HK1168122B - Novel fluorescent dyes and uses thereof - Google Patents

Description

Novel fluorescent dyes and their use

Technical Field

The present invention relates to fluorescent dyes, and more particularly to ultraviolet excitable fluorescent dye compositions, compounds and conjugates, and methods of their use and manufacture.

Background

Fluorescent dyes are widely used to label, detect, and/or quantify components in a sample. Various methods for such detection and/or quantification include fluorescence microscopy, fluorescence immunoassay, flow cytometric analysis of cells, and a variety of other applications. In general, for many applications that utilize fluorescent dyes as detection tools, it is necessary to conjugate the fluorescent dye to ligands such as proteins, antibodies, enzymes, nucleotides, nucleic acids, and other biological and non-biological molecules to prepare the dye-labeled ligand. Dye-labeled ligands are important reagents that confer specificity for subsequent biochemical reactions in which fluorescent dyes provide a means for detecting and/or quantifying the reaction.

The choice of fluorescent dye is particularly important in applications where multiple, multi-color assays are employed, as well as in a variety of other applications, such as fluorescence microscopy, fluorescence immunoassay, flow cytometry. Notably, certain detection applications require fluorophores that are ultraviolet excitable with a particular excitation range.

In particular, there is a need for a fluorescent dye that: which can be efficiently excited by a 405nm violet laser in a multi-color flow cytometer. The target dyes used in these applications should have essentially the following characteristics: 1) its excitation spectrum has a maximum around 405nm, 2) a strong spectrally resolvable emission maximum, 3) a large Stokes' shift, preferably at least 50nm, and 4) the ability of the fluorochrome to couple to the biomolecule through a reactive group. To date, there has been a lack of ultraviolet excitable fluorophores that can be conjugated to different ligands to provide dye-labelled reagents with optically and electronically separable fluorescence spectra.

The fluorescent dyes of the present invention are similar in structure to firefly luciferin compounds. These compounds have previously been used as chemiluminescent reagents by which light is generated by the oxidative catalysis of fluorescein. Luciferin is a substrate of the enzyme luciferase, which is oxidized in the presence of luciferase to produce oxyluciferin and energy, which is released as light. For these types of assays, the luciferase-luciferin reaction provides the basis for a simple, rapid, sensitive assay for a wide variety of substances (Karicka, lj., Analytical Biochemistry, 175, 14-21 (1988)).

Although these chemiluminescent dyes based on the structure of firefly luciferin have gained widespread use, such use does not demonstrate the ability of compounds with a structure similar to firefly luciferin to: having the function of a fluorescent dye that is excitable in a specific part of the ultraviolet spectrum. Such ultraviolet excitable fluorescent dyes are particularly advantageous for a wide range of applications including, but not limited to, those utilizing multiple, multi-color fluorescence assays.

Disclosure of Invention

The present invention relates to novel ultraviolet excitable fluorescent dyes based generally on the structure of firefly luciferin, compositions containing such fluorescent dyes, intermediates to such fluorescent dyes, and methods of their use.

In one aspect, there is provided a composition of fluorescent dyes having the following general formula (I):

wherein the content of the first and second substances,

X₁and X₂Independently is S or O;

Y₁and Y₂Independently is halogen, alkyl, haloalkyl, alkoxy, or alkenyl, or Y₁Or Y₂One of which is H and the other is haloalkyl;

wherein each W is independently H or alkyl;

Z₁is H or alkyl;

wherein Z₁And one W group may be deleted, and if deleted, they are replaced by C in the ring C₄And C₅Additional keys in between, which are indicated as dashed lines;

Z₂comprising a tagged substituent of formula-L-RG, wherein L is a bond or a linking group; and is

RG is a reactive group that enables a fluorescent dye to bind to another molecule.

In another aspect, synthetic intermediates useful in the synthesis of the fluorescent dyes of the present invention are provided. In one embodiment, the synthetic intermediates are represented by the following general formula.

Wherein the content of the first and second substances,

Y₁and Y₂Independently a halogen, alkyl, haloalkyl, alkoxy, or alkenyl,

or Y₁Or Y₂One of which is H and the other is haloalkyl.

In another aspect, dye-conjugates comprising a fluorescent dye conjugated to a biomolecule are provided.

In another aspect, methods are provided for using fluorescent dyes and dye-conjugates to localize or detect the presence or interaction of an analyte or ligand in a sample. In particular embodiments, methods of using the dye-conjugates for detecting the presence of complementary biomolecules bound to the dye-conjugates are provided.

In other embodiments, reagents and kits are provided comprising a fluorescent dye, dye intermediate, or dye-conjugate for detecting the presence of a complementary biomolecule bound to a dye-conjugate of the invention.

Drawings

FIG. 1 depicts the general structure of the UV excitable fluorescent dye of the present invention.

FIGS. 2A-2D depict synthetic schemes for fluorescein derivatives.

Figures 3A and 3B depict a synthetic scheme for dehydrofluorescein acid derivatives.

FIG. 4 depicts a histogram using two-color flow cytometry showing the relative performance of CD4-Dye2 conjugated to a CD4-Pacific Orange conjugate.

FIG. 5 depicts a histogram using ten color flow cytometry showing the relative performance of CD4-Dye3 conjugated to a CD4-Pacific Orange conjugate.

Detailed Description

Definition of

All scientific and technical terms used in the present application have the meanings commonly used in the technical field unless otherwise specified. As used in this application, the following text or terms have a limiting meaning.

The term "activated ester" as used herein refers to an ester that reacts spontaneously with an amino group. The activated ester generally has the formula-COR, where R is a good leaving group. Activated esters include, but are not limited to, succinimidyloxy (-OC)₆H₄O₂) Also known as N-hydroxy-succinimide ester; sulfosuccinimidyloxy (-OC)₆H₃O₂-SO₃H) -1-oxybenzotriazolyl (-OC)₆H₄N₃)。

The term "alkyl" as used herein refers to a branched, straight or cyclic hydrocarbon group containing from 1 to 20 carbons. The term "alkyl" includes "lower alkyl" groups typically containing up to 8 carbons.

The term "alkenyl" as used herein refers to a branched, straight or cyclic hydrocarbon group containing at least one C ═ C double bond and containing 2 to 20 carbons. The term "alkenyl" includes "lower alkenyl" groups typically containing up to 8 carbons.

The term "alkynyl" as used herein refers to a branched or straight chain hydrocarbyl group containing at least one C.ident.C triple bond and containing from 2 to 20 carbons. The term "alkynyl" includes "lower alkynyl" groups typically containing up to 8 carbons.

The term "aryl" as used herein refers to an aromatic ring-containing group containing 1 to 5 carbocyclic aromatic rings, which can be substituted with 1 or more substituents other than H.

The term "analyte" as used herein refers to a substance: its presence or amount in the sample will be measured by the assay. The analyte may comprise a reactive group such as a group via which the dye of the invention can be conjugated to the analyte. Analytes include organic molecules and biomolecules to which specific binding partners (specific binding partners) have specific binding affinities. Exemplary analytes include, but are not limited to: single or double stranded DNA, RNA, DNA-RNA complexes, oligonucleotides, antibodies, antibody fragments, antibody-DNA chimeras, antigens, haptens, proteins, lectins, avidin, streptavidin and biotin. Other exemplary analytes also include drugs and hormones.

The term "conjugated molecule" or "CM" as used herein refers to a biological or non-biological component linked to at least one dye of the present invention. These components include, but are not limited to: antigens, antibodies, carbohydrates (e.g., monosaccharides, oligosaccharides, and polysaccharides), proteins, peptides, haptens, nucleosides, nucleotides, oligonucleotides, nucleic acids, polymers, viruses, microorganisms, or cells or cellular components. "conjugated molecules" also include solid supports (e.g., synthetic supports, chromatographic supports, membranes, or beads).

The term "fluorescent dye" as used herein refers to a compound that: which absorbs light in the ultraviolet and violet regions of the electromagnetic spectrum and re-emits light in the blue region, thereby producing a detectable signal. "fluorescent dyes" also include fluorescent compounds having chemically reactive groups that facilitate attachment of the dye to the molecule being conjugated.

The term "dye-conjugate" as used herein refers to a molecule in which a fluorescent dye is attached to a biological or non-biological component. Such biological or non-biological components are also referred to as "conjugated molecules". In one embodiment, the attachment of the fluorescent dye to the molecule to be conjugated is via a covalent bond (covalent linkage). Examples of dye-conjugates include, but are not limited to, conjugates of: an antigen, an antibody, a carbohydrate, a protein, a peptide, a hapten, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a polymer, a solid support (e.g., a synthetic support, a chromatographic support, a membrane, or a bead), a virus, a microorganism, or a cell or cellular component.

The term "fluorophore" as used herein refers to a molecule that: which is capable of absorbing energy in one wavelength range and releasing energy in another wavelength range outside the absorption range. The term "excitation wavelength" refers to the wavelength range over which the fluorophore absorbs energy. The term "emission wavelength" refers to the wavelength range in which the fluorophore releases energy or emits fluorescence.

The term "halogen" as used herein refers to a fluorine, chlorine, bromine or iodine atom. Similarly, the term "halo" refers to a fluoro, chloro, bromo, or iodo group.

The term "haloalkyl" as used herein refers to an alkyl group wherein one or more hydrogen atoms are replaced by one or more halogen atoms. In one embodiment, the haloalkyl can be substituted with 1, 2, or 3 halo groups. The term "haloalkyl" also includes perfluoroalkyl. Examples of haloalkyl groups include trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl and the like.

The term "linking group" or "L" as used herein refers to a group used to attach a dye to a reactive group (or RG). The linking group may be a chemical bond, an atom, or another divalent or multivalent group. The linking group can comprise a linear or branched chain of atoms, some of which can be part of a cyclic structure. The linking group typically contains from 1 to about 50 non-hydrogen atoms, more typically from 1 to about 30 non-hydrogen atoms. The atoms comprising the chain are selected from the group consisting of C, O, N, S, P, Si, B, and Se atoms, preferably from the group consisting of C, O, N, P and S atoms that can covalently attach the fluorescent dye to another moiety such as a chemically reactive group. The halogen atoms may be present as substituents on the chain or ring. Typical functional groups containing attached substituents include: alkenyl, alkylene, arylene, alkenylene, ether, peroxide, carbonyl such as ketone, ester, carbonate, thioester, or amide groups, amine, amidine, carbamate, urea, imine, diimide salt, carbodiimide, hydrazine, diazo, phosphodiester, phosphotriester, phosphate ester, thioether, disulfide, sulfoxide, sulfone, sulfonate, sulfate, and thiourea groups. Examples of linking groups include substituted or unsubstituted polymethylene, arylene, alkylarylene, aralkylene, or arylthio groups.

A linking group according to the invention for linking a dye to a reactive group has the general structure R₁C(O)R₂Wherein R is₁Is a chemical bond or C attached to a dye_1-10Methylene (CH)₂) n; c (O) is carbonyl; r₂Is OH or a chemical bond attaching a carbonyl group to a reactive group RG. Examples of such linking groups include:

in another embodiment, the linking group has the general structure R, as shown below₁C(O)A₁R₃C(O)R₂Wherein R is₁Is a chemical bond or G as defined above_1-10A methylene group; a. the₁Or NH, S, or O; r₃Is alkenyl (CH)₂) n, a five-membered or six-membered ring having at least one unsaturated bond, or C_1-10Methylene (CH)₂) n in combination with a five-membered ring or a six-membered ring; r₂Is OH or a chemical bond attaching the terminal carbonyl group to the reactive group RG. Examples of such linking groups include:

the term "reactive group" or "RG" as used herein is an atom or group that: its presence may facilitate bonding to another molecule by covalent attachment or physical force. Examples of alternative reactive groups are shown in table 1. In some embodiments, for example, when the reactive group is a leaving group such as a halogen atom or a tosylate group, and the fluorescently labeled compound is covalently attached to another compound by a nucleophilic displacement reaction, attachment of the fluorescent compound of the invention to another compound will involve the loss of more than one atom from the reactive group. In other embodiments, when an addition reaction, such as Michael addition, occurs, or a cycloaddition reaction, such as diels-alder reaction, occurs, or when the reactive group is an isocyanate or isothiocyanate group, attachment of the fluorescently labeled compound to another compound through formation of a covalent bond will involve reconfiguration of the chemical bond within the reactive group. In yet other embodiments, the attachment does not involve covalent bond formation, but involves physical forces, in which case the reactive groups remain unchanged. And the physical force is: attractive forces such as hydrogen bonding, electrostatic or ionic attractive forces; hydrophobic attractions such as base stacking; and specific avidity interactions such as biotin-streptavidin interactions, antigen-antibody interactions, and nucleotide-nucleotide interactions.

TABLE 1

Reactive groups for chemically binding dyes to conjugated molecules

Reactive dyes are particularly useful for preparing dye conjugates using molecules containing more than one amino group, such as proteins, peptides, nucleotides, oligonucleotides, or haptens, when the reactive group is a moiety such as a carboxylic acid or an activated ester of a carboxylic acid. Reactive dyes are particularly useful for conjugation with thiol-containing molecules if the reactive group is maleimide or haloacetamide. If the reactive group is a hydrazide, the reactive dye is particularly useful for conjugation to periodate-oxidized carbohydrates and glycoproteins.

In one embodiment, the reactive group comprises a carboxylic acid, a succinimide ester of a carboxylic acid, an isothiocyanate, a haloacetamide, a hydrazine, a fatty amine, and a maleimide group. Methods for preparing each of these reactive groups are well known to those skilled in the art, and the use of these reactive groups for specific purposes is within the ability of those skilled in the art to recognize.

Bifunctional coupling agents can also be used to couple labels to organic and biomolecules using intermediate reactive groups (see L.J.Kricka, ligand-conjugate assay, published by Marcel Dekker, New York, 1985, pages 18-20, Table 2.2; and T.H.Ji, "bifunctional reagents", methods in enzymology, 91, 580-609 (1983)). There are two types of bifunctional agents: bifunctional reagents that are incorporated into the final structure, and bifunctional reagents that are not incorporated into the final structure and are used only for coupling two reactants.

The term "sample" as used herein refers to a liquid containing or suspected of containing more than one analyte to be assayed. Typical samples for analysis using fluorescent dyes are biological samples including body fluids such as blood, plasma, serum, urine, semen, saliva, cell lysates, tissue extracts, and the like. The sample may also include diluents, buffers, detergents, admixtures, and other components typically found in body fluids.

The term "specific binding pair" as used herein refers to two substances that exhibit binding affinity for each other. Examples include antigen-antibody pairings, hapten-antibody pairings, antibody-antibody pairings, complementary oligonucleotides or polynucleotides, avidin-biotin, streptavidin-biotin, enzyme-substrates, hormone-hormone receptors, lectin-carbohydrates, IgG-protein A, IgG-protein G, nucleic acid-nucleic acid binding proteins, and nucleic acid-anti-nucleic acid antibodies (Table 2).

TABLE 2

The term "(substituted) as used herein means that at least one hydrogen atom on the group is replaced by another atom or a group having 1 to 50 atoms selected from the group consisting of: C. o, N, S, P, Si, F, C1, Br, I. By (substituted) group is meant herein a group in which a plurality of substitution sites may be present.

Compositions of the invention

The present invention provides a class of fluorescent dyes that are based on the structure of firefly luciferin and have specific functional advantages. For example, the fluorescent dyes of the present invention are suitable for excitation at shorter wavelengths (between 340 and 450 nm), with emission maxima typically between 500 and 550nm wavelengths, narrower emission bandwidths, large Stokes shifts of at least about 50nm, and other favorable fluorescence characteristics.

The fluorescent dyes of the present invention are particularly useful with violet 405nm lasers. The fluorescent dyes of the present invention, despite having similar absorbance, exhibit surprising and unexpectedly bright fluorescence compared to existing violet laser excitable dyes (e.g., Pacific Orange). The fluorescent dyes of the present invention can be advantageously used for coupling to conjugated molecules via reactive groups. In addition, the dye-conjugates of the present invention, particularly protein conjugates, may exhibit bright fluorescence even at relatively high degrees of dye substitution. These properties make the dyes of the present invention particularly suitable for use in multi-color, multi-fold applications.

Fluorescent dyes of the general formula:

wherein the content of the first and second substances,

X₁and X₂Independently is S or O;

Y₁and Y₂Independently halogen, alkyl, haloalkyl, alkoxy, or alkenyl, or

Y₁Or Y₂One of which is H and the other is haloalkyl;

wherein each W is independently H or alkyl;

Z₁is H or alkyl;

wherein Z₁And one W group may be deleted, and if deleted, they are replaced by C in the ring C₄And C₅Additional keys in between, which are indicated as dashed lines;

Z₂a labeling substituent comprising a labeling substituent of the formula-L-RG, wherein L is a bond or a linking group that can link the dye to a reactive group RG, wherein the linking group can comprise a covalent bond comprising 1 to 50 non-hydrogen atoms selected from the group consisting of C, N, O, S, P and a halogen atom, and consists of any combination of any of the following bonds: single, double, triple, or aromatic carbon-carbon, carbon-oxygen, carbon-sulfur, carbon-nitrogen, and nitrogen-nitrogen bonds; and wherein RG includes carboxylic acids, carboxylic acidsActivated esters, anhydrides, acid chlorides, acid azides, acid halides, aldehydes, chloroformates, amines, hydroxyls, hydrazines, isocyanates, isothiocyanates, sulfonyl halides, tosyls, maleimides, N-hydroxy-succinimidyl esters, aziridines, imines, and disulfide groups.

In one embodiment of the fluorescent dye of formula (I), X₁And X₂Are both S, of the general formula:

wherein the content of the first and second substances,

Y₁and Y₂Independently halogen, alkyl, haloalkyl, alkoxy, or alkenyl, or

Y₁Or Y₂One of which is H and the other is haloalkyl;

Z₁is H or alkyl;

wherein each W is independently H or alkyl;

in L-RG, L is a chemical bond or a linking group that can link the dye to a reactive group RG, wherein the linking group can comprise a covalent bond containing 1-50 non-hydrogen atoms selected from C, N, O, S, P and halogen atoms and consists of any combination of single, double, triple, or aromatic carbon-carbon bonds, carbon-oxygen bonds, carbon-sulfur bonds, carbon-nitrogen bonds, and nitrogen-nitrogen bonds; and is

Wherein RG can comprise carboxylic acids, activated esters of carboxylic acids, anhydrides, acid chlorides, acid azides, acid halides, aldehydes, chloroformates, amines, hydroxyl groups, hydrazines, isocyanates, isothiocyanates, sulfonyl halides, tosyl, maleimide, N-hydroxy-succinimidyl esters, aziridines, imines, and disulfide groups.

Alternative embodiments of the dyes of the general formula (I-A) are shown in Table 3

TABLE 3

In other embodiments, the fluorescent dye has the following general formula:

wherein the content of the first and second substances,

X₁and X₂Independently is S or O;

Y₁and Y₂Independently is halogen, alkyl, haloalkyl, alkoxy, or alkenyl; or

Y₁Or Y₂One of which is H and the other is haloalkyl;

w is H or alkyl;

l is a chemical bond or a linking group that can link the dye to the reactive group RG, wherein the linking group can comprise a covalent bond containing 1-50 non-hydrogen atoms selected from C, N, O, S, P and a halogen atom and consists of any combination of single, double, triple, or aromatic carbon-carbon bonds, carbon-oxygen bonds, carbon-sulfur bonds, carbon-nitrogen bonds, and nitrogen-nitrogen bonds; and is

Wherein RG can comprise carboxylic acids, activated esters of carboxylic acids, anhydrides, acid chlorides, acid azides, acid halides, aldehydes, chloroformates, amines, hydroxyl groups, hydrazines, isocyanates, isothiocyanates, sulfonyl halides, tosyl, maleimide, N-hydroxy-succinimidyl esters, aziridines, imines, and disulfide groups.

In one embodiment of the fluorescent dye of formula (I-B), X₁And X₂Is S, when the formula is:

wherein the content of the first and second substances,

Y₁and Y₂Independently is halogen, alkyl, haloalkyl, alkoxy, or alkenyl; or

Y₁Or Y₂One of which is H and the other is haloalkyl;

w is H or alkyl;

l is a bond or a linking group, wherein the linking group comprises a covalent bond containing from 1 to 50 non-hydrogen atoms selected from C, N, O, S, P and a halogen atom, and consists of any combination of single, double, triple, or aromatic carbon-carbon bonds, carbon-oxygen bonds, carbon-sulfur bonds, carbon-nitrogen bonds, and nitrogen-nitrogen bonds; and is

RG is a reactive group, where RG can comprise carboxylic acid, activated ester of carboxylic acid, anhydride, acid chloride, acid azide, acid halide, aldehyde, chloroformate, amine, hydroxyl, hydrazine, isocyanate, isothiocyanate, sulfonyl halide, tosyl, maleimide, N-hydroxy-succinimide ester, aziridine, imine, and disulfide groups.

Alternative embodiments of the dyes of the general formula (I-B) are shown in Table 4.

TABLE 4

Dye material

X1

X2

Y1

Y2

W

L

RG

I-B-1

S

S

Cl

Cl

H

C(O)

OH

I-B-2

S

S

Cl

Cl

H

C(O)

NHS

I-B-3

S

S

Cl

Cl

Me

C(O)

OH

I-B-4

S

S

Cl

Cl

Me

C(O)

NHS

I-B-5

S

S

F

F

Me

C(O)

OH

I-B-6

S

S

F

F

Me

C(O)

NHS

I-B-7

S

S

F

F

H

C(O)

OH

I-B-8

S

S

F

F

H

C(O)

NHS

The present invention further provides synthetic intermediate compounds that can be used to synthesize the fluorescent dyes of the present invention. In one embodiment, the intermediate compound is represented by the following general formula (II):

wherein the content of the first and second substances,

Y₁and Y₂Independently halogen, alkyl, haloalkyl, alkoxy, or alkenyl, or

Y₁Or Y₂One of which is H and the other is haloalkyl.

In another embodiment, the intermediate compound is represented by the following general formula (III):

wherein the content of the first and second substances,

X₂is S or O;

wherein each W is independently H or alkyl;

Z₁is H or alkyl; and is

LH is OH or NH (CH)₂) nCOOH (n is 1 to 10).

The fluorescent dyes of the present invention may be covalently or non-covalently linked to more than one conjugated molecule "CM". Covalent attachment may be by various mechanisms, including attachment of the conjugated molecule CM to the fluorescent dye via a linking group L as described above, and may involve a covalent linkage (covalent linkage).

When a dye is non-covalently linked to more than one molecule, the linkage may occur by a variety of mechanisms, including incorporation of the dye or synthetic intermediate compound into or onto a solid or semi-solid substrate such as a bead, or surface; or by non-specific interactions such as hydrogen bonding, ionic bonding, or hydrophobic interactions such as van der waals forces. The linked molecules may be selected from the group consisting of: polypeptides, polynucleotides, polysaccharides, beads, microwell surfaces, metal surfaces, semiconductor and insulating surfaces, nanoparticles, and other solid surfaces. The linked or conjugated molecules may be linked or conjugated to more than one fluorescent dye, which may be the same or different. Generally, methods for preparing dye-conjugates of biological substances are well known in the art. See, for example (Greg T. Hermanson, "bioconjugate technology", 1996 edition, academic Press, Chapter 8, pp 298-. Typically, the fluorophore is attached to or conjugated to a dye-conjugate to impart the spectral properties of the fluorophore to the molecule.

In one embodiment, the fluorescent dye of the present invention is attached to the molecule CM to be conjugated via a covalent bond, the dye-conjugate having the general formula:

wherein the content of the first and second substances,

X₁and X₂Independently is S or O;

Y₁and Y₂Independently represents H, halogen, alkyl, haloalkyl, alkoxy, or alkenyl;

wherein each W is independently H or alkyl;

Z₁is H or alkyl;

wherein Z₁And one W group may be deleted, and if deleted, they are replaced by C in the ring C₄And C₅Additional keys in between, which are indicated as dashed lines;

l is independently a chemical bond or a linking group, wherein the linking group comprises a covalent bond containing from 1 to 50 non-hydrogen atoms selected from C, N, O, S, P and a halogen atom, and consists of any combination of single, double, triple, or aromatic carbon-carbon bonds, carbon-oxygen bonds, carbon-sulfur bonds, carbon-nitrogen bonds, and nitrogen-nitrogen bonds; and is

Wherein CM is a conjugated molecule.

Conjugated molecules CM that may be used to prepare dye-conjugates according to the present invention include, but are not limited to: amino acids, peptides, proteins, nucleosides, nucleotides, nucleic acids, carbohydrates, enzymes, lipids, non-biological polymers, cells, and cellular components. The molecules to be conjugated may be protected on more than one functional group to facilitate conjugation, or to safeguard subsequent reactivity.

When the molecule is a peptide, the peptide may be a dipeptide or larger, typically comprising 5 to 50 amino acids. When the molecule being conjugated is a protein, it may be an enzyme, antibody, catalytic antibody, kinase, lectin, glycoprotein, histone, albumin, lipoprotein, avidin, streptavidin, protein a, protein G, hormone, toxin, growth factor. Typically, the protein to be conjugated is an antibody, an antibody fragment, avidin, streptavidin, a lectin, or a growth factor.

The molecule to be conjugated may be a nucleic acid polymer such as a DNA oligonucleotide, an RNA oligonucleotide (or a hybrid thereof), or a single-stranded DNA, a double-stranded DNA, a triple-stranded DNA, or a quadruple-stranded DNA, or a single-stranded RNA or a double-stranded RNA. The conjugated molecule may also include carbohydrate polysaccharides, such as dextran.

The linked or conjugated molecule may be a member of a specific binding pair and may therefore be used as a detection reagent for a complementary member of a specific binding pair. Dye-conjugates of specific binding pair members may be used to detect, and optionally quantify, the presence of a complementary specific binding pair member in a sample by methods well known to those skilled in the art.

Representative specific binding pairs can include, but are not limited to, ligands and receptors, and can include, but are not limited to, the following pairs: antigen-antibody, biotin-avidin, biotin-streptavidin, IgG-protein A, IgG-protein G, carbohydrate-lectin, enzyme-enzyme substrate, DNA-complementary strand DNA, and RNA-complementary strand RNA, hormone-hormone receptor (table 2).

Method for preparing fluorescent dye compound

In one aspect of the present invention, there is provided a method of preparing a fluorescent dye of formula (I):

wherein the content of the first and second substances,

X₁and X₂Independently is S or O;

Y₁and Y₂Independently represents H, halogen, alkyl, haloalkyl, alkoxy, or alkenyl;

wherein each W is independently H or alkyl;

Z₁is H or alkyl;

wherein Z₁And one W group may be deleted, and if deleted, they are replaced by C in the ring C₄And C₅Additional keys in between, which are indicated as dashed lines;

wherein Z₂Comprising a labeled substituent of the formula-L-RG, wherein,

l is independently a chemical bond or a linking group, wherein the linking group comprises a covalent bond containing from 1 to 50 non-hydrogen atoms selected from C, N, O, S, P and a halogen atom, and consists of any combination of single, double, triple, or aromatic carbon-carbon bonds, carbon-oxygen bonds, carbon-sulfur bonds, carbon-nitrogen bonds, and nitrogen-nitrogen bonds; and is

Wherein RG is a reactive group selected from the group consisting of: carboxylic acids, activated esters of carboxylic acids, anhydrides, acid chlorides, acid azides, acid halides, aldehydes, chloroformates, amines, hydroxyls, hydrazines, isocyanates, isothiocyanates, sulfonyl halides, tosyls, maleimides, N-hydroxy-succinimidyl esters, aziridines, imines, and disulfide groups.

In one embodiment, the fluorescent dyes of the present invention having the general formula below can be synthesized using the synthetic schemes shown in FIGS. 2A-2D.

Wherein，Y₁And Y₂Independently represents H, halogen, alkyl, haloalkyl, alkoxy, or alkenyl;

X₂is S or O;

wherein each W is independently H or alkyl;

Z₁is H or alkyl; and is

L is O or NH (CH)₂) nCOO (n is 1-10).

The method comprises the following steps:

(a) using the method shown in fig. 2B, a cyanobenzothiazole intermediate of the dye having the following general formula was formed:

wherein the content of the first and second substances,

Y₁and Y₂Independently represents H, halogen, alkyl, haloalkyl, alkoxy, or alkenyl;

(b) by reacting a cyanobenzothiazole intermediate with a modified amino acid of the general formula:

wherein the content of the first and second substances,

X₂is S or O;

wherein each W is independently H or alkyl;

Z₁is H or alkyl; and is

LH is OH or NH (CH)₂) nCOOH (n is 1 to 10);

forming a carboxyl derivative of the dye, thereby producing a fluorescein derivative of the general formula:

wherein the content of the first and second substances,

X₂is S or O;

Y₁and Y₂Independently represents H, halogen, alkyl, haloalkyl, alkoxy, or alkenyl;

wherein each W is independently H or alkyl;

Z₁is H or alkyl; and is

LH is OH or NH (CH)₂) nCOOH (n is 1 to 10);

(c) the hydroxyl function of the carboxyl group in LH is replaced by a reactive group.

In another embodiment, the fluorescent dyes of the present invention having the general formula below are synthesized using the synthetic scheme shown in FIG. 3A:

wherein the content of the first and second substances,

Y₁and Y₂Independently represents H, halogen, alkyl, haloalkyl, alkoxy, or alkenyl; and is

R is H or alkyl.

In another embodiment, the fluorescent dyes of the present invention having the following general formula can be synthesized by using the synthetic scheme shown in fig. 3B (Bagley, MC., et al, j.am.chem.soc., 122, 3301-flac 3313 (2000)):

wherein the content of the first and second substances,

Y₁and Y₂Independently represents H, halogen, alkyl, haloalkyl, alkoxy, or alkenyl; and is

R is H or alkyl.

In another aspect, the present invention provides a method for making the dye-conjugates of the present invention. The method comprises mixing a fluorescent dye of the present invention comprising a reactive group with a molecule to be conjugated. The conjugated molecule may be an amino acid, a protein, a peptide, an antibody fragment, a nucleoside, a nucleotide, a nucleic acid polymer. The conjugated molecule may comprise a carbohydrate polysaccharide, such as dextran. Typically, conjugation of a fluorophore to a conjugated molecule imparts the spectral properties of the fluorophore to the molecule.

Methods for preparing dye-conjugates are well known in the art. For the preparation of peptide or protein conjugates, the method typically comprises: the protein to be conjugated is first dissolved in an aqueous buffer solution at a concentration of about 1-10mg/ml below room temperature (typically 4-25 ℃). Borate or carbonate/bicarbonate buffers (pH about 8.0-9) are particularly suitable for reaction with succinimidyl esters, phosphate buffers (pH about 4.0-8) are suitable for reaction with thiol-reactive functional groups, and carbonate or borate buffers (pH about 9.0-9.8) are suitable for reaction with isothiocyanates and dichlorotriazines. A suitable reactive dye is dissolved in a non-aqueous solvent (typically DMSO or DMF) in an amount sufficient to provide a suitable degree of conjugation, and added to a solution of the protein to be conjugated. Suitable levels of reactive dye to be used for any protein or other component are typically predetermined by experiments in which varying levels of reactive dye are added to the protein to be conjugated. The reactive dye is typically used in a 5-100 fold excess over the protein to be conjugated. After the reactive compound is added to the component solution, the mixture is incubated for a suitable period of time (typically about 1 hour at room temperature to several hours on ice) and the dye-conjugate is separated from the unconjugated dye by gel filtration, dialysis, HPLC (high performance liquid chromatography), or other suitable means. The dye-conjugate can be stored in solution or lyophilized until tested in the desired application. This method is generally applicable to the preparation of dye-conjugates using antibodies, antibody fragments, avidin, lectins, enzymes, protein a and protein G, cellular proteins, albumin, histones, growth factors, hormones, and other proteins.

Application method

The present invention provides a method of detecting a complementary member of a specific binding pair in a sample, comprising: mixing said sample with a dye-conjugate of the invention that specifically binds to said complementary member; and detecting a complex formed from the mixture to detect the complementary member. The method of the invention may comprise: fluorescent dyes, including the reactive dyes and dye-conjugates of the invention, are used to detect and/or quantify analytes in a sample. In particular embodiments, the sample comprises a heterogeneous mixture of components including whole cells, cell extracts, bacteria, viruses, organelles, and mixtures thereof. In other embodiments, the sample comprises a single component or a homogeneous group of components, for example, a biopolymer such as an amino acid polymer, a nucleic acid polymer, a carbohydrate polymer, or a lipid membrane complex.

In particular embodiments, the fluorescent dyes of the present invention, including reactive dyes and dye-conjugates, are used to stain or label an analyte in a sample to enable the analyte to be identified or quantified. For example, fluorescent dyes, including the dye-conjugates of the invention, may be used as detectable tracer elements in biological or non-biological fluids in assays for target analytes.

In other embodiments, fluorescent dyes, including reactive dyes and dye-conjugates, are used to detect samples containing ligands that are complementary members of a specific binding pair (e.g., see table 2) for the molecule being conjugated. Thus, the ligand is one member of a specific binding pair, while the molecule being conjugated is the other member. Fluorescent dyes, including dye-conjugates, are also used to recognize interactions between the dye-conjugate and complementary binding molecules.

In the first step of the method, fluorescent dyes, including the reactive dyes and dye-conjugates of the present invention, are generally used under conditions selected to produce a detectable optical reaction by combining the dyes of the present invention with a sample of interest, as described above. The dye-conjugate typically forms a covalent or non-covalent association with a component in the sample, or forms a complex, or simply exists within the confines of the sample or a portion of the sample. The sample is then irradiated at a selected wavelength to cause an optical reaction. Typically, the sample is stained for the purpose of determining a specific characteristic of the sample by further comparing the optical response to a standard.

In one aspect of the invention, the dye-conjugate is a labeled protein, such as an antibody, antibody fragment, avidin or streptavidin, or the like. In this embodiment, the dye-conjugate is used to detect a complementary specific binding pair, typically an antigen, hapten or biotin. These dye-conjugates of the present invention can be used to detect an analyte in a sample by employing a variety of applications. Major applications include immunofluorescence, Fluorescence In Situ Hybridization (FISH), flow cytometry, labeling of receptors, and tracking of labeled cells. For particular detection methods, e.g., detection methods based on cell counts that inherently involve spatial separation/resolution of bound versus unbound conjugate, it is not necessary to remove unbound dye-conjugate from the assay mixture. However, for other detection methods, for example immunoassays that do not involve spatial separation/resolution of bound versus unbound conjugate, it may be necessary to remove unbound dye-conjugate from the assay mixture prior to detection of the optical reaction.

In another aspect, the fluorescent dyes and dye-conjugates of the present invention may have application as laser dyes. Since the dyes and dye-conjugates of the present invention can be excited by ultraviolet or violet laser light, these dyes and dye-conjugates can be used in multiplex assays, especially with other dyes that are excited at longer wavelengths. In one embodiment, these dyes and dye-conjugates are excited at about 350-405nm and, when used with dyes excited at longer wavelengths (e.g., 480nm and longer), their emission spectra are distinguishable from dyes excited at longer wavelengths.

In another aspect of the invention, the dye-conjugates of the invention can be used in a multicolor method for detecting more than one analyte in a sample. In one embodiment, multiple dye-conjugates can be used to detect multiple analytes in a sample using a multi-color assay. The method includes incubating a sample with a composition of the invention comprising a plurality of dye-conjugates, e.g., a first dye-conjugate and a second dye-conjugate. In the composition, a component of the first dye-conjugate is a binding partner for the first analyte and a component of the second dye-conjugate is a binding partner for the second analyte. The incubation is continued under conditions suitable to induce an interaction between the first analyte and the first dye-conjugate. During this incubation, it is generally preferred that a similar interaction occurs between the second analyte and the second dye-conjugate, however, incubation conditions may be varied within the scope of the invention as desired to drive the formation of a dye-conjugate-analyte complex between the second dye-conjugate and the second analyte. After at least a first dye-conjugate-analyte complex is formed, the sample is illuminated with light of a wavelength suitable to cause the complex to fluoresce, thereby detecting the first analyte. The second analyte is detected in a similar manner and may be detected simultaneously with the first analyte or by sequentially illuminating the sample with wavelengths suitable for inducing fluorescence of each fluorochrome-conjugate. In a particular aspect, the irradiating and/or detecting step comprises flow cytometry.

Alternatively, different characteristics of the analyte can be detected using a plurality of dye-conjugates, preferably dye-conjugates that fluoresce at different wavelengths. For example, epitopes of cells or analytes can be labeled with dye-conjugates of different colors, targets detected and their identity confirmed by employing co-localization of each color on each target.

The dyes and dye-conjugates of the invention may be used for diagnostic purposes. For example, the dyes and dye-conjugates of the invention can be used to detect the presence of a pathogenic organism (e.g., a bacterial pathogenic organism, a viral pathogenic organism, a fungal pathogenic organism) in an individual. Alternatively, the presence of a particular analyte and/or the amount of a particular analyte and/or variant of a particular analyte may be related to a pathological condition in the individual. The dyes and dye-conjugates of the invention that bind to such analytes may have utility for determining the presence and/or amount of a particular analyte and/or variant thereof, and thus may be useful for diagnosing the presence and/or extent of a pathological condition.

Kit of the invention

One aspect of the invention is the use of any of the dyes of the invention as described above to formulate kits that facilitate various assay practices. The kits of the invention typically comprise a fluorescent dye of the invention either as a chemically reactive label that can be used to prepare a dye-conjugate or as a dye-conjugate in which the molecule being conjugated is a member of a specific binding pair. The selected conjugated molecules include, but are not limited to, polymers of biological molecules such as proteins, nucleic acids, or carbohydrates. In particular embodiments, the dyes of the present invention listed in tables 3 and 4 are particularly suitable for preparing such kits.

Preferably, the kit comprises more than one fluorescent dye listed in table 5.

TABLE 5

The kit can also comprise more than one Dye-conjugate Dye1, Dye2, Dye3, Dye4, Dye5, Dye6, or Dye 7.

In an exemplary embodiment, the kit comprises a reactive dye of the present invention and instructions for conjugating the dye to a molecule having a suitable functional group, and optionally, for recovering the dye-conjugate.

In another exemplary embodiment, the kit includes instructions for directing an assay for detecting an analyte or ligand in a sample. For example, in one embodiment, instructions are provided for detecting a cell surface receptor, or enzyme, or other ligand capable of binding an antibody.

The kit also optionally comprises one or more buffers, typically present as an aqueous solution. The kits of the invention optionally further comprise additional detection reagents, purification media for purifying the resulting labeled molecules, fluorescence standards, enzymes, enzyme inhibitors, organic solvents, or instructions for performing the methods of the invention.

Various embodiments of the present invention are further illustrated by the following exemplary examples. These examples are provided to illustrate the invention and are not intended to limit the scope or the rights of the invention in any way.

Example 1

The following example describes a general method for the synthesis of dihalo derivatives of firefly luciferin and their respective NHS esters.

Preparation of Appel salt (Appel salt)

A500 mL round bottom flask was charged with chloroacetonitrile (20mL), sulfur monochloride (120mL), and CH under argon shield₂Cl₂(110 mL). The reaction was stirred under argon for 3 days while a brown precipitate formed. The precipitate was collected by suction filtration and washed with CH₂Cl₂(300mL) and hexane (300 mL). The washed solid was dried in vacuo to give the product as a brown solid (75% yield).

2. Preparation of 2-cyanobenzothiazole

The following procedure was used to prepare 5, 7-dichloro-6-hydroxy-2-cyanobenzothiazole. The same procedure was followed to prepare 5, 7-difluoro-6-hydroxy-2-cyanobenzothiazole with very similar results.

A5L round bottom flask was charged with 1, 3-dichloro-2-methoxynitrobenzene (0.33 mole), ammonium chloride (3.3 moles), and ethanol (1200 mL). Zinc powder (3.3 moles) was added quickly to the stirred mixture. After a few minutes, the reaction mixture was warmed to near reflux, thenThen it was allowed to cool to room temperature. After stirring overnight, the suspension was filtered to remove residual zinc and ammonium chloride. The filtrate was concentrated under reduced pressure and the resulting solid was resuspended in CH₂Cl₂(1500 mL). The suspension was filtered, and the filtrate was concentrated under reduced pressure to give substituted aniline (0.33 mol) as a white solid.

1A Y＝Cl ¹H NMR(400MHz，CDCl₃): δ 6.601(s, 2H), 3.811(s, 3H), 3.6-3.7 (broad, s, 2H)

1B Y＝F ¹H NMR(400MHz，CDCl₃): δ 6.22(m, 2H), 3.860(s, 3H), 3.6-3.7 (broad, s, 2H)

A 2L three-neck round bottom flask was oven dried and cooled under a stream of argon. The flask was charged with 1, 3-dichloro-2-methoxyaniline (0.33 mol), pyridine (75mL), and CH₂Cl₂(550 mL). Appel salt (0.33 mol) was added in portions over 5 minutes, causing the solution to reflux. TLC showed complete disappearance of aniline after 15 minutes. The reaction mixture was concentrated to give an orange solid. The solid was dissolved in 2.5L of ethyl acetate. The organic solution was washed with type 1water (type 1water) (2X 1000mL) and brine (1000 mL). The washed organic layer was washed with anhydrous Na₂SO₄Dried and concentrated to give the Appel salt adduct as a solid.

2A Y＝Cl ¹H NMR(400MHz，CDCl₃)：δ7.203(s，2H)，3.933(s，3H)

2BY＝F ¹H NMR(400MHz，CDCl₃)：δ6.87(m，2H)，4.030(s，3H)

In a 500mL three-necked round bottom flask was mounted an argon introduction line and a water condenser with a bubbler connected to the outlet. The flask was charged with the Appel salt adduct (0.134 moles) and heated to a surface temperature of 140 ℃ for 1 hour. Toluene (50mL) was added and the reaction was cooled to 90 ℃. Adding CH₂Cl₂(100mL) and silica (50 g). Filtering the resulting slurry, andby CH₂Cl₂The filter cake was washed (200 mL). The combined filtrate and washings were concentrated and purified by flash Chromatography (CH)₂Cl₂Hexane) to give 5, 7-dichloro-6-methoxy-2-cyanobenzothiazole (0.031 moles) as a solid.

3A Y＝Cl ¹H NMR(400MHz，CDCl₃)：δ8.194(s，1H)，4.027(s，3H)

3B Y＝F ¹H NMR(400MHz，CDCl₃)：δ7.981(dd，J＝10Hz & 1.6Hz，1H)，4.156(s，3H)

A250 mL round bottom flask was charged with pyridinium hydrochloride (160 mmol) and 5, 7-dichloro-6-methoxy-2-cyanobenzothiazole (8.2 mmol). The reaction was charged under argon and then heated at 195 ℃ for 3 hours. After cooling to room temperature, type 1water (150mL) was added and the mixture was sonicated to dissolve the solids. The aqueous solution was extracted with ethyl acetate. Anhydrous Na for ethyl acetate₂SO₄Dried and concentrated to give the product as a yellow solid (2.8 mmol).

4A Y＝Cl ¹H NMR (400MHz, acetone-d₆)：δ8.313(s)

4B Y＝F ¹H NMR (400MHz, acetone-d₆)：δ8.385(dd，J＝10.4Hz & 1.6Hz)

3. Preparation of amino acids

Synthesis of modified Penicillium Amines

A2000 mL round bottom flask was charged with DL-penicillamine (10g) and acetone (1000 mL). The reaction was refluxed until all the solid had dissolved (approximately 24 hours). The reaction mixture was filtered hot and cooled to room temperature overnight. A small amount of crystals had formed overnight. The reaction mixture was kept at-20 ℃ overnight. The resulting solid was collected by suction filtration and washed with acetone (300 mL). The thiazole product (11.9g) was dried under argon.¹H NMR(400MHz，DMSO-d₆)：δ3.750(s，1H)，1.556(s，6H)，1.438(s，3H)，1.185(s，3H)。

A250 mL round bottom flask was charged with thiazolic acid (11.9g) and sodium formate (4.3g) under argon. Formic acid (98%, 100mL) was added and the reaction mixture was stirred under argon. The reaction mixture was cooled to 0 ℃ in an ice water bath. Acetic anhydride (33.3mL) was added dropwise over 45 minutes while carefully maintaining the temperature below 5 ℃. The ice-water bath was removed and the reaction was stirred at room temperature overnight. The solvent was removed under vacuum at 35 ℃ to yield a white solid. The solid was stirred in type 1water (200mL) and collected by suction filtration. The solid was washed with type 1water (100mL) and air dried (7.4 g). The combined filtrate and washings were extracted with ethyl acetate (3X 300 mL). Mixing the extractive solutions, adding Na₂SO₄Drying and filtering. The filtrate was concentrated to give a pale yellow solid (6.8 g).¹H NMR showed that the two solids were an isomeric mixture of the target compound (about 85: 15).¹H NMR(400MHz，DMSO-d₆12.916(S，1H)，8.446(s，0.86H)，8.195(s，0.14H)，4.641(s，0.15H)，4.502(s，0.85H)，1.828(s，6H)，1.590(s，3H)，1.362(s，3H)。

A round bottom flask was charged with formylated thiazolic acid (1 equivalent), triethylamine (2 equivalents), and CH₂Cl₂. The reaction mixture was stirred under argon in an ice-water bath at 0 ℃. Isobutyl chloroformate (1 equivalent) was added via syringe. After the reaction mixture had been stirred for 1 hour, the amino ester (1 equivalent) was added. The ice water bath was removed and the reaction mixture was stirred at room temperature overnight. The reaction mixture was washed with 1.0M HCl (75mL), 5% NaHCO₃(75mL), and type 1water (75 mL). The organic layer was concentrated under reduced pressure and subjected to flash column chromatography (50: 50 ethyl acetate: n-hexane) to obtain the objective product (yield 60-70%).¹H NMR showed an isomeric mixture of the product.

n＝0 ¹H NMR(400MHz，CDCl₃)：8.389(s，0.88H)，8.328(s，0.12H)，6.932(m，0.15H)，6.383(m，0.82)，4.620(s，0.82H)，4.367(s，0.15H)，4.030(dd，J＝18.4Hz & 5.2Hz，1H)，3.91(m，1H)，2.00-1.94(m，6H)，1.70-1.59(m，3H)，1.47(m，12H)

n＝1 ¹H NMR(400MHz，CDCl₃)：8.358(s，0.79H)，8.291(s，0.15H)，6.973(m，0.15H)，6.486(m，0.78)，4.418(s，0.80H)，4.313(s，0.13H)，3.6-3.4(m，2H)，2.43(m，2H)，2.00-1.94(m，6H)，1.68-1.60(m，3H)，1.44(m，12H)

A round bottom flask was charged with formylated thiazole ester, and dioxane: 2.0M HCl (50: 50). The reaction mixture was heated at 85 ℃ under argon atmosphere overnight. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure to give the modified penicillamine as a gummy solid.

n＝0 ¹H NMR showed the isomeric mixture of the product.¹H NMR(400MHz，D₂O)：4.128 &4.083(s，1H)，4.043 & 4.000(s，2H)，1.557(s，3H)，1.481(s，3H)

n＝1 ¹H NMR(400MHz，D₂O)：3.702(s，1H)，3.45-3.38(m，1H)，3.27-3.19(m，1H)，2.441(m，2H)，1.295(s，3H)，1.233(s，3H)

Synthesis of methylated cysteine

Cysteine methyl ester (8.6g), pivalaldehyde (11mL), and triethylamine (8mL) were stirred in pentane. The reaction mixture was refluxed while removing water via Dean-Stark trap (Dean-Stark trap). After 36 hours, the reaction was cooled to room temperature, forming a solid. The solid was collected by suction filtration and washed with diethyl ether.

A100 mL round bottom flask was charged with thiazole ester (4.0g), formic acid (98%, 30mL) and sodium formate (1.5 g). The reaction mixture was stirred under argon and cooled to 0 ℃ in an ice water bath. Acetic anhydride (5.7mL) was added dropwise over 30 minutes while carefully maintaining the temperature below 5 ℃. The ice-water bath was removed and the reaction was stirred at room temperature overnight. The solvent was removed under vacuum and the resulting solid was stirred in type 1water (100 mL). By adding solid NaHCO₃And adjusting the pH value to 7-8. The aqueous solution was extracted with diethyl ether (3X 100 mL). Mixing the extractive solutions, adding Na₂SO₄Drying and filtering. The filtrate was concentrated to give a solid. The solid was recrystallized from ethyl acetate/hexane.¹H NMR(400MHz，CDCl₃)：8.317(s，1H)，4.858(t，J＝8.8Hz，1H)，4.708(s，1H)，3.739(s，3H)，3.258(m，2H)，1.000(s，9H)。

A solution of lithium diisopropylamine (12 mmol) in dry THF (tetrahydrofuran) (100mL) was stirred at-78 deg.C under argon. 1, 3-dimethyl-3, 4, 5, 6-tetrahydro-2 (1H) -pyrimidinone (DMPU, 10mL) was added and the reaction mixture was stirred for 90 minutes. Formylated thiazole ester (10 mmol) was added and the reaction mixture was stirred at-78 ℃ for 1 hour. MeI (15 mmol) was added,and the reaction mixture was stirred at-78 ℃ for 2 hours. The dry ice acetone bath was removed and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and the residue was kept in brine. The aqueous solution was extracted with diethyl ether (100 mL). With Na₂SO₄The extract was dried and filtered. The filtrate was concentrated to give a solid. The solid was purified by flash column chromatography with 10% ethyl acetate/hexanes.¹H NMR showed a diastereomeric mixture of the product.¹H NMR(400MHz，CDCl₃) The main products are as follows: 8.252(s, 1H), 5.263(s, 1H), 3.738(s, 3H), 3.293(d, J ═ 11.6Hz, 1H), 2.695(d, J ═ 11.2Hz, 1H), 1.038(s, 9H); minor products: 8.377(s, 0.4H), 5.274(s, 0.4H), 3.790(s, 1.3H), 3.612(d, J-12 Hz, 0.5H), 2.831(d, J-12 Hz, 1H), 0.929(s, 4.6H).

Methylated thiazole ester (1.8g) and 5M HCl (30mL) were heated at 105 ℃ for 3 days. The reaction mixture was cooled to room temperature and extracted with ethyl acetate (2 × 100 mL). The aqueous phase was concentrated under reduced pressure to give a gummy solid.

4. Preparation of 6-hydroxyfluorescein acid

This is a typical process for preparing basic fluorescein structures.

A100 mL round bottom flask was charged with cyanobenzothiazole (0.5 mmol) and methanol (25 mL). The stirred reaction mixture was bubbled with argon for 1 hour. Preparation and addition of pH 9 (by addition of Na)₂CO₃pH adjusted) of an aqueous solution of an amino acid (1 mmol) bubbled with argon. The reaction mixture was stirred for 2 hours under argon shieldTime, and TLC (thin layer chromatography) indicated the reaction was complete. The reaction was concentrated to approximately 10mL under reduced pressure and diluted with type 1water (40 mL). The aqueous solution was extracted with ethyl acetate (3X 75mL) and the extract was left. The aqueous solution was acidified to pH 2 by dropping high concentration of HCl. The acidified aqueous solution was extracted again with ethyl acetate (3X 75 mL). The 6 extracts were combined and washed with anhydrous Na₂SO₄Drying followed by concentration gave the product as a solid (40-90% yield).

6A ¹H NMR(400MHz，CD₃OD)：7.688(dd，J＝11Hz & 1.4Hz，1H)，5.017(dd，J＝10.2Hz & 8.2Hz，1H)，4.774(m，2H)

6B ¹H NMR(400MHz，CDCl₃)：7.795(dd，J＝10Hz & 1.6Hz，1H)，3.932(d，J＝11.8Hz，1H)，3.434(d，J＝11.8Hz，1H)，1.688(s，3H)

6C ¹H NMR(400MHz，CD₃OD)：7.646(dd，J＝10.4Hz & 1.2Hz，1H)，4.935(s，1H)，1.778(s，3H)，1.488(s，3H)

6D ¹H NMR(400MHz，CD₃OD): 8.379 (broad peak, s, 1H), 7.658(d, J ═ 10Hz, 1H), 4.758(s, 1H), 4.000(d, J ═ 6Hz, 2H), 1.829(s, 3H), 1.448(s, 3H)

6E ¹H NMR(400MHz，CD₃OD): 8.190 (broad peak, s, 1H), 7.647(d, J ═ 10.4Hz, 1H), 4.705(s, 1H), 3.56-3.50(m, 2H), 2.558(t, J ═ 6.6Hz, 2H), 1.814(s, 3H), 1.395(s, 3H)

6F ¹H NMR(400MHz，CD₃OD): 8.271 (broad peak, s, 1H), 8.093(s, 1H), 4.794(s, 1H), 3.610(m, 2H), 2.640(t, J ═ 6.6Hz, 2H), 1.898(s, 3H), 1.478(s, 3H)

6G ¹H NMR (400MHz, acetone-d₆)：9.838(s，1H)，7.810(dd，J＝10.8Hz，& 1.6Hz，1H)，5.504(t，J＝9.2Hz，1H)，3.860(m，2H)

6H ¹H NMR(400MHz，DMSO-d₆)：8.266(s，1H)，5.007(s，1H)，1.725(s，1H)，1.447(s，1H)

6I ¹H NMR (400MHz, acetone-d₆)：8.149(s，1H)，5.505(t，J＝9.2Hz，1H)，3.855(m，2H)

Compound 6J (L ═ NH) was prepared in three steps starting from compound 6H₂(CH₂)₅COOH). Reaction of carboxylic acid 6H with N-hydroxysuccinimide and DCC produced NHS ester 7H coupled with methyl 6-aminocaproate. The resulting methyl ester was hydrolyzed with base to give 6J.

5. Preparation of fluorescein NHS ester

This is a typical process for the preparation of fluorescein NHS esters.

A10 mL round bottom flask was purged with argon and charged with fluorescein (0.3 mmol), N-hydroxysuccinimide (0.3 mmol), and DCC (0.305 mmol). Anhydrous THF (4mL) was added and the reaction mixture was stirred for 3 hours. The solvent was removed under reduced pressure and a small volume of CH was added₂Cl₂. A white precipitate formed and was removed by filtration. The filtrate was concentrated under reduced pressure to form a gummy solid. The solid was dissolved in a minimum amount of THF and the solid was reformed by the addition of hexane. The solvent was decanted and the procedure repeated an additional 2 times to give the product as a yellow solid (60% yield).

7B ¹H NMR(400MHz，CDCl₃)：7.650(m，1H)，4.093(d，J＝11.4Hz，1H)，3.481(d，J＝11.4Hz，1H)，1.814(s，3H)

7C ¹H NMR(400MHz，CDCl₃)：7.660(dd，J＝10.2Hz & 1.4Hz，1H)，5.153(s，1H)，2.858(s，4H)，1.874(s，3H)，1.653(s，3H)

7D ¹H NMR(400MHz，CDCl₃): 7.678(d, J ═ 10Hz, 1H), 7.608 (broad, s, 1H), 4.713(s, 1H), 4.614(dd, J ═ 18Hz& 6.6Hz，1H)，4.389(dd，J＝18Hz & 5.4Hz，1H0，2.829(s，4H)，1.907(s，3H)，1.447(s，3H)

7E ¹H NMR(400MHz，CDCl₃): 7.670(d, J ═ 10Hz, 1H), 7.616 (broad peak, s, 1H), 4.660(s, 1H), 3.82-3.65(m, 2H), 2.899(t, J ═ 6Hz, 2H), 2.789(s, 4H), 1.899(s, 3H), 1.412(s, H)

7F ¹H NMR(400MHz，CDCl₃): 8.073(s, 1H), 7.653 (broad, s, 1H), 4.699(s, 1H), 3.9-3.7(m, 2H), 2.932(t, J ═ 6.0Hz, 2H), 2.820(s, 4H), 1.940(s, 3H), 1.451(s, 3H)

7G ¹H NMR (400MHz, acetone-d₆)：7.840(dd，J＝10.0Hz & 1.6Hz，1H)，5.962(dd，J＝10.2Hz & 8.2Hz，1H)，4.127(t，J＝10.8Hz，1H)，3.914(dd，J＝11.6Hz & 8Hz，1H)，2.935(s，4H)

7H ¹H NMR (400MHz, acetone-d₆)：8.13(s，1H)，5.59(s，1H)，2.33(s，4H)，1.89(s，3H)，1.64(s，3H)

7J ¹H NMR (400MHz, acetone-d₆): 9.53 (broad, s, 1H), 8.10(s, 1H), 7.78(m, 1H), 4.69(s, 1H), 3.22-3.42(m, 2H), 2.81(s, 4H), 2.62(t, 2H), 1.86(s, 3H), 1.76-1.69(m, 2H), 1.63-1.56(m, 2H), 1.50-1.44(m, 2H), 1.42(s, 3H)

6. Preparation of fluorescein maleimide derivative

Fluorescein NHS ester (22mg) was stirred in anhydrous THF. N- (2-aminoethyl) maleimide-trifluoroacetic acid (13mg) was added followed by NaHCO₃An aqueous solution. The mixture was stirred for 4 hours and then concentrated under reduced pressure. The mixture was dissolved in 5mL of water and extracted with ethyl acetate. Extracting with anhydrous Na₂SO₄Dried and concentrated under reduced pressure. The solid was dissolved in acetone and hexanes were added to give an air-dried precipitate (13 mg).¹H NMR(400MHz，DMSO-d₆)：8.118(m，1H)，7.886(d，J＝10.8Hz，1H)，6.972(s，2H)，4.670(s，1H)，3.38(m，2H)，3.33(m，2H)，1.678(s，3H)，1.264(s，3H)

Preparation of fluorescent Dye1 (7H):

the fluorescent Dye1(LD 1; 7H) was synthesized using the synthetic procedure described above. In brief, 2-cyano-5, 7-dichloro-6-hydroxybenzothiazole (4A) synthesized by using the above-described method was reacted with D, L-penicillamine (5C) under the conditions described in detail above, to give 5, 7-dichloro-6-hydroxyfluorescein acid (6H).

To synthesize 5, 7-dichloro-6-hydroxyfluorescein NHS ester, dichloro-6-hydroxyfluorescein acid (6H) was reacted with N-hydroxysuccinimide in the presence of DCC as described above. The NHS ester (7H) obtained was purified and characterized by NMR as described above.

Preparation of fluorescent Dye2 (7J):

the fluorescent Dye2(LD 2; Compound 7J) was synthesized by employing the synthetic procedure as described above, except that 2-cyano-5, 7-dichloro-6-hydroxybenzothiazole (4A) was reacted with Compound 5F under the conditions described in detail above to prepare 5, 7-dichloro-6-hydroxyfluorescein acid (6J).

Next, dichloro-6-hydroxyfluorescein acid (6J) was reacted with N-hydroxysuccinimide in the presence of DCC as described above to produce the NHS ester (7J), which was purified as described above and analyzed by NMR.

Preparation of fluorescent Dye3 (7C):

the fluorescent Dye3(LD 3; 7C) was synthesized by employing the synthetic procedure as described above, except that 2-cyano-5, 7-difluoro-6-hydroxybenzothiazole (4B) was reacted with D, L-penicillamine (5C) under the conditions described in detail above to prepare 5, 7-difluoro-6-hydroxyfluorescein acid (6C).

Next, difluoro-6-hydroxyfluorescein acid (6C) was reacted with N-hydroxysuccinimide in the presence of DCC as described above to produce 5, 7-difluoro-6-hydroxyfluorescein NHS ester (7C), which was purified as described above and analyzed by NMR.

Preparation of fluorescent Dye4 (7B):

the fluorescent Dye4(LD 4; 7B) was synthesized by employing the synthetic procedure as described above, except that 2-cyano-5, 7-difluoro-6-hydroxybenzothiazole (4B) was reacted with compound 5B under the conditions described in detail above to prepare 5, 7-difluoro-6-hydroxyfluorescein acid (6C). Subsequently, 5, 7-difluoro-6-hydroxyfluorescein acid (6C) was reacted with N-hydroxysuccinimide in the presence of DCC as described above to produce 5, 7-difluoro-6-hydroxyfluorescein NHS ester (7C), which was purified as described above and analyzed by NMR.

Preparation of fluorescent Dye5(7E)

The fluorescent Dye5(LD 5; 7E) was synthesized by employing the synthetic procedure as described above, except that 2-cyano-5, 7-difluoro-6-hydroxybenzothiazole (4B) was reacted with compound 5E under the conditions described in detail above to prepare 5, 7-difluoro-6-hydroxyfluorescein acid (6E). Subsequently, 5, 7-difluoro-6-hydroxyfluorescein acid (6E) was reacted with N-hydroxysuccinimide in the presence of DCC as described above to produce 5, 7-difluoro-6-hydroxyfluorescein NHS ester (7E), which was purified as described above and analyzed by NMR.

Preparation of fluorescent Dye6(9B)

The fluorescent Dye6(LD6) was synthesized by using the process flow chart described below.

N-Boc-L-glutamic acid gamma-tert-butyl ester (4.3g) in CH₂Cl₂Stirring at 0 ℃. Triethylamine (2mL) was added via syringe followed by ethyl chloroformate (1.4 mL). The reaction mixture was stirred for 60 minutes, filtered, and the solid was washed with anhydrous THF. The filtrate and washings were combined and transferred to the addition funnel. The solution was added dropwise to stirred NaBH at 0 deg.C₄At H₂O (20mL) for 30 minutes. The reaction mixture was stirred at 0 ℃ for 5 hours before being stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with ethyl acetate. The solution was washed with water and brine, and anhydrous Na₂SO₄Dried and concentrated under reduced pressure. Flash column chromatography (20: 80 ethyl acetate: n-hexane) afforded the product as an oil.¹H NMR(400MHz，CDCl₃): 4.814 (broad, s, 1H), 3.62-3.49(m, 3H), 2.496 (broad, s, 1H), 2.290(q, J ═ 6.8Hz, 2H), 1.83-1.69(m, 2H), 1.406(s, 18H).

Amino alcohol (3.5g) was stirred in pyridine (25mL) at 0 ℃ under argon. Tosyl chloride (3.0g) was added and the reaction mixture was stirred at 0 ℃ for 6 hours. The solution was warmed to room temperature and stirred overnight. The reaction mixture was concentrated under reduced pressure, and the resulting solid was dissolved in ethyl acetate. With saturated NaHCO₃The solution was washed, followed by brine. The washed solution was washed with anhydrous Na₂SO₄Dried and concentrated under reduced pressure. Flash column chromatography gave the product as an oil.¹H NMR(400MHz，CDCl₃): 7.744(d, J ═ 8.8Hz, 2H), 7.311(d, J ═ 8.8Hz, 2H), 4.623(d, J ═ 8.4Hz, 1H), 4.00-3.91(m, 2H), 3.739 (broad peak, s, 1H), 2.411(s, 3H), 2.224(m, 2H), 1.719(m, 2H), 1.390(s, 9H), 1.350(s, 9H).

A solution of tosylate (3.9g) and potassium thioacetate (2.9g) in anhydrous DMF was stirred at room temperature for 3 days. The reaction mixture was diluted with ethyl acetate (300mL) and washed with brine (3 × 100 mL). The washed solution was washed with anhydrous Na₂SO₄Dried and concentrated under reduced pressure. Flash column chromatography (15: 85 ethyl acetate: n-hexane) afforded the product as an oil.¹H NMR(400MHz，CDCl₃): 4.522(d, J ═ 8Hz, 1H), 3.704 (broad peak, s, 1H), 3.07-2.94(m, 2H), 2.311(s, 3H), 2.264(t, J ═ 7.2Hz, 2H), 1.80-1.60(m, 2H), 1.404(s, 9H), 1.386(s, 9H).

Thioacetate (2.5g) was heated in degassed 6N HCl at 105 ℃ overnight. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to give a red oil.¹H NMR(400MHz，D₂O)：

A flask was charged with 2-cyano-5, 7-difluoro-6-hydroxybenzothiazole (110mg) and methanol (25 mL). The stirring reaction mixture was bubbled with argon for 1 hour. Preparation of pH 9 (by addition of Na)₂CO₃pH was adjusted) of an aqueous solution of an amino acid (300mg) bubbled with argon, and added to the reaction mixture three times at 1-hour intervals. The reaction mixture was stirred overnight. The reaction was concentrated to approximately 10mL under reduced pressure and diluted with type 1water (40 mL). The aqueous solution was extracted with ethyl acetate (50mL), and the extract was left. The aqueous solution was acidified to pH 2 by dropping high concentration of HCl. The acidified aqueous solution was extracted again with ethyl acetate (2X 50 mL). The 2 extracts were combined and washed with anhydrous Na₂SO₄Dried and concentrated to give the product as a solid (130 mg).¹H NMR(400MHz，CD₃OD)：7.641(d，J＝10.4Hz，1H)，4.694(m，1H)，3.612(t，J＝8.8Hz，1H)，3.179(t，J＝9.4Hz，1H)，2.550(m，2H)，2.037(m，2H)

The flask was purged with argon, and charged with deoxyfluorescein acid (41mg), N-hydroxysuccinimide (15mg), and DCC (26 mg). Anhydrous THF (4mL) was added and the reaction mixture was stirred for 3 hours. The reaction solution was filtered to remove solids. Will be provided withN-hexane was added to the precipitate solid collected by suction filtration. The solid was redissolved in THF, and n-hexane was added to the precipitated solid. The solid was collected by suction filtration and air dried.¹H NMR(400MHz，CDCl₃)：7.666(dd，J＝10.4Hz & 1.6Hz，1H)，4.772(m，1H)，3.612(m，1H)，3.158(dd，J＝11Hz & 8.6Hz，1H)，3.00-2.86(m，2H)，2.821(s，4H)，2.188(m，2H)。

Preparation of dehydrofluorescein (10)

The following reaction description is for example 10A, and similar reaction conditions are used in case 10B. Giving both cases¹H NMR data.

2-cyano-5, 7-dichloro-6-hydroxybenzothiazole (2.8 mmol) was stirred in pyridine (20mL) and triethylamine (0.3 mL). Hydrogen sulfide was bubbled through the solution for 4.5 hours. The addition of hydrogen sulfide was stopped and the reaction mixture was stirred at room temperature overnight. The reaction mixture was stripped under reduced pressure, and the resulting solid was recrystallized from methanol.¹H NMR(400MHz，CD₃OD)：8.000(s，1H)。

The above benzothiazole product (55mg) was suspended in methanol (5 mL). Ethyl bromopyruvate (100mg) was added to the solution, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was heated at 80 ℃ for 7 hours. The reaction mixture was cooled to room temperature, thereby forming a solid. The solid was collected by suction filtration, washed with methanol, and air-dried (35 mg).

R＝H ¹H NMR(400MHz，CDCl₃)：8.295(s，1H)，7.992(s，1H)，4.436(q，J＝7Hz，2H)，1.414(t，J＝7Hz，3H)

Process for preparing R-Me methyl ester¹H NMR(400MHz，CDCl₃)：7.964(s，1H)，3.951(s，3H)，2.837(s，3H)

Dehydrofluorescein ethyl ester (30mg) was suspended in methanol and 1N NaOH. The reaction was stirred at room temperature for 2 days. The reaction mixture was acidified to pH 2 with 1N HCl and extracted with ethyl acetate (2 × 50 mL). The combined extracts were washed with anhydrous Na₂SO₄Drying and concentrating under reduced pressure to obtain a solid product.

10A ¹H NMR(400MHz，CD₃OD)：8.518(s，1H)，8.001(s，1H)

10B ¹H NMR(400MHz，CD₃OD)：7.965(s，1H)，2.810(s，3H)

Preparation of 5-chloro-4-hydroxyfluorescein acid (11A) and NHS ester (11B)

The synthetic route for 4-hydroxyfluorescein acid and NHS esters is the same as that used to prepare 6-hydroxyfluorescein acid and NHS esters. The NMR data are given for the above compounds.

3-chloro-2-methoxyaniline Appel salt:¹H NMR(400MHz，CDCl₃)：7.224(m，1H)，7.068(t，J＝8.2Hz，1H)，6.988(d，J＝8Hz，1H)，3.809(s，3H)

2-cyano-5-chloro-4-methoxybenzothiazole:¹H NMR(400MHz，CDCl₃)：7.591(d，J＝8.4Hz，1H)，7.521(d，J＝8.8Hz，1H)，4.349(s，3H)

2-cyano-5-chloro-4-hydroxybenzothiazole:¹H NMR(400MHz，CDCl₃)：7.576(d，J＝8.4Hz，1H)，7.416(d，J＝8.4Hz，1H)，6.702(s，1H)

5-chloro-4-hydroxydimethylfluorescein acid (11A):¹HNMR(400MHz，CDCl₃)：7.451(d，J＝8.4Hz，1H)，7.370(d，J＝9.2Hz，1H)，6.689(s，1H)，4.903(s，1H)，1.886(s，3H)，1.528(s，3H)

5-chloro-4-hydroxydimethylfluorescein NHS ester (11B):¹H NMR(400MHz，CDCl₃)：7.425(d，J＝8.8Hz，1H)，7.354(d，J＝8.4Hz，1H)，6.707(s，1H)，5.157(s，1H)，2.855(s，4H)，1.882(s，3H)，1.659(s，3H)

preparation of 9.6-chloro-5-hydroxyfluorescein acid (12D)

The synthetic route for 5-hydroxyfluorescein acid is the same as that used to prepare 6-hydroxyfluorescein acid. The NMR data are given for the above compounds.

4-chloro-3-methoxyaniline Appel salt (12A):¹H NMR(400MHz，CDCl₃)：7.396(d，J＝8.8Hz，1H)，6.78-6.76(m，2H)，3.882(s，3H)

2-cyano-6-chloro-5-methoxybenzothiazole (12B):¹H NMR(400MHz，CDCl₃)：7.929(s，1H)，7.636(s，1H)，3.988(s，3H)

2-cyano-6-chloro-5-hydroxybenzothiazole (12C):¹H NMR(400MHz，CDCl₃)：7.927(s，1H)，7.792(s，1H)，5.852(s，1H)

6-chloro-5-hydroxydimethylfluorescein acid (12D):¹H NMR(400MHz，CD₃OD)：7.980(s，1H)，7.511(s，1H)，4.927(s，1H)，1.778(s，3H)，1.489(s，3H)

10.preparation of 5, 7-dimethyl-6-hydroxydimethylfluorescein acid (13E)

The synthetic route for 5, 7-dimethyl-6-hydroxydimethylfluorescein acid is the same as that used to prepare 6-hydroxyfluorescein acid. The NMR data are given for the above compounds.

3, 5-dimethyl-4-methoxyaniline (13A):¹H NMR(400MHz，CDCl₃)：6.320(s，2H)，3.615(s，3H)，2.167(s，6H)

3, 5-dimethyl-4-methoxyaniline Appel salt (13B):¹H NMR(400MHz，CDCl₃)：6.905(s，2H)，3.711(s，3H)，2.278(s，6H)

2-cyano-5, 7-dimethyl-6-methoxybenzothiazole (13C):¹H NMR(400MHz，CDCl₃)：7.845(s，1H)，3.782(s，3H)，2.488(s，3H)，2.429(s，3H)

2-cyano-5, 7-dimethyl-6-hydroxybenzothiazole (13D):¹H NMR(400MHz，CDCl₃): 7.805(s, 1H), 5.2 (broad, s, 1H), 2.443(s, 3H), 2.401(s, 3H)

5, 7-dimethyl-6-hydroxydimethylfluorescein acid (13E):¹H NMR(400MHz，CD₃OD)：7.641(s，1H)，4.905(s，1H)，2.401(s，3H)，2.333(s，3H)，1.777(s，3H)，1.491(s，3H)

11.preparation of fluorescein acid substituted with trifluoromethyl (14E) and NHS ester (fluorescent Dye 7; 14F):

the synthetic route for fluorescein acid and NHS esters substituted with trifluoromethyl is the same as that used for the preparation of 6-hydroxyfluorescein acid and NHS esters. The NMR data are given for the above compounds.

4-methoxy-3-trifluoromethylaniline Appel salt (14)A)：¹H NMR(400MHz，CDCl₃)：7.522(d，J＝2.4Hz，1H)，7.424(dd，J＝8.6Hz & 2.6Hz，1H)，7.064(d，J＝9.2Hz，1H)，3.916(s，3H)

2-cyano-5-trifluoromethyl-6-methoxybenzothiazole (14B):¹H NMR(400MHz，CDCl₃)：8.384(s，1H)，7.451(s，1H)，4.033(s，3H)

2-cyano-7-trifluoromethyl-6-methoxybenzothiazole (14C):¹H NMR(400MHz，CDCl₃)：8.313(d，J＝8.8Hz，1H)，7.374(d，J＝9.2Hz，1H)，3.997(s，3H)

2-cyano-5-trifluoromethyl-6-hydroxybenzothiazole (14D):¹H NMR(400MHz，CDCl₃)：8.368(s，1H)，7.492(s，1H)

5-trifluoromethyl-6-hydroxydimethylfluorescein acid (14E):¹H NMR(400MHz，CD₃OD)：8.168(s，1H)，7.479(s，1H)，4.927(s，1H)，1.779(s，3H)，1.492(s，3H)

5-trifluoromethyl-6-hydroxydimethylfluorescein NHS ester (14F):¹H NMR(400MHz，CDCl₃)：8.278(s，1H)，7.445(s，1H)，5.148(s，1H)，2.861(s，4H)，1.874(s，3H)，1.653(s，3H)

fluorescence measurement of fluorescein compounds

In HORIBA Jobin Yvon-3 fluorescence measurement experiments were performed on a spectrophotometer. Typically, the excitation wavelength is set to 405nm, unless otherwise specified. The excitation and emission slit was 1 nm. A20 μ M solution of the dye in 25mM Tris pH 8.0 buffer was used to obtain fluorescence spectra and to determine the intensity at the maximum emission wavelength shown in Table 6.

TABLE 6

Example 2

The following examples describe methods for conjugating the fluorescent dyes of the present invention to antibody molecules.

In a typical experiment, antibody conjugates of fluorescein derivatives were prepared as follows. The antibody of interest was prepared at a concentration of 4mg/mL in 50mM borate pH 9.0 buffer. The dye reagent was dissolved in anhydrous DMSO at a concentration of 5 mg/mL. A predetermined amount of dye in DMSO was slowly added to the antibody solution and mixed. The reaction was incubated at room temperature for 60 minutes. The reaction was then quenched by the addition of a solution of glycylglycine (200 molar excess to dye used) at a concentration of 75mg/mL in PBS, 2mM EDTA. The dye-antibody conjugate was isolated by desalting on a Sephadex G-50 column after equilibration with PBS. The absorbance of the elution process at 280nm was monitored and antibody-containing zones were collected from the column. The degree of substitution (F/P value) was determined by measuring the absorbance value of the dye-conjugate at 280nm and at the absorption maximum of the dye peak. The degree of substitution was calculated by using the extinction coefficient of the dye used for conjugation, as shown in table 7.

TABLE 7

The following table (table 8) provides data for 6 of the dyes of the present invention conjugated to various conjugates of representative antibodies.

TABLE 8

^aND means not determined

Example 3:

the following examples provide methods of using antibody-dye conjugates in a flow cytometer.

Flow analysis

Optimal amounts of various combinations of the antibody-dye conjugates of the invention (determined by titration alone) were independently combined with whole blood-containing biological samples (0.1mL), incubated for 10-15 minutes, by using VersaLyse^TMCytolytic reagents are processed using standard procedures and analyzed by using a flow cytometer. Briefly, the treated biological sample is mixed with VersaLyse^TMReagent/0.2% formaldehyde (1mL) were combined for 10 minutes, the treated sample was spun down, the supernatant aspirated, the pellet resuspended in PBS (2mL), the cell suspension spun down, and the pellet resuspended in PBS/0.1% formaldehyde (1mL) for analysis. Lymphocyte populations were selected by flow cytometry based on forward scatter and side scatter characteristics. Each subpopulation is identified by the use of specific monoclonal antibodies. The fluorescence signal is collected by using a suitable band-pass filter selected based on the fluorescence emission spectrum of the conjugate.

Similarly, the antibody-Pacific Orange was evaluated^TMA conjugate. In selected cases, the flow data results for each fluorescein derivative dye-antibody conjugate were compared to the corresponding antibody-Pacific Orange dye-conjugate. The following table summarizes these results (table 9). The antibody-fluorochrome conjugates of the present invention show higher signal/noise values compared to the antibody-Pacific Orange conjugate.

TABLE 9

Two-color flow analysis:

in the selected case, the CD8-Pacific Blue of the invention, using a 405nm laser line^TMDye-conjugate and CD 4-fluorescein derivative dye, were similarly run in a two-color flow assay. The results were compared with the corresponding properties of the CD8-Pacific Blue dye conjugate and CD4-Pacific Orange dye (Invitrogen corporation). As shown in FIG. 4, when combined with the antibody, Pacific Orange^TMantibody-Dye 2 conjugates showed a brighter signal when compared to the conjugate.

Multicolor flow analysis:

for one of the dyes of the present invention (Dye3), ten color applications on a 3 laser 10 color PMT instrument equipped with a 405nm laser, a 488nm laser, and a 635nm laser are also shown. An 550/40nm bandpass filter was used to collect the fluorescence output into the photomultiplier tube. The following antibody reagents were used:

1.CD45(RA)-FITC

2.CD56-PE

3.CD45(RO)-ECD

4.CD25-PC5

5.CD19-PC7

6.CD3-APC

7.CD27-APCA700

8.CD5-APCCy7

9.CD8-Pacific Blue

10.CD4-Pacific Orange/CD4-Dye3.

the histogram shown in FIG. 5 shows that in the ten color flow assay, the CD4-Dye3 conjugate gave a brighter signal when compared to the CD4-Pacific Orange conjugate.

Claims (22)

1.A fluorescent dye having the general formula:

wherein the content of the first and second substances,

X₁and X₂Independently is S or O;

Y₁and Y₂Independently of one another, halogen, or Y₁Or Y₂One of which is H and the other isIs a haloalkyl group;

wherein each W is independently H or alkyl;

Z₁is H or alkyl;

wherein Z₁And one W group may be deleted, and if deleted, they are replaced by C in the ring C₄And C₅Substituted with another bond in between;

l is independently a bond or a linking group; and is

RG is a reactive group which is a reactive group,

wherein the linking group comprises a covalent bond having from 1 to 50 non-hydrogen atoms selected from C, N, O, S, P and a halogen atom, and the linking group consists of a single bond, a double bond, a triple bond, or any combination of aromatic carbon-carbon bonds, carbon-oxygen bonds, carbon-sulfur bonds, carbon-nitrogen bonds, and nitrogen-nitrogen bonds; and is

Wherein RG comprises a reactive group selected from the group consisting of: carboxylic acids, activated esters of carboxylic acids, anhydrides, acid chlorides, acid azides, acid halides, aldehydes, chloroformates, amines, hydroxyls, hydrazines, isocyanates, isothiocyanates, sulfonyl halides, tosyls, maleimides, N-hydroxy-succinimidyl esters, aziridines, imines, and disulfide groups.

2. The fluorescent dye of claim 1, wherein the linking group comprises the formula:

R₁C(O)R₂wherein R is₁Is a chemical bond or C attached to the dye_1-10Methylene (CH)₂) n; c (O) is carbonyl; r₂Is OH or a chemical bond attaching a carbonyl group to a reactive group RG; or

R₁C(O)A₁R₃C(O)R₂Wherein R is₁Is a chemical bond or C_1-10A methylene group; c (O) is carbonyl; a. the₁Or NH, S, or O; r₃Is alkenyl (CH)₂) n, a five-membered or six-membered ring having at least one unsaturated bond, or C_1-10Methylene (CH)₂) n in combination with a five-membered ring or a six-membered ring; r₂Is OH or attaches a terminal carbonyl group to a reactive groupChemical bonds on RG.

3. A fluorescent dye according to claim 1 or claim 2, wherein RG comprises a carboxylic acid, a succinimide ester of a carboxylic acid, a hydrazide, an amine, an isothiocyanate, or a maleimide.

4. The fluorescent dye of claim 1, having the following formula:

wherein the content of the first and second substances,

r represents OH, succinimide oxy, NH (CH)₂)nCOOH、NH(CH₂) nCO-succinimide, or NH (CH)₂) n-maleimide, wherein n ═ 1 to 10.

5. The fluorescent dye of claim 1, having the following formula:

6. the fluorescent dye of claim 1, having the following formula:

wherein the content of the first and second substances,

r represents OH, succinimide oxy, NH (CH)₂)nCOOH、NH(CH₂) nCO-succinimide, or NH (CH)₂) n-maleimide, wherein n ═ 1 to 10.

7. The fluorescent dye of claim 1, having the following formula:

8. the fluorescent dye of claim 1, having the following formula:

9. the fluorescent dye of claim 1, having the following formula:

10. the fluorescent dye of claim 1, having the following formula:

wherein the content of the first and second substances,

r represents OH, succinimide oxy, NH (CH)₂)nCOOH、NH(CH₂) nCO-succinimide, or NH (CH)₂) n-maleimide, wherein n ═ 1 to 10.

11. The fluorescent dye of claim 1, having the following formula:

wherein the content of the first and second substances,

X₁and X₂Independently is S or O;

Y₁and Y₂Independently of one another, halogen, or Y₁Or Y₂One of which is H and the other is haloalkyl;

w is H or alkyl;

l is independently a chemical bond or a linking group, wherein the linking group comprises a covalent bond having from 1 to 50 non-hydrogen atoms selected from C, N, O, S, P and a halogen atom, and the linking group consists of any combination of single, double, triple, or aromatic carbon-carbon bonds, carbon-oxygen bonds, carbon-sulfur bonds, carbon-nitrogen bonds, and nitrogen-nitrogen bonds; and is

Wherein RG is a reactive group selected from the group consisting of: carboxylic acids, activated esters of carboxylic acids, anhydrides, acid chlorides, acid azides, acid halides, aldehydes, chloroformates, amines, hydroxyls, hydrazines, isocyanates, isothiocyanates, sulfonyl halides, tosyls, maleimides, N-hydroxy-succinimidyl esters, aziridines, imines, and disulfide groups.

12. The fluorescent dye of claim 1, which is excitable at a wavelength of 340nm to 450 nm.

13. A dye-conjugate having the general formula:

wherein the content of the first and second substances,

X₁and X₂Independently is S or O;

Y₁and Y₂Independently represent halogen, or Y₁Or Y₂One of which is H and the other is haloalkyl;

wherein each W is independently H or alkyl;

Z₁is H or alkyl;

wherein Z₁And one W group may be deleted, and if deleted, they are replaced by C in the ring C₄And C₅Substituted with another bond in between;

l is independently a chemical bond or a linking group, wherein the linking group comprises a covalent bond having from 1 to 50 non-hydrogen atoms selected from C, N, O, S, P and a halogen atom, and the linking group consists of any combination of single, double, triple, or aromatic carbon-carbon bonds, carbon-oxygen bonds, carbon-sulfur bonds, carbon-nitrogen bonds, and nitrogen-nitrogen bonds; and is

Wherein the CM is a conjugated molecule comprising a peptide, protein, polysaccharide, enzyme, lipid, nucleotide, oligonucleotide, or nucleic acid polymer.

14. The dye-conjugate of claim 13, wherein the conjugated molecule is a peptide or a protein.

15. The dye-conjugate of claim 13 or claim 14, wherein the conjugated molecule is an antibody, or antibody fragment.

16. A method of detecting a complementary member of a specific binding pair in a sample, comprising:

a) mixing the sample with the dye-conjugate of any one of claims 13 to 15 that can specifically bind to the complementary member; and

b) detecting complexes formed from the mixture so as to detect the complementary members.

17. The method of claim 16, wherein the complementary member is a protein or peptide.

18. The method of claim 16 or 17, wherein the complementary member is present on a bacterium, virus or animal cell.

19. The method of claim 16 or 17, wherein the complex is detected by its fluorescent response.

20. The method of claim 19, wherein the fluorescent response is detected using a flow cytometer, a fluorometer, a fluorescence microscope, or a fluorescent plate reader.

21. The method of claim 19, further comprising: the fluorescent response is distinguished from the fluorescent response of a second fluorophore having a detectably different optical characteristic.

22. The method of claim 19, further comprising: separating the complex from other sample components based on the fluorescent response.