyl, heteroaryl, alkylthio, aryl, alkyl, ether, dialkylamine, alkylarylamine, cycloalkyl or cycloalkyl amine, ester, active ester, carboxyl, quaternary amine, phosphonium, sulfonate,
  • R 3 and R 6 are selected from the substitutents
  • substituents may be substituted with additional groups comprising (this term includes the cases where the substituent is adjacent to, or remote from, the parent substituent) substituents such as carboxyl, amine, secondary amine, tertiary amine, quaternary amine, phosphonium or sulfonate.
  • R' 6 is selected from 0 or NQ ⁇ Q 2 where Qi and Q 2 are selected from H, alkyl, alkenyl, alknyl, cycloalkyl or aryl, or Q ⁇ Q 2 may constitute a cycloalkyl or cycloalkenyl group.
  • substituents aside from 0 may be substituted with additional groups comprising substituents such as carboxyl, amine, secondary amine, tertiary amine, quaternary amine, phosphonium or sulfonate.
  • RiO Rii r R12 is selected from the substitutents hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, arylalkyl, acyl, sulfo, amino, alkylamine, dialkylamine, cycloalkylamine, isothiocyanate, carboxyl, amide, ester, active ester, ether, fused aryl or alkythio.
  • the R groups can be further functionalized with a chemically reactive group selected from the substitutents carboxyl, active ester, hydroxyl, amine, haloalkyl, sulfhydryl, anhydride, acylhalide, imidazole, maleimide, isothiocyanate, aldehyde, hydrazide, phenol, sulfonylhalide, hydrazine or oxyamine. In this way a reactive site is established in the compound.
  • this reactive site is set up at the 3' or 3 position.
  • Primary to quaternary amine groups are amino, alkyl-or aryl-amino, dialkylamino, alkylarylamino, trialkylamino and tetraalkylamino.
  • General examples of phosphonium groups are trialkylphosphonium and tri-arylphosphonium.
  • the sulfonates may be alkyl- or aryl-sulfonates.
  • the counterions for the primary to tertiary amines in a protonated (cationic) form, or for the quaternary amines or the phosphonium groups may be selected broadly including halides, nitrate, bisulfate, tosylate, perchlorate, and bicarbonate.
  • the counterions for the sulfonate group may be selected broadly including alkali metal ions such as sodium ion, protonated tertiary amines, and quaternary amines.
  • xanthamide dyes (3-8) were prepared as shown in Figure 2 by means of a synthetic scheme that begins with fluorescein. As seen, the synthetic scheme enables a functional carboxyl group to be placed either at the 3 ' position (compound 5) or the 3 position (compound 8) of a xanthamide dye, thus making these positions the reactive site of the dye. (Note, in compound 8, the carboxy group is a part of the pi ⁇ eridine-4-carboxylic (isonipecotic) acid moiety that is at the 3- position of the dye.) Similar reactions can be employed to place this functional group at neither or both of these positions, or to similarly install a diversity of other functional or nonfunctional groups at these two, readily-accessible positions.
  • Carboxyxanthamide dye analogs of fluorescein such as rhodamines, naphthofluoresceins and compounds comprising the generalized structure shown in Fig. 7A also can be subjected to the reactions of this scheme, or to related reactions that produce secondary amides analogous to those shown in Figure 2. It is preferred to subject such carboxyxanthenes first to reactions a and e or a and b in this scheme, or to more generic versions of these reactions in which the carboxyxanthene dye is first converted into an active ester, and then this active ester in turn is reacted with a secondary amine.
  • reaction a fluorescein was reacted with dicyclohexyl-carbodiimide and N-hydroxysuccinimide (NHS) to form the NHS ester, 2.
  • Compound 2 was converted into 3 by reaction with dimethyl amine hydrochloride in the presence of triethylamine (reaction b) , and also into 6 by reaction with isonipecotic acid in the presence of diisopropylethylamine (reaction e) .
  • reaction e diisopropylethylamine
  • Compound 4 was obtained by reacting 3 with methylbromoacetic acid in the presence of potassium carbonate (reaction c) , and 4 in turn was converted into 5 by alkaline hydrolysis
  • reaction f reaction f
  • reaction g alkaline hydrolysis
  • Compound 10 a nonfluorescent chemical, is important as both a free radical scavenger, and as an indicator or quencher of reactive oxygen species such as hydroxyl radicals since 10 can form the fluorescent product 3 when exposed to such species.
  • Reactive oxygen species are of great interest in the biomedical field since they are important in both normal metabolism and also some disease processes.
  • 8 has absorbance maxima especially at 459 and 483 nm, which means that its fluorescence can be stimulated by using the same lasers that are commonly employed in tests with fluorescein and BODIPY dyes. This adds to the practicality of 8.
  • xanthamide dyes can provide pH-independent fluorescence. It is usually a nuisance when the fluorescence of a dye varies with the pH, so this property of xanthamide dyes adds to their usefulness.
  • an inert alkyl group such as methyl at the 3'-0 position, creating an ether, yields a moderately polar, nonionic xanthenyl moiety, as in 8, which is anticipated to have minimal nonspecific binding interactions with many macromolecules .
  • This enables the binding of a ligand molecule which is attached via the secondary amide group of 8 to control the interaction of this ligand with its binding partner, such as a receptor or antibody, without interference from the other part of the dye.
  • Xanthamide dyes also can be prepared with pH- dependent fluorescence since this property sometimes is useful.
  • Fluorescent dyes are widely employed in studies of chemical and biological systems including microscopic and visual imaging of cellular and tissue samples. They are also employed in other areas such as fabrics, safety, visual displays, lasers and communications. For example, blue fluorescent dyes are employed as fabric brighteners . Fluorescent dyes are used to make clothing, vehicle and road markers more visible at night for safety or other purposes. Increasingly fluorescent dyes are being employed as substitutes for radioisotopes in chemical and biological analysis because of the cost and safety problems of the latter substances. However, the dyes available currently for these purposes have some shortcomings, depending on the application. For example, viewing time for imaging applications is limited since fluorescent dyes necessarily are destroyed (bleached) during the viewing process.
  • our invention impacts on xanthene-based dyes not only by conveniently enhancing the photostability of existing dyes, but also by extending their structural properties in a novel way.
  • the central carboxyl group at the 3 position of these dyes is considered to be a relatively useless or inaccessible feature, and functional groups are placed elsewhere in the molecule such as on the 5 or 6 positions of this same ring.
  • our invention converts the 3 position into a functional site so that xanthamide dyes with quite different structural features for this ring can be attached in a new way to other substances of interest.
  • This new orientation and surface properties of xanthene- based dyes provides additional control over their physicochemical properties which may be useful, for example; to reduce nonspecific binding or fluorescence quenching problems; to enhance reaction coupling yields; to improve fluorescence energy transfer processes, and to modify solubility properties. Further, as demonstrated by the preparation of xanthamide dyes 5 and 8, the availability of two sites where functional groups can be installed is especially useful for varying the physicochemical properties, while maintaining high photostability.
  • one site can be used to install a reactivity group so that the dye can be attached covalently to a substance of interest, while the other site can be used to incorporate a diversity of chemical groups ranging from polar to nonpolar, bulky to nonbulky, and nonionic to ionic where the latter includes both anionic and cationic groups including quaternary amines, alkyl-or aryl-substituted phosphonium groups, and sulfonates.
  • Xanthamide dyes can have a reactive group at two sites, forming a cross-linking, dendrimer - forming, combinatorial scaffold, or polymer forming reagent, including cases where two, different reactive groups are selected.
  • xanthamides in a dye form are used to enhance the sensitivity of detecting a substance not only by fluorescence but also by mass spectrometry. It is well known that the presence of cationic groups such as protonated tertiary amines or quaternary amines, or anionic groups such as sulfonates on a substance can enhance its response by electrospray mass spectrometry or matrix-assisted laser desorption ionization mass spectrometry, for example. Our xanthamide dyes can easily be prepared with one or more of these polar groups without compromising the dye properties.
  • an ionic dye of this type for example, a dye containing the core structural component shown in Figure 7B, where R is a C-attached secondary amide, and one or more ionic groups are present
  • the labeled substance then can be detected with high sensitivity by both fluorescence and mass spectrometry. This can enhance not only method development, but help in the quality control of an analytical method.
  • This invention also applies to placing other bulky groups at the 3 position of fluorescein, or the corresponding position of fluorescein analogs, especially to enhance photostability in the same way that a secondary amide there enhances this property.
  • These other compounds are termed X ⁇ 3-bulkyxanthenes" , and these other bulky groups include N-attached secondary amide, ether, alkyl, cycloalkyl, cycloalkenyl, alkenyl, alkynyl, aryl, heteroaryl, alkylamine, dialkylamine, alkylarylamine, cycloalkylamine, thioether, sulfonamide, and fused aryl.
  • fluorescent xanthene compounds by activating the carboxyl group at the 3 position of fluorescein, or the corresponding position of a fluorescein derivative, to an active ester, and reacting the resulting active ester with a secondary amide to form a secondary amide fluorescein or secondary amide fluorescein derivative.
  • the following examples are intended to further illustrate, and not limit, the invention .
  • Example 1 Preparation of 2- (6-Hydroxy-3-oxo-3#-xanthen- 9-yl) -benzoic acid 2, 5-dioxo-pyrrolidin-l-yl ester, 2. Fluorescein 1 (3.32 g, 10 mmol, unpurified commercial grade), N-hydroxysuccinmide (1.17g, 10 mmol), and dicyclohexylcarbodiimide (2.10 g, 10 mmol) in 15 ml dry DMF were heated to 70°C ⁇ 80°C under nitrogen for one hour. Upon cooling with ice-bath, dicyclohexylurea was filtered off.
  • Example 3 Preparation of [9- (2-Dimethylcarbamoyl- phenyl) -6-oxo-6H-xanthen-3-yloxy] -acetic acid methyl ester, 4. 3 (72 mg, 0.2 mmol), methyl b omoacetate (95 ⁇ l, lmmol) and potassium carbonate (138 mg, lmmol) in 2.5 ml DMF were heated to 70°C for two hours. Upon cooling, the reaction mixture was isolated by flash chromatography using ethyl acetate/methanol (80/20, v/v) . The yield was 73 mg (85%) of 4.
  • Example 4 Preparation of [9- (2-Dimethylcarbamoyl- phenyl) -6-oxo-627-xanthen-3-yloxy] -acetic acid, 5. 4 (35 mg, 0.008 mmol) was dissolved in 3 ml methanol and 1 ml 1 N sodium hydroxide and stirred half-hour at room temperature. The mixture was neutralized with 1 N HC1, concentrated and isolated by flash chromatography using ethyl acetate/methanol (50/50, v/v) . The yield was 20 mg (59%) of 5.
  • Example 6 Preparation of 1- [2- (6-Methoxy-3-oxo-3i ⁇ - xanthen-9-yl) -benzoyl] -piperidine-4-carboxylic acid methyl ester, 7. 6 (90 mg, 0.2 mmol) in 5 ml DMF, potassium carbonate (278 mg, 2 mmol) and 1ml methyl iodide were mixed and heated to 60°C for 30 minutes under nitrogen. Upon cooling, the mixture was subject to flash chromatography using ethyl acetate/methanol (80/20, v/v) . The yield was 44 mg (47%) of 7.
  • Example 7 Preparation of 1- [2- (6-Methoxy-3-oxo-3iJ- xanthen-9-yl) -benzoyl] - piperidihe-4-carboxylic acid, 8. 7 (24 mg, 0.05 mmol) was dissolved in 1 ml methanol and 1 ml water. 0.4 ml IN sodium hydroxide was added. The mixture was stirred at room temperature for two hours and neutralized with IN HCl. The reaction mixture was subject to flash chromatography using ethyl acetate/methanol/acetic acid (40/59/1, v/v) . 10 mg (43%) of 8 was obtained.
  • Compound 8 was also synthesized as shown in Figure 8.
  • Compound 2 was reacted with isonipecotic acid tert- butyl ester 15 in the presence of three equivalents of triethylamine in dimethylformamide (reaction k) , yielding compound 11, which in turn was reacted with methyl iodide/potassium carbonate in dimethylformamide at 60 degree (C) (reaction 1) to form compound 12.
  • the tert- butyl group on 12 was removed with trifluoroacetic acid to form compound 8 (reaction m) .
  • Example 8 Preparation of 2- (3, 6-Dihydroxy-9H-xanthen- 9yl)-benzoic acid, 9. Fluorescein (700 mg, 2 mmol, unpurified commercial grade) , sodium hydroxide (480 mg, 12 mmol) and zinc (650 mg, 10 mmol) in 20 ml water were refluxed one hour. Upon cooling zinc was filtered off. The solution was acidified by IN HCl amd extracted with ethyl ether. The organic phase was washed by water and saturated brine and dried over anhydrous sodium sulfate, evaporated to a yellow solid, gave 630 mg (94%) of 9.
  • Example 9 Preparation of 2- (3, 6-Dihydroxy-9H-xanthen-9- yl) -N,N-dimethyl-benzamide, 10. 2 (2.73 g, 8.15 mmol), N-hydroxysuccinimide (1.06g, 9 mmol), and dicyclohexylcarbodiimide (1.68 g, 8.15 mmol) in 20 ml acetonitrile and 5 ml tetrahydrofuran were stirred one hour at 0°C (ice bath) and overnight at room temperature. The precipitated dicyclohexylurea was filtered off with suction.
  • Example 10 Conversion of 10 to 3. 10 (650 mg, 1.5 mmol) was dissolved in 4 ml methyl sulfoxide. Two ml of Jones reagent (prepared by combining 2.57 g of chromium oxide, 7.5 ml of water and 0.9 ml of sulfuric acid) was added drop by drop in half-hour at room temperature and continued to ' stir half-hour. The reaction was quenched by adding 10 ml water. The red precipitate was filtered off and isolated by flash chromatography using ethyl acetate/methanol (80/20, v/v) . The yield was 371 mg (57%) of 3. *H NMR (CD 3 OD, ppm): 7.76-7.68 (m, 2H) , 7.67-
  • a cationic xanthamide dye can be prepared by reacting compound 2 sequentially with isonipecotic acid t-butyl ester, bromoacetic acid methyl ester, sodium hydroxide (to convert the latter ester to a carboxylic acid), dicyclohexylcarbodiimide, Girard' s Reagent T or D, and trifluoroacetic acid.
  • a xanthamide dye bearing a reactive amino functional group can be prepared by reacting compound 8 with dicyclohexycarbodiimide in the presence of N- hydroxysuccinimide ester followed by addition of ethylenediamine.
  • a rhoda ine xanthamide dye can be prepared by reacting rhodamine B (Aldrich, R95-3) sequentially with a carbodiimide, N-hydroxysuccinimide ester, and isonipecotic acid.
  • Example 14 A very long-wavelength excitation and emission xanthamide dye can be prepared by subjecting naphthofluorescein sequentially to reactions a and e of Figure 2.
  • Example 15 The photostability study was conducted by irradiating a 50 nM pH 10 sample solution of each dye (1, BODIPY-FL, 5 and 8) using a Hotspot lamp (Cheltenham, PA) with a 200W soft white bulb (General Electric, Canada) , positioned at a distance of 10 cm between the sample and the bulb. The sample solution was cooled by circulating room temperature water. Fluorescence spectra were recorded on aliquots taken as a function of time.
  • a Hotspot lamp Cheltenham, PA
  • 200W soft white bulb General Electric, Canada
  • Example 16 The synthesis of compound 15 is shown in Figure 9. Isonipecotic acid was reacted with three equivalents of trifluoroacetic acid anhydride (reaction n) to form compound 13, which was reacted with isobutylene in the presence of a catalytic amount of sulfuric acid (reaction o) to form compound 14. Compound 14 was reacted with potassium carbonate in methanol/water (reaction p) to form isonipecotic acid tert-butyl ester, 15.

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  • Organic Chemistry (AREA)
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Abstract

L'invention porte sur des dérivés de xanthène d'amide secondaire, appelés 'xanthamides' et sur d'autres dérivés de xanthène qui sont produits sous forme de colorants fluorescents pouvant avoir une photostabilité supérieure à celles de colorants apparentés tels que la fluorescéine et BODIPY-FL. Selon une réalisation de cette invention, la synthèse débute par 5-carboxyfluorescéine, un réactif bon marché. Des carboxyxanthènes apparentés utilisés dans d'autres colorants tels que les rhodamines peuvent également être utilisés comme matériaux de départ. Des étapes synthétiques simples permettent de produire les composés de cette invention. Parmi leurs propriétés notables, les colorants de xanthamide peuvent éventuellement avoir une fluorescence indépendante du pH et une facilité à contrôler les groupes fonctionnels et autres groupes. Ce qui signifie qu'il est possible de préparer divers colorants de xanthamide et des colorants de xanthène apparentés ayant une grande variété de propriétés physico-chimiques pour renforcer l'utilité de la fluorescence dans des analyses chimiques et biologiques et dans d'autres domaines. En variante, les xanthamides peuvent être utilisés comme précurseurs de colorants de xanthamide, notamment comme indicateurs ou extincteurs de luminescence de l'oxygène réactif ou d'espèces de radicaux libres.
PCT/US2002/000801 2001-01-12 2002-01-10 Derives de xanthene Ceased WO2002055512A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004055117A2 (fr) 2002-12-18 2004-07-01 Atto-Tec Gmbh Colorants substitues par carboxamide pour applications analytiques
CZ297332B6 (cs) * 2004-09-01 2006-11-15 Azacycles S. R. O. Chemoselektivní metoda monoaminomethylace sulfoftaleinových a sulfofluoresceinových derivátu
WO2007071927A3 (fr) * 2005-12-19 2007-09-07 Cambridge Entpr Ltd Complexes et colorants fluorescents
EP1740100A4 (fr) * 2004-04-13 2009-09-16 Biosearch Technologies Inc Colorants au xanthene
US8178360B2 (en) 2006-05-18 2012-05-15 Illumina Cambridge Limited Dye compounds and the use of their labelled conjugates
US9228225B2 (en) 2004-02-03 2016-01-05 Biosearch Technologies, Inc. Xanthene dyes
KR101792214B1 (ko) * 2015-12-08 2017-11-20 (주)바이오액츠 염료 화합물 및 이의 제조방법
CN108369235A (zh) * 2015-12-18 2018-08-03 丹麦科达有限公司 生色过氧化物酶底物
CN110713484A (zh) * 2018-07-12 2020-01-21 罗宇峰 易于结合的呫吨类荧光化合物及制备与应用
EP3604292A1 (fr) * 2018-08-01 2020-02-05 Delmar Chemicals Inc. Procédé de préparation de forme quinoïde de fluorescéine
CN111004200A (zh) * 2019-11-11 2020-04-14 广州中医药大学(广州中医药研究院) 一种氢化二氯荧光素二乙酰胺衍生物及其制备方法和应用
CN120289410A (zh) * 2025-06-13 2025-07-11 浙江材华科技有限公司 一种呫吨染料的合成方法

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US5625081A (en) * 1986-07-02 1997-04-29 E. I. Du Pont De Nemours And Company Fluorescent dye intermediates
US6025505A (en) * 1996-06-27 2000-02-15 The Perkin-Elmer Corporation 4,7-Dichlororhodamine dyes
US5846737A (en) * 1996-07-26 1998-12-08 Molecular Probes, Inc. Conjugates of sulforhodamine fluorophores with enhanced fluorescence

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004055117A3 (fr) * 2002-12-18 2004-08-19 Atto Tec Gmbh Colorants substitues par carboxamide pour applications analytiques
US7935822B2 (en) 2002-12-18 2011-05-03 Atto-Tec Gmbh Carboxamide-substituted dyes for analytical applications
US8530660B2 (en) 2002-12-18 2013-09-10 Atto-Tec Gmbh Carboxamide-substituted dyes for analytical applications
WO2004055117A2 (fr) 2002-12-18 2004-07-01 Atto-Tec Gmbh Colorants substitues par carboxamide pour applications analytiques
US9228225B2 (en) 2004-02-03 2016-01-05 Biosearch Technologies, Inc. Xanthene dyes
EP1740100A4 (fr) * 2004-04-13 2009-09-16 Biosearch Technologies Inc Colorants au xanthene
CZ297332B6 (cs) * 2004-09-01 2006-11-15 Azacycles S. R. O. Chemoselektivní metoda monoaminomethylace sulfoftaleinových a sulfofluoresceinových derivátu
WO2007071927A3 (fr) * 2005-12-19 2007-09-07 Cambridge Entpr Ltd Complexes et colorants fluorescents
USRE49362E1 (en) 2006-05-18 2023-01-10 Illumina Cambridge Limited Dye compounds and the use of their labelled conjugates
US8178360B2 (en) 2006-05-18 2012-05-15 Illumina Cambridge Limited Dye compounds and the use of their labelled conjugates
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