Fluorescent labelling complexes with large stokes shifts formed by coupling together cyanine and other fluorochromes capable of resonance energy transfer

IS 2 170 204 T3

DESCRIPTION

Fluorescent labeling complexes with wide stoke shifts formed by coupling cyanine and other fluorochromes capable of transferring resonance energy.

The present invention relates to fluorescent labeling complexes and more particularly to low molecular weight fluorescent complexes with long Stokes shifts and their use in the preparation of fluorescent derivatives of target materials.

Fluorescence marking is an important technology for detecting biological molecules. For example, antibodies can be labeled with fluorescent dyes. In this way, the binding of antibodies to their specific target molecules can be tracked, based on a fluorescence signal that can be detected with a spectrometer, immunofluorescence instrument, flow cytometer, or fluorescence microscope. Similarly, DNA sequences can be detected by fluorescence detection instruments once the DNA has hybridized to a complementary DNA sequence that has been labeled with a fluorescent dye.

Energy transfer complexes containing covalently linked donor and acceptor molecules are known. For example, Stryer and Haugland have developed a model system for studying the dependence of singlet to singlet energy transfer on distance (Stryer, L. and Haugland, RP, Proc. Nat. Acad. Sci, vol., 58 , pp. 720-26 (1967)). The synthesis and properties of novel photochromic model compounds containing a cyanine dye and a porphyrin have been described (Lindsey et al., Tetrahedron, vol. 45, No. 15, pp. 4845-66, (1989)). Complexes containing fluorescent acceptor and donor chromophores have been described as substrates for the study and kinetic assay of hydrolotic enzymes (Carmel et al., FEBS Letters, vol. 30, N^° 1, pp 11 (1973)).

In European patent application N^° 609894 discloses a labeling complex that includes a three-core dye represented by the general formula (1):

<img file="ES2170204T3_D0001.tif" />

wherein Xa, Xb and Xc are independently unsubstituted or substituted hetercoclic rings containing one to three heteroatoms and La and Lb are conjugated methine chains. One between La and Lb can be omitted for binding the heterocycles directly. Compounds of structure (1) may include a reactive group to form a covalent linkage between the three-core dye and a biological substance. Compounds of such a formula have been reported to have a long Stokes shift (50-100 nm). However, it is not believed that resonance energy transfer is involved in the fluorescence process with these dyes.

In European patent application N^° 601889 nucleic acid probes are described that contain a target binding sequence, a region thereof being capable of forming one or more imperfect hairpins, and at least one donor marker and at least one acceptor marker covalently linked to the nucleootide sequence. , so that when one or more hairpin structures are formed, one of the acceptor fractions and one of the donor fractions are in close proximity to allow the transfer of resonance energy between them. The labels are preferably fluorophores. A method is also described for the use of said probes to detect a polynucleotide target, so that when the probe interacts with a target sequence, the distance between the fluorescent groups is changed, resulting in a change in fluorescence emission. of the probe.

In Lee et al., Nucleic Acids Research, 20 (10), 2471-2483 (1992) refers to fluorescent dye-labeled terminators for use in DNA sequences. A ddG-bifluorine dye is disclosed consisting of a fluoresceone and rhodamine dye covalently linked to each other and to a purine nucleotide. The maximum absorption of the dye complex is 498 nm and 554 nm and the maximum emission is 580.

WO 88/04777 describes monomeric water-soluble metallized phthalocyanine compounds which are conjugated to biochemical moieties such as antibodies or ligands. Among the derivatives

ES 2 170 204 T3 of phthalocyanine includes substituents that provide water solubility or a suitable captive bond for conjugation with another reagent.

US 5410030 refers to nucleic acid spots which are dimers of non-symmetric cyanine dyes. At least one dimer cyanine dye contains a pyridinium moiety. The other dye unit contains a pyridinium moiety or quinolinium ring. Non-symmetric cyanine dyes are linked by a bridging unit in which additional positive charges are preferably incorporated. The dyes are used as nucleic acid labeling agents that bind by non-covalent bonding.

In European patent application No. 601889 nucleic acid probes containing a target binding sequence are described, a region of which is capable of forming one or more imperfect hairpins and at least one donor marker and at least one acceptor marker. covalently linked to the nucleotide sequence, so that when one or more hairpin structures are formed, one of the donor fractions and one of the acceptor fractions are in close proximity to allow a transfer of resonance energy between them. The markers are preferably fluorophores. A method is also described to use said probes to detect a polynucleootide target, so that when the probe interacts with a target sequence, the distance between the fluorescent groups is changed, resulting in a change in the emission of the fluorescence. of the probe.

Lee et al., Nucleic Acids Research, 20 (10), 2471-2483 (1992) refers to fluorescent dye-labeled terminators for use in DNA sequences. A ddG-bifluoror dye is disclosed consisting of a fluoresceone and a rhodamine dye covalently linked to each other and to a purine nucleotide. The maximum absorption of the dye complex is 498 nm and 554 nm and the maximum emission is 580.

The analysis of several paraometers using fluorescent marks with clearly different emission wavelengths further increases the importance of this technology as it provides a powerful instrument for correlating several antigoenic or genoetic paraometers in individual cells. In epifluorescence microscopy, a continuous light source with different emission and excitation filter apparatus is used to excite and detect each fluorescent species. This method works particularly well when the absorption and emission wavelengths of each fluorophore are relatively close (eg, Stokes shifts of 15-30 nm). Most highly fluorescent low molecular weight fluorophores, such as cyanines and xanthenes, have narrow absorption and emission peaks and small Stokes shifts. Up to 5 different fluorescent marks have been analyzed in the same specimen by microscope using epifluorescence filter kits, as described by DeBiasio et al., Journal of Cell Biology, vol. 105, pp. 1613-1622 (1987).

While it is easy to find a fluorophore uonic that can be efficiently excited at a particular laoser wavelength, it is difficult to find additional fluorescent labels with Stoke shifts long enough to provide a well differentiated emission from that of the first fluorophore. Natural phycobiliproteones are a class of multi-chromophoric fluorescent photosystem proteones that have long wavelength shifts; see Oi, VT, Glazer, AN and Stryer, L. Journal of Cell Biology, vol. 93, pp. 981-986 (1982). These can be covalently coupled with antibodies and have been used extensively in flow cytometry for a two-color lymphocyte subset analysis. R-phycoerythrin (R-PE), a photosystem proteon containing 34 bilin fluorophores that can be excited at 488 nm with the readily available argon ion laser, has proven especially useful. It has a maximum fluorescence at 575 nm. R-PE and fluoresceon can both be excited at 488 nm, but R-PE can be easily discriminated with optical bandpass interference filter apparatus from the fluorescein signal appearing at 525 nm. Recently, 3-color immunofluorescence by flow cytometry has been possible through the development of row conjugated labeling reagents containing a reactive fluorescent dye that is excited at 488 nm and has fluorescence at 613 nm, and is commercially available. under the brand name Duochrome, see US Patent No.^° 4876190. With another fluorophore in a row the energy transfer from excited R-PE to the bound cyanine dye, known as Cy-5 leads to fluorescence at 670 nm (Wagoner et al., Ann. NY Acad. Sci., Vol. 677 , pp. 185-193, (1993)).

Phycobiliprotein-based labels are highly fluorescent and provide excellent signals in 2- and 3-parameter experiments for the detection of cell surface antigens. However, these reagents have not been widely used to measure cytoplasmic antigens or for the detection of chromosome markers by fluorescence in situ hybridization since their large size (MW 210,000 Dalton) limits penetration into dense cell structures.

IS 2 170 204 T3

Despite all the above, there are still no low molecular weight fluorescent compounds that can be used as labels for the covalent labeling of target molecules and that provide multi-color fluorescent detection using uine wavelength excitation. There is still a need to find various such fluorescent labels that can be individually excited in the most optimal way at a particular laser wavelength, but which fluoresce at significantly different emission wavelengths. We have found a new class of low molecular weight fluorescent labels that provide multi-color fluorescence detection using single wavelength excitation.

Accordingly, the present invention relates to a low molecular weight fluorescent labeling complex that includes:

i) a first fluorochrome having a first absorption and emission spectrum;

ii) a second fluorochrome that has a second absorption and emission spectrum, the wavelength of the maximum emission of said second fluorochrome being longer than the wavelength of the maximum emission of said first fluorochrome and a portion of the spectrum of absorption of said fluorochrome with a portion of the emission spectrum of said first fluorochrome.

iii) at least one bonding group for covalent bonding of said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;

iv) at least one target binding group capable of forming a covalent bond with a target compound;

at least one of said first or second fluorochromes being a cyanine dye and the combined molecular weight of said first and second fluorochrome and said binding group being less than 20,000 Daltons.

The binding agent can be rigid or flexible to orient the transition moments of the donor and acceptor chromophores. For optimal energy transfer to occur, the transition moments of the first and second fluorochrome are oriented relative to each other in a non-perpendicular direction, ie, generally located parallel or cascading relative to each other. The transition moments of the flexibly bound fluorochromes change as the binding agent is bent, but as long as the donor and acceptor transition moments are not perpendicular during the life of the donor in the excited state, energy transfer would occur. . The complexes prepared described herein demonstrate 50% to 99% efficient energy transfer. The efficiency of energy transfer depends on several factors such as spectral overlap, the spatial separation between the donor and the acceptor, the relative orientation of the donor and acceptor molecules, the quantum performance of the donor and the duration in the excited state of the donor. . In one of the preferred embodiments, the fluorochromes may be separated by a distance that provides efficient energy transfer, preferably 75%.

The close proximity of the donor and acceptor fluorochromes should enhance energy transfer, since the efficiency of energy transfer varies according to the sixth inverse power of separation of the chromophore centers according to the Forster equation

ET α K² φD J / R⁶ id where ET is the energy transfer rate constant, K² is the relative orientation of the donor and acceptor transition moments, φD is the quantum yield of the donor molecule, R is the distance between the centers of the donor and acceptor fluorochromes, J is the overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor fluorochromes and id is the duration in the excited state of the donor molecule. See, Forster, T. "Intermolecular Energy Transfer and Fluorescence", Ann. Physik, vol. 2 P. 55 (1948). The distance R between the centers of the donor and acceptor fluorochromes may preferably be between 10 and 80 Angstroms. The bonding agent should allow the transfer of resonance energy between the fluorochromes.

Fluorochromes should not chemically interact or form secondary bonds with each other.

The binding agent may preferably have a bond length of 2 to 20. For example, if the binding agent contains an alkyl chain, - (CH2) n-, the carbon number "n" may be 1 to about 15. The bonding agent may include part of the constituents that extend from

ES 2 170 204 T3 fluorochrome. That is, the binding agent is bound to the dye chromophore but not part of it. As regards the binding agents listed in Table 2, some of them range from the nitrogen of the ring in one cyanine to a functional group in the benzene ring of another cyanine. Some linking agents extend between the functional groups on the benzene rings of attached dyes. However, in these examples, none of the unioan agents include a network of double bonds that allows conjugation of donor and acceptor. With a relatively short binding agent and optimal orientation, efficient resonance energy transfer can occur even when the spectrum overlap becomes small. Consequently, it is possible to obtain long wavelength shifts even when only two chromophores are used in the complex.

Suitable unioan agents are selected from the group consisting of alkyl chains containing 1 to 20 carbon atoms which may optionally include 1 to 8 oxygen atoms such as polyether linkages, or 1 to 8 atoms. nitrogen, such as polyamine linkages, or 1 to 4 CO-NH groups such as polyamide linkages, up to 2 bicyclo [2,2,2] octyl groups and up to 10 nucleaotide units.

The complexes of the present invention include a target binding group capable of forming a covalent bond with a target compound to allow the complex to mark the target, such as a carrier material or a biologic compound. The target binding group may be a reactive group for reaction with a functional group on the target material. Alternatively, the complex may contain a functional group and the target may contain the reactive constituent.

Suitably, the reactive group is selected from the group consisting of succinimidal asters, isothiocyanates, dichlorotriazine, isocyanates, haloacetamide, maleimide, sulfonyl halides, acid halides, alkylamido esters, arylimido asters, substituted hydrazines, substituted hydroxylamine phosphors, and substituted hydroxylamides.

Suitably, the functional group is selected from the group consisting of amino, sulfhydryl, carboxyl, hydroxyl, carbonyl, thiophosphate.

Suitably, the halo- and halide is selected from chlorine, bromine and iodine, or chlorine, bromine and iodine.

Suitably, the target materials may include antibodies, antigens, proteans, carbohydrates, gravestones, nucleaotides that are derivatized to contain one of an amino, hydroxyl, sulfhydryl, carboxyl, or carbonyl group and derivatized oxy or deoxy polynucleic acids for contain an amino hydroxyl, thiophosphoryl, sulfhydryl, carboxyl or carbonyl group, cells, polymer particles, or glass beads. In one of the alternative embodiments, the target can be derived to contain the reactive groups that have been identified to form covalent bonds with the functional groups in the complex.

In a second embodiment, the fluorescent complexes of the invention can contain a polymerizable group suitable for the formation of a polymer containing the complex. Suitable polymerizable groups are selected from acrylate, methacrylate, and acrylamide. The polymerization can be carried out with a complex that forms derivatives according to the present invention used in combination with a second polymerizable monoomer starting material, such as styrene or vinyl toluene, to form a copolymer containing the fluorescent complex.

Alternatively, the fluorescent complexes of the invention need not have a reactive group when used without covalently bonding to another material. For example, the complex can be incorporated during polymerization or particle formation or it can be absorbed into polymer particles.

The complex may also include water-solubilizing constituents bound to impart a hydrophilic characteristic to the complex. Preferably, they are attached to the cyanine fluorochrome aroma ring system. If the cyanine dye does not contain the water-solubilizing constituent, then the other dye or the fraction of the unioan agent may contain a water-solubilizing group. The water-solubilizing constituents must not be reactive with the target-binding group of the complex. Suitable solubilizing constituents can be selected from the group consisting of amide, sulfonate, sulfate, phosphate, quaternary ammonium, hydroxyl, guanidinium, and phosphonate. Sulfonate or sulfonic acid groups attached directly to the aromatic ring of the cyanine fluorochrome are particularly preferred. Water solubility may be required when labeling proteans and oxy and deoxy nucleic acids that are derivatized with amino groups or sulfhydryl groups in aqueous solutions. Alternatively, a less hydrophilic polar form of the transfer compound may be non-covalently attached.

ES 2 170 204 T3 energy to DNA by intercalation between base pairs or by interaction in the minor groove of DNA. Such compounds can be useful for the quantitative determination of DNA and its location.

In addition to the embodiment of the invention that includes a single donor and single acceptor fluorochrome, the fluorescent labeling complex can include other fluorochromes. The additional fluorochromes must have absorption or emission spectra that allow energy transfer to take place. For example, a third fluorochrome can be attached to the second fluorochrome. In this example, the wavelength of the emission spectrum of the third fluorochrome is longer than the emission spectrum of the second fluorochrome and a portion of the emission spectrum of the second fluorochrome overlaps with a portion of the absorption spectrum of the third fluorochrome. to transfer the absorbed energy from the first fluorochrome to the second fluorochrome to the third fluorochrome.

In another of the embodiments of the present invention, the complex can include a plurality of the first fluorochromes, each one of them covalently linked by a fraction of the binding agent to the second fluorochrome and each of them being capable, after excitation with light, to transfer energy to the second fluorochrome. In a further embodiment of the present invention, the complex can include a plurality of second fluorochromes, each one of them being covalently linked by a fraction of the binding agent to a first fluorochrome and each one of them being capable of accepting energy from the first fluorochrome when the first fluorochrome is excited with light. The plurality of the first and second fluorochromes can be the same molecule or different. For example, there may be several donor fluorochromes that can each be excited to different wavelengths to accommodate different excitation light sources.

In another embodiment of the present invention, the complex may include a plurality of the second fluorochromes, each of them being covalently linked by a fraction of a binding agent to a first fluorochrome or to a plurality of them and each being linked. one of them covalently through a binding agent fraction to a third fluorochrome. Energy transfer takes place in parallel in these embodiments.

The first fluorochrome preferably has an extinction coefficient greater than 20,000 liters / mole. cm, with more than 50,000 liters / mol.cm being more preferable. The second fluorochrome has a fluorescence quantum yield greater than or equal to about 0.05. The quantum efficiency is generally related to the rigidity or flatness of the molecule and indicates the propensity of the molecule to fluorescence, that is, to give off energy in the form of light, rather than in the form of heat when energy is supplied to the molecule.

The complexes of the present invention preferably include at least one cyanine fluorochrome, preferably at least one polymethine cyanine dye. Cyanines are particularly useful because of the wide range of available structural variations and spectral properties that can be obtained by varying the number of carbon atoms in the methine bridge, and heteroatoms or other constituents of cyanine dyes. It is possible to synthesize dyes with specific excitation wavelengths that correspond to a specific excitation source, such as a laser, eg a HeNe laser or a diode laser. Consequently, energy transfer marks can be obtained that absorb and emit efficiently at most wavelengths in the visible region of the spectrum. The excitation sources used normally excite in the 488 nm laser line. Although such excitation wavelength was used to describe the present invention, those skilled in this field should understand that other energy transfer marks can be obtained for specific excitation sources without departing from the framework of the invention.

Examples of dyes that can be used as donor and acceptor fluorochromes in the fluorescent labeling complexes of the present invention are depicted in Formulas 2 and 3.

IS 2 170 204 T3

<img file="ES2170204T3_D0002.tif" />

and in the formula (4)

<img file="ES2170204T3_D0003.tif" />

where X is selected from C (CH₃)₂, sulfur and oxygen, R¹ and R² are independently selected from the group consisting of CH2NH2, SO3-, CH2COOH, and NCS, P is selected from SO3-, NH2, and COOH, and n is an integer from 1 to 5.

Other cyanines used in the complexes of the invention are the stiffened monomethine cyanines described in the co-pending application by Wagoner et al. entitled "Rigidized Monomethine Cyanines", registered on the same date. The stiffened monomethine dyes have the following general formula (5)

<img file="ES2170204T3_D0004.tif" />

optionally substituted by one to six R groups² aR⁷; where T is a union group so that:

IS 2 170 204 T3

<img file="ES2170204T3_D0005.tif" />

it is a ring of six or seven links;

X and Y are selected from bis substituted carbon, oxygen, sulfur, selenium, and -CH = CH-, and -NW where N is nitrogen and W is selected from hydrogen and a group - (CH2) n R⁸ where n is an integer from 1 to 26 and selecting R⁸ among hydrogen, amino, aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl, sulfonate, sulfate, carboxylate, substituted amino, quaternary amino, nitro, primary amide, substituted amide and groups reactive with amino, hydroxyl, aldehyde, phosphoryl or sulfhydryl;

the Z groups¹ and Z² they represent the atoms necessary to complete a ring, two or three condensed aromatic rings, each ring having five or six atoms, the atoms being selected from among carbon atoms and, optionally, not more than two oxygen, nitrogen and sulfur atoms; and R² yR³ They are attached to the carbon atoms of T when T contains carbon atoms.

The stiffened monomethine cyanine dyes have clearly differentiated absorption and emission signals, which are photostable. Certain stiffened monomethine cyanine dyes absorb and maximally emit light at wavelengths between 300 and 500 nm.

Low molecular weight fluorochromes other than cyanine fluorochromes can be selected from fluoresceones, pyrene trisulfonates (commercially available under the trademark "Cascade Blue"), rhodamines, and bis-boron difluoride dye derivatives. pyrrometin, as 3,3 ', 5,5'-tetramethyl-2,2'-pyrromethene-1,1'-boron difluoride, available commercially under the trademark BODIPY by Molecular Probes Inc. In US Pat. USA N^° 4774339, 5187223, 5248782 and 5274113 (Haugland and Kang), as well as in the "Handbook of Fluorescent Probes and Research Chemicals" published by Molecular Probes Inc., analogues of BODIPY are described.

To obtain exceptionally long excitation-emission wavelength shifts, it is possible to use sequential energy transfer steps within the complex. For example, three chromophores have been linked to provide maximum emission at the wavelength of a cyanine dye. , heptamethine cyanine, CY7, (compound 4, X = C (CH3) 2, R¹, R²= -SO3^-; P = COOH, n = 5; m = 3), above 780 nm with excitation at 488 nm. The initial donor was fluoresceone isothiocyanate and the intermediate fluorophore of the complex was the trimethine cyanine dye designated CY3 (compound 4, X = C (CH3) 2, R¹= R²= CH2NH2, P = SO3^-, n = 4; m = 1). Fluoresceon was excited at 488 nm and practically 100% of its energy in the excited state was transferred to trimethine cyanine, which in turn transferred approximately 90% of its energy in the excited state to CY7 fluorescent at 782 nm. The same efficacy was observed when a CY5 pentamethine cyanine was used instead of CY7, with a fluorescence at 667 nm. The development of such multi-chromophor complexes is particularly useful for multi-color detection systems.

Although several of the complexes exhibit efficient energy transfer, the overall quantum performance of these labeling complexes can be further improved. For example, the use of acceptor dyes with a higher quantum yield than CY5 will improve the overall gloss of the complex.

The fluorescent labeling complexes of the invention have low molecular weights and can be easily conjugated with antibodies, other proteones and DNA probes. Low molecular weight, as used herein, means that the combined molecular weight of the fluorochromes and the binding agent of the complex is preferably between about 500 and 10,000 Daltons, and for the complex of two fluorochromes, preferably within the range of 1000 to 25000 Daltons. Consequently, these tagged species will have much greater penetration into intracellular environments than is possible with the large phycobiliproteon tags currently in use. The low molecular weight fluorescent complexes of the present invention should be of value not only for flow cytometry, but also for laoser confocal microscopy and for other detection systems that require multi-color detection with single wavelength excitation.

The invention includes a reagent and a method for obtaining said reagent that includes incubation of the water-soluble fluorescent labeling complex that has been described with a carrier material.

IS 2 170 204 T3

The present invention also provides processes for the preparation of fluorescent marking complexes consisting of the covalent bonding of fluorochromes, such as cyanine fluorochromes, with cyanines and other fluorochromes, through methods known to those skilled in this field to form donor-acceptor energy transfer complexes.

For example, complexes according to the present invention can be prepared in which the binding contains an amide or an ester by reacting a compound of formula (6) with a compound of formula (7):

R- (M) -COA B- (N) -R '(6) (7) where R and R' are different fluorochromes; COA is an activated or activatable carboxyl group; Bes NH2 or OH; and M and N are independently C1-12 alkyl-containing aliphatic moieties and optionally including one or more phenyl, naphthyl, amide, eoster, or bonding ether functions. See, for example, Mujumdar, RB et al., Bioconjugate Chemistry, vol. 4, pp. 105-111 (1993); US Patent No.^° 5268486 to Wagoner et al., The disclosure of which is incorporated herein by reference. Suitable A groups include halogen, for example chlorine or bromine, para-nitrophenoxy, N-hydroxysuccinimido or OCOR "where R" is C1-6 alkyl.

The complexes of the present invention in which the linkage contains an amino, ether or thioether group can be prepared by reacting a compound of formula (8) with a compound of formula (9);

R- (M) -B 'C- (N) -R' (8) (9) where R, R ', M and N are as defined above; B 'is OH, NH2, O, or SH; and C is a displaceable group, such as iodine, or para-toluenesulfonate. The reaction is suitably carried out in the presence of a base.

Alternatively, complexes can be prepared according to the present invention by first coupling two dye precursors using an unconjugated binding agent to give an intermediate represented by structure (10).

Xa- (L) -Xb (10) wherein Xa and Xb are independently substituted or unsubstituted heterococlic precursors and (L) is an unconjugated linking group consisting of C1-12 alkyl, optionally including one or more groups of phenyl, naphthyl, bicyclo [2,2,2] octyl, ether, amine, ester or amide linkage, or combinations thereof. In Table 1, suitable heterococlic precursors, Xa and Xb, Compound I and II are shown. By way of example, the synthesis of intermediate (10) in which the binding agent consists of an alkyl chain attached to the nitrogen atoms of two indolenine units, can be carried out by reaction with an α, ω- dihaloalkane, such as 1,6-dibromohexane, in a one- or two-stage reaction process. The reaction is suitably carried out at an elevated temperature such as about 100-110^°C, in an inert solvent such as xylene. See for example, Hamer, FM "The Cyanine Dyes and Related Compounds", p.676, Willey Interscience (1964), the description of which is incorporated herein by reference.

The intermediate (10) can be used as a precursor in the formation, through the methods known in the art, of complexes containing two different fluorophores connected by the binding agent. See, for example, Hamer, FM "The Cyanine Dyes and Related Compounds," p. 118-119, Wiley Interscience (1964), the disclosures of which are incorporated herein by reference.

The examples set forth below serve to illustrate the preparation of the complexes of the present invention and their spectral properties.

IS 2 170 204 T3

Example 1

Preparation of the CY5-CY7 complex

<img file="ES2170204T3_D0006.tif" />

Cyanuric chloride (trichlorotriazine) (5 mg), sodium bicarbonate (2 mg), and purified dimethylformamide (DMF) (0.25 ml) were mixed at 0 ° C. 5 mg of an aminocyanin dye (Mujumdar et al., Cytometry, Vol. 10 pp. 11-19 (1989)), represented in the formula by the box containing CY5, was added to this solution, and the mixture was shaken at 0^°C for 10 minutes. Stirring was continued overnight at room temperature. Thin layer chromatography (TLC) revealed one major spot and two minor spots; these last two were determined as impurities.

The reaction mixture was treated by precipitation with ether. A dark blue powder was obtained. DMF (0.3 ml) was added to dissolve the powder. Sodium bicarbonate (2 mg) and 4.7 mg of amino-CY7 dye represented by the box containing CY7 were added to this solution. The mixture was stirred at room temperature for 24 hours. The product was precipitated and washed several times with ether to provide a dark powder. The complex exhibited an absorption spectrum with peaks for the individual fluorochromes at 650 nm (CY5) and 761 nm (CY7), indicating that no new chromophores had been generated.

Example 2

Synthesis of complex 1 and related compounds (see table 2)

i) General methods

a) Purification of dyes

Purification of fluorochromes was carried out on a Spectra-Physics model SP8700 HPLC analyzer unit equipped with a C8-RP column. Purification could also be achieved by flash column or conventional chromatography on commercial C18-RP powder. Water / methanol mixtures were used for elution in all experiments. The dyes were recovered from the fractions by rotary evaporation at 60-70^°C without appreciable loss. For further purification, the fluorochrome, with an indeterminate counter ion composition, was passed through a Dowex-50 W column (hydrogenic form).

b) Measurements of copy spectra and analytical determinations

Ultraviolet / visible spectra were measured with a Hewlett-Packard HP8452 serial diode spectrophotometer. Protoonic NMR spectra were obtained with a 300 FT-NMR spectroometer.

ES 2 170 204 T3 of IBN using D₂Or, CD₃DO or DMSO-d₆ as solvents. NMR signals are described in δ using s for singlet, d for doublet, t for triplet, q for quartet and m for multiplet. Fluorescence measurements were performed using a SPEX Fluorolog 2 system. Quantum yields were determined through known techniques as described by Mujumdar RB, et al., "Cyanine Dye Labeling Reagents Containing isothiocyanate Groups", Cytometry, vol. 10, pp. 11-19 (1989).

c) Cell preparation and flow cytometry

Mononuclear leukocytes were obtained by Histopaque separation, density 1.077, from healthy volunteers. The lymphocyte population was selected by flow cytometry based on the characteristics of forward and lateral dispersion. Subpopulations were identified using specific monoclonal antibodies (CD4, staining helper T cells and CD3, tray T cell population). The optimal concentration of complex 1-tagged antibody was determined by analysis of the results of a series of dilutions. Direct immunofluorescence was carried out by incubating the recommended amount of the labeled antibody with 1-2 x 10⁶ cells for 45 minutes at 4 C. The samples were then washed twice in a salt solution balanced with Hank's solution (HBSS) containing 2% fetal calf serum and 0.1% sodium azide. After the final wash, the cells were resuspended in 1 ml of HBSS containing 1% paraformaldehyde and analyzed within a week. Flow cytometric measurements were performed with a Becton Dickinson FACS 440 dual laser flow cytometer equipped with a Consort 40 data analysis system. The argon ion laser provided 400 mW of excitation at 488 nm. Complex 1 and R-phycoerythrin fluorescence signals were collected using the 670 / 13.5 nm and 575/26 nm bandpass filters, respectively.

d) Calculation of donor extinction efficiency (DQE)

Resonance energy transfer efficiencies were estimated from the extinction of the donor fluorescence intensities. The absorption and fluorescence spectra of the donor (alone) and the fluorescent labeling complex were obtained in order to determine the relative concentrations of each in the fluorescence experiments. Donor excitation was used to obtain the emission spectra of both compounds. Afterwards, the DQE was calculated using the formula:

% DQE = (1-F^ET A / FA^ET) x 100 where F is the fluorescence intensity of the donor alone, F^ET is the fluorescence intensity of the complex at the donor wavelength, A is the absorbance at the excitation wavelength of the donor alone, and A^ET is the absorbance at the excitation wavelength of the fluorescent labeling complex.

e) Synthesis of fluorochromes

Amino cyanines (CY3 NH2, CY3 (NH2) 2 and CY3NH2SO3) and carboxyalkyl cyanines (CY5COOH, CY30 (SO3) 2, CY5 (SO3) 2 and CY7 (SO3) 2) needed as precursors for energy transfer fluorochromes to Through the methods previously described in Ernst, LA et al., "Cyanine Dye Labeling Reagents for Sulphydryl Groups", Cytometry, vol. 10, pp. 3-10 (1989), Hammer, FM, "The Cyanine Dyes and Related Compounds", (Wiley, pub. New York 1964), Mujumdar, RB et al., "Cyanine Dye Reagents Containing Isothiocianate Groups" Cytometry, vol.10, pp 11-19 (1989); Mujumdar, RB et al. "Cyanine Dye Labeling Reagents: Sulphoindocyanine succinimidyl ester", Bioconjugate Chemistry, vol., 4, pp. 105-111 (1993); Southwick, PL et al. "Cyanine Dye Labeling Reagents: Carboxymethylindocyanine succinimidyl esters", Cytometry, vol. 11, pp. 418-430, (1990). Next, the synthesis and properties of an amino-cyanine fluorochrome, CY3NH2SO3 and its conjugation with the sucinimidelic ester of CY5 (SO3) 2 to form complex 1 are described. Tables 3 and 4 describe the spectral properties of all fluorochromes. Non-symmetrical trimetine-carbocyanine CY3NH2SO3 was synthesized in four steps. Is hac and reference to Table 1 for the structures (I) - (VI).

IS 2 170 204 T3

<img file="ES2170204T3_D0007.tif" />

1.5.1 Synthesis of 5-phthalimidomethyl-1 - (- carboxypentyl) -2,3,3-trimethylindole (III)

5-Phthalimidomethyl-2,3,3-trimethylindolenine (II) was synthesized according to the procedure of Gale and Wilshire, "The Amidomethylation and Bromination of Fischer's Base. The Preparation of Some New Polymethine Dyes ”, Aust. J. Chem. Vol. 30, pp. 689-694 (1977). Powdered N-hydroxymethylphthalimide (70 g, 0.4 mol) was added in small portions over a period of 45 minutes to a stirring solution of 2,3,3-trimethyl- (3H) -indolenine (I) (70 g 0.44 mole) in concentrated sulfuric acid (360 ml) at room temperature. The solution was stirred for 70 hours at room temperature before being poured into ice water. Alkalinization of the solution with concentrated amoanic hydroxide gave a yellow powder which was filtered and dried (111 g, 80% yield, mp 180-182 ° C). ¹H-NMR (DMSO- d₆), δ 7.8-7.95 (m, 4H, phthalimido), 7.4 (s, 1H, 4-H), 7.38 (d, 1H, J = 9.0 Hz, 6-H) , 7.2 (d, 1H, J = 9.0 Hz, 7-H), 4.7 (s, 2H, -CH2), 2.2 (s, 3H, CH3), 1.2 (s, 6H, - (CH3) 2).

IS 2 170 204 T3

This dry powder (10 g, 0.03 mol) and 6-bromohexanoic acid (9.1 g, 0.05 mol) were mixed in

1,2-dichlorobenzene (25 ml) and heated at 125 ° C for 12 hours under nitrogen. The mixture was cooled.

1,2-Dichlorobenzene was decanted and the solid mass was triturated with isopropanol until obtaining a free powder (11 g, 80% yield, mp 124-126^°C). ¹H-NMR (DMSO- d6), δ 7.8-7.95 (m, 4H, phthalimido), 7.4 (s, 1H, 4-H), 7.38 (d, 1H, J = 9, 0Hz, 6-H), 7.2 (d, 1H, J = 9.0 Hz, 7-H), 4.7 (s, 2H, -CH2), 4.5 (t, 2H, J =

7.5Hz, a- CH₂), 2.3 (t, 2H, J = 7Hz, e- CH₂), 1.99 (m, 2H, e-CH₂), 2.3-1.7 (m, 4H, γ-CH₂ and δ- CH₂melted with s of 6H- (CH3) 2).

1.5.2 Synthesis of 1- (e-carboxypentyl) -2,3,3-trimethylindoleninium-5-sulfonate (IV)

Compound (IV) was synthesized according to the procedure described above by Mujumdar, RB et al., Bioconjugate Chemistry (1993), supra. The potassium salt of 2,3,3-trimethylindoleninium-5-sulfonate (11 g, 0.04 mol) and 6-bromohexanoic acid (9.8 g, 0.05 mol) were mixed in 1,2-dichlorobenzene (100 ml) and heated to 110^°C for 12 hours under nitrogen. The mixture was cooled. 1,2-Dichlorobenzene was decanted and the solid mass was triturated with isopropanol until obtaining the free powder (11 g, 80% yield), λ max (water) 275 nm:¹H-NMR (D2O), δ 8.13 (s, 1H, 4-H), 8.03 (dd, 1H, J = 9.0, 1.1 Hz, 6-H), 7.2 (d , 1H, J = 9.0 Hz, 7-H), 4.51 (t, 2H, J = 7.5 Hz, a-CH2), 2.25 (t, 2H, J = 7.5 Hz, γCH₂), 1.99 (m, 2H, β-CH₂), 1.35-1.66 (m, 4H, δ-CH₂, y-CH₂), 1.61 (s, 6H, - (CH₃)₂). Rf = 0.55 (C-18, water-methanol, 25%).

1.5.3 Synthesis of the intermediary (V)

A solution of 1- (e-carboxypentyl) -2,3,3-trimethylindoleninium-5-sulfonate (IV) (10 g, 0.03 mol) and N, N-dimethylformamide (7.2 g, 0, 04 moles) in acetic acid (20 ml) at reflux for 1 hour. Acetic acid was removed on a rotary evaporator and the product was washed with ethyl acetate (3 x 50 ml), after which a dark brown solid was obtained. λ max (water) 415 nm Rf = 0.32 (C-18, 25% methanol in water). The crude product obtained was used for the next reaction without further purification. The solid (3.8 g) was dissolved in a mixture of acetic anhydride (10 ml) and pyridine (5 ml). 5-Phthalimidomethyl-1- (e-carbopentyl) -2,3,3-trimethylindole (III) (2.5 g, 6 mmol) was added and the reaction mixture was heated to 110^°C for 1 hour. The solution was cooled and diluted with dietary ether (500 ml). The product was separated as a red powder from which the supernatant fluid was separated by decantation. It was dissolved in a minimum volume of methanol and reprecipitated with 2-propanol. The product was collected on filter paper and dried to yield 5.3 g of compound (V). It was purified by flash column chromatography on reverse phase C-18 using a mixture of water and methanol as eluent (1.6 g, 30% yield). λ max (water) 554 nm, e 1.3x10⁵ L / mol.cm. ¹H-NMR (CD₃OD), δ, 8.5 (t, 1H, J = 14 Hz, β-proton of the bridge), 7.8-8.0 (m, 6H, 4 protons of the phthalimido group and 4-H and 6-H sulfoindole ring), 7.55 (s, 2H, 4'-H), 7.6 (d, 1H, J = 12Hz, 6'-H), 7.3 (two d, 2H, 7-H and 7'-H), 6.1-6.3 (t, 2H, a, a'- bridge protons), 4.1 (m, 4H, a, a'- CH2-), 2.9 (t , 2H, J = 7Hz, -CH2COOH), 1.4-2.0 (m, 21H, three -CH2, one -CH3, and two - (CH3) 2, the methyl protons of the phthalimidomethyl group fuse into one water signal at 4.8.

1.5.4 Hydrolysis of (V) to give (VI)

Compound (V) (1 g, 1.1 mmol) was dissolved in concentrated hydrochloric acid (5 ml) and heated under reflux for 12 hours. After cooling, the crystalline phthalic acid was removed by filtration. The filtrate was concentrated with a rotary evaporator and then slowly neutralized with concentrated ammonium hydroxide while keeping the temperature below 30^°C. Pure fluorochrome CY3NH2SO3 (VI) was obtained by reverse phase column chromatography using a mixture of water and methanol as eluent. λ max (methanol) 552 nm.¹H-NMR (DMSO-d6), δ, 8.45 (t, J = 7.2 Hz, 1H, 9-H), 7.3-7.9 (m, 6H, aromaotic protons), 6.55 (dd, 2H, 8 and 8'-H), 4.5 (m, 4H, N-CH2), 4.1 (s, 2H, CH2NH2), 2.15 (t, 2H, CH2COOH), a, a'-bridge protons), 4.1 (m, 4H, a, a'-CH2-), 2.9 (t, 2H, J = 7Hz, -CH2COOH), 1.25-1.8 (broad m, 24H, two - (CH2) 2 and 6-C- (CH3) 2. Rf = 0.415 (RP C 18.16% methanol in water).

1.55 Synthesis of complex 1

CY5 succinimidyl ester dry powder (SO₃)₂ (425 mg, 0.26 mmol) prepared by the method of Mujumdar et al., Bioconjugate Chemistry, vol. 4, pp. 105-111 (1993) in small portions to a well stirred solution of CY3NH₂SW₃ (200 mg, 0.26 mmol) in 10 ml of carbonate-bicarbonate buffer (0.1 M, pH 9.4). Stirring was continued for 30 more minutes, after which the reaction was purified by flash column chromatography on C-18 inversion powder using

ES 2 170 204 T3 water-methanol (6.3: 3.7) as eluent. 5 ml fractions were collected and monitored by TLC: Fractions containing CY5 (SO3) 2 acid and CY3NH2SO3 were discarded. The violet fractions were checked by ultraviolet light in methanol and the fractions containing complex 1 fluorochrome were pooled (Table 2). Evaporation of the solvent produced complex 1 as a violet powder, (37% yield). Rf = 0.45 (RP 37% methanol-water). The spectrum of¹H-NMR recorded in D₂Or it showed broad signals that were difficult to map. The fluorochrome was purified on a strongly acidic ion exchange column (Dowex 50, form H⁺). High resolution BAR mass spectrometry showed ion (M + H)⁺ at 1391.83 (C73H91N5 O16S3 + H requires 1391.73). 1.5.6 Energy Transfer Cyanine Dye Succinimidyl Ester

Complex 1 (60 mg, 0.04 mmol) was dissolved in a mixture of dry DMF (1 ml) and dry pyridine (0.05 ml). Disuccirnmidyl carbonate (DSC) (46 mg, 0.18 mmol, 1.5 equivalents / carboxyl group) was added and the mixture was stirred at 55-60^°C for 90 minutes under nitrogen. After diluting the mixture with dry diethyl ether (20 ml), the supernatant was decanted. The product was washed several times with ether, filtered and vacuum dried. The formation of the active succinimidolic ester was confirmed by its reaction with benzylamine in DMF or its reaction with taurine in a bicarbonate buffer of pH 9.4. It was developed by reverse phase C-18 TLC stained with the conjugate, the succinimidiolic ester and the hydrolyzed carboxylate product for comparison with the water-methanol mixture (1: 1). Rf = 0.78 (Acid), 0.3 (Benzylamine adduct).

1.5.7. Reaction of succinimidyl ester with antibody and streptavidin

A stock solution of complex 1 fluorochrome succinimidolic active ester in dry DMF (1 mg / 100: 1) was obtained. In one of the samples, one milligram of sheep globulin was dissolved in 0.25 ml of carbonate / bicarbonate buffer (approximately 6.45 nmol / 0.25 ml). In another example, streptavidin (1 mg) was dissolved in 0.25 ml of the carbonate / bicarbonate buffer. Appropriate volumes of the fluorochrome stock were added to 0.25 ml portions of each proteone solution to produce the desired starting fluorochrome to antibody ratios and each reaction mixture was stirred at room temperature for 30 minutes. The protein conjugate was separated from the unreacted fluorochrome in each mixture by gel filtration chromatography on Sephadex G-50 (0.7 x 20 cm column), using PBS, pH 7.4, containing 0.1% azide. . The dye conjugated proteones eluted as well separated color bands from the unreacted fluorochrome. The normalized excitation spectrum of the complex conjugate 1-streptavidin in PBS is shown in Figure 4. Figure 5 shows the absorbance spectrum of the sheep 1-IgI complex in PBS. Figure 6 shows the flow cytometric analysis of the 1-streptavidin complex used to detect the CD3 antibody.

Other energy transfer donor and acceptor complexes according to the invention were prepared from cyanine fluorochromes in order to investigate the energy transfer efficiency of said compounds. Table 2 shows the structures of these analogues.

Table 3 shows the spectral properties of the precursor cyanines and in Table 4, those of the complexes.

IS 2 170 204 T3

TABLE 2

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IS 2 170 204 T3

TABLE 2 (Continued)

<img file="ES2170204T3_D0009.tif" />

"A" designates the fluorochrome that acted as an energy acceptor and "D" designates the fluorochrome that acted as an energy donor.

The energy transfer complexes represented in table 2 are the following, complex 1, CY3NH2 SO3 (donor) + CY5 (SO3) 2 (acceptor); complex 2, CY3-O (SO3) 2 (donor) + CY3NH2 (acceptor); complex 3, CY3NH2 (donor) + CY5COOH (acceptor); complex 4, CY3NH2 (donor) + CY5 (SO3) 2 (acceptor); complex 5, CY3 (NH2) 2 (donor) + CY7 (SO3) 2 (acceptor); complex 6, 2 CY3NH2 SO3 (donor) + CY5 (SO3) 2 (acceptor).

TABLE 3

Spectrum properties of cyanine dyes used as precursors for the fluorescent energy transfer complexes of the invention

<td>Colorant</td><td>Solvent</td><td>Maximum absorption (nm)</td><td>Maximum emission (nm)</td><td>Quantum yield (φ)</td>

<td colspan="5">Cyanine dyes containing amine</td>

<td>CY3 NH₂</td><td>Methanol</td><td> 552</td><td> 569</td><td> 0,05</td>

<td></td><td>PBS</td><td> 548</td><td> 563</td><td> 0,05</td>

<td>CY3 (NH2)₂</td><td>Methanol</td><td> 552</td><td> 569</td><td> 0,05</td>

<td></td><td>PBS</td><td> 548</td><td> 653</td><td> 0,05</td>

<td>CY3 NH₂ SW₃</td><td>Methanol</td><td> 556</td><td> 573</td><td> 0,08</td>

<td></td><td>PBS</td><td> 548</td><td> 653</td><td> 0,09</td>

IS 2 170 204 T3

TABLE 3 (Continued)

<td>Colorant</td><td>Solvent</td><td>Maximum absorption (nm)</td><td>Maximum emission (nm)</td><td>Quantum yield (φ)</td>

<td colspan="5">Cyanine dyes containing carboxyl</td>

<td>CY5COOH</td><td>Methanol</td><td> 658</td><td> 685</td><td> 0,22</td>

<td></td><td>PBS</td><td> 648</td><td> 667</td><td> 0,13</td>

<td>CY5 (SO3) 2</td><td>Methanol</td><td> 658</td><td> 677</td><td> 0,4</td>

<td></td><td>PBS</td><td> 650</td><td> 667</td><td> 0,27</td>

<td colspan="5">Cyanine dyes containing carboxyl</td>

<td>CY3-O (SO3) 2</td><td>Methanol</td><td> 492</td><td> 506</td><td> 0,2</td>

<td></td><td>PBS</td><td> 486</td><td> 500</td><td> 0,09</td>

<td>CY7 (SO3) 2</td><td>Methanol</td><td> 758</td><td> 789</td><td>ND^to</td>

<td></td><td>PBS</td><td> 750</td><td> 777</td><td>ND^to</td>

^to ND means not determined. PBS stands for Phosphate Buffered Saline.

Energy transfer efficiency was estimated by calculating the amount of donor fluorescence quenching that occurs (DQE) when the acceptor binds. Some extinction may occur in ways other than resonance energy transfer when the acceptor is attached. However, the preferred cyanine donors for the fluorescent labeling complexes of the present invention are relatively intensive for their molecular environment. On the other hand, the addition of large substituents to trimethine cyanines tends to increase, rather than decrease, their fluorescence. Therefore, the DQE can be equal to the energy transfer efficiency. Estimated energy transfer efficiencies based on DQE measurements can be equal to energy transfer efficiency. Estimated energy transfer efficiencies based on DQE measurements ranged from 50% to 99% and the wavelength shifts between maximum donor absorption and maximum acceptor terminal emission (DI) ranged from 83 nm to 294 nm. nm.

Two of complexes 1 and 6 are capable of absorbing light at the argon laoser wavelength, 488 nm. Complex 1 contains a single donor and a single acceptor and Complex 6 contains 2 donors per acceptor. Complex 1 has 3 carboxyl groups and Complex 6 has 4 carboxyl groups. These are converted to active succinimidyl osters upon activation. The absorption spectra of complex 1 and complex 6 in methanol are shown in Figure 2.

Complex 1 was selected for further study. As shown in Figures 3 (a) and 3 (b), the absorbance (continuous line) of Complex 1 varies slightly in phosphate buffered saline (Figure 3 (b)) and in methanol (Figure 3 (a) ), on the other hand the fluorescence remains unchanged. The emission from the donor component at 572 nm is very weak compared to the emission from the acceptor at 675 nm, as would be expected when energy transfer is efficient.

In figure 5 it is shown that sheep antibodies can be easily labeled with activated complex 1. Conjugates obtained from complex 1 conjugated to sheep IgG were tested at various dye: proteon ratios. The minimum dye: proteone ratio is represented by the lon having its first peak (at approximately 270 nm) at 0.8 and the maximum dye: proteon ratio is represented by the lon having its first peak (at approximately 270 nm). less than 0.4. No dimer formation was observed in which acceptor or donor fluorochromes participated with increasing dye: proteone ratios. Each of Complex 1 contains up to 3 groups

ES 2 170 204 T3 reagents. More reactive groups can be used as long as crosslinking does not occur. Labeling conditions should be used that avoid protein cross-linking that quenches fluorescence. Southwick PL et al. "Cyanine Dye Labeling Reagents: Carboxymethylindocyanine succinimidyl esters", Cytometry, vol. 11, pp. 418-430 (1990) has previously observed cross-linking by doubly activated cyanines, and it can be minimized by limiting the concentration of the protein to be labeled to about 1 mg / ml.

Upon binding to the antibodies, the quantum yield of the complex was improved threefold, as shown in Table 4. This is believed to occur because the non-radiation deactivation pathway of the CY3 and CY5 components of complex 1 is reduced. due to their restricted mobility when attached to the surface of the protein. Other mechanisms of conformational mobility restriction are known to increase the fluorescence efficiency of cyanine fluorochromes, as described in Mujumdar, RB et al., "Cyanine Dye Labeling Reagents: Sulphoindocyanine Succinmidyl Ester", Bioconjugate Chemistry, vol. 4 pp. 105-111 (1993). In fact, when complex 1 was dissolved in glycerin, the quantum yield increased several times, as shown in Table 4.

Activated complex 1 can be used as a fluorescent label for two-color flow cytometry experiments with excitation at 488 nm. Figure 6 shows the scatter plot. Human T lymphocytes were used to compare the complex 1 label with another two-color reagent, R-focoerythrin, which is also excited at 488 nm and emits at 575 nm. Complex 1 (fluorochrome / protein 4) -labeled streptavidin was used to detect biotinylated CD3 antibody, which labels all T cells. In the same lymphocyte sample, phycoerythrin (PE) -labeled anti-CD4 was used to label the cell subgroup cell collaborators T. Thus, in the total population of lymphocytes there is a population of cells that do not contain CD3 or CD4 (that is, CD3 and CD4 negative, represented in the lower left population of the 2-dimensional scatter plot in Figure 6), a subgroup of complex 1-labeled CD3 positive cells that do not have a phycoerythrin signal (i.e. CD3 positive and CD4 negative, represented in the upper left population of figure 6) and a third subgroup consisting of cells labeled with complex 1 that are stained with phycoerythrin (i.e. DC3 and CD4 positive, represented in the upper right population of figure 6). It is clear that complex 1 gave a baseline separation of positive and negative cell populations, and that there was minimal spread of complex 1 fluorescence in the phycoerythrin channel. Complex 1 fluorochrome gave a signal three times brighter when fluorochrome was excited at 514 nm.

TABLE 4

Spectrum properties of energy transfer complexes

<td>Colorant</td><td>Abs max (nm)</td><td>Wavelength of excitement (nm)</td><td>Em max (nm)</td><td>Performance quantum (Φ)</td><td>Energy transferred (%)</td><td>Wavelength shift^and (nm)</td>

<td>Complex</td><td> 556 (9,5)</td><td> 488</td><td> 675</td><td> 0,32</td><td> 91</td><td> 119</td>

<td> 1^to</td><td> 652(14,3)</td><td> 514</td><td> 676</td><td> 0,37</td><td> 92</td><td> 120</td>

<td></td><td></td><td> 600</td><td> 673</td><td> 0,49</td><td> -</td><td> -</td>

<td>Complex</td><td> 536 (16)</td><td> 488</td><td> 675</td><td> 0,03</td><td> 89</td><td> 139</td>

<td> 1^b</td><td> 658 (16)</td><td> 514</td><td> 673</td><td> 0,04</td><td> 89</td><td> 137</td>

<td></td><td></td><td> 600</td><td> 668</td><td> 0,21</td><td> -</td><td> -</td>

<td>Complex</td><td> 558,658</td><td> 488</td><td> 674</td><td> 0,11</td><td> 95</td><td> 116</td>

<td> 1^c</td><td></td><td> 514</td><td> 673</td><td> 0,13</td><td> 95</td><td> 116</td>

<td>(PBS)</td><td></td><td> 600</td><td> 676</td><td> 0,14</td><td> -</td><td> -</td>

<td>Complex</td><td> 562,658</td><td> 488</td><td> 674</td><td> 0,19</td><td>ND</td><td>ND</td>

<td> 1^d</td><td></td><td> 514</td><td> 674</td><td> 0,32</td><td>ND</td><td>ND</td>

<td></td><td></td><td> 600</td><td> 674</td><td> 0,39</td><td>ND</td><td>ND</td>

IS 2 170 204 T3

TABLE 4 (Continued)

<td>Colorant</td><td>Abs max (nm)</td><td>Wavelength of excitement (nm)</td><td>Em max (nm)</td><td>Performance quantum (Φ)</td><td>Energy transferred (%)</td><td>Wavelength shift^and (nm)</td>

<td>Complex</td><td> 490(13)</td><td> 466</td><td> 571</td><td> 0,15</td><td> 89</td><td> 81</td>

<td> 2^to</td><td> 554(9,5)</td><td></td><td></td><td></td><td></td><td></td>

<td>Complex</td><td> 545(9,5)</td><td> 514</td><td> 679</td><td> 0,08</td><td> 83</td><td> 133</td>

<td> 3^to</td><td> 658(14,3)</td><td></td><td></td><td></td><td></td><td></td>

<td>Complex</td><td> 550 (9,4)</td><td> 514</td><td> 674</td><td> 0,2</td><td> 96</td><td> 124</td>

<td> 4^to</td><td> 656(14,2)</td><td></td><td></td><td></td><td></td><td></td>

<td>Complex</td><td> 445(9,5)</td><td> 520</td><td> 782</td><td>ND</td><td> 99</td><td> 226</td>

<td> 5^to</td><td> 754(14,4)</td><td></td><td></td><td></td><td></td><td></td>

<td>Complex</td><td> 556(9,5)</td><td> 488</td><td> 674</td><td> 0,23</td><td> 49</td><td> 118</td>

<td> 6^to</td><td> 652(14,4)</td><td> 514</td><td> 674</td><td> 0,24</td><td> 50</td><td> 118</td>

<td></td><td></td><td> 600</td><td> 674</td><td> 0,34</td><td> -</td><td> -</td>

<td>Complex</td><td> 548(20,0)</td><td> 488</td><td> 566</td><td> 0,05</td><td> 43</td><td> 118</td>

<td> 6^b</td><td> 652(15,0)</td><td> 514</td><td> 564</td><td> 0,05</td><td> 38</td><td> 116</td>

<td></td><td></td><td> 600</td><td> 668</td><td> 0,23</td><td> -</td><td> -</td>

^to = in methanol ^b = in PBS c = complex 1 in streptavidin, d / p = 4 ^d = in glycerin ^and = difference between Emmax (A) - Abmax (D)

ND means not determined

Example 3

Various other complexes were synthesized with the general structure shown in formula (10) below. Table 5 shows its spectral properties in methanol solution.

IS 2 170 204 T3

<img file="ES2170204T3_D0010.tif" />

This series of spectra demonstrates efficient energy transfer with long Stoke shifts. Each emission spectrum presents substantially all of the emission from the final acceptor fluorochrome of each series with only minimal emission from the donor fluorescein, or the intermediate cyanine.

Multi-parameter analysis of various samples can be carried out to detect the presence of target biological compounds. Each of the samples is marked by well known marking methods with a different complex. For example, a sample that presumably contains a target biological compound is incubated with a uenic fluorochrome, such as fluorescein, Cascade Blue, a BODIPY dye, or one of the stiffened monomethine dyes, or CY3O (SO3) 2 or CY3 (SO3 ) 2, which all emit at a wavelength range of 500 to 575 nm (green to orange). A second sample that presumably contains the target biological compound (the same compound or another compound different from that of sample 1) is incubated with a complex of the invention, for example, fluoresceinCY3NH2, which will absorb light at 488 nm and fluoresce at 574 nm (orange). Additional samples that contain presumably another target compound are incubated with other labeling complexes according to the invention, such as fluorescein CY3-CY5 and fluorescein-CY3-CY7, which both emit light at 488 nm, but fluoresce at 672 and 782 nm. nm respectively (from red to near infrared). After an adequate period of time to allow the fluorescent labels to bind with the target compounds, the unbound label is washed away and the labeled samples mixed. Detection is possible with a single wavelength excitation source, that is, at 488 nm from the laser line. Each differently labeled sample will fluoresce with a different color at the emission wavelength of its particular brand. Those skilled in this field will recognize that the fluorescent labeling complexes of the present invention can be used for various immunofluorescence techniques, including direct and indirect immunoassays, and other known fluorescence detection methods. The conditions of each incubation, eg, pH, temperature and time, are those known to those of skill, although room temperature is generally preferable. If reacted with an amine, a pH of 9.4 is preferable. The pH is adjusted depending on the optimal reaction conditions for the particular reactive groups according to known techniques.

IS 2 170 204 T3

TABLE 5

<td>Complex</td><td>Excite- cion (nm)</td><td>Fluoro- chrome absorption max. #1</td><td>Fluoro- chrome absorption max. #two</td><td>Fluoro- chrome absorption max. #3</td><td>Emi- Zion (nm)</td><td>Yield I lie αιΐιι- Tico (φ)</td><td>Stokes displacement (nm)</td><td>Energy transfer efficiency (%)</td>

<td>Fluoresceon- CY3 (NH₂)</td><td> 488</td><td> 500</td><td> 558</td><td> -</td><td> 574</td><td> 0,041</td><td> 74</td><td> 98,3</td>

<td>Fluorescein- CY3 (NH2) 2- CY5 (SO3) 2</td><td> 488</td><td> 500</td><td> 560</td><td> 650</td><td> 672</td><td> 0,1566</td><td> 172</td><td> 99</td>

<td>Fluorescein- CY3 (NH2) 2- CY7 (SO3) 2</td><td> 488</td><td> 500</td><td> 560</td><td> 754</td><td> 782</td><td> -</td><td> 282</td><td> 99</td>

Fluorescent labeling complexes can be used to form reagents by covalently binding the complexes to a carrier material, such as polymer particles, cells, glass beads, antibodies, proteones, enzymes, carbohydrates, lopids and nucleotides or nucleic acids (DNA and RNA) and analogs that have formed derivatives to include at least a first reactive group capable of forming a covalent bond with the functional group in the marking complex (or a functional group capable of forming a binding covalent with a reactive group in the complex, as described above) and at least one second reactive group (or functional group, as the case may be) that has specificity to form and be capable of forming a covalent bond with a target biologic compound, such as antibodies, cells, drugs, antigens, bacteria, viruses and other microorganisms. When the carrier has functional groups, it can be an antibody or DNA suitable for binding to an antigen or a complementary DNA sequence, respectively. When the carrier material has reactive groups on it, the carrier may be a polymer particle or an antigen suitable for DNA binding or an antibody for example. Techniques for covalently attaching fluorochromes to carrier molecules such as those mentioned are known in the art and are available in the literature. The carrier material may include further nucleotides that form derivatives to contain an amino, sulfhydryl, carboxyl, carbonyl, or hydroxyl group, and oxy- or deoxypolinucleic acids that form derivatives to contain an amino, triphosphoryl, sulfhydryl, carboxyl, carbonyl, or hydroxyl group. Functional groups in the carrier material that are complementary to the reactive groups of the labeling complexes of the invention, ie, form covalent bonds with them, include amino, sulfhydryl, carboxyl, carbonyl, and hydroxyl groups.

In Table 6 below, a comparison of the energy transfer complexes of the present invention with conventional R-phycoerythrin dyes is set forth.

IS 2 170 204 T3

TABLE 6

Complex 2 against R-phycoerythrin

<td></td><td>R-phycoerythrin</td><td>Complex 2</td>

<td>Excitation wavelength (nm)</td><td> 488</td><td> 488</td>

<td>Emission wavelength (nm)</td><td> 580</td><td> 578</td>

<td>488 laser line flow cytometer</td><td>PE fluorescence was greatly reduced at pH 8.5 and estinguted at pH 9.5</td><td>The signals were stable throughout the entire pH range.</td>

<td>P.M</td><td> 240000</td><td> 1667</td>

<td>Stained</td><td>Does not easily penetrate intracellular tissues to reach the target antigen</td><td>The labeled antibody penetrates intracellular tissues to reach the target antigen</td>

<td>Bonding speed</td><td>The antigen binding rate is low</td><td>Quick join</td>

The energy transfer complexes of the invention provide a valuable set of fluorescent labels that are particularly useful for multi-parameter analysis and, most importantly, have a sufficiently low molecular weight to allow materials labeled with the complexes. fluorescents penetrate all structures. As such, the complexes are well suited for use as DNA probes. The complexes of the present invention and the reagents that can be obtained from them offer a wide variety of fluorescent labels with long Stokes shifts. Those skilled in the art will recognize that the complexes of the invention can be used in various fluorescence applications over a wide range of the visible spectrum.

Figures (edit)

Figure 1 is a schematic illustration of the overlap of the absorption and emission spectra of four cyanine fluorochromes that can be used in the energy transfer marking complexes of the present invention.

Figure 2 illustrates the absorption spectra of two fluorescent labeling complexes, complex 1 (continuous lone) in methanol, consisting of a cyanine donor and a cyanine acceptor, and complex 6 (discontinuous lone), in methanol. consisting of two cyanine donors and one cyanine acceptor.

Figures 3 (a) and (b) illustrate the absorbance (continuous line) and emission (discontinuous line) spectra of complex 1 of the invention obtained from cyanine dyes of trimethine and pentamethine in (a) methanol and (b) PBS.

Figure 4 illustrates the normalized excitation spectra of complex 1 in PBS (continuous line), methanol (_ _), glycerol (___) and complex conjugate 1-streptavidin in PBS (-).

Figure 5 illustrates the absorbance spectra in PBS of conjugates of sheep IgG-complex 1 at various ratios of dye molecule: proteon (1 - 4: 1) which shows that the dimer formation in which it participates is not evident. either the donor or the acceptor by increasing the relationship between

ES 2 170 204 T3 dye: protein.

Figure 6 illustrates the two-color flow cytometry analysis of human lymphocytes labeled with CD4-PE and anti-CD3-streptavidin-complex 1 to label the T cell helper cell subgroup and the total T cell subgroup, respectively, showing a subset of complex 1 labeled cells without the PE signal and a second subset of complex 1 labeled cells that are stained with PE.