JP3815051B2 - Novel peptide derivatives - Google Patents

Description

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【図面の簡単な説明】
【図１】実施例６で得られた、本発明の基質を用いたプロテインチロシンホスファターゼ活性に基づく蛍光強度変化を示す図である。
【図２】実施例７で得られた、本発明の基質を用いたプロテインチロシンホスファターゼ活性に基づく蛍光強度変化を示す図である。
【図３】実施例８で得られた、本発明の基質を用いたプロテインチロシンホスファターゼ活性に基づく蛍光強度変化を示す図である。
【図４】実施例８で得られたプロテインチロシンホスファターゼの濃度と脱リン酸化率（％）との関係を示す検量線である。
【図５】実施例９で得られた、本発明の基質を用いたプロテインチロシンホスファターゼ活性に基づく蛍光強度変化を示す図である。
【図６】実施例１０で得られた、基質濃度（横軸）と所定基質濃度で得られた初速度（縦軸）との関係を表すグラフである。[0001]
[Field of the Invention]
The present invention relates to a peptide derivative useful for measuring protein phosphatase activity and a method for measuring protein phosphatase activity using the peptide derivative as a substrate.
[0002]
BACKGROUND OF THE INVENTION
Protein phosphorylation by protein kinase plays an important role in information transmission between cells, control of cell growth and differentiation, etc. (Bishop, J.M., Cell (1991),64, 235-248). This reaction is reversibly controlled by dephosphorylation by protein phosphatase. In this sense, protein phosphatase research is important along with protein kinase research.
[0003]
In order to study protein phosphatase, a substrate for measuring its activity and a measuring method are required. Conventionally used substrates and measuring methods are as follows.
[0004]
1) Using a peptide derivative containing a phosphorylated amino acid residue as a substrate, the phosphate ion released by the action of protein phosphatase is quantified by a colorimetric method (Cho, H. et al., Biochemistry (1991),306210-6216). The detection limit of phosphate ions by this method is about 1 nanomolar, but there is a problem that the measurement is disturbed if phosphate ions are mixed in the enzyme sample.
[0005]
2) Using a peptide derivative containing a phosphorylated amino acid residue as a substrate, the absorbance or fluorescence intensity that increases with the dephosphorylation reaction by the action of protein phosphatase is measured (Jao, Z. et al., Analytical Biochemistry (1992),201, 361-366). The detection limit of the dephosphorylation reaction in this method is about 1 nmole by the absorbance method and about 10 picomoles by the fluorescence method. This method has a drawback that when measuring a sample derived from a living body, it cannot be accurately measured due to the influence of various coexisting substances mixed in the sample.
[0006]
3) Using a peptide derivative or protein labeled with radioactive phosphate as a substrate, the radioactivity of phosphate ions released by the action of protein phosphatase is measured (Tonks, N.K. et al., J. Biological Chemistry (1988),263, 6722-6730). If the specific activity of radioactive phosphate is 1,000 counts per minute per picomole, the detection limit of phosphate ions in this method is about 0.1 picomolar. Although this method has the advantage of being able to measure even a weak enzyme activity because of its high detection sensitivity, it has the disadvantages that the preparation of the substrate is complicated and that the substrate cannot be stored for a long time due to the decay of radioactivity.
[0007]
As described above, the protein phosphatase activity measurement methods that have been adopted so far have advantages and disadvantages, which are not always satisfactory, and it is desirable to develop a simple, highly accurate and safe measurement method that does not use radioactive substances. It was.
[0008]
[Problems to be Solved by the Invention]
The present invention has been made in view of the above situation, and an object thereof is to provide a peptide derivative useful as a substrate for protein phosphatase activity measurement, a protein phosphatase activity measurement method using the peptide derivative as a substrate, and a reagent therefor.
[0009]
  The present inventionGeneral formula [1]
                  A ₁ -X-A ₂ [1]
(In the formula, X is an amino acid residue having a phosphate group introduced therein, and A ₁ And A ₂ Is an amino acid residue or peptide chain as shown below, one of which has a quenching group at its terminal or side chain part and the other has its own fluorescence at its terminal or side chain part. And having a fluorescent group having the property that the fluorescence is quenched by the quenching group in the molecule, ₁ Is an amino acid residue or a peptide chain consisting of 2 to 20 amino acid residues bonded to X, and the amino group at the amino terminus thereof is amino-protected when no fluorescent group or quenching group is bonded. An amino group protected by a group or a free amino group, and A ₂ Is a peptide chain consisting of amino acid residues or 2 to 20 amino acid residues bonded to X, and the carboxyl group at the carboxyl terminus is carboxyl-protected when no fluorescent group or quenching group is bonded. A carboxyl group protected by a group or a free carboxyl group. And a peptide derivative having 1 to 39 amino acid residues between an amino acid residue having a fluorescent group and an amino acid residue having a quenching group, wherein the amino acid sequence of the peptide derivative is a fluorescent group One of the peptide bonds existing between the amino acid residue having an amino acid residue and the amino acid residue having a quenching group is the phosphate group of the amino acid residue into which the phosphate group represented by X is introduced by protein phosphatase Peptide derivatives that are sequences that can be selectively cleaved by proteases when dephosphorylatedAnd a method for measuring the activity of protein phosphatase using the same as a substrate, and a reagent therefor.
[0010]
That is, the present inventor has conducted intensive research to develop a safe protein phosphatase activity measuring method that is simple, highly accurate, and does not use radioactive substances. As a result, if the peptide derivative as described above is used as a substrate, dephosphorylation is possible. Until then, the fluorescence is quenched by the quenching group, but after dephosphorylation, fluorescence is generated by reacting with an appropriate protease to cleave the fluorescent group and the quenching group, and the fluorescence intensity is reduced. It has been found that protein phosphatase activity can be measured simply and with high accuracy if used, and the present invention has been completed.
[0011]
The fluorescent group according to the present invention is a group having fluorescence and the property that the fluorescence is quenched by the quenching group in the molecule, and varies depending on the type of quenching group present in the molecule. For example, (7-methoxycoumarin-4-yl) acetyl group, 5-[(2-aminoethyl) amino] naphthalene-1-sulfonic acid group, 9,10-dioxa-syn-3,4,6,7 -Tetramethylbimane group etc. are mentioned. These fluorescent groups are appropriately added to the α-amino group or α-carboxyl group of the terminal amino acid or the side chain of the amino acid residue, such as an amino group, a carboxyl group, and a thiol group, according to the type of the fluorescent group. What is necessary is just to combine. An appropriate spacer may be used for bonding.
[0012]
The quenching group according to the present invention is only required to quench the fluorescence caused by the fluorescent group present in the molecule. Specific examples thereof include, for example, a dinitrophenyl group, a 4- (4-dimethylaminophenylazo) benzoyl group. , 4- (4-dimethylaminophenylazo) sulfonyl group and the like. These quenching groups are appropriately added to the α-amino group or α-carboxyl group of the terminal amino acid or the side chain of the amino acid residue, for example, amino group, carboxyl group, thiol group, etc. according to the type of quenching group. What is necessary is just to combine. An appropriate spacer may be used for bonding.
[0013]
  Examples of the peptide derivative of the present invention include those represented by the following general formula.
General formula [1]
                  A₁-X-A₂ [1]
(WhereX is an amino acid residue having a phosphate group introduced, and A ₁ And A ₂ Is an amino acid residue or peptide chain as shown below, one of which has a quenching group at its terminal or side chain part and the other has its own fluorescence at its terminal or side chain part. And having a fluorescent group having the property that the fluorescence is quenched by the quenching group in the molecule, ₁ Is an amino acid residue or a peptide chain consisting of 2 to 20 amino acid residues bonded to X, and the amino group at the amino terminus thereof is amino-protected when no fluorescent group or quenching group is bonded. An amino group protected by a group or a free amino group, and A ₂ Is a peptide chain consisting of amino acid residues or 2 to 20 amino acid residues bonded to X, and the carboxyl group at the carboxyl terminus is carboxyl-protected when no fluorescent group or quenching group is bonded. A carboxyl group protected by a group or a free carboxyl group. And a peptide derivative having 1 to 39 amino acid residues between an amino acid residue having a fluorescent group and an amino acid residue having a quenching group, wherein the amino acid sequence of the peptide derivative is a fluorescent group One of the peptide bonds existing between the amino acid residue having an amino acid residue and the amino acid residue having a quenching group is the phosphate group of the amino acid residue into which the phosphate group represented by X is introduced by protein phosphatase A peptide derivative that is a sequence that can be selectively cleaved by a protease when dephosphorylated.
[0014]
In general formula [1], A₁Or A₂The amino acid residue having the quenching group represented by the above may be protected as long as it is an amino acid residue not having a phosphate group and a fluorescent group and having a quenching group bonded thereto. . Further, the peptide chain having the quenching group may be any peptide chain including the amino acid residue having the quenching group as described above and having no phosphate group and no fluorescent group. A₁Or A₂When is a peptide chain, the position of the amino acid residue having a quenching group is not particularly limited. Furthermore, the number of amino acid residues in the peptide chain is preferably 2 to 20, including 2 to 10, more preferably 2 to 5 including amino acid residues having a quenching group.
[0015]
In general formula [1], A₁Or A₂The amino acid residue having a fluorescent group represented by the above may be protected as long as it is an amino acid residue having neither a phosphate group nor a quenching group and having a fluorescent group bonded thereto. . The peptide chain having a fluorescent group may be any peptide chain including an amino acid residue having a fluorescent group as described above and having no phosphate group and no quenching group. A₁Or A₂When is a peptide chain, the position of the amino acid residue having a quenching group is not particularly limited. Furthermore, the number of amino acid residues in the peptide chain is preferably 2 to 20, more preferably 2 to 10, more preferably 2 to 5, including amino acid residues having a fluorescent group.
[0016]
In the peptide derivative of the present invention, examples of the protecting group of the amino acid residue or peptide chain which may be protected include a carboxyl terminal protecting group and an amino terminal protecting group. Examples of the carboxyl terminal protecting group include lower alkoxy groups such as methoxy group, ethoxy group, and t-butoxy group, and aromatic hydrocarbon groups such as benzyl group and pentamethyldihydrobenzofuranyl group. The terminal carboxyl group may be an amide group in order to avoid the influence of carboxypeptidase that may be present in the biological sample. Specific examples of the amino-terminal protecting group include t-butoxycarbonyl group, benzyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl group, 2-phenyl-2-propyl group, acetyl group and the like.
[0017]
Examples of X in the general formula [1] include phosphorylated tyrosine residues, phosphorylated serine residues, phosphorylated threonine residues, and the like phosphorylated amino acid residues having a hydroxyl group in the side chain. .
[0018]
  The amino acid sequence of the peptide derivative of the present invention only needs to meet the purpose of the present invention. More specifically, the type of protein phosphatase to be measured, the amino acid residue having a fluorescent group, and the amino acid residue having a quenching group It may be appropriately selected according to the type of protease that selectively cleaves the peptide bond between and. The total number of amino acid residues of the peptide derivative of the present invention is usually 3 to 3.41The number is preferably 3 to 22, more preferably 3 to 12.
[0019]
These amino acid sequences may be replicated within an appropriate range including the site of phosphorylation of a naturally occurring protein that is phosphorylated by a protein kinase. It should be noted that a replica of such a naturally occurring material may be used as it is, but it is desirable to use it by appropriately modifying it.
[0020]
That is, for example, when chymotrypsin having a property of selectively cleaving the carboxy terminal side of an amino acid residue having an aromatic ring such as a tyrosine residue is used as a protease, the peptide derivative of the present invention includes, for example, phosphorylated tyrosine. Residues etc. are necessary, but other amino acid residues may react with chymotrypsin, such as tyrosine residues, tryptophan residues, phenylalanine residues, leucine residues, isoleucine residues, It is desirable not to include asparagine residues, threonine residues, methionine residues, glutamine residues, valine residues, etc., especially tyrosine residues, tryptophan residues, phenylalanine residues, leucine residues, isoleucine residues. . When the natural type contains such amino acid residues, it is desirable to incorporate amino acid residues that are difficult to react with chymotrypsin, such as alanine residues, in place of these amino acid residues.
[0021]
In the peptide derivative of the present invention, if the number of amino acid residues present between the amino acid residue having a quenching group and the amino acid residue having a fluorescent group is too large, the quenching action of the quenching group is weakened. Therefore, the number of amino acid residues present between the amino acid residue having a quenching group and the amino acid residue having a fluorescent group is 1 to39It is desirable that the number is 1, 20, more preferably 1-10.
[0022]
The amino acid sequence of the peptide derivative of the present invention is the amino acid residue having a fluorescent group after the phosphate group of the amino acid residue introduced with the phosphate group represented by X is desorbed and dephosphorylated by protein phosphatase. One of the peptide bonds existing between the group and the amino acid residue having a quenching group is a sequence that can be selectively cleaved by a protease.
Examples of the protein phosphatase in the present invention include protein tyrosine phosphatase, protein serine phosphatase, protein threonine phosphatase and the like.
The protease varies depending on the type of amino acid residue having a phosphate group, and examples thereof include chymotrypsin, trypsin, papain, and plasmin.
[0023]
Specific examples of the peptide derivative of the present invention include the following.
Flu-Gly-Asp-Ala-Glu-pTyr-Ala-Ala-Lys (Inh) -Arg-NH₂
Flu-Gly-Glu-Gly-Thr-pTyr-Gly-Lys (Inh) -Arg-NH₂
Flu-Gly-Glu-Val-Asn-pTyr-Glu-Glu-Lys (Inh) -Arg-NH₂
Flu-Gly-Glu-Pro-Gln-pTyr-Gln-Pro-Lys (Inh) -Arg-NH₂
Flu-Gly-Glu-Lys-Glu-pTyr-His-Ala-Lys (Inh) -Arg-NH₂
Flu-Gly-Asp-Gly-Val-pTyr-Ala-Ala-Lys (Inh) -Arg-NH₂
Flu-Gly-Ser-Ala-pTyr-Gly-Lys (Inh) -Arg-NH₂
Flu-Gly-Gly-Ser-pTyr-Ser-Lys (Inh) -Arg-NH₂
Flu-Gly-Arg-Val-Phe-pSer-Lys (Inh) -Arg-NH₂
Flu-Gly-Asp-Arg-Phe-pThr-Lys (Inh) -Arg-NH₂
Flu-Gly-Arg-Lys-Phe-pThr-Lys (Inh) -Arg-NH₂
Flu-Gly-Pro-Gly-Phe-pSer-Lys (Inh) -Arg-NH₂
Flu-Gly-Thr-Arg-Phe-pSer-Lys (Inh) -Arg-NH₂
Flu-Gly-Lys-Arg-Phe-pSer-Lys (Inh) -Arg-NH₂
(Flu: fluorescent group, pTyr: O-phosphotyrosine residue, pSer: O-phosphoserine residue, pThr: O-phosphothreonine residue, Inh: quenching group, Lys (Inh): quenching group-bound lysine residue. The abbreviation is an abbreviation for a conventional amino acid residue.)
[0024]
The peptide derivative of the present invention uses a post-phosphorylation method or a pre-phosphorylation method, which is a conventional method in the synthesis of a peptide derivative containing a phosphorylated amino acid residue, using an amino acid having a fluorescent group and an amino acid having a quenching group as starting materials. It can be synthesized by the method. The Post phosphorylation method is a method of phosphorylating hydroxy amino acid residues after peptide chain construction, and the Pre phosphorylation method is a method of synthesizing a pre-protected phosphoamino acid derivative and incorporating it into a peptide chain as a building block. In general, however, the pre-phosphorylation method is desirable, particularly when the peptide chain contains amino acid residues that are vulnerable to oxidation, such as methionine residues, cysteine residues, tryptophan residues, and the like.
[0025]
As a method for phosphorylating amino acid residues, 1 to 20 equivalents of phosphoric acid halides such as sulfonyl chloride and phosphorus trichloride are phosphorylated with respect to the amino acid residue to be phosphorylated and the free hydroxyl group of the amino acid residue. The agent is reacted in a solvent (anhydrous) such as dimethylformamide (DMF) or dimethylsulfoxide (DMSO), for example, with an alkaline substance such as pyridine, triethylamine or NaH as a catalyst at 0 to 40 ° C. for 1 to 24 hours. A method is mentioned.
[0026]
The peptide chain may be constructed in accordance with a conventional method usually used in this field, and amino acids that may be protected are bonded one by one from the carboxyl terminal side or amino terminal side of the main chain. Alternatively, those synthesized by dividing into several fragments may be combined.
[0027]
The activation of the carboxyl group for generating a peptide bond may be performed according to a conventional method usually used in this field, such as an azide method, a mixed acid anhydride method, a dicyclohexylcarbodiimide method, or an active ester method.
[0028]
The reaction temperature is usually −30 to 50 ° C., preferably −10 to 25 ° C.
While the reaction time varies depending on the synthesis method, it is generally 10 minutes to 24 hours, preferably 30 minutes to 2 hours.
[0029]
For example, Mca-Gly (Mca: (7-methyloxycoumarin-4-yl) acetyl group) is used as an amino acid having a fluorescent group, and Fmoc-Tyr (PO is used as a phosphorylated amino acid._ThreeH₂) (Fmoc: 9-fluorenylmethyloxycarbonyl group) and Fmoc-Lys (DNP) (DNP: dinitrophenyl group) is used as an example of an amino acid having a quenching group.
[0030]
Mca-Gly is an example of a method for synthesizing Mca-Pro-Leu (C.G. Knight et al., FEBS Letters (1992)., 296, 263-266). Fmoc-Tyr (PO_ThreeH₂) May be a commercially available product (for example, manufactured by Nova Biochem).
[0031]
Fmoc-Lys (DNP) is a conventional method (H. Nagase et al., Journal of Biological Chemistry (1994),269, 20952-20957). The peptide chain of the whole peptide derivative of the present invention is constructed by a method of constructing a peptide chain without protecting the phosphate group (E.A.Ottinger, L.L.Shekels, D.A.Bernlohr and G.Barany, Biochemistry (1993))., 32, 4353-4361 etc.) and is similarly carried out by solid phase synthesis. Operations other than those described above and post-treatment methods may be performed in accordance with methods known per se.
[0032]
In the peptide derivative of the present invention, the fluorescent group having fluorescence itself is bonded to the amino acid residue constituting the peptide derivative, and the fluorescence is quenched by the quenching group present in the peptide derivative molecule. ing. However, the phosphoryl group is eliminated from the peptide derivative by dephosphorylation by protein phosphatase, and then between the amino acid residue having a fluorescent group and the quenching group of the dephosphorylated peptide derivative. When the peptide bond is cleaved by a protease, the fluorescent group and the quenching group are cleaved, and the quenching group does not coexist in the peptide molecule or amino acid residue to which the fluorescent group is bound. , The fluorescence originally possessed by the fluorescent group is restored, and fluorescence is emitted.
[0033]
When the peptide derivative of the present invention is used as a substrate, activities of protein tyrosine phosphatase, protein serine phosphatase, protein threonine phosphatase and the like in biological samples such as serum, plasma, and cerebrospinal fluid can be easily and accurately measured.
[0034]
The activity measurement may be performed as follows, for example.
That is, an appropriate buffer containing an appropriate amount of the peptide derivative of the present invention is incubated in advance at an appropriate temperature, and a biological sample such as serum, plasma, cerebrospinal fluid is added to this, and further 1-30 at the same temperature. Incubate for minutes. Next, a protein phosphatase inhibitor such as sodium vanadate solution is added thereto to terminate the enzyme reaction. When the phosphate group of the phosphorylated amino acid residue contained in the peptide derivative, such as chymotrypsin, trypsin, papain and plasmin, is removed from this solution, an amino acid residue having a quenching group and a fluorescent group are bound. A protease that specifically cleaves one of the peptide bonds existing between the amino acid residues is added, and the resulting fluorescence intensity is measured. By applying the obtained fluorescence intensity to a calibration curve representing the relationship between the fluorescence intensity and the protein phosphatase activity obtained in the same manner except that a protein phosphatase solution with a known concentration was used as a sample, the protein in the sample Phosphatase activity can be determined.
[0035]
The concentration of the peptide derivative of the present invention at this time varies depending on how much the calibration range is set, but is usually 0.1 to 100 μM, preferably 1 to 10 μM, as the concentration at the time of reaction with protein phosphatase. Although the pH at the time of making protein phosphatase react may be the range which can implement the activity measurement of protein phosphatase, it is 4-9 normally, Preferably it is 5-8. Moreover, the temperature to incubate is 20-40 degreeC normally. The concentration of the protein phosphatase inhibitor varies slightly depending on the type of inhibitor, but is usually 0.01 to 10 mM, preferably 0.1 to 1 mM as the concentration during the reaction. The concentration of the protease varies slightly depending on the type of protease, but the concentration during the reaction is usually 0.1 to 100 μg / ml, preferably 1 to 50 μg / ml.
[0036]
In addition, when the protein phosphatase to be measured is not inactivated by the protease used, the protein phosphatase activity can also be obtained by the following method.
[0037]
That is, an appropriate buffer containing an appropriate amount of the peptide derivative of the present invention and a protease having the above-mentioned properties is incubated at an appropriate temperature in advance, and a biological sample such as serum, plasma, spinal fluid or the like is added thereto. The amount of change in fluorescence intensity per unit time is determined while further incubating at the same temperature. By fitting the obtained fluorescence intensity change amount to a calibration curve representing the relationship between the fluorescence intensity change amount and the protein phosphatase activity obtained in the same manner as above except that a protein phosphatase solution with a known concentration was used as a sample. Protein phosphatase activity in the sample can be determined.
[0038]
The concentration of the peptide derivative of the present invention at this time varies depending on how much the calibration range is set, but is usually 0.1 to 100 μM, preferably 1 to 10 μM, as the concentration at the time of reaction with protein phosphatase. Although the pH at the time of making protein phosphatase react may be the range which can implement the activity measurement of protein phosphatase, it is 4-9 normally, Preferably it is 5-8. The incubation temperature is usually 20 to 40 ° C. The concentration of protease varies slightly depending on the type of protease, but the concentration during the reaction is usually 0.1-100 μg / ml, preferably 1-50 μg / ml.
[0039]
The reagent for measurement of the present invention contains the peptide derivative of the present invention as described above as a main component, and is used for measuring protein phosphatase activity in biological samples such as serum and plasma and tissue extracts, for example. Therefore, preferred aspects and specific examples of the respective constituent elements are as described above.
[0040]
In addition, the reagent can be provided in a lyophilized state or in a solution state, and in addition to the above-mentioned surfactants and antiseptics that are usually used in this field, as long as they do not inhibit the protein phosphatase activity measurement. An agent, a buffering agent and the like may be contained, and the concentration used thereof may be appropriately selected according to the concentration usually used in this field.
[0041]
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
In the examples, the following abbreviations were used.
Fmoc: 9-fluorenylmethyloxycarbonyl group
Mca: (7-methoxycoumarin-4-yl) acetyl group
pTyr: O-phosphotyrosine residue
Lys (DNP): Nε-dinitrophenyllysine residue
Pbf: Pentamethyldihydrobenzofuranyl group
^tBu: t-butyl group
PyBOP: Benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
HOBt: 1-hydroxybenzotriazole
DIEA: Diisopropylethylamine
TFA: trifluoroacetic acid
[0042]
【Example】
Example 1. Mca-Gly-Asp-Ala-Glu-pTyr-Ala-Ala-Lys (DNP) -Arg-NH₂Synthesis of (peptide derivative of the present invention)
Mca-Gly, Fmoc-Asp (O^tBu), Fmoc-Ala, Fmoc-Glu (O^tBu), Fmoc-Tyr (PO_ThreeH₂), Fmoc-Lys (DNP) and Fmoc-Arg (Pbf), and using PyBOP, HOBt and DIEA as a binding reagent, a method for constructing a peptide chain without protecting the phosphate group (EAOttinger, LLShekels, DA Bernlohr and G. Barany, Biochemistry, (1993),32, 4353-4361 etc.). That is, first, 10 equivalents on 10 mg of 4- (2 ′, 4′-dimethoxyphenyl-Fmoc-aminomethyl) -phenoxyacetamido-norleucyl-methylbenzhydrylamine resin (manufactured by Watanabe Chemical Co., Ltd.) (hereinafter abbreviated as MBHA resin). A DMF solution containing a predetermined protected amino acid residue, 10 equivalents of PyBOP, 10 equivalents of HOBt, and 20 equivalents of DIEA were added, and amino acid residues were sequentially introduced by performing a coupling reaction at room temperature for 1 hour. The order of the introduced amino acid residues is Fmoc-Arg (Pbf) (Watanabe Chemical Co., Ltd.), Fmoc-Lys (DNP) (references (H. Nagase et al., J. Biological chemistry, (1994) ,269, 20952-20957). ), Fmoc-Ala (Watanabe Chemical Co., Ltd.), Fmoc-Ala, Fmoc-Tyr (PO_ThreeH₂) (Manufactured by Nova Biochem), Fmoc-Glu (O^tBu) (Watanabe Chemical Co., Ltd.), Fmoc-Ala, Fmoc-Asp (O^tBu) (manufactured by Watanabe Chemical Co., Ltd.), Mca-Gly (references (C.G. Knight, etc., FEBS Letters, (1992),296, 263-266). )Met. After completion of the reaction, the MBHA resin was washed with methanol, 1 ml of a mixed solution of TFA: water: thioanisole: phenol: ethanedithiol (84: 5: 5: 3: 3) was added, and the mixture was stirred at room temperature for 2 hours. Simultaneously cleaving the polypeptide derivative of^tThe Bu group (protecting group for the side chain carboxyl group of Glu and Asp) and the Pbf group (protecting group for the side chain guanidino group of Arg) were cleaved. Next, the resin was removed by filtration, and the filtrate was blown with nitrogen gas and concentrated, and then ether was added to precipitate the desired product. The precipitate was collected and dried with a desiccator to obtain 7.0 mg (97% yield) of the peptide derivative of the present compound. The obtained peptide derivative was found to be the target compound by amino acid analysis and mass spectrometry.
[0043]
Example 2 Mca-Gly-Glu-Gly-Thr-pTyr-Gly-Lys (DNP) -Arg-NH₂Synthesis of (peptide derivative of the present invention)
Mca-Gly, Fmoc-Glu (O^tBu), Fmoc-Gly, Fmoc-Thr (^tBu), Fmoc-Tyr (PO_ThreeH₂), Fmoc-Lys (DNP) and Fmoc-Arg (Pbf) were synthesized in the same manner as in Example 1.
Fmoc-Gly and Fmoc-Thr (^tBu) is manufactured by Watanabe Chemical Industries, Ltd., and the order of the introduced amino acid residues is Fmoc-Arg (Pbf), Fmoc-Lys (DNP), Fmoc-Gly, Fmoc-Tyr (PO_ThreeH₂), Fmoc-Thr (^tBu), Fmoc-Gly, Fmoc-Glu (O^tBu), Mca-Gly.
The yield was quantitative, and the obtained peptide derivative was found to be the target compound by amino acid analysis and mass spectrometry.
[0044]
Example 3 FIG. Mca-Gly-Asp-Gly-Val-pTyr-Ala-Ala-Lys (DNP) -Arg-NH₂Synthesis of (peptide derivative of the present invention)
Mca-Gly, Fmoc-Asp (O^tBu), Fmoc-Val, Fmoc-Tyr (PO_ThreeH₂), Fmoc-Ala, Fmoc-Lys (DNP) and Fmoc-Arg (Pbf) were synthesized in the same manner as in Example 1.
Fmoc-Val is manufactured by Watanabe Chemical Co., Ltd., and the order of the introduced amino acid residues is Fmoc-Arg (Pbf), Fmoc-Lys (DNP), Fmoc-Ala, Fmoc-Ala, Fmoc-Tyr (PO_ThreeH₂), Fmoc-Val, Fmoc-Gly, Fmoc-Asp (O^tBu), Mca-Gly.
The yield was quantitative, and the obtained peptide derivative was found to be the target compound by amino acid analysis and mass spectrometry.
[0045]
Example 4 Mca-Gly-Ser-Ala-pTyr-Gly-Lys (DNP) -Arg-NH₂Synthesis of (peptide derivative of the present invention)
Starting materials: Mca-Gly, Fmoc-Ser (^tBu), Fmoc-Ala, Fmoc-Tyr (PO_ThreeH₂), Fmoc-Lys (DNP) and Fmoc-Arg (Pbf) were synthesized in the same manner as in Example 1.
Fmoc-Ser (^tBu) is manufactured by Watanabe Chemical Co., Ltd., and the order of the introduced amino acid residues is Fmoc-Arg (Pbf), Fmoc-Lys (DNP), Fmoc-Gly, Fmoc-Tyr (PO_ThreeH₂), Fmoc-Ala, Fmoc-Ser (^tBu), Mca-Gly.
The yield was quantitative, and the obtained peptide derivative was found to be the target compound by amino acid analysis and mass spectrometry.
[0046]
Example 5 FIG. Mca-Gly-Gly-Ser-pTyr-Ser-Lys (DNP) -Arg-NH₂Synthesis of (peptide derivative of the present invention)
Starting materials: Mca-Gly, Fmoc-Gly, Fmoc-Ser (^tBu), Fmoc-Tyr (PO_ThreeH₂), Fmoc-Lys (DNP) and Fmoc-Arg (Pbf) were synthesized in the same manner as in Example 1.
The order of the introduced amino acid residues is Fmoc-Arg (Pbf), Fmoc-Lys (DNP), Fmoc-Ser (^tBu), Fmoc-Tyr (PO_ThreeH₂), Fmoc-Ser (^tBu), Fmoc-Gly and Mca-Gly.
The yield was quantitative, and the obtained peptide derivative was found to be the target compound by amino acid analysis and mass spectrometry.
[0047]
Example 6 Measurement of protein tyrosine phosphatase activity
(reagent)
Substrate solution: Mca-Gly-Asp-Ala-Glu-pTyr-Ala-Ala-Lys (DNP) -Arg-NH synthesized in Example 1₂10 mM dimethylglutarate buffer (pH 6.6) containing 1 μM was used as a substrate solution.
Chymotrypsin solution: 1 mM hydrochloric acid (pH 3) containing 0.2% chymotrypsin (manufactured by Sigma) was used as the chymotrypsin solution.
Protein tyrosine phosphatase solution: A protein tyrosine phosphatase solution (pH 6.6) containing 1 U of protein tyrosine phosphatase (derived from Yersinia enterocolitica, New England Biolabs) per 1 ml was used as the protein tyrosine phosphatase solution. 1U means 1 nanomole of paranitrophenyl per minute when protein tyrosine phosphatase is added to 20 mM dimethyl glutarate buffer (pH 6.6) containing 10 mM paranitrophenyl phosphate and reacted at 25 ° C. The amount of enzyme that hydrolyzes phosphate (the same applies hereinafter).
(Operation method)
1 ml of the substrate solution and 10 μl of chymotrypsin solution were added to a fluorescence measuring cell (10 mm × 10 mm) and incubated at 25 ° C. for 5 minutes. Subsequently, 1 μl of protein tyrosine phosphatase solution was added thereto, and the fluorescence intensity change was measured with a fluorometer (Hitachi F-3000, excitation wavelength: 328 nm, fluorescence wavelength: 395 nm) while incubating at 25 ° C.
(result)
The results are shown in FIG.
As is apparent from FIG. 1, it can be seen that the protein tyrosine phosphatase activity can be accurately measured in real time by using the peptide derivative of the present invention as a substrate.
[0048]
Example 7
(reagent)
-Sodium vanadate solution: A solution obtained by dissolving sodium vanadate to 100 mM in water was used as a sodium vanadate solution.
The substrate solution, chymotrypsin solution and protein tyrosine phosphatase solution are the same as in Example 6.
(Operation method)
1 ml of the substrate solution was added to the fluorescence measurement cell and incubated at 25 ° C. for 5 minutes. Next, 1 μl of protein tyrosine phosphatase solution was added thereto, and reacted for 10 minutes while incubating at 25 ° C. Next, 1 μl of sodium vanadate solution was added thereto to inactivate protein tyrosine phosphatase, 10 μl of chymotrypsin solution was added, and the fluorescence intensity change was measured with a fluorimeter (Hitachi F-3000, excitation wavelength: 328 nm, fluorescence wavelength: 395 nm). Measured with
(result)
The results are shown in FIG.
As is clear from FIG. 2, it can be seen that by using the peptide derivative of the present invention as a substrate, fluorescence intensity corresponding to protein tyrosine phosphatase activity can be obtained, in other words, the activity can be measured with high accuracy.
[0049]
Example 8 FIG. Measurement of protein tyrosine phosphatase activity
(reagent)
Substrate solution: Mca-Gly-Asp-Ala-Glu-pTyr-Ala-Ala-Lys (DNP) -Arg-NH synthesized in Example 1₂As a substrate solution, 20 mM dimethyl glutarate buffer (pH 6.6) containing 1 μM was used.
-Chymotrypsin solution: 1 mM hydrochloric acid (pH 3) containing 5% of chymotrypsin (manufactured by Worthington) was used as the chymotrypsin solution.
-Sodium vanadate solution: A solution obtained by dissolving sodium vanadate to 10 mM in water was used as a sodium vanadate solution.
Protein tyrosine phosphatase solution: 20 mM dimethyl glutarate buffer (pH 6.6) containing a predetermined unit (U) of protein tyrosine phosphatase (derived from Yersinia enterocolitica, manufactured by New England Biolabs) was used as the protein tyrosine phosphatase solution.
(Operation method)
200 μl of the substrate solution was added to a fluorescence measurement cell (5 mm × 5 mm) and incubated at 25 ° C. for 2 minutes. Next, 2 μl of a predetermined protein tyrosine phosphatase solution was added thereto, and reacted for 1 minute while incubating at 25 ° C. Next, 2 μl of sodium vanadate solution was added thereto to inactivate the protein tyrosine phosphatase, 2 μl of chymotrypsin solution was added, and the fluorescence intensity change was measured with a fluorimeter (Hitachi F-3000, excitation wavelength: 328 nm, fluorescence wavelength: 395 nm). Measured with
(result)
The results are shown in FIGS. 3 and 4, respectively. FIG. 3 shows the result of measuring the change in fluorescence intensity, and FIG. 4 shows the concentration (U / ml) in the protein tyrosine phosphatase solution obtained from the result of FIG. It is a calibration curve showing the relationship with the phosphorylation rate (%).
As is clear from these results, by using the peptide derivative of the present invention as a substrate, fluorescence intensity corresponding to the protein tyrosine phosphatase concentration (U / ml) in the sample can be obtained, in other words, the concentration can be accurately determined. It can be measured.
[0050]
Example 9 Measurement of protein tyrosine phosphatase activity
(reagent)
Substrate solution: Mca-Gly-Glu-Gly-Thr-pTyr-Gly-Lys (DNP) -Arg-NH synthesized in Example 2₂20 mM dimethylglutarate buffer (pH 6.6) containing 2 μM was used as a substrate solution.
-Chymotrypsin solution: 1 mM hydrochloric acid (pH 3) containing 0.2% of chymotrypsin (manufactured by Worthington) was used as the chymotrypsin solution.
Protein tyrosine phosphatase solution: 20 mM dimethyl glutarate buffer (pH 6.6) containing 30 U / ml of protein tyrosine phosphatase (derived from Yersinia enterocolitica, manufactured by New England Biolabs) was used as the protein tyrosine phosphatase solution.
(Operation method)
200 μl of substrate solution and 2 μl of chymotrypsin solution were added to a fluorescence measurement cell (5 mm × 5 mm) and incubated at 25 ° C. for 2 minutes. Subsequently, 1 μl of protein tyrosine phosphatase solution was added thereto, and the fluorescence intensity change was measured with a fluorometer (Hitachi F-3000, excitation wavelength: 328 nm, fluorescence wavelength: 395 nm) while incubating at 25 ° C.
(result)
The results are shown in FIG.
As is apparent from FIG. 5, it can be seen that the activity of protein tyrosine phosphatase can be continuously measured with high accuracy in real time by using the peptide derivative of the present invention as a substrate. This result also shows that this method can be an effective means for analyzing the reaction between protein tyrosine phosphatase and a substrate kinetically.
[0051]
Example 10 Measurement of maximum rate (Vmax) and Michaelis constant (Km) of protein tyrosine phosphatase
(reagent)
Substrate solution: Mca-Gly-Asp-Ala-Glu-pTyr-Ala-Ala-Lys (DNP) -Arg-NH synthesized in Example 1₂20 mM dimethyl glutarate buffer solution (pH 6.6) containing a predetermined concentration of was used as a substrate solution.
-Chymotrypsin solution: 1 mM hydrochloric acid (pH 3) containing 5.0% of chymotrypsin (manufactured by Worthington) was used as the chymotrypsin solution.
Protein tyrosine phosphatase solution: A protein tyrosine phosphatase solution (pH 6.6) containing 20 U per ml of protein tyrosine phosphatase (derived from Yersinia enterocolitica, manufactured by New England Biolabs) was used as the protein tyrosine phosphatase solution.
(Operation method)
200 μl of a predetermined substrate solution and 2 μl of chymotrypsin solution were added to a fluorescence measurement cell (5 mm × 5 mm) and incubated at 25 ° C. for 2 minutes. Subsequently, 1 μl of protein tyrosine phosphatase solution was added thereto, and the fluorescence intensity change was measured with a fluorometer (Hitachi F-3000, excitation wavelength: 328 nm, fluorescence wavelength: 395 nm) while incubating at 25 ° C. The initial rate (change in fluorescence intensity per minute) at various substrate concentrations was determined from the obtained reaction time course.
(result)
The results are shown in FIG. FIG. 6 is a graph showing the relationship between the substrate concentration (horizontal axis) and the initial velocity (vertical axis) obtained at a predetermined substrate concentration.
Based on this result, the maximum rate (Vmax) and Michaelis constant (Km) of protein tyrosine phosphatase were determined by conventional methods, and the following values were obtained.
Vmax: 1.69nmol / unit · min
Km: 11.0 μM
As is clear from the above, it can also be seen that the kinetic study of protein phosphatase can be easily performed by using the present invention.
[0052]
【The invention's effect】
As described above, the present invention provides an unprecedented peptide derivative and a method for measuring protein phosphatase activity using the peptide derivative as a substrate, and has the following effects.
1) Since measurement is performed using fluorescence intensity, protein phosphatase activity can be measured with high sensitivity.
2) Since the reagent of the present invention does not contain a radioactive phosphorus compound, it can be stored stably for a long period of time.
3) Since the activity measurement method of the present invention is not a method for measuring free phosphate ions, it can also be measured for samples containing phosphate ions.
4) By appropriately selecting the fluorescent group, the excitation wavelength at the time of measuring the fluorescence intensity can be set to a wavelength different from the absorption wavelength of the protein, so that a crude tissue extract can also be used as a sample.
5) If the protein phosphatase to be measured is resistant to the protease having the above-mentioned properties used for the measurement, the substrate dephosphorylation reaction can be followed in real time.
[0053]
[Sequence Listing]
[0054]
[SEQ ID NO: 1]

[0055]
[SEQ ID NO: 2]

[0056]
[SEQ ID NO: 3]

[0057]
[SEQ ID NO: 4]

[0058]
[SEQ ID NO: 5]

[0059]
[SEQ ID NO: 6]

[0060]
[SEQ ID NO: 7]

[0061]
[SEQ ID NO: 8]

[0062]
[SEQ ID NO: 9]

[0063]
[SEQ ID NO: 10]

[0064]
[SEQ ID NO: 11]

[0065]
[SEQ ID NO: 12]

[0066]
[SEQ ID NO: 13]

[0067]
[SEQ ID NO: 14]

[Brief description of the drawings]
1 is a graph showing changes in fluorescence intensity based on protein tyrosine phosphatase activity using a substrate of the present invention obtained in Example 6. FIG.
2 is a graph showing changes in fluorescence intensity based on protein tyrosine phosphatase activity using the substrate of the present invention obtained in Example 7. FIG.
3 is a graph showing changes in fluorescence intensity based on protein tyrosine phosphatase activity obtained in Example 8 using the substrate of the present invention. FIG.
4 is a calibration curve showing the relationship between the protein tyrosine phosphatase concentration and the dephosphorylation rate (%) obtained in Example 8. FIG.
5 is a graph showing changes in fluorescence intensity based on protein tyrosine phosphatase activity using the substrate of the present invention obtained in Example 9. FIG.
6 is a graph showing the relationship between the substrate concentration (horizontal axis) obtained in Example 10 and the initial velocity (vertical axis) obtained at a predetermined substrate concentration. FIG.

Claims (10)

General formula [1]
A ₁ -X-A ₂ [1]
(In the formula, X is an amino acid residue having a phosphate group introduced therein, and A ₁ And A ₂ Is an amino acid residue or peptide chain as shown below, one of which has a quenching group at its terminal or side chain part, and the other has its own fluorescence at its terminal or side chain part. And having a fluorescent group having the property that the fluorescence is quenched by the quenching group in the molecule, ₁ Is an amino acid residue or a peptide chain consisting of 2 to 20 amino acid residues bonded to X, and the amino group at the amino terminus thereof is amino-protected when no fluorescent group or quenching group is bonded. An amino group protected by a group or a free amino group, and A ₂ Is a peptide chain composed of amino acid residues or 2 to 20 amino acid residues bonded to X, and the carboxyl group at the carboxyl terminus is carboxyl-protected when no fluorescent group or quenching group is bonded. A carboxyl group protected by a group or a free carboxyl group. ), A peptide derivative having 1 to 39 amino acid residues between an amino acid residue having a fluorescent group and an amino acid residue having a quenching group, and the amino acid sequence of the peptide derivative is a fluorescent group One of the peptide bonds existing between an amino acid residue having an amino acid residue and an amino acid residue having a quenching group is that the phosphate group of the amino acid residue into which the phosphate group represented by X is introduced is converted by protein phosphatase A peptide derivative that is a sequence that can be selectively cleaved by a protease when dephosphorylated. The amino protecting group is t - Butoxycarbonyl group, benzyloxycarbonyl group, 9- A fluorenylmethyloxycarbonyl group, 2- Phenyl -2- The peptide derivative according to claim 1, which is a propyl group or an acetyl group, and the carboxyl protecting group is a methoxy group, an ethoxy group, a t-butoxy group, a benzyl group, a pentamethyldihydrobenzofuranyl group or an amide group. The fluorescent group is ( 7- Methoxycoumarin -Four- Yl) acetyl group, Five- [( 2- Aminoethyl) amino] naphthalene -1- Sulfonic acid groups, 9,10- Dioxa -syn-3,4,6,7- The tetramethylbiman group is selected, and the quenching group is a dinitrophenyl group, Four- ( Four- Dimethylaminophenylazo) benzoyl group, Four- ( Four- The peptide derivative according to claim 1, which is selected from a dimethylaminophenylazo) sulfonyl group. Amino acid residues phosphate group Ru is introduced, an amino acid residue having a hydroxyl group, the peptide derivative of claim 1. Amino acid residues phosphate group Ru is introduced is a tyrosine residue, a serine residue or threonine residue, the peptide derivative of claim 1. Amino acid sequence
-Gly-Asp-Ala-Glu-Tyr-Ala-Ala-Lys-Arg- (SEQ ID NO: 1),
-Gly-Glu-Gly-Thr-Tyr-Gly-Lys-Arg- (SEQ ID NO: 2),
-Gly-Glu-Val-Asn-Tyr-Glu-Glu-Lys-Arg- (SEQ ID NO: 3),
-Gly-Glu-Pro-Gln-Tyr-Gln-Pro-Lys-Arg- (SEQ ID NO: 4),
-Gly-Glu-Lys-Glu-Tyr-His-Ala-Lys-Arg- (SEQ ID NO: 5),
-Gly-Asp-Gly-Val-Tyr-Ala-Ala-Lys-Arg- (SEQ ID NO: 6),
-Gly-Ser-Ala-Tyr-Gly-Lys-Arg- (SEQ ID NO: 7),
-Gly-Gly-Ser-Tyr-Ser-Lys-Arg- (SEQ ID NO: 8),
-Gly-Arg-Val-Phe-Ser-Lys-Arg- (SEQ ID NO: 9),
-Gly-Asp-Arg-Phe-Thr-Lys-Arg- (SEQ ID NO: 10),
-Gly-Arg-Lys-Phe-Thr-Lys-Arg- (SEQ ID NO: 11),
-Gly-Pro-Gly-Phe-Ser-Lys-Arg- (SEQ ID NO: 12),
-Gly-Thr-Arg-Phe-Ser-Lys-Arg- (SEQ ID NO: 13), or
-Gly-Lys-Arg-Phe-Ser-Lys-Arg- (SEQ ID NO: 14)
The peptide derivative according to claim 1, comprising: A method for measuring the activity of protein phosphatase using the peptide derivative according to claim 1 as a substrate. The peptide derivative according to claim 1 is reacted with protein phosphatase, and then the phosphate group of the amino acid residue into which the phosphate group represented by X contained in the peptide derivative is introduced is converted into protein phosphatase. When dephosphorylated, a protease having a property of selectively cleaving one of peptide bonds existing between an amino acid residue having a fluorescent group and an amino acid residue having a quenching group is reacted, A method for measuring the activity of protein phosphatase, characterized by measuring a change in fluorescence intensity caused thereby. A reagent for measuring protein phosphatase activity, comprising the peptide derivative according to claim 1. When the phosphate group of the peptide derivative according to claim 1 and the amino acid residue introduced with a phosphate group represented by X contained in the peptide derivative is dephosphorylated by protein phosphatase, a fluorescent group is formed. A reagent for measuring protein phosphatase activity, comprising a protease having a property of selectively cleaving one of peptide bonds existing between an amino acid residue having a quenching group and an amino acid residue having a quenching group .

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