JP2005233854A - Peptide derivative and intermolecular interaction detecting method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To rapidly and quantitatively measure the intermolecular interaction between a physiologically active molecule of protein, a nucleic acid, a sigar chain or the like and a target molecule. <P>SOLUTION: The intermolecular interaction detecting method of the target molecule has a process (a) for bringing a substance having a photochromic compound bonded thereto in its molecule into contact with the target molecule and a process (b) for measuring the optical isomerization speed constant of the photochromic compound. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a peptide derivative and an intermolecular interaction detection method. The peptide derivative of the present invention is used for a method for detecting an interaction between molecules, and the method for detecting an interaction between molecules of the present invention can easily detect an interaction between a substance and a target molecule.

  Today, more than 100 biological genomes, including the human genome, have been decoded or are in progress. Now that we have entered the post-genomic era, a vast amount of genetic information is available. In accordance with the central dogma principle of DNA-> RNA-> protein, it is necessary to have a technology that matches all DNA, RNA and protein, elucidates their functions, and leads to applied technology. Since the correlation between the expression level of mRNA, which is an index of the expression level of DNA, and the abundance of protein is considered to be low, development of a technique capable of directly detecting protein is urgently required. .

Today, antibodies, aptamers, fusion proteins, phage display peptides, etc. are used as supplemental molecules for detecting proteins, and chemiluminescence, fluorescence, mass spectrometry, surface plasmon resonance, etc. are used as detection methods. ing.
For example, by using a group of fluorescent dye-labeled peptides as supplementary molecules, labeling of an analyte can be avoided, and a change in fluorescence intensity based on the interaction with a protein is known as a “protein fingerprint”. A technique that allows easy identification of proteins in a sample by comparison with a protein (Construction of a Protein-Detection System Using a Loop Peptide Library with a Fluorescence Label ”M. Takahashi, K. Nokihara, H. Mihara Chem. Biol. 2003, 10, 53-60) The method disclosed in this document is a simple and highly sensitive method and can be carried out at low cost. Since it is bound to a peptide, it has the disadvantage that the fluorescence background is relatively large, so there is a need for optimization of the compound that labels the peptide.

Construction of a Protein-Detection System Using a Loop Peptide Library with a Fluorescence Label ”M. Takahashi, K. Nokihara, H. Mihara Chem. Biol. 2003, 10, 53-60

  The difference between detection of so-called DNA-DNA interaction and detection of protein-protein interaction in a protein chip is that the difference in the degree of binding of protein interaction is extremely large, on the order of about 10 to the fourth power. It is necessary to detect the intensity difference. An object of the present invention is to provide a method for quickly and quantitatively measuring the intermolecular interaction between a target molecule and a physiologically active molecule such as a protein, nucleic acid or sugar chain.

  In order to achieve the above object, the present inventors have intensively studied, and as a result, have found that the above object can be achieved by using a substance formed by binding a photochromic compound. In addition, the present inventors have completed the present invention by paying attention to a change in the photoisomerization rate constant when the above substance and the target molecule are brought into contact with each other.

That is, this invention provides the peptide derivative formed by couple | bonding a photochromic compound in a molecule | numerator.
The present invention also provides a method for detecting an intermolecular interaction of a target molecule, comprising the following steps.
(A) contacting the target molecule with a substance formed by binding a photochromic compound in the molecule; and (b) measuring the photoisomerization rate constant of the photochromic compound;
The present invention also provides a method for detecting an intermolecular interaction of a target molecule, comprising the following steps.
(A) contacting a peptide derivative formed by binding a photochromic compound in the molecule with a target molecule; and (b) measuring a photoisomerization rate constant of the photochromic compound;

  According to the peptide derivative of the present invention, the intermolecular interaction between a molecule such as a protein and a target molecule can be measured. In addition, according to the intermolecular interaction detection method of the present invention, the interaction between the substance and the target molecule can be easily detected.

Hereinafter, the peptide derivative of the present invention will be described first.
The peptide derivative of the present invention is formed by binding a photochromic compound in the molecule.
The photochromic compound means a compound that causes a phenomenon called photochromism. Photochromism means a phenomenon in which a single chemical species reversibly reciprocates between two different states with a large change in absorption spectrum, and at least one conversion is caused by light irradiation. Specific examples of the photochromic compound include fulgides, diallylethenes, spiropyrans, spirooxazines and the like. Specific examples of these photochromic compounds are shown below.

  Since the isomerization reaction of the photochromic compound is performed with a substantial restructuring of the molecular structure, the photoisomerization rate constant is considered to depend on the microenvironment change around the photochromic compound. That is, a change in the photoisomerization rate constant can be regarded as a change in the surrounding microenvironment resulting from the intermolecular interaction. Therefore, the peptide derivative can be used in a method for detecting an intermolecular interaction as described below. The use of the peptide derivative of the present invention will be described later.

  The peptide derivative of the present invention is formed by binding a photochromic compound in the molecule. The photochromic compound is, for example, a lysine residue of a peptide chain in the molecule, or an ornithine residue or a diaminobutanoic acid residue instead of a lysine residue. And a peptide derivative in which a diaminopropionic acid residue is introduced and a photochromic compound is bound to the residue, and a peptide derivative in which a photochromic compound is inserted into the peptide main chain.

The peptide derivative of the present invention is used for detecting an intermolecular interaction, as will be described later. Therefore, when the target molecule for detecting this intermolecular interaction is an enzyme, peptide derivatives include those obtained by binding a photochromic compound to a substrate peptide of the enzyme that is the target molecule. Moreover, even if it is a peptide other than the substrate peptide of the enzyme which is a target molecule, it should just be a thing couple | bonded with a target molecule.
Specific examples of the peptide derivative of the present invention are shown below.

Ac-Cys (Acm) -Gly-Lys (SP) -Gly-Ile-Tyr-Gly-Glu-Phe-Lys-Lys-Lys-Gly-NH ₂ (1)
Ac-Cys (Acm) -Gly-Lys (SP) -Gly-Ile-Tyr-Ala-Ala-Pro-Lys-Lys-Lys-Gly-NH ₂ (2)
Ac-Cys (Acm) -Gly-Lys (SP) -Gly-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH ₂ (3)
Ac-Cys (Acm) -Gly-Lys (SP) -Gly-Lys-Arg-Thr-Leu-Arg-Arg-Gly-NH ₂ (4)
Ac-Cys (Acm) -Gly- (amino-SP) -Gly-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH ₂ (5)
Ac-Cys (Acm) -Gly-Gly-Leu-Arg- (amino-SP) -Arg-Ala-Ser-Leu-Gly-NH ₂ (6)
Ac-Cys (Acm) -Gly-Gly-Leu-Arg-Arg-Ala-Ser- (amino-SP) -Leu-Gly-NH ₂ (7)
Ac-Cys (Acm) -Gly-Orn (SP) -Gly-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH ₂ (8)
Ac-Cys (Acm) -Gly-Dab (SP) -Gly-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH ₂ (9)
Ac-Cys (Acm) -Gly-Dap (SP) -Gly-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH ₂ (10)
Ac-Cys (Acm) -Gly-Lys (amino-SP) -Gly-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH ₂ (11)
SP-Cys (Acm) -Gly-Ala-Gly-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH ₂ (12)
In the above formulas (1) to (12), SP is a photochromic compound.

In the peptide derivatives represented by the above formulas (1) to (4), Lys is arranged via Gly on the N-terminal side of substrate peptides of c-Src kinase, c-Abl tyrosine kinase, protein kinase A and protein kinase C, respectively. And, it is a peptide derivative in which a spiropyran molecule that is a photochromic compound is bound to the Lys side chain.
The peptide derivatives represented by the above formulas (5) to (7) each reduce the nitro group of the spiropyran molecule to obtain an aminated spiropyran, and the resulting aminated spiropyran molecule is converted into the main chain skeleton of the substrate peptide of protein kinase A. Is a peptide derivative inserted in

  In the peptide derivatives represented by the above formulas (8) to (10), Lys to which the spiropyran molecule of the peptide derivative represented by the formula (3) binds is converted to ornithine (Orn), diaminobutanoic acid (Dab), and diaminopropionic acid (Dap). The peptide derivative represented by the formula (11) is a peptide derivative obtained by replacing the spiropyran molecule bonded to the Lys side chain of the peptide derivative represented by the formula (3) with an aminated spiropyran. The peptide derivative shown in 12) is a peptide derivative in which a spiropyran molecule is bonded to the N-terminal side instead of Lys (SP) of the peptide derivative shown in formula (3).

The method for producing the peptide derivative will be described below.
First, the manufacturing method of the peptide derivative shown to Formula (1)-(4) is demonstrated. First, spiropyran is activated, and a peptide derivative is produced using the activated spiropyran molecule.
The activation of spiropyran will be described with reference to FIG. Spiropyran was synthesized according to the method described in Raymo, FM; Giordani, SJ Am. Chem. Soc. 2001, 123, 4651-4652 and Raymo, FM; Giordani, SJ Am. Chem. Soc. 2002, 124, 2004-2007 (Compound 1 in FIG. 1). The spiropyran is then activated with p-nitrochloroformate in methylene chloride in the presence of a base such as diisopropylethylamine (DIAE) to give compound 2.

  Next, as shown in FIG. 2, the peptide is elongated by Fmoc solid-phase synthesis, reacted with compound 2 on the resin, and a spiropyran molecule is selectively introduced into the Lys side chain. After removal of the protecting group and elimination from the resin, the peptide derivative can be obtained by purifying the obtained peptide derivative using reverse phase HPLC.

  The peptide derivatives represented by the formulas (5) to (7) are peptide derivatives in which an aminated spiropyran molecule is inserted into the main chain skeleton of the peptide. To produce this peptide derivative, an activated aminospiropyran is synthesized as shown in FIG. As shown in FIG. 3, the nitro group of spiropyran 1 is reduced with stannous chloride, and Fmoc-OSu (succinimide ester) is added to obtain Fmocated aminospiropyran 3. This Fmocated amino spiropyran 3 is allowed to react with p-nitrophenyl chloroformate in the presence of a base to obtain 4 compounds. Using the compound of 4 above, the peptide is extended by the Fmoc solid phase synthesis method to obtain peptide derivatives represented by the formulas (5) to (7).

  In order to produce the peptide derivatives represented by the formulas (8) to (10), as shown in FIG. 4, Fmoc-L-Orn (SP) -OH, Fmoc-L-Dab (SP) -OH and Fmoc-L -Dap (SP) -OH is synthesized. FIG. 4 is a diagram showing a production process of a compound for producing peptide derivatives represented by formulas (8) to (10). As shown in FIG. 4, Fmoc-L-Orn (Mtt) -OH (Mtt is 4-methyltrityl group), Fmoc-L-Dab (Mtt) -OH or Fmoc-L-Dap (Mtt) -OH side The chain protecting group is removed with TFA (trifluoroacetic acid) and bound to the compound 2 shown in FIG. 2, and Fmoc-L-Orn (SP) -OH, Fmoc-L-Dab (SP) -OH or Fmoc- L-Dap (SP) -OH is obtained. Using the obtained compound, the peptide is elongated by the Fmoc solid phase synthesis method to obtain peptide derivatives represented by the formulas (8) to (10).

The peptide derivative of the present invention may be bound to a solid phase. As the solid phase to be used, for example, a well of a microplate, or a recess such as a groove or a hole provided in a chip such as a slide glass can be used.
Examples of a method for binding a peptide derivative to a solid phase include a method in which an amino group, a carboxyl group, or the like of a peptide is reacted with an amino group, a carboxyl group, or the like on the solid phase and covalently bonded. It can also be carried out by binding biotin or avidin to a peptide derivative and binding it to avidin or biotin immobilized on a solid phase.

The amount of the peptide derivative bound on the solid phase is not limited in any way, and is appropriately set depending on the application, but is, for example, about 1 fmol to 1000 nmol.
The peptide derivative can be bound to the solid phase by physical adsorption. In this case, the peptide derivative is bound by bringing a solution obtained by dissolving the peptide derivative in a buffer solution into contact with a solid phase. In this case, the binding reaction can be performed overnight, for example, at room temperature for 15 minutes to 2 hours at a temperature of 4 ° C., as in the conventional case. The concentration of the peptide derivative solution at the time of binding to the solid phase can be appropriately selected according to the type of peptide derivative and the type and concentration of the substance in the sample to be measured, for example, about 1 ng / mL to 100 μg / mL. preferable.

  When binding the peptide derivative of the present invention to a solid phase, it is preferable to bind a plurality of types of peptide derivatives to a specific region on one solid phase. By doing so, it is possible to simultaneously measure a plurality of samples in the specimen. The number of types of peptide derivatives is not limited in any way, but about 10 to 10,000 types may be bound on one solid phase.

Next, the intermolecular interaction detection method for target molecules of the present invention will be described.
The method for detecting an interaction between target molecules of the present invention has the following steps.
(A) a step of contacting a target molecule with a substance formed by binding a photochromic compound in the molecule; and (b) a step of measuring a photoisomerization rate constant of the photochromic compound.

In the present invention, the target molecule means a molecule that interacts with a substance formed by binding a photochromic compound in the molecule, and examples thereof include proteins, nucleic acids, sugar chains, and low molecular compounds.
In addition, the interaction is usually an action caused by a force acting between molecules generated from at least one of a covalent bond, a hydrophobic bond, a hydrogen bond, a van der Waals bond, and an electrostatic force bond between the target molecule and the peptide derivative. However, this term should be construed in the broadest sense and should not be construed as limiting in any way. The covalent bond includes a coordination bond and a dipole bond. Moreover, the coupling | bonding by an electrostatic force contains an electrical repulsion other than an electrostatic coupling. In addition, a binding reaction, a synthesis reaction, and a decomposition reaction resulting from the above action are also included in the interaction.

  Specific examples of the interaction include, for example, binding and dissociation of an enzyme and a substrate, binding and dissociation of an antigen and an antibody, binding and dissociation of a protein receptor and a ligand, binding and dissociation between an adhesion molecule and a counterpart molecule. And binding and dissociation of a nucleic acid and a protein bound thereto, binding and dissociation of proteins in an information transmission system, binding and dissociation of a glycoprotein and a protein, and binding and dissociation of a sugar chain and a protein.

Hereinafter, the intermolecular interaction detection method of the present invention will be described taking the binding and dissociation of an enzyme and a substrate peptide as an example.
In the intermolecular interaction detection method of the present invention, first, a target molecule is brought into contact with a substance formed by binding a photochromic compound in a molecule. As the substance formed by binding a photochromic compound in the molecule, the above-described peptide derivative of the present invention can be used. In the intermolecular interaction detection method of the present invention, it is preferable to bind the peptide derivative to a solid phase. The method for binding the peptide derivative to the solid phase is as described above.

  In the method for detecting an interaction between molecules of the present invention, any method for bringing a peptide derivative and a target molecule into contact with each other may be used as long as the two molecules are brought into contact with each other to a sufficient extent. Preferably, it is preferable to prepare and use a solution in which the peptide derivative and the target molecule are dissolved in a buffer solution commonly used biochemically at an appropriate concentration. After contacting the peptide derivative with the target molecule, it is preferable to wash the excess peptide derivative and target molecule with a buffer solution in which the peptide derivative and target molecule are dissolved.

  Next, in the intermolecular interaction detection method of the present invention, the interaction between the peptide derivative and the target molecule is detected by measuring the photoisomerization rate constant of the photochromic compound. . As described above, a photochromic compound is a compound that causes photochromism, and this photochromism is a reversible reciprocation of a single chemical species between two different states with a large change in absorption spectrum, and at least one of them. Conversion is a phenomenon caused by light irradiation. In the present invention, after bringing the peptide derivative into contact with the target molecule, the chemical species is irradiated with light having a wavelength that causes conversion, and the rate at which the converted chemical species returns to the one chemical species is the photoisomerization rate. The measurement is performed by measuring a constant, and the interaction between the peptide derivative and the target molecule is determined.

This point will be briefly described with reference to FIG. 5 by taking spiropyran as an example.
Spiropyran, which is a photochromic compound, has a spiropyran body and a merocyanine body as photoisomers (in FIG. 5, the left side is a merocyanine body and the right side is a spiropyran body). The merocyanine body has absorption in the visible region (500 to 600 nm) and emits fluorescence around 600 nm. Spiropyran bodies have a feature that they absorb light of 400 nm or less but do not emit fluorescence. When a peptide derivative formed by binding a protein and a photochromic compound interacts, it is considered that the photoisomerization rate constant to the spiropyran body in the peptide chain is affected. Excitation with light having a wavelength in the absorption band (510 nm) of the merocyanine body, and the fluorescence intensity at 600 nm is measured. The photoisomerization rate is considered to be different depending on whether the peptide derivative interacts with the target molecule (k _{bound in} FIG. 5) or not (k _{unbound in} FIG. 5). By measuring the constant, it is possible to detect whether or not the peptide derivative and the target molecule are interacting with each other. In addition, when the peptide derivative is bound to the target molecule, the photoisomerization rate constant may be larger than when the peptide derivative is not bound to the target molecule, and vice versa. How the photoisomerization rate constant changes depends on the combination of the peptide derivative and the target molecule.

  For example, when the peptide derivative represented by the formula (1) interacts with protein kinase A, α-amylase, β-galactosidase, lysozyme, and S-100 protein, the photoisomerization rate constant increases and hexokinase and Conversely, when it interacts, it becomes smaller. In addition, the peptide derivative represented by the formula (2) has a high photoisomerization rate constant when interacting with protein kinase A, β-galactosidase, lysozyme, hexokinase, and S-100 protein, but α-amylase and There is little change when interacting. The peptide derivative represented by the formula (3) has a higher photoisomerization rate when interacting with protein kinase A, but interacts with α-amylase, β-galactosidase, lysozyme, hexokinase, and S-100 protein. In some cases, the photoisomerization rate constant is small. The peptide derivative represented by the formula (4) has a small photoisomerization rate constant when interacting with protein kinase A, α-amylase, β-galactosidase, lysozyme, hexokinase, and S-100 protein.

  The intermolecular interaction detection method of the present invention can be used to determine whether or not a specific molecule is contained in a specimen using a specimen that may contain a target molecule as a sample. Examples of such specimens include animal cell disruption fluid, plant cell disruption fluid, bacterial cell disruption fluid, virus disruption fluid, and fractionated components, blood, serum, plasma, urine, stool, saliva, tissue fluid, spinal fluid And body fluids, and various foods and beverages.

  The intermolecular interaction detection method of the present invention can be used not only for detection of an initial target molecule but also for screening for an unknown target molecule. That is, peptide derivatives in which photochromic compounds are bound to various constituent amino acid side chains are prepared, bound to a solid phase, reacted with various proteins, and the photoisomerization rate constant is measured. By making the results into a database, it is possible to detect extremely different types of target molecules. By doing so, it is thought that it will greatly contribute to the diagnosis of various diseases and the development of new drugs.

  The intermolecular interaction detection method of the present invention described above is performed for a plurality of peptide derivatives, and the difference is converted into data that can be recognized with the naked eye according to the values measured for each peptide derivative. The target molecules in the test sample can be detected easily and simply, and the target molecules contained in the unknown test sample can be identified.

Hereinafter, the present invention will be described in more detail with reference to examples. Needless to say, the scope of the present invention is not limited to such examples.
Example 1
To a methylene chloride solution (20 mL) of 1.00 g (2.84 mmol) of a compound represented by the following formula (13) and diisopropylethylamine (DIEA) (2.0 mL) at 0 ° C., p-nitrophenyl chloroformate (572 mg, 2 .84 mmol) of methylene chloride (5.0 mL) was added dropwise over 1 minute, and the resulting mixed solution was stirred at 0 ° C. for 1 hour. To the reaction mixture, p-nitrophenyl chloroformate (572 mg, 2.84 mmol) in methylene chloride (5.0 mL) was added dropwise at 0 ° C. over 1 minute, and the resulting mixture was stirred at 0 ° C. for 1 hour. did. This operation was repeated once more, followed by concentration under reduced pressure. The residue was purified by silica gel column chromatography [ethyl acetate / petroleum ether (1: 2)], decantation [ethyl acetate / petroleum ether (1: 2)], and then solidified [ethyl acetate / petroleum ether (1: 2) The compound represented by the formula (14) was obtained as a pale pink powder. Yield: 827 mg (56%): MALDI-MS (SA, sinapinic acid) calcd 517.5; obsd 517.9 [M + H] ⁺

Fmoc-Gly-Rink amide MBHA resin (254 mg, 30.0 μmol, sub = 0.118 mmol / g) and literature (Chen, WC; White, PD “Fmoc solid phase peptide synthesis: A practical approach” Oxford University Press: New York, 2000) according to the conventional method of Fmoc solid-phase synthesis described in Ac.Cys (Acm) -Gly-Lys (Mtt) -Gly-Ile-Tyr ( ^t Bu) -Gly-Glu (O ^t Bu) -Phe-Lys (Boc) -Lys (Boc) -Lys (Boc) -Gly-NH ₂ was obtained. Half of the obtained resin with a protected peptide (15.0 μmol) was treated with TFA (trifluoroacetic acid) / TIS (triisopropylsilane) / DCM (methylene chloride) (1/5/94) to remove the Mtt group. . The obtained resin was washed with 10% DIEA / NMP (N-methylpyrrolidone) (x 1) and NMP (x 3), and the compound represented by the formula (14) (23 mg, 45 μmol) and DIEA (16 μL, 90 μmol) Of NMP (1.0 mL) was added and shaken at room temperature. After shaking for 6 hours, DIEA (16 μL, 90 μmol) was added and further shaken for 12 hours. After washing with NMP (x 3) and chloroform (x 3), the dried resin was washed with TFA / EDT (ethanedithiol) / TA (thioanisole) / m-cresol 2.0 mL (10: 0.75: 0.75). : 0.25) to remove the protecting group and cleave from the resin. The reaction mixture was filtered and concentrated under reduced pressure, and ether precipitation was performed to obtain a crude peptide. The obtained crude peptide was purified by RP-HPLC [(C18 semi-preparative column, AcCN: 35 → 60% (30 min) containing 0.1% TFA)], lyophilized, and then expressed by the formula (1). The indicated peptide derivative was obtained as a yellow powder. Yield: 5.6 mg (19% from Fmoc-Gly-resin): MALDI-MS (SA) calcd 1905.2; obsd 1906.1 [M + H] ⁺ , 1891.2 [M-Me] ⁺ , 1875.3 [M-(2 x Me )] ⁺

Example 2
The crude peptide obtained by the same operation as in Example 1 was purified by RP-HPLC ((C18 semi-preparative column, AcCN: 30 → 55% (30 minutes) containing 0.1% TFA)) Lyophilization gave the peptide derivative of formula (2) as a yellow powder, yield: 6.7 mg (25% from Fmoc-Gly-resin): MALDI-MS (SA) calcd 1811.1; + H] ⁺ , 1797.5 [M-Me] ⁺ , 1782.0 [M-(2 x Me)] ⁺

Example 3
After purifying the crude peptide obtained by the same operation as in Example 1 by RP-HPLC ((C18 semi-preparative column, AcCN: 35 → 60% (30 minutes) containing 0.1% TFA)) The peptide derivative represented by the formula (3) was obtained as a yellow powder, yield: 6.7 mg (28% from Fmoc-Gly-resin): MALDI-MS (SA) calcd 1607.8; M + H] ⁺ , 1592.8 [M-Me] ⁺ , 1577.6 [M-(2 x Me)] ⁺

Example 4
After purifying the crude peptide obtained by the same operation as in Example 1 by RP-HPLC ((C18 semi-preparative column, AcCN: 30 → 55% (30 minutes) containing 0.1% TFA)) The product was freeze-dried to obtain the peptide derivative represented by the formula (4) as a yellow powder, yield: 4.6 mg (28% from Fmoc-Gly-resin): MALDI-MS (SA) calcd 1722.0; + H] ⁺ , 1707.9 [M-Me] ⁺

Example 5
A concentrated hydrochloric acid solution (9.0 mL) of the compound represented by the formula (13) (500 mg, 1.41 mmol) and SnCl ₂ × 2H ₂ O (955 mg, 4.23 mmol) was stirred at room temperature overnight. This solution was neutralized with 50% NaOH, and Fmoc-OSu (524 mg, 1.55 mmol) in acetonitrile (20 mL) and water were added to prepare a water / acetonitrile ratio of (1: 1). And stirred at room temperature for 1 hour. A new acetonitrile solution (5 mL) of Fmoc-OSu (262 mg, 0.78 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After adding 300 mL of ethyl acetate to the reaction mixture and washing the organic layer with water, it was dried over MgSO ₄ . The resulting crude product is purified by silica gel column chromatography [chloroform / methanol (49: 1)] and then solidified (ether / petroleum ether) to obtain the compound represented by the following formula (15) as a light brown powder. It was. Yield: 355 mg (46%): MALDI-MS (SA) calcd 544.2; obsd 545.5 [M + H] ⁺

To a methylene chloride solution (5 mL) of the compound represented by the formula (15) (300 mg, 0.551 mmol) and DIEA (0.210 mL) obtained as described above, p-nitrophenyl chloroformate (124 mg) was added at room temperature. 0.613 mmol) of methylene chloride (3.0 mL) was added dropwise over 1 minute, and the resulting mixed solution was stirred at room temperature for 2 hours. A methylene chloride solution (3.0 mL) of p-nitrophenyl chloroformate (124 mg, 0.613 mmol) was added dropwise to the reaction mixture solution at room temperature over 1 minute, and the resulting mixture solution was stirred at room temperature for 2 hours. This operation was repeated once more, followed by concentration under reduced pressure. To the residue was added 100 mL of ethyl acetate, washed with 4% NaHCO ₃ (x 1) and water (x 3), and dried over MgSO ₄ . The obtained crude product was purified by silica gel column chromatography [ethyl acetate / petroleum ether (1: 3)], then solidified (ethyl acetate / petroleum ether), and the compound represented by the following formula (16) was pale green Obtained in powder form. Yield: 212 mg (54%): MALDI-MS (SA) calcd 709.7; obsd 711.3 [M + H] ⁺ , 695.6 [M-Me] ⁺

Example 6
Using Fmoc-Gly-Rink amide resin (54 mg, 30 μmol, sub = 0.56 mmol / g), the same operation as in Example 1 was performed to extend the peptide chain. To condense the compound represented by formula (16) into the peptide chain, add DIEA (26 μL, 150 μmol) to NMP solution (64 mg, 90 μmol) of the compound represented by formula (16) and shake at room temperature for 8 hours. It went by. The obtained crude peptide was purified by RP-HPLC [(C18 semi-preparative column, AcCN: 20 → 30% (30 min) containing 0.1% TFA)], lyophilized, and expressed by the formula (5) The indicated peptide derivative was obtained in the form of an orange powder. Yield: 8.2 mg (19% from Fmoc-Gly-resin): MALDI-MS (SA) calcd 1449.7; obsd 1450.8 [M + H] ⁺

Example 7
After the crude peptide obtained by performing the same operation as in Example 6 was purified by RP-HPLC [(C18 semi-preparative column, AcCN: 20 → 30% (30 min) containing 0.1% TFA)] The peptide derivative represented by the formula (6) was obtained as an orange powder. Yield: 6.3 mg (14% from Fmoc-Gly-resin): MALDI-MS (SA) calcd 1449.7; obsd 1449.0 [M + H] ⁺

Example 8
After the crude peptide obtained by performing the same operation as in Example 6 was purified by RP-HPLC [(C18 semi-preparative column, AcCN: 20 → 30% (30 min) containing 0.1% TFA)] The peptide derivative represented by the formula (7) was obtained as an orange powder. Yield: 6.3 mg (14% from Fmoc-Gly-resin): MALDI-MS (SA) calcd 1449.7; obsd 1451.20 [M + H] ⁺

Example 9
A solution of Fmoc-L-Orn (Boc) -OH (175 mg, 0.386 mmol) in TFA (3.0 mL) was stirred at 0 ° C. for 30 minutes. The reaction mixture was concentrated under reduced pressure, 50 mL of ether / petroleum ether (1: 2) was added to the residue, solidified, and vacuum dried. A DMF solution (3.0 mL) of the compound represented by the formula (14), (100 mg, 0.193 mmol) and DIEA (134 iL, 0.772 mmol) was stirred at 0 ° C. for 30 minutes, and then stirred at room temperature for 1 hour. did. 50 mL of ethyl acetate was added to the reaction mixture, washed with 10% aqueous citric acid (x 1) and water (x 3), and dried over MgSO ₄ . The obtained crude product was purified by silica gel column chromatography [chloroform / methanol / acetic acid (90: 10: 1)] and then solidified (ether / petroleum ether). Add 50 mL of ethyl acetate and wash with 1N HCl (x 1) and water (x 3). Concentrate the organic layer and solidify (ether / petroleum ether). N ^α- (9-Fluorenylmethoxycarbonyl) -N ^ε- (SP) -La-ornithine (Fmoc-L-Orn (SP) -OH) was obtained as a pale yellow powder. Yield: 57 mg (40%): MALDI-MS (SA) calcd 732.8; obsd 733.4 [M + H] ⁺ , 717.6 [M-Me] ⁺ , 703.4 [M-(2 x Me)] ⁺

Using Fmoc-Gly-Rink amide resin (54 mg, 30 mmol, sub = 0.56 mmol / g), the same operation as in Example 1 was carried out to extend the peptide chain. The condensation of Fmoc-L-Orn (SP) -OH in the peptide chain was carried out by adding Fmoc-L-Orn (SP) -OH (33 mg, 45 mmol) and DIEA (16 mL, 90 mmol) in 2.0 mL of NMP. In addition, it was performed by shaking at room temperature for 1 hour. The obtained crude peptide was purified by RP-HPLC [(C4 semi-preparative column, AcCN: 10 → 10% (10 minutes) → 60% (30 minutes) containing 0.1% TFA)] and then lyophilized Thus, the peptide derivative represented by the formula (8) was obtained as a yellow powder. Yield 1.8 mg (3.6% from Fmoc-Gly-resin): MALDI-MS (SA) calcd 1593.8; obsd 1596.8 [M + H] ⁺ , 1581.3 [M-Me] ⁺ , 1562.6 [M-(2 x Me)] ⁺

Example 10
N ^α- (9-Fluorenylmethoxycarbonyl) was carried out in the same manner as in Example 9 except that Fmoc-L-Dab-OH (79 mg, 0.232 mmol) was used instead of Fmoc-L-Orn (Boc) -OH. -N ^epsilon - give (SP) -La-diaminobutylic acid of (Fmoc-Dab (SP) -OH ) as a pale yellow powder. Yield: 95 mg (68%): MALDI-MS (SA) calcd 718.8; obsd 718.4 [M + H] ⁺ , 702.2 [M-Me] ⁺ , 688.4 [M-(2 x Me)] ⁺ .

Subsequently, the same operation as in Example 9 was performed to condense Fmoc-Dab (SP) -OH, and the resulting crude peptide was RP-HPLC [(C18 semi-preparative column, AcCN: 30 → 60% ( 30 minutes) containing 0.1% TFA) and lyophilized to obtain the peptide derivative represented by the formula (9) as a yellow powder. Yield: 1.8 mg (3.8% from Fmoc-Gly-resin): MALDI-MS (SA) calcd 1579.8; obsd 1582.3 [M + H] ⁺ , 1566.0 [M-Me] ⁺

Example 11
N ^α- (9-Fluorenylmethoxycarbonyl) was carried out in the same manner as in Example 9 except that Fmoc-L-Dap-OH (80 mg, 0.232 mmol) was used instead of Fmoc-L-Orn (Boc) -OH. -N ^epsilon - the (SP) -La-diaminopropionic acid ( Fmoc-Dap (SP) -OH), to give a pale yellow powder. Yield: 83 mg (61%): MALDI-MS (SA) calcd 704.7; obsd 704.6 [M + H] ⁺ , 688.8 [M-Me] ⁺ , 674.5 [M-(2 x Me)] ⁺

Next, the same operation as in Example 9 was performed to condense Fmoc-Dap (SP) -OH, and the resulting crude peptide was RP-HPLC [(C18 semi-preparative column, AcCN: 30 → 60% ( 30 minutes) containing 0.1% TFA) and lyophilized to obtain the peptide derivative represented by the formula (10) as a yellow powder. Yield: 1.9 mg (4.0% from Fmoc-Gly-resin): MALDI-MS (SA) calcd 1565.8; obsd 1566.6 [M + H] ⁺ , 1551.0 [M-Me] ⁺

Example 12
Using a half amount (15.0 μmol) of the resin with a protected peptide obtained in Example 1, the same procedure as in Example 1 was performed to remove the peptide and remove the Mtt protecting group on the Lys side chain. The condensation of the Lys side chain in the peptide chain and the compound represented by the formula (16) was performed by adding the compound represented by the formula (16) (30 mg, 45 μmol) and DIEA (15 μL, 90 μmol) to 2.0 mL of NMP for 2 hours at room temperature. The mixture was shaken, DIEA (15 μL, 90 μmol) was added, and the mixture was shaken overnight at room temperature. The obtained crude peptide was purified by RP-HPLC [(C4 semi-preparative column, AcCN: 20 → 50% (30 min) containing 0.1% TFA)], lyophilized, and represented by the formula (11) The peptide derivative was obtained as a yellow powder. Yield: 11.8 mg (50% from Fmoc-Gly-resin): MALDI-MS (SA) calcd 1577.9; obsd 1580.4 [M + H] ⁺

Example 13
Using Fmoc-Gly-Rink amide resin (54 mg, 30 μmol, sub = 0.56 mmol / g), the same procedure as in Example 1 was performed to extend the peptide chain. Condensation of the compound represented by the formula (14) with the N-terminal in the peptide chain was performed by adding the compound represented by the formula (14) (47 mg, 90 μmol) and DIEA (31 μL, 180 μmol) to 2.0 mL of NMP for 2 hours at room temperature. The mixture was shaken, and DIEA (31 μL, 180 μmol) was added, followed by shaking overnight at room temperature. The obtained crude peptide was purified by RP-HPLC [(C4 semi-preparative column, AcCN; 30 → 60% (30 min) containing 0.1% TFA)], lyophilized, and represented by the formula (12) The peptide derivative was obtained as a yellow powder. Yield: 3.3 mg (7.3% from Fmoc-Gly-resin): MALDI-MS (SA) calcd 1508.7; obsd 1509.9 [M + H] ⁺ , 1494.9 [M-Me] ⁺ , 1478.2 [M-(2 x Me)] ⁺

Example 14
The peptide derivatives represented by the formulas (1) to (4) were brought into contact with various proteins as target molecules, and the photoisomerization of spiropyran from the merocyanine form to the spiropyran form in the peptide derivative was examined. The measurement was performed as follows. As the protein, protein kinase A, α-amylase, β-galactosidase, lysozyme, hexokinase, and S-100 protein were used.

1 mL of a sample solution of 20 mM Tris HCl (pH 7.4) and 150 mM NaCl containing 2.0 to 2.2 μM of the peptide derivative represented by the formulas (1) to (4) and 0.76 mg / mL of protein was stored in the dark. The sample solution was left overnight in a cool dark place (4 ° C.). This sample solution was transferred into a measurement cell of a fluorescence spectrophotometer in the dark, and left at 4 ° C. for 30 minutes in the dark. At the same time when the shutter of the measurement sample chamber was opened, the time change of the fluorescence intensity at 600 nm caused by 510 nm excitation light was measured at 4 ° C. with a Hitachi F-2500 fluorescence spectrophotometer. The obtained attenuation curve was fitted with F = (F ₀ −F _∞ ) exp (−kt) + F _∞ to obtain the photoisomerization rate constant. As an index of intermolecular interaction, changes in photoisomerization rate constant (k / k ₀ , where k = photoisomerization rate constant when protein is added, k ₀ = photoisomerization rate constant when no protein is added, ) And the rate of change [(k−k ₀ ) = Δk / k ₀ ]. The results for the peptide derivative represented by formula (1) are shown in FIG. 6, the results for the peptide derivative represented by formula (2) are shown in FIG. 7, and the results for the peptide derivative represented by formula (3) are shown in FIG. The results for the peptide derivative represented by formula (4) are shown in FIG.
Each figure is a graph showing the results of examining the photoisomerization rate constants of peptide derivatives and various proteins, and the vertical axis shows k / k0. k is a photoisomerization rate constant when protein is added, and k0 is a photoisomerization rate constant when no protein is added.

As shown in FIG. 6, when the peptide derivative represented by the formula (1) interacts with protein kinase A, α-amylase, β-galactosidase, lysozyme, and S-100 protein, the photoisomerization rate constant is When it interacts with hexokinase, it becomes smaller.
As shown in FIG. 7, the peptide derivative represented by the formula (2) has a high photoisomerization rate constant when interacting with protein kinase A, β-galactosidase, lysozyme, hexokinase, and S-100 protein. There is almost no change when interacting with α-amylase.
As shown in FIG. 8, when the peptide derivative represented by the formula (3) interacts with protein kinase A, the photoisomerization rate increases, but α-amylase, β-galactosidase, lysozyme, hexokinase, S When interacting with -100 protein, the photoisomerization rate constant decreases.
Further, as shown in FIG. 9, the peptide derivative represented by the formula (4) is photoisomerized when interacting with protein kinase A, α-amylase, β-galactosidase, lysozyme, hexokinase, and S-100 protein. The rate constant becomes smaller.

  From the above results, the change rate Δk / k0 of the photoisomerization rate constant in each protein is shown in FIG. FIG. 10 is a graph showing the rate of change of the photoisomerization rate constant when various peptide derivatives interact with various proteins. As shown in FIG. 10, protein kinase A (+ / + / + / −), α-amylase (− / + / 0 / −), β in the order of the peptide derivatives represented by formulas (1) to (4). -Galactosidase (-/ + / + /-), lysozyme (-/ + / + /-), hexokinase (-/-/ + /-), S-100 protein (-/ + / + /-) The results characterizing the protein were obtained. From this, the usefulness of the “protein fingerprint” produced using the method of the present invention was shown.

Example 15
The protein concentration dependency of the photoisomerization rate constant of the peptide derivative represented by the formula (1) from the merocyanine form to the spiropyran form was examined. The operation was carried out in the same manner as in Example 14, and the protein concentrations were measured at 0, 0.095, 0.19, 0.38 and 0.76 mg / mL. Protein kinase A and α-amylase were used as proteins. The results are shown in FIG. FIG. 11 is a graph showing the results of examining the protein concentration dependence of the photoisomerization rate constant of a peptide derivative from a merocyanine form to a spiropyran form, with the horizontal axis representing the protein concentration and the vertical axis representing the change in the photoisomerization rate constant. Indicates the rate. As shown in FIG. 11, when protein kinase A was added, the rate constant increased with increasing concentration. On the other hand, when α-amylase was used, the rate constant decreased with increasing concentration. From this result, it was shown that the protein concentration is quantitative by the method of the present invention.

Example 16
The effect on the photoisomerization rate constant when two kinds of proteins coexist was examined. The measurement conditions were the same as in Example 14. The peptide derivative represented by the formula (1) was used as the peptide derivative, and protein kinase A and α-amylase were used as the proteins. The results are shown in FIG. As shown in FIG. 12, when protein kinase A was used, a change rate of +0.046 was shown, and when α-amylase was used, a change rate of -0.071 was shown. When protein kinase A and α-amylase were used, a change rate of −0.0051 was shown. The sum of when protein kinase A is used and when α-amylase is used is -0.025, and the value is smaller when both are mixed. It was found that the acceleration / deceleration effect of the photoisomerization rate constant is distributed depending on the affinity of the peptide. From this, it was shown that by adding a known protein to the sample, the protein can be detected by a multidimensional “protein fingerprint”.

It is a figure which shows the synthesis | combination of activated spiropyran. It is a simplification figure of the manufacturing method of a peptide derivative. It is a figure which shows the amination of spiropyran and the synthesis | combination of activated amino spiropyran. It is a figure which shows the manufacturing process of the compound for manufacturing the peptide derivative shown to Formula (8)-(10). It is a figure which shows the interaction mode of a peptide derivative and protein. It is a graph which shows the result of having investigated interaction with a peptide derivative and protein. It is a graph which shows the result of having investigated interaction with a peptide derivative and protein. It is a graph which shows the result of having investigated interaction with a peptide derivative and protein. It is a graph which shows the result of having investigated interaction with a peptide derivative and protein. It is a graph which shows the change rate of a photoisomerization rate constant when various peptide derivatives and various proteins interact. FIG. 11 is a graph showing the results of examining the protein concentration dependence of the photoisomerization rate constant of a peptide derivative from a merocyanine form to a spiropyran form. It is a figure which shows the change rate of the photoisomerization rate constant at the time of interaction with a peptide derivative and a protein mixed system.

Claims (11)

A peptide derivative formed by binding a photochromic compound in the molecule. The peptide derivative according to claim 1, wherein the photochromic compound is spiropyran. The peptide derivative according to claim 1 or 2, which is bound to a solid phase. A method for detecting an intermolecular interaction of a target molecule, comprising the following steps.
(A) contacting the target molecule with a substance formed by binding a photochromic compound in the molecule; and (b) measuring the photoisomerization rate constant of the photochromic compound; The method according to claim 4, wherein the target molecule is a protein, nucleic acid, sugar chain, or low molecular weight compound. The method according to claim 4 or 5, wherein the photochromic compound is spiropyran. The method according to any one of claims 4 to 6, wherein a substance formed by binding a photochromic compound in the molecule is immobilized on a solid phase. A method for detecting an intermolecular interaction of a target molecule, comprising the following steps.
(A) contacting a peptide derivative formed by binding a photochromic compound in the molecule with a target molecule; and (b) measuring a photoisomerization rate constant of the photochromic compound; The method according to claim 8, wherein the target molecule is a protein, a nucleic acid, a sugar chain, or a low molecular weight compound. The method according to claim 8 or 9, wherein the photochromic compound is spiropyran. The method according to any one of claims 8 to 10, wherein a peptide derivative formed by binding a photochromic compound in the molecule is immobilized on a solid phase.

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