JP2010119382A - Method for fluorescently labeling protein - Google Patents

Abstract

前記式（Ｉ）中、Ｘは、蛍光性基であり、Ｙは、消光性基であり、Ｌ¹は、Ｘのためのリンカーであり、Ｌ²は、Ｙのためのリンカーであり、Ｒ¹は、Ｈ、低級アルキルカルボニルオキシで置換された低級アルキレン基または低級アルキル基である。
【選択図】なしPROBLEM TO BE SOLVED: To provide a fluorescent labeling method for proteins capable of labeling in vivo molecules.
The method includes obtaining a fusion protein of a target protein to be labeled and a mutant β-lactamase, and contacting the target protein with a compound represented by the following formula (I) to fluorescently label the target protein: Protein fluorescent labeling method.

In the formula (I), X is a fluorescent group, Y is a quenching group, L ¹ is a linker for X, L ² is a linker for Y, R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy.
[Selection figure] None

Description

  The present invention relates to a method for fluorescently labeling proteins.

  In recent years, bioimaging for observing the functions of biomolecules such as proteins in living organisms in the state of living organisms has attracted attention. The observation of the biomolecule function is generally performed by labeling the biomolecule with fluorescence and visualizing the localization and movement of the biomolecule in the organism. As a method for labeling a biomolecule with fluorescence, a method in which a fluorescent protein is co-expressed at the end of a target biomolecule (protein) using a genetic engineering technique is generally used. This fluorescent protein has disadvantages such as large molecular size and difficulty in observation of near-infrared fluorescence with high tissue permeability. For this reason, attention has been focused on small organic molecules having a smaller molecular size than fluorescent proteins and excellent fluorescence characteristics. Labeling using HaloTag (registered trademark) (Promega Corporation) (see, for example, Non-Patent Document 1) and SNAP-tag (registered trademark) (for example, see Non-Patent Document 2) as a technique for labeling a small organic molecule to a target protein The kit has been commercialized. In these labeling methods, a target biomolecule is labeled with fluorescence using an enzyme reaction. Therefore, the specificity when labeling a small organic molecule to a target biomolecule is very high, but fluorescence derived from the small organic molecule that is not bound to the biomolecule is observed as a background signal. In order to reduce this background signal, it is necessary to remove the unreacted organic small molecules by fluorescently labeling the target biomolecules with organic small molecules and then washing them. However, it is generally difficult to clean biomolecules present in cells or in vivo, and in many cases cannot be cleaned. Therefore, the above-described labeling method is not versatile for observing the function of biomolecules in cells or in vivo. In addition, a method of labeling a biomolecule with fluorescence using a peptide having a specific sequence that links an organic small molecule with a target biomolecule has also been developed. There is a problem that is low.

Qureshi, M.H., et al., J. Biol. Chem., 2001, 276, p.46422-8 Proc. Natl. Acad. Sci. USA 2004, 101, p.9955-9959

  Therefore, an object of the present invention is to provide a method for fluorescently labeling proteins, which can be used to label biomolecules (proteins) in cells or in vivo with fluorescence.

  The present invention is a method of fluorescently labeling a protein, obtaining a fusion protein of a protein to be labeled and a mutant β-lactamase, and reacting the fusion protein with a compound represented by the following formula (I) or a salt thereof The mutated β-lactamase opens the β-lactam ring of the compound represented by the formula (I), and as a result, the formula (I) It is a protein that liberates Y of the represented compound and can be covalently bonded to the portion of the compound opened by the β-lactam ring (hereinafter sometimes referred to as “first fluorescent labeling method”).

In the formula (I), X is a fluorescent group,
Y is a quenching group,
L ¹ is a linker for X
L ² is a linker for Y;
R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy.

  The method of fluorescently labeling the protein of the present invention exhibits strong fluorescence only when the target protein is labeled. Therefore, the fluorescence background can be minimized without removing organic small molecules that are not labeled with proteins. Therefore, it is possible to perform fluorescence observation with high accuracy. Further, since it is not necessary to remove unreacted organic small molecules, it is suitable for observing biomolecules in vivo.

FIG. 1A shows a fluorescence image (i) and gel-stained image (ii) when E166NTEM-1 is labeled with CA, and a fluorescence image (iii) when only CA is used, and FIG. , A fluorescent image (i) and a gel-stained image (ii) when E166NTEM-1 is labeled with a CCD, and a fluorescent image (iii) when only a CCD is used. 2A shows a fluorescence image (left) and a gel-stained image (right) when CA is added to WTTEM-1, and FIG. 2B shows a CCD image on WTTEM-1. A fluorescence image (left) and a gel-stained image (right) are shown. FIG. 3 (a) shows an excitation spectrum at a fluorescence wavelength of 450 nm obtained for WT (wild strain), and FIG. 3 (b) shows a fluorescence spectrum at an excitation wavelength of 410 nm. FIG. 4A shows an excitation spectrum at a fluorescence wavelength of 450 nm obtained for E166N TEM-1, and FIG. 4B shows a fluorescence spectrum at an excitation wavelength of 410 nm. FIG. 5 shows a fluorescent image (left) and a gel-stained image (right) when MBP-E166NTEM-1 is labeled with a CCD in a mammalian cell disruption solution. FIG. 6 shows a fluorescence image (left) and a gel-stained image (right) when MBP-E166N TEM-1 is labeled with compound CA. FIG. 7A shows an absorption spectrum of the compound csDAm coordinated with Tb ³⁺ , and FIG. 7B shows an absorption spectrum of the compound csDAm coordinated with Eu ³⁺ . 7C is an enlarged view of FIG. 7A, and FIG. 7D is an enlarged view of FIG. 7B. 8A shows the excitation spectrum of the compound csDAm coordinated with Tb ³⁺ , and FIG. 8B shows the excitation spectrum of the compound csDAm coordinated with Eu ³⁺ . FIG. 9A shows a fluorescence spectrum obtained by steady-state fluorescence measurement when E166N TEM-1 is labeled with a compound csDAm coordinated with Tb ³⁺ , and FIG. 9B shows a fluorescence spectrum obtained by time-resolved fluorescence measurement. . FIG. 10A shows a fluorescence spectrum obtained by steady-state fluorescence measurement when E166N TEM-1 is labeled with a compound csDAm coordinated with Eu ³⁺ , and FIG. 10B shows a fluorescence spectrum obtained by time-resolved fluorescence measurement. . FIG. 11A is a spectrum showing the fluorescence lifetime of the compound csDAm coordinated with Tb ³⁺ , and FIG. 11B is a spectrum showing the fluorescence lifetime of the compound csDAm coordinated with Eu ³⁺ . FIG. 12 shows a fluorescence image and a gel-stained image when E166NTEM-1 is labeled with the compound csDAm coordinated with Ln. FIG. 13 shows a fluorescence image and a gel-stained image when compound csDAm in which Ln is coordinated is added to WTTEM-.

  One of the β-lactamases, TEM-1, is known to hydrolyze β-lactam antibiotics such as penicillin through a reaction as shown in Scheme 1 below.

  As shown in Scheme 1 above, TEM-1 has a 70-position Ser that undergoes a nucleophilic attack on the lactam ring to form an acyl-enzyme complex. The complex is then hydrolyzed resulting in hydrolysis of the β-lactam ring. It is known that the hydrolysis is promoted by 166-position Glu of TEM-1. And it has been known that the mutant β-lactamase in which the 166th position Glu is changed to another amino acid greatly reduces the hydrolysis rate of the acyl-enzyme complex. For example, in the case of a mutant β-lactamase in which 166th position Glu is changed to Asn, hydrolysis of the acyl-enzyme intermediate does not occur as shown in the following scheme 2. Therefore, such mutant β-lactamase binds irreversibly to the substrate β-lactam ring.

  In the present invention, for example, a mutant β-lactamase obtained by changing the 166th Glu to another amino acid and a compound represented by the formula (I) (hereinafter referred to as “compound of the formula (I)”) Another feature is to use a salt thereof. As the mutant β-lactamase, the amino acid mutation site of the natural β-lactamase is not limited, and the β-lactam ring of the compound represented by the formula (I) is opened, and as a result, the mutant β-lactamase is represented by the formula (I). Any protein can be used as long as it releases Y of the compound and can be covalently bonded to the portion of the compound having the β-lactam ring opened. The compound of formula (I) has X as a fluorescent group and Y as a quenching group in the intramolecular structure. Therefore, the compound of formula (I) has a very low fluorescence intensity despite having a fluorescent group.

  In the first fluorescent labeling method of the present invention, for example, a fusion protein obtained by fusing this mutant β-lactamase and a protein to be labeled is reacted with a compound of the formula (I). Then, the mutant β-lactamase of the fusion protein nucleophilically attacks the β-lactam ring of the compound of formula (I) to open the β-lactam ring (see, for example, Scheme 3). Electron transfer occurs through the vinyl group in the resulting acyl-enzyme intermediate, thereby liberating Y from the compound of formula (I). It should be noted that the resulting acyl-enzyme intermediate cannot be hydrolyzed, ie, the mutated β-lactamase and the ring-opened portion of the compound of formula (I) remain covalently bonded. Since the complex of the mutant β-lactamase and the compound of the formula (I) does not contain Y as a quencher, the fluorescent X contained in the compound of the formula (I) emits fluorescence. Then, the compound of the formula (I) bound to the labeling target protein via the mutant β-lactamase emits strong fluorescence, and the compound of the formula (I) not bound to the labeling target protein via the mutant β-lactamase is The fluorescence intensity is extremely low. That is, since the compound of formula (I) does not emit fluorescence when not bound to the protein to be labeled, the background signal is low. As a result, the protein to be labeled (biomolecule) is labeled with the compound of formula (I) and can be accurately detected without being disturbed by the background signal without washing. .

  Examples of the polynucleotide encoding the mutant β-lactamase include, for example, any one of the nucleotide sequences of SEQ ID NOs: 5 to 8 in the sequence listing, 1 to several bases of any of the nucleotide sequences of SEQ ID NOs: 5 to 8 in the sequence listing Wherein the β-lactam ring of the compound of the formula (I) or a salt thereof is opened in the fusion protein, and as a result, Y of the compound is liberated. And a nucleotide sequence that encodes an amino acid sequence that can be covalently bonded to the portion of the compound having the β-lactam ring opened, and a nucleotide sequence of any one of SEQ ID NOS: 5 to 8 in the sequence listing has a homology of 70% or more. Wherein the β-lactam ring of the compound of formula (I) or a salt thereof is opened in the fusion protein, so that Y of the compound is liberated, and the β-lactam of the compound is Examples thereof include a base sequence selected from the group consisting of a base sequence encoding an amino acid sequence that can be covalently bonded to a portion where a tom ring is opened. For example, the base sequence of SEQ ID NO: 5 is a base sequence of mutant TEM-1 in which glutamic acid at position 166 is changed to asparagine (see Adachi et al., The Journal of Biochemistry, 1991, 266, 3186-3191).

  In order to obtain a plasmid or vector capable of expressing a fusion protein, such a plasmid or vector is prepared according to a conventional method using a polynucleotide encoding a protein to be labeled, a polynucleotide encoding a mutant β-lactamase, or the like. be able to. In the present invention, a vector refers to a polynucleotide for introducing a polynucleotide, preferably a polynucleotide encoding a fusion protein, into a cell, and includes a plasmid. The vector may be a plasmid vector or a viral vector. Those skilled in the art can appropriately select a sequence capable of expressing the fusion protein according to the type of cell to be introduced. Plasmids and vectors may contain sequences that regulate the expression of the fusion protein (eg, expression-inducible promoters and control sequences).

  Expression of the fusion protein in the cell can be performed according to a usual method using a polynucleotide encoding the fusion protein or a plasmid or vector capable of expressing the fusion protein.

  Isolation of the expressed fusion protein can be performed according to conventional methods.

  The fusion protein of the protein to be labeled and the mutant β-lactamase can be obtained by obtaining a polynucleotide encoding the fusion protein, obtaining a plasmid or vector capable of expressing the fusion protein, expressing the fusion protein in the cell, or expressing the fusion protein. Can be obtained by a method comprising one or more steps of isolating the fusion protein, or a method comprising all steps. That is, obtaining a fusion protein includes not only isolating the protein but also expressing it in cells or in vivo.

As described above, the compound of the formula (I) used in the first fluorescent labeling method of the present invention is such that X is a fluorescent group, Y is a quenching group, and L ¹ is X. L ² is a linker for Y, and R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy.

  In the compound of the present invention, the fluorescent group is irradiated with visible light, ultraviolet light, X-rays, etc., and when the energy is absorbed, electrons are excited, and extra energy is converted into electromagnetic waves when it returns to the ground state. It means a group having a releasing property. Specifically, the fluorescent group is derived from a coumarin dye, xanthene dye, cyanine dye, acridine, isoindole, dansyl dye, aminophthalic acid hydrazide, aminophthalimide, aminonaphthalimide, aminobenzofuran, aminoquinoline, dicyanohydroquinone, etc. Or a group derived from a coumarin dye or xanthene dye and a light-emitting rare earth complex are preferred.

Examples of the light-emitting rare earth complex include complexes of groups represented by the following formulas (ii) and (xi) to formula (xxviii) with rare earth ions. Examples of the rare earth ions include Tb ³⁺ , Eu ³⁺ , Dy ³⁺ and Sm ³⁺ .

  In the compounds of formula (I), quenching groups that reduce fluorescence are fluorescence resonance energy transfer (FRET), photoinduced electron transfer, enhanced paramagnetic intersystem crossing, Dexter exchange coupling and dark complex It is a group that extinguishes fluorescence of a fluorescent group by a mechanism including exciton coupling such as formation. Specifically, the quenching group is

Examples thereof include dimethylaminoazobenzene, tetramethylrhodamine, QSY quencher dye (manufactured by Invitrogen), and dimethylaminoazobenzene is preferred.

In the compound of the present invention, the linker L ¹ for X is a group for connecting the cephalosporin skeleton of the compound of the formula (I) or the compound of the formula (III) and X or the compound of the formula (II). This is a group for linking the penicillin skeleton and X. Specifically, L ¹ _{is, - (CH 2) n1 CONR} 2 (CH 2) n2 -, - (CH 2) n1 NR 2 CO (CH 2) n2 -, - (CH 2) n1 NR 3 CONR 2 _{(CH 2) n2 -, -} (CH 2) n1 NR 3 CSNR 2 (CH 2) n2 -, - (CH 2) n1 CONR 3 (CH 2) n3 CONR 2 (CH 2) n2 -, - (CH 2 _{^{) n1 CONR 3 (CH 2)}} n3 (CHR 4) n4 CONR 2 (CH 2) n2 -, - (CH 2) n1 -, - (CH 2) n1 S (CH 2) n2 -, - (CH 2) _{n1 O (CH 2) n2 -} , - (CH 2) n1 NR 2 (CH 2) n2 -, - (CH 2) n1 CO 2 (CH 2) n2 - , and the like, - (CH _{2) n1} CONR ³ (CH ₂ ) _n3 CONR ² (CH ₂ ) _n2 — is preferred. In the formula, n1, n2, n3, n4 each is an integer of 0 or 1 to 5, R ^2, R ³ and R ⁴ are each a hydrogen atom, a lower alkyl group or a phenyl group. The structure of the linker for X is not limited as long as it is a linker that is not cleaved under the conditions in the method for fluorescently labeling the protein of the present invention.

In the compound of formula (I), the linker L ² for Y is a group for connecting Y to the cephalosporin skeleton of the compound of formula (I). Furthermore, the linker for Y is the β-lactam ring of the compound of formula (I) opened by the mutant β-lactamase, resulting in electron transfer through the vinyl group in the resulting acyl-enzyme intermediate, A linker that liberates Y is preferred. Specifically, L ² _{is, -CH 2 O (CH 2)} n1 -, - CH 2 S (CH 2) n1 -, - CH 2 NR 2 (CH 2) n1 -, - CH 2 N + R 2 ₂ (CH ₂ ) _n1 —, —CH ₂ OCONR ² (CH ₂ ) _n1 —, —CH ₂ OCO (CH ₂ ) _n1 —, —CH ₂ CSNR ² (CH ₂ ) _n1 —, —CH ₂ SCSO (CH _{2 N1} −, —CH═CH—,

Etc.

Is preferred. In the formula, n1, n2 are each an integer of 0 or 1 to 5, R ² is a hydrogen atom or a lower alkyl group.

In the compounds of the present invention, the cephalosporin skeleton, penicillin skeleton, X, Y, L ¹ and L ² may have one or more asymmetric centers, and therefore the compounds of formula (I) Compounds of formula (II), compounds of formula (III) may exist as racemates or pure stereoisomers. Further, when an alkenyl group is contained in X, Y, L ¹ and / or L ² , it can exist as a cis or trans isomer. In any case, the present invention includes both mixtures and isomers.

  However, in the compound of the formula (I), the cephalosporin skeleton preferably has a steric form represented by the following formula because the specificity to the mutant β-lactamase increases.

In the above formula, L ¹ , L ² and R ¹ are as defined in the compound of formula (I).

  In the compound of the present invention, examples of the lower alkyl group include those having 1 to 6 carbon atoms. Specifically, as the lower alkyl group, methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, i-butyl group, sec-butyl group, t-butyl group, n-pentyl Group, i-pentyl group, sec-pentyl group, t-pentyl group, 2-methylbutyl group, n-hexyl group, 1-methylpentyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group 1-ethylbutyl group, 2-ethylbutyl group, 1,1-dimethylbutyl group, 2,2-dimethylbutyl group, 3,3-dimethylbutyl group, and 1-ethyl-1-methylpropyl group Or a branched alkyl group can be mentioned, A C1-C3 thing is mentioned suitably.

  In the compounds of the present invention, examples of the lower alkylene group include those having 1 to 6 carbon atoms. Specifically, examples of the lower alkyl group include linear or branched alkyl groups such as a methylene group, an ethylene group, a propylene group, a butylene group, a pentylene group, and a hexylene group. 1-3 are mentioned.

  In the compound of the present invention, examples of the lower alkylene group substituted with lower alkylcarbonyloxy include t-butoxymethyl, acetoxymethyl and the like, and among them, acetoxymethyl is preferable.

  In the present invention, the salt of the compound of formula (I), the salt of the compound of formula (II) and the salt of the compound of formula (III) may be a salt with an acid or a base. Examples of such salts include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium and magnesium, salts with inorganic bases such as ammonium salts, and triethylamine, pyridine, picoline, ethanolamine, and triethanol. Organic amine salts such as amine, dicyclohexylamine, N, N′-dibenzylethyleneamine, and inorganic acid salts such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and formic acid, acetic acid, trifluoroacetic acid, maleic acid, Salts or acids with organic carboxylates such as tartaric acid, and sulfonic acid addition salts such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and basic or acidic amino acids such as arginine, aspartic acid and glutamic acid Addition salts are mentioned.

  In the present invention, the compound of formula (I), the compound of formula (II) and the compound of formula (III) may take the form of solvates, which are also included in the scope of the present invention. Preferred solvates include hydrates and ethanol solvates.

  The compound of formula (I), the compound of formula (II) and the compound of formula (III) used in the fluorescent labeling method of the present invention may be made in-house with reference to known literatures in the prior art or commercially available. You may obtain it.

  Obtaining a fusion protein of a protein to be labeled and a mutant β-lactamase used in the fluorescent labeling method of the present invention includes, for example, obtaining a polynucleotide encoding the fusion protein, and a plasmid or vector capable of expressing the fusion protein. Obtaining expression of the fusion protein in the cell, or isolating the expressed fusion protein. The polynucleotide encoding the fusion protein may be a polynucleotide encoding the protein to be labeled, a polynucleotide encoding the mutant β-lactamase, etc., and a plasmid or vector capable of expressing the fusion protein may be prepared according to a conventional method. it can.

  In the first fluorescent labeling method of the present invention, the step of reacting the fusion protein with the compound of formula (I) may be carried out in a cell or in a living body expressing the fusion protein. You may carry out in vitro using protein. When labeling is performed in vitro, for example, it may be performed in a buffer solution (pH 6.8 to 7.4) at a temperature of 20 to 37 ° C.

In the method of fluorescently labeling the protein of the present invention (first fluorescent labeling method), the amino acid sequence of the mutant β-lactamase in the fusion protein is:
(I) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 in the sequence listing,
(Ii) an amino acid sequence in which one to several amino acids of the amino acid sequences of SEQ ID NOs: 1 to 4 in the sequence listing are deleted, substituted, or added, and in the fusion protein, An amino acid sequence capable of opening the β-lactam ring of the compound represented, thereby liberating Y of the compound and covalently binding to the moiety of the β-lactam ring of the compound, and
(Iii) an amino acid sequence having 70% or more homology with any one of SEQ ID NOs: 1 to 4 in the Sequence Listing, wherein the β-lactam ring of the compound represented by the above formula (I) in the fusion protein Is an amino acid sequence selected from the group consisting of an amino acid sequence that liberates Y of the compound and as a result is capable of covalently binding to a portion where the β-lactam ring of the compound is opened. preferable.

  The present invention is also a composition for use in the first fluorescent labeling method of the present invention comprising the compound represented by the formula (I) or a salt thereof.

In the formula (I), X is a fluorescent group,
Y is a quenching group,
L ¹ is a linker for X
L ² is a linker for Y;
R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy.

The present invention also provides a plasmid or vector for use in the first fluorescent labeling method of the present invention,
The plasmid or vector is a plasmid or vector for cloning a polynucleotide encoding a fusion protein of a protein to be labeled and a mutant β-lactamase or for expressing the fusion protein,
The plasmid or vector is
A nucleotide sequence in which one to several bases of the nucleotide sequence of any one of SEQ ID Nos. 5 to 8 in the sequence listing are deleted, substituted, or added. Opening the β-lactam ring of the compound represented by the formula (I) or a salt thereof in the fusion protein, thereby liberating Y of the compound, and the β-lactam ring of the compound or a salt thereof A base sequence encoding an amino acid sequence that can be covalently bonded to the opened ring; and
A base sequence having 70% or more homology with any one of SEQ ID NOs: 5 to 8 in the Sequence Listing, wherein the compound represented by the formula (I) or a salt thereof in the fusion protein is a β-lactam ring Selected from the group consisting of: a base sequence encoding an amino acid sequence capable of liberating Y of the compound and, as a result, liberating Y of the compound and capable of covalently binding to a portion where the β-lactam ring of the compound or a salt thereof is opened The base sequence to be included.

  The present invention also provides a fluorescent label for a protein comprising a composition for use in the method of the present invention comprising the compound represented by the formula (I) or a salt thereof, and a plasmid or vector for use in the method of the present invention. Method kit.

  The kit for fluorescent labeling method of a protein of the present invention preferably further includes a host cell for introducing the plasmid or vector and an instruction manual for the kit.

  In the first fluorescent labeling method of the present invention, when X in the formula (I) is a luminescent rare earth complex, it is preferable to detect the fluorescently labeled target protein by a time-resolved fluorescence measurement method. In such a compound of formula (I), since X is a luminescent rare earth complex, the emitted fluorescence has a long lifetime. Then, fluorescence with a short lifetime (fluorescence derived from endogenous fluorescent substances such as riboflavin and NADH) observed as autofluorescence in a living body can be eliminated, and the target protein can be detected with high sensitivity at a high S / N ratio. .

  In the first fluorescent labeling method, composition and kit of the present invention, a group represented by the following formula (i) wherein X is derived from coumarin:

And Y is

L ¹ is — (CH ₂ ) _n1 CONR ³ (CH ₂ ) _n3 CONR ² (CH ₂ ) _n2 —, and L ² is

Or a compound of formula (I) wherein -CH = CH- is preferred,
A compound CCD represented by the following formula is more preferred.

  The present invention also relates to a compound represented by the general formula (I ′) (hereinafter referred to as “compound of formula (I ′)”) or a salt thereof.

In the formula (I), X is a group derived from a fluorescent group, preferably a coumarin dye or a xanthene dye,
Y is a quenching group, preferably dimethylaminoazobenzene,
L ¹ is a linker for X, preferably — (CH ₂ ) _n1 CONR ³ (CH ₂ ) _n3 CONR ² (CH ₂ ) _n2 —
L ² '

(In the above formula, n1 and n2 are each 0 or an integer of 1 to 5,
R ² is a hydrogen atom or a lower alkyl group)
Or -CH = CH-
A linker for Y, preferably represented by

And
R ¹ is H or a lower alkyl group.

  The compound of the formula (I ′) is produced by using a cephalosporin derivative protected with a protecting group represented by the following formula as a starting material and referring to known literature in the prior art and a method for producing a compound CCD. Can do.

  In the above formula, Pro and Pro 'each represent a protecting group, and Hal represents a halogen atom.

The present invention also provides a method for fluorescently labeling a protein,
By obtaining a fusion protein of a protein to be labeled and a mutant β-lactamase, and reacting the fusion protein with a compound represented by the following formula (II) or a compound represented by the following formula (III) or a salt thereof, Including fluorescently labeling the target protein, wherein the mutant β-lactamase opens the β-lactam ring of the compound represented by the formula (II), the compound represented by the following formula (III), or a salt thereof, In addition, it is a protein fluorescent labeling method that is a protein that can be covalently bonded to a portion of the compound or a salt thereof opened by a β-lactam ring (hereinafter, sometimes referred to as “second fluorescent labeling method”). .

In the formula (II) and formula (III),
X is a fluorescent group,
L ¹ is a linker for X
R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy,
R ⁴ is H or lower alkyl.

  As described above, when the fusion protein is reacted with a compound of the following formula (II) or a compound of the following formula (III) or a salt thereof, the mutant β-lactamase of the fusion protein becomes a compound of the formula (II) or Nucleophilic attack on the β-lactam ring of the compound of formula (III) opens the β-lactam ring. Since the adduct between β-lactam and the compound of formula (II) or the compound of formula (III) is not hydrolyzed after ring opening, the mutant β-lactamase is a compound of formula (II) or a compound of formula (III) Will be fluorescently labeled. This method has the advantage that the mutant β-lactamase can be selectively fluorescently labeled. β-lactamase is a bacterial enzyme and is not present in mammalian cells. Therefore, this method has the advantage that there is no fear of labeling endogenous proteins in mammalian cells.

In the compound of the formula (II) and the compound of the formula (III) used in the second fluorescent labeling method, X is a fluorescent group and L ¹ is a linker for X as described above. , R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy, and R ⁴ is H or a lower alkyl. The fluorescent group, the linker for X, the lower alkylene group substituted with lower alkylcarbonyloxy, and the lower alkyl group are as described above for the compound of formula (I).

  Obtaining a fusion protein of a protein to be labeled and a mutant β-lactamase used in the second fluorescent labeling method of the present invention includes, for example, obtaining a polynucleotide encoding the fusion protein and capable of expressing the fusion protein. It may include obtaining a plasmid or vector, expressing the fusion protein in a cell, or isolating the expressed fusion protein. The polynucleotide encoding the fusion protein may be a polynucleotide encoding the protein to be labeled, a polynucleotide encoding the mutant β-lactamase, etc., and a plasmid or vector capable of expressing the fusion protein may be prepared according to a conventional method. it can.

  In the second fluorescent labeling method of the present invention, the step of reacting the fusion protein with the compound of the formula (II) or the compound of the formula (III) is carried out in a cell or in vivo expressing the fusion protein. Alternatively, it may be performed in vitro using the isolated fusion protein. When labeling is performed in vitro, for example, it may be performed in a buffer solution (pH 6.8 to 7.4) at a temperature of 20 to 37 ° C.

In the second fluorescent labeling method of the present invention, the amino acid sequence of the mutant β-lactamase in the fusion protein is:
(I) the amino acid sequence of any one of SEQ ID NOs: 1 to 4 in the sequence listing,
(Ii) an amino acid sequence obtained by deleting, substituting, or adding one to several amino acids of any one of SEQ ID NOs: 1 to 4 in the sequence listing, wherein the fusion protein is represented by the above formula (II) Opening the β-lactam ring of the compound represented by formula (III) or a salt thereof, thereby liberating Y of the compound or salt thereof, and the β-lactam ring of the compound or salt thereof An amino acid sequence that can be covalently bonded to the ring-opened portion, and
(Iii) an amino acid sequence having 70% or more homology with any one of SEQ ID NOs: 1 to 4 in the Sequence Listing, wherein the compound represented by the above formula (II) or the formula (III) in the fusion protein The β-lactam ring of the compound represented by the formula (1) or a salt thereof, thereby opening Y of the compound or the salt thereof, and covalently bonding to the portion where the β-lactam ring of the compound or the salt thereof is opened An amino acid sequence selected from the group consisting of possible amino acid sequences is preferred.

  The second fluorescent labeling method of the present invention utilizes mutant β-lactamase. Since β-lactamase is a bacterial enzyme and does not exist in mammalian cells, it does not label endogenous proteins. Therefore, the method of fluorescently labeling the protein of the present invention can selectively label the target protein instead of the endogenous protein.

  In the second fluorescent labeling method of the present invention, when X is a luminescent rare earth complex in the formulas (II) and (III), the fluorescently labeled target protein is detected by a time-resolved fluorescence measurement method. Is possible. In such a compound of formula (II) or a compound of formula (III), since X is a luminescent rare earth complex, the emitted fluorescence has a long lifetime. Then, fluorescence with a short lifetime (fluorescence derived from endogenous fluorescent substances such as riboflavin and NADH) observed as autofluorescence in a living body can be eliminated, and the target protein can be detected with high sensitivity at a high S / N ratio. .

  The present invention is also a composition for use in the method of the present invention comprising the compound represented by the formula (II), the compound represented by the following formula (III) or a salt thereof.

In the formula (II) and formula (III),
X is a fluorescent group,
L ¹ is a linker for X
R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy,
R ⁴ is H or lower alkyl.

The present invention is a plasmid or vector for use in the second fluorescent labeling method of the present invention,
The plasmid or vector is a plasmid or vector for cloning a polynucleotide encoding a fusion protein of a protein to be labeled and a mutant β-lactamase or for expressing the fusion protein,
The plasmid or vector is
A nucleotide sequence in which one to several bases of the nucleotide sequence of any one of SEQ ID Nos. 5 to 8 in the sequence listing are deleted, substituted, or added. Opening the β-lactam ring of the compound represented by formula (II) or the compound represented by formula (III) or a salt thereof in the fusion protein, thereby liberating Y of the compound, and the compound Or a base sequence that encodes an amino acid sequence that can be covalently bonded to a portion of the salt whose β-lactam ring is opened, and
A compound represented by the formula (II) or a compound represented by the formula (III) in the fusion protein, the nucleotide sequence having 70% or more homology with any one of the sequence numbers 5 to 8 in the sequence listing Alternatively, it encodes an amino acid sequence that opens the β-lactam ring of a salt thereof, thereby liberating Y of the compound and that can be covalently bonded to the opened portion of the β-lactam ring of the compound or a salt thereof. A base sequence selected from the group consisting of base sequences.

  The present invention also provides a composition for use in the method of the present invention comprising the compound represented by the formula (II), the compound represented by the following formula (III) or a salt thereof, and the method for use in the method of the present invention. A kit for fluorescent labeling of proteins comprising a plasmid or a vector.

  The kit for fluorescent labeling method of a protein of the present invention preferably further includes a host cell for introducing the plasmid or vector and an instruction manual for the kit.

  In the second fluorescent labeling method, composition and kit of the present invention, as the compound represented by the general formula (II), X represents a group represented by the following formula (i) or a group represented by the formula (ii) and a rare earth Complex with ions:

And L ¹ is — (CH ₂ ) _n1 CONR ³ (CH ₂ ) _n3 CONR ² (CH ₂ ) _n2 — or — (CH ₂ ) _n1 CONR ³ (CH ₂ ) _n3 (CHR ⁴ ) _n4 CONR ² (CH _{2) n2} - (in the formula, n1, n2, n3, n4 each is an integer of 0 or 1 to 5, R ^2, R ³ and R ⁴ are each a hydrogen atom, a lower alkyl group or a phenyl group .)
And a compound in which R ¹ is H, and a compound in which a rare earth ion forms a complex with the following compound CA and the following compound csDAm is more preferable.

  The present invention also relates to a compound represented by the general formula (II ') or a salt thereof.

In the formula (II ′),
X ′ is a luminescent rare earth complex,
L ¹ is a linker for X
R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy.

  Examples of the compound represented by the general formula (II ′) include a complex of a group represented by the following formula (ii) and a rare earth ion:

And L ¹ is — (CH ₂ ) _n1 CONR ³ (CH ₂ ) _n3 CONR ² (CH ₂ ) _n2 — or — (CH ₂ ) _n1 CONR ³ (CH ₂ ) _n3 (CHR ⁴ ) _n4 CONR ² (CH _{2) n2} - (in the formula, n1, n2, n3, n4 each is an integer of 0 or 1 to 5, R ^2, R ³ and R ⁴ are each a hydrogen atom, a lower alkyl group or a phenyl group .)
And a compound in which R ¹ is H is preferable, and a compound in which a rare earth ion forms a complex with the following compound csDAm is more preferable.

  The compounds of the formula (II) and the formula (II ′) are obtained from a penicillin derivative protected with a protecting group represented by the following formula as a starting material, and by referring to known literature in the prior art and a method for producing the compound CA, Can be manufactured.

Wherein L ¹ is a linker for X;
R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy.

[Example]
The following abbreviations are used in the description of the present specification.
DMSO: dimethyl sulfoxide HOBt: 1-hydroxybenzotriazole DMF: dimethylformamide WSCD: water-soluble carbodiimide: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide Ar: argon TEA: triethylamine BOC: t-butoxyoxycarbonyl [compound And equipment]
The compounds were of the highest grade available and were purchased from Tokyo Chemical Industry Co., Ltd., Wako Pure Chemical Industries, Ltd., and Sigma-Aldrich Japan Co., Ltd. and used without further purification. 7-amino-3-chloromethyl-3-cephem-4-carboxylic acid p-methoxybenzyl ester hydrochloride (ACLE.HCl) was purchased from Otsuka Chemical Co., Ltd. Restriction endonuclease and PrimeSTAR® HS DNA polymerase were purchased from Takara Bio Inc. Kanamycin and inorganic salts were purchased from Wako Pure Chemical Industries or Nacalai Tesque. Plasmid DNA was isolated using QIAprep Spin Miniprep kit (trade name) (Qiagen Co., Ltd.).

  XL10-Gold (Stratagene) was used as an intermediate host for site-directed mutagenesis experiments. E. coli BL21 (DE3) (Novagen) was used as a host for target gene expression. Luria-Bertani (LB) medium or Terrific Broth (TB) was used as the growth medium for all strains. Bacto Agar (Wako Pure Chemical Industries, Ltd.) was used for the preparation of solid media at concentrations up to 1.5%.

NMR spectra were measured at 400 MHz for ¹ H and 100.4 MHz for ¹³ NMR using a JEOL JNM-AL400 apparatus using tetramethylsilane as an internal standard. Mass spectrum (FAB) was measured by JEOL JMS-700. ESI-TOF MS was measured with Waters LCT-Premier XE (Nihon Waters Co., Ltd.). MALDI-TOF MS was measured with Applied Biosystems Voyager RP (Applied Biosystems Japan Co., Ltd.). The UV-visible absorption spectrum was measured using an ultraviolet-visible spectrophotometer UV1650PC spectrometer (Shimadzu Corporation). The fluorescence spectrum was measured using a Hitachi spectrofluorometer F-4500. The slit width was 2.5 nm for both excitation and emission. The photomultiplier tube voltage was 700V. All probes were dissolved in (Biochemical grade, Wako Pure Chemical Industries, Ltd.) before fluorescence measurement. Silica gel column chromatography was performed using BW-300 (Fuji Silysia Chemical Ltd.).

[Reference Example: Synthesis of Compound CA]
(1) Synthesis of compound (11)

  2,4-Dihydroxybenzaldehyde (3.0 g, 22 mmol) and diethyl malonate (4.2 g, 26 mmol) were dissolved in absolute EtOH (30 mL), and then piperidine (370 mg, 4.3 mmol) was added. The reaction mixture was heated at reflux for 12 hours and then cooled in an ice bath. The precipitate was collected and washed with cold EtOH to obtain compound (11) (2.5 g, yield 49%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.29 (t, J = 6.9 Hz, 3H), 4.28 (q, J = 6.9 Hz, 2H), 6.69 (d, J = 1.8 Hz, 1H), 6.85 (dd, J = 1.8, 8.7 Hz), 7.74 (d, J = 8.7 Hz. 1H), 8.67 (s, 1H), 11.1 (brs, 1H);
¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 15.2, 61.9, 102.9, 111.5, 113.1, 115.1, 133.1, 150.4, 157.5, 158.1, 164.0, 165.2;
HRMS (ESI +) m / z: 257.0406 (calculated [M + H] +: 257.0426).

  (2) Synthesis of compound (12)

  Compound (11) (0.39 g, 1.6 mmol) was dissolved in 2N NaOH aqueous solution (5 mL) and stirred at room temperature for 12 hours. The mixture was acidified with 2N aqueous HCl. The precipitate was then collected and washed with water to give compound (12) (0.29 g, yield 87%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 6.74 (d, J = 2.1 Hz, 1H), 6.84 (dd, J = 2.1, 8.7 Hz, 1H), 7.74 (d, J = 8.7 Hz, 1H) , 8.68 (s, 1H), 11.1 (brs, 1H), 12.8 (brs, 1H);
¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 102.4, 111.7, 113.6, 115.1, 133.1, 150.5, 158.1, 158.6, 165.0, 165.3;
HRMS (ESI +) m / z: 206.0215 (calculated [M + H] +: 206.0220).

  (3) Synthesis of compound (13)

  Compound (12) (0.25 g, 1.2 mmol) and N-hydroxysuccinimide (0.21 g, 1.8 mmol) were dissolved in DMF (5 mL), and then WSCD · HCl (0.35 g, 1.8 mmol) was added. Added at 0 ° C. The mixture was stirred at 0 ° C. for 3 hours under Ar atmosphere. The reaction mixture was diluted with ethyl acetate, washed with 10% aqueous citric acid solution and water, and dried over sodium sulfate. Evaporation gave compound (13) (0.24 g, 66% yield).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.91 (s, 4H), 6.77 (d, J = 1.8 Hz, 1H), 6.89 (dd, J = 1.8, 8.7 Hz, 1H), 7.87 (d, J = 8.7 Hz, 1H), 9.00 (s, 1H), 11.4 (brs, 1H);
¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 102.4, 111.7, 113.6, 115.1, 133.1, 150.5, 158.1, 158.6, 165.0, 165.3;
HRMS (ESI +) m / z: 326.0263 (calculated value [M + H] +: 326.0277).

  (4) Synthesis of compound CA

  Ampicillin (0.35 g, 0.99 mmol) was dissolved in DMF (20 mL) and then diisopropylethylamine (DIEA) (1 drop) and compound (13) (0.20 g, 0.66 mmol) were added thereto at 0 ° C. did. The mixture was stirred for 10 hours. Then 10% aqueous citric acid solution was added and the mixture was extracted with ethyl acetate. The organic phase was washed with 10% aqueous citric acid solution and water and dried over sodium sulfate. Evaporation gave compound CA (coumalinyl ampicillin) (0.31 g, 88% yield).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ1.41 (s, 3H), 1.55 (s, 3H), 4.21 (s, 1H), 5.40 (d, J = 4.0 Hz, 1H), 5.56 (dd , J = 3.0 Hz, 7.7 Hz, 1H), 5.92 (d, J = 7.6 Hz, 1H), 6.83 (s, 1H), 6.88 (d, J = 8.4 Hz, 1H), 7.35 (m, 7H), 7.83 (d, J = 8.43 Hz, 2H), 9.38 (d, J = 7.6 Hz, 2H), 9.62 (d, J = 7.6 Hz, 2H), 11.1 (s, 1H), 13.4 (brs, 1H).

Synthesis of Compound CCD (1) Synthesis of Compound (1)

7-amino-3-chloromethyl-3-cephem-4-carboxylic acid p-methoxybenzyl ester hydrochloride (ACLE.HCl) (1.0 g, 2.5 mmol), BOC-glycine (480 mg, 2.7 mmol) and HOBt monohydrate (760 mg, 4.9 mmol) was dissolved in DMF (5 mL) followed by the addition of triethylamine (250 mg, 2.5 mmol) and WSCD.HCl (570 mg, 3.0 mmol) at 0 ° C. The mixture was stirred at 0 ° C. for 6 hours under Ar atmosphere. The reaction mixture was diluted with ethyl acetate and washed with saturated aqueous NaHCO ₃ , 10% aqueous citric acid and water, then dried over sodium sulfate. After evaporation, the residue was purified by silica gel column chromatography eluting with hexane / ethyl acetate (1: 1) to give compound (1) (1.25 g, yield 96%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.46 (s, 9H), 3.48 (d, J = 18.4 Hz, 1H), 3.65 (d, J = 18.4 Hz, 1H), 3.81 (s, 3H), 3.83 (m, 2H), 4.43 (d, J = 12.0 Hz, 1H), 4.54 (d, J = 12.0 Hz, 1H), 4.97 (d, J = 5.2 Hz, 1H), 5.23 (s, 2H), 5.84 (dd, J = 5.2 Hz, 8.8 Hz, 1H), 6.89 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H);
¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 27.1, 28.2, 43.2, 44.1, 55.2, 57.5, 58.9, 68.2, 80.5, 113.9, 125.5, 126.2, 126.6, 130.6, 156.0, 159.9, 161.1, 164.6, 170.2 , 171.2;
HRMS (FAB +) m / z: 526.1426 (calculated [M + H] +: 526.1415).

  (2) Synthesis of compound (2)

A mixture of compound (1) (560 mg, 1.1 mmol) and sodium iodide (1.6 g, 11 mmol) in acetone was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and diluted with ethyl acetate. The organic phase was washed with water and then dried over sodium sulfate. The obtained iodine derivative (light orange amorphous) was used in the next reaction without purification. Iodine derivative (400 mg, 0.65 mmol) was dissolved in DMF and then NaHCO ₃ (80 mg, 0.95 mmol) was added. A DMF solution of p-aminothiophenol (120 mg, 0.95 mmol) was added dropwise thereto. The resulting mixture was stirred at room temperature under Ar atmosphere for 5 hours. The mixture was diluted with ethyl acetate and washed with saturated aqueous NaHCO ₃ , 10% aqueous citric acid and water, then dried over sodium sulfate. After evaporation, the residue was purified by silica gel column chromatography eluting with hexane / ethyl acetate (1: 1) to give compound (2) (260 mg, yield 54% (2 steps)).

¹ H NMR (400 MHz, CDCl ₃ ) δ1.46 (s, 9H), 3.33 (d, J = 18.0 Hz, 1H), 3.59 (d, J = 13.6 Hz, 1H), 3.67 (d, J = 18.0 Hz, 1H), 3.75 (brs, 2H), 3.81 (s, 3H), 3.84 (m, 2H), 4.17 (d, J = 13.6 Hz, 1H), 4.87 (d, J = 4.9 Hz, 1H), 4.98 (d, J = 11.7 Hz, 1H), 5.07 (d, J = 11.7 Hz, 1H), 5.17 (brs, 1H), 5.84 (dd, J = 4.9 Hz, 8.8 Hz, 1H), 6.53 (d, J = 8.3 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 7.04 (d, J = 8.8 Hz, 1H), 7.15 (d, J = 8.3 Hz, 2H), 7.30 (d, J = 8.8 Hz, 2H);
¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 28.3, 28.6, 39.2, 44.3, 55.3, 57.5, 58.7, 67.6, 80.6, 113.9, 115.5, 120.6, 124.0, 127.0, 130.6, 130.7, 136.0, 147.1, 156.0 , 159.8, 161.4, 164.4, 170.0;
HRMS (FAB +) m / z: 615.1940 (calculated [M + H] +: 615.1947).

  (3) Synthesis of compound (3)

Compound (2) (270 mg, 0.44 mmol) and 4-dimethylaminoazobenzene-4′-carboxylic acid (130 mg, 0.48 mmol) were dissolved in pyridine (5 mL) and then POCl ₃ (74 mg, 0.48 mmol). Was added dropwise at −20 ° C. over 5 minutes. The mixture was stirred at −20 ° C. for 20 minutes under Ar atmosphere. The reaction mixture was diluted with ethyl acetate, washed with saturated aqueous NaHCO ₃ , 10% aqueous citric acid and water and dried over sodium sulfate. After evaporation, the residue was purified by silica gel column chromatography eluting with MeOH / CH ₂ Cl ₂ (0% → 1%) to give compound (3) (76 mg, 20% yield).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.45 (s, 9H), 3.11 (s, 6H), 3.40 (d, J = 18.0 Hz, 1H), 3.63 (d, J = 18.0 Hz, 1H), 3.80 (m, 3H), 4.15 (d, J = 13.3 Hz, 1H), 4.86 (d, J = 4.8 Hz, 1H), 5.04 (d, J = 11.8 Hz, 1H), 5.08 (d, J = 11.8 Hz , 1H), 5.24 (brs, 1H), 5.71 (dd, J = 4.8 Hz, 9.0 Hz, 1H), 6.76 (d, J = 9.2 Hz, 2H), 6.82 (d, J = 8.6 Hz, 2H), 7.09 (d, J = 9.0 Hz, 1H), 7.28 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 8.6 Hz, 2H), 7.54 (d, J = 8.6 Hz, 2H), 7.92 ( m, 6H), 8.03 (brs, 1H);
¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 28.3, 28.3, 38.0, 40.3, 44.3, 55.2, 57.5, 58.8, 67.7, 80.6, 111.5, 113.9, 120.6, 122.4, 124.4, 125.5, 126.9, 128.1, 128.4 , 129.4, 130.6, 134.3, 134.4, 138.1, 143.6, 152.9, 155.4, 156.0, 159.8, 161.4, 164.3, 165.2, 170.0;
HRMS (FAB +) m / z: 866.3000 (calculated [M + H] +: 866.3006).

  (4) Synthesis of compound (4)

Compound (3) (40 mg, 46 μmol) was dissolved in anhydrous CH ₂ Cl ₂ (4 mL), and thioanisole (800 μL) and trifluoroacetic acid (TFA) (2.4 mL) were added at 0 ° C. The mixture was stirred at 0 ° C. for 4 hours and then added to cold diethyl ether (15 mL). The precipitate was collected and washed with diethyl ether to obtain compound (4) (trifluoroacetate salt) (36 mg, quantitative).

¹ H NMR (400 MHz, CD ₃ OD) δ3.12 (s, 6H), 3.55 (d, J = 17.6 Hz, 1H), 3.76 (m, 3H), 3.93 (d, J = 13.6 Hz, 1H) , 4.33 (d, J = 13.6 Hz, 1H), 5.06 (d, J = 4.9 Hz, 1H), 5.72 (d, J = 4.9 Hz, 1H), 6.85 (d, J = 9.3 Hz, 2H), 7.45 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 8.8 Hz, 2H), 7.88 (t, J = 9.3 Hz, 4H), 8.04 (d, J = 9.3 Hz, 2H);
¹³ C NMR (100 MHz, DMF-d ₇ ) δ 28.4, 35.8, 40.2, 41.6, 58.4, 59.9, 112.3, 121.3, 121.4, 122.4, 125.9, 126.0, 129.5, 129.7, 132.2, 133.1, 135.9, 140.0, 143.9 , 154.0, 155.5, 162.9, 163.8, 165.0, 165.8, 167.9;
HRMS (FAB +) m / z: 646.1915 (calculated [M + H] +: 646.1906).

  (5) Synthesis of compound CCD

  Compound (4) (10 mg, 13 μmol) was dissolved in DMF (500 μL), and then triethylamine (TEA) (1 drop) and compound (13) (4.4 mg, 15 μmol) were added at room temperature. The mixture was stirred for 1 hour, then the solvent was evaporated and 100 mM aqueous ammonium formate (15 mL) was added. The precipitate was collected and washed with a 100 mM aqueous ammonium formate solution and water to obtain a compound CCD (10 mg, yield 91%).

¹ H NMR (400 MHz, DMF-d ₇ ) δ 3.13 (s, 6H), 3.61 (d, J = 18.1 Hz, 1H), 3.83 (d, J = 18.1 Hz, 1H), 4.02 (d, J = 13.2 Hz, 1H), 4.27 (d, J = 3.9 Hz, 2H), 4.34 (d, J = 13.2 Hz, 1H), 5.20 (d, J = 4.9 Hz, 1H), 5.83 (dd, J = 4.9 Hz , 8.8 Hz, 1H), 6.87 (d, J = 2.0 Hz, 1H), 6.91 (d, J = 9.3 Hz, 2H), 6.97 (dd, J = 2.4 Hz, 8.8 Hz, 1H), 7.47 (d, J = 8.8 Hz, 2H), 7.90 (m, 7H), 8.20 (d, J = 6.8 Hz, 2H), 8.84 (s, 1H), 9.01 (d, J = 8.8 Hz, 1H), 9.18 (t, J = 5.4 Hz, 1H), 10.5 (s, 1H), 11.5 (brs, 1H);
¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 27.3, 36.9, 39.8, 42.3, 57.7, 58.8, 101.8, 111.0, 111.6, 113.1, 114.4, 120.8, 121.6, 125.2, 128.9, 129.0 (two carbons), 129.6 , 131.6, 132.1, 134.9, 138.4, 142.7, 148.3, 152.9, 154.3, 156.4, 160.9, 161.6, 163.0, 163.9, 164.2, 164.9, 169.2;
HRMS (FAB +) m / z: 834.2010 (calculated [M + H] +: 834.2016).
[Plasmid and point mutation]
Omp-A WT (wild type) TEM-1 vector S1 (Thomas, VL; Golemi-Kotra, D .; Kim, C .; Vakulenko, SB; Mobashery, S .; Shoichet, BK Biochemistry. 2005, 44, 9330− 9338) was provided by Professor S. Mobashery (Georgia Institute of Technology). Point mutation in Omp-A WT TEM-1 is achieved by using QuikChange (registered trademark) Site-Directed Mutagenesis Kit (Stratagene) and using mutagenic primers of SEQ ID NOs: 9 and 10 according to the manufacturer's protocol. Plasmid pET-24a (+)-E166N TEM-1 was obtained. Sequenase DNA sequencing kit (BigDye Terminator) was used for sequencing of the point mutation double-stranded plasmid DNA containing the obtained pET-24a (+)-E166N TEM-1 (Glu at position 166 of TEM-1 was replaced with Asn). version 3.1; Applied Biosystems) and custom internal primers according to the manufacturer's protocol.

[Purification of mutant TEM-1 β-lactamase]
Escherichia coli E. coli in LB medium supplemented with 20 μg / mL kanamycin. In E. coli BL21 (DE3), a point mutation double-stranded plasmid containing E166N TEM-1 was cultured overnight. For enzyme purification, the culture was diluted 100-fold with Terrific Broth supplemented with 20 μg / mL kanamycin, 500 mM sorbitol and 2.5 mM betaine. After incubation for 4-5 hours with shaking (260-280 rpm) at 37 ° C. until the optical density of the culture reached 0.6 at 600 nm, isopropyl-β-D-thiogalactopyranoside (IPTG) was added. A final concentration of 0.4 mM was added and the culture was stirred at 25 ° C. overnight. Cells were pelleted by centrifugation and the supernatant containing the enzyme was concentrated by Vivaspin (membrane molecular weight cut-off, 10,000; Vivascience) and replaced with 10 mM Tris-HCl buffer (pH 7.0). The concentrated protein was loaded onto a DEAE Sephadex column equilibrated with the same buffer. The enzyme was eluted with a gradient TEM-1 β-lactamase using a gradient eluent (10-100 mM Tris-HCl buffer).

[Detection of protein label by SDS-PAGE]
WT (wild type) TEM-1 β-lactamase or E166N TEM-1 β-lactamase (20 μM) at 25 ° C. in a compound CA (30 μM) solution or compound CCD (30 μM) in 10 mM Tris-HCl buffer (pH 7.0). ) Added to the solution. After 30 minutes, the fluorescently labeled protein and the non-fluorescently labeled protein were dissolved in SDS gel loading buffer (1% SDS, 20% glycerol, 10% mercaptoethanol, pH 6.8), and SDS-PAGE. Separated by. After electrophoresis, a fluorescent image of the gel was taken with a digital camera under a fluorescence of 365 nm with a UV lamp. After taking a fluorescent image, the gel was stained with Coomassie Brilliant Blue, and then the stained gel image was taken. The obtained results are shown in FIG. 1 and FIG. Fig. 1 (a) shows the fluorescence image (i) and gel-stained image (ii) when E166NTEM-1 is labeled with CA, and Fig. 1 (b) shows the fluorescence when E166NTEM-1 is labeled with CCD. An image (i) and a gel-stained image (ii) are shown. Further, only compound CA (30 μM) or compound CCD (30 μM) was dissolved in SDS gel loading buffer (1% SDS, 20% glycerol, 10% mercaptoethanol, pH 6.8) and separated by SDS-PAGE. After electrophoresis, a fluorescent image of the gel was taken with a digital camera under a fluorescence of 365 nm with a UV lamp. The obtained fluorescence images are shown in FIG. 1 (a) (iii) for the compound CA and in FIG. 1 (b) (iii) for the compound CCD.

  2 (a) shows the fluorescence image (left) and gel-stained image (right) when CA is added to WTTEM-1, and FIG. 2 (b) shows the case where CCD is added to WTTEM-1. A fluorescence image (left) and a gel-stained image (right) are shown.

As shown in FIG. 1, it was confirmed that E166NTEM-1 was labeled when compound CCD was present or when compound CA was present. In addition, when E166NTEM-1 was labeled with Compound CA, in addition to the labeled protein-derived band, a band derived from the probe that was automatically hydrolyzed in the unreacted probe and buffer solution could be confirmed. On the other hand, when E166NTEM-1 was labeled with the compound CCD, almost no fluorescence other than the labeled protein could be confirmed. Therefore, it was confirmed that the labeling of E166NTEM-1 with CCD was very small in background such as unreacted probe as compared with CA. As shown in FIG. 2, compound CA and CCD were reacted with WTTEM-1. It was confirmed that both compounds were hydrolyzed by WTTEM-1.

[Fluorescence spectroscopy]
All fluorescent probes were dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. The relative fluorescence quantum yield of the probe is the area under the corrected fluorescence spectrum of the sample in 100 mM HEPES buffer (pH 7.4) and the area under the corrected fluorescence spectrum of quinine disulfate in 100 mM H ₂ SO ₄ aqueous solution. (According to the document Dawson, RW; Windsor, WMJ Phys. Chem. 1968, 72, 3251-3260, the quantum yield is 0.55). The obtained results are shown in Table 1.

[Enzyme assay]
Enzymatic assays of WT (wild type) and E166N TEM-1 were performed in 100 mM HEPES buffer (pH 7.4) at room temperature. The purified enzyme solution (15 μM, 5 μL) was added to the probe (1 μM, 500 μL) in HEPES buffer. The sample was excited at 410 nm and the fluorescence intensity at 450 nm was measured. The emission spectrum at 450 nm obtained for WT (wild strain) is shown in FIG. 3 (a), and the excitation spectrum at 410nm is shown in FIG. 3 (b). The emission spectrum at 450 nm obtained for E166N TEM-1 is shown in FIG. 4 (a), and the excitation spectrum at 410nm is shown in FIG. 4 (b).

  As shown in FIG. 3, when WT (wild type) β-lactamase and compound CCD were reacted, the fluorescence intensity increased with time. On the other hand, as shown in FIG. 4, when E166N TEM-1β lactamase is reacted with compound CCD, the fluorescence intensity increases with time. However, the degree of enhancement is higher than that of WT (wild type) β-lactamase. It was quite gradual. Therefore, E166N TEM-1β-lactamase is covalently labeled with compound CCD, whereas WT (wild-type) β-lactamase has a large fluorescence due to its catalytic hydrolysis. It was confirmed that it increased.

[MALDI-TOF MS analysis]
CCD stock solution (10 mM, 0.2 μL) was added at 25 ° C. to E166N TEM-1 solution (15 μM, 19.8 μL) in 10 mM Tris-HCl buffer (pH 7.0). After 16 hours, unreacted compound CCD was removed using Sephadex G-50 quick spin column (DNA-grade; GE Healthcare). Thereafter, a sample for MS was prepared using α-cyano-4-hydroxycinnamic acid as a matrix. The obtained results are shown in Table 2.

  As shown in Table 2, the molecular weight of E166N TEM-1β lactamase itself and the molecular weight of E166N TEM-1 fluorescently labeled with the compound CCD were confirmed. Therefore, the structure of these substances could be confirmed.

[Expression and Purification of Fusion Protein MBP-E166N TEM-1]
The DNA fragment of MBP was amplified by PCR from pMAL-c2 (New England Biolabs) and then treated with restriction enzymes with NdeI and NheI. Plasmid pET-21b (+)-MBP was obtained by ligating the MBP DNA fragment with pET-21b (+) subjected to the same restriction treatment. Next, pET-24a (+)-E166N TEM-1 to E166N TEM-1 nucleotide sequence was amplified by PCR and subjected to restriction enzyme treatment with EcoRI and HindIII. Similarly, the target plasmid pET-21b (+)-MBP-E166N TEM-1 was obtained by ligation with pET-21b (+)-MBP treated with restriction enzymes with EcoRI and HindIII. pET-21b (+)-MBP-E166N TEM-1 was obtained from E. coli in LB medium supplemented with 100 μg / mL ampicillin. After adding IPTG (100 μM) to growth cell culture medium (OD ₆₀₀ = 0.6 to 1.0) in E. coli BL21 (DE3), expression was performed at 25 ° C. for 20 hours. Cells were harvested by centrifugation, resuspended in column buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4) and lysed by sonication. The lysate was loaded onto an amylose resin column (New England Biolabs) and the fusion protein was eluted using elution buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 10 mM maltose, pH 7.4). Mammalian cell disruption solution and CCD (30 μM) were added to the obtained MBP-E166NTEM-1 (10 μM) and reacted at 25 ° C. in 10 mM Tris-HCl buffer (pH 7.0). After 45 minutes, the fluorescently labeled protein and the non-fluorescently labeled protein were dissolved in SDS gel loading buffer (1% SDS, 20% glycerol, 10% mercaptoethanol, pH 6.8), and SDS-PAGE. Separated by. After electrophoresis, a fluorescent image of the gel was taken with a digital camera under a fluorescence of 365 nm with a UV lamp. After taking a fluorescent image, the gel was stained with Coomassie Brilliant Blue, and then the stained gel image was taken. The obtained results are shown in FIG. FIG. 5 shows a fluorescence image (left) and a gel-stained image (right) when MBP-E166NTEM-1 is labeled with a CCD in a mammalian cell disruption solution.

  As shown in FIG. 5, when a mammalian cell disruption solution was added to the fusion protein MBP-E166N TEM-1 and labeled with a CCD, the CCD selectively labeled only the fusion protein. From these results, it was confirmed that even when E166NTEM-1 was fused with other proteins, its function was maintained and it did not react non-specifically with contaminating proteins.

[Labeling of Fusion Protein MBP-E166N TEM-1 by Compound CA]
Compound CA (30 μM) was added to MBP-E166N TEM-1 (10 μM) obtained as described above and reacted at 25 ° C. in 10 mM Tris-HCl buffer (pH 7.0). After 45 minutes, the fluorescently labeled protein and the non-fluorescently labeled protein were dissolved in SDS gel loading buffer (1% SDS, 20% glycerol, 10% mercaptoethanol, pH 6.8), and SDS-PAGE. Separated by. After electrophoresis, a fluorescence image of the gel was taken with a digital camera under irradiation of excitation light of 365 nm with a UV lamp. After fluorescent image capturing, the gel was stained with Coomassie Brilliant Blue, and the stained gel image was captured with a digital camera. The obtained result is shown in FIG. FIG. 6 shows a fluorescence image (left) and a gel-stained image (right) when MBP-E166N TEM-1 is labeled with compound CA.

  As shown in FIG. 6, it was confirmed that Compound CA can selectively fluorescently label mutant β-lactamase.

[Synthesis of Compound csDAm]
(1) Synthesis of 7-amino-4-methylquinolin-2 (1H) -one (compound cs124)

  1,3-phenylenediamine (3.22 g, 29.8 mmol) and ethyl acetoacetate (3.8 mL, 30.1 mmol) were heated to reflux at 130 ° C. for 48 hours. The mixture gradually solidified. Ethanol (5 mL) was added to the mixture, ground and filtered to give the crude product. The crude product was crystallized with methanol to give the title compound (2.52 g, 14.5 mmol, yield 48.5%, yellow needles.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ11.1 (s, 1H, 1), 7.33 (d, J = 8.3 Hz, 1H, 4), 6.44 (dd, J = 8.3 Hz, 2.0 Hz, 1H, 5), 6.36 (d, J = 2.0 Hz, 1H, 7), 5.93 (s, 1H, 2), 5.71 (s, 2H, 6, 2.27 (s, 3H, 3);
MS (ESI ⁺ ) Calculated C ₁₀ H ₁₁ N ₂ O 175.08, Measured 175.08.

  (2) Synthesis of compound (csDAm)

DTPA dianhydride (1.04 g, 2.91 mmol) and triethylamine (TEA, 275 mL) were dissolved in DMF (275 mL) to which TEA (2 mL), compound cs124 (0.572 g, 3.29 mmol) and A solution of ampicillin (Ampicillin, 1.09 g, 3.13 mmol) in DMF (50 ml) was added dropwise. The solvent was removed from the mixture under reduced pressure. The resulting product was purified by reverse phase HPLC as follows: The solution was loaded onto an Inertsil® ODS-3 column (10 × 250 mm), detected at a wavelength of 330 nm, and 0.1% formic acid. A 15-30% acetonitrile / H ₂ O gradient solution containing was eluted at a flow rate of 4.726 mL / min in 30 minutes. The product eluted at 29.5 minutes.

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.79 (s, 1H, 5), 7.66 (d, J = 8.8 Hz, 1H, 3), 7.50 (d, J = 8.8 Hz, 1H, 4), 7.24 ~ 7.34 (m, 6H, 1,16,1718,19,20), 5.48 (s, 1H, 15), 5.46 (d, J = 3.6 Hz, 1H, 22) 5.36 (dd, J = 1.2 Hz, 3.8 Hz, 1H, 21), 4.28 (s, 1H, 25), 3.06 to 3.73 (m, 28H, 6, 7, 8, 9, 10, 11, 12, 13, 14), 2.42 (s, 3H, 2 ), 1.47 (s, 3H, 23), 1.39 (s, 3H, 24);
MS (ESI +) Calculated C ₄₀ H ₄₉ N ₈ O ₁₃ S 881.31, Measured 881.32,
Calculated C ₄₀ H ₅₀ N ₈ O ₁₃ S 441.16, measured 441.16.

[Production and fluorescence measurement of compound in which rare earth ions are coordinated to compound csDAm]
The coordination of rare earth ions is adjusted in 0.1M HEPES buffer (pH 7.4) so that the final concentration of each compound csDAm and Ln (NO ₃ ) ₃ .6H ₂ O (Ln = Tb, Eu) is 10 μM. And vortexed at 25 ° C. for 1 hour. The measurement was performed after vortexing and diluted to a final probe concentration of 1 μM. FIG. 7A shows an absorption spectrum obtained when Ln is Tb ³⁺ , and FIG. 7B shows an absorption spectrum obtained when Ln is Eu ³⁺ . An enlarged view of FIG. 7A is shown in FIG. 7C, and an enlarged view of FIG. 7B is shown in FIG. 7D. As shown in FIGS. 7A to 7D, the absorption of the compound cs124 can be confirmed around 330 nm in the absorption spectrum. Therefore, the production | generation of the compound which the rare earth ion coordinated to compound csDAm has been confirmed.

FIG. 8A shows an excitation spectrum obtained when Ln is Tb ³⁺ , and FIG. 8B shows an excitation spectrum obtained when Ln is Eu ³⁺ . In FIG. 8A, λ _Em is 546 nm, and in FIG. 8B, λ _Em is 615 nm. As shown in FIG. 8A, similarly to the absorption spectrum of FIG. 7, it has a maximum value near 330 nm, and it was confirmed that rare earth ions were excited by the compound cs124 by this absorption. Further, as shown in FIG. 8B, similarly to the absorption spectrum of FIG. 7, it has a maximum value near 321 nm, and it was confirmed that the rare earth ions were excited by the compound cs124 by this absorption.

[Time-resolved fluorescence spectrum]
The compound csDAm and Ln (NO ₃ ) ₃ .6H ₂ O (Ln = Tb, Eu) were each adjusted to a final concentration of 10 μM in 0.1 M HEPES buffer (pH 7.4), and then at 25 ° C. for 1 hour. Vortexed. The measurement was performed after vortexing and diluted to a final probe concentration of 1 μM. When the sample is excited at 330 nm, the fluorescence spectrum by the steady state fluorescence measurement is shown in FIG. 9A, and the fluorescence spectrum by the time-resolved fluorescence measurement is shown in FIG. 9B. At that time, the delay time was measured at 60 μs and the gate time was measured at 2 ms. As shown in FIG. 9A, a peak derived from the compound cs124 was observed in the vicinity of 340 to 450 nm, and a sharp peak derived from the terbium ion was observed in the vicinity of 500 to 620 nm. Moreover, as shown in FIG.9 (b), the peak derived from compound cs124 of 350-450 nm vicinity disappeared, and it has confirmed that only the peak derived from the terbium ion which is rare earth ions was observed. A sharp peak derived from terbium ions around 500 to 620 nm is significantly stronger than a peak derived from compound cs124 around 340 to 450 nm. This is considered to originate from the high energy transfer and luminous efficiency from the compound cs124 to europium.

Further, by using _{Tb (NO 3) 3 · 6H} 2 O in place of _{Eu (NO 3) 3 · 6H} 2 O, the fluorescence spectrum was measured in the same manner by exciting at 321 nm. FIG. 10A shows a fluorescence spectrum obtained by steady-state fluorescence measurement, and FIG. 10B shows a fluorescence spectrum obtained by time-resolved fluorescence measurement. At that time, the delay time was measured at 60 μs and the gate time was measured at 2 ms. As shown in FIG. 10A, a peak derived from the compound cs124 was observed in the vicinity of 340 to 450 nm, and a sharp peak derived from the europium ion was observed in the vicinity of 500 to 620 nm. Moreover, as shown in FIG.10 (b), it has confirmed that the peak derived from the compound cs124 of 350-450 nm vicinity disappeared, and only the peak derived from the europium ion which is rare earth ions was observed.

[Fluorescence lifetime]
Compounds csDAm (1 mM, 20 μL) and Tb (NO ₃ ) ₃ .6H ₂ O (1 mM, 20 μL) were adjusted to a final concentration of 0.5 mM and vortexed at 25 ° C. for 1 hour. The measurement was performed after vortexing and diluted to a final probe concentration of 1 μM. Samples were excited at 330 nm and the fluorescence lifetime at 546 nm was measured. The result is shown in FIG. Further, Eu (NO ₃ ) ₃ .6H ₂ O was used instead of Tb (NO ₃ ) ₃ .6H ₂ O, excitation was performed at 321 nm, and the fluorescence lifetime at 615 nm was measured. The result is shown in FIG. From FIG. 11A, it was confirmed that the fluorescence lifetime when Ln is Tb was 1.46 ms, and from FIG. 11B, the fluorescence lifetime when Ln was Eu was 0.587 ms. That is, it was confirmed that when Ln is Tb or Eu, it has a long fluorescence lifetime on the order of ms.

[Detection of protein label by SDS-PAGE]
The final concentrations of WT (wild-type) TEM-1 β-lactamase or E166N TEM-1 β-lactamase, compounds csDAm and Ln (NO ₃ ) ₃ .6H ₂ O are 27.5 μM, 55.0 μM and 18.3 μM, respectively. Thus, WT (wild type) TEM-1 β-lactamase or E166N TEM-1 β-lactamase in 10 mM Tris-HCl buffer (pH 7.0) was treated with 10 mM Tris-HCl buffer (pH 7.0) at 25 ° C. ) And the Ln (NO ₃ ) ₃ .6H ₂ O solution in 10 mM Tris-HCl buffer (pH 7.0) to prepare a reaction solution. The reaction solution was incubated at 25 ° C. for 60 minutes, then dissolved in SDS gel loading buffer (1% SDS, 20% glycerol, 10% mercaptoethanol, pH 6.8) and separated by SDS-PAGE. After electrophoresis, a fluorescent image of the gel was taken with a digital camera under a fluorescence of 365 nm with a UV lamp. After taking a fluorescent image, the gel was stained with Coomassie Brilliant Blue, and then the stained gel image was taken. The obtained results are shown in FIG. 12 and FIG. FIG. 12 shows fluorescence images (left 4 lanes) and gel-stained images (right 4 lanes) when E166NTEM-1 is labeled with the compound csDAm coordinated with Ln, and FIG. 13 shows WT (wild strain). A fluorescence image (left two lanes) and a gel-stained image (right two lanes) when TEM-1 is labeled with a compound csDAm coordinated with Ln are shown.

  Samples in each lane in FIG. 12 were prepared by mixing the components as shown in Table 3 below.

  Samples in each lane in FIG. 13 were prepared by mixing the components as shown in Table 4 below.

  As shown in FIG. 12, in the vicinity of 29 kDa, which is the molecular weight of β-lactamase serving as the tag protein, green fluorescence derived from Tb ions in lane 1 and red fluorescence derived from Eu ions in lane 2 were confirmed. From this, it was confirmed that the compound csDAm coordinated with Ln was labeled with β-lactamase. In lanes 1 and 2, green fluorescence is also observed in the vicinity of 15 kDa, which is considered to be fluorescence of the compound [csDAm-Tb] coordinated with free Tb. Since the compound [csDAm-Eu] coordinated with Eu is weaker than the compound [csDAm-Tb] coordinated with Tb, the fluorescence derived from the compound [csDAm-Eu] coordinated with free Eu is used. Is not seen.

  As shown in FIG. 13, it can be confirmed that the β-lactamase of WT is not labeled with any of the compound [csDAm-Tb] coordinated with Tb and the compound [csDAm-Eu] coordinated with Eu. It was.

  The method of fluorescently labeling the protein of the present invention can be applied to cell imaging and the like.

SEQ ID NO: 1 E166N TEM-1 amino acid sequence SEQ ID NO: 2 E166A TEM-1 amino acid sequence SEQ ID NO: 3 E166D TEM-1 amino acid sequence SEQ ID NO: 4 E166Q TEM-1 amino acid sequence SEQ ID NO: 5 E166D TEM-1 amino acid sequence SEQ ID NO: 6 E166A Base sequence encoding amino acid sequence of TEM-1 SEQ ID NO: 7 E166D Base sequence encoding amino acid sequence of TEM-1 SEQ ID NO: 8 E166Q Base sequence encoding amino acid sequence of TEM-1 SEQ ID NO: 9 mutagenic primer SEQ ID NO: 10 mutagenic primer

Claims (14)

A method for fluorescently labeling proteins,
Obtaining a fusion protein of the target protein to be labeled and a mutant β-lactamase, and reacting the fusion protein with a compound represented by the following formula (I) or a salt thereof to fluorescently label the target protein: ,
The mutant β-lactamase opens the β-lactam ring of the compound represented by the formula (I) or a salt thereof, and as a result, liberates Y of the compound represented by the formula (I) or a salt thereof, And the fluorescent labeling method of protein which is a protein which can be covalently bonded with the part which the beta lactam ring of the said compound or its salt opened.

In the formula (I), X is a fluorescent group,
Y is a quenching group,
L ¹ is a linker for X
L ² is a linker for Y;
R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy. The amino acid sequence of the mutant β-lactamase in the fusion protein is
The amino acid sequence of any one of SEQ ID NOs: 1 to 4 in the Sequence Listing,
A compound represented by the above formula (I) in the fusion protein, wherein one to several amino acids of the amino acid sequences of SEQ ID NOs: 1 to 4 in the sequence listing are deleted, substituted, or added. Or an amino acid sequence capable of opening a β-lactam ring of a salt thereof, thereby releasing Y of the compound or a salt thereof, and covalently binding to a portion where the β-lactam ring of the compound or a salt thereof is opened ,as well as,
A β-lactam ring of a compound represented by the above formula (I) or a salt thereof in the fusion protein, the amino acid sequence having 70% or more homology with any one of SEQ ID NOs: 1-4 in the sequence listing Selected from the group consisting of an amino acid sequence capable of liberating Y of the compound or a salt thereof and capable of covalently bonding to a portion where the β-lactam ring of the compound or a salt thereof is opened. The method for fluorescent labeling a protein according to claim 1, which is an amino acid sequence.
Obtaining the fusion protein includes obtaining a polynucleotide encoding the fusion protein, obtaining a plasmid or vector capable of expressing the fusion protein, expressing the fusion protein in a cell, or expressing the fusion protein. The method for fluorescent labeling a protein according to claim 1 or 2, comprising isolating the fusion protein.
The composition for using in the method in any one of Claims 1-3 containing the compound or its salt represented by the said formula (I).

In the formula (I), X is a fluorescent group,
Y is a quenching group,
L ¹ is a linker for X
L ² is a linker for Y;
R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy.
A plasmid or vector for use in the method of any one of claims 1-3,
The plasmid or vector is a plasmid or vector for cloning a polynucleotide encoding a fusion protein of a protein to be labeled and a mutant β-lactamase or for expressing the fusion protein,
The plasmid or vector is
A nucleotide sequence in which one to several bases of the nucleotide sequence of any one of SEQ ID Nos. 5 to 8 in the sequence listing are deleted, substituted, or added. Opening the β-lactam ring of the compound represented by formula (I) or a salt thereof in the fusion protein, thereby releasing Y of the compound and opening the β-lactam ring of the compound or salt thereof. A base sequence encoding an amino acid sequence that can be covalently bonded to the ring portion; and
A base sequence having 70% or more homology with any one of SEQ ID NOs: 5 to 8 in the Sequence Listing, wherein the β-lactam ring of the compound represented by formula (I) or a salt thereof in the fusion protein Selected from the group consisting of a nucleotide sequence that encodes an amino acid sequence that can be ring-opened, thereby liberating Y of the compound and covalently binding to a portion of the compound or salt thereof that is open at the β-lactam ring. A plasmid or vector containing the nucleotide sequence.

In the formula (I), X is a fluorescent group,
Y is a quenching group,
L ¹ is a linker for X
L ² is a linker for Y;
R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy.
A kit for a method for fluorescent labeling of a protein comprising the composition according to claim 4 and the plasmid or vector according to claim 5.
A compound represented by the general formula (I ′) or a salt thereof.

In the formula (I), X is a fluorescent group,
Y is a quenching group,
L ¹ is a linker for X
L ² '

Or -CH = CH-
A linker for Y represented by:
R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy.
A method for fluorescently labeling proteins,
By obtaining a fusion protein of a protein to be labeled and a mutant β-lactamase, and reacting the fusion protein with a compound represented by the following formula (II) or a compound represented by the following formula (III) or a salt thereof, Including fluorescently labeling the protein of interest,
The mutant β-lactamase opens the β-lactam ring of the compound represented by the formula (II) or the compound represented by the following formula (III) or a salt thereof, and the β-lactam ring of the compound or a salt thereof. A method for fluorescently labeling proteins, wherein is a protein that can be covalently bound to a ring-opened moiety.

In the formula (II) and formula (III),
X is a fluorescent group,
L ¹ is a linker for X
R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy,
R ⁴ is H or lower alkyl. The amino acid sequence of the mutant β-lactamase in the fusion protein is
The amino acid sequence of any one of SEQ ID NOs: 1 to 4 in the Sequence Listing,
A compound represented by the above formula (II) in the fusion protein, wherein one to several amino acids of the amino acid sequence of SEQ ID NO: 1 to 4 in the sequence listing are deleted, substituted, or added. Alternatively, the β-lactam ring of the compound represented by formula (III) or a salt thereof is opened, and as a result, Y of the compound or the salt thereof is liberated, and the β-lactam ring of the compound or the salt thereof is opened. An amino acid sequence that can be covalently bound to the moiety, and
An amino acid sequence having 70% or more homology with any one of the amino acid sequences of SEQ ID NOs: 1 to 4 in the sequence listing, represented by the compound represented by the above formula (II) or the formula (III) in the fusion protein An amino acid capable of opening a β-lactam ring of a compound or a salt thereof, thereby liberating Y of the compound or a salt thereof, and covalently binding to a portion where the β-lactam ring of the compound or a salt thereof is opened The protein fluorescent labeling method according to claim 8, wherein the protein is an amino acid sequence selected from the group consisting of:   Obtaining the fusion protein includes obtaining a polynucleotide encoding the fusion protein, obtaining a plasmid or vector capable of expressing the fusion protein, expressing the fusion protein in a cell, or expressing the fusion protein. The method for fluorescent labeling a protein according to claim 8 or 9, comprising isolating the fusion protein. A composition for use in the method according to any one of claims 8 to 10, comprising the compound represented by the formula (II), the compound represented by the following formula (III), or a salt thereof.

In the formula (II) and formula (III),
X is a fluorescent group,
L ¹ is a linker for X
R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy,
R ⁴ is H or lower alkyl. A plasmid or vector for use in the method of any of claims 8-10,
The plasmid or vector is a plasmid or vector for cloning a polynucleotide encoding a fusion protein of a protein to be labeled and a mutant β-lactamase or for expressing the fusion protein,
The plasmid or vector is
A nucleotide sequence in which one to several bases of the nucleotide sequence of any one of SEQ ID Nos. 5 to 8 in the sequence listing are deleted, substituted, or added. Opening the β-lactam ring of the compound represented by formula (II) or the compound represented by formula (III) or a salt thereof in the fusion protein, thereby liberating Y of the compound, and the compound Or a base sequence that encodes an amino acid sequence that can be covalently bonded to a portion of the salt whose β-lactam ring is opened, and
A compound represented by the formula (II) or a compound represented by the formula (III) in the fusion protein, the nucleotide sequence having 70% or more homology with any one of the sequence numbers 5 to 8 in the sequence listing Alternatively, it encodes an amino acid sequence that opens the β-lactam ring of a salt thereof, thereby liberating Y of the compound and that can be covalently bonded to the opened portion of the β-lactam ring of the compound or a salt thereof. A plasmid or vector comprising a base sequence selected from the group consisting of a base sequence.

In the formula (II) and formula (III),
X is a fluorescent group,
L ¹ is a linker for X
R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy,
R ⁴ is H or lower alkyl.   A kit for a method for fluorescent labeling of a protein, comprising the composition according to claim 11 and the plasmid or vector according to claim 12. A compound represented by the general formula (II ′) or a salt thereof.

In the formula (II ′),
X ′ is a luminescent rare earth complex,
L ¹ is a linker for X
R ¹ is H, a lower alkylene group or a lower alkyl group substituted with lower alkylcarbonyloxy.

Images