Protein labeling with cyanobenzothiazole conjugates

Related application This application takes precedence under US Patent Law §119 (e) over US Provisional Patent Application No. 61 / 040,073 (incorporated herein by reference) filed March 27, 2008. Claim the right.

Site-specific labeling of biomolecules with fluorophores often requires careful selection of labeling chemistry, optimization of labeling reactions for labeling efficiency and characterization of labeled biomolecules, site specificity, and maintenance of functional groups. And. The two most commonly used approaches for proteins are based on chemical coupling to sulfhydryl groups or primary amines, which are characteristic of cysteine (Cys) and lysine residues in a given protein. As a result of the content and distribution of, it results in different labeling patterns on different proteins. The most common method for site-specific labeling of proteins with fluorophores is Cys-specific labeling with thiol reaction reagents. During this reaction, proteins with surface-exposed Cys residues are covalently modified by maleimide, iodoacetamide, or other reactive conjugates of the fluorophore (Waggoner, Methods Enzymol., 246: 362 (1995); Haugland, Handbook of Fluorescent Probes and Research Products, 8th Edition, 2002; Selvin, Methods Enzymol., 246: 300 (1995)). This is the method of choice for small proteins (less than about 200 residues), as cysteine is a rare amino acid and can be easily replaced with other amino acids using site-directed mutagenesis (less than about 200 residues). Kunkel et al., Methods Enzymol., 205: 125 (1991)).

If the protein of interest lacks Cys residues, the site of label uptake is selected after examination of high-resolution tertiary structure (created using X-ray crystallography or nuclear magnetic resonance). Labeling should not disrupt enzyme activity or spatial arrangement of protein sequences (also known as "protein folding"). Subsequently, the amino acids present at the selection site, preferably having a charge, size, and hydrophobic side chain similar to the side chain of Cys, are replaced by Cys using site-directed mutagenesis. (Kunkel et al., 1991).

If the unmodified protein has a single existing Cys, structural information measures the surface accessibility of Cys side chains (Kapanidis et al., J. Mol. Biol., 312: 453 (2001)). Together, it can be determined whether the existing Cys can be used for labeling, otherwise the existing Cys can be converted to the structurally similar amino acid serine, followed by the Cys-free protein. Can be followed.

Expressed Protein Ligation (EPL) (Muir, Annu. Rev. In a recently developed approach called Biochem., 72: 249 (2003)), proteins are expressed in C-terminal fusions with intein domains and affinity tags. The resulting fusion protein can be separated from the expression host protein on the affinity matrix. Treatment of the immobilized protein with a high concentration of thiol leads to cleavage of the peptide bond between the intein and the target protein. The cleaved protein has a thioester group on the C-terminus that can be coupled to a peptide with Cys at the N-terminus (or in fact any molecule) by a native chemical ligation that produces a native peptide bond at the coupling site. (Dawson et al., Science, 266: 776 (1994)). This approach has been successfully used for protein engineering, but its drawbacks are the need to express large fusion proteins that can affect the solubility and folding of the target protein, as well as the effect of adjacent residues of the target protein. Due to different intein splicing efficiencies (Zhang et al., Gene, 275: 241 (2001)).

An alternative strategy is to couple a thioester-conjugated functional group such as a fluorophore onto the N-terminal Cys of the recombinant protein (Schuler et al., Bioconjugate Chem., 13:1039 (2002)). In some cases, efficiency varies between proteins, but Cys at position 2 is N-terminal to methionine cleavage by the expression host aminopeptidase (Gentle et al., Bioconjugate Chem., 15: 658 (2004)). ). In another strategy, N-terminal Cys is produced by self-cleavage of the intein domain fused to the target protein at the N-terminus. Alternatively, the N-terminal Cys residue can be produced by proteolytic cleavage of a properly engineered protease cleavage site. Proteases that have been shown to allow Cys at the +1 position at the cleavage site include Factor X, Precision Proteases, and TEV Proteases (Cotton et al., Chem. Biol., 7: 253 (2000); Tolbert et al., Angew. Chem. Int. Ed., 41: 2171 (2002)).

Therefore, there is a need for compounds, compositions and methods that support the ligation of site-specific chemical groups to specific sites of proteins or peptides.

The present invention relates to fluorophores or other detectable groups capable of assisting in the identification, quantification and / or purification of biomolecules such as proteins, such as reporter moyets, affinity moyets, antigens, quencher compounds, light. Provided is a cyanobenzothiazole linked to a crosslinked protein, or solid support. For example, such cyanobenzothiazole derivatives are capable of rapid and specific reactions with proteins containing a cysteine (Cys) residue at the N-terminus. By reacting with a protein that has an N-terminal Cys residue, the cyanobenzothiazole derivative can introduce a reporter moisture or other functional group at the N-terminal of the protein. In one embodiment, the reaction can proceed by nucleophilic attack of Cys thiol on the cyano group of the cyanobenzothiazole derivative, followed by concomitant cyclization.

Cyclization provides a stable thiazolin ring, thereby covalently attaching a benzothiazole derivative to the N-terminus of the protein of interest. Derivatives can include at least one reporter or affinity yeti of cyanobenzothiazole, eg, covalently linked to the 6'position. The addition of the cyanobenzothiazole derivative to the internal Cys side chain can be easily removed. Removal can be achieved by adding a Cys solution and incubating typically for about 1 to about 10 minutes, often about 2 to about 5 minutes.

The present invention also provides a method of introducing a reporter yeti or other functional group at the N-terminus of the protein of interest using the cyanobenzothiazole derivative of the present invention, thereby providing a labeled protein. The Cys-containing protein can be a protein having or engineered to have Cys at position 2, a protein prepared by an intein-mediated method, or a protein prepared by a suitable protease. In one embodiment, the derivative has the following general structure XLM, where X is the reporter or affinity yeti, L is the optional linker, and M is the cyanobenzothiazole. In one embodiment, L can be photocleaved. In one embodiment, L is the enzyme recognition site. In one embodiment, L cannot be photocut. In one embodiment, L is not the enzyme recognition site. In one embodiment, XLM is not detectable, but the product of the reaction between XLM and the N-terminal Cys-containing protein is detectable. In one embodiment, XLM is detectable, but the product of the reaction between XLM and the N-terminal Cys-containing protein is not detectable. In another embodiment, the XLM and the product of the reaction between the XLM and the N-terminal Cys-containing protein can be distinguished, for example, optically.

Applications of the method include, but are not limited to, site-specific labeling of proteins for the detection or analysis of protein quality structure and function. The present invention also provides a method of performing an analysis to detect a molecule such as an enzyme of interest using a derivative of 2-cyanobenzothiazole. Enzymes of interest include, but are not limited to, kinases, phosphatases, peroxidases, sulfatases, peptidases, glycosidases, and proteases such as proteases involved in apoptosis. The present invention further provides novel compounds and compositions that can be used in such analyses.

Accordingly, the present invention provides an in vitro method of labeling a protein at the N-terminus according to a particular embodiment. The method is a mixture comprising a derivative of cyanobenzothiazole comprising at least one reporter or affinity moisity and at least one protein with terminal Cys, eg, N-terminal Cys, eg, a protein extract (lysate). Includes contacting, purified proteins, or components of the protein synthesis system. The cyanobenzothiazole derivative can include at least one reporter or affinity yeti attached to the benzomois of cyanobenzothiazole. For example, a reporter or affinity yeti can be attached to the 4', 5', 6'or 7'position of cyanobenzothiazole. In certain embodiments, the reporter or affinity yeti is attached to the 6'position of cyanobenzothiazole.

In one embodiment, the mixture containing the terminal Cys protein is a cell lysate, eg, a cell lysate from a commercially available library. In one embodiment, the mixture comprises at least one nucleic acid molecule, such as mRNA or tRNA, amino acid and / or charged tRNA, ribosome, one or more translation initiation factors, one or more elongation factors, and one or more termination factors. For example, a protein synthesis system can be included. The mixture can also be a combined eukaryotic transcription / translation mixture.

Cyanobenzothiazole derivatives can be any in vitro translation system (eg wheat germ extract, insect cell lysate, rabbit reticulocyte lysate), eukaryotic (eg S30) E. coli, frogs. Can be used with eukaryotic translation systems, including, but not limited to, egg mother cell lysates, canine pancreatic lysates, human cell lysates, or a mixture of purified or semi-purified eukaryotic translation factors. it can. In one embodiment, the protein that then has a reporter or affinity yeti at the N-terminus is detected, isolated, and / or quantified.

One embodiment provides a method of specifically attaching a selected chemical group (eg, a fluorophore or other detectable group) to the C-terminus of a protein, peptide, or other carboxy-containing molecule. The method comprises contacting a benzothiazole derivative, eg, a compound of formula I, with a mixture having a labeled molecule. Unstable derivatives can optionally be removed with chase reagents, thereby producing a mixture with molecules with stable and detectable groups.

The benzothiazole compound of the present invention comprises a compound of formula I.<chemistry num="1"><img id="000002" he="26" wi="52" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

Variable Z is H, F, Cl, Br, I, CN, amino, alkylamino, dialkylamino, alkyl ester (eg-CO).₂(Alkyl)), carboxy, carboxylate, alkylamide (-C (= O) NH (alkyl)), phosphate (-OPO (OH))₂), Alkylphosphonate, Sulfate (-OSO₃H), alkyl sulfonate, nitro, or optionally unsaturated, optionally substituted with amino, hydroxy, oxo (= O), nitro, thiol or halo (C)₁-C₁₀) Can be alkyl. Group Z can be located at the 4', 5'or 7'position of cyanobenzothiazole. In certain embodiments, Z is located at the 7'position.

Each R¹Independently, H, F, Cl, Br, I, CN, (C₁-C₆) Alkyl, (C₁-C₆) Alkoxy, or (C₁-C₆) Alkylthios, each alkyl, alkoxy, or alkylthio can be F, Cl, Br, I, amino, alkenyl, alkynyl, cycloalkyl, aryl, alkylsulfonate, or CO.₂Arbitrarily substituted with M (where M is H, organic or inorganic cation); n is 0, 1 or 2. Group R¹The (s) can be located at the 4', 5'or 7'position of the cyanobenzothiazole. In certain embodiments, Z can be located in the 7'position.

Group Y is one or more (eg, 1, 2, 3, 4, 1-5, or 1-6) halos, hydroxys, oxos, (C).₁-C₆) Alkyl, or (C₁-C₆) N (R) optionally substituted with alkoxy and greater than or equal to 1 (eg, 1, 2, 3, 4, 1-5, or 1-6)¹), O, S or -NC (= O)-arbitrarily interrupted at the group (C)₁-C₁₆) It can be a linking group containing alkyl, or Y can be absent. The term "arbitrarily interrupted" is one or more of the linking groups, including one or both terminal carbons of the linking group, eg, 1, 2, 3, 4, 1-5, or 1-6 carbon atoms. , O, N (R)¹), S, or -NC (= O) -means that it can be replaced by a group. In some embodiments, for example, X is an azide (N).₃), Y may not exist arbitrarily. For example, in some embodiments, Y is-(C.₁-C₆) Alkyl-, -O- (C₁-C₆) Alkyl-, -O- (C₁-C₆) Alkyl-O-, -O- (C₁-C₆) Alkyl-NH-, -O- (C₁-C₆) Alkyl- (CO) NH-, -NH- (C₁-C₆) Alkyl-NH-, -NH- (CO) (C₁-C₆) Alkyl-NH-, -NH- (CO) (C₁-C₆) Alkyl- (CO) -NH- or -O- (C₁-C₆) Alkyl- (CO) NH- (C₁-C₆) Alkyl-can be.

Group X is Reporter Yeti, Affinity Yeti, Quencher, Photocrosslinking Yeti, Solid Support, N₃, H, or OH. In certain embodiments, where X is H or OH, the compound of formula I is, for example, in the absence of Y, the radioactive moisture or isotope of any atom that is not the carbon or nitrogen atom of the 2-nitrile yeti. Includes body variants.

The functional group attached to the cyanobenzothiazole can be any molecule (or part thereof) that can be detected, detected, or isolated. These moities are nucleic acid molecules (ie DNA or RNA, eg oligonucleotides), drugs, proteins, peptides (eg epitopes recognized by ligands), haptens (eg keyhole limpet hemosinen (KLH)). , But not limited to, carbohydrates, biotins, resins, substrates for enzymes, fluorophores, chromophores, and the like, or combinations thereof. For example, nucleic acid reporter moities can be detected by hybridization, amplification, binding to nucleic acid binding proteins specific for nucleic acid reporters, enzymatic analysis (eg, if the nucleic acid molecule is a ribozyme), or If the nucleic acid molecule contains a molecule that is or can be detected by itself (eg, radiolabeled or biotin), it can be detected by analysis appropriate for that molecule.

Nucleic acid reporters can be useful for protein detection and / or isolation in microarrays or ribosome displays. ImmunoPCR and immunodetection by amplification with T7 polymerase (IDAT) are also applicable to the detection of nucleic acid reporters. For example, a nucleic acid reporter attached to cyanobenzothiazole is used to label the N-terminal Cys-containing protein, the nucleic acid is amplified, for example, in the presence of fluorescent nucleotides, and then the amplified nucleic acid is detected (Published US Application 2002/0028450 (Greene). et al.)). Protein-based or peptide-based reporter or affinity moities can be detected by ligand binding (eg, binding to a protein or peptide-specific antibody), or biochemical, enzymatic or luminescent activity. For example, a protein-based protein can be a transport domain, antibody, caspase, luciferase or green fluorescent protein (GFP).

Affinity moietti and their corresponding ligands (eg, maltose and maltose binding proteins, biotin and avidin or streptavidin, and histidine tags and metals such as cobalt, zinc, nickel or copper) can be, for example, beads, resins, or mulch. There are specific uses in the detection and isolation of proteins on solid supports such as wells in well plates. Fluorescent (or bioluminescent) reporters, such as those located at the N-terminus of the protein, which can be detected by UV-excited and / or visible-excited fluorescence detection, are used to detect changes in the system in real time, such as phosphorylation. Can be used. In addition, metal ion chemical sensors such as Cu²⁺Fluorescent molecules such as 9-carbonyl-anthracene-modified glycyl-histidyl-lysine, or a pair of fluorescent molecules, such as fluorescein and rhodamine, can be used in labeled proteins to form protein biosensors.

Bioluminescent or fluorescent reporters, such as BODIPY, Rhodamine Green, GFP, or Infrared Dyes, are also used in interaction studies using, for example, BRET, FRET, LRET or electrophoresis, such as capillary electrophoresis. .. For interaction studies, one or more specific molecules are combined with the labeled protein (either before or after isolation) to form a mixture containing the labeled protein and one or more molecules.

Then, the interaction between one or more molecules and one or more labeled proteins can be detected. Thus, human proteins, viral proteins, including such proteins, such as recombinant gene products, gene fusion products, enzymes, cytokines, hormones, immunogenic proteins, carbohydrate binding proteins, lipid binding proteins, nucleic acid binding proteins, and fragments thereof. , Bacterial proteins, and parasitic proteins can be used in protein synthesis or in mixtures of synthetic proteins to detect, isolate, and quantify derivatives of cyanobenzothiazole.

Proteins encoded by naturally occurring genes or recombinant genes can be labeled with moyetti using the methods of the invention. The protein containing moyetti can then be detected and / or isolated by methods known in the art. For example, proteins can be electrophoresed or gel filtered, high pressure liquid chromatography or high performance liquid chromatography, mass spectrometry, affinity chromatography, ion exchange chromatography, chemical extraction, magnetic bead separation, precipitation, hydrophobic interaction chromatography. It can be detected and / or isolated by taking advantage of the unique properties of the moyetti, such as the specific spectral properties of the moyetti, by any means, including imaging (HIC), or any combination thereof. Isolated proteins can be used for structural and functional studies, development of diagnostic applications, preparation of biological or pharmaceutical reagents as tools for drug development, and study of protein interactions or protein complexes. It can be used for separation and characterization.

The present invention also considers kits. In one embodiment, the kit comprises a derivative of cyanobenzothiazole having a reporter or affinity moisture, and optionally one or more reagents for detection, identification, and / or purification of labeled proteins (eg, beads, etc.). Reagents such as resins, columns, and similar). In another embodiment, the kit comprises a derivative of cyanobenzothiazole and at least one reagent. In certain embodiments, the reagents and derivatives are in separate containment means (eg, tubes, vials, and the like). Some kits include an immobilized derivative of cyanobenzothiazole, or a reagent that immobilized a cyanobenzothiazole derivative. Such kits are useful for preparing one or more N-terminally labeled proteins, optionally isolated from cell or cell-free translation systems.

<figref num="1">It is a figure which illustrated the specific specific compound which is useful in the composition and method of this invention described in various embodiments.</figref><figref num="2(a)">It is a figure which illustrated the specific specific compound which is useful in the composition and method of this invention described in various embodiments.</figref><figref num="2(b)">It is a figure which illustrated the specific specific compound which is useful in the composition and method of this invention described in various embodiments.</figref><figref num="3">Ambis Imaging, which detects fluorescent radiation from fluorescent species present on TLC plates when collected through a filter that blocks UV light present on the imaging camera when exposed to UV light. It is a figure which showed the image of the thin layer chromatography (TLC) plate taken in by system set).</figref><figref num="4">The relative degree of labeling observed when the compounds of the present invention were incubated with a protein having an N-terminal cysteine residue is illustrated. This relativity is for the same reaction in which a protein with an N-terminal Cys residue is replaced with an equal amount of a second protein (a protein that differs from the N-terminal Cys protein only in that it has an N-terminal alanine residue). Both proteins can be produced by cleavage with TEV protease, as described in Example 5.</figref><figref num="5">The fluorescent gel image and the Coomassie-stained gel prepared according to Example 5 are shown, and it is a figure which showed that the protein by N-terminal cysteine can be prepared by the cleavage of the fusion construct by TEV protease. The protein can be labeled at the N-terminus with a cyanobenzothiazole reagent and then selectively cleaved with a second protease in subsequent steps.</figref><figref num="6">FIG. 5 is a diagram illustrating a Coomassie-stained gel of a peptide chain cleaved and labeled only at the N-terminus according to the procedure of Example 5. The abbreviation CN-BT is a cyanobenzothiazole derivative reagent as described herein; HT = halotag; GST = glutathione-S-transferase; TMR = tetramethylrhodamine; UC = uncut; and FXa = factor Xa. Shows cleavage by protease.</figref><figref num="7">FIG. 5 is a diagram illustrating a Coomassie-stained gel of peptide chain cleavage and non-specifically labeled peptides (both internal cysteine and N-terminal cysteine) described in the procedure of Example 5. The abbreviation CN-BT is a cyanobenzothiazole derivative reagent as described herein; HT = halotag; GST = glutathione-S-transferase; TMR = tetramethylrhodamine; UC = uncut; and FXa = factor Xa. Shows cleavage by protease.</figref><figref num="8(a)">FIG. 5 illustrates the results of labeling used to detect protein-protein interactions as described in Example 6.</figref><figref num="8(b)">FIG. 5 illustrates the results of labeling used to detect protein-protein interactions as described in Example 6.</figref>

Definition As used herein, the following terms and expressions have the indicated meanings. It will be appreciated that the compounds of the invention contain asymmetrically substituted carbon atoms and can be isolated as optically active or racemic. For example, methods of preparing an optically active substance by dividing the racemate or synthesizing it from an optically active starting material are well known in the art. All chiral, diastereomeric, racemic and structural isomers are part of the present invention.

The specific values listed below for radicals, substituents, and ranges are for illustration purposes only, and they are other defined values, or others within the defined range for radicals and substituents. Do not exclude the value of.

As used herein, the term "replaced" refers to one or more of the bases (eg, 1, 2, 3, 4, or 5, 1, 2 or 3, in some embodiments, and others. In the embodiment of 1 or 2), the hydrogen of 1 or 2) is replaced by one or more "substituents" (eg, one or more selections of suitable groups known to those skilled in the art), provided that the standard atom of the indicated atom. It is intended to show that the substitution does not exceed the valence and results in a stable compound. Suitable substituents are, for example, alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, tri. Fluoromethylthio, difluoromethyl, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, heteroarylsulfinyl, heteroarylsulfonyl, complex Includes ring sulfinyl, heterocyclic sulfonyl, phosphoric acid, sulfuric acid, hydroxylamine, hydroxyl (alkyl) amine, and cyano. In addition, the appropriate indicated groups are, for example, -X, -R, -O-, -OR, -SR, -S-, -NR.₂, -NR₃, = NR, -CX₃, -CN, -OCN, -SCN -N = C = O, -NCS, -NO, -NO₂, = N₂, -N₃, NC (= O) R, -C (= O) R, -C (= O) NRR -S (= O)₂O, -S (= O)₂OH, -S (= O)₂R, -OS (= O)₂OR, -S (= O)₂NR, -S (= O) R, -OP (= O) O₂RR, -P (= O) O₂RR -P (= O) (O-)₂, -P (= O) (OH)₂, -C (= O) R, -C (= O) X, -C (S) R, -C (O) OR, -C (O) O-, -C (S) OR, -C (O) ) SR, -C (S) SR, -C (O) NRR, -C (S) NRR, -C (NR) NRR (In the formula, each X is independently halogen ("halo"): F, Cl, Br, or I; each R can independently contain H, alkyl, aryl, heteroaryl, heterocycle, protecting group or prodrug yeti). As will be readily appreciated by those skilled in the art, if the substituents are keto (= O) or thioxo (= S), or of the same type, the two hydrogen atoms of the substituted atom will be replaced. In some embodiments, one or more of the aforementioned groups can be clearly excluded from the embodiments.

The terms "stable compound" and "stable structure" refer to a compound that is robust enough to leave the isolate from the reaction mixture to a useful degree of purity. Although only stable compounds are claimed in the present invention, certain unstable compounds (eg, those that cannot be easily isolated) can be used in the methods described herein.

Some biased left and right isomers may exhibit superior properties or activity compared to others. If necessary, material separation of racemic compounds is performed by HPLC using a chiral column, or camphonic chloride as described by Tucker et al., J. Med. Chem., 37: 2437 (1994). ) Can be achieved by splitting with a splitting agent such as). Chiral compounds can also be synthesized directly using chiral catalysts or chiral ligands (eg, Huffman et al., J. Org. Chem., 60: 1590 (1995)).

As used herein, the term "alkyl" has, for example, 1 to 20 carbon atoms, and often 1 to about 12, 1 to about 6, or 1 to about 4 carbon atoms. Refers to branched hydrocarbons, non-branched hydrocarbons, or cyclic hydrocarbons. Specific examples are methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl (t-butyl), 1-pentyl, 2 -Pentyl, 3-Pentyl, 2-Methyl-2-butyl, 3-Methyl-2-butyl, 3-Methyl-1-butyl, 2-Methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl , 2-Methyl-2-pentyl, 3-Methyl-2-pentyl, 4-Methyl-2-pentyl, 3-Methyl-3-pentyl, 2-Methyl-3-pentyl, 2,3-Dimethyl-2-butyl , 3,3-Dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl, and the like, but not limited to these. Alkyl can be unsubstituted or substituted. Alkyl can further optionally be partially or completely unsaturated. Therefore, the list of alkyl groups includes both alkenyl and alkynyl groups. As described and exemplified above, the alkyl can be a monovalent hydrocarbon radical or a divalent hydrocarbon radical (ie, alkylene).

The term "alkenyl" refers to a monoradical branched or unbranched partially unsaturated hydrocarbon chain (ie, carbon-carbon sp).²Double bond). In one embodiment, the alkenyl group can have 2 to 10 carbon atoms, or 2 to 6 carbon atoms. In another embodiment, the alkenyl group has 2-4 carbon atoms. Specific examples include, but are not limited to, ethylene or vinyl, allyl, cyclopentenyl, 5-hexenyl, and the like. Alkenyl can be unsubstituted or substituted.

The term "alkynyl" refers to a monoradical branched or unbranched hydrocarbon chain (ie a carbon-carbon sp triple bond) with a point of complete unsaturatedity. In one embodiment, the alkynyl group can have 2 to 10 carbon atoms, or 2 to 6 carbon atoms. In another embodiment, the alkynyl group can have 2-4 carbon atoms. The term is by groups such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1-octynyl, and the like. Illustrated. The alkynyl is unsubstituted or can be substituted.

The term "cycloalkyl" refers to a cyclic alkyl group of 3 to 10 carbon atoms having a single cyclic ring or multiple fused rings. Such cycloalkyl groups include, for example, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the same, or multiple ring structures such as adamantanyl, and the same. Cycloalkyl is unsubstituted or can be substituted. The cycloalkyl group can be monovalent or divalent and can be optionally substituted for the alkyl group as described above. The cycloalkyl group can optionally contain one or more unsaturated sites, eg, the cycloalkyl group is, for example, cyclohexene, 1,3-cyclohexadiene, 1,4-cyclohexadiene, and the like. Can contain one or more carbon-carbon double bonds.

The term "alkoxy" refers to the group alkyl-O- (in the formula, alkyl is as defined herein). In one embodiment, the alkoxy group is, for example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentaoxy, n-hexaoxy, 1,2-dimethylbutoxy. , And similar ones. Alkoxy can be unsubstituted or substituted.

As used herein, "aryl" refers to an aromatic hydrocarbon group derived from the removal of a single hydrogen atom from a single carbon atom in the parent aromatic ring system. Radicals can be present at saturated or unsaturated carbon atoms in the parent ring system. Aryl groups can have 6 to 20 carbon atoms. Aryl groups can have a single ring (eg, phenyl) or multiple fused (fused) rings, where at least one ring is aromatic (eg, naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Is. Typical aryl groups include, but are not limited to, radicals derived from benzene, naphthalene, anthracene, biphenyl, and the like. Aryl can be unsubstituted or optionally substituted as described above for alkyl groups.

The term "halo" refers to fluoro, chloro, bromo, and iodine. Similarly, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

The term "haloalkyl" refers to an alkyl as defined herein, substituted with one or more halo groups (which may be the same or different) as defined herein. In one embodiment, the haloalkyl can be substituted with 1, 2, 3, 4 or 5 halo groups. In another embodiment, the haloalkyl can be substituted with 1, 2 or 3 halo groups. The term haloalkyl also includes perfluoro-alkyl groups. Typical haloalkyl groups are, for example, trifluoromethyl, 3-fluorododecyl, 12,12,12-trifluorododecyl, 2-bromooctyl, 3-bromo-6-chloroheptyl, 1H, 1H-perfluorooctyl, And similar ones. The haloalkyl can be optionally substituted for the alkyl group as described above.

The term "heteroaryl" is a monocyclic, bicyclic ring system containing one, two or three aromatic rings and containing at least one nitrogen, oxygen or sulfur atom in the aromatic ring. , Or as a tricyclic ring system, which is unsubstituted or, as described above in the definition of "substituted", for example, one or more (particularly 1 to 3) substituents. Can be replaced with. A typical heteroaryl group contains 2 to 20 carbon atoms in addition to one or more heteroatoms. Specific examples of heteroaryl groups include 2H-pyrrolyl, 3H-indrill, 4H-quinolinidinyl, acridinyl, benzo [b] thienyl, benzothiazolyl, β-carbolinyl, carbazolyl, chromenyl, cinnolinyl, dibenzo [b, d] flanyl, flazanyl, Frill, imidazolyl, imidazolyl, indazolyl, indolicinyl, indrill, isobenzofuranyl, isoindrill, isoquinolyl, isothiazolyl, isooxazolyl, naphthylidine, oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenalsadinyl, phenazinyl, phenothiazine Inyl, phenoxadinyl, phthalazinyl, pteridinyl, prynyl, pyranyl, pyrazinyl, pyrazolyl, pyridadinyl, pyridyl, pyrimidinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, tetrazolyl, and xan. , Not limited to these. In one embodiment, the term "heteroaryl" refers to 5 or 6 ring atoms containing carbon, as well as non-oxygen peroxide, sulfur and N (Z) (in the formula, Z is absent or H, O, alkyl, aryl or (C₁-C₆) (Alkyl) aryl) means a monocyclic aromatic ring containing 1, 2, 3 or 4 heteroatoms, selected independently. In another embodiment, the heteroaryl fuses an ortho-fused bicyclic heterocycle of about 8-10 ring atoms derived from it, particularly a benzo derivative, or a propylene diradical, trimethylene diradical or tetramethylene diradical with it. It means something that is induced by.

The term "heterocycle" contains at least one heteroatom selected from the group oxygen, nitrogen and sulfur and is a group of one or more as defined herein under the term "substituted". Refers to an arbitrarily substituted, saturated or partially unsaturated ring system. The heterocycle can be a monocyclic group, a bicyclic group, or a tricyclic group containing one or more heteroatoms. The heterocyclic group can also contain an oxo group (= O) or a thioxo group (= S) attached to the ring. Non-limiting examples of heterocyclic groups include 1,3-dihydrobenzofuran, 1,3-dioxolan, 1,4-dioxane, 1,4-ditian, 2H-pyran, 2-pyrazoline, 4H-pyran, chromanyl, Includes imidazolidinyl, imidazolinyl, indolinyl, isochromanyl, isoindolinyl, morpholine, piperazinyl, piperidine, piperidil, pyrazolidine, pyrazoridinyl, pyrazolinyl, pyrrolidine, pyrroline, quinuclidine, and thiomorpholin.

For illustration purposes and without limitation, the term "heterocycle" is used in Paquette, Leo A .; Principles of Modern Heterocyclic Chemistry (WA Benjamin, New York, 1968), especially Chapters 1, 3, 4, 6, 7 and 9; The Chemistry of Heterocyclic Compounds, "A Series of Monographs" (John Wiley &amp; Sons, New York, 1950-present), especially 13, 14, 16, 19 and 28 It can contain heterocyclic monoradicals, as described in Volume; and J. Am. Chem. Soc., 82: 5566 (1960). In one embodiment, the "heterocycle" includes a "carbon ring" as defined herein, wherein one or more (eg, 1, 2, 3 or 4) carbon atoms are heteroatoms (eg, 1, 2, 3 or 4). For example, it has been replaced by O, N, or S).

Specific examples of heterocycles for purposes of illustration, but not limited to, are dihydroxypyridyl, tetrahydropyran (piperidyl), thiazolyl, tetrahydrothiophenyl, tetrahydrothiophenyl sulfur oxide, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, Piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidnyl, pyrrolinyl, tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2, 5-Thiadiazinyl, 2H, 6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxatinyl, 2H-pyrrolyl, isothiazolyl, isooxazolyl, pyrazinyl, pyridadinyl, indolidinyl, isoindrill , 3H-indazoly, 1H-indazoly, prynyl, 4H quinolinyl, phthalazinyl, naphthyldinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, , Phenothiazinyl, Frazanyl, Phenoxazinyl, Isochromanyl, Chromanyl, Imidazolydinyl, Imidazolinyl, Pyrazolydinyl, Pyrazolinyl, Piperazinyl, Indolinyl, Isoindrinyl, Kinucridinyl, Morphorinyl, Oxazolidinyl, Bentriazolyl, benzoisooxazolyl, , Isatinoyl, and bis-tetrahydrofuranyl.

For illustration purposes and without limitation, the carbon-bonded heterocycles are the 2, 3, 4, 5 or 6 positions of pyridine, the 3, 4, 5 or 6 positions of pyridazine, the 2, 4, 5 or 6 positions of pyrimidine. 6th place, 2,3,5 or 6th place of pyrazine, 2,3,4 or 5th place of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, 2,4 or 5th place of oxazole, imidazole or thiazole, iso Oxazole, pyrazole or isothiazole at position 3, 4 or 5, aziridine at position 2 or 3, azetidine at position 2, 3 or 4, quinoline at position 2, 3, 4, 5, 6, 7 or 8 or isoquinolin Combined at 1, 3, 4, 5, 6, 7 or 8 positions. Carbon-bonded heterocycles are 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, Includes 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, and the like.

For illustration purposes and without limitation, the nitrogen-bonded heterocycles include aziridine, azetidine, pyrrole, pyrroline, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazolin, 3-imidazolin, pyrazoline, pyrazoline. , 2-Pyrazoline, 3-Pyrazoline, Piperidine, Piperazin, Indol, Indoline, 1H-Indazole 1st, Isoindor or Isoindoline 2nd, Morpholine 4th, and Carbazole or β-Carbolin 9th sell. In one embodiment, the nitrogen-bonded heterocycle comprises 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

The term "carbon ring" is a saturated ring, unsaturated ring or aromatic having 3 to 8 carbon atoms as a monocycle, 7 to 12 carbon atoms as a dicyclic ring, and up to about 30 carbon atoms as a polycycle. Refers to the ring. Monocyclic carbocycles typically have 3 to 6 ring atoms, and even more typically 5 or 6 ring atoms. Bicyclic carbocycles are 7 to 12 ring atoms (eg, arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] system), or bicyclo. It has 9 or 10 ring atoms arranged as a [5,6] or [6,6] system. Specific examples of the carbocycle include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopenta-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-. Includes cyclohex-2-enyl, 1-cyclohex-3-enyl, phenyl, spiryl and naphthyl. The carbocycle can be optionally substituted for the alkyl group as described above.

The term "alkanoyl" or "alkylcarbonyl" refers to -C (= O) R (in the formula, R is an alkyl group as defined above).

The term "acyloxy" or "alkylcarboxyl" refers to -OC (= O) R (in the formula, R is an alkyl group as defined above). Specific examples of acyloxy groups include, but are not limited to, acetoxy, propanoyloxy, butanoyloxy, and pentanoyloxy. Any alkyl group as defined above can also be used to form the acyloxy group.

The term "alkoxycarbonyl" refers to -C (= O) OR (or "COOR") (where R is an alkyl group as defined above).

The term "amino" is -NH₂Point to. Amino groups can be optionally substituted as defined herein for the term "substituted". The term "alkylamino" is -NR₂(In the formula, at least one R is alkyl and the second R is alkyl or hydrogen). The term "acylamino" refers to N (R) C (= O) R (wherein each R is independently hydrogen or alkyl or aryl).

The term "amino acid" refers to naturally occurring amino acid residues of type D or L (eg, Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, In addition to Phe, Pro, Ser, Thr, Trp, Tyr, and Val), non-naturally occurring amino acids (eg, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, γ-carboxyglutamic acid; horseuric acid, octahydroindole-2 -Carboxylic acid, statin, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citrulin, α-methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert -Butylglycine) is included. The term refers to natural and non-naturally occurring amino acids with conventional amino protecting groups (eg, acetyloxycarbonyl or benzyloxycarbonyl), as well as naturally occurring and non-naturally occurring amino acids protected at the carboxy terminus. Also includes (eg (C)₁-C₆) As an ester or amide of alkyl, phenyl or benzyl; or as α-methylbenzylamide). Other suitable amino-protecting and carboxy-protecting groups are known to those of skill in the art (eg, Greene, TW; Wutz, PGM Protecting Groups In Organic Synthesis, 2nd Edition, John Wiley &amp; Sons, New York (1991). ), And the references cited therein).

The term "peptide" describes the sequence of 2-3 amino acids (eg, as defined above) or peptidyl residues. The sequence can be linear or circular. For example, cyclic peptides can be prepared or can result from the formation of disulfide bridges between two cysteine residues in the sequence. Preferably the peptide comprises 3 to 20 or 5 to 15 amino acids. Peptide derivatives can be prepared as disclosed in US Pat. Nos. 4,612,302; 4,853,371; and 4,684,620, or as described herein below in the examples. The peptide sequences specifically listed herein are written with an amino terminus on the left and a carboxy terminus on the right.

The term "saccharide" refers to sugars or other carbohydrates, especially monosaccharides. Saccharide is C₆-Polyhydroxy compounds (typically C₆-Pentahydroxy) and often cyclic glycers. The term includes known monosaccharides and their derivatives, as well as polysaccharides with two or more monosaccharide residues. As mentioned above in the definition of amino acids, saccharides can contain protecting groups on the hydroxyl groups. The hydroxyl group of saccharides can be replaced with one or more halo or amino groups. Furthermore, one or more carbon atoms can be oxidized to, for example, a ketone group or a carboxyl group.

The term "interrupted" does not exceed the standard valence of each indicated atom, and if interruption results in a stable compound, the term "interrupted" is used in the expression of a particular carbon chain referred to. Two adjacent carbon atoms (and the hydrogen atom to which they are attached (eg, methyl (CH))₃), Methylene (CH)₂) Or methine (CH))) to indicate that another group is inserted. Suitable groups capable of interrupting the carbon chain are, for example, one or more non-oxidic peroxides (-O-), thio (-S-), imino (-N (H)-) methylenedioxy (-OCH).₂O-), carbonyl (-C (= O)-), carboxy (-C (= O) O-), carbonyldioxy (-OC (= O) O-), carboxylate (-OC (= O)) -), Imine (C = NH), Sulfinyl (SO) and Sulfonyl (SO)₂)including. Alkyl groups can be interrupted by one or more (eg, 1, 2, 3, 4, 5 or about 6) suitable groups as described above. The site of interruption can also be between the carbon atom of the alkyl group and the carbon atom to which the alkyl group is attached. In certain embodiments, one or more of the aforementioned groups are excluded from the embodiments.

With respect to any of the above groups (containing one or more substituents), it is of course understood that such groups do not contain sterically impractical and / or synthetically impractical substitutions or substitution patterns. .. Furthermore, the compounds of the present invention include all stereochemical isomers resulting from substitutions of these compounds. In certain embodiments, the compounds of the invention do not include the compounds disclosed in US Pat. No. 5,424,440 (Klem et al.).

Selected substituents within the compounds described herein are present to a recursive degree. In this context, "recursive exhibiting" means that a substituent can enumerate another example of itself. Due to the recursive nature of such substituents, in theory there can be many in any given claim. Those skilled in the art of medicinal chemistry and organic chemistry will understand that the total number of such substituents is reasonably limited by the desired properties of the intended compound. Such properties include, but are not limited to, physical properties such as molecular weight, solubility or logP, applicability such as activity against intended targets, and practical properties such as ease of synthesis. ..

Recursive substituents are an intended embodiment of the present invention. Those skilled in the art of medicinal chemistry and organic chemistry will understand the wide range of uses of such substituents. To the extent that recursive substituents are present in the claims of the present invention, the total number is determined as described above.

As used herein, the term "linker" is either covalently attached to two chemical groups and contains a substrate for an enzyme or an enzyme that can be cleaved by another molecule, or is photosensitive. It is an atomic chain (typically a carbon chain). The chain is optionally interrupted by one or more nitrogen atoms, oxygen atoms, carbonyl groups, (substituted) aromatic rings, or peptide bonds, and / or one of these groups forms a linker. Can occur at one or both ends of. Many linkers are well known in the art and can be used to link the compounds or formulas described herein to other groups such as solid supports or resins. For example, described by Sewald and Jakubke in Peptides: Chemistry and Biology, Wiley-VCH, Weinheim (2002), pp. 212-223; and by Dorwald in Organic Synthesis on Solid Phase, Wily-VCH, Weinheim (2002). See linkers and solid supports.

As used herein, a "fluorofore" includes molecules capable of absorbing energy in one wavelength region and emitting energy in a wavelength region other than the absorption range. In certain embodiments, fluorophores are molecules capable of absorbing energy at about 250 nm to about 900 nm and emitting energy in the wavelength range of about 260 nm to about 910 nm. The term "excitation wavelength" refers to the range of wavelengths at which a fluorophore absorbs energy. The term "radiation wavelength" refers to the range of wavelengths at which a fluorophore emits energy or fluorescence.

Fluorescein includes fluorosane, Texas red, DAPI, PI, acrydin orange, Alexafluol (eg Alexa 350, Alexa 405 or Alexa 488), cyanine pigments such as Cy3, Cy5 and Cy7, coumarin, ethidium bromide, fluorescein, BODIPY, Rhodamine, Rox, 5-carboxyfluorescein, 6-carboxyfluorescein, anthracene, 2-amino-4-methoxynaphthalene, phenalenone, acridone, fluorinated xanthene derivatives, α-naphthol, β-naphthol, 1-hydroxypyrene, coumarin (For example, 7-amino-4-methylcoumarin (AMC) or 7-amino-4-trifluoromethylcoumarin (AFC)), Rhodamine (eg, tetramethylrhodamine, Rhodamine-110, Carboxyrodamine), cresyl violet, Or in addition to Resolfin, US Pat. No. 6,420,130 (Makings, et al.), Including, but not limited to, the fluorophores disclosed in (this disclosure is incorporated herein by reference). Fluorophore is the formula Ar- [CH = CH]_n-[CH =]_mAr (in the formula, Ar is an aryl or heteroaryl group; n is 1, 2, 3 or 4; m is 0 or 1; in the formula, each Ar is quaternary nitrogen or quaternary via resonance. Contains cyanine pigments such as compounds (including nitrogen that can be graded). Specific examples of such aryl or heteroaryl groups include dimethyl-aminophenyl, imidazole, pyridine, pyrrole, quinoline, thiazole, and indole, each of which is optionally substituted. The fluorophore can be a compound that is fluorescent in nature or exhibits a change in fluorescence upon binding to a biological compound, i.e., it can be fluorescent, or its intensity can be reduced by quenching. it can. Fluorophores can contain substituents that alter the solubility, spectroscopic or physical properties of the fluorophore. Various fluorophores are known to those of skill in the art and are known to those skilled in the art, in addition to benzofurans, quinolines, quinazolinones, indoles, benzoazoles, borapolyazaindacenes, and xanthenes (including fluoroscein, rhodamine and lordole), as well as invitrogen molecular probes. (Invitrogen Molecular Including other fluorophores described in Richard P. Haugland's The Handbook, A Guide to Fluorescent Probes and Labeling Technologies (10th edition, 2005), which describes a large number of fluorophores available from Probes). Not limited to these.

A "fluorescence-generating analysis" or "fluorescence-generating reaction" includes a reaction in which the product of the reaction is fluorescent. The "fluorescence-generating analytical reagent" can include, in addition to the substrate, other molecules (s) such as cofactors (s) or proteins (eg, enzymes) for the fluorescence-generating reaction.

The term "solid support" refers to a support that can be isolated from the reaction mixture in solid form, such as silicato or polymer particles. Solid supports include various particles and surfaces such as beads, microtiter plates, Eppendorf tubes, and slides. The surface can be a polymer such as cepharose, cellulose, alginate, polystyrene or other plastics, and / or other surfaces including membranes and glass. Many common solid supports are available in Peptides: Described by Sewald and Jakubke in Chemistry and Biology, Wiley-VCH, Weinheim (2002), pp. 212-223; and by Dorwald in Organic Synthesis on Solid Phase, Wiley-VCH, Weinheim (2002). Cyanobenzothiazole is applied to the solid support non-specifically (via adsorption to the surface) or specifically (through capture by an antibody specific for cyanobenzothiazole, as in a "sandwich" ELISA). It can be connected by either. The detection antibody on the surface of the solid support can be covalently linked to the enzyme or can itself be detected by a secondary antibody linked to the enzyme via bioconjugation. See Schuurs and van Weemen, J. Immunoassay 1980; 1: 229-49 for specific examples of ELISA "sandwich" test procedures.

The term "reporter moisture" refers to a portion of a molecule that can be detected in a biological or non-biological mixture (eg, fluorophore, chromophore, or radioactive element). The reporter yeti allows the reporter molecule to function as a member of an energy transfer pair when attached to a ligand analog, retaining its innate properties (eg, spectroscopic properties, conformation, and / or activity). It is possible and can be the molecule used in the methods disclosed herein. The reporter yeti can be a reporter molecule linked to cyanobenzothiazole. Specific examples of reporter molecules include nucleic acids, volapolyazaindacenes, coumarins, xanthenes, cyanines, and luminescent molecules including dyes, fluorescent proteins, chromophores, and chemiluminescent compounds capable of producing detectable signals upon proper activation. Including, but not limited to. The term "dye" refers to a compound that emits light that produces an observable and detectable signal. "Dyes" include, but are not limited to, pigments, fluorophores, chemiluminescent compounds, luminescent compounds and chromophores, including phosphorescent compounds, fluorescent compounds and non-fluorescent compounds. The term "chromophore" is visible space can be observed without the aid of equipment refers to labels that emit and / or reflect light spectrum.

The terms "affinity moisture", "affinity label", and / or "affinity molecule" are molecules that can be effectively attached to a molecule, biomolecule, or material of interest in a non-covalent or covalent bond. Refers to the part of (eg, biomolecule, His tag, or chitin). Affinity moisture can be a molecule containing an acceptor group. Thus, a cyanobenzothiazole derivative containing an affinity moiety may selectively interact with another molecule (eg, a molecule that binds to an affinity moiety that may be of biological or non-biological origin). As such, it can be used to facilitate the identification and separation of labeled molecules or complexes.

The term "quencher" or "quenching moisture" is a strong photon absorber that is non-fluorescent or essentially non-fluorescent and effectively fluoresces other molecules around it. Refers to a molecule or part of a molecule that is quenched. Quenching moisture is a moisture that can absorb energy from an energy donor that is not re-emitted (non-fluorescent) or is re-emitted at a wavelength different from the energy emitted by the donor molecule so that it can be detected. Can be. In this regard, in certain embodiments, the quencher can be non-fluorescent or fluorescent in nature. Some specific examples of quencher moisture that can be linked to cyanobenzothiazole include xanthene, xanthene derivatives, cyanine, cyanine derivatives, dimethylaminoazosulfonic acid (DABSYL), and dimethylaminoazo-carboxylic acid (DABCYL). Numerous quenching yetis are well known in the art, xanthenes, cyanines, and Richard P. Includes other compounds disclosed in Haugland's The Handbook, A Guide to Fluorescent Probes and Labeling Technologies (10th edition, 2005). Quenching and Fluorescence Quenching is available in Principles of Fluorescence Spectroscopy, 2nd Edition, New York: Kluwer Academic / Plenum (1999). Further described by Lakowicz; in particular, Chapter 8 ("Quenching of Fluorescence") pp. 237-264; and 3rd Edition, New York: Springer Science (2006), pp. 8 278-327, and its See references cited in. In certain embodiments, the quencher can be part of a chromophore molecule or compound that can reduce radiation from the fluorescent donor when attached to the donor. Quenching can also occur by any of several mechanisms, including exciton coupling such as fluorescence resonance energy transfer, photoinduced electron transfer, paramagnetic enhancement of intersystem crossing, Dexter exchange coupling, and dark complex formation. sell.

The term "acceptor" refers to a quencher that operates through energy transfer. The acceptor can re-radiate the transition energy as fluorescence, which is the "acceptor fluorescence moisture". Specific examples of acceptors include coumarins and related fluorophores, xanthenes (such as fluorescein, rhodamine and rhodamine), resorphins, cyanines, difluoroborasia zaindacenes, and phthalocyanines. The chemical classes of other acceptors generally do not re-radiate transitional energy as light. Specific examples include some indigo, benzoquinone, anthraquinone, azo compounds, nitro compounds, indian aniline, and diphenylmethane and triphenylmethane.

The term "photocrosslinking moisture" refers to a portion of a molecule that can be covalently attached to another molecule, biomolecule or material of interest upon photoexcitation. For example, compounds containing photocrosslinking moitty can be used to crosslink proteins upon photoexcitation.

The term "enzyme of interest" refers to any enzyme that can be labeled using the methods of the invention or other methods known in the art. Enzymes of interest include, for example, kinases, phosphatases, peroxidases, sulfatases, peptidases, glycosidases, proteases (eg, proteases involved in apoptosis), hydrolase, oxidoreductases, lyases, transferases, isomerases, ligases, protein kinases, protein phosphatases, etc. Includes esterases, isomerases, glycosylases, kinases, dehydrogenases, oxidases, reductases, methylases, and the like. Further targeted enzymes include those involved in the production or hydrolysis of esters (both organic and inorganic), glycosylation, and hydrolysis of amide bonds. In any class, as in kinases, there can be further subdivisions, and kinases can be specific for phosphorylation of serine, threonine and / or tyrosine residues in peptides and proteins.

Other enzymes of interest include any protein exhibiting enzymatic activity, such as lipase, phospholipase, sulfatase, urease, peptidase, protease, and esterase, including acid phosphatase, glucosidase, glucuronidase, galactosidase, carboxylesterase, and luciferase. Including. In one embodiment, the enzyme is a hydrolase. Specific examples of hydrolases include alkaline phosphatase and acid phosphatase, esterase, decarboxylase, phosphoripase D, P-xylosidase, β-D-fucosidase, thioglucosidase, β-D-galactosidase, α-D-galactosidase, α-D. -Includes glucosidase, β-D-glucosidase, β-D-glucuronidase, α-D-mannosidase, β-D-mannosidase, β-D fructofuranosidase, and β-D-glucosiduronase.

Further target enzymes are hydrolase, carboxylic acid ester hydrolase, thiol ester hydrolase, phosphoric acid monoester hydrolase, phosphoric acid diester hydrolase, triphosphate monoester hydrolase, sulfate ester hydrolase, diphosphate monoester hydrolase, phosphoric acid. Triester hydrolase, exodeoxyribonuclease that produces 5'-phosphomonoester, exoribonuclease that produces 5'-phosphomonoester, exoribonuclease that produces 3'-phosphomonoester, ribonucleic acid or deoxyribonucleic acid Active exonucleases, exonucleases active in either ribonucleic acid or deoxyribonucleic acid, endodeoxyribonucleases that produce 5'-phosphomonoesters, endodeoxyribonucleases that produce non-5'-phosphomonoesters, modified Base-specific site-specific endodeoxyribonucleases, endoribonucleases that produce 5'-phosphomonoesters, endoribonucleases that produce non-5'-phosphomonoesters, and endos that are active in either ribonucleic acid or deoxyribonucleic acid. Enzymes that act on ester bonds, such as endoribonucleases that are active in ribonucleases, ribonucleic acid or deoxyribonucleic acid glycosylases; enzymes that hydrolyze glycosidases (eg, O-glycosyl compounds and S-glycosyl compounds, and N-glycosyl compounds). Degrading enzymes); Enzymes that act on ether bonds, such as trialkylsulfonium ribolase or etherhydrolase;Aminopeptidase, dipeptidase, dipeptidylpeptidase and trypeptidylpeptidase, peptidyldipeptidase, serine-type carboxypeptidase, metallocarboxypeptidase, cysteine-type carboxypeptidase, omegapeptidase, omegapeptidase, serineendopeptidase, Enzymes that act on peptide binding (peptide hydrolase), such as threonine endopeptidases, and endopeptidases of unknown catalytic mechanism; peptides such as linear amides, cyclic amides, linear amidins, cyclic amidins, nitriles, or other compounds. Enzymes that act on carbon-nitrogen bonds other than bonds; Enzymes that act on acid proteases, such as those in phosphite-containing and sulfonyl-containing anhydrides; Enzymes that act on acid anhydrides (catalyst intermembrane migration) ); Enzymes that act on acid proteases or are involved in cell migration and subcellular migration; enzymes that act on carbon-carbon bonds (eg, in ketone substances); halide bonds (eg C-halogen) Enzymes that act on (in the protease), enzymes that act on phosphorus-nitrogen bonds; enzymes that act on sulfur-nitrogen bonds; enzymes that act on carbon-phosphorus bonds; and enzymes that act on sulfur-sulfur bonds, but these Not limited to.Enzymes that act on halide bonds (eg in C-halide compounds), enzymes that act on phosphorus-nitrous bonds; enzymes that act on sulfur-nitrogen bonds; enzymes that act on carbon-phosphorus bonds; and sulfur-sulfur bonds Including, but not limited to, enzymes that act on.Enzymes that act on halide bonds (eg in C-halide compounds), enzymes that act on phosphorus-nitrous bonds; enzymes that act on sulfur-nitrogen bonds; enzymes that act on carbon-phosphorus bonds; and sulfur-sulfur bonds Including, but not limited to, enzymes that act on.

The term "poly-histidine tract" or "His tag" refers to a molecule containing 2-10 histidine residues, eg, a 5-10 residue poly-histidine tract. The poly-histidine tract is shared on an immobilized metal (eg, nickel, zinc, cobalt or copper) chelate column or through interaction with another molecule (eg, an antibody reactive with a His tag). Allows affinity purification of bound molecules.

Linker A linker strategy is used to ligate a reporter moity (eg, a fluorophore, an affinity moity, or another labeling group such as biotin, resin, carbohydrate, oligopeptide, dye or drug moity) to the cyanobenzothiazole and N-terminus. It produces a cyanobenzothiazole derivative capable of reacting with a protein having cysteine. The use of linkers or "linking groups" is well known in the art.

The linking group is (C₁-C₆) Alkyl group or (C₁-C₆) It can be an alkyl chain such as an alkoxy group or an alkoxy chain. The chain can have a substituent R of one or more electron-attracting groups, such as an aldehyde group, an acetyl group, a sulfoxide group, a sulfone group, a nitro group, a cyano group or a combination thereof. Specific linkers and methods for the preparation of covalent bonds are described, for example, in US Pat. No. 7,282,339 (Beechem et al.); Peptides: Chemistry and Biology by Sewald and Jakubke, Wiley-VCH, Weinheim (2002), 212. -On page 223; and in Organic Synthesis on Solid Phase by Dorwald, Wily-VCH, Weinheim (2002), which are incorporated herein by reference.

In certain embodiments, the linking group is a divalent radical of formula WA (where A is (C).₁-C₆) Alkyl, (C₂-C₆) Alkenyl, (C₂-C₆) Alkyne, (C₃-C₈) Cycloalkyl, or (C₆-C₁₀) Aryl; W is -N (R) C (= O)-, -C (= O) N (R)-, -OC (= O)-, -C (= O) O-,- O-, -S-, -S (O)-, -S (O) 2-, -N (R)-, -C (= O)-, or direct coupling; each R is independent H, (C₁-C₆) Can be alkyl or protecting group); the linker group links together two other molecular yeti such as the reporter yeti (eg, cyanobenzothiazole yeti and group X as defined above).

Immobilization The present invention includes an immobilized cyanobenzothiazole derivative and a method for immobilizing a cyanobenzothiazole derivative. Some immobilized cyanobenzothiazoles include those linked to a solid support at a position on the benzo ring (eg, at the 6'-position). Other immobilized cyanobenzothiazoles are solid supports by non-covalent interactions with bioactive groups (eg, biotin, avidin, streptavidin, or derivatives thereof), antibodies or antigens, or His tags on nickel columns. Including those bound to the surface of. In addition, the other immobilized cyanobenzothiazole has the appropriate antibody on the surface of the solid support to which the terminal Cys-containing protein of interest reacts with cyanobenzothiazole and subsequently binds the protein of interest. It can be prepared by binding to a solid support.

Marking method The present invention includes methods for labeling and detecting proteins and / or isolating labeled proteins from cell or cell-free translation systems. The isolated protein can be used directly or can be further purified and / or manipulated. In one embodiment, the method is a protein comprising one or more undefined proteins, such as those used with a defined protein (eg, a defined population of proteins) or obtained from a cell or expression library. Can be used with a set of.

One or more proteins are eukaryotic cells (eg, yeast cells, avian cells, plant cells, insect cells, or human cells, monkey cells, mouse cells, dog cells, bovine cells, horse cells, cat cells, sheep cells, etc. It can be from a sample containing cells from, but not limited to, goat cells or pig cells) or prokaryotic cells, or cells from two or more different organisms, or a cell lysate or supernatant thereof.

The methods disclosed herein include labeling a protein with Cys at the N-terminus with any detectable or detectable molecule. Can Cys be added to the protein via recombinant techniques (eg, exposing Cys, such as those in fusion proteins by intein-mediated splicing, or inserting appropriate protease sites)? , Can be naturally present at position 2 in proteins sensitive to N-terminal aminopeptidases, or can be added by various synthetic techniques of synthesis (eg, via peptide ligation, or reverse proteolysis) (eg, Wehofsky). , J. Amer. Chem. Soc. 125: 6126 (2003) and Chang, PNAS See 91: 12,544 (1994)). For example, a protein preparation, one of which has an N-terminal Cys, is reacted with a derivative of a cyanobenzothiazole having a fluorophore to form at least one protein containing a fluorophore covalently linked to the N-terminus. To do. The linker group can be used to facilitate the ligation of the reporter or affinity yeti to cyanobenzothiazole.

A. Illustrative moisture for signs The label comprises a detectable or detectable molecule (moietti), and in one embodiment, a useful moetty in the compounds and methods described herein is Cys via a derivative of cyanobenzothiazole. It is a molecule that can be covalently linked to the amino group of. Moyetti useful in compounds and methods has one or more properties that facilitate the detection and optionally quantification and / or isolation of proteins containing moyetti. One physical property is a characteristic electromagnetic spectral property such as radiation or absorption, magnetism, electron spin resonance, capacitance, permittivity or conductivity. In certain embodiments, the moyetti can have ferromagnetism, paramagnetism, diamagnetism, luminescence, electrochemical luminescence, fluorescence, phosphorescence, stainability, antigenicity, or a unique mass.

Related moities are nucleic acid molecules (ie, DNA or RNA, eg, oligonucleotides or nucleotides such as those with nucleotide analogs, DNA to which proteins can bind, single or double strands corresponding to the gene of interest. Stranded DNA, RNA corresponding to the gene of interest, mRNA lacking termination codon, aminoacylated initiation tRNA, aminoacylated amber suppressor tRNA, or double-stranded RNA for RNAi), proteins (eg, luminescent proteins), peptides, Peptide nucleic acids, epitopes recognized by ligands (eg, biotin or streptavidin), hapten, amino acids, lipids, lipid bilayers, solid supports, fluorophores, chromophores, reporter molecules, radioactive nuclei (eg used in radiometric measurements) Radioisotopes or stable isotopes, etc.), electron-impermeable molecules, X-ray contrast reagents, MRI or X-ray contrast agents (eg, barium, iodine, manganese, gadolinium (III) or iron oxide particles), and Includes, but is not limited to, the same species.

In one embodiment, the moyetti is an electron opaque as a glycoprotein, polysaccharide, triple term sensitizer (eg CALI), drug, toxin, lipid, biotin, or solid support (self-assembling monomolecular layer). , Etc.), nanoparticles, enzymes, substrates for enzymes, inhibitors of enzymes (eg suicide substrates), cofactors (eg NADP), coenzymes, succinimidyl esters or aldehydes, glutathione, NTA , Biotin, cAMP, phosphatidylinositol, ligands for cAMP, metals, nitroxides or nitrons used as spin traps (detected by electron spin resonance (ESR)), metal chelators (eg for use as contrast agents) , In time-resolved fluorescence, or for metal capture), photo-caged compounds (eg, when irradiation releases caged compounds such as fluorophores), intercalators (eg, solarene or another intercalator useful for DNA binding). Etc., or molecules that can be photoactivated), triphosphates or phosphoamidite (eg, allowing the uptake of the substrate into DNA or RNA), antibodies, heterobifunctional cross-linking agents (conjugated proteins or other Mixed disulfides such as hydrazide, aryl azide, maleimide, iodoacetamide / bromoacetamide, N-hydroxysuccinimidyl ester, pyridyldisulfide, glyoxal / phenylglioxal, vinylsulfone / vinylsulfoneamide, etc. Is it a lipid containing acrylamide, boronic acid ester, hydroxamic acid, imidate ester, isocyanate / isothiocyanate, or chlorotriazine / dichlorotriazine), glycoproteins, polysaccharides, lipid bilayers;Or a solid support (eg, Sepharose beads or cellulose beads), membrane, glass (eg, slide glass), cellulose, alginate, plastic polymer or other synthetically prepared polymer (eg, Eppendorf tube or multi-well plate wells). ), Self-assembled monolayers, surface plasmon resonance chips, or solid supports with electron-conducting surfaces, drugs, aminoacylated tRNAs (such as aminoacylated initiation tRNAs or aminoacylated amber suppressor tRNAs), Ca²⁺The molecule that binds to, K⁺The molecule that binds Na⁺Molecules that bind to, pH-sensitive molecules, radioactive nuclei, electron-impermeable molecules, molecules that fluoresce or are sensitive to active oxygen in the presence of NO, non-protein substrates for enzymes, inhibitors of enzymes, reversible Of inhibitors, chelating agents, cross-linking groups (eg, succinimidyl esters or aldehydes), glutathione, biotin or other avidin binding molecules, avidin, streptavidin, phosphatidylinositol, hem, cAMP, which are either target or irreversible. For, in one embodiment, one or more dyes (eg, xanthene dyes), calcium sensitive dyes (eg, 1- [2-amino-5- (2,7-dichloro-6-)). Hydroxy-3-oxy-9-xanthenyl) -phenoxy] -2- (2'-amino-5'-methylphenoxy) ethane-N, N, N', N'-tetraacetic acid (Fluo-3)), sodium Sensitive dyes (eg, 1,3-benzenedicarboxylic acid, 4,4'-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis (5-methoxy-6,,) 2-benzofuranyl)] bis (PBFI)), NO sensitive dyes (eg 4-amino-5-methylamino-2', 7'-difluoresane), or other fluorophores. In one embodiment, the moyetti is not a radionuclide. In another embodiment, the moyetti is a radionuclide containing molecules useful in diagnostic methods (eg,,³H,¹⁴C,³⁵S,¹²⁵I,¹³¹I).

Exemplary moyetti are haptens (eg, molecules useful in promoting immunogenicity such as keyhole limpet hemoscein (KLH)), cleavable moyets (eg, photocleavable biotin), and fluorescent moyets (eg, photocleavable biotin). , N-hydroxy-xantheneimide (NHS) -modified coumarin, and succinimide or sulfonosuccinimide-modified BODIPY (which can be detected by UV and / or visible light excitation fluorescence detection), Rhodamine (eg R110), Rhodamine, CRG6 , Texas Methyl Red (carboxytetramethylrhodamine), 5-carboxy-X-rhodamine, or fluoroscein, fluorescein derivatives (eg 7-aminocoumarin and 7-hydroxycoumarin), 2-amino-4-methoxynaphthalene, 1- Hydroxanthene containing hydroxypyrene, resorphin, phenalenone or benzophenalenone (US Pat. No. 4,812,409), acridinone (US Pat. No. 4,810,636), anthracene, and derivatives of α-naphthol and β-naphthol, fluorinated fluorescein and rhodamine. Includes derivatives (eg, US Pat. No. 6,162,931), bioluminescent molecules (eg, luciferin, serenterazine, luciferase), chemiluminescent molecules (eg, stabilized dioxetane), and electrochemiluminescent molecules.

Specific examples of affinity moietti are immunogenic molecules (eg, epitopes of proteins, peptides, carbohydrates or lipids, i.e. any molecule useful for preparing antibodies specific for that molecule); biotin, avidin, Includes molecules such as streptavidin and derivatives thereof; metal binding molecules; and fragments and combinations of these molecules. Exemplary affinity molecules are His5 (HHHHH) (SEQ ID NO: 1), His × 6 (HHHHHH) (SEQ ID NO: 2), C-myc (EQKLISEEDL) (SEQ ID NO: 3), Flag (DYKDDDDK) ( SEQ ID NO: 4), Stept tag (WSHPQFEK) (SEQ ID NO: 5), HA tag (YPYDVPDYA) (SEQ ID NO: 6), thioredoxin, cellulose-binding domain, chitin-binding domain, S-peptide, T7 peptide, carmodulin-binding peptide , C-End RNA tag, metal binding domain, metal binding reactive group, amino acid reactive group, intain, biotin, streptavidin, and protein binding maltose.

For example, the presence of biotin at the N-terminus of a protein is selective for avidin molecules (eg, avidin molecules coated on a surface (eg beads, microwells, nitrocellulose and the like)). It becomes possible to combine. Suitable surfaces include resins for chromatographic separation, plastics (tissue culture surfaces for binding plates, microtiter dishes and beads, etc.), ceramics and glass, particles containing magnetic microparticles, polymers and other matrices. The treated surface is washed, for example, with phosphate buffered saline (PBS) to remove non-neoplastic proteins and other translation reagents and isolated nascent proteins. In some cases, these materials can be part of biomolecule detectors such as optical fibers, ChemFETs, and plasmon detectors.

Another specific example of an affinity molecule is dancil lysine. Are antibodies that interact with the Dansyl ring system available commercially (Sigma Chemical; St. Louis, Missouri), or Antibodies: It can be prepared using known protocols such as those described in the A Laboratory Manual (Harlow and Lane, 1988). For example, the anti-dancil antibody is immobilized on the packing material of the chromatographic column. This method (affinity column chromatographie) immobilizes a complex between an immobilized antibody and a substrate to be retained on the column (eg, a benzothiazole derivative linked to an affinity moyetti). It is formed due to its interaction with the antibody, but other molecules carry out separation by passing through a column. The complex can then be released by disrupting the antibody-antigen interaction. Specific chromatographic column materials such as ion exchange resins or affinity sepharose resins, sephadex resins, sephadex resins and other chromatographic resins are commercially available (Sigma Chemicals; St. Louis, Missouri; Pharmacia Biotech). Pharmacia Biotech); Piscataway, NJ). Dancil lysine has fluorescent properties and can be conveniently detected.

When using an antibody as the acceptor molecule, separation is also performed via other biochemical separation methods such as immunoprecipitation and immobilization of the antibody on a filter or other surface (such as beads, plates or resins). be able to. The beads are often separated from the mixture using a magnetic field.

Another class of moyetti contains molecules that can be detected using electromagnetic radiation, in addition to xanthenefluorofore, dansylfluorofore, coumarin and coumarin derivatives, fluorescent acridinium moyety, benzopyrene-based fluorophore, 7- Includes nitrobenz-2-oxa-1,3-diazole, as well as 3-N- (7-nitrobenz-2-oxa-1,3-diazol4-yl) -2,3-diamino-propionic acid. Not limited. Preferably, the fluorescent molecule has a high quantum yield fluorescence at a wavelength different from that of the native amino acid, and more preferably a high quantum yield fluorescence that can be excited in both the visible, or UV and visible parts of the spectrum. Has. Upon excitation at a preselected wavelength, molecules can be detected at low concentrations either with the naked eye or by using conventional fluorescence detection methods. Electrochemiluminescent molecules such as ruthenium chelate and its derivatives or nitroxide amino acids and their derivatives can be detected in the femtomoler range and below.

In one embodiment, the optional moetities can be detected by one of the following:<chemistry num="2"><img id="000003" he="136" wi="118" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>(In the formula, R¹Is, for example, (C₁-C₈) Alkyl, optionally substituted with one or more substituents).

Methods that can be used to isolate and / or detect moyetti-labeled proteins include gel filtration chromatography, high-speed pressure chromatography or high-pressure liquid chromatography, reverse-phase chromatography, affinity chromatography, ion exchange chromatography, electrophoresis. , Capillary electrophoresis, and chromatographic techniques including isoelectric point electrophoresis. Other methods of separation (eg, electrophoresis, isoelectric focusing and mass spectrometry) are also useful for detection and subsequent isolation.

Separation can also be performed via other biochemical separation methods such as immunoprecipitation and immobilization of antibodies on filters or other surfaces (such as beads, plates or resins). For example, proteins can be isolated by coating paramagnetic particles with protein-specific antibodies. A magnetic field is used to separate the beads from the protein translation extract.

Many devices designed to detect proteins are based on the interaction of a specific acceptor molecule (eg, an immobilized acceptor molecule) with a target protein. Biodetectors based on sensing changes in surface plasmons, light scattering, and electronic properties of materials that change due to the interaction of target molecules with immobilized acceptor groups if the protein contains affinity moietic. Such devices, such as, can also be used to detect proteins.

B. Quenching of excess labeling reagents In one embodiment, the fluorescence of excess unreacted label (eg, compound 3028) can be quenched by the addition of a quenching reagent to the labeling reaction. The quenching reagent can be, for example, any β-mercaptoethyramine conjugated to a known fluorescent quencher. One possible embodiment of such a quenching reagent is compound 3191.<chemistry num="3"><img id="000004" he="32" wi="77" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>β-Mercaptoetyramine can react with the cyanobenzothiazole moyetti of any unreacted labeling reagent, thereby conjugating the quencher to the fluorophore of the labeling reagent. This type of quenching reagent does not react with or quench any label already conjugated to the protein.

Moity that can be detected using electromagnetic radiation Moieties that can be detected using electromagnetic radiation include dansylfluorophore, coumarin and coumarin derivatives, fluorescent acridinium moyetti, benzopyrene-based fluorophore, plus 7-nitrobenz-2-oxa-1,3-diazole, Also include, but are not limited to, 3-N- (7-nitrobenz-2-oxa-1,3-diazol4-yl) -2,3-diamino-propionic acid. In one embodiment, the fluorescence moisture has a high quantum yield of fluorescence at a wavelength different from that of the native amino acid and is excited by either the UV or visible portion of the spectrum, or both the UV and visible portion of the spectrum. Has high quantum yield fluorescence that can be achieved. Upon excitation at a preselected wavelength, moisture can be detected at low concentrations, either macroscopically or using conventional fluorescence detection methods. Electrochemiluminescent labels such as ruthenium chelate and its derivatives or nitroxide amino acids and their derivatives can be detected in the femtomoler range and below.

In addition to fluorescent moyties, various moyties with physical properties based on the interaction and response of moyets to electromagnetic fields and radiation can be used to detect protein production. These properties include absorption of the electromagnetic spectrum into the UV, visible and infrared regions, Raman activity and the presence of chromophores that can be enhanced by resonance Raman spectroscopy, electron spin resonance activity and nuclear magnetic resonance activity. Also includes molecular mass (eg, by mass spectrometer). These electromagnetic spectroscopic properties of optics preferably do not retain the native amino acids or can be easily distinguished from the properties of the native amino acids.

Fluorescent and other moyties with detectable electromagnetic spectroscopic properties can be detected by a variety of instruments (such as spectrometers or fluorometers and the like) and distinguished from the electromagnetic spectroscopic properties of native amino acids. Spectrometers include fluorescence spectrometers, Raman spectrometers, absorption spectrometers, electron spin resonance spectrometers, visible spectrometers, infrared spectrometers and ultraviolet spectrometers. Other moities, such as those with distinct electrical properties, can be detected by devices such as ammeters or voltmeters or other spectrometers. Moiety's physical properties regarding the unique interaction of electromagnetic fields and labels can be easily detected using instruments such as fluorescence spectrometers, Raman spectrometers, absorption spectrometers, or electron spin resonance spectrometers. Moieties can also carry out chemical, biochemical, electrochemical or photochemical reactions such as color changes in response to external stresses or agents (such as electromagnetic fields or reactant molecules) that allow detection.

Regardless of which class of fluorescent compound is used, detection can include physical separation of proteins from other biomolecules present in cellular or cell-free protein systems. Protein separation can be performed using, for example, gel electrophoresis or column chromatography. Detection of proteins containing fluorophores by gel electrophoresis can be performed using conventional fluorescence detection methods. After protein synthesis in a cell-free system, the reaction mixture, which contains the protein in addition to all the biomolecules required for protein synthesis, is loaded onto a gel consisting of polyacrylamide or agarose. Following the loading of the reaction mixture, a voltage is applied that spatially separates the proteins on the gel in the direction of the applied electric field. The proteins are separated and appear as a set of separated or overlapping bands that can be visualized using pre-gel staining techniques or post-gel staining techniques such as Coomassie blue staining. The movement of the protein band on the gel is a function of the molecular weight of the protein, and the increase in distance from the loading position is a function of the decrease in the molecular weight. The band on the gel containing the N-terminal Cys-labeled protein exhibits fluorescence when excited at the appropriate wavelength. These bands can be detected with the naked eye, photographic or spectroscopically, and the protein is purified from the gel section if desired.

The molecular weight and amount of a protein can be determined by comparing the band position on the gel with a set of bands of labeled (eg, fluorescently labeled) protein of a given molecular weight. For example, in known amounts and known molecular weights (bovine serum albumin, 66 kD; porcine heart fumarase, 48.5 kD; carbonic anhydrase, 29 kD; β-lactoglobulin, 18.4 kD; α-lactoglobulin, 14.2 kD; sigma chemicals. A protein with a molecular weight of 25,000 can be determined from its relative position on the gel compared to a calibration gel containing a standard commercially available marker protein with (St. Louis, Missouri). The calibration protein can contain similar moities for convenience of detection using the same method as proteins with moities. This can be accomplished in many cases by directly reacting the calibration protein with a molecule that is similar or identical to Moyetti. Thus, one or more calibration proteins (protein markers), such as those selected based on pI or molecular weight, should be labeled by the methods of the invention, for example, using fluorescein, rhodamine, BODIPY or infrared type moisture. Can be arbitrarily isolated.

For example, calibration proteins can be modified with Dancil Chloride, or NHS-modified BODIPY FL, to obtain their fluorescent equivalents. These fluorescent proteins can be analyzed using PAGE. Detection in combination of these fluorescence calibration proteins with sample proteins containing fluorescence moitty can accurately determine both the molecular weight and amount of the synthesized protein. If necessary, the amount of moisture within each calibration and protein can be determined for accurate quantification. A protein having a predetermined level of fluorescence moyity can be advantageously used to quantify a sample protein having moyity.

Other methods of protein separation, including capillary electrophoresis, isoelectric focusing, low pressure chromatography and high performance liquid chromatography or high performance liquid chromatography, are the detection of sample proteins containing moisture that can be detected by electromagnetic radiation. Also useful for subsequent isolation and purification. In these cases, the individual proteins are separated into fractions that can be analyzed individually by a fluorescence detector at the emission wavelength of Moyetti. Alternatively, online fluorescence detection can be used to detect the protein as it emerges from the column fractionation system. Fluorescence graphs as a function of retention time provide information for both the amount and purity of protein produced.

Use of labeled protein and exemplary detection methods Moiety-containing proteins prepared by the methods of the invention can detect the amount or presence of a particular protein, isolate the protein, facilitate high-throughput or low-throughput screening, protein-protein interactions, protein-DNA interactions or Detection of other protein-based interactions (eg, using protein microarrays that use moyetti to bind proteins to the array or detect bound proteins), promote protein immunogenicity (eg, detect proteins) The N-terminal of one or more proteins can be labeled with a hapten that promotes the production of antibodies against the protein and further promotes antigen purification prior to immunization), at specific cell locations or below the cellular level of the protein. Targeting (eg, a label that is a protein localization domain can target a protein to a nucleus, chloroplast or mitochondria, or to a specific cell, eg, via a liver-specific antibody), of the protein. Providing site-specific orientations (eg, ligands for motities are attached to semi-solid or solid surfaces, or to semi-solid or solid surfaces (such as glass), of any length of linker (eg, glass). Preparation of chimeric proteins, preparation of chimeric proteins containing, for example, polyethylene glycol (organic linkers such as "PEG"), reporter labels (eg, luciferase) and proteins of interest (eg, CYP450), preparation of protein markers, or on proteins. It is useful for any purpose including, but not limited to, mapping of proteins, antigen epitopes and binding sites of.

In addition, labeled proteins have uses in protein display technology and directional evolution. Ribosome display technology, nucleic acid-protein fusion display technology and phage display technology are widely used to study protein-protein interactions and directional evolution. In ribosome-related display technology, in vitro lysate expression systems are used for the production of mRNA-protein-ribosome complexes or mRNA-protein / cDNA-protein / DNA-protein fusion products (published US Patent Application No. 1). See 2001/0046680 and US Pat. No. 6,194,550; 6,207,446; and 5,922,545). N-terminal labeling of proteins using derivatives of the invention assists in the isolation, identification and selection of targets. This approach is also useful for detecting protein interactions in ribosome / nucleic acid-protein display-based protein microarrays. Therefore, the derivatives of the present invention are particularly useful for the isolation, characterization and identification of protein targets.

The use of in vitro lysate-based protein expression in phage display is described in published US Patent Application No. 2001/0029025. The cDNA / mRNA library is expressed in cell lysates along with the derivatives of the invention, and the cDNA-expressing phage display library is screened for proteins expressed in vitro. The interacting proteins can be easily identified by N-terminal labeling without the need for cloning steps. This approach is also useful for protein variant selection in directional evolution. In addition, labeled proteins synthesized in vitro using derivatives of the invention can be used, for example, with phage display-based protein microarray / bead technology to detect protein interactions involving phage display. Can be done. Multiplexing is also possible.

Another approach for directional evolution, in which in vitro transcription and translation in cell lysates is used for directional evolution of proteins, is described in published US Patent Application No. 2001/0039014. In this approach, N-terminal labeling can be used with derivatives of the invention to facilitate isolation, purification, and characterization of mutant proteins with improved function.

In addition, the labeled protein can be introduced into cells via, for example, endocytosis, permeabilization or microinjection.

Mass spectrometry measures the mass of a molecule. The use of mass spectrometry in biology is advancing rapidly and is applied in a variety of areas, including analysis of carbohydrates, proteins, nucleic acids, and biomolecular complexes. For example, the development of matrix-assisted laser desorption / ionization (MALDI) mass spectrometry (MS) has provided important tools for the analysis of biomolecules, including proteins, oligonucleotides and oligosaccharides. The success of this technique stems from its ability to determine the molecular weight of large biomolecules and non-covalent complexes (&gt; 500,000 Da) with high accuracy (0.01%) and high sensitivity (amount less than femtomolar). So far, it has been found to be applicable in a variety of areas of biology and medicine, including rapid DNA sequencing, screening for bioactive peptides and analysis of membrane proteins.

Surface plasmon resonance (SPR) can be used to study protein / protein interactions. SPR is based on changes in optical properties, especially the index of refraction of the surface after bonding. This change (which can be measured very accurately) can then be used to detect both the degree and rate of binding. For example, incident light that hits the back surface of a thin gold layer having a ligand monolayer on the front surface at a variable angle penetrates the ligand monolayer. The interaction is caused by surface plasmons and the reflected light is reduced to a minimum due to plasmon resonance at a particular angle. The smallest position is detected and the refractive index can be calculated. Binding of molecules by ligands changes the index of refraction. Measurement of protein adsorption on polymer surface, protein binding to DNA, protein interaction with self-assembled monolayer on gold surface, protein interaction with phospholipid layer, and antibody-antigen interaction SPR has been used to do this. For example, the sensor chip of the carboxymethylated dextran matrix (Biocore) is preimmobilized with streptavidin. The biotin-labeled protein prepared by the method of the invention was contacted with one or more proteins to determine the optical properties before and after contact.

Electrophoresis can be used to detect and / or isolate proteins, or to detect protein-molecule interactions translated in a translation system. Many proteins can interact with multiple protein partners at the same time. For example, a protein can have proteins that interact within up to 86 cells. The use of labeled proteins in combination with electrophoresis (eg, labeled with affinity labels, fluorophore labels, luminescent labels or bioluminescent labels) involves an infinite number of concurrent protein-protein interactions and / or protein-nucleic acid interactions. Allows observation of action.

Therefore, the methods of the invention provide a very rapid screen for possible interactions with proteins expressing a specific gene, even if the protein has not been isolated or its function has not been identified. Allows for a large number of molecules. It also enables a library of proteins expressed by a pool of genes that will be rapidly screened for interactions with molecules (eg, compounds) without the need to isolate the protein. For example, a library of molecules can be screened to identify those that act as ligands for specific target proteins. The molecule can be part of a combinatorial library of compounds or can be present in a complex biological mixture such as a natural sample that can contain a therapeutic compound. Molecules can interact with nascent proteins by binding, or can cause changes in the structure or other properties of nascent proteins by chemical or enzymatic modifications.

The interaction of a specific molecule can be determined by comparing the presence or absence of the nascent protein exposed to the specific molecule with a similar analysis or measurement of the unexposed nascent protein. The binding strength of the molecule is then measured by changing the concentration of the specific molecule added to the protein synthesis system and by measuring the relative strength of the uncomplexed nascent protein and the band assigned to the complexed nascent protein. , Can be confirmed. Detection and / or isolation of complexed or uncomplexed proteins involves capillary electrophoresis (CE), in addition to gel electrophoresis (which measures the degree of electrophoresis of proteins in gels such as polyacrylamide gels). (See, for example, US Pat. No. 5,571,680 (Chen)). CE measures the migration time of protein electrophoresis, which is proportional to the charge-to-mass ratio of a molecule.

In one embodiment, the various labels are introduced into two or more proteins of interest, eg, at the N-terminus of each protein, or inside the N-terminus of one protein and the other protein. For example, each different protein contains a label that emits light at a wavelength different from that on the various proteins. The two labeled proteins are mixed under favorable conditions for binding. The binding mixture is then subjected to CE and added to the complex of the two proteins to detect uncomplexed proteins. In addition, labeling a potential ligand for a protein of interest with a second label (sensitive to the proximity of the first label) (eg, using FRET, BRET or LRET) can be used. Allows detection of the proximity of two markers.

One form of CE (sometimes called affinity capillary electrophoresis) is highly sensitive to protein interactions with other molecules, including small ligands, as long as the binding results in a change in the charge-to-mass ratio of the protein after the binding event. It has been found that there is. For example, nascent protein-antibody interactions can be detected due to changes in the effective electrophoresis degree of the formed complex. However, the highest sensitivity can be obtained if the protein has a label with specifically detectable electromagnetic spectral properties, such as a fluorescent dye. Detection of peaks in electrophoretic chromatograms is primarily carried out by laser-induced radiation of visible light. Specific examples of fluorescent dyes useful for CE include Fluorescein, Rhodamine, Texas Red and BODIPY.

Interactions that result in modification of the labeled protein, including but not limited to phosphorylation, proteolysis, and glycosylation, in addition to the interactions involved in the binding of one or more molecules to the labeled protein, use electrophoresis. Can be detected.

To determine the concentration of the protein of interest in the sample, the sample can be mixed with the corresponding labeled protein of interest and the protein that binds to both the labeled protein of interest and the unlabeled protein of interest. .. The mixture can then be subjected to CE to determine the concentration of the protein of interest (see, eg, US Pat. No. 5,571,680 (Chen)). As in "sandwich" ELISA, this technique is also used, for example, through capture with antibodies specific for cyanobenzothiazole, eg, using a technique similar to ELISA, for capture of labeled and / or unlabeled proteins. can do. See Schuurs and van Weemen, J. Immunoassay 1980; 1: 229-49 for specific examples of ELISA "sandwich" test procedures.

General synthesis method Labeling and detectable moities (eg, covalently linked to cyanobenzothiazole or derivatives thereof) allow immediate detection of the molecule in a complex mixture after reaction with the peptide of interest. The label can be added to the cyanobenzothiazole core by chemical synthesis according to the techniques described herein or by techniques well known to those of skill in the art. For example, the attachment of a fluorescent label or other label onto a core molecule can be accomplished by chemical modification. See Greg T. Hermanson, Bioconjugate Techniques, Academic Press, San Diego, California (1996). For additional information on common synthetic methods that can be used to prepare the compounds described herein, see Michael B. et al. March's Advanced Organic Chemistry Reactions, Mechanisms, and Structure by Smith and Jerry March, 5th Edition, John Wiley and Sons; and Wuts et al. (1999), Protective Groups in Organic Synthesis, 3rd Edition, John. It can be found throughout Wiley and Sun.

The method of preparing the compounds of the present invention can give rise to isomers in certain embodiments. The methods of the present invention do not always require the separation of these isomers, but if desired, such separation can be carried out by methods known in the art. For example, preparative high performance liquid chromatography can be used for isomer purification, for example by the use of columns with chiral packing.

Common methods of linking cyanobenzothiazole to linking group Y to form compounds of formula I are typically well known in the art. Such "coupling" or "coupling" reactions are standard techniques. Techniques used to couple linking groups to various benzothiazole derivatives can be found in standard handbooks such as Hermanson's Bioconjugate Techniques. Of course, those skilled in the art will not only react between the appropriate cyanobenzothiazole and the group YX, but also the appropriately functionalized group X (such as group X with the appropriate proelectron or nucleophilic molecule). You will also recognize that the reaction between and the cyanobenzothiazole-Y group can also prepare compounds of formula I. For example, a primary hydroxyl group on a linking group can be converted to a leaving group such as a toluenesulfonyl group, which is then replaced with a deprotonating molecule (eg deprotonated 6'-hydroxycyanobenzothiazole). be able to. A specific example of forming a cyanobenzothiazole-Y group is Zhou (J. Amer. Chem. Soc. 2006, 128 (10), 3122).

A number of succinimidyl esters useful for the preparation of compounds of formula I are commercially available, for example, from Invitrogen. In addition, one of ordinary skill in the art can generally use reagents and conditions for the preparation of succinimidyl esters. Hermanson's Bioconjugate Techniques provides a broad description of ligation reactions that can be used in the preparation of compounds of formula I, in particular Part I (describes "functional targets" and "chemistry of reactive groups") (pages 1-416). ) Provided in. For example, a common reagent used to prepare succinimidyl esters is N-hydroxysuccinimide (NHS, J. Am. Chem. Soc., 86: 1839 (1964)), and carbodiimide activators such as dicyclohexyl-carbodiimide (DCC) or 1,3-dimethylaminoprproply-ethylcarbodiimide. Alternatively, the "self-activated" NHS derivative is N-trifluoroacetyl-succinimide ("TFA-NHS"), N, N-discusin imidazole carbonate (Tetrahedron Lett., 22: 4817 (1981)), or O- (N-succinimidel) -N, N, N', N'-bis (tetramethylene) uranium hexafluorophosphate and the like can be used. Depending on the reactivity and solubility of the benzothiazole or the activated linking group, the conditions can range from organic solvents to aqueous solvents. For example, a suitable organic solvent can be dimethylformamide (DMF). These reactions can be carried out in the presence of bases such as hindered amine bases (eg, triethylamine, diethylisopropylamine), but aqueous conditions can include adjusting the pH to the range of about 6.5 to about 8.5.

If the group XY contains amines, along with cyclic nucleotides, nucleic acids or many chemotherapeutic agents and proteins, the succinimidyl ester of the next group can be used in the coupling reaction. If group X contains an acid, along with many citrates, proteins, chemotherapeutic agents and avidin, then the acid is converted to succinimidyl ester and the amine terminal Y previously linked to cyanobenzothiazole. Can be combined with a group. Other active groups such as sulfosuccinimidyl esters, tetrafluorophenyl esters, sulfodichlorophenol esters, isothiocyanates, sulfonyl chlorides, dichlorotriazines, aryl halides, or acyl azides can be linked to amines in succinimidyl esters. Can be used instead. In addition, one of ordinary skill in the art can readily convert a particular organic moist to a suitable amine or acid using standard transformations, including oxidation, reduction and substitution reactions. In addition, protecting groups can be used to simplify the preparation of specific compounds of formula I. The use of protecting groups is well known in the art (see, eg, Greene, Protecting Groups In Organic Synthesis; Wily: New York, 1981).

The following examples are intended to illustrate the invention described above, and should not be construed to narrow its scope. Those skilled in the art will readily recognize that the examples suggest many other methods in which the present invention may be practiced. It is understood that many modifications and modifications can be made if they remain within the scope of the present invention.

<p num="0122">Example 1 Preparation of cyanobenzothiazole derivatives Part A. 4- (3- (2-Cyanobenzo [d] thiazole-6-yloxy) propylcarbamoyl) -2- (3- (dimethylamino) -6- (dimethyliminio) -6H-xanthene-9-yl) benzoart Synthesis of; "2-cyano- (6-oxopropylamide tetramethyl-5'-carboxyrhodamine) benzothiazole" (Compound 3028):<chemistry num="4"><img id="000005" he="42" wi="104" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry></p><p num="0123">Method A: 100 mg of 6- (N-Boc-3-aminopropyloxy) -2-cyano-benzothiazole (or "tert-butyl 3- (or" tert-butyl3- (") in dichloromethane (1 mL), trifluoroacetic acid (1 mL) and anisole (250 μL) A flask containing "2-cyanobenzo [d] thiazole-6-yloxy) propylcarbamate" (W. Zhou, J. Amer. Chem. Soc. 2006, 128 (10), 3122)) is stirred at 0 ° C. did. The solvent was evaporated after 2 hours. Ether (2 mL) was added to precipitate the product. The white solid was washed twice with 2 mL diether ether and dried under vacuum. The solid was used without further purification.</p><p num="0124"> To the flask containing the solid described above was added 6-TAMRA SE (138 mg, 0.3 mmol, 1 eq) dissolved in 1 mL DMF and DIPEA (50 μL). After 24 hours at room temperature, the solvent was removed under reduced pressure. The residue was eluted through silica with 90% heptane / 10% methanol eluent. Appropriate fractions were combined and evaporated. The film was dissolved in 1 mL of acetone and precipitated with 6 mL of diethyl ether to give 10 mg of solid compound 3028.¹1 H NMR (300 MHz, DMSO) δ 8.78 (t, 1H, J = 5.6), 8.20 (td, 2H, J = 4.1, 8.3), 8.03 (d, 1H, J = 9.0), 7.78 (s, 2H) , 7.20 (dd, 1H, J = 2.5, 9.1), 6.92 (d, 5H, J = 23.2), 4.09 (t, 2H, J = 5.9), 3.41 (dd, 3H, J = 6.1, 11.9), 3.19 (s, 12H), 2.43 (d, 10H, J = 1.7), 1.98 (dd, 2H, J = 6.0, 12.1).</p><p num="0125"> Alternatively, the compound can be purified by preparative reverse phase HPLC. Similar compounds with fluorescein, Alexa 633, biotin and IC-5 labels are suitable FAM-SE (Sigma), Biotin SE (Sigma), Alexa-633-SE (Invitrogen) or IC. Synthesized using method A, replacing TAMRA-SE with -5-SE (Biosearch Technologies, catalog number FC-1065S-25). Cyanbenzothiazole derivatives linked to other groups of interest, including reporter moyets, affinity labels, quencher moyets, photocrosslinked moyets, or solid supports, as will be readily recognized by those of skill in the art. A similar technique can be used to prepare the.</p><p num="0126">Part B. The following compounds were synthesized using Method A, which utilizes the appropriate 5,6FAM-SE instead of 6-TAMARA SE, or Bodipy488-SE, biotin-SE, or IC-5-SE. 4 (and 5)-(3- (2-cyanobenzo [d] thiazole-6-yloxy) propylcarbamoyl) -2- (3-hydroxy-6-oxo-6H-xanthene-9yl) benzoic acid (eg, compound 3066) ):<chemistry num="5"><img id="000006" he="43" wi="85" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry> Mixture of isomers (66%: 35%);¹1 H NMR (300 MHz, DMSO) δ 10.12 (s), 8.90 (t), 8.76 (t), 8.44 (d), 8.22 (dd), 8.11 (m), 7.89 (d), 7.82 (d), 7.66 (s), 7.33 (m), 7.21 (dd), 6.66 (d), 6.54 (m), 4.18 (t), 4.08 (t), 3.50 (dd), 3.37 (t), 2.07 (m), 1.97 (m), 1.22 (s), 0.83 (t) .C₃₂H₂₁N₃O₇MS for S: Calculated 592.1; Measured 592.</p><p num="0127">(Z) -N- (3- (2-cyanobenzo [d] thiazole-6-yloxy) propyl) -3- (1- (difluoroboryl) -5-((3,5-dimethyl-2H-pyrrole-2) -Ilidene) Methyl) -1H-Pyrrole-2-yl) Propanamide (Compound 3226):<chemistry num="6"><img id="000007" he="33" wi="97" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>¹1 H NMR (300 MHz, DMSO) δ 8.17 (d, 1H, J = 9.1), 8.07 (t, 1H, J = 5.7), 7.87 (d, 1H, J = 2.5), 7.67 (s, 1H), 7.35 (dd, 1H, J = 2.5, 9.1), 7.08 (d, 1H, J = 3.9), 6.56 (s, 0H), 6.39 (d, 1H, J = 4.0), 6.33 (s, 1H), 4.12 ( t, 2H, J = 6.3), 3.30 (dd, 2H, J = 6.4, 12.2), 3.12 (t, 2H, J = 7.5), 2.50 (s, 3H), 2.28 (s, 3H), 1.95 (p) , 2H, J = 6.4). C₂₅H₂₄BF₂N₅O₂MS calculated value 508 for S; measured value 507.</p><p num="0128">N- (3- (2-Cyanobenzo [d] thiazole-6-yloxy) propyl) -5-((3aS, 4S, 6aR) -2-oxo-hexahydro-1H-thieno [3,4-d] imidazole- 4-yl) Pentaneamide (Compound 3167):<chemistry num="7"><img id="000008" he="30" wi="113" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>¹1 H NMR (300 MHz, DMSO) δ 8.18 (d, 1H, J = 9.1), 7.92 (dd, 2H, J = 4.1, 6.2), 7.36 (dd, 1H, J = 2.6, 9.1), 6.44 (s, 2H), 4.32 (dd, 1H, J = 4.4, 7.7), 4.14 (m, 3H), 3.26 (q, 2H, J = 6.5), 3.10 (m, 1H), 2.83 (dd, 1H, J = 5.1) , 12.4), 2.60 (d, 1H, J = 12.3), 2.10 (t, 2H, J = 7.3), 1.94 (t, 2H, J = 6.4), 1.52 (m, 4H), 1.33 (m, 2H) . C₂₁H₂₅N₅O₃S₂MS calculated value 460.1; measured value 460.4.</p><p num="0129">2-((1E, 3E, 5E) -5- (1- (6- (3- (2-cyanobenzo [d] thiazole-6-yloxy) propylamino) -6-oxohexyl) -3,3-dimethyl Indoline-2-iriden) Penta-1,3-dienyl) -1-ethyl-3,3-dimethyl-3H-Indolium chloride (Compound 3272):<chemistry num="8"><img id="000009" he="40" wi="130" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>¹1 H NMR (300 MHz, DMSO) δ 8.31 (t, 2H, J = 13.1), 8.11 (d, 1H, J = 9.1), 7.84 (m, 2H), 7.60 (d, 2H, J = 7.0), 7.29 (m, 6H), 6.55 (t, 1H, J = 12.3), 6.26 (dd, 2H, J = 4.0, 13.8), 4.08 (m, 7H), 3.84 (s, 15H), 3.54 (s, 0H) , 3.19 (d, 2H, J = 5.9), 2.49 (dt, 4H, J = 1.8, 3.7), 2.30 (s, 0H), 2.05 (m, 2H), 1.85 (m, 2H), 1.66 (d, 12H, J = 2.8), 1.52 (dd, 2H, J = 7.3, 14.7), 1.33 (dd, 2H, J = 7.3, 14.9), 1.24 (t, 3H, J = 7.1). C₄₄H₅₀N₅O₂S⁺MS calculated value 712.4; measured value 712.</p><p num="0130">Part C. 4- (6- (2-Cyano-5-fluorobenzo [d] thiazole-6-yloxy) hexylcarbamoyl) -2- (3- (dimethylamino) -6- (dimethyliminio) -6H-xanthene-9 -Il) Synthesis of benzoate (Compound 3086):<chemistry num="9"><img id="000010" he="42" wi="123" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry></p><p num="0131"> In a microwave oven 5-fluoro-6-hydroxybenzo [d] thiazole-2-carbonitrile (200 mg) with acetone (2 mL), potassium carbonate (284 mg) and tert-butyl 6-bromohexyl carbamate (265 μL) Heated to 65 ° C for 40 minutes at 50 W. Then another 150 μL of tert-butyl 6-bromohexyl carbamate was added and the reaction was heated at 75 W for 23 minutes to 80 ° C. The reaction was partitioned between ethyl acetate and bicarbonate, washed with citric acid water and brine and evaporated. The crude product was eluted through a silica column with a mixture of heptane: ethyl acetate (3: 1). Yield 78%.</p><p num="0132"> Cooling tert-butyl 6- (2-cyano-5-fluorobenzo [d] thiazole-6-yloxy) hexyl carbamate (200 mg) with dichloromethane (3 mL), trifluoroacetic acid (3 mL) and anisole (300 μL) ( 0 ° C) Add to solution. After 15 minutes most of the solvent evaporated and 30 mL of diethyl ether was added. The precipitate was isolated (165 mg).</p><p num="0133"> 6- (6-Aminohexyloxy) -5-fluorobenzo [d] thiazole-2-carbonitrile (50 mg) was stirred with 6-TAMRA-SE (65 mg) as in Method A above. Yield 10 mg.¹1 H NMR (300 MHz, DMSO) δ 8.67 (t, 1H, J = 5.8), 8.17 (q, 2H, J = 8.2), 8.05 (dd, 2H, J = 9.7, 21.1), 7.78 (s, 1H) , 6.90 (d, 5H, J = 26.3), 4.08 (t, 2H, J = 6.4), 3.17 (s, 11H), 1.73 (m, 2H), 1.48 (m, 2H), 1.35 (s, 4H) .C₃₉H₃₆FN₅O₅MS calculated value 706.2 for S; measured value 706.</p><p num="0134">Part D. 4- (6- (2-Cyano-7-nitrobenzo [d] thiazole-6-yloxy) hexylcarbamoyl) -2- (3- (dimethylamino) -6- (dimethyliminio) -6H-xanthene-9- Il) Synthesis of benzoate (Compound 3087):<chemistry num="10"><img id="000011" he="45" wi="122" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry></p><p num="0135"> 6-Hydroxybenzo [d] thiazole-2-carbonitrile (352 mg), ZrO (NO)₃)₂× H₂Heated in a microwave oven at 100 ° C (200 W) for 10 minutes with O (462 mg) and acetone (7 mL). The product was extracted with dichloromethane and eluted with heptane: ethyl acetate (1: 1) through silica. Yield 222 mg</p><p num="0136"> 6-Hydroxy-7-nitrobenzo [d] thiazole-2-carbonitrile (100 mg) in a microwave oven with acetone (2 mL), potassium carbonate (125 mg) and tert-butyl 6-bromohexyl carbamate (139 mg). It was heated to 70 ° C for 30 minutes at 50 W. Then another 150 μL of tert-butyl 6-bromohexyl carbamate was added and the reaction was heated at 75 W for 30 minutes to 80 ° C. Then an additional 300 μL of tert-butyl 6-bromohexyl carbamate, cesium carbonate (162 mg) and diglyme (1 mL) were added and the reaction was heated to 100 ° C. at 75 W for 250 minutes. The reaction was partitioned between ethyl acetate and bicarbonate, washed with citric acid water and brine and evaporated. The crude product was eluted through a silica column with a mixture of heptane: ethyl acetate (2: 1). Yield 44%.</p><p num="0137"> Cooling (0) of tert-butyl 6- (2-cyano-7-nitrobenzo [d] thiazole-6-yloxy) hexyl carbamate (50 mg) with dichloromethane (1 mL), trifluoroacetic acid (1 mL) and anisole (99 μL). ° C) Added to solution. After 30 minutes, most of the solvent evaporated and 30 mL of diethyl ether was added. The precipitate was isolated and used without further purification.</p><p num="0138"> 6- (6-Aminohexyloxy) -7-nitrobenzo [d] thiazole-2-carbonitrile (51 mg) was stirred with 6-TAMRA-SE (50 mg) as in Method A above. Yield 13 mg¹1 H NMR (300 MHz, DMSO) δ 8.72 (t, 1H), 8.58 (d, 1H, J = 7.3), 8.22 (d, 2H, J = 8.0), 7.83 (s, 2H), 6.98 (d, 5H) ), 4.39 (t, 2H), 3.24 (s, 15H), 1.82 (m, 2H), 1.52 (m, 4H), 1.40 (m, 2H). C₃₉H₃₆N₆O₇MS calculated value 733.2 for S; measured value 733.6.</p><p num="0139">Part E. 4- (6- (2-Cyanobenzo [d] thiazole-6-ylamino) -6-oxohexylcarbamoyl) -2- (3- (dimethylamino) -6- (dimethyliminio) -6H-xanthene-9- Il) Synthesis of benzoate (Compound 3082):<chemistry num="11"><img id="000012" he="42" wi="120" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry></p><p num="0140"> 6- (tert-Butyloxycarbonylamino) hexanoic acid (316 mg), anhydrous THF (10 mL), 6-aminobenzo [d] thiazole-2-carbonitrile (200 mg), iso-butylchloroformate (193 μL), and N. -Mixed with methylmorpholine (314 μL) at -4 ° C. The reaction was allowed to stand overnight at room temperature. The reaction was partitioned between ethyl acetate and bicarbonate. The ethyl acetate layer was evaporated and the residue was eluted through silica with heptane: ethyl acetate (1: 2). Yield 354 mg.</p><p num="0141"> Cooling (0) of tert-butyl 6- (2-cyanobenzo [d] thiazole-6-ylamino) -6-oxohexyl carbamate (350 mg) with dichloromethane (4 mL), trifluoroacetic acid (4 mL) and anisole (400 μL). ° C) Added to solution. After 135 minutes, most of the solvent evaporated and 10 mL of acetonitrile and 30 mL of diethyl ether were added. The mixture was left overnight. The precipitate was isolated and used without further purification.</p><p num="0142"> 6-Amino-N- (2-cyanobenzo [d] thiazole-6-yl) hexaneamide (100 mg) was stirred with 6-TAMRA-SE (122 mg) as in Method A above. Yield (22 mg).¹1 H NMR (300 MHz, DMSO) δ 10.31 (s, 1H), 8.67 (dd, 2H, J = 3.8, 7.2), 8.12 (m, 3H), 7.77 (s, 1H), 7.63 (dd, 1H, J = 2.1, 9.1), 3.17 (s, 14H), 2.31 (t, 2H, J = 7.4), 1.57 (m, 2H), 1.47 (m, 2H), 1.31 (m, 2H) .C₃₉H₃₆N₆O₅MS for S: Calculated: 701.2; Measured 701.6.</p><p num="0143">Example 2 Synthesis of (E) -N- (2- (2-amino-3-mercaptopropaneamide) ethyl) -4-((4- (dimethylamino) phenyl) diazenyl) benzamide (Compound 3191):<chemistry num="12"><img id="000013" he="36" wi="99" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry></p><p num="0144"> (E) -2,5-dioxopyrrolidine-1-yl 4-((4- (dimethylamino) phenyl) diazenyl) -benzoate (200 mg), dimethylformamide (5 mL), tert-butyl 1- (2) -Mixed with aminoethylamino) -1-oxo-3- (tritilthio) propan-2-ylcarbamate (333 mg) and diisopropylethylamine (285 μL). After 12 hours, the reaction was partitioned between ethyl acetate and aqueous citric acid. The organic layer was washed with bicarbonate and then brine. After evaporation, the residue was eluted through silica with heptane: ethyl acetate (1: 1). Yield: 242 mg.</p><p num="0145"> (E) -tert-Butyl 1-(2- (4-((4- (dimethylamino) phenyl) diazenyl) benzamide) -ethylamino) -1-oxo-3- (tritylthio) propan-2-ylcarbamate (240 mg) was added to a cooling solution of trifluoroacetic acid (10 mL), water (500 μL) and triisopropylsilane (100 μL). After 5 hours, diethyl ether (50 mL) was added to the precipitate product to give compound 3191. The product was further purified by preparative reverse phase HPLC. Yield 60 mg.¹1 H NMR (300 MHz, DMSO) δ 8.56 (m, 2H), 8.18 (s, 3H), 7.92 (d, 2H, J = 8.5), 7.75 (d, 4H, J = 8.3), 6.78 (d, 2H) , J = 8.2), 3.85 (m, 1H), 3.35 (m, 3H), 3.20 (m, 1H), 3.01 (s, 6H), 2.85 (m, 2H), 2.50 (s, 1H).</p><p num="0146">Example 3 N-terminal peptide labeling with cyanobenzothiazole derivatives In this example, the cyanobenzothiazole-rhodamine reagent is added to a solution containing various concentrations of cysteine residue-containing material. The solution is incubated with the cyanobenzothiazole-rhodamine reagent under conditions that allow the formation of adducts between the N-terminal cysteine residue and the reagent. After incubation with the reagent, the presence of the newly labeled species is detected by fractionation of a portion of the reaction mixture on a silica thin layer chromatography (TLC) plate and examination of the presence of fluorescent species.</p><p num="0147"> Five different materials containing cysteine residues were reacted with the cyanobenzothiazole-rhodamine reagent. Tocinic acid (Sigma) is a peptide containing amino-terminal cysteine residues and carboxy-terminal cysteine residues linked by disulfide bonds (peptide sequence: Cys-Tyr-Ile-Gln-Asn-Cys) (SEQ ID NO: 7). ). To reduce disulfide bonds in peptides, 50 μl of 1 mM tocinic acid, 5 μl of 1 M hepes (pH 8.0) and 0.25 μl of bond breaker (Pierce) Chemical) and 46 μl of water were mixed. To produce an oxidized tocinic acid solution, 50 μL of tocinic acid (1 mM) was mixed with 5 μL of 1 M Hepes (pH 8.0) and 45 μl of water. Bachem H4696 (Bachem Biosciences, King of Prasha, Pennsylvania) is a peptide containing an internal cysteine residue (Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys) (SEQ ID NO: : 8). A solution of Buckem H4696 was made by mixing 48 μl of 5 mM Buckem H4696 with 0.25 μl of bond breaker and 1 μl of Hepes (pH 8.0). Buckem H4702 is a peptide containing an N-terminal cysteine residue (Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr) (SEQ ID NO: 9). A solution of Buckem H4702 was made by mixing 48 μl of 5 mM Buckem H4702 with 0.25 μl of bond breaker and 1 μl of 1 M Hepes (pH 8.0). An implementation solution of 10 mM Cys-Gly dipeptide (Sigma; 15 mg) and 20 mM cysteine (Sigma) was also prepared.</p><p num="0148"> Reaction buffer for the cyanobenzothiazole labeling reaction was made by mixing 500 μL of 1 M Hepes (pH 7.5) with 7.5 mL of water. Six sets of reactions were constructed to allow testing of various molar ratios of reagents to peptides. Each set contained 6 reactions involving different cysteine materials. Peptide: The relative molar ratio of the reagents tested varied from 0.3: 1 to 2: 1. 75 μl of reaction buffer was added to all reactions. Different amounts of water were added to each reaction in each set. That is, 16.7 μl in the first set, 13.3 μl in the second set, 10 μl in the third set, 6.7 μl in the fourth set and 3.3 μl in the fifth set. The sixth set of reaction tubes did not add water, as was the highest ratio of cysteine material to the cyanobenzothiazole reagent. One reaction tube was constructed as a peptide-free control with 20 μl of water added.</p><p num="0149"> Cysteine (2.5 μl of 20 mM cysteine made into 100 μl with water), Buckem H4696 (10 μl of the above solution made into 100 μl with water), Buckem H4702 (10 μl of the above solution made into 100 μl with water), and Cys- A diluted solution of Gly (5 μl of the above solution made 100 μl with water) was added to different reaction tubes to give a final reaction volume of 95 μl for each of the 6 sets. Oxidized tocinic acid and reduced tocinic acid were also added to different reaction tubes to bring the final reaction volume of each of the 6 sets to 95 μl. Overall, each of the 6 sets of reaction tubes contained a different cysteine solution.</p><p num="0150"> To all reaction tubes, 5 μl of 1 mM cyanobenzothiazole-rhodamine reagent was added and mixed. After a 15 minute incubation at room temperature, 1 μl from each reaction tube was spotted on a silica gel TLC plate. Plates were developed in a mixture of 90 volumes of EtOH, 10 volumes of water and 1 volume of glacial acetic acid. After development, the plate was air-dried and visualized under ultraviolet light to confirm that the reaction had progressed and was completed.</p><p num="0151"> Peptide-free control reactions containing only buffer and cyanobenzothiazole-rhodamine reagent produced strong fluorescent spots at Rf values of ~ 0.8 and weak spots at Rf values of ~ 0.4. This identifies the mobility of the original unreacted cyanobenzothiazole reagent and demonstrates that the reagent is not affected by the reaction conditions.</p><p num="0152"> Figure 3 shows an Ambis imaging system that detects fluorescence emission from fluorescent species present on a TLC plate when exposed to UV light and collected through a filter that blocks the UV light present on the imaging camera. An image of a thin layer chromatography (TLC) plate captured on the set is shown. Reactions with increased cysteines (lanes 2-7) show the presence of new strong fluorescent adducts with a mobility of Rf values of ~ 0.42, along with the presence of small amounts of other species (adducts). The amount of new fluorescent species increases until the molar amounts of cysteine and benzothiazole are approximately equal (lanes 4-5). This is because 1) the reaction between cysteine and the cyanobenzothiazole-rhodamine reagent is rapid (~ 15 minutes), and 2) about 1 molar equivalent of cysteine is required to react with 1 mol of the cyanobenzo-thiazole reagent. Demonstrate what is required for the reaction.</p><p num="0153"> Reactions with increased doses of Buckem H4696 (peptide with internal cysteine) (lanes 8-13) showed essentially the same (lane 1) fluorescence mobility patterns as those seen with the cyanobenzothiazole reagent alone. .. This is because either 1) the internal cysteine cannot react with the cyanobenzothiazole reagent, 2) a large amount of peptide is required to react with the cyanobenzothiazole reagent, or 3) the adduct formed. Demonstrate that either unstable and returning to the starting material, rapidly making adduct detection almost impossible, is most likely.</p><p num="0154"> In contrast, reactions with increased amounts of Buckem H4702 (a peptide containing N-terminal cysteine) showed significant amounts of very low mobility adducts (lanes 14-19). The amount of new adduct formation increased and the corresponding unreacted cyanobenzothiazole reagent decreased. Therefore, the cyanobenzothiazole reagent can form a stable adduct with a peptide having an N-terminal cysteine. This is most likely caused by the formation of cyclic benzothiazole products through the attack of N-terminal amino groups on amino acids, similar to that seen in the formation of luciferin through reaction with cysteine. However, if the N-terminal cysteine group is involved in a disulfide bond, as in the reaction with oxidized tocinic acid (lanes 20-25), no new adduct is formed and the unreacted cyanobenzothiazole reagent is reduced. Does not occur even if the amount of peptide is increased.</p><p num="0155"> If the disulfide bond in tocinic acid is reduced to free cysteine (reduced tocinic acid described above), the resulting N-terminal cysteine is available for reaction with the cyanobenzothiazole reagent (lanes 26-31). New low mobility adducts are formed. The new adduct is formed in a manner similar to that seen in the cysteine reaction, i.e. the amount of adduct formed depends on the ratio of 1 molar equivalent of reduced tocinic acid to 1 molar cyanobenzothiazole reagent. is there. Reactions with the dipeptide Cys-Gly (lanes 32-37) also demonstrate that the N-terminal cysteine is rapidly labeled with the cyanobenzothiazole reagent, even in small peptides.</p><p num="0156"> Thus, this example demonstrates that the cyanobenzothiazole reagent can easily label the available N-terminal cysteine-containing peptides, large or small.</p><p num="0157">Example 4 Labeling proteins with cyanobenzothiazole reagents In this example, proteins with N-terminal cysteine residues were labeled with the compounds of the invention. Comparison of labeled amounts to other proteins in solution and to parallel reactions in which the target protein was replaced with a protein that was identical to the target protein except that the N-terminal cysteine residue was replaced with an alanine residue. did. The examples demonstrate that: 1) Fusion protein constructs can be constructed such that when digested by TEV protease, the construct produces the protein of interest with N-terminal cysteine; Other proteins in the reaction that do not have an N-terminal cysteine residue, when exposed, acquire little or no fluorescence, but a protein that has an N-terminal cysteine residue (TEV of a well-designed fusion protein construct). Exposure to exposure to (such as those produced by protease digestion) becomes highly fluorescent, and; 3) a fusion construct digested to the same TEV as the one exposing the cysteine residue during TEV digestion (but it is an alanine residue). In parallel reactions containing), there is little or no labeling of the protein in the solution containing the protein that is identical to the protein with N-terminal cysteine but with the N-terminal cysteine replaced by alanine.</p><p num="0158"> To demonstrate these points, experiments (discussed in detail below) were performed in the following steps. A) Recombinant DNA clones are designed and constructed to express fusion proteins in Escherichia coli, i) N-terminal affinity protein tags of intact fusion constructs for rapid and easy purification of fusion protein species. Following [GST], it has ii) a protein sequence encoding a TEV protease recognition site, and iii) another protein segment. One construct exposes a cysteine residue at the new N-terminus of the protein produced by TEV cleavage of the fusion construct, while the other construct exposes two constructs that differ only in that they expose the alanine residue at the new N-terminus. Generated; B) expression of recombinant DNA in E. coli, and confirmation that the bacterium expressed the fusion protein; C) purification of the fusion protein from the E. coli lysate by using an affinity protein tag, and; D) fusion. Digestion of the construct, followed by exposure of both the intact and cleaved fusion proteins to PBI compound 3128 (see Figure 2 (a)), followed by analysis to detect labeling of the protein species in the reaction mixture.</p><p num="0159">Step A). Construction of recombinant DNA species encoding fusion protein pairs. A plasmid species encoding a prokaryotic promoter and translation initiation region, followed by an in-frame coding sequence for glutathione S transferase [GST, affinity protein tag] was designed. One version had a GST, followed by a coding sequence containing a recognition sequence for TEV protease in frame, followed by cysteine, followed by another protein in frame. The second plasmid species is identical to the plasmid described above, but designed to replace the cysteine encoded after the TEV site with alanine. The protein coding region of beetle luciferase is then fused in-frame with the end of the coding sequence encoding the TEV protease site so that there is one continuous coding region encoding all these polypeptide segments. It was.</p><p num="0160"> The plasmid was confirmed by DNA sequence analysis. The amino acid and nucleotide sequences of GST-Luc (Ala and Cys) are as follows.</p><p num="0161">Amino acid sequence GST- (TEV-Cys) -Luc (SEQ ID NO: 10)</p><p num="0162">Nucleotide sequence of GST- (TEV-Cys) -Luc atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttgaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgtttcaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatccggaggtggtggcggagaaaacctgtacttccaatgcatcgccATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCTCTAGAGGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTCCTCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTACGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTTCATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAAACGCTTCCATCTTCCAGGGATACGACAAGGATAT(SEQ ID NO: 11)11)11)11)11)</p><p num="0163">Amino acid sequence of GST- (TEV-Ala) -Luc (SEQ ID NO: 12)</p><p num="0164">Nucleotide sequence of GST- (TEV-Ala) -Luc atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttgaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgtttcaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatccggaggtggtggcggagaaaacctgtacttccaagcgatcgccATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCTCTAGAGGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTCCTCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTACGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTTCATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAAACGCTTCCATCTTCCAGGGATACGACAAGGATAT(SEQ ID NO: 13)13)13)13)13)</p><p num="0165">Process B). Expression of fusion proteins. Cultures transformed with the confirmed plasmid were grown and protein expression was induced. After growth of the culture, expression of expected size fusion protein production was confirmed by SDS PAGE fractionation of cell samples by Coomassie blue staining to detect protein bands. Expression of the fusion protein was estimated to be 1-5% of the total soluble protein in the cell lysate.</p><p num="0166">Process C). Purification of fusion proteins. After growth of the culture, cells were harvested by centrifugation and frozen at -20 ° C until ready for treatment. Once ready for purification, thaw the cell precipitate and re-sprinkle in buffer A (1 x PBS (pH 7.3), 1 mM PMSF, Roche complete protease tablet per 50 mL). The cells became turbid and the cells were resuspended in this buffer at a ratio of 8-10 mL of buffer per gram of cell paste. The cells were then sonicated and insoluble cell debris was precipitated by centrifugation of the lysed cells at 4 ° C for 10 minutes at 3900 x G.</p><p num="0167"> After centrifugation, the supernatant above the precipitate was carefully removed and applied to a column of glutathione sepharose (from GE Healthcare) equilibrated in 1 x PBS (pH 7.3). After application, the column is washed with 10-20 column volumes of 1 x PBS buffer (pH 7.3), then a solution containing 10-15 mM glutathione in 50 mM Tris HCl buffer (pH 8.0) is applied. The protein was eluted. Fractions of material eluted during this process were collected and small fractions were analyzed by SDS PAGE. As expected, the fusion protein was highly concentrated in the fraction where glutathione-containing column buffer was eluted from the column. A fraction of the highly concentrated fusion protein was pooled and dialyzed against 10 mM Hepes buffer (pH 7.5, 50 mM NaCl).</p><p num="0168">Process D). Digestion and labeling of fusion proteins. The protein concentration of the dialyzed fusion protein was determined by the use of Pierce's Coomassie Plus protein reagent as per the manufacturer's protocol. Equal amounts of a pair of protein constructs were diluted to ~ 1 μM in ProTEV protease buffer. ProTEV was added and the fusion protein was digested overnight at 4 ° C. The ProTEV buffer was 50 mM Hepes (pH 7.0), 0.5 mM EDTA, 1 mM DTT. Digested samples were analyzed by SDS PAGE. Fusion constructs with alanine after the TEV cleavage site and constructs with cysteine after the TEV cleavage site both have the emergence of new protein species (the size expected for cleavage of the construct at the TEV site) and intact fusion protein bands. Digested up to 90% or more based on the disappearance of.</p><p num="0169"> Two digest samples can then be placed in a fresh tube containing 10 mM hepes (pH 7.5) and a sample of PBI compound 3028 in stock of 2 mM acetonitrile (as in Figure 3; DMSO can also be used). In addition to produce PBI 3028 with a final concentration of 10 μM. When using DMSO as the solvent, a 6.5 mM stock solution was used. At set time intervals, samples of these labeling reactions were added to a new tube containing the reagent and reacted with the labeling reagent cyanobenzothiazole moyeti to terminate the labeling reaction. This solution contained cysteine HCl (final concentration 1-5 mM in the termination reaction) and equal concentrations of TCEP. It was even found to be effective even at 10-fold lower concentrations of TCEP. This stop solution may be prepared in ~ 200 mM Hepes (pH 8.0) to reduce acidity and inhibit protein precipitation. Previous studies have shown that the reaction of this solution with reagents results in the rapid conversion of labeled reagents to the desired species.</p><p num="0170"> After all timed samples were collected, the samples were analyzed by SDS PAGE electrophoresis and subsequently imaged by Typhoon. After imaging, the gel was stained with SimplyBlue SafeStain (Invitrogen) to visualize protein bands. Fluorescent scanning of gels before Coomassie staining and comparison of gel images obtained after Coomassie staining were performed (Coomassie-stained gels are not shown). The fluorescent gel image is shown in FIG. Cys in incubation of benzothiazole dye conjugates, as seen in fluorescent gel images The N-terminal protein partner became highly fluorescent, but other protein species in these reactions (visible on Coomassie-stained gels) were barely or completely unlabeled (digestion samples with Cys after TEV). Digestive sample with Ala after vsTEV). In addition, reactions in which cleavage by TEV protease resulted in a new protein species with amino-terminal alanine were not highly fluorescently labeled. Finally, if the fusion protein construct is not digested by TEV protease, little or no labeling is seen (uncleaved Ala and Cys versions of the fusion protein). Therefore, cysteine residues that allow the digested fusion protein construct to be strongly labeled are not highly labeled if exposed to reagents with internal cysteine residues. The large dark spot at the bottom right of the figure arises from the colored protein standard loaded into the lane.</p><p num="0171"> These observations demonstrate that there is at least very strong selectivity for labeling of N-terminal cysteine residues by compounds of the invention, if not completely specific labeling. In addition, the label is dependent on the N-terminus of the protein, which is a cysteine residue, which protein can be produced by digestion of the fusion protein construct with a protease.</p><p num="0172">Example 5 Confirmation of N-terminal labeling of fluorescently tagged protein with cyanobenzothiazole labeling reagent In this example, a highly specific fusion protein construct is labeled with various cyanobenzothiazole labeling reagents. The second time, the protein is exposed to a second site-specific protease that cleaves only a few amino acids from the new amino terminus of the TEV digestion protein. This cleavage allows testing of the specificity of the label achieved prior to the second digestion. Therefore, if the protein is labeled only at the new amino terminus, all fluorescence on the digested protein must be eliminated by the action of the second protease. However, if the protein is labeled at multiple sites, treatment of the labeled protein with a second protease produces a second protein species that is slightly smaller than the first product and yet highly fluorescent. Let's go.</p><p num="0173"> In the order of the GST-TEV protease site-Cys-Factor Xa protease site-Halotag (version 2) segment, a fusion construct with two protease cleavage sites between the two protein partners was produced. The protein was expressed in E. coli and purified by use of an affinity resin for the GST fusion protein (typically GE Healthcare Glutathione Sepharose 4 First Flow) according to the supplier's instructions.</p><p num="0174"> The purity of the isolated fusion protein was examined by SDS PAGE electrophoresis and found to contain a large amount of the desired full length protein. The protein was dialyzed and then digested with ProTEV protease. After cleavage, the digest sample was labeled with a cyanobenzothiazole labeling agent with different attached dye segments. Individual samples were labeled with a halotag TMR ligand, indicating that the halotag protein was well labeled within the protein sequence. For information on HaloTag® technology and the use of Halotag TMR ligands, see Technical Manual HaloTag (r) Technology: Focus on Imaging, Part Number TM260 (http://www.promega.com/tbs/tbs.htm;). Available from Promega), and M.M. Urh et al., "Halolink (tm) Resin For Protein Pull-Down And Analysis" Cell Notes 2006, 14, 15-19, (available from Promega at http://www.promega.com/cnotes/) reference.</p><p num="0175"> After labeling, samples of purified protein labeled with each drug were digested with factor Xa. After factor Xa digestion, samples of undigested labeled protein and factor Xa digested labeled protein were fractionated on SDS PAGE gels, which were imaged on a Typhoon imager. After imaging, the gel was stained with Coomassie Brilliant Blue to visualize the protein using a fluorescence-independent method of the protein. Fluorescent gel image and Coomassie-stained gel image are shown in FIG.</p><p num="0176"> As expected, the fusion construct is labeled with a halotag ligand (thus labeling the protein partner expected to be labeled with the cyanobenzothiazole reagent, but with a label approximately in the center of the protein segment) and factor Xa. When digested with, the labeled protein changed only slightly in size and remained fluorescent (first two lanes of gel in Figure 5). However, when digesting a sample labeled with a cyanobenzothiazole reagent, almost all of the fluorescence associated with the labeled protein band, except for very small amounts of fluorescence associated with the protein species with the mobility of the first labeled protein. It was removed from the protein (the pair of lanes illustrated in FIG. 5 for fluorescence loss are 3028 TMR, 3168 Alexa, and 3272).</p><p num="0177"> When this segment of the gel was stained with Kumashi, it was found that proteins slightly smaller than the labeled protein were abundant in the lane where the fusion protein was treated with factor Xa (bottom panel, Figure 5). .. Fluorescent labeling is on the few amino acids digested from the construct (the amino terminus of the labeled protein species) because this protein band is not fluorescent and results from the removal of only a few amino acids from the amino terminus of the labeled protein. It must have been.</p><p num="0178"> Figures 5, 6 and 7 illustrate gels representing cleavage results, as well as expected results from labeling only at the N-terminus and non-specific labeling at cysteine residues, respectively.</p><p num="0179"> If the cyanobenzothiazole-labeled reagent labels only the N-terminus, not the internal cysteine, as illustrated in FIG. 6, on the protein of interest to be removed by cleavage of the protein at the protease site downstream from the label. It is expected that there is a cyanobenzothiazole label. Internal labels such as the halotag ligand control were not removed by cleavage of the protein of the second protease. Fluorescence scans of SDS-PAGE gels on these samples show the disappearance of fluorescence from the cyanobenzothiazole-labeled band after cleavage of the second protease. The halotagligand-labeled protein shows a size shift, but remains fluorescent because the fluorescent label was not removed and is illustrated in the fluorescent gel illustration of FIG.</p><p num="0180"> If the cyanobenzothiazole-labeled reagent is attached to the internal cysteine as illustrated in FIG. 7, cleavage of the labeled protein of the second protease is of lower molecular weight (substantially similar to the halotag ligand-labeled control protein). It is expected to leave a fluorescent band (which appears to be), which is illustrated in the fluorescent gel illustration of FIG.</p><p num="0181">Amino acid sequence of GST- (TEV-Cys-FXa) -HaloTag MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSGGGGGENLYFQCIAMIEGRAMGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYLWRNIIPHVAPSHRCIAPDLIGMGKSDKPDLDYFFDDHVRYLDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIACMEFIRPIPTWDEWPEFARETFQAFRTADVGRELIIDQNAFIEGALPMGVVRPLTEVEMDHYREPFLKPVDREPLWRFPNELPIAGEPANIVALVEAYMNWLHQSPVPKLLFWGTPGVLIPPAEAARLAESLPNCKTVDIGPGLFLLQEDNPDLIGSEIARWLPGLV (SEQ ID NO: 14)</p><p num="0182">Nucleotide sequence of GST- (TEV-Cys-FXa) -HaloTag atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttgaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgtttcaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatccggaggtggtggcggagaaaacctgtacttccaatgcatcgctatgatagagggtagagctatgggatccgaaatcggtacaggcttccccttcgacccccattatgtggaagtcctgggcgagcgtatgcactacgtcgatgttggaccgcgggatggcacgcctgtgctgttcctgcacggtaacccgacctcgtcctacc(SEQ ID NO: 15)15)15)15)15)15)15)15)15)</p><p num="0183">Example 6 Use of labeled proteins in protein interaction reactions This example demonstrates that proteins labeled with benzothiazole dye conjugates can be used in protein interaction studies. To facilitate the identification of expressed proteins in cell-free expression systems independent of labeling of the compounds of the invention, one of the set of reactions was FluoroTect Green Liz In vitro Translation. Labeling System (Green Lys in vitro Translation Labeling) A parallel protein expression reaction containing System) (Promega) was carried out. The protein expressed by Fluorotect is fluorescently labeled with a dye added from the inside to the end of the protein. Labeling is detected by exposing a sample of protein to light at 488 nm and detecting synchrotron radiation above 510 nm. The use of this particular second labeling method allows for easy distinction between signals from the compounds of the invention used for N-terminal labeling of proteins and protein labeling by fluorotect. Proteins labeled at the N-terminus by the compounds of the invention are less excited by light at 488 nm, but are strongly excited by light at 633 nm. On the other hand, the fluorotect dye is not so excited by the light of 633 nm, but is strongly excited by the light of 488 nm. Therefore, proteins made in an in vitro protein synthesis reaction from protein species labeled using the compounds of the invention by scanning samples from the reactions below using separate excitation wavelengths of 488 nm and 633 nm. Species can be distinguished.</p><p num="0184"> By adding 20 μg of indicated DNA to the reaction using SP6 TnT High Yield Extract (Promega, Madison, Wisconsin) assembled in a reaction as recommended by the supplier 3 Two 250 μL translation reactions were performed. The three added constructs code for: 1) A fusion protein between a halotag and a catalytic subunit of protein kinase A; 2) a construct expressing a metal-binding peptide, followed by a TEV cleavage site, followed by a cysteine residue and a regulatory subunit of protein kinase A (as RIα). Also known), and; 3) The third reaction is identical to the second reaction, but also contains 10 μL of fluorotect (Promega). The reaction was incubated at 25 ° C for 120 minutes.</p><p num="0185"> Three 225 μL translation reaction samples were treated after 120 minutes of incubation through a microbiospin column (BioRad) according to the manufacturer's recommendations. After microbiospin treatment, 12 μL of 20 × ProTEV buffer, 2.4 μL of 0.1 M DTT, and ~ 10 U of ProTEV protease (Promega) were added at room temperature and the tubes were incubated for 60 minutes. After TEV treatment, proteases were removed using 30 μL of Magnet His. To the treated lysate, 2.8 μL 2 mM TCEP and 3.8 μL 125 μM PBI 3168 were added and the tubes were incubated for 60 minutes at room temperature. Finally, freshly reduced cysteine is added to these tubes to react with excess benzothiazole reagent.</p><p num="0186"> A sample of 400 μL HaloLink magnetic beads (Promega Catalog No. G9311) was washed, resuspended in 300 μL according to the manufacturer's recommendations, and then again 50 μL of slurry as described by the manufacturer. It is used to capture the halotag fusion protein from 50 μL of SP6 translation reaction. Other halolink resins such as Promega catalog number G1911 or catalog number G1912 can also be used. See M. Urh et al., Halolink (tm) Resin For Protein Pull-Down And Analysis, Cell Notes 2006, 14, 15-19 (available at www.promega.com/cnotes/). After washing, the resin is treated with kinase buffer (40 mM Tris HCl (pH 7.5), 20 mM MgCl).₂, 0.1 mg / mL BSA) and the resin was incubated with cyanobenzothiazole-labeled prey protein. After these incubations and washing with 1x wash buffer, samples of protein solution were fractionated on SDS PAGE gels, analyzed by SDS PAGE electrophoresis and gels, and imaged using laser excitation at 633 nm.</p><p num="0187"> Visualization of the image generated by laser scanning for the detection of fluorotect products showed a pull-down specific protein (Fig. 8 (b)). Scanning of the gel in Figure 8 with a 633 nm laser shows the detection of pull-downs with red cyanobenzothiazole-derived pigments. Higher interactions were observed in the sample than in the control resin, demonstrating that the label can be used to detect protein-protein interactions.</p><p num="0188"> Figure 8 (a) (scanned at wavelengths showing only red cyanobenzothiazole dye labels) shows scanning of the fluorescence pull-down of PBI 3168 (Alexa 633 SE and the corresponding primary amine linked to cyanobenzothiazole). Prepared from; emits fluorescence after excitation by 633 nm light). In Figure 8 (b), the same gel was scanned for fluorotect. Everything is pulled down and the baits and plays are as follows: Lane 1 + 4, bait = buffer, play = RI-α. Lane 2 + 5, bait = TNT high yield lysate (no translation), play = RI-α. Lane 3 + 6, bait = halo tag-PKA, play = RI-α. Samples in lanes 1-3 were labeled with fluorotect during translation, while 4-6 were unlabeled. Far infrared (633 nm) was used because it separates the spectrum from fluorotect, so no crosstalk between dyes was observed. In FIG. 8 (b), a small amount of RI-α was not cleaved by TEV, so a double line of RI-α is visible. The much weaker high molecular weight bands on the gel are probably oligomers and are also present in Figure 8 (a). Although there are some non-specific labeled species in this cyanobenzothiazole labeled example, the ability of the cyanobenzothiazole labeling method to detect protein-protein interactions is clearly demonstrated.</p><p num="0189"> Scanning the gel with a 488 nm or 523 nm laser allowed a specific pull-down of RIα by the immobilized PKA. It is confirmed that when RIα is added to the control resin, the capture of this protein is much less, thus resulting in these results as a result of the well-known interaction between PKA and the PKA regulatory subunit.</p><p num="0190"> All publications, patents and patent documents are incorporated herein by reference as they are incorporated by reference individually. The present invention has been described with respect to various specific and preferred embodiments and techniques. However, it should be understood that many modifications and modifications may be made, as long as they are within the spirit and scope of the invention.<u style="single"> As one aspect of the present invention, for example, there are the following.</u><u style="single">[1] Compound of formula I</u><img id="000014" he="34" wi="150" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /><u style="single">(During the ceremony</u><u style="single"> Z is H, F, Cl, Br, I, CN, amino, alkylamino, dialkylamino, alkyl ester, carboxy, carboxylate, alkylamide, phosphate, alkylphosphonate, sulfate, alkylsulfonate, nitro, or optionally It is unsaturated and optionally substituted with amino, hydroxy, oxo (= O), nitro, thiol or halo (C).</u>_{<u style="single">1</u>}<u style="single">-C</u>_{<u style="single">10</u>}<u style="single">) Alkyl;</u><u style="single"> Each R</u>^{<u style="single">1</u>}<u style="single">Independently, H, F, Cl, Br, I, CN, (C</u>_{<u style="single">1</u>}<u style="single">-C</u>_{<u style="single">6</u>}<u style="single">) Alkyl, (C</u>_{<u style="single">1</u>}<u style="single">-C</u>_{<u style="single">6</u>}<u style="single">) Alkoxy, or (C</u>_{<u style="single">1</u>}<u style="single">-C</u>_{<u style="single">6</u>}<u style="single">) Alkylthios, each alkyl, alkoxy, or alkylthio can optionally be F, Cl, Br, I, amino, alkenyl, alkynyl, cycloalkyl, aryl, alkylsulfonate, or CO.</u>_{<u style="single">2</u>}<u style="single">Substituted with M (in the formula, M is H, an organic cation, or an inorganic cation);</u><u style="single"> n is 0, 1, or 2;</u><u style="single"> Y is 1 or more halos, oxo (= O), (C</u>_{<u style="single">1</u>}<u style="single">-C</u>_{<u style="single">6</u>}<u style="single">) Alkyl, or (C</u>_{<u style="single">1</u>}<u style="single">-C</u>_{<u style="single">6</u>}<u style="single">) Arbitrarily substituted with alkoxy and one or more N (R)</u>^{<u style="single">1</u>}<u style="single">), O, S, or -NC (= O)-arbitrarily interrupted at the group (C)</u>_{<u style="single">1</u>}<u style="single">-C</u>_{<u style="single">16</u>}<u style="single">) Alkyl-containing linking group or X is N</u>_{<u style="single">3</u>}<u style="single">If Y does not exist arbitrarily; and</u><u style="single"> X is Reporter Yeti, Affinity Yeti, Quencher, Photocrosslinking Yeti, Solid Support, N</u>_{<u style="single">3</u>}<u style="single">, H, or OH, where X is H or OH, the compound of formula I contains isotopic variants of atoms other than the carbon or nitrogen atom of the radioactive or 2-nitrile yeti).</u><u style="single">[2] Z is H or F, and R</u>^{<u style="single">1</u>}<u style="single">The compound according to the above [1], wherein is H or F.</u><u style="single">[3] The compound according to the above [1], wherein Z is hydrogen, fluorine, nitro, or alkyl sulfonate.</u><u style="single">[4] The compound according to the above [3], wherein X is a fluorophore.</u><u style="single">[5] The compound of formula I</u><img id="000015" he="131" wi="150" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /><u style="single">The compound according to the above [4].</u><u style="single">[6] The compound according to [3] above, wherein X is a reporter yeti, an affinity yeti, a quencher, a photocrosslinked yeti, or a solid support.</u><u style="single">[7] The compound of formula I</u><img id="000016" he="33" wi="150" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /><u style="single">The compound according to the above [6].</u><u style="single">[8] X is N</u>_{<u style="single">3</u>}<u style="single">, Alexa-663,</u><img id="000017" he="39" wi="150" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /><u style="single">The compound according to the above [1].</u><u style="single">[9] The compound of formula I</u><img id="000018" he="37" wi="150" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /><u style="single">The compound according to the above [1].</u><u style="single">[10] A method of labeling the N-terminus of a protein.</u><u style="single"> A mixture having a molecular population containing one or more different proteins and at least one of which has a cysteine at the N-terminus is the compound according to [1] above, wherein X in Formula I is a reporter. A step of contacting a compound that is a Yeti or Affinity Moyety to result in one or more proteins including the Reporter Moyety or the Affinity Moyety covalently attached to the cysteine.</u><u style="single">How to include.</u><u style="single">[11] The method according to [10] above, further comprising a step of detecting the reporter yeti.</u><u style="single">[12] The method according to [10] above, further comprising the step of isolating the protein containing the affinity moietti.</u><u style="single">[13] A method for detecting a protein labeled at the N-terminus.</u><u style="single"> a) The compound according to [1] above, wherein X in Formula I is one or more different proteins contacted with a compound that is a reporter or affinity protein, at least one of which is N-terminal. In the step of providing a mixture having a molecular population containing a protein having a cysteine in the above, a step in which one or more of the proteins contain the reporter moyety or the affinity moyety covalently bound to the cysteine.</u><u style="single"> b) The step of detecting the presence or absence of the reporter yeti or the affinity yeti in the mixture.</u><u style="single">How to include.</u><u style="single">[14] A method for isolating a protein labeled at the N-terminus.</u><u style="single"> a) A protein according to the above [1], which is one or more different proteins contacted with a compound in which X in the formula I is an affinity moietti, and at least one of which has a cysteine at the N-terminus. A step of providing a mixture having a molecular population comprising, the step of comprising the affinity moyity in which one or more of the proteins are covalently attached to the cysteine.</u><u style="single">b) The step of isolating the one or more proteins containing the affinity moietti,</u><u style="single">How to include.</u><u style="single">[15] The method according to [10], [13] or [14] above, wherein the mixture comprises a cell-free translation system.</u><u style="single">[16] The cell-free translation system is a wheat germ extract, an insect cell lysate, a rabbit reticulocyte lysate, a frog egg mother cell lysate, a canine pancreatic lysate, a human cell lysate, a purified or semi-purified true. The method according to [15] above, which is a mixture of eukaryotic translation factors or a combination thereof.</u><u style="single">[17] The method according to [10], [13] or [14] above, wherein the mixture comprises intact eukaryotic cells.</u><u style="single">[18] The method according to [17] above, wherein the cell is a tissue culture cell or a primary cell, and the cell is optionally a human cell.</u><u style="single">[19] The method according to [10] or [13] above, further comprising the step of isolating the protein containing the reporter yeti.</u><u style="single">[20] The protein containing the moisture is a recombinant gene product, a gene fusion product, an enzyme, a cytokine, a carbohydrate-binding protein, a lipid-binding protein, a nucleic acid-binding protein, a hormone, an immunogenic protein, a human protein, a viral protein, or a bacterium. The method according to [10], [13] or [14] above, which is a protein, a parasitic protein, or a fragment thereof.</u><u style="single">[21] The method according to [10] or [13] above, wherein the reporter yeti is a fluorophore.</u><u style="single">[22] The method according to [10], [13] or [14] above, wherein the affinity moisture is a peptide of three or more amino acids.</u><u style="single">[23] The method of [22] above, wherein the peptide comprises an epitope or three or more contiguous histidine residues.</u><u style="single">[24] The method according to [10], [13] or [14] above, wherein the moisture is a nucleic acid.</u><u style="single">[25] The method according to [24] above, wherein the moisture is RNA or DNA.</u><u style="single">[26] The method according to [10], [13] or [14] above, wherein the affinity moisture is a hapten.</u><u style="single">[27] The method according to [10], [13] or [14] above, wherein the moeti contains biotin, and the biotin is a biotin moeti capable of optionally photocleaving.</u><u style="single">[28] A method for detecting a protein labeled at the N-terminus.</u><u style="single"> A compound according to a) above [1], X in formula I is a reporter moiety or affinity Moiete I compounds wherein, reacted with one or more a different protein but of which at least one A step of providing a mixture having a molecular population containing an N-terminally labeled protein, which comprises a cysteine at the N-terminus.</u><u style="single"> b) This interaction is compounded by combining a mixture containing the N-terminally labeled protein with a sample containing a second protein that is selected or suspected to interact with the different protein. The process of bringing the body and providing a second mixture, and</u><u style="single"> c) The step of detecting the presence of the reporter yeti or the affinity yeti in the complex,</u><u style="single">How to include.</u><u style="single">[29] The method according to [28] above, further comprising the step of isolating one complex from the second mixture.</u><u style="single">[30] The method according to [28] above, wherein the second protein is a fusion protein.</u><u style="single">[31] The method according to [30] above, wherein the fusion protein comprises a third protein that binds a synthetic substrate.</u><u style="single">[32] The method according to [31] above, wherein the third protein comprises a mutant dehalogenase.</u><u style="single">[33] The method according to [32] above, wherein the synthetic substrate comprises a solid support and a dehalogenase substrate.</u><u style="single">[1'] Compound of formula I</u><img id="000019" he="34" wi="150" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /><u style="single">(During the ceremony</u><u style="single"> Z is H, F, Cl, Br, I, CN, amino, alkylamino, dialkylamino, alkyl ester, carboxy, carboxylate, alkylamide, phosphate, alkylphosphonate, sulfate, alkylsulfonate, nitro, or optionally It is unsaturated and optionally substituted with amino, hydroxy, oxo (= O), nitro, thiol or halo (C).</u>_{<u style="single">1</u>}<u style="single">-C</u>_{<u style="single">10</u>}<u style="single">) Alkyl;</u><u style="single"> Each R</u>^{<u style="single">1</u>}<u style="single">Independently, H, F, Cl, Br, I, CN, (C</u>_{<u style="single">1</u>}<u style="single">-C</u>_{<u style="single">6</u>}<u style="single">) Alkyl, (C</u>_{<u style="single">1</u>}<u style="single">-C</u>_{<u style="single">6</u>}<u style="single">) Alkoxy, or (C</u>_{<u style="single">1</u>}<u style="single">-C</u>_{<u style="single">6</u>}<u style="single">) Alkylthios, each alkyl, alkoxy, or alkylthio can optionally be F, Cl, Br, I, amino, alkenyl, alkynyl, cycloalkyl, aryl, alkylsulfonate, or CO.</u>_{<u style="single">2</u>}<u style="single">Substituted with M (in the formula, M is H, an organic cation, or an inorganic cation);</u><u style="single"> n is 0, 1, or 2;</u><u style="single"> Y is 1 or more halos, oxo (= O), (C</u>_{<u style="single">1</u>}<u style="single">-C</u>_{<u style="single">6</u>}<u style="single">) Alkyl, or (C</u>_{<u style="single">1</u>}<u style="single">-C</u>_{<u style="single">6</u>}<u style="single">) Arbitrarily substituted with alkoxy and one or more N (R)</u>^{<u style="single">1</u>}<u style="single">), O, S, or -NC (= O)-arbitrarily interrupted at the group (C)</u>_{<u style="single">1</u>}<u style="single">-C</u>_{<u style="single">16</u>}<u style="single">) Alkyl-containing linking group or X is N</u>_{<u style="single">3</u>}<u style="single">If Y does not exist arbitrarily; and</u><u style="single"> X is Reporter Yeti, Affinity Yeti, Quencher, Photocrosslinking Yeti, Solid Support, N</u>_{<u style="single">3</u>}<u style="single">, H, or OH, where X is H or OH, the compound of formula I contains isotopic variants of atoms other than the carbon or nitrogen atom of the radioactive or 2-nitrile yeti).</u><u style="single">[2'] Z is H or F, R</u>^{<u style="single">1</u>}<u style="single">The compound according to the above [1'], wherein is H or F.</u><u style="single">[3'] The compound according to the above [1'], wherein X is a fluorophore.</u><u style="single">[4'] The compound of formula I</u><img id="000020" he="123" wi="159" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000021" he="31" wi="150" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /><u style="single">Or</u><img id="000022" he="29" wi="150" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /><u style="single">The compound according to the above [1'].</u><u style="single">[5'] X is N</u>_{<u style="single">3</u>}<u style="single">, Alexa-633, Alexa-488,</u><img id="000023" he="38" wi="150" file="JP5774471B2_D0001.tif" img-format="tif" img-content="drawing" /><u style="single">The compound according to the above [1'].</u><u style="single">[6'] A method of labeling the N-terminus of a protein.</u><u style="single"> A mixture having a molecular population comprising one or more different proteins, at least one of which has a cysteine at the N-terminus, is the compound according to [1'] above, where X in Formula I is a reporter. A step of contacting a compound that is a moyet or affinity moyety to result in one or more proteins, including the reporter moyety or the affinity moyety covalently attached to the cysteine.</u><u style="single">How to include.</u><u style="single">[7'] The method according to [6'] above, further comprising a step of detecting the reporter yeti.</u><u style="single">[8'] The method according to [6'] above, further comprising the step of isolating the protein containing the affinity moietti.</u><u style="single">[9'] The method according to [6'] above, wherein the mixture comprises a cell-free translation system.</u><u style="single">[10'] The method according to [6'] above, wherein the mixture comprises intact eukaryotic cells.</u><u style="single">[11'] The protein containing the moisture is a recombinant gene product, a gene fusion product, an enzyme, a cytokine, a carbohydrate-binding protein, a lipid-binding protein, a nucleic acid-binding protein, a hormone, an immunogenic protein, a human protein, a viral protein, The method according to [6'] above, which is a bacterial protein, a parasitic protein, or a fragment thereof.</u><u style="single">[12'] The N-terminally labeled protein is combined with a sample containing a second protein that is selected or suspected to interact with the different protein, and this interaction is a complex. And the process of providing a second mixture, and</u><u style="single"> The step of detecting the presence of the reporter yeti or the affinity yeti in the complex,</u><u style="single">The method according to the above [6'], further comprising.</u><u style="single">[13'] The method according to [12'] above, further comprising the step of isolating one complex from the second mixture.</u><u style="single">[14'] The method according to [12'] above, wherein the second protein is a fusion protein.</u><u style="single">[15'] The method according to [14'] above, wherein the fusion protein comprises a mutant dehalogenase.</u></p>