WO2000017650A1 - Compositions a transfert d'energie comprenant des proteines phycobiline - Google Patents

Compositions a transfert d'energie comprenant des proteines phycobiline Download PDF

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Publication number
WO2000017650A1
WO2000017650A1 PCT/US1999/022193 US9922193W WO0017650A1 WO 2000017650 A1 WO2000017650 A1 WO 2000017650A1 US 9922193 W US9922193 W US 9922193W WO 0017650 A1 WO0017650 A1 WO 0017650A1
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composition
dye
salt
energy transfer
alkyl
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PCT/US1999/022193
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English (en)
Inventor
Richard P. Haugland
Rosaria P. Haugland
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Molecular Probes, Inc.
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Priority claimed from PCT/US1998/019921 external-priority patent/WO1999015517A1/fr
Application filed by Molecular Probes, Inc. filed Critical Molecular Probes, Inc.
Priority to AU64002/99A priority Critical patent/AU6400299A/en
Publication of WO2000017650A1 publication Critical patent/WO2000017650A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the invention relates to fluorescent energy transfer compositions comprising one or more fluorescent dyes and a fluorescent protein, particularly a phycobi protein.
  • Fluorescence Resonance energy transfer is a process whereby a first fluorescent dye (the "donor” dye) is excited, typically by illumination, and transfers its absorbed energy to a second fluorescent dye (the “acceptor” dye) that has a longer wavelength and therefore lower energy emission
  • the efficiency of the energy transfer is governed by several factors, including the distance between the donor dye and the acceptor dye, and the degree of spectral overlap between the emission wavelengths of the donor dye and the absorption wavelengths of the acceptor dye.
  • the difference between the excitation wavelength and the emission wavelength of a fluorescent dye is called the "Stokes shift" of that dye
  • Some artificial fluorescent labels utilize FRET to produce large effective Stokes shifts, the magnitude of which is rarely seen in a single fluorescent dye Pairs of dissimilar dyes tethered by a covalent linkage such that they undergo energy transfer typically possess utility as labels for specific binding parr members, such as antibodies and oligonucleotides
  • Such "bifluorophore" labels permit the use of a single excitation source (frequently a laser) to simultaneously excite a varietv of fluorescent labels, that may then be detected at widely separated wavelengths
  • Single phycobihprotems have been used as fluorescent labels for a variety of biomolecules
  • useful fluorescent labels have been prepared that utilize energy transfer pairs incorporating two phycobihprotems or a phycobihprotein and an organic fluorescent dye
  • the emission intensity of the conjugate is typically less intense than could be obtained using a phycobihprotein as the ultimate emitter and taking advantage of the exceptionally high absorbance and high fluorescence quantum yields of phycobihprotems
  • the present invention describes energy transfer compositions comprising a synthetic dye and a fluorescent protein where the fluorescent protein acts as an energy acceptor
  • the compositions of the present invention are brightlv fluorescent, undergo efficient energy transfer, and have exceptionallv large effective Stokes shifts
  • the subject energy transfer compositions comprise an intrinsically fluorescent protein that has been chemicallv crosslinked acting as an acceptor for a synthetic donor dye
  • the energy transfer compositions comprise an intrinsically fluorescent protein that acts as an acceptor for a synthetic donor dye, where the donor dye is sulfonated one or more times
  • the energy transfer compositions comprise an intrinsically fluorescent protein that acts as an acceptor from a synthetic donor dye, where the compositions exhibit an effective Stokes shift of greater than 100 nm
  • the energy transfer compositions comprise an lntrinsicallv fluorescent protein that acts as an acceptor from a synthetic donor dye, where the compositions further comprise one or more additional synthetic dyes that act as intermediate energy transfer dyes
  • the energy transfer compositions comprise an intrinsically fluorescent protein that acts as an acceptor from a synthetic donor dye, where the compositions further comprise one or more additional synthetic dyes or fluorescent proteins that act as final emitter dyes (ultimate acceptors), yielding even greater effective Stokes shifts
  • the energy transfer compositions comprise an mtrinsicallv fluorescent protein that acts as an acceptor from a synthetic donor dye, where the compositions are further substituted by chemically reactive functional groups, or by covalently bound conjugated substances
  • FIG. 1 The fluorescence emission spectra of an energy transfer composition utilizing ALEXA FLUOR 488 dye and crosslinked allophycocyanm (XL-APC) Fluorescence emission is plotted as a function of the number of ALEXA FLUOR 488 fluorophores on the protein (degree of substitution, or DOS)
  • FIG. 2 The fluorescence emission spectra of an energy transfer composition utilizing fluorescem isothiocyanate (FITC) dye and XL-APC Fluorescence emission is plotted as a function of the fluorescem DOS.
  • FITC fluorescem isothiocyanate
  • Figure 7 A) The fluorescence emission spectrum of XL-APC when excited at 488 nm.
  • B) The fluorescence emission spectrum of an energy transfer composition utihzmg ALEXA FLUOR 488 dye and XL-APC (DOS 16), when excited at 488 nm.
  • FIG. 9 A comparison of the fluorescence emission spectra of A) an energy transfer composition utilizing R-phycoerythrm (R-PE) and TEXAS RED dye, and B) an energy transfer composition utihzmg CASCADE BLUE dye, R-phycoerythrin (R-PE), and TEXAS RED dye. Both compositions were excited at 375 nm. The spectra show improved emission from the TEXAS RED dye when a fluorophore with improved absorbance at shorter wavelengths is also conjugated to the phycobihprotein
  • FIG. 10 A comparison of the fluorescence emission spectra of A) ALEXA FLUOR 488 dye. and B) The ALEXA FLUOR 488 dye-XL-APC composition shows that extensive energy transfer occurs between the ALEXA FLUOR 488 dye and the phycobihprotein (excitation at 485 nm).
  • Figure 11 The excitation spectra of A) XL-APC alone and B) the ALEXA FLUOR 488 dye- XL-APC composition, showing the enhanced excitation of the energy transfer composition in the 480-510 nm range, relative to the phycobihprotein alone
  • Figure 12 A comparison of A) the fluorescence emission spectrum of XL-APC, when excited at 640 nm, and B) the fluorescence emission spectrum of ALEXA FLUOR 488 dye-XL-APC, when excited at 640 nm (as described in Example 6)
  • the spectrum of the energy transfer composition shows little effect on the quantum yield of the phycobihprotein due to its chemical modification.
  • Figure 13 The effect of degree of substitution on FRET efficiency Increasing dye:prote ⁇ n molar ratios during the preparation of the energy transfer compositions of the invention yields increasing FRET efficiency.
  • C Fluorescence emission spectra of ALEXA FLUOR 488 dye-XL-APC prepared at a dye-protem ratio of 60 (Example 7)
  • Figure 14 The effect of salt and chemical cross-linking on phycobihprotein stability.
  • Figure 15 A comparison of the absorption spectra of the energy transfer compositions A) ALEXA FLUOR 488 dye-APC. and B) ALEXA FLUOR 488 dye-XL-APC (Example 9).
  • Figure 16 A comparison of flow cytometric analvsis of Jurkat cells with streptavidin- modified ALEXA FLUOR 488 dve-XL-APC and with streptavidm-RED 670 (Example 13)
  • Figure 17 Flow cytometric analysis of Jurkat cells using three-color analysis SYTOX GREEN nucleic acid stain (green fluorescent) for evaluation of cell viability, R-PE labeled ant ⁇ -CD4 (orange fluorescent) and streptavidm-modified ALEXA FLUOR 488 dye-XL-APC in combination with biot ylated ant ⁇ -CD3 (red to infrared fluorescent)
  • Figure 17A shows the total population of cells under analysis, based on light scattering properties
  • Figure 17B shows the portion of the cells analyzed that were not stained by any of the three fluorescent probes
  • Figure 17C shows the cell population that exhibited low green fluorescence (or viable cells)
  • Figure 17D shows the cell population that was stained by all three fluorescent probes
  • the present invention describes novel energy transfer compositions comprising a synthetic dye and a fluorescent protein
  • the synthetic dye is optionally a sulfonated dye
  • the fluorescent protein is optionally a phycobihprotein In one embodiment the phycobihprotein subunits have been chemically cross-linked
  • the energy transfer compositions of the invention optionally further comprise additional synthetic dyes or fluorescent proteins that act as intermediate energy transfer dyes or ultimate emitter dyes
  • the energy transfer compositions of the invention are optionally substituted by chemically reactive functional groups, or by covalently bound conjugated substances
  • the energy transfer compositions of the invention typically have the formula
  • A is an intrinsically fluorescent protein and D is a fluorescent moiety having a molecular weight less than 2,000 that is covalently bound to A
  • D has a fluorescence emission maximum at a shorter wavelength than the fluorescence emission maximum of A, and acts as an energy donor, while A acts as an energy acceptor
  • the integer p is the number of molecules of the fluorescent moiety D that are covalently attached to one molecule of the fluorescent protein A, where p is typically an integer from 1 to 30.
  • fluorescent protein is meant a protein that has intrinsic fluorescence the visible or infrared region of the electromagnetic spectrum, with a fluorescence emission maximum at a wavelength beyond about 380 nm
  • Preferred fluorescent proteins are those of abundant natural origin, such as from algae or an animal, or their genetically modified forms, and typically has a molecular weight less than 500,000, more typically less than 300,000
  • the fluorescent proteins are easily purified, reasonably stable in solution and have high molar absorptivity
  • the fluorescent proteins exhibit extinction coefficients greater than 10 5 cm *M l . often greater than 10 b cm 'M l .
  • the fluorescent proteins typically exhibit a fluorescence emission maximum ( ⁇ Em ma ) at a wavelength beyond 450 nm, preferably bevond about 560 nm, and in some cases beyond about 650 nm
  • the fluorescent proteins of the invention are phycobihprotems, such as phycoerythrins, phycocyanms or allophycocyan s
  • B- or R- phycoerythrms for maximum emission wavelengths at less than about 600 nm
  • allophycocyanms APC
  • Other intrinsically fluorescent proteins that emit maximally beyond 400 nm include peridmm chlorophyll protein (PerCp) and natural and genetically modified green fluorescent protein (GFP)
  • subumts of the fluorescent protein of the invention are chemicallv cross-hnked (Examples 1, 2, and 3 and Table 2)
  • Chemical crosslinking of protein subumts of phycobihprotems significantly improves the fluorescence of the compositions, particularly in the case of allophycocyanm Energy transfer compositions that incorporate a crosshnked protein exhibit enhanced fluorescence, relative to compositions that incorporate the native protein Cross-linking also helps prevent dissociation of the phycobihprotein upon conjugation to a high number of dyes
  • the energy transfer compositions of the invention comprise multiple identical donor dyes attached to a single fluorescent protein
  • the fluorescent protein A is covalently attached to 1 - 30 identical fluorescent moieties that function as donor dyes for energy transfer to the fluorescent protein
  • the optimal DOS of a given donor dye on a particular fluorescent protein is readily determined by experimentation, using methods well known in the art (Example 1, Figures 1-6)
  • preferred fluorescent donor dves are those having strong absorption bands at the output wavelengths of commonlv utilized excitation sources
  • Preferred donor dyes are those that are readily excited at 350-365 nm, 400-410 nm, 442 nm, 488 nm, 514 nm, 532 nm, 540-550 nm, 568 nm 590-600 nm oi 630-650 nm, and possess an extinction coefficient m at least one of these wavelength ranges that is greater than about 15,000 cm X M ⁇ more
  • the fluorescent moiety D has a molecular weight of less than about 2,000 preferably less than 1,000
  • the fluorescence emission maximum ( ⁇ m max) of the fluorescent moiety is typically at least 25 nm lower than the maximum absorbance wavelength ( ⁇ Ab max) of the fluorescent protein more preferably more than 50 nm lower, and even more preferablv more than 100 140 or even 200 nm lower than that of the fluorescent protein
  • donor dves that possess intrinsic fluorescence quantum yields when covalently bound to a non-fluorescent protein (for example an lmmunoglobulm) of greater than about 0 1, more preferably greater than about 0 2, yet more preferably greater than about 0 4, and the most preferred dyes have a quantum yield on proteins of greater than about 0 6
  • the preferred dyes of the invention do not exhibit significant fluorescence quenching even when 4 or more molecules of the dye, to as many as 10 dyes per molecule, are conjugated to an lmmunoglobulm having a molecular weight of greater than about 1
  • Preferred donor d ⁇ es also exhibit relatively prompt emission, that is they have excited state lifetimes of less than about 100 nsec
  • the fluorescence of such donor dyes typically can be detected during the transit of the dye or dye-conjugate through a beam of exciting light For this reason the use of rare earth dye complexes such as those of terbium and europium as donors is not preferred, and in some applications may not be suitable
  • the donor dye-fluorescent protein composition exhibits efficient FRET, that is most of the light absorbed at the excitation wavelength of the donor dye results in emission at or near the emission maximum of the fluorescent protein
  • FRET emission that is much less than quantitative
  • the energy transfer of the instant compositions is greater than about 50% efficient as measured bv the decrease in donor dye fluorescence (Example 5)
  • Other preferred energy transfer compositions exhibit transfer efficiencies of greater than about 60%, 70%, 80% 90%, 95%, or most preferably greater than about 98% when measured against the free unconjugated dye in the same medium at the same absorbance intensity
  • the intensity of the donor dye emission is less than half the fluorescence intensity of the long wavelength emission, more preferablv less than 30%, 20%, 15%, 10%, 5%, or most preferably less than 2% the fluorescence intensity of the long wavelength emission peak.
  • the most preferred energy transfer compositions exhibit a maximum fluorescence emission at greater than about 560 nm (more preferably greater than about 620 nm and yet more preferably greater than about 650 nm), exhibit a short w avelength donor dye emission that is less than about 20% of the intensity of the longer wavelength peak (more preferably less than about 10%. and yet more preferably less than about 5%, and most preferably less than about 2%)
  • compositions of the invention typically exhibit greater fluorescence at the maximal emission wavelength of the fluorescent protein when excited at the excitation maximum of the donor dye than the fluorescent protein alone exhibits when excited at the same wavelength (See Figure 7)
  • fluorescence enhancements may be useful, a relative fluorescence increase of at least 30% upon conjugation to the donor dye is typical, and enhancement of greater than about 50% is preferred
  • a two-fold enhancement in relative fluorescence upon conjugation is useful, a five-fold enhancement is preferred a ten-fold enhancement is more preferable, a twenty-fold enhancement is vet more preferably, and a forty-fold enhancement is exceptionally useful
  • the energy-transfer compositions of the invention incorporate one or more additional fluorescent moieties that exhibit maximal absorption and emission wavelengths that are intermediate between those of the first fluorescent donor dye and the fluorescent protein
  • These intermediate "transfer dyes” typically improve the efficiency of FRET between the initial donor dye and the acceptor fluorescent protein, and results in decreased residual fluorescence from the first donor dye (Example 2)
  • addition of the transfer dye also results in enhanced fluorescence emission from the protein acceptor
  • the intermediate transfer fluorophores are covalently bound to the fluorescent protein
  • the degree of substitution is readily optimized by experimentation, using methods known in the art (Example 1)
  • the energy transfer compositions of the invention typically comprise 1-10 distinct types of transfer dyes, there being from 1-30 individual dyes of each type bound to the fluorescent protein. Typically, 1-3 types of transfer dyes are used, and typically the degree of substitution of the transfer dye is less than that of the initial donor dye.
  • the transfer dyes are each distinct from the initial donor dye, and typically have both a maximum absorption wavelength ( ⁇ Ab max) and a fluorescence emission maximum wavelength ( ⁇ Ab max) that e between the maximum absorption wavelength of the donor dye and the fluorescence emission wavelength of the fluorescent protein
  • the additional fluorescent moiety is a fluorescem, a rhodol, a rhodamme, a cyanme, a polyaza dacene, or an oxazine dye
  • the energy transfer composition further comprises one or more additional dyes that have absorption maxima that overlap the emission of the fluorescent protein and exhibit maximal fluorescence emission at longer wavelengths than that of the fluorescent protein. That is, these additional dyes function as final acceptor dyes, receiving energy from the fluorescent protein and emitting fluorescence at an even longer wavelength
  • these compositions typically exhibit an exceptionally high shift in the emission relative to the exciting light.
  • a second fluorescent protein may be used as an ultimate acceptor, the ultimate acceptor is preferably a synthetic dye of molecular weight less than about 2,000 that has maximal absorption that overlaps the emission of the protein dye
  • these dyes are cyanmes, including sulfonated cyanmes
  • the energy transfer compositions of the invention optionally further comprise a chemically reactive functional group that is covalently attached to the fluorescent protein, in order to permit the covalent conjugation of the energy transfer composition to a surface, a carrier, or a targeting molecule (typically a specific binding pair member) or a tracer.
  • the chemically reactive functional group is typically one that will react with an amino, a thiol, an aldehyde, a ketone, a hydrazine, or a hydroxylamine derivative
  • the fluorescent moietv is typically a synthetic dve that acts as an energy donor
  • additional fluorescent moieties may be utilized as intermediate transfer dyes, or even as an ultimate emitter, receiving energy transfer from the fluorescent protein A.
  • 'synthetic dye' is used to refer to fluorescent moieties useful as donor dyes, intermediate dyes or ultimate emitter dves
  • One of ordinarv skill in the art is capable of determining the utility of a given fluorescent moiety as a donor dye vs an intermediate dye by examination of that moiety's maximal excitation and emission bands with respect to the desired fluorescent protein
  • the most preferred candidates for the synthetic dyes of the invention possess extinction coefficients greater than about 60,000 cm l M l in the wavelength range of 485 nm to 515 nm
  • Their conjugates with nonfluorescent immunoglobuhns exhibit a quantum yield of greater than 0 2, preferably greater than 0 4, and they do not exhibit significant fluorescence quenching upon conjugation to immunoglobuhns unless more than 6,
  • the synthetic dye (whether a donor dye, intermediate transfer dye, or ultimate emitter dye) is a pyrene, an anthracene, a naphthalene, an acridme, a stilbene, an mdole or benzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a 4-ammo-7- n ⁇ trobenz-2-oxa-l,3-d ⁇ azole (NBD), a cyanine a carbocyanme, a carbostyryl, a porphynn, a sahcylate, an anthramlate, an azulene a perylene a pyridme a qumohne, a coumarm (including hvdroxycoumarms and aminocoumarins and fluormated derivatives thereof (as described in U S Patent No 5,830,912 to Gee et al (1998)),
  • the synthetic dye is a xanthene
  • the synthetic dye is optionally a fluorescem, a rhodol (US Patent 5,227 487 to Haugland et al (1993)), or a rhodamme
  • fluorescem includes benzo- or dibenzofluorescems, semmaphthofluorescems, or naphthofluorescems
  • rhodol includes semmaphthorhodafluors (US Patent 4,945, 171 to Haugland. et al. (1990)) Fluormated xanthene dyes have also been described previously (Int Publ. No. WO 97/39064.
  • oxazmes include resorufins. ammooxazmones. diaminooxazmes, and their benzo-substituted analogs
  • the synthetic dye is a sulfonated dye.
  • the sulfonation of fluorescent dyes typically results in enhanced dye solubihty in aqueous solutions.
  • the sulfonated dye of the invention is a sulfonated pyrene, coumarm, cyanme, or xanthene (including sulfonated fluorescems, rhodols and rhodammes).
  • the sulfonated synthetic dye is a pyrene, a coumarm, a carbocyanme, or a xanthene dye that has been sulfonated one or more times (as described in U.S. Patent No. 5,132,432 to Haugland et al., (1992); U.S. Patent No. 5,696, 157 to Wang et al. (1997); U.S. Patent No. 5.268,486 patent to Waggoner et al. (1993), and Int. Publ. No.
  • Sulfonated pyrenes and coumarms are typically excited at wavelengths below about 450 nm Sulfonated xanthenes.
  • sulfonated rhodamme dyes are particularly preferred as the fluorescent donor dyes of the invention Sulfonated dyes that absorb maximally beyond about 515 nm. such as sulfonated rhodammes. are also preferred, as are sulfonated cyanmes
  • At least one synthetic dye of the invention is a sulfonated xanthene.
  • Sulfonated xanthene includes fluorescems, rhodammes and rhodols that are substituted one or more times by -SO3X or -CH2SO3X, where X is H (sulfonic acid), or a counterion (salt of a sulfonic acid).
  • X is a counterion
  • it is typically a cation that is not toxic as used, and does not have a substantially deleterious effect on biomolecules
  • suitable cations include without limitation K + , Na + , Cs + , , Ca 2+ , Mg 2+ , ammonium, alkylammonium or alkoxyammonium salts, or pyridmium salts.
  • the counterion of the sulfonic acid mav form an inner salt with a positively charged atom on the xanthene dye itself, typically the quaternary nitrogen atom of a rhodamme dye
  • each synthetic dye is attached to the fluorescent protein (A) via a covalent linkage, L
  • the covalently bound fluorescent protein is represented herein as an -L-A moiety
  • the covalent linkage L binds the sulfonated xanthene to the fluorescent protein either directly (L is a single bond) or with a combination of stable chemical bonds, optionally including single, double, triple or aromatic carbon-carbon bonds, as well as carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, and phosphorus-nitrogen bonds.
  • L typically includes ether, thioether, carboxamide, sulfonamide, urea, urethane or hydrazine moieties
  • Preferred L moieties have 1-20 nonhydrogen atoms selected from the group consisting of C N, O, P, and S, and are composed of any combination of ether, thioether, amine ester carboxamide. sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds
  • L is or incorporates a carboxamide, sulfonamide or thiourea moiety
  • the sulfonated xanthene dyes have the formula
  • Substituents R 2 , R 3 , R 4 and R 5 are independently H, F, Cl, Br, I, CN; or Ci-Cis alkyl, or Ci-Cis alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxyhc acid ester of a Ci-Ce alcohol.
  • one of R 2 , R 3 , R 4 and R 5 is -L-A).
  • R 3 and R 4 are
  • Substituents R 1 and R 6 are H, or R 1 taken in combination with R 2 , or R 5 taken in combination with R ⁇ , or both, form a fused aromatic six membered ring, that is optionally substituted by one or more -SO3X moieties
  • R 2 , R 3 , R 4 and R 5 are independently H, F, Cl, Br, I or Ci-Cis alkyl
  • R 1 , R 2 R 5 and R 6 are H
  • R 2 and R 5 are each F or Cl
  • J moiety is OR 7 where R 7 is H, Ci-Cis alkyl, or is -L-A
  • J is NR 8 R 9 where R 8 and R 9 are independently H, C ⁇ -C 6 alkyl, C ⁇ -C 6 carboxyalkyl, Ci-C ⁇ sulfoalkyl, a salt of Ci-Ce carboxyalkyl, or a salt of Ci-Ce sulfoalkyl, where the alkyl portions each are independently and optionally substituted by ammo, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a Ci-Ce alkyl
  • R 8 m combination with R 9 forms a saturated 5- or 6-membered heterocycle that is a piperidme, a morpho ne, a pyrrolidme or a piperazme, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic
  • R 8 m combination with R 2 , or R 9 in combination with R 3 , or both form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted bv one or more Ci-Ce alkyls or -CH2SO3X moieties
  • the K moiety is O or N + R 18 R 19 , where R 18 and R 19 are independently H, C ⁇ -C 6 alkyl,
  • R 18 m combination with R 19 forms a saturated 5- or 6-membered heterocycle that is a piperidme a morphohne, a pyrrolidme or a piperazme, each of which is optionally substituted bv methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a Ci-C ⁇ alkyl
  • one of R 18 and R 19 is -L-A
  • R 18 in combination with R 4 , or R 19 in combination with R 3 or both form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more Ci-Ce alkyls or -CH2SO3X moieties
  • R 9 and R 18 are independently H, or carboxyalkyl, salt of carboxyalkyl, sulfoalkyl or a salt of sulfoalkyl, each having 1-6 carbons
  • R 9 and R 18 are H, methyl or ethyl
  • R 8 in combination with R 2 and R 19 in combination with R 3 independently form 5- or 6-membered rings that are saturated or unsaturated and are optionally substituted by one or more alkyl groups having 1-6 carbons, or by one or more -CH2SO3X moieties
  • R 8 in combination with R 2 and R 19 in combination with R 3 independently form 5- or 6-membered rings that are saturated, and are substituted by one or more -CH2SO3X moieties
  • the substituent R 10 is H, F, CN, a carboxyhc acid, a salt of carboxylic acid, or a carboxylic acid ester of a Ci-Ce alcohol.
  • R 10 is a saturated or unsaturated Ci-Cis alkyl that is optionally substituted one or more times by F, Cl, Br, carboxyhc acid, a salt of carboxylic acid, a carboxylic acid ester of a Ci-Ce alcohol, -SO3X, ammo, alkylamino, or dialkylamino, the alkyl groups of each substituent having 1-6 carbons R 10 is optionally -L-A
  • R 10 is an aryl substituent having the formula
  • R 12 , R 13 , R 14 , R 15 and R 16 substituents are independently H, F, Cl, Br, I, -SO3X, a carboxylic acid, a salt of carboxy c acid, CN, hydroxy, amino, or hydrazmo
  • one pair of adjacent substituents R 13 and R 14 , R 14 and R 13 or R 15 and R 16 when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxy c acid, or a salt of carboxyhc acid.
  • R 1 1 , R 15 , and R 16 are independently H, Cl, F, ammo, nitro, -SO?X, a carboxyhc acid, a salt of carboxyhc acid, or a carboxy - substituted alkylthio having the formula -S-(CH2)nCOOH, where n is 1-15
  • at least three of R 13 , R 14 , R 15 , and R 16 are F or Cl.
  • one of R 14 and R 15 is a carboxyhc acid, a salt of a carboxyhc acid, or -S-(CH 2 )nCOOH, where n is 1-15, and the other of R 14 and R 15 is H, F or Cl.
  • J is OR 7 , K is O, R 10 is aryl and R 12 is carboxy or -SO3X, the described dye is a fluorescem Where J is NR 8 R 9 , K is O, R 10 is aryl and R 12 is carboxy, the described dye is a rhodol Where J is NR 8 R 9 .
  • the described dye is a rhodamme
  • the dyes of the invention are fluorescems, they are preferably sulfonefluorescems (wherein R 1 - is -SO3X)
  • the dyes of the invention are rhodammes or rhodols, more preferably rhodammes
  • R 2 , R 3 , R', and R ⁇ is -SO3X. preferably R 3 and R 4 are -SO?X.
  • R 1 taken in combination with R 2 , or R 3 taken in combination with R 6 ,or both, form a fused aromatic six- membered ring that is substituted by at least one -SO3X moiety
  • R 8 in combination with R 2 , or R 9 in combination with R 3 , or R 18 m combination with R 1 , or R 19 in combination with R 5 form a 5- or 6-membered ring that is saturated or unsaturated, and is substituted by at least one -CH2SO3X moiety
  • R 8 combination with R and R 19 m combination with R 5 form a 5- or 6-membered ring that is saturated or unsaturated. and is substituted by at least one -CH2SO3X moiety
  • Spectral properties of some selected dyes are given Table
  • Table 1 Spectral properties of selected fluorophores useful for the preparation of energy transfer compositions of the invention.
  • the energy transfer composition contains a chemically reactive group (R x ), it is typically attached to the fluorescent protein by a covalent linkage L, as defined above
  • Each covalent linkage L present in the energy transfer composition is optionally the same or different
  • the conjugation reaction between the chemically reactive fluorescent protein and a substance to be conjugated results in one or more atoms of the reactive group Rx to be incorporated into a new linkage L attaching the energy transfer composition to the conjugated substance S c
  • the covalent linkage L binds the reactive group R x or conjugated substance Sc to the fluorescent protein, either directly (L is a single bond) or with a combination of stable chemical bonds, as described above
  • R x is an acrylamide, an activated ester of a carboxyhc acid, hydroxy, an aldehyde, an alkyl hahde, a sulfonate ester, an amine, an anhydride, an aniline, an aryl hahde, an azide, an aziridine, a boronate, a carboxyhc acid, an epoxide, a glycol, a haloacetamide, a halot ⁇ azme, a hydrazine, a hydroxylamine, an isothiocyanate, a ketone, a maleimide, a thiol, or a disulfide group More preferably, R x is an activated ester of a carboxyhc acid, hydroxy, an aldehyde, an alkyl hahde, a sulfonate ester, an amine, an anhydride, an aniline, an aryl hahde, an azide, an
  • the reactive functional group is typically present as 1-5 functional groups having the same chemical structure each of which is covalently bound to the fluorescent protein, A
  • the chemically reactive energy transfer compositions of the invention are useful for the preparation of any conjugated substance that possess a suitable functional group for covalent attachment of the fluorescent protein
  • particularly useful conjugates include, among others, conjugates of antigens, steroids, vitamins, drugs, haptens, metabohtes, toxins, environmental pollutants, peptides, proteins, nucleic acids, nucleic acid polymers, carbohydrates, and non-biological polymers
  • these are conjugates of cells, cellular systems, cellular fragments, or subcellular particles
  • examples include, among others, virus particles, bacterial particles, virus components, biological cells (such as animal cells, plant cells, bacteria, yeast, or protists), or cellular components
  • the reactive energy transfer compositions typically label reactive sites at the cell surface, in cell membranes, organelles, or cytoplasm
  • the conjugated substance is a peptide, protein, tyramme, polysaccha ⁇ de, nucleic acid polymer, hapten drug, hormone, ion chelator polymer, polymeric microp article
  • Preferred protein conjugates include enzymes, antibodies, lectins, gly cop ro terns, histones, albumins, poproteins, avidin, streptavidin, protein A, protein G, phycobihprotems and other fluorescent proteins, hormones, toxins and growth factors
  • the conjugated polypeptide or protein is an antibody, an antibody fragment, an avidin, a streptavidin, a toxin, a lectm, a hormone, a cytokme, or a growth factor
  • the conjugated substance is a polypeptide, it is biologically active peptide such as a neuropeptide or a toxin
  • nucleic acid polymer typically a complex of the nucleic acid is formed with a fluorescent conjugate as described above
  • a complex of a biotmylated nucleic acid with an APC -streptavidin The nucleic acid polymer of the complex is single- or multi-stranded, natural or synthetic DNA or RNA, DNA or RNA oligonucleotides, or DNA/RNA hybrids, or incorporate an unusual hnker such as morphohne derivatized phosphates, or peptide nucleic acids such as N-(2- am ⁇ noethy )glycme units
  • the nucleic acid is an ohgonucleotide, it typically contains fewer than 50 nucleotides, more typically fewer than 25 nucleotides
  • the phycobihprotein is attached via one or more purme or py ⁇ midme bases through an amide, ester, ether or thioether bond, or is attached to the
  • the conjugated substance (Sc) is a carbohydrate that is typically a monosaccharide or a polysaccharide, such as a dextran, FICOLL, heparin, glycogen, amylopectin, mannan, lnu n, starch, agarose and cellulose
  • the carbohydrate is a polysaccharides that is a hpopolysaccharide
  • conjugates of non-biological materials include dye-conjugates or dye- complexes of organic or inorganic polymers, polymeric films, polymeric wafers, polymeric membranes, polvmeric particles, polymeric microp articles including magnetic and non- magnetic microspheres conducting and non-conducting metals and non-metals, and glass silicon or plastic surfaces, particles and chips
  • Conjugates are typically prepared by chemical modification of a polymer that contains functional groups with suitable chemical reactivity, and complexes are typiclly formed by modification of the material with a hapten or biotm, and complexation with a dye-protein conjugate of the invention
  • the conjugated substance is a glass or sihca, which may be formed into an optical fiber or other structure
  • Labeled members of a specific binding pair are typically used as fluorescent probes for the complementary member of that specific binding pair, each specific binding pair member having an area on the surface or in a cavity that specifically binds to and is complementary with a particular spatial and polar organization of the other Preferred specific binding pair members are proteins that bind non-covalently to low molecular weight ligands, such as biotm, drug-haptens and fluorescent dyes (such as an anti- fluorescein antibodv) Representative specific binding pairs are shown m Table 3
  • IgG is an immunoglobuhn t aDNA and aRNA are the antisense (complementary) strands used for hybridization
  • the energy transfer compositions of the invention are useful as detection reagents and as fluorescent tracers in a wide variety of apphcations, most of which have been described for similar protem-based fluorescent dyes that do not have the useful spectral properties of the compositions of this invention
  • These useful spectral properties include a large effective Stokes shift when excited near the absorption maximum of the donor dye(s), combined with their high absorbance at this excitation wavelength This large Stokes shift permits extremely sensitive detection with minimal autofluorescence or scattered hght from either the excitation source or via Raman scattering.
  • the care and handling of the instant energy transfer compositions are substantially the same as for well known phycobihprotein labels (see for example, MOLECULAR PROBES HANDBOOK, supra, Section 6.4 and references therein)
  • the energy transfer compositions of the invention are generally utilized by combining a composition as described above with the sample of interest under conditions selected to yield a detectable optical response
  • the energy transfer composition typically forms a covalent or non-covalent association or complex with an element of the sample, or is simply present within the bounds of the sample or portion of the sample
  • the sample is then illuminated at a wavelength selected to ehcit the optical response Typically, staining the sample is used to determine a specified characteristic of the sample bv further comparing the optical response with a standard or expected response
  • the compositions of the invention are typically used in an aqueous, mostly aqueous or aqueous-miscible solution prepared according to methods generally known in the art The exact concentration of the composition is dependent upon the experimental conditions and the desired results, but typically ranges
  • the sample is optionally washed after staining to remove residual, excess or unbound energy transfer compositions
  • the sample is optionally combined with one or more other solutions in the course of staining, including wash solutions, permeabihzation and/or fixation solutions, and solutions containing additional detection reagents
  • An additional detection reagent typically produces a detectable response due to the presence of a specific cell component, mtracellular substance, or cellular condition, according to methods generally kno n in the art Where the additional detection reagent has, or yields a product with, spectral properties that differ from those of the subject dye compounds multi-color applications are possible
  • the compositions of the invention that are conjugates are used according to methods extensively known in the art e g use of antibody conjugates m microscopy and lmmunofluorescent assays, and ohgonucleotide conjugates for nucleic acid hybridization assays
  • the fluorescent conjugates of the invention may be used as secondary detection reagents, for example by using goat-
  • the other fluorescent labels are typically conjugates of other biopolvmers such as of proteins or nucleic acid probes or they are lower molecular weight stains such as nucleic acid stains, organelle stains, products of fluorogenic enzvme substrates, probes for receptors, other tracers or other fluorescent labels
  • the sample is illuminated with a wavelength of light selected to give a detectable optical response, and observed with a means for detecting the optical response.
  • Illumination sources include, but are not limited to, hand-held ultraviolet lamps, mercury arc lamps, xenon lamps, lasers and laser diodes These illumination sources are optionally integrated into laser scanners, fluorescence microplate readers, standard or minifluorometers, or chromatographic detectors. Alternatively, the source of illumination is energy transfer from a chemilummescent species.
  • a detectable optical response means a change in, or occurrence of, an optical signal that is detectable either by observation or instrumentally
  • the detectable response is a change in fluorescence, such as a change in the intensity, excitation or emission wavelength distribution of fluorescence, fluorescence lifetime, fluorescence polarization, or a combination thereof
  • the degree and/or location of staining, compared with a standard or expected response indicates whether and to what degree the sample possesses a given characteristic
  • the optical response is optionally detected by visual inspection, or by use of any of the following devices- CCD cameras, video cameras, photographic film, laser-scanning devices, fluorometers, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, fluorescence microplate readers, or by means for amplifying the signal such as photomultipher tubes
  • examination of the sample optionally includes sorting components of the sample according to their fluorescence response.
  • kits wherein one or more kit components is a reagent of this invention
  • kit components optionally comprise one or more of the following: additional detection reagents, buffers, fluorescence standards, and instructions for performing one or more assays using the kit.
  • the fluorescence standards may comprise single fluorescent compounds, or preferably, fluorescent-labeled polymeric microspheres, more preferably microspheres labeled internally with fluorescent dyes.
  • a solution of the desired fluorescent protein such as a phycobihprotein, is prepared at a concentration of 10 mg/mL in O l M phosphate, 0 1 M NaCl, pH 7 5
  • the selected synthetic dye is dissolved in anhydrous DMF at a concentration of 10 mg/mL
  • the dye solution is then added to the fluorescent protein solution with stirring
  • the molar ratio of dye to protein in the reaction mixture is adjusted so as to yield a composition exhibiting relatively low residual fluorescence emission of the donor dye or donor dyes and relatively high fluorescence intensity of the fluorescent protein that is the FRET acceptor
  • the reaction mixture is incubated at room temperature for 1 hour, the reaction is stopped by addition of 1 5 M hydroxvlamine, pH 8 0 m a volume corresponding to 1/10 of the volume of the reaction mixture and the reaction mixture is incubated for an additional 30 minutes
  • the reaction mixture is purified by size-exclusion chromatography on BioGel P-30 (BioRad) The degree of labehng (mole
  • Mol of dye/mol of PRO [(Absdye - CF )/E dy e] x (E M p R0 /Absmax of PRO)
  • CF is the correction factor corresponding to the contribution by the unconjugated protein's absorption at the absorption maximum of the donor dye (s) This correction factor is determined as follows
  • the energy transfer compositions of Table 2 are prepared using this general synthetic method, by careful selection of appropriate synthetic dyes and fluorescent proteins
  • the synthetic dye is an ALEXA FLUOR 488 dye and the fluorescent protein is cross-linked allophycocyanm (XL-APC)
  • the results in Figure 1 are obtained Additional examples of the spectra of energy transfer compositions given for FITC-XL-APC in Figure 2, OREGON GREEN 488 dye-XL-APC in Figure 3, CY3 dye-XL- APC m Figure 4, OREGON GREEN 488 dye-R-PE in Figure 5, and ALEXA FLUOR 350 dye-R-PE in Figure 6
  • Example 2 Conjugation of a transfer dve to a preformed donor dve-labeled p hvcobi pr otem
  • a composition comprising ALEXA FLUOR 488 dye conjugated to XL-APC (Table 2) is dissolved m a pH 7 5 buffer (as in Example 1) and incubated with variable amounts of the succimmidyl ester of ALEXA FLUOR 568 dye Following purification and characterization as described in Example 1, the resulting composition exhibits decreased residual fluorescence of the first donor dye ( Figure 7)
  • the transfer dye(s) can be conjugated simultaneously with or preceding conjugation of the first donor dye
  • a fluorescent protein that has been modified with a donor dye or dyes such as ALEXA FLUOR 488 dye can be further conjugated to a dye that has spectral overlap with the emission of the fluorescent protein but has overall a longer wavelength emission maximum
  • XL-APC that has been first modified by conjugation to ALEXA FLUOR 488 dye (Compound 4 of Table 1) can be further modified by CY7 dye (Compound 17 of Table 1) essentially as described in US Patent No 5,268,486 to Waggoner et al or with LaserPro 790 succimmidyl ester dye (Molecular Probes, Eugene OR), giving conjugates that can be excited at or near 488 nm with emission detected beyond -700 nm
  • R-phycoerythrm that has been modified by CASCADE BLUE dye
  • Figure 8 can be further modified by conjugation to a TEXAS RED dye, e g such as described in US Patent 5,798,276 to Haugland et al (1998) or with CY5 dye as described in US Patent No 5,268,486 to give a conjugate that can be excited in the ultraviolet but that has principal fluorescence emission beyond -600 nm ( Figure 9)
  • a TEXAS RED dye e g such as described in US Patent 5,798,276 to Haugland et al (1998) or with CY5 dye as described in US Patent No 5,268,486 to give a conjugate that can be excited in the ultraviolet but that has principal fluorescence emission beyond -600 nm ( Figure 9)
  • ALEXA FLUOR 488 dye-XL-APC composition is biotmylated with biotm-X, succimmidyl ester (Molecular Probes) according to standard protocols ("Coupling of Antibodies with Biotm", Haugland et al , THE PROTEIN PROTOCOLS HANDBOOK, J M Walker ed , 1996, pg 293)
  • the conjugate is purified by size-exclusion column chromatography
  • the resulting complex can be combined with labeled or unlabeled avidms
  • Example 5 Determination of FRET efficiency for the first donor dye The efficiency of energy transfer from the shortest wavelength emitting donor dye to the fluorescent acceptor protein is determined by comparing the fluorescence of the donor dye excited at or near its maximal absorption in the conjugate versus that of the same or a similar dye that is not conjugated to the protein For instance, when ALEXA FLUOR 488 dye is conjugated to XL-APC to give an energy transfer composition (Table 1), the energy transfer efficiency for a particularly useful conjugate is typically above 95% ( Figure 10) The conjugation of additional transfer dyes to the composition improves energy transfer efficiency to an even greater percentage
  • the emission efficiency (quantum yield) of a fluorescent moiety is an indication of how much of the absorbed excitation energy is re-emitted as fluorescence
  • the emission efficiency of the fluorescent protein is not substantially altered by the conjugation of donor dyes
  • the chemical modification of the phycobihprotein does not appreciably interfere with the protein's absorbance or fluorescence quantum yield
  • FRET efficiency between the donor dye and the fluorescent protein may be measured by exciting the energy transfer composition at the optimal excitation wavelength of the donor dye and measuring the ratio of the long wavelength emission to that of the short wavelength emission
  • FRET efficiency varies as a function of the degree of substitution of the donor dye(s) ( Figure 13)
  • Figure 13 A high ratio is preferred so as to reduce spectral compensation in the conjugate's multicolor applications
  • Example 8 FRET takes place efficiently without performing the conjugation of the dve with phvcobihproteins at high salt concentrations
  • Conjugation of the sulfonated dye to the fluorescent protein need not be performed in high salt as specified in US Patent No 5,272,257 to Gupta (1993)
  • Conjugation of native allophycocyanm (APC) and crosslinked allophycocyanm (XL-APC) to the succimmidyl ester of carboxyfluorescem or to the succimmidyl ester of ALEXA FLUOR 488 dye is performed exactly as described in the Gupta patent in presence or absence of 10% sodium sulfate
  • the use of high salt (reportedly in order to expose more hydrophobic regions of the molecule) is not necessary, and may even be inhibitory, when conjugating the proteins to dyes
  • Example 9 Conjugation of chemically cross-linked phycobihprotems yields energv transfer compositions having advantageous spectral and chemical properties Energy transfer compositions comprising ALEXA FLUOR 488 dye and either native
  • APC or cross-hnked APC are prepared using the procedure of Example 1, wherein the molar ratio of dye to protein is 30
  • Figure 15 shows that the composition containing native APC exhibits an altered absorption spectrum, having an absorption maximum at -630 nm instead of -650 nm
  • the composition containing XL-APC has maintained an absorption spectrum characteristic of the native protein, with an absorption maximum at -650 nm
  • the relative fluorescence yield of the conjugate made from native APC is much lower than the relative yield of the conjugate prepared from XL-APC ( Figure 15)
  • Chemical crosslinking of the phycobihprotein subumts permits the preparation of energy transfer compositions that retain the integritv and high fluorescence intensity of native phycobihprotein
  • Example 10 Protein labeling using energy transfer compositions
  • a labeled phycobihprotein (as prepared in Examples 1, 2 or 3) may be further modified by conjugation of 1-2 moles of SPDP (3-(2-pyr ⁇ dyld ⁇ th ⁇ o)prop ⁇ on ⁇ c acid succimmidyl ester), to give a pyridyldisulfide-modified protein Following reduction of the disulfide, such as with dithiothreitol (DTT), the thiolated protein is readily conjugated to any thiol- re active second protein, such as one that has been modified bv SMCC (4-(N- male ⁇ m ⁇ domethvl)-cvclohexane- l-carboxvhc acid succinimidvl ester)
  • SPDP 3-(2-pyr ⁇ dyld ⁇ th ⁇ o)prop ⁇ on ⁇ c acid succimmidyl ester)
  • Example 11 Using the energy transfer compositions to stain biological samples
  • the use of specific binding pair members labeled with energy transfer compositions is typically analogous to well-known applications of the particular specific binding pair member used
  • the instant compositions are highly useful m applications such as staining cells blots targets immobilized on DNA chips and the like
  • BPAEC Permeabihzed and fixed bovme pulmonary artery endothehal cells
  • Example 12 Multicolor flow cvtometrv with a single excitation wavelength
  • Tll-FITC Cytostat Coulter # 6603863, 10 ⁇ L/test
  • T4-RPE Coulter #6602864, 5 ⁇ L test
  • biotmylated ant ⁇ -CD3 Immunotech Coulter #1301
  • streptavidm-modified ALEXA FLUOR 488 dye-XL-APC (1 ⁇ g/mL) is added
  • the cells are incubated for an additional 30 minutes on ice and analyzed by flow cytometry using appropriate single-color controls and negative controls Analysis of dot plots clearly demonstrates the utihty of streptavidm-modified ALEXA FLUOR 488 dye-XL- APC in performing three color analysis using a single laser for excitation
  • the fluorescent signal from a dot stained with the energy transfer composition is approximately 42 times brighter than a
  • Example 14 Staining cells in combination with a nucleic acid stam

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Abstract

La présente invention concerne des compositions à transfert d'énergie renfermant une ou plusieurs teintures fluorescentes et une protéine fluorescente, en particulier une teinture fluorescente sous forme de teinture sulfonée et une protéine fluorescente sous forme de protéine phycobiline. Les compositions à transfert d'énergie selon l'invention peuvent par ailleurs contenir d'autres teintures fluorescentes ou protéines fluorescentes qui font office de teintures à transfert d'énergie intermédiaire ou de teintures émettrices ultimes. Les compositions à transfert d'énergie peuvent également être remplacées par des groupes fonctionnels chimiquement réactifs, ou des substances conjuguées à liaison covalente. Les substances selon l'invention conviennent pour les applications les plus diverses, biologiques notamment, en tant que réactifs de détection ou marqueurs fluorescents.
PCT/US1999/022193 1998-09-23 1999-09-23 Compositions a transfert d'energie comprenant des proteines phycobiline WO2000017650A1 (fr)

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PCT/US1998/019921 WO1999015517A1 (fr) 1997-09-23 1998-09-23 Derives de xanthene sulfone
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WO2002079183A1 (fr) * 2001-04-02 2002-10-10 Theratechnologies Inc. Derives halogenes de la rhodamine et leurs applications
WO2006134319A1 (fr) * 2005-06-11 2006-12-21 University Court Of The University Of Edinburgh Procede d'analyse
EP1947095A1 (fr) * 2007-01-22 2008-07-23 Pierce Biotechnolgoy, Inc. Dérivés de sulfamide de xanthènes utiles comme réactifs fluorecsents de détection de biomolécules
US7491545B2 (en) * 2002-10-01 2009-02-17 Richard Thompson Excitation ratiometric fluoroscent biosensor for zinc ion at picomolar levels
US7560574B2 (en) 2001-04-02 2009-07-14 Celmed Biosciences Inc. Halogenated rhodamine derivatives and applications thereof
CN103408555A (zh) * 2013-07-19 2013-11-27 陕西学前师范学院 一种罗丹明b衍生物及其制备和应用
CN104370927A (zh) * 2014-10-28 2015-02-25 齐鲁工业大学 一种希夫碱类荧光探针化合物及其制备
WO2017198334A1 (fr) * 2016-05-18 2017-11-23 Biocytex Nouvelle propriete optique d'un marqueur fluorescent

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US4666862A (en) * 1984-08-14 1987-05-19 Ortho Diagnostic Systems Inc. Fluorescent energy transfer with phycobiliproteins
WO1994005701A1 (fr) * 1992-09-03 1994-03-17 Coulter Corporation Procede de marquage preferentiel de la phycobiliproteine a l'aide d'un colorant aminoreactif destine a etre utilise dans un titrage multicolore
EP0747700A2 (fr) * 1995-06-07 1996-12-11 Carnegie-Mellon University Complexe avec un grand décalage de stokes pour marquage fluorescent formé par couplage de cyanine et autres fluorochromes capables de transfert d'énergie résonance
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Cited By (16)

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US9636363B2 (en) * 2001-04-02 2017-05-02 Kiadis Pharma Canada Inc. Halogenated rhodamine derivatives and applications thereof
US7560574B2 (en) 2001-04-02 2009-07-14 Celmed Biosciences Inc. Halogenated rhodamine derivatives and applications thereof
US20110301573A1 (en) * 2001-04-02 2011-12-08 Abdelkri Habi Halogenated rhodamine derivatives and applications thereof
US8383672B2 (en) 2001-04-02 2013-02-26 Kiadis Pharma Canada Inc. Halogenated rhodamine derivatives and applications thereof
WO2002079183A1 (fr) * 2001-04-02 2002-10-10 Theratechnologies Inc. Derives halogenes de la rhodamine et leurs applications
US20140314720A1 (en) * 2001-04-02 2014-10-23 Kiadis Pharma Canada Inc. Halogenated rhodamine derivatives and applications thereof
US7491545B2 (en) * 2002-10-01 2009-02-17 Richard Thompson Excitation ratiometric fluoroscent biosensor for zinc ion at picomolar levels
WO2006134319A1 (fr) * 2005-06-11 2006-12-21 University Court Of The University Of Edinburgh Procede d'analyse
EP1947095A1 (fr) * 2007-01-22 2008-07-23 Pierce Biotechnolgoy, Inc. Dérivés de sulfamide de xanthènes utiles comme réactifs fluorecsents de détection de biomolécules
US7745645B2 (en) 2007-01-22 2010-06-29 Pierce Biotechnology, Inc. Sulfonamide derivatives of xanthene compounds
CN103408555A (zh) * 2013-07-19 2013-11-27 陕西学前师范学院 一种罗丹明b衍生物及其制备和应用
CN103408555B (zh) * 2013-07-19 2016-06-08 陕西学前师范学院 一种罗丹明b衍生物及其制备和应用
CN104370927B (zh) * 2014-10-28 2016-12-07 齐鲁工业大学 一种希夫碱类荧光探针化合物及其制备
CN104370927A (zh) * 2014-10-28 2015-02-25 齐鲁工业大学 一种希夫碱类荧光探针化合物及其制备
WO2017198334A1 (fr) * 2016-05-18 2017-11-23 Biocytex Nouvelle propriete optique d'un marqueur fluorescent
FR3051556A1 (fr) * 2016-05-18 2017-11-24 Biocytex Utilisation de marqueurs fluorescents pour la detection de cible biologique dans un echantillon

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