WO2008027075A2 - Sulfhydryl-reactive, water soluble dyes - Google Patents

Abstract

The invention relates to novel dye compounds and to methods of joining compounds, such as the dye compounds of the invention, to ulfhydryl-containing compounds.

Description

SULFHYDRYL-REACTIVE, WATER SOLUBLE DYES

Related Application

This application claims benefit of the filing date of U.S. Provisional Ser. No. 60/743,305, filed February 16, 2006, the contents of which are incorporated herein by reference.

Government Funding

The invention described herein was made with United States Government support under Grant Numbers ROl -AG- 15430 and R01-GM-57464 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

Field of the Invention The invention relates to novel fluorescent dye compounds and to processes for making compounds that are reactive with sulfhydryl moieties, including cysteine residues in proteins. The invention also relates to biosensor molecules that include the dyes of the invention, and methods for detecting target biomolecules and protein activities using the present biosensors, for example, within living cells.

Background of the Invention

Cell behavior is regulated through transient activation of protein activities at specific subcellular locations. Our ability to study translocation of proteins has been greatly increased by advances in the microscopy of fluorescent protein analogues within living cells. However, in many cases, localized protein activities are controlled not by translocating proteins to the site of action, but by localized activation of a small portion of the protein pool. Hahn, K.; Toutchkine, A. Curr. Opin. Cell Biol. 2002, 14, 167-172; Wouters, F. S.; Verveer, P. J.; Bastiaens, P. I. Trends Cell Biol. 2001, 11, 203-211. Such behaviors are not apparent when studying protein translocations or when using in vitro biochemical approaches. Furthermore, the outcome of signaling protein activation can depend on subtle variations in activation kinetics that are not discernible in the population averages generated by biochemical techniques. For precise quantification of rapid activation kinetics and the level of protein activation, it is also necessary to measure protein activity in living cells. Wouters, F. S.; Verveer, P. J.; Bastiaens, P. I. Trends Cell Biol. 2001 , 11, 203- 21 1 ; Williams, D. A.; Fogarty, K. E.; Tsien, R. Y.; Fay, F. S. Nature 1985, 318, 558- 561 ; Berridge, M. J. J Biol. Chem. 1990, 265, 9583-9586.

Protein activity in living cells has occasionally been observed using FRET (fluorescence resonance energy transfer). Similarly, the interactions between two proteins have been observed by tagging each with different fluorophores that undergo FRET when the proteins associate. FRET biosensors have also been built that bind to a protein only when it adopts a specific confoπnation. These approaches can be useful, but FRET-based techniques suffer from limitations that prevent the study of many important targets. For example, proteins undergoing conformational changes often cannot be "sampled" by a biosensor because the protein is bound to a competing ligand or is incorporated in a multi-protein complex, where it is blocked from biosensor access. However, it is precisely such large, unstable complexes that are difficult to reproduce in vitro and whose transient formation in specific locations must be studied in intact cells. Moreover, even when protein interactions are not sterically blocked by dye or fluorphore, derivatization with a FRET fluorophore near regions of conformational change within the protein can affect its biological activity. Finally, because FRET is generated through indirect excitation, it produces a relatively weak fluorescence signal. Such a low signal leads to low sensitivity and the need for complicated methods to differentiate the real signal from auto-fluorescence or fluorescence of the FRET donor. Therefore, a need exists for tools that can do more than monitor protein movements, and do so without the above-mentioned disadvantages of FRET. As such, there is a need for new dyes and biosensors that can be used to observe and/or quantify diverse protein activities, including changing subcellular locations, conformational changes, posttranslational modifications, and/or small ligand binding of proteins in vivo. Summary of the Invention

The invention relates to quick and easy methods for synthesizing water- soluble, sulfhydryl-reactive compounds. In some embodiments, the methods are used to synthesize novel water-soluble, sulfhydryl-reactive fluorescent dyes. In general, the method includes reaction of electron donor heterocycles with bromo-alkylamine hydrobromide followed by one-pot coupling of the resulting amino-containing compound with an acceptor heterocycle in the presence of chloroacetyl anhydride. The present methods permit quick synthesis of water-soluble compounds that are reactive with sulfhydryl groups, including the cysteine residues on proteins. The present methods are simpler, faster, and consequently more economical that currently available procedures that involve at least three protection-deprotection steps. Moreover, attachment of dyes having the sulfhydryl-reactive moiety provided herein to other molecules is simple and requires only mild reaction conditions.

Another aspect of the invention is a fluorescent dye with advantageous properties for live cell imaging and high throughput screening. The dyes of the invention fluoresce with excitation wavelengths greater than 450 nm. The present dyes also exhibit solvent-dependent changes in fluorescence wavelengths and/or fluorescence brightness. The present dyes are very bright, enabling use of these dyes as sensors of protein behavior, enzyme activity and biological status both in vitro and in vivo. The dyes of the invention can also have the sulfhydryl-reactive groups of the invention. Thus, the present fluorescent dyes can easily be attached to other molecules, including proteins and nucleic acids. Biosensors generated by attachment of the present dyes to proteins and nucleic acids can be used to report changes in the environment of the biosensor or a complex between the biosensor and other molecules. Dyes of the invention are therefore useful in numerous ways, including the formation of biosensors, as detectable labels in biological diagnostic assays, for high-throughput screening, and for monitoring protein activity in living cells.

Thus, one aspect of the invention is a fluorescent compound of formula I, II, III or IV: Ringι=CH-(-CH=CH-)_m-CH=Ring2 I

Ring , ==CH-(-CH==CH-) _m-Ring₂ II

Hal-CH2-CO-NRi-(CH₂)n-Ringι=CH-(-CH==CH-)_m-CH=Ring2 III Hal-CH₂-CO-NR|-(CH₂)_n-Ring,==CH-(-CH==CH-) _m-Ring₂ IV wherein:

Ringi is an aryl, a heteroaryl, cycloalkyl, cycloalkenylene or heterocyclic ring that can be substituted with hydrogen, lower alkyl, oxygen (O), hydroxy (OH), sulfonate (SO₃ ^") or sulfate (SO₄ ^');

Ring? is an aryl, a heteroaryl, cycloalkyl, cycloalkenylene or heterocyclic ring that can be substituted with 0-6 substituents separately selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, oxygen (O), hydroxy (OH), sulfur (S), halogen, sulfonate (SO₃ ^"), sulfate (SO₄ ^"), aryl, cyclopentyl, cyclohexyl or Hal-CH₂-CO-NR|-(CH₂)_n-, wherein each of the lower alkyl, aryl, cyclopentyl or cyclohexyl substituents can be substituted with halogen, sulfonate (SO₃ ^") or sulfate (SO₄ ^"), cyano or lower alkoxy

Hal is halogen;

Ri is hydrogen or -C(0)-(CH₂)_n-Hal; n is an integer of from 2 to 20; and m is an integer of from O to 4.

Examples of aryl groups that can used in the compounds of the invention include phenyl, benzyl or phenylalkylene. In some embodiments, the heteroaryl or heterocyclic ring can have one to three heteroatoms in the ring, wherein the heteroatoms are selected from the group consisting of nitrogen, sulfur or oxygen. Such sulfur heteroatoms can be substituted sulfur atoms, for example, -SO₂-.

Nitrogen heteroatom can also be substituted with lower alkyl, phenyl, benzyl, phenyl, HaI-CH₂-CO-NR i -(CH₂)_n-, or phenyl that is substituted with halogen, alkoxy or cyano. Examples of Ring] moieties that can be used in the compounds of the invention include radicals such as those depicted below, where the asterisks identifies an attachment site for the remainder of the compound:

2,3-Dihydro- lH-indole 3H-Indole Benzothiazole

2,3-Dihydro-thiazole 2,3-Dihydro-[ l ,3,4]thiadiazole

2,3-Dihydro-benzothiazole

2,3-Dihydro-benzooxazole 1 ,4-Dihydro-quinoline 1 ,2-Dihydro-pyridine

Examples of Ring₂ moieties that can be used in the compounds of the invention include the radicals shown below, where the asterisks identifies an attachment site for the remainder of the compound:

2,3-Diliydio-benzo[/₃]thiophene

Indan

Indan- 1,3-dione

Benzo[ύ]thiophen-3-one 2,3-Dihydro-benzo[6]thiophene 1,1-dioxide Hexahydio-pyπmidine

Tetrahydro-pyπmidin-2-one Tetrahydro-pyπmιdin-2-one

1 -Dioxo- 1 ,2-dihydro-l λ -benzo[Z)]thiophen-3-one

Hexahydro-pyπmidine Dihydro-pyπmidine-4,6-dione Dihydro-pyrimidine-4,6-dione

3H-Indole

Pyrimidine-2,4,6-trione 2-Thioxo-dihydro-pyrimidine-4,6-dione

2,3-Dihydro- lH-indole 2,3-Dihydro-benzothiazole Benzothiazole

and wherein: Ri₀ is a substituent selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, oxygen (O), hydroxy (OH), sulfur (S), halogen, sulfonate (SO₃ ^"), sulfate (SO₄ ^"), aryl, cyclopentyl, cyclohexyl or HaI-CH₂-CO-NR ₁ -(CH₂)_n-, wherein each of the lower alkyl, aryl, cyclopentyl or cyclohexyl substituents can be substituted with halogen, sulfonate (SO₃ ^") or sulfate (SO₄ ^"), cyano or lower alkoxy. Another aspect of the invention is a compound of formula V:

wherein:

D is an atom, which is an electron donor and which can be substituted by one or two hydrogen atoms;

Hal is halogen;

W is hydrogen, an electron donating group or a group to enhance the solubility of the fluorescent dye in water;

Z is hydrogen, a sulfhydryl-reactive group, an electron accepting group or a substituent that can enhance the solubility of the fluorescent dye in water; and q and v are integers, each separately selected from 0 to 6.

In some embodiments, D is a primary amine. Similarly, Hal can be Br, Cl, I, or F. Examples of W substituents that can be used in the present compounds include hydrogen, lower alkyl, oxygen (O), hydroxy (OH), sulfonate (SO₃ ^") or sulfate (SO₄ ^"). Examples of Z substituents that can be used in the compounds of the invention include hydrogen, lower alkyl, lower alkoxy, oxygen (O), hydroxy (OH), sulfur (S), sulfonate (SO₃ ^"), sulfate (SO₄ ^"), halogen, aryl, cyclopentyl, cyclohexyl or HaI-CH₂-CO-NRi -(CH₂)_n-, wherein each of the lower alkyl, aryl, cyclopentyl or cyclohexyl substituents can be substituted with halogen, sulfonate (SO₃ ^"), sulfate (SO₄ ^"), cyano or lower alkoxy.

In some embodiments, the ring attached to W is a Ringi moiety, defined as described above. Similarly, in some embodiments the ring to which Z is attached is a Ring₂ radical, as defined above. For example, compounds of the invention can have any one of the following structures:

wherein: Hal is Cl or I; Z is hydrogen or SO₃ ^"; Rio is methyl or (CH₂)₃SO₃ ^"; and Rn is C(CH₃)₂ or sulfur.

Another aspect of the invention is a biosensor comprising a binding entity and a compound of the invention. Such binding entities include peptides, proteins, carbohydrates or nucleic acids. For example, the binding entity can be an antibody, antibody fragment, leucine zipper, histone, enhancer, complementary determining region (CDR), a single chain variable fragment (scFv), receptor, ligand, aptamer, or lectin.

Another aspect of the invention is a method for making sulfhydryl-reactive compound, which comprises:

(a) obtaining a compound with a tertiary amine;

(b) activating the tertiary amine on the compound with an L-(CH₂)_n-D activator to form an activated first reactant as shown below:

wherein: L is a leaving group and D is an electron donating group; and

(c) reacting the activated first reactant with a 2-halo-acetamido moiety which is linked to a leaving group to thereby attach a sulfhydryl- reactive group to the compound as shown below:

wherein: D is an electron donating group, L is a leaving group, and Hal is a halogen.

In some embodiments, the compound with a tertiary amine is a compound of the invention. However, any compound with a tertiary amine can be used in the present method for making sulfhydryl-reactive compounds. Thus, the compound with a tertiary amine can be a biological molecule such as a peptide, protein or a nucleic acid. Examples of other biological molecules that can be used in the present methods includes antibodies, antibody fragments, leucine zippers, histones, enhancers, complementary determining regions (CDRs), single chain variable fragments (scFvs), receptors, ligands, aptamers, or lectins.

Another aspect of the invention is a method of detecting a selected target molecule's activity and/or location within a cell comprising (a) contacting the cell with a biosensor; and (b) observing a change in signal produced by the biosensor and/or observing a location of the signal, wherein the biosensor comprises a binding domain and a dye or fluorescent compound of the invention. For example, the selected target molecule can be a protein, receptor, ligand or enzyme. Thus, the selected target molecule's activity can involve the selected molecule's phosphorylation state, subcellular location, interaction with subcellular structures or interaction with cellular proteins.

Another aspect of the invention is a method of detecting an interaction between a selected endogenous target biomolecule and a cellular entity, the method comprising:

(a) identifying a cell comprising a selected endogenous target biomolecule and a recognizable cellular entity; (b) providing a biosensor comprising (i) a binding entity with specific binding affinity for a binding site on the target biomolecule and (ii) a compound of any one of claims 1-16;

(c) incubating the biosensor with the cell; (d) observing if a background signal is detectable from the biosensor and optionally subtracting the background signal; and (e) detecting a signal change from the biosensor to thereby detect an interaction between the target biomolecule and a cellular entity.

For example, the cellular entity can be a sub-cellular organelle, nucleic acid, protein, peptide, enzyme, receptor, cytokine, cytoskeleton and signal transduction protein. Binding entities can have a specific affinity for a particular target conformation, target-ligand interaction, or posttranslational modification of the target biomolecule. Binding entities can bind to the target biomolecule, for example, at or by a phosphorylation site. In some embodiments, the compounds employed have an excitation or emission light wavelength of about 600 nm or more. In general, the compound does not substantially interfere with binding between the binding entity and the target biomolecule.

In some embodiments, the method of detecting a target can also include introducing the biosensor into a selected cell, for example, by using electroporation, transduction, microporation, microinjection, surfactants, or projectiles. Moreover, in some embodiments, the signal change involves at least a 50% increase in fluorescence. Detecting a signal change can be accomplished by use of fluorimetry, by quantifying fluorescence levels, and/or by locating a cellular entity. For example, detecting a signal change can involve detecting an increase in the signal or a change in wavelength of the signal. In some embodiments, the signal change reflects a change in conformation of the protein, in activation of the protein, or in phosphorylation state of the protein. Such a signal change can result from a change in hydrophobicity, hydrogen bonding, polarity, polarization, phosphorylation, polypeptide folding, hydration, ligand binding, or subunit interaction of the target biomolecule upon interaction with the cellular entity. Another aspect of the invention is a method of attaching a haloacetoamido compound of the invention to a sulfhydryl-containing compound, comprising reacting the haloacetoamido compound with the sulfhydryl-containing compound in an aqueous solvent to form a reaction mixture and incubating the reaction mixture for a time sufficient to generate a product consisting of the compound of claim 9 covalently linked to a sulfhydryl-containing compound. For example, this method can proceed by the following reaction:

nd

For example, in some embodiments the sulfhydryl-containing compound is a polypeptide or nucleic acid. Description of the Figures

FIG. IA-B illustrates one method for generating solvent-sensitive merocyanine dyes of the invention that are also water-soluble and cysteine-reactive. FIG. IA illustrates a reaction between propane sultone and 3-bromopropylamine hydrobromide in the presence of triethylamine to produce 3-

[3 (bromopropyl)amino]propane-l -sulfonic acid (BAS). FIG. IB illustrates quarternization of electron-donor heterocycles with BAS to produce water-soluble heterocycles bearing secondary amines. Synthesis of a series of solvent sensitive merocyanine dyes was accomplished in a one-pot coupling reaction between water soluble, amine-containing electron donor heterocycles and electron-acceptor intermediates in the presence of chloroacetyl anhydride and sodium acetate.

FIG. 2 illustrates the absorption spectra of the protein EGFP-Erk2 labeled with dyes 9a (solid line) and 9b (dashed line).

Detailed Description of the Invention

The invention relates to novel fluorescent dye compounds with improved properties and to methods for attaching sulfhydryl-reactive groups to compounds. In some embodiments the sulfhydryl reactive groups can be attached to the fluorescent dye compounds of the invention.

Definitions

The following definitions are used, unless otherwise described: halo (or halogen) is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc. denote both straight and branched groups; but reference to an individual radical such as "propyl" embraces only the straight chain radical, a branched chain isomer such as "isopropyl" being specifically referred to. Aryl denotes a phenyl radical or an ortho- fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. Heteroaryl encompasses a radical attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non- peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O, (Ci-C₄)alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto. "Alkyl" is a hydrocarbon having up to 25 carbon atoms. Alkyls can be branched or unbranched radicals, for example methyl, ethyl, propyl, isopropyl, n- butyl, sec-butyl, isobutyl, tert-butyl, 2-ethylbutyl, n-pentyl, isopentyl, 1 -methylpentyl, 1,3-dimethylbutyl, n-hexyl, 1-methylhexyl, n-heptyl, isoheptyl, 1,1,3,3- tetramethylbutyl, 1-methylheptyl, 3-methylheptyl, n-octyl, 2-ethylhexyl, 1,1,3- trimethylhexyl, 1,1,3,3-tetramethylpentyl, nonyl, decyl, undecyl, 1-methylundecyl, dodecyl, 1,1,3,3 ,5,5-hexamethylhexyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, icosyl or docosyl.

"Alkenyl" is an alkyl with at least one site of unsaturation, i.e. a carbon- carbon double bond.

"Alkene" is a hydrocarbon having 2 to 25 carbon atoms and at least one double bond.

"Alkylene" is a saturated, branched or straight chain or cyclic hydrocarbon radical of 1-25 carbon atoms. An alkylene has two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Examples of alkylenes include methylene, ethylene, propylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, decamethylene, dodecamethylene or octadecamethylene.

Lower alkyl is an alkyl having 1 to 6 carbon atoms, i.e., lower alkyl is (Cr C₆)alkyl. (Ci-C₆)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec- butyl, pentyl, 3-pentyl, or hexyl; (C₃-C₆)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C₃-C₆)cycloalkyl(C|-C₆)alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2- cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexyl ethyl; (C|-C₆)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3- pentoxy, or hexyloxy; (C₂-C₆)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl, 1- butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1- hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl; (C₂-C₆)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2- pentynyl, 3-pentynyl, 4-pentynyl, 1- hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5- hexynyl; (Ci-C₆)alkanoyl can be acetyl, propanoyl or butanoyl; halo(Ci-C₆)alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2- chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl; hydroxy(Ci- C₆)alkyl can be hydroxymethyl, 1 -hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2- hydroxypropyl, 3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl; (Ci-C₆)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl; (Ci-C₆)alkylthio can be methylthio, ethylthio, propylthio, isopropylthiό, butylthio, isobutylthio, pentylthio, or * hexylthio; (C₂-C₆)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy; aryl can be phenyl, indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N- oxide) or quinolyl (or its N-oxide).

Specific and preferred values listed herein for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents.

It will be appreciated by those skilled in the art that compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine fluorescence using the standard tests described herein, or using other similar tests which are well known in the art.

Fluorescent Dyes of the Invention One aspect of the invention is a fluorescent dye compound. Fluorescent dye compounds of the invention generally have structures shown in Formula I or II: Ring,=CH-(-CH==CH-)_m-CH==Ring₂ I

Ring,=CH-(-CH==CH-) _m-Ring2 II

In other embodiments, the invention is directed to fluorescent dye compounds having at least one sulfhydryl-reactive group. The sulfhydryl-reactive moieties on the compounds of the invention are generally 2-halo-acetamide moieties. Fluorescent dye compounds with at least one sulfhydryl-reactive group are of formula III or IV:

Hal-CH₂-CO-NRi-(CH₂)_n-Ring,=CH-(-CH==CH-)_m-CH==Ring2 III Hal-CH₂-CO-NR,-(CH₂)_n-Ringι=CH-(-CH=CH-) _m-Ring₂ IV wherein:

Ringi is an aryl, a heteroaryl, cycloalkyl, cycloalkenylene or heterocyclic ring that can be substituted with hydrogen, lower alkyl, oxygen (O), hydroxy (OH), sulfonate (SO₃ ^") or sulfate (SO₄ ^"); Ring₂ is an aryl, a heteroaryl, cycloalkyl, cycloalkenylene or heterocyclic ring that can be substituted with 0-6 substituents separately selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, oxygen (O), hydroxy (OH), sulfur (S), halogen, sulfonate (SO₃ ^"), sulfate (SO₄ ^"), aryl, cyclopentyl, cyclohexyl or Hal-CH₂-CO-NRi-(CH₂)_n-, wherein each of the lower alkyl, aryl, cyclopentyl or cyclohexyl substituents can be substituted with halogen, sulfonate (SO₃ ^") or sulfate (SO₄ ^"), cyano or lower alkoxy Hal is halogen;

R₁ is hydrogen or -C(O)-(CH₂)_n-Hal; n is an integer of from 2 to 20; and m is an integer of from O to 4. In some embodiments, the fluorescent dyes of the invention have formula V, shown below:

. wherein: D is an electron donor, for example, D can be a primary amine;

Hal is halogen, for example, Hal can be Br, Cl, I, or F; W is hydrogen, an electron donating group or a group on Ringi to enhance the solubility of the fluorescent dye in water, for example, W can be hydrogen, lower alkyl, oxygen (O), hydroxy (OH), sulfonate (SO₃ ^") or sulfate (SO₄-);

Z is hydrogen, a sulfhydryl-reactive group, an electron accepting group or a substituent that can enhance the solubility of the fluorescent dye in water, for example, Z can be hydrogen, lower alkyl, lower alkoxy, oxygen (O), hydroxy (OH), sulfur (S), halogen, sulfonate (SO₃ ^"), sulfate (SO₄ ^"), aryl, cyclopentyl, cyclohexyl or HaI-CH₂-CO-NR i-(CH₂)_n-, wherein each of the lower alkyl, aryl, cyclopentyl or cyclohexyl substituents can be substituted with halogen, sulfonate (SO₃ ^') or sulfate (SO₄ ^"), cyano or lower alkoxy; q and v are integers, each separately selected from O to 6. Specific examples of Ringi (electron donor ring) include but are not limited to the following:

2,3-Dihydro-lH-indole 3H-Indole Benzothiazole

2,3-Dihydro-thiazole 2,3-Dihydro-[ 1 ,3,4]thiadiazole

2 ,3 -Dihydro-benzothiazole

2,3-Dihydro-benzooxazole ^l ,4-Dihydro-quinoline j ,2-Dihydro-pyridine wherein the asterisks identifies an attachment site for the remainder of the dye compound.

Specific examples of Ring2 (electron acceptor ring) include but are not limited to the following:

2,3-Dihydro-benzo[ft]thiophene

Indan

Indan- 1,3-dione

Benzo[ά]thiophen-3-one 2,3-Dihydro-benzo[6]thiophene 1,1 -dioxide Hexahydro-pyrimidine

Tetrahydro-pyrimidin-2-one Tetrahydro-pyrimidin-2-one

, 1 -Dioxo- 1 ,2-dihydro- 1 λ -benzo[ft]thiophen-3-one

Hexahydro-pyrimidine Dihydro-pyrimidine-4,6-dione Dihydro-pyrimidine-4,6-dione

3//-Indole

Pyrimidine-2,4,6-trione 2-Thioxo-dihydro-pyrimidine-4,6-dione

2,3-Dihydro- l //-indole 2,3-Dihydro-benzothiazole Benzothiazole

wherein the asterisks identifies an attachment site for the remainder of the compound; and Rio is a substituent selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, oxygen (O), hydroxy (OH), sulfur (S), halogen, sulfonate (SO₃ ^'), sulfate (SO₄ ^"), aryl, cyclopentyl, cyclohexyl or Hal-CH₂-CO-NRi-(CH₂)_n-, wherein each of the lower alkyl, aryl, cyclopentyl or cyclohexyl substituents can be substituted with halogen, sulfonate (SO₃ ^") or sulfate (SO₄ ^"), cyano or lower alkoxy. Specific examples of dyes of the invention are shown below.

Dye Structures (Merocyanines, Chloroacetamides).

Dye Structures (Merocyanines, Iodoacetamides):

Additional Dyes Structures:

The dyes of the invention are many times brighter than other dyes used previously to study antibody-antigen and other protein-protein interactions in vitro or in vivo. See Renard, M., Belkadi, L. & Bedouelle, H. Deriving Topological Constraints from Functional Data for the Design of Reagentless Fluorescent Immunosensors; JMoI Biol 326, 167-175 (2003); Sloan, D.J. & Hellinga, H.W. Structure-based Engineering of Environmentally Sensitive Fluorophores for Monitoring Protein-protein Interactions. Protein Eng 11, 819-823 (1998); Iwatani, S., Iwane, A. H., Higuchi, H., Ishii, Y. & Yanagida, T. Mechanical and Chemical Properties of Cysteine-modified Kinesin Molecules. Biochemistry 38, 10318-10323 (1999).

The quantum yields for many of the dyes of the invention are very high in hydrophobic environments. For example, for many of the present dyes in a hydrophobic environment or solvent, the quantum yield is greater than about 0.4, or greater than about 0.5, or greater than about 0.6, or greater than about 0.7, or greater than about 0.80. In some embodiments, the dyes of the invention have a quantum yield that is greater than about 0.90. The dyes of the invention also have high extinction coefficients. Such high extinction coefficients and quantum yields provide strong direct signals that can be detected inn biological samples and from within subcellular locations. These high quantum yields and extinction coefficients can allow detections from very small amounts of dye, minimally perturbing the biological activity of the endogenous proteins being studied, and enabling high resolution kinetic studies of sensitive biological interactions by obtaining many images before photo-bleaching.

The fluorescent dye compounds of the invention are generally environmentally sensitive dyes. Such a dye is "environmentally sensitive" because a signal from the dye changes when the dye is exposed to a change in environment, for example, a hydrophobicity, hydrogen bonding, polarity, or conformational change. Thus, a signal from an environmentally sensitive dye of the invention detectably changes upon exposure to a change in solvent, change in hydrogen bonding, change in the hydrophobicity of the environment, changed polarity or polarization, or change affecting the conformation of the dye. For example, the environmentally sensitive dye can have a detectable change in signal intensity, for example, of 50%, 100%, 200%, 500%, 1000%, or more. Alternatively, the environmentally sensitive dyes can have a detectable change in the wavelength of the signal.

In one embodiment, the signal provided by the environmentally sensitive dye increases when the dye is exposed to an environment that is more hydrophobic. In another embodiment, the signal provided by the environmentally sensitive dye increases when the dye is exposed to an environment where there is increased hydrogen binding between the dye and a component of the environment. Such an increase in hydrophobicity or an increase in hydrogen bonding can occur when a biosensor of the invention binds to a target protein or subcellular component.

In other embodiments, the signal provided by the environmentally sensitive dye decreases when the dye is exposed to an environment that is more hydrophilic. In further embodiments, the signal provided by the environmentally sensitive dye decreases when the dye is exposed to an environment that has less hydrogen binding. Such an increase in hydrophilicity or a decrease in hydrogen binding can occur when a biosensor of the invention is exposed to an aqueous environment or when such a biosensor becomes unbound from a target protein or subcellular component.

Use of biosensors with environmentally-sensitive dyes can provide important advantages over currently available affinity probes, such as those that involve imaging antibodies labeled with non-environmentally sensitive dyes. See, Nizak, C. et al. Science 300, 984-987 (2003). For example, a change in fluorescence intensity or wavelength of emission provided by the present dyes can be quantified in near real time, as compared to visualization of target retrospectively by radioactive or ELISA formats. In many cases the fleeting presence of target cannot be observed by previously available methods with because those methods involve delayed detection.

Dyes of the invention therefore have many properties that make them particularly suitable for detection of targets and molecular interactions within living cells. The dyes are, for example, bright, emitting long wavelengths outside of cellular auto-fluorescence background frequencies that are often damaging to cells. Addition or deletion of parts of the aromatic system can shift excitation and/or emission wavelengths of the dyes so that several related dyes are generated with different emission wavelengths permitting more than one event to be monitored in a cell at the same time.

The dyes of the invention are also designed to have enhanced water solubility, e.g., by attaching W and Z groups that sterically block aggregation without unduly increasing hydrophobicity. This is in contrast to the old and less desirable technique of enhancing water solubility using highly charged groups that can affect protein interaction. Dyes of the invention can be detected in cells by observing changes in intensity, a change in the shape or maxima of the excitation or emission peak, and/or a change in dye lifetime, to permit ratio imaging and other techniques that can eliminate effects of uneven illumination, cell thickness and the like.

Several dyes with excellent spectral properties were previously developed by the inventors, but reaction between these dyes and proteins (e.g., to make a biosensor) led to attachment of multiple dyes, even when labeling was done at low dye concentrations. Moreover, the fluorescence of the "over-labeled" conjugates was weak. This suggested that the dyes were forming non-fluorescent H-aggregates in water, as reported previously for other merocyanines. Wurthner et al., Angewandte Chemie, International Edition in English 39, 1978-1981 (2000); Lu et al., L., J. Am. Chem. Soc. 121, 8146-8156 (1999); Valdes-Aguilera et al., Ace. Chem. Res. 22, 171- 177 (1989). The essentially planar dyes previously developed are thought to be aggregating to reduce the exposure of their hydrophobic surfaces to water. In the present invention, such aggregation was greatly decreased by incorporating bulky, non-planar W and Z substituents, which can have tetragonal geometries, in the aromatic rings, to make stacking unfavorable. This innovation led to dyes with good water solubility while retaining substantial hydrophobic character. These dyes are responsive to protein conformational changes induced within or by the protein itself, conformational changes in target molecules that associate with a labeled biosensor of the invention, binding of biosensors to target molecules, protein-protein reactions, and the like.

Dyes having, for example, formulae I- V, are an aspect of the invention. The invention provides these unique dyes as general structures with generally described ring structures and with generally described W and Z groups, and the invention provides these dyes as specific structures with preferred ring structures and preferred W and Z groups. Preferred dyes of the invention have one or more W or Z groups arranged in tetragonal geometry from rings of the dye. In many cases, the W and Z groups include groups with one or more carbons providing significant steric hindrance to ring stacking. The W and Z groups also have a polar to weak ionic character to enhance water solubility. Dyes of the invention are therefore water- soluble and can be used in the aqueous environments, chemistries or aqueous/organic solvent combinations used to detect and analyze biomolecules.

The dyes of the invention can also have sulfhydryl-reactive groups to facilitate attachment of the dye to biological molecules such as proteins and nucleic acids. Biosensors of the invention, comprising, e.g., dyes linked to binding entities, can be compatible with and move freely in intracellular and/or extracellular environments of living sells. Dyes on the biosensors can exist, for example, in or near binding regions between a sensor and target to provide a detectable signal without significantly interfering with binding.

Synthetic Methods

The invention further relates to a method for making water-soluble, sulfhydryl-reactive compounds by activating a first reactant and coupling the activated first reactant to a sulfhydryl-reactive moiety to thereby form a water-soluble, sulfhydryl-reactive compound. As indicated above, the sulfhydryl-reactive moieties on the compounds of the invention are generally 2-halo-acetamido moieties. Using the methods provided herein, such 2-halo-acetamido moieties can be placed on a variety of molecules including peptides, proteins, nucleic acids and a variety of other small or large molecules (e.g. drugs). Thus, the "first reactant" is a protein, nucleic acid, polysaccharide, lipid, drug or other molecule to which dye will be attached.

As specifically provided herein, the 2-halo-acetamide moieties are coupled to fluorescent compounds to provide the water-soluble, sulfhydryl-reactive fluorescent compounds described herein. Such 2-halo-acetamide moieties are conveniently attached to a selected first reactant, so that a sulfhydryl-reactive compound is formed after such attachment. An activating agent can be used to activate the first reactant and thereby facilitate attachment of the 2-halo-acetamide moiety. The activating agent employed depends to some extent upon the substituents present on the first reactant. One convenient substituent that can be activated on the first reactant to permit attachment of the 2- halo-acetamido moiety is a secondary or tertiary amine. Such secondary and tertiary amines are present on a variety of biological molecules including, for example, amino acids (e.g. arginine, histidine and tryptophan), peptides, proteins, purine and pyrimidine bases, nucleosides, nucleotides, oligonucleotides, and nucleic acids (RNA and DNA). Therefore, the first reactant can be any of these secondary or tertiary amines.

The activating agent employed to activate the first reactant can include a spacer, a leaving group and an electron donating group. Thus, for example, the activating agent can be of formula VI:

L-lower alkyl-D where L is a leaving group and D is an electron donor. A non-aqueous organic solvent is typically used for this reaction, for example, xylene, toluene, benzene or related solvents.

The following reaction further illustrates activation of a first reactant that contains, for example, a tertiary amine:

wherein: L is a leaving group and D is an electron donating group.

As illustrated, the leaving group in this reaction is displaced by the tertiary amine, to form a quarternary amine. The leaving group can be any convenient leaving group available to of skill in the art. Suitable leaving groups include, for example, halogen, acetyl, haloacetyl, trifiate, dimethylsulfonium, trifluoromethane sulfonyl group, sulfonyl halides, aryl-sulfonyl halides (e.g., tosyl-halides), alkyl- sulfonyl halides (e.g., methane sulfonyl halide), halo-alkyl-sulfonyl halides (e.g., trifluoroethane sulfonyl halides), halopyrimidines (e.g., 2-fluoro-l-methylpyridinium toluene-4-sulfonate), triflate and the like. In some embodiments, the leaving group is a halogen.

One example of an activating agent that can be used to activate first reactants is a haloalkylamine, for example, a halogen salt of a haloalkylamine such as Hal-(CH₂)_n-NH₃ ⁺ Br^", where Hal is halogen and n is integer of from 2 to 20. As illustrated herein, activation can readily be accomplished using bromoalkylamine hydrobromide.

When the first reactant contains a tertiary amine and a halogen is used as a leaving group, the activated first reactant may form a salt with the free halogen, which can form a crystalline product. The crystalline form of the activated first reactant is easily purified, for example, by recrystallization from methanol, or by reverse-phase chromatography on Cl 8 column using water-methanol as eluent. In some cases, the crystalline form of the activated first reactant is hygroscopic. To prevent water absorption that might inhibit further reaction, crystalline activated first reactants can be kept in a dessicator.

To form the water-soluble, sulfhydryl-reactive compound, a sulfhydryl- reactive reagent is used to generate the sulfhydryl-reactive group at the site of the electron donor on the activated first reactant. A sulfhydryl-reactive reagent is a 2- halo-acetamido moiety linked to a leaving group. The leaving group is one of those described herein, preferably a haloacetyl group. Examples of sulfhydryl-reactive groups that can be used to attach the sulfhydryl-reactive group to the electron donor include, for example, haloacetic acid anhydride, haloacetyl chloride, chloroacetyl anhydride, or trifluoroacetyl chloride. In some embodiments, the sulfhydryl-reactive reagent is haloacetic acid anhydride, preferably chloroacetic acid anhydride. The activated first reactant and the sulfhydryl-reactive reagent are dissolved in a suitable solvent, for example, dimethylformamide (DMF) or an alcohol (e.g. methanol). Thus, for example, the following reaction can be used for coupling a sulfhydryl-reactive moiety to an electron donor site on the activated first reactant:

wherein: D is an electron donating group, L is a leaving group, and Hal is a halogen. In some embodiments a salt is used during the reaction, for example, sodium iodide or sodium acetate. After the reaction has proceeded to completion, the product can be dried and purified, for example, by column chromatography using a SiO₂ separation material and a suitable solvent, for example, a chloroform-methanol mixture. In some embodiments, the sulfhydryl-reactive compound can also be purified by crystallization from alcohol, for example, a mixture of methanol and isopropanol. Thus, a sulfhydryl-reactive compound is formed by the methods of the invention. As described above, sulfhydryl reactive moieties can be placed on a large variety of small and large molecules. In some embodiments, the sulfhydryl reactive moiety is attached to a dye of the invention.

The methods of the invention for making sulfhydryl reactive compounds can readily be incorporated into methods for making dye compounds of the invention. When synthesizing the fluorescent dye compounds of the invention, the first reactant is a ring with an electron donor. An activated electron donor ring can thus be formed using the activating reagents described above. For example, activation of the electron donor ring can proceed as follows:

wherein: L is a leaving group and D is an electron donating group.

The activated electron donor ring can then be coupled to the sulfhydryl- reactive moiety as described above to form the sulfhydryl-reactive fluorescent dye of the invention.

However, according to the invention, sulfhydryl-reactive fluorescent compounds of the invention that have two rings can readily be generated in a one-pot reaction between the activated electron donor reactant (Ringl), the sulfhydryl- reactive reagent and an electron acceptor ring (Ring2). This permits formation of the water-soluble, sulfhydryl-reactive fluorescent dye compound in a single reaction vessel.

The electron donor reactant is a Ring i -containing reactant and the electron acceptor ring is a Ring₂-containing reactant. Coupling the activated Ringi- containing reactant to a Ring₂-containing reactant and formation of the sulfhydryl- reactive moiety can be performed in one step as illustrated below:

wherein:

D is an electron donor, for example, D can be a primary amine,

L is a leaving group;

Hal is halogen, for example, Hal can be Br, Cl, I or F; W is hydrogen, an electron donating group or a group on Ringi to enhance the solubility of the fluorescent dye in water, for example, W can be hydrogen, lower alkyl, oxygen (O), hydroxy (OH), sulfonate (SO₃ ^") or sulfate (SO₄-); Z is hydrogen, a sulfhydryl-reactive group, an electron accepting group or a substitute that can enhance the solubility of the fluorescent dye in water, for example, Z can be hydrogen, lower alkyl, lower alkoxy, oxygen (O), hydroxy (OH), sulfur (S), halogen, sulfonate (SO₃ ^"), sulfate (SO₄ ^'), aryl, cyclopentyl, cyclohexyl or HaI-CH₂-CO-NR |-(CH₂)_n ^~, wherein each of the lower alkyl, aryl, cyclopentyl or cyclohexyl substituents can be substituted with halogen, sulfonate (SO₃ ^") or sulfate (SO₄ ^"), cyano or lower alkoxy; q and v are integers, each separately selected from O to 6. Suitable solvents for this reaction include non-aqueous organic solvents such as xylene, toluene and benzene. Thus, in some embodiments the reactions for synthesizing dyes of the invention can be carried out in one reaction vessel. A number of water soluble, cysteine-reactive fluorescent dyes were synthesized using the one-pot coupling methods of the invention. One specific example of a reaction between an electron donor heterocyclic ring with bromo- alkylamine hydrobromide followed by one-pot coupling of the resulting amino- compound with an acceptor heterocycle in the presence of chloroacetyl anhydride, is shown below.

O, S

wherein:

X is a heteroatom (e.g. O, N or S) or -C(CH₃)?; A is a Ring2 electron acceptor ring, shown here as a diketone.

Biosensors

The dyes of the invention can be incorporated into biosensors. Such biosensors include a binding entity (e.g., an antibody or a nucleic acid probe) or a target molecule of interest and at least one dye of the invention. In the case of biosensors consisting of a target molecule of interest, the dyes of the invention can be linked to the target at a position that permits detection of a change in signal by the dye when, for example, the target changes conformation, binds a ligand, interacts with another protein, undergoes phosphorylation, or becomes post-translationally modified. Similarly, the dyes of the invention can be linked to binding entities that bind to targets of interest - when bound the present dyes again provide a change in signal when, for example, the target changes conformation, binds a ligand, interacts with another protein, undergoes phosphorylation, or becomes post-translationally modified. The dyes of the invention can be linked to a binding entity or target of interest using the sulfhydryl-reactive moieties of the invention. As used herein, "binding entities" include any molecule that can specifically bind to a target molecule. Binding entities are typically binding regions of affinity molecules known in the biological sciences including, but not limited to, antibodies, antibody fragments, leucine zippers, histones, enhancers, complementary determining regions (CDRs), single chain variable fragments (scFv's), receptors, ligands, aptamers, lectins, nucleic acid probes and the like. Binding entities can simply comprise, for example, either member of a pair of proteins in a protein-protein interaction, where the "binding entity" member is the member introduced into an assay system to probe for its binding partner (or target). Binding entities of the invention can include binding regions that are generated, for example, of full sized versions of an affinity molecule, fragments of an affinity molecule, or the smallest portion of the affinity molecule providing binding that is useful in the detection of a target of interest. In many embodiments, the binding entities can have specific affinity for endogenous (e.g., constitutive or inducible, but not recombinant) polypeptides in a cell.

Biosensors of the invention can react with diverse targets. Biosensors of the invention can be designed with binding entities that bind only to a particular target protein or state of a targeted protein. When the biosensor binds to the target, a fluorescence change in a dye included in the biosensor can be detected to reveal, for example, the level and/or location of protein in the targeted state. Changes over time can be monitored. Biosensors can incorporate binding entities of naturally occurring protein domains with specific binding activity for a target. The binding entities can optionally, for example, be full length affinity proteins, members of protein-protein interaction pairs (or portions thereof), Fv antibody fragments, aptamers, Vh antibody fragments, and the like. Signals from the biosensors can depend on FRET systems or, preferably, employ a single environmentally-sensitive dye. Single chain variable fragment (scFv) binding entities can be particularly useful in modular biosensors of the invention in which binding entity and/or target modules connected with a linker can be replaced with alternate versions to provide new desired specificities to the sensor. In another preferred aspect, scFvs can be attached to environmentally sensitive dyes of the invention to form biosensors useful for probing living cells. The dyes of the invention can be attached to a part of a protein of interest for forming a biosensor that is subject to changed phosphorylation states and/or protein- ligand interactions, where the ligand can be a small molecule or a second protein. Such a dye-protein is a relatively simple biosensor of the invention. In some embodiments, dyes of the invention are used. The biosensor can then emit a signal that is correlated with the phosphorylation state of the biosensor that signals that the biosensor is involved in a protein-protein interaction. This type of detection is in contrast to previously described detections (Hahn et al., Solvent-sensitive Dyes to Report Protein Conformational Changes in Living Cells, J Am Chem. Soc 125, 4132- 4145 (2003)) where the biosensors detect a conformational change in the target protein induced by the action of a third element. For example, in Hahn, K. M. et al., J. Biol. Chem. 265, pp 20335-20345, (1990), a conformational change was induced by calcium in a calmodulin target and that was detectable as a signal from an attached dye.

In the present invention, detection is extended to conformational changes induced by phosphorylation, but does not necessarily require induction of a conformational change in the protein of interest. An important advantage of this technique is that proteins within multi-protein complexes can be monitored in situations where previously available types of detection methods, for example, those requiring a large antibody to find and bind to the target protein, would be blocked. Environmentally sensitive dyes of the invention are particularly well suited to such biosensor applications. Previous dyes were not suitable for many proteins because the dyes were insoluble except in particular aqueous or organic solvents.

Furthermore, single molecules of the dyes of the invention can be detected, so that there is no need to use two fluorophores, as in FRET. Thus, small amounts of the present dyes can be detected directly and with a brighter signal. In another embodiment, the biosensor includes a dye attached to a binding entity. Binding entities can comprise polypeptide or nucleic acid sequences. For example, binding entities can be single stranded DNA (sDNA), double stranded DNA (dsDNA), RNA, nucleic acids with modified bases, and the like. In one embodiment, the binding entity is an oligonucleotide probe and the target is a complimentary target nucleic acid. In another embodiment, the binding entity is a dsDNA strand specific to a target enhancer protein target. In a further embodiment, the binding entity is a polypeptide. Environmentally sensitive dyes of the invention can be linked to proteins through cysteine residues present in the protein. Environmentally sensitive dyes of the invention can be linked to nucleic acids by modifying the nucleic acid to include a sulfhydryl group. In other embodiments, the sulfhydryl group can be incorporated into the nucleic acid by in vitro synthesis of the nucleic acid using modified bases that contain one or more sulfhydryl groups. The sulfhydryl group- containing nucleic acid can then be linked to a sulfhydryl-reactive dye of the invention. Thus, the dyes of the invention can be attached to any compound that has or can be modified to have a sulfhydryl group. The following reaction scheme illustrates how the dyes of the invention can be attached to a compound with a sulfhydryl group.

wherein W, Z, D, q and v are as described above.

In some preferred embodiments, the binding entity comprises a polypeptide or peptide sequence. The sulfhydryl-reactive dyes of the invention are easily attached to cysteine residues in proteins as shown below.

Dyes with sulfhydryl reactive groups can be attached to selected molecules by reaction of the dye with the selected molecule at room temperature using a suitable solvent (e.g., an aqueous buffer). The dye can initially be dissolved in a suitable organic solvent, for example, dimethyl sulfoxide (DMSO) and aliquots of the dye solution can be added to the selected molecule. Optimal dye attachment is achieved in some embodiments by using a molar excess of dye. For example, the ratio of selected molecule to dye can be about 1 :2, or 1 :3, or 1 :4 or 1 :5 or 1 :10. In some embodiments, the reaction is performed in an aqueous buffer at neutral or slightly alkaline pH. For example, a buffer such as a sodium phosphate buffer with a pH of about 7.4 can be used The dye-molecule mixture is then incubated at room temperature with gentle mixing. The reaction will proceed to yield a good quantity of labeled product within about 1 to about 10 hours. In some embodiments, a good quantity of labeled product is obtained within about two hours. The reaction can be stopped by addition of a sulfur-containing compound, for example, β- mercaptoethanol. The unreacted dye can be removed from the labeled product by gel filtration column chromatography. Therefore, molecules such as polypeptides and nucleic acids that have an exposed sulfur group can quickly and easily be labeled with the dyes of the invention under mild conditions that do not denature or otherwise adversely affect those polypeptides and nucleic acids.

A more detailed description of one example of a protein labeling procedure is provided as follows.

The selected dye can be dissolved in lOOμL DMSO and an aliquot of the dye solution can be diluted 1 : 5000 in DMSO. The absorbance of the diluted dye aliquot is then measured to determine concentration of dye. Optimal dye attachment using a dye with a -N-CO-CH2-I functional group is achieved when the protein to dye ratio was about 1 :5. A 300μL reaction volume (buffer: 5OmM sodium phosphate, pH 7.4) is used with about lOOμM of protein (~5mg/ml). The dye-protein mixture is incubated with mixing (using a shaker or rotating platform) at room temperature for 2 hours. The reaction can be stopped by addition of 5μL β-mercaptoethanol with mixing and incubation at room temperature for 5 min. The unreacted dye can be removed from the labeled protein using G- 15 or G-25 gel filtration column chromatography. The labeling efficiency can be determined by dilution of the labeled protein in DMSO and measurement of the absorbance at the wavelength of maximal absorption of the dye (to determine dye concentration) combined with protein concentration determination by SDS-PAGE.

After attachment of the dye, the conjugated dye-molecule, or biosensor, can be used in any assay or experiment selected by one of skill in the art. For example, biosensors that have a peptide binding entity can be used to detect and monitor a target molecule of interest. The affinity and specificity of peptide binding entities for a target can be provided by a short sequence of amino acids (e.g., 3 to 20 residues), or the specificity can rely on contributions of amino acid side chains brought in proximity by the primary, secondary, tertiary, and/or quaternary structural conformations of one or more affinity proteins. Binding entities made from peptides can have natural amino acid side chains, modified side chains, or the like that provide reactive groups specifically reactive with sulfhydryl -reactive groups on dyes of the invention.

In a one embodiment of the invention, the biosensors comprise binding entities which are members of the immunoglobulin family of proteins, or derivatives thereof. For example, the binding entity can be a complete immunoglobulin, fragment, single chain variable fragment (scFv), a heavy or light chain variable region, a CDR peptide sequence, and/or the like.

Antibody molecules belong to a family of plasma and cell surface proteins called immunoglobulins, whose basic building block, the immunoglobulin fold or domain, is used in various forms in many molecules of the immune system and other biological recognition systems. A standard antibody is a tetrameric structure consisting of two identical immunoglobulin heavy chains and two identical light chains and has a molecular weight of about 150,000 Daltons.

The heavy and light chains of an antibody consist of different domains. Each light chain has one variable domain (VL) and one constant domain (CL), while each heavy chain has one variable domain (VH) and three or four constant domains (CH). See, e.g., Alzari, P. N., Lascombe, M.-B. & Poljak, R. J. (1988) Three-dimensional structure of antibodies. Annu. Rev. Immunol. 6, 555-580. Each domain, consisting of about 110 amino acid residues, is folded into a characteristic β-sandwich structure formed from two β-sheets packed against each other, the immunoglobulin fold. The VH and VL domains each have three complementarity determining regions (CDRl -3) that are loops, or turns, connecting β-strands at one end of the domains. The variable regions of both the light and heavy chains generally contribute to antigen specificity, although the contribution of the individual chains to specificity is not always equal. Antibody molecules have evolved to bind to a large number of molecules through these six randomized loops (CDRs). Immunoglobulins can be assigned to different classes depending on the amino acid sequences of the constant domain of their heavy chains. There are at least five (5) major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM. Several of these may be further divided into subclasses (isotypes), for example, IgG-I , IgG-2, IgG-3 and IgG-4; IgA-I and IgA-2. The heavy chain constant domains that correspond to the IgA, IgD, IgE, IgG and IgM classes of immunoglobulins are called alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), respectively. The light chains of antibodies can be assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on the amino sequences of their constant domain. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

The term "variable" in the context of variable domain of antibodies, refers to the fact that certain portions of variable domains differ extensively in sequence from one antibody to the next. The variable domains are for binding and determine the specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed through the variable domains of antibodies. Instead, the variability is concentrated in three segments called complementarity determining regions (CDRs), also known as hypervariable regions in both the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called framework

(FR) regions. The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from another chain, contribute to the formation of the antigen-binding site of antibodies.

An antibody that is contemplated for use in the present invention thus can be in any of a variety of forms, including a whole immunoglobulin, an antibody fragment such as Fv, Fab, and similar fragments, a single chain antibody which includes the variable domain complementarity determining regions (CDR), and the like form^'s, all of which fall under the broad term "antibody", as used herein. The present invention contemplates the use of any specificity of an antibody, polyclonal or monoclonal. In many embodiments, in the context of methods described herein, an antibody, or fragment thereof is used that is immunospecific for a selected target. The term "antibody fragment" refers to a portion of a full-length antibody, generally the antigen binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments. Papain digestion of antibodies produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Fab fragments thus have an intact light chain and a portion of one heavy chain. Pepsin treatment yields an F(ab')₂ fragment that has two antigen binding fragments that are capable of cross-linking antigen, and a residual fragment that is termed a pFc¹ fragment. Fab' fragments are obtained after reduction of a pepsin digested antibody, and consist of an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained per antibody molecule. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region.

Fv is a small antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (V_H -V _L dimer). It is in this configuration that the three CDRs of each -variable domain interact to define an antigen binding site on the surface of the V_H -V _L dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. Antibody fragments usefully incorporated into biosensors of the invention can include, e.g., single CDRs, V_H regions, V _L regions, Fv fragments, F(ab) and F(ab')₂ fragments

Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Antibody fragments used in binding entities of the invention can include, e.g., natural, synthetic, or recombinant versions. Single chain antibodies are genetically engineered molecules containing the variable region of a light chain and a variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Such single chain antibodies are also referred to as "single-chain Fv" or "scFv" antibody fragments. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds. Springer- Verlag, N.Y., pp. 269-315 (1994).

In some embodiments of the invention, any antibody or antibody fragment can be used in a binding entity to which dyes can be attached to form a biosensor. In one embodiment, single chain variable fragments are used as binding entities. Any available scFv can be used so long as it binds to a selected target with sufficient affinity to permit detection of a complex formed between the scFv and the selected target. A fluorescent dye can be attached to the selected scFv at any convenient site, for example, at a cysteine residue on the scFv.

Administration of Biosensors

Biosensors of the invention can be used in vitro and/or in vivo to detect target molecules of interest. In many cases, the biosensors can simply be added to test samples in an assay, and no addition of multiple reagents and/or wash steps are required before detection of the target. '

Test samples for in vitro assays can, for example, be molecular libraries, cell lysates, bodily fluids (urine, blood, serum, saliva, etc.), analyte eluates from chromatographic columns, and the like. The in vitro assay often takes place in a chamber, such as, e.g., a well of a multiwell plate, a test tube, an Eppendorf tube, a spectrophotometer cell, conduit of an analytical system, channels of a microfluidic system, and the like. In an exemplary in vitro assay of the invention, an enzyme protein of interest is coated to the bottom of 96-well dishes also containing solutions representing a library of possible enzyme substrates. A biosensor of the invention with specific affinity for enzyme-substrate complex is added to each well. A multiwell scanning fiuorometer is used to observe each well for fluorescence. Wells containing enzyme substrate can be identified as those in which fluorescent emissions at the wavelength of the biosensor dye. That is, in this example, the binding entity of the biosensor only binds to enzyme acting on substrate; the binding placing the dye into a binding pocket environment that significantly changes the emissions intensity of the dye.

Where biosensors of the invention are administered to living cells, binding can take place with targets on the cell surface, or the biosensor is transferred into the cell to make contact with an intracellular target molecule. In some cases, the biosensor can penetrate a cell suspected of containing a selected target passively by mere exposure of the cell to a medium containing the biosensor. In other embodiments, the biosensor is actively transferred into the cell by mechanisms known in the art, such as, e.g., poration, injection, transduction along with transfer peptides, and the like.

In some embodiments, one of skill in the art may choose to incorporate a translocation functionality on the biosensor in order to facilitate the translocation or internalization of that biosensor from the outside to inside the cell. As used herein, the term "translocation functionality" refers to a chemical compound, group or moiety that increases the cell's ability to internalize another compound or material, for example, a biosensor. Examples of such translocation functionalities include peptide recognition/transport sequences, liposomal compositions, or the like. Alternative translocation methods and compositions are also utilized in accordance with the present invention to induce uptake of the second component, including, e.g., electroporation, cell permeating compositions containing, e.g. PEG, porins, saponins, streptolysin or the like.

Techniques useful for promoting uptake of biosensors include optoporation, for example, as described in Schneckenburger, H., Hendinger, A., Sailer, R., Strauss, W. S. & Schmitt, M. Laser-assisted optoporation of single cells. JBiomed Opt 7, 410- 6 (2002); or Soughayer, J. S. et al., Characterization of Cellular Optoporation with Distance. Anal Chem 72, 1342-7 (2000). A variety of transduction peptides are also useful for promoting uptake of biosensors including those described in Zelphati, O. et al., Intracellular Delivery of Proteins with a New Lipid-mediated delivery System. J Biol Chem 276, 35103-10 (2001); Yang, Y., Ma, J., Song, Z. & Wu, M., HIV-I TAT- Mediated Protein Transduction and Subcellular Localization Using Novel Expression Vectors. FEBS Lett 532, 36-44 (2002); and Torchilin, V. P. et al., Cell Transfection in Vitro and In Vivo with Nontoxic TAT Peptide-liposome-DNA Complexes. Proc Natl Acad Sci USA 100, 1972-7 (2003). Additional techniques such as electroporation can also be used. Examples of electroporation procedures are provided in Glogauer, M. & McCulloch, C. A., Introduction of Large Molecules into Viable Fibroblasts by Electroporation: Optimization of Loading and Identification of Labeled Cellular Compartments. Exp Cell Res 200, 227-34 (1992); Teruel, M. N. & Meyer, T., Parallel Single-cell Monitoring of Receptor-triggered Membrane Translocation of a Calcium-sensing Protein Module. Science 295, 1910-2 (2002); and Teruel, M. N., Blanpied, T. A., Shen, K., Augustine, G. J. & Meyer, T., A Versatile Microporation Technique for the Transfection of Cultured CNS Neurons. J Neurosci Methods 93, 37-48 (1999).

Another procedure for introducing molecules such as biosensors into cells is the osmotic shock procedure. Examples of osmotic shock procedures include those described in Okada, C. Y. & Rechsteiner, M., Introduction of Macromolecules into Cultured Mammalian Cells by Osmotic Lysis of Pinocytic Vesicles. Cell 29, 33-41 (1982); and Park, R. D., Sullivan, P. C. & Storrie, B., Hypertonic Sucrose Inhibition of Endocytic Transport Suggests Multiple Early Endocytic Compartments. J Cell PAys/o/ 135, 443-50 (1988).

One of skill in the art may also employ bead/syringe loading to introduce the biosensors of the invention into cells. Bead/syringe loading procedures are described in McNeil, P. L., Murphy, R. F., Lanni, F. & Taylor, D. L., A Method for Incorporating Macromolecules into Adherent Cells, J. Cell Biol. 98, 1556-1564 (1984); and McNeil, P. L. & Warder, E., Glass Beads Load Macromolecules into Living Cells. Journal of Cell Science 88, 669-678 (1987).

Nucleic acids encoding binding entities of the invention can optionally be introduced into cells in expression plasmids, e.g., by transduction or other forms of transformation. Once inside the living cells, the binding entity can be translated from the nucleic acid to a functional peptide. Dyes of the invention can enter the cell, e.g., by injection or diffusion to become linked to the expressed binding entity to generate a biosensor in situ.

Detection of Target-Biosensor Binding Reactions

A wide variety of binding reactions can be detected and monitored using the present biosensors, for example, protein-protein interactions, receptor-ligand interactions, nucleic acid interactions, protein-nucleic acid interactions, and the like. Detection of a target molecule can include, for example, observation of the presence of the target molecule, identification of a specified state of a target molecule, quantification of the target molecule, and/or localization of the target molecule. Multiple measurements peπnit determination of the kinetics of target molecule interactions, target molecule conformational changes, target molecule enzyme activities and the like. Moreover, as described herein, multiple targets can be detected and/or monitored. The ability to monitor multiple targets permits the balance between different signaling activities to be monitored. In the intracellular environment, many of these types of reactions are involved in the multiplicity of steps that comprise signal transduction within cells. For example, activation of a particular cellular event is often triggered by the interaction between a cell surface receptor and its ligand. The signal from the receptor is often transmitted along via the binding of enzymes to other proteins, for example, kinases, which then pass the signal on through the cell until the ultimate cell system response is achieved. In many cases, the signal or ultimate response can be detected using biosensors of the invention. For example, signal transduction often involves phosphorylation of system molecules that can be detected directly with the phosphate involved in the binding site, or indirectly through conformational changes induced by the phosphorylation. In one embodiment, the invention provides methods for identifying the activation status of endogenous proteins in living cells. Biosensors of the invention can permit identification, quantification, and resolution of the spatial, temporal and compartmental regulation of receptor phosphorylation and activation during various processes, for example, endocytosis. In another embodiment, the biosensors and methods of the invention can permit observation of epidermal growth factor receptor (EGFR) effects on the development and progression of breast cancer. In a further embodiment, complex formation between HIV gpl20 and CD4 cell receptors can be monitored.

In accordance with the present invention, binding interactions can occur between a biosensor and one or more target molecules or components of the cell. A "target molecule of interest" is a molecule that is known by, or available to, one of skill in the art and is selected for interaction with a biosensor of the invention. A target molecule often comprises an endogenous unlabeled and/or untagged component of a test solution or cell. Endogenous components can, for example, be expressed by the cell naturally, or be present in a cell as a result of introduction of an appropriate genetic construct within the cell. For example, nucleic acid or protein target molecules can be expressed in the cell, either naturally (e.g., constitutively) or by induction of an appropriate genetic construct introduced into the cell line. Cells Subject to Biosensor Detections The methods and biosensors of the present invention can be useful in detection of target molecules in or on the surface of virtually any type of biological cell, including, mammalian, bacterial, fungal, yeast, insect, and plant cells. In some embodiments, target molecules can be detected in freshly isolated cells from mammals (e.g., humans), insects, fungal, or bacterial cells. For example, blood cells, such as B cells, T cells, monocytes, and neutrophils, and the like, can be probed with biosensors of the invention. In other embodiments, stably maintained cell lines such as CHO, HEK-293, L-cells, 3T3 cells, COS, or THP-I cells can be investigated using methods of the invention.

Useful information can be obtained from any type of cell using the biosensors and methods of the invention. For example, mammalian cells, such as human cells or animal cells, that naturally or recombinantly express human proteins can be evaluated to identify potential human therapeutics, observed for interactions between biomolecules, and/or studied for the effects of ligands, drugs, and other molecules on mammalian and human systems. In another example, bacterial or fungal cells can be used to screen for potential antibiotic or anti-fungal agents.

In some embodiments, well characterized cell lines known to provide predictive models of human cell functions can be used to obtain results correlated with human systems in pharmaceutical and medical research. Exemplary cell lines useful in such research include, for example, COS cells, CHO cells, HEK-293 cells, RBL-I , Jurkat, U937, and YB-I cells. The cells to be monitored can be provided in either immobilized foπn or as a suspension culture. Immobilized cells, such as, for example, cell lawns, tissue slices, or libraries, can be monitored by procedures available to one of skill in the art. Such procedures include, for example, microscopy, scanners, or other imaging systems. The target molecules in immobilized cells can be monitored while the cells are alive, or after fixation of the cells.

In many embodiments, cells subject to target molecule detection with biosensors of the invention are in suspension. Suspended cells can be cells from suspension cell culture or cells liberated from tissues or lawns. Suspended cells are particularly well suited to handling and monitoring in fluidic systems, such as cell sorters, cell counters, and microfluidic systems. Cell suspensions can be provided at cell densities appropriate to the handling system and detection method that is being employed. Determination of optimal cell densities is routine for one of ordinary skill in the art. In the case of flow-through embodiments of the invention, cell densities of monitored suspensions generally range from about 1 cell/nl to about 30 cells/nl in , e.g., a reaction vessel or detection channel. In the case of test tube or multiwell plate based reactions, cell densities typically range from about 1,000 cells/mm² to about 100,000 cells/mm². Of course, these ranges can vary depending upon, e.g., the cell types used, the type of biosensor employed, the type of interaction to be studied, the relative adherence of the cells to the vessel surfaces, as well as each other factors. Biosensor Kits

The invention further provides a packaged composition such as a kit or other container for detecting, monitoring or otherwise observing a target molecule. The kit or container can hold a biosensor of the invention and instructions for using the biosensor for detecting, monitoring or otherwise observing a target molecule. The biosensor can include at least one binding entity and a dye. Alternatively, the kit or container holds a dye of the invention and instructions for using the dye. In some embodiments, kits containing dyes can contain instructions for attaching a dye to a molecule selected by one of skill in the art. Solvents and reagents for facilitating dye attachment can also be included.

The kits of the invention can also comprise containers with solutions or tools useful for manipulating or using the dyes or biosensors of the invention. Such tools include buffers, reaction tubes, reagents for coupling dyes of the invention to selected binding entities and the like. In one embodiment, the kit can contain solvents and/or buffers to facilitate coupling of a dye of the invention to a selected molecule and/or a solution of mercaptoethanol for quenching the conjugation reaction. The kit can also contain a container of buffer at roughly neutral pH (e.g. sodium phosphate buffer, pH 7.5).

The following examples are illustrative of the present invention, but are not limiting. Numerous variations and modifications on the invention as set forth can be effected without departing from the spirit and scope of the present invention.

EXAMPLE 1: Synthesis of 3-[(3-bromopropyl)ammonio]propane-l -sulfonate

(BAS)

To a mixture of bromopropylamine hydrobromide (0.5 g, 2.3 mmol) and [1 ,2]oxathiolane 2,2-dioxide (0.4 ml, 4.6 mmol) in ethanol (EtOH, 5 ml), triethylamine (Et₃N, 0.32 ml, 2.3 mmol) was added and reaction mixture was stirred at room temperature for 24 h. The residue was filtered and crystallized from methanol to produce 0.27 g (45%) of desired product, 3-[(3- bromopropyl)ammonio]propane-l -sulfonate (BAS). M.p. = 184-186⁰C.

EXAMPLE 2: General Procedure for the synthesis of cysteine reactive water soluble dyes

wherein: X is a heteroatom (e.g. O, N or S) or -C(CH₃)₂.

The conditions employed were: (a) Xylene, 15O⁰C, BAS; (b) dimethylformamide (DMF), room temperature (r.t.), sodium acetate (AcONa), chloroacetic anhydride, and an Electron Acceptor Intermediate, where the Electron Acceptor Intermediate was generally of the following structure:

A mixture of 2,3,3-trimethylindolenine (6.19 ml, 39 mmol) with BAS (1 g, 3.9 mmol) was stirred in xylene at 200 ⁰C in a sealed reaction vessel for 30 min. A glassy substance formed on the walls. Excess 2,3,3-trimethylindolenine was decanted and the residue was triturated with diethyl ether. The resulting crystals were filtered to produce 0.8 g (50%) of desired product, which was used in the next step without purification. Note that the ionic character of the product compound results in very hygroscopic crystals, therefore the resulting compound was kept in a dessicator, to ensure completion of the next step.

To a suspension of the product from the previous reaction (0.3 Ig, 0.74 mmol) in dimethylformamide, solid chloroacetic anhydride (0.63g, 3.7 mmol, 5 eq) was added in one portion, followed by immediate addition of sodium acetate (0.182g, 2.2 mmol, 3 eq). Solid electron acceptor intermediate (the enol ether shown above, 0.198 g, 0.74 mmol) was added immediately in one portion. Stirring continued at room temperature for 24 h and then the solvent was evaporated in vacuo. The residue was separated by column chromatography (SiO₂, CH₂Cl₂/Me0H = 5/1) to produce 0.149 g of desired compound, yield 32%. Analytical samples were obtained by crystallization from a mixture of methanol and isopropanol (MeOH:iPrOH = 1 : 1).

EXAMPLE 3: Synthesis of cysteine-reactive water-soluble dye I-SO-s-CA

A cysteine-reactive, water-soluble dye named I-SO-s-CA was synthesized as depicted above and described below. A mixture of 2,3,3-trimethylindolenine (6.19 ml, 39 mmol) with BAS (1 g, 3.9 mmol) was stirred in xylene at 200 ⁰C in a sealed reaction vessel for 30 min. A glassy substance formed on the walls. Excess 2,3,3-Trimethylindolenine was decanted and residue was triturated with diethyl ether. The resulting crystals were filtered to produce 0.8 g (50%) of desired product that was used in the next step without purification. Due to the ionic character of the compounds the resulting hygroscopic crystals were kept in dessicator.

To a suspension of the product from the previous reaction (0.31 g, 0.74 mmol) in DMF, solid chloroacetic anhydride (0.63g, 3.7 mmol, 5 eq) was added in one portion, followed by immediate addition of sodium acetate (0.182g, 2.2 mmol, 3 eq). Solid electron acceptor intermediate (an enol ether, 0.198 g, 0.74 mmol) was added immediately in one portion. Stirring continued at room temperature for 24 h. The solvent in the reaction mixture was evaporated in vacuo and the resulting residue was separated by column chromatography (SiO₂, CH₂Cl₂ZMeOH = 5/1 ) to produce 0.149 g of desired compound, yield 32%. Analytical samples were obtained by crystallization from a mixture of methanol and isopropanol (MeOH:iPrOH = 1 :1).

EXAMPLE 4: Synthesis of water soluble merocyanine and cyanine dyes having halogenacetamide reactive group.

Using procedures like those described in Examples 1-3, many merocyanine and cyanine dyes were synthesized that had halogen acetamide reactive groups (Dye- NHCOCH₂L, where L is Cl, Br, or I; and Dye is a fluorescent dye molecule). The methods employed included quarternization of substituted indole with bromoalkylamine hydrobromide having the formula Br-(CH₂)_H-NH₃ ⁺ Br^" where n can be any number in the range 2-20 (2<n<20). The following activation reaction scheme was employed:

where R is a substituent such as H, halogen, alkyl, aryl, or sulfonate (SO₃ ^"). Using R as a sulfonate group is preferred for synthesis of water soluble dyes.

The resulting indole salt formed as shown above was purified by recrystallization from methanol, or by reverse-phase chromatography on Cl 8 column using water-methanol as eluent. In general, recrystallization from methanol was preferred.

The indole salt reacted with dye intermediates of structure 1 (preparation of cyanine dyes) or dye intermediates of structure 2 (preparation of merocyanine dyes), which are shown below, when using dimethylformamide (DMF) as solvent in presence of halogenacetamide and sodium iodide. Typical ratios employed were indole salt: intermediate 1 or 2: halogenacetoamide: sodium acetate = 1 : 1.2: 2:3. The resulting dye was purified by column chromatography on SiO₂ using acetone- methanol as eluent or by reverse phase chromatography on Cl 8 using water-methanol as eluent. In general, purification by column chromatography on SiO₂ using acetone- methanol as eluent was preferred. When using chloroacetamide the preferred reaction temperature was 25 ⁰C, when using bromo or iodoacetamides the preferred temperature was 0 ⁰C. Intermediates 1 and 2 were prepared using available published methods.

Preparation of Cvanines:

Scheme 1. Synthesis of Cyanine dyes.

R = -SO₃

R₂ = S, O, C(CH₃)₂

R₃ = alkyl, alkylenesulfonate such as SO₃-(CH₂)n- where n is 2, 3 or 4. A sulfonate group is preferred for preparation of water soluble dyes.

R₄ = alkyl, aryl, halogeno, alkoxy, sulfonate (SO₃). A sulfonate group is preferred for synthesis of water soluble dyes.

An additional method for preparation of iodoacetamides involved heating the chloroacetamido dye prepared as on Scheme 1 with methanolic solution of sodium iodide at 70 ⁰C for 24-48 hours in argon atmosphere in the dark. The resulting iodoacetamido dye can be purified by chromatography on SiO₂.

Preparation of Merocvanines:

Scheme 2. Preparation of Merocyanines. R₅ is SO₂ or CO

R₆ is any substituent required to complete the cyclic ring (e.g., barbituric acid, thiobarbituric acid, l,l -dioxo-l,2-dihydro-benzothiophen-3-one, indole, 3H-indole, or 2.3-dihydro- lH-indole)

barbituric acid thiobarbituric acid 1 , 1 -Dioxo- 1 ,2-dihydro- 1 -benzothiophen-3-one

2,3-Dihydro-l//-indole 3H-Indole

1. Dye Structures (Merocyanines, Chloroacetamides).

Structures (Merocyanines, Iodoac

Dye Structures (Cyanines)

Additional Dye Structures:

EXAMPLE 5: Preparation of Chloro- and Iodo-acetamido-Substituted Dyes.

This Example illustrates the one-pot-procedures of the invention for preparation of certain protein-reactive, water soluble merocyanine and cyanine dyes. The l-(3-ammoniopropyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate bromide (1) was used as a common starting intermediate. The method allows easy preparation of dyes with chloro and iodoacetamide side chains for covalent attachment to cysteine. By placing a sulfonato groups on the aromatic ring, dyes with high fluorescence quantum yields in water were generated. Both iodo- and chloroacetamido- derivatives were shown to be useful in protein labeling. In some cases, the less reactive chloroacetamides may be preferred to permit more selective labeling of the most reactive cysteines.

In general, the one-pot methods include preparation of a key intermediate 1- (3-ammoniopropyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate (1), followed by its conversion to merocyanine and cyanine dyes. The reaction of this intermediate (1) with hemicyanine 2 (Ernst et al. Cytometry 10: 3-10 (1989)) or 3-methoxyprop-2- enyl-l -idene-2-thiobarbituric acid 3 (Toutchkine et al. J Am Chem Soc 125, 4132-45 (2003)) in dimethylformamide in the presence of sodium acetate and chloroacetic acid anhydride (CAA) gave the cyanine or merocyanine dyes, respectively. The pure dyes were obtained by chromatography of crude reaction mixtures on silica gel using acetone-methanol as eluent. The chloroacetamides were cleanly converted into more reactive iodoacetamides by reaction with sodium iodide in methanol/chloroform.

Materials. Analytical grade reagents were purchased from Sigma- Aldrich Co.

Potassium 2,3,3-trimethyl-3H-indole-5-sulfonate (Mason et al., Journal of Organic Chemistry 70, 2939-2949 (2005)), 3-{2-[(l£,3£>4-anilinobuta-l,3-dien-l-yl]-l,3- benzothiazol-3-ium-3-yl}propane-l -sulfonate (3) (Brooker et al., Journal of the American Chemical Society 63, 3192-203 (1941), (2Z)-2-[(2E)-3-methoxyprop-2-en- l-ylidene]-l-benzothiophen-3(2H)-one 1,1-dioxide (5) (Toutchkine et al. J Am Chem Soc 125, 4132-45 (2003)), and 5-[(2£)-3-methoxyprop-2-en-l-ylidene]-l,3-dimethyl- 2-thioxodihydropyrimidine-4,6(l/-/,5H)-dione (4) (Toutchkine et al. J Am Chem Soc 125, 4132-45 (2003)) were prepared as previously described.

Methods. Absorption spectra were recorded on a Hewlett-Packard UV-Vis spectrophotometer, and fluorescence measurements were taken on a Spex Fluorolog 2 spectrofluorometer. NMR spectra were obtained on Varian Mercury 300 MHz or on a Bruker 500 MHz DRX 500 spectrometer. Mass spectra were obtained on a Hewlett- Packard 5890 gas chromatograph equipped with a 5971A mass selective detector (MS-EI). Quantum yields were measured using merocyanine 540 (Onganer et al., J. Phys. Chem. 97, 2344-2354 (1993)) or Cy5 (Gruber et al., Bioconjug Chem 11, 161-6 (2000)) as an internal standards (Demas & Crosby, J. Phys. Chem. 75, 991-1024 (1971)). Synthesis of l-(3-ammoniopropyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate bromide (1).

To a suspension of 2.77 g of potassium 2,3,3-trimethyl-3H-indole-5-sulfonate in dichlorobenzene was added 2.18 g of 3-bromopropylamine hydrobromide. The mixture was stirred at 130° C under nitrogen for 12 hours. It was then cooled and the solid was filtered. The solid was stirred with 50 mL of hot methanol for 10 minutes, filtered and dried. The yield was 2.45 g (65%).

NMR (400 MHz, D₂O-DMSO-^o) 1.48 (s, 6H, 2^χCH₃), 2.13 (p, ³J_H-_H = 6.2 Hz, 2H, CH₂-CH₂-CH₂), 3.00 (t, ³J_H-H = 6.2 Hz, 2H, CH₂-CH₂-NH₃ ⁺), 4.45 (t, ³J_H._H= 6.2 Hz, 2H, CH₂-N), 7.81-7.87 (m, 2H), 7.94 (s, IH). ESI-MS: 297 (M⁺, positive ion detection).

Compounds 2, 3, 4, and 5 served as electron acceptor rings and are also referred to herein as Ring₂-containing reactants. The structures of compounds 2, 3, 4, and 5 are shown below.

General Methods for preparation of the chloroacetamido-substituted dyes.

Compounds 6 and 7 were synthesized by combining compound 1 with compound 2 or 3, respectively, as described below. For compound 6, substituent Z was SO₃ ^", Rio was methyl, Ri i was C(CH₃)₂, and Hal is chloride. For compound 7, substituent Z was hydrogen, Ri₀ was (CH₂)₃SO₃, Rn was sulfur, and Hal is chloride (compound 7a) or iodide (compound 7b).

Substituent Hal was chloride for compounds 8a and 9a, and iodide for compounds 8a and 9a.

Compounds 6, 7, 8 and 9 were prepared as follows. To a stirred suspension of 377 mg (1.00 mmol) of l-(3-ammoniopropyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate bromide (1) in 10 mL of DMF was added 425 mg (2.50 mmol) chloroacetic anhydride (CAA), 1.20 mmol of acceptor intermediate (e.g., 2, 3, 4 or 5) and 200 mg (2.50 mmol) of sodium acetate. The mixture was stirred at room temperature for 1 hour. The DMF was removed under vacuum and the residue was purified by silica gel column chromatography using acetone-methanol as eluent.

The following compounds were made by this procedure: Sodium (2-((lE,3E,5E)-5-(l-(3-(2-chloroacetamido)propyl)-3,3-dimethyl-5- sulfonatoindolin-2-ylidene)penta-l, 3-dienyl)-l, 3, 3-trimethyl-3H-indolium-5- sulfonate) or CyS-CAA (6). The yield was 369 mg (54%). NMR (300 MHz, DMSO- d6): δ 1.69 (s, 12H, 4xCH₃), 1.85 (p, V_H = 6.8 Hz, 2H, CH₂-CH₂-CH₂), 3.21 (q, ³J₁₁. H= 6.8 Hz, 2H, CH₂-CH₂-NH), 3.61 (s, 3H, N-CH₃), 4.08 (m, 4H, CH₂-N and CH₂Cl), 6.27 (m, 2H), 6.54 (t, V_H = 12.3 Hz, IH), 7.30 (d, V_H = 8.4 Hz, IH), 7.33 (d, ³J_H._H = 8.4 Hz, IH), 7.60-7.66 (m, 2H), 7.80 (d, V_H = 1.5 Hz, I H), 7.82 (d, V_H = 1.5 Hz, IH), 8.30-8.41 (m, 3H). ESI-MS: 660 ((M-Na)^", negative ion detection).

(2-((JE,3E,5E)-5-(l-(3-(2-chloroacetamido)propyl)-3,3-dimethyl-5- sιdfonatoindolin-2-ylidene)penta-l,3-dienyl)-3-ethylbenzo[d]thiazol-3-ium-6- sulfonate) or CySS-CAA (7a). The yield was 369 mg (63%). NMR (300 MHz, DMSO-J6): δ 1.64 (s, 6H, 2^χCH₃), 1.87 (p, ³J_H-H = 6.8 Hz, 2H, CH₂-CH₂-CH₂), 2.03 (p, ³J_H-H = 6.8 Hz, 2H, CH₂-CH₂-CH₂), 2.60 (t, ³J_H-_H = 6.8 Hz, 2H, CH₂-SO₃) 3.25 (q, ³J_H-_H = 6.8 Hz, 2H, CH₂-CH₂-NH), 3.96 (t, ³J_H-_H = 6.8 Hz, 2H, CH₂-N), 4.06 (2Η, CH₂-N, CH₂Cl), 4.67 (t, ³J_H-_H = 6.8 Hz, 2H, CH₂-N), 6.07 (d, ³J_Η-_Η = 13.0 Hz, IH), 6.51 (t, ³JH-H = 13.0 Hz, IH), 6.98 (d, ³J_H._H = 13.0 Hz, IH), 7.15 (d, ³J_H._H = 8.1 Hz, IH), 7.50-7.70 (m, 4H), 7.98-8.15 (m, 4H), 8.42 (t, ³J_H-_H = 6.0 Hz, IH, NH). ESI-MS: 678 ((M-Na)^", negative ion detection).

Sodium (E)-l-(3-(2-chloroacetamido)propyl)-2-((E)-4-(l, 3-dimethyl-4, 6- dioxo-2-thioxotetrahydropyrimidin-5(6H)-ylidene)but-2-enylidene)-3,3- dimethylindoline-5 ^'-sulfonate or I-TBA-CAA (8). The yield was 308 mg (51%). NMR (500 MHz, DMSO-rf6): δ 1.64 (s, 6H, 2xCH₃), 1.82 (p, ³J_H-H = 7.0 Hz, 2H, CH₂- CH₂-CH₂), 3.24 (q, 2H, ³J_H-H = 7.0 Hz, CH₂-CH₂-NH), 3.60 (s, 6H, 2^χCH₃), 4.06 (s, 2H, CH₂Cl), 4.12 (t, ³JH-H= 7.0 Hz, 2H, CH₂-N), 6.40 (d, ³J_H-H = 14 Hz, IH), 7.31 (d, ³JH-H= 8.5 Hz, IH), 7.58 (d, ³J_H-H= 8.5 Hz, IH), 7.73 (d, ³J_H._H= 1.5 Hz, IH), 7.81 (t, ³JH-H= 13.0 Hz, IH), 8.19 (d, ³J_H-H= 13.5 Hz, IH), 8.27 (t, ³J_H-H= 13.5 Hz, IH), 8.39 (t, ³J_H-H = 6.0 Hz, IH, NH). ESI-MS: 579 ((M-Na)^", negative ion detection).

Sodium (E)-I -(3-(2-chloroacetamido)propyl)-3, 3-dimethyl-2-((2E, 4Z)-4-(3- oxobenzo[b]thiophen-l,l-dioxide-2(3H)-ylidene)but-2-enylidene)indoline-5-sulfonate or ISO-CAA (9a). The yield was 313 mg (55%). NMR (300 MHz, DMSO-^6): δ 1.64 (s, 6H, 2^χCH₃), 1.83 (p, ³J_H-H = 6.5 Hz, 2H, CH₂-CH₂-CH₂), 3.24 (q, ³J_H-H = 6.5 Hz, 2H, CH₂-CH₂-NH), 4.03 (t, ³J_H-H = 6.5 Hz, 2H, CH₂-N), 4.06 (s, 2H, CH₂Cl), 6.32 (d, IH, ³JH-H = 13 Hz), 6.68 (t, IH, ³J_H-H= 13 Hz), 7.21 (d, IH, ³J_H-H = 8.5 Hz), 7.58 (d, IH, ³J_H-_H = 8.5 Hz), 7.69 (s, I H), 7.70-8.05 (m, 5H), 8.25 (t, I H, ³J_H._H = 13 Hz), 8.39 (t, IH, ³J_H-_H = 5.5 Hz, NH). ESI-MS: 589 ((M-Na)^", negative ion detection).

Methods for conversion of chloroacetamido substituted dyes into iodoacetamido substituted dyes

A solution of 0.5 mmol of chloro-substituted dye and 1 g of sodium iodide in 10 mL of methanol-chloroform mixture was refluxed for 24 hours under nitrogen. The mixture was filtered and evaporated. The iodoacetamide compounds were purified on HPLC using Cl 8 column (VydacTP 152022) with water-acetonitrile gradient.

The following compounds were made by this procedure: Sodium (E)-I -(3-(2-chloroacetamido)propyl)-3, 3-dimethyl-2-((2E, 4Z)-4-(3- oxobenzo[b]thiophen-l,l-dioxide-2(3H)-ylidene)but-2-enylidene)indoline-5-sulfonate or ISO-IAA (9b). The yield was 211 mg (60 %). NMR (300 MHz, DMSO-^6): δ 1.64 (s, 6H, 2^χCH₃), 1.79 (p, ³J_H-_H = 6.5 Hz, 2H, CH₂-CZZ₂-CH₂), 3.21 (q, ³J_H._H = 6.5 Hz, 2H, CH₂-CZZ₂-NH), 3.64 (s, 2H, CH₂I), 4.02 (t, ³J_H-_H = 6.5 Hz, 2H, CH₂-N), 6.31 (d, ³JH-H = 13 Hz, IH), 6.69 (t, ³J_H._H = 13 Hz, IH), 7.21 (d, ³JH-H = 8.5 Hz, IH), 7.58 (d, ³J_H-H = 8.5 Hz, IH), 7.69 (s, IH), 7.70-8.05 (m, 5H), 8.25 (t, ³J_H-H= 13 Hz, IH), 8.39 (t, ³J_H-_H = 5.5 Hz, IH, NH). ESI-MS: 681 ((M-Na)^", negative ion detection).

(2-((1E, 3E, 5E)S-(I -(3-(2-iodooacetamido)propyl)-3 , 3-dimethyl-5- sulfonatoindolin-2-ylidene)penta-l,3-dienyl)-3-ethylbenzo[d]thiazol-3-ium-6- sulfonate) or CySS-IAA (7b). The yield was 258 mg (76 %). NMR (300 MHz, DMSCW6): S 1.64 (s, 6H, 2^χCH₃), 1.85 (p, ³J_H-_H = 6.8 Hz, 2H, CH₂-CH₂-CH₂), 2.07 (p, ³JH-H = 6.8 Hz, 2H, CH₂-CH₂-CH₂), 2.60 (t, ³J_H-H = 6.8 Hz, 2H, CH₂-SO₃) 3.20 (q, ³JH-H= 6.8 Hz, 2H, CH₂-CH₂-NH), 3.65 (2H, CH₂-N, CH₂I), 3.96 (t, ³J_H-H= 6.8 Hz, 2H, CH₂-N), 4.67 (t, ³J_H-_H = 6.8 Hz, 2H, CH₂-N), 6.07 (d, ³J_Η-Η = 13.0 Hz, IH), 6.51 (t, ³JH-H = 13.0 Hz, IH), 6.98 (d, ³J_H-H = 13.0 Hz, IH), 7.15 (d, ³J_H-H = 8.1 Hz, IH), 7.50-7.70 (m, 4H), 7.98-8.15 (m, 4H), 8.42 (t, ³J_H-H = 6.0 Hz, IH, NH). ESI-MS: 770 ((M-Na)^", negative ion detection). Results

As illustrated above, cyanine and merocyanine dyes were prepared using a facile one-pot procedure from the key intermediate, l -(3-ammoniopropyl)-2,3,3- trimethyl-3//-indolium-5-sulfonate (intermediate 1). This inteπnediate was prepared by reaction of potassium 2,3,3-trimethyl-3//-indole-5-sulfonate with 3- bromopropylamine hydrobromide in dichlorobenzene at 130° C. The intermediate 1 precipitates during the reaction and can be easily purified from unreacted starting compounds by simple washing with hot methanol, as the intermediate is practically insoluble in that solvent. The reaction of intermediate 1 with 3-methoxyprop-2-enyl- l-idene-2-thiobarbituric acid 4 or with 3-methoxyprop-2-enyl-l-idene-2- benzothiophen-3-one 1,1 -dioxide 5 in the presence of sodium acetate and chloroacetic acid anhydride (CAA) resulted in the formation of merocyanine dyes bearing chloroacetamido reactive groups. Use of the same reaction conditions with hemicyanines 2 or 3 produced the cyanine dyes as shown above. The dyes were purified by silica gel chromatography of crude reaction mixtures using acetone- methanol as an eluent. Iodoacetamides were prepared by the reaction of chloroacetamides with sodium iodide in methanol/chloroform mixture. The yields of both merocyanine and cyanine chloroacetamido dyes were in the range of 50-65%, and the substitution of iodide for chloride occurred with 60-80 % yield. The reaction mechanism likely proceeds by deprotonation of 1 by sodium acetate with formation of enamine 10, as illustrated below.

The enamine than reacts with 3-methoxyprop-2-enyl-l-idene-2-benzothiophen-3-one 1,1 -dioxide 4 to give amino-substituted dye 11. The dye 11 is not stable and its solution rapidly loses color, as Michael addition of free amino group to the polymethine chain destroys the dye fluorophore system. In the presence of chloroacetic anhydride, the free amine is quickly trapped as the chloroacetamide, as shown in the reaction scheme above. This mechanism is supported by trapping of unstable dye intermediate 10 with amino-reactive compounds, including acetic anhydride or acetic acid succinimidyl ester.

These reactions generated novel, bright merocyanine and cyanine dyes, as illustrated in Table 1.

Table 1. Absorption and fluorescence data for merocyanine and cyanine dyes

Dye Solvent λ_max(Abs)/nm λ_ma,(Em)/nm^c Φ

6 water 215000 644 660 0.24 methanol 215000 646 658 0.27 n-butanol 215000 656 671 0.39

7a water 185000 636 662 0.11 methanol 185000 643 668 0.22 n-butanol 190000 652 676 0.34

8 water 160000 590 610 0.04 methanol 180000 591 610 0.27 n-butanol 180000 596 616 0.39

9a water 140000 594 616 0.02 methanol 120000 584 620 0.12 n-butanol 120000 590 624 0.32

I SO-IAA' water 143000 599 630 0.004 methanol 138000 601 634 0.01 n-butanol 150000 607 639 0.06 neo-Cy^ methanol 200000 648 665 0.22

α Extinction coefficient. ^b Absorption maximum. ^c Fluorescence maximum. Fluorescence quantum yield. ^e From ref. 6/ From ref. 9.

The merocyanine and cyanine dyes had high extinction coefficients, in the range of 120,000-215,000, and moderate fluorescence quantum yields of 0.1-0.4. The absorption characteristics of dyes 6 and 9a were similar to those previously observed for other compounds made by the inventors, including neoCy5 (Toutchkine et al., Bioconjug Chem 13, 387-91 (2002)) and I-SO-IAA (Toutchkine et al., J Am Chem Soc 125, 4132-45 (2003)) shown below.

Substitution with a sulfonate group (-SO₃) at the aromatic ring in the new merocyanine dye 9a and in the new cyanine dye 6 significantly increased the fluorescence quantum yields in polar solvents (H₂O, MeOH) compared to I-SO-IAA and neo-Cy5. For the cyanine, the quantum yield in water more than doubled, and for the merocyanine it increased five fold. Brightness in polar solvents is especially valuable for imaging applications, where dyes are conjugated to proteins and exposed to water. Similar increases in dye fluorescence upon ring-substitution have been reported for rhodamines (Panchuk-Voloshina et al. J Histochem Cytochem 47, 1179- 88 (1999) and cyanines (Mujumdar et al., Bioconjugate Chemistry 7, 356-362 (1996).

EXAMPLE 6: Attachment of Dyes to Binding Entities or Target Molecules

This Example illustrates procedures for attaching a dye of the invention to a selected protein or polypeptide having an exposed cysteine residue. Methods

A fusion protein of enhanced green fluorescent protein and extracellular regulated kinase 2 (EGFP-Erk2) was incubated with a 10-fold molar ratio of the 9a or 9b dye (FIG. 2) in 25 mM Hepes (pH 7.4), 50 mM NaCl (0.15 mL 20 μM EGFP- Erk2). To this protein solution 13.5 μL of 2 mM dye stock solution in DMSO was added. The mixture was incubated at room temperature with gentle agitation for 2 hours and then was spun at 11750 g (Eppendorf 5415C centrifuge) for 1 min to remove any precipitate that might have formed during labeling. The supernatant was then purified using a G25 Sepharose gel filtration column, pre-equilibrated with 25 mM Hepes (pH 7.4). The dye-protein adduct was clearly separated from free dye during gel-filtration. Purity of the conjugates was confirmed by SDS-PAGE electrophoresis. No free dye was seen in the purified protein conjugates. Control samples of free dye were clearly visible on the gel at lower MW than protein. Conjugates formed single, highly colored fluorescent protein bands with molecular weight corresponding to EGFP-Erk2. The degree of labeling D/P (dye-to-protein ratio) was calculated using the following formula:

D/P = (A_dye/εdye)/(Ap_rot/ε_pi.o,)

A_dye - absorbance at the absorption maximum of the dye

A_p10I- absorbance at the absorption maximum of EGFP (490 nm) £_dy_e - extinction coefficient of the dye in H₂O ε_p,ot - extinction coefficient of EGFP (61000)

Results

All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.

The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "an antibody" includes a plurality (for example, a solution of antibodies or a series of antibody preparations) of such antibodies, and so forth. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

WHAT IS CLAIMED:

1. A fluorescent compound of formula I, II, III or IV:

Ringi==CH-(-CH==CH-)_ni-CH==Ring2 I

Ring i ==CH-(-CH==CH-) _m-Ring₂ II

Hal-CH₂-CO-NR|-(CH₂)_n-Ringi==CH-(-CH==CH-)_m-CH==Ring₂ III Hal-CH₂-CO-NR,-(CH₂)_n-Ringι==CH-(-CH==CH-) _m-Ring₂ IV wherein: Ringi is an aryl, a heteroaryl, cycloalkyl, cycloalkenylene or heterocyclic ring that can be substituted with hydrogen, lower alkyl, oxygen (O), hydroxy (OH), sulfonate (SO₃ ^") or sulfate (SO₄ ^");

Ring₂ is an aryl, a heteroaryl, cycloalkyl, cycloalkenylene or heterocyclic ring that can be substituted with 0-6 substituents separately selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, oxygen (O), hydroxy (OH), sulfur (S), halogen, sulfonate (SO₃ ^"), sulfate (SO₄ ^"), aryl, cyclopentyl, cyclohexyl or Hal-CH₂-CO-NRi-(CH₂)_n-, wherein each of the lower alkyl, aryl, cyclopentyl or cyclohexyl substituents can be substituted with halogen, sulfonate (SO₃ ^") or sulfate (SO₄ ^"), cyano or lower alkoxy

Hal is halogen;

Ri is hydrogen or -C(O)-(CH₂)_n-Hal; n is an integer of from 2 to 20; and m is an integer of from O to 4. 2. The compound of claim 1, wherein the aryl is phenyl, benzyl or phenylalkylene.

3. The compound of claim 1, wherein the heteroaryl or heterocyclic ring has one to three heteroatoms in the ring, wherein the heteroatoms are selected from the group consisting of nitrogen, sulfur or oxygen. 4. The compound of claim 3, wherein a sulfur heteroatom is a substituted sulfur atom.

5. The compound of claim 4, wherein the substituted sulfur atom is -SO₂-.

6. The compound of claim 3, wherein a nitrogen heteroatom is substituted with lower alkyl, phenyl, benzyl, phenyl, HaI-CH₂-CO-NR]-(CH₂),!-, or phenyl that is substituted with halogen, alkoxy or cyano.

7. The compound of claim 1 , wherein Ringi is a radical selected from the following radicals, where the asterisks identifies an attachment site for the remainder of the compound:

2,3

iazole

2,3-Dihydro-benzothiazole

2,3-Dihydro-benzooxazole 1 ,4-Dihydro-quinoline 1 ,2-Dihydro-pyridine

8. The compound of claim 1, wherein R^g₂ is a radical selected from the following radicals, where the asterisks identifies an attachment site for the remainder of the compound:

2,3-Dihydro-benzo[ό]thiophene

Indan

Indan- 1,3-dione

Benzo[6]thiophen-3-one 2,3-Dihydro-benzo[6]thiophene 1 ,1 -dioxide Hexahydro-pyπmidine

Tetrahydro-pyπmidin-2-one Tetrahydro-pyπmidin-2-one

1 , 1 -Dioxo-1 ,2-dihydro- 1 λ⁶-benzo[i]thiophen-3-one

Hexahydro-pyπmidine Dihydro-pyπmidine-4,6-dione Dihydro-pyrimidine-4,6-dione

3H-lndole

Pyrimidine-2,4,6-trione 2-Thioxo-dihydro-pyrimidine-4,6-dione

2,3-Dihydro- lH-indole 2,3-Dihydro-benzothiazole Benzothiazole

and wherein: Ri₀ is a substituent selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, oxygen (O), hydroxy (OH), sulfur (S), halogen, sulfonate (SO₃ ^'), sulfate (SO4^"), aryl, cyclopentyl, cyclohexyl or Hal-CH₂-CO-NRi-(CH₂)_n-, wherein each of the lower alkyl, aryl, cyclopentyl or cyclohexyl substituents can be substituted with halogen, sulfonate (SO₃ ^") or sulfate (SO₄ ^"), cyano or lower alkoxy.

9. The compound of claim 1 comprising formula V:

wherein:

D is an atom, which is an electron donor and which can be substituted by one or two hydrogen atoms;

Hal is halogen;

W is hydrogen, an electron donating group or a group to enhance the solubility of the fluorescent dye in water;

Z is hydrogen, a sulfhydryl-reactive group, an electron accepting group or a substituent that can enhance the solubility of the fluorescent dye in water; and q and v are integers, each separately selected from 0 to 6.

10. The compound of claim 9, wherein D is a primary amine.

1 1. The compound of claim 9, wherein Hal is Br, Cl, I, or F.

12. The compound of claim 9, wherein W is hydrogen, lower alkyl, oxygen (O), hydroxy (OH), sulfonate (SO₃ ^") or sulfate (SO₄ ^").

13. The compound of claim 9, wherein Z is hydrogen, lower alkyl, lower alkoxy, oxygen (O), hydroxy (OH), sulfur (S), sulfonate (SO₃ ^"), sulfate (SO₄ ^"), halogen, aryl, cyclopentyl, cyclohexyl or HaI-CH₂-CO-NR i-(CH2)_n-, wherein each of the lower alkyl, aryl, cyclopentyl or cyclohexyl substituents can be substituted with halogen, sulfonate (SO₃ ^"), sulfate (SO₄ ^"), cyano or lower alkoxy.

14. The compound of claim 9, wherein the ring to which W is attached is a Ringi selected from one of the following radicals, where the asterisks identifies an attachment site for the remainder of the compound:

2,3-Dihydro- lH-indole 3H-Indole Benzothiazole

2,3-Dihydro-thiazole 2,3-Dihydro-[ 1 ,3,4]thiadiazole

2,3-Dihydro-benzothiazole

2,3-Dihydro-benzooxazole 1 ,4-Dihydro-quinoline 1 ,2-Dihydro-pyridine

15. The compound of claim 9, wherein the ring to which Z is attached is a Ring₂ radical selected from one of the following radicals, where the asterisks identifies an attachment site for the remainder of the compound:

2.3-Dihydro-benzo[5]thiophene

Indan

Indan- 1,3-dione

Benzo[i]thiophen-3-one 2,3-Dihydro-benzo[ά]thiophene 1,1 -dioxide Hexahydro-pyrimidine

Tetrahydro-pyrimidin-2-one Tetrahydro-pyπmidin-2-one

1 , 1 -Dioxo- 1 ,2-dihydro- 1 λ -benzo[6]thiophen-3-one

Hexahydro-pyπmidine Dihydro-pyπmidine-4,6-dione Dihydro-pyrimidine-4,6-dione

3H-Indole

Pyrimidine-2,4,6-trione 2-Thioxo-dihydro-pyrimidine-4,6-dione

2,3-Dihydro- lH-indole 2,3-Dihydro-benzothiazole Benzothiazole

and wherein: R]₀ is a substituent selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, oxygen (O), hydroxy (OH), sulfur (S), halogen, sulfonate (SO₃ ^"), sulfate (SO₄ ^"), aryl, cyclopentyl, cyclohexyl or HaI-CH₂-CO-NR i-(CH₂)_n-, wherein each of the lower alkyl, aryl, cyclopentyl or cyclohexyl substituents can be substituted with halogen, sulfonate (SO₃ ^") or sulfate (SO₄ ^"), cyano or lower alkoxy.

6. The compound of claim 1 or 9, having any one of the following structures:

wherein: Hal is Cl or I; Z is hydrogen or SO₃ ^"; Ri₀ is methyl or (CH₂)₃SO₃ ^'; and R| i is C(CH₃)₂ or sulfur.

17. A biosensor comprising a binding entity and a compound of any one of claims

1-16.

18. The biosensor of claim 17, wherein the binding entity is a peptide, protein or a nucleic acid. 19. The biosensor of claim 17, wherein the binding entity is an antibody, antibody fragment, leucine zipper, histone, enhancer, complementary determining region (CDR), a single chain variable fragment (scFv), receptor, ligand, aptamer, or lectin.

20. A method for making sulfhydryl-reactive compound, which comprises: (a) obtaining a compound with a tertiary amine;

(b) activating the tertiary amine on the compound with an L-(CH₂)_n-D activator to form an activated first reactant as shown below:

wherein: L is a leaving group and D is an electron donating group; and (c) reacting the activated first reactant with a 2-halo-acetamido moiety which is linked to a leaving group to thereby attach a sulfhydryl- reactive group to the compound as shown below:

wherein: D is an electron donating group, L is a leaving group, and Hal is a halogen.

21. The method of claim 20, wherein the compound with a tertiary amine is a compound of any one of claims 1-16.

22. The method of claim 20, wherein the compound with a tertiary amine is a biological molecule.

23. The method of claim 20, wherein the biological molecule is a peptide, protein or a nucleic acid. 24. The method of claim 22, wherein the biological molecule is an antibody, antibody fragment, leucine zipper, histone, enhancer, complementary determining region (CDR), a single chain variable fragment (scFv), receptor, ligand, aptamer, or lectin.

25. A method of detecting a selected target molecule's activity and/or location within a cell comprising (a) contacting the cell with a biosensor; and (b) observing a change in signal produced by the biosensor and/or observing a location of the signal, wherein the biosensor comprises a binding domain and a compound of any one of claims 1-16.

26. The method of claim 25, wherein the selected target molecule is a protein, receptor, ligand or enzyme.

27. The method of claim 25, wherein the selected target molecule's activity comprises the selected molecule's phosphorylation state, subcellular location, interaction with subcellular structures or interaction with cellular proteins.

28. A method of detecting an interaction between a selected endogenous target biomolecule and a cellular entity, the method comprising: a. identifying a cell comprising a selected endogenous target biomolecule and a recognizable cellular entity; b.providing a biosensor comprising (i) a binding entity with specific binding affinity for a binding site on the target biomolecule and (ii) a compound of any one of claims 1-16; c. incubating the biosensor with the cell; d. observing if a background signal is detectable from the biosensor and optionally subtracting the background signal; and e. detecting a signal change from the biosensor to thereby detect an interaction between the target biomolecule and a cellular entity.

29. The method of claim 28, wherein the cellular entity is selected from the group consisting of a sub-cellular organelle, nucleic acid, protein, peptide, enzyme, receptor, cytokine, cytoskeleton and signal transduction protein. 30. The method of claim 28, wherein the binding entity can bind to the target biomolecule by a phosphorylation site. 31. The method of claim 28, wherein the binding entity has specific affinity for a particular conformation, ligand interaction, or posttranslational modification of the target biomolecule. 32. The method of claim 28, wherein the compound comprises an excitation or emission light wavelength of about 600 nm or more.

33. The method of claim 28, wherein the compound does not substantially interfere with binding between the binding entity and the target biomolecule.

34. The method of claim 28, wherein the method further comprises introducing the biosensor into the cell by using electroporation, transduction, micro poration, microinjection, surfactants, or projectiles.

35. The method of claim 28, wherein the signal change comprises an at least 50% increase in fluorescence.

36. The method of claim 28, wherein detecting a signal change comprises fluorimetry, quantifying fluorescence levels, locating a cellular entity. 37. The method of claim 28, wherein detecting a signal change comprises an increase in the signal or a change in wavelength of the signal. 38. The method of claim 28, wherein the signal change reflects a change in conformation of the protein, in activation of the protein, or in phosphorylation state of the protein. 39. The method of claim 28, wherein the signal change results from a change in hydrophobicity, hydrogen bonding, polarity, polarization, phosphorylation, polypeptide folding, hydration, ligand binding, or subunit interaction of the target biomolecule upon interaction with the cellular entity. 40. A method of attaching the compound of claim 9 to a sulfhydryl-containing compound, comprising reacting the compound of claim 9 with the sulfhydryl- containing compound in an aqueous solvent to form a reaction mixture and incubating the reaction mixture for a time sufficient to generate a product consisting of the compound of claim 9 covalently linked to a sulfhydryl- containing compound. 41. The method of claim 40, wherein the method proceeds by the following reaction:

pound

42. The method of claim 40, wherein the sulfhydryl-containing compound is a polypeptide or nucleic acid.