JP2010030918A - epsilon-N-LUCIFERYLLYSINE COMPOUND, PEPTIDE-LABELING AGENT OBTAINED BY USING THE SAME AND PEPTIDE-LABELING METHOD - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a peptide-labeling technique by means of a new ε-N-luciferyllysine compound that can label a peptide or a protein with a label permitting detection utilizing luminescence by a firefly luciferin-photogenic beetle luciferase photogenic system and can adjust the amount of luminescence. <P>SOLUTION: The ε-N-luciferyllysine compound is represented by the general formula of the figure. A peptide labeled with luciferin is prepared by peptide synthesis using the compound as a peptide-labeling agent. Thus, detection of luminescence in the presence of an active substance making luciferin dissociate from the peptide can be performed. R in the general formula is a methyl group and Pv is a hydrogen atom or a fluorenylmethoxycarbonyl group. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a novel ε-N-luciferyl lysine compound having a structure of firefly luciferin, which is a luminescent substrate of a luminescent system by luminescent beetle luciferase, a peptide labeling agent and a peptide labeling method using the compound. More specifically, a novel ε-N-luciferyl lysine compound in which the ε-amino group of lysine is bound to luciferin, a peptide labeling agent using the compound, and a peptide label that enables detection by luciferase luminescence Regarding the method.

  Firefly luminescence, which is famous as bioluminescence, is due to the reaction of firefly luciferin-luminescent beetle luciferase luminescence system (firefly bioluminescence system), and the luminescent substrate firefly luciferin is activated by firefly luciferase in the presence of ATP and magnesium ions. Light is emitted by being converted to oxyluciferin, which is a light emitter. The firefly bioluminescence system has very high luminescence efficiency, and research is also underway on the interpretation of the molecular mechanism of bioluminescence. Gene expression and gene transfer efficiency can be achieved by introducing a luminescent beetle luciferase gene into a gene recombination vector or cell. It is known that it can be used for analysis of cell growth and cell growth monitoring. For this reason, attention has been paid in various fields such as biochemistry, medicine, pharmacy, and immunology, and its application is being studied, and a wide variety of luminescent materials using a firefly bioluminescence system are provided by various companies.

  In recent years, visualization of events and phenomena in monitoring has become important, and the labeling technology has been required to diversify with the expansion of the visualization target. In particular, molecular imaging has developed greatly in conjunction with advances in diagnostics and testing instruments, and for example, its application to advanced technologies such as personalized medicine such as cancer and heart disease has been energetically studied. In addition, with the advancement of measurement technology, the need for more sensitive and high performance instruments and labeling materials is rapidly increasing. On the other hand, product demand for reasonable systems with reasonable performance for a wide range of users is also expected, and the need for inexpensive new light-emitting materials that support this cannot be overlooked.

  Conventional technologies related to bioluminescent labeling materials include a system that lacks any of the elements required for the luminescent reaction (luminescent substrate, ATP, luminescent enzyme, etc.), and the aforementioned shortage when a target phenomenon occurs. There is a system that is configured to emit light by supplementing the elements to be monitored, and the target phenomenon is monitored by light emission. For example, a system using a compound in which galactose is bound to the phenolic hydroxyl group of luciferin is commercially available, and this compound has a luciferin that emits light by luciferase when the galactosidase enzyme acts and the bond with galactose is cleaved. The presence of galactosidase or an enzyme activity that acts equivalently is detected and visualized (for example, see Patent Document 1 below).

Further, in Patent Document 2 below, in order to quantify antigens, haptens or antibodies, an enzyme-ligand complex is formed using these as ligands, and luciferin that is released by the enzyme when a luciferin derivative acts on this is converted to luciferase. It is described that an enzyme is detected (that is, a ligand is detected) by using it to emit light. Several luminescent substrate-bound amino acid compounds in which the phenolic hydroxyl group or carboxyl group of luciferin is bound to an amino acid have been presented as luciferin derivatives.
Japanese Patent Laid-Open No. 11-332593 JP-T 63-501571

  Amino acids are constituents of living bodies, and peptides and proteins that are aggregates of amino acids have various physiological activities. Therefore, monitoring of phenomena related to peptides and proteins is useful in analyzing various biological reaction mechanisms. In order to monitor peptides and proteins by bioluminescence, it is necessary to prepare peptides or proteins labeled with a bioluminescent labeling material. For any peptide or protein, a peptide or protein labeled with an arbitrary labeling amount is obtained. It is important to develop bioluminescent labeling technology.

  The present invention can easily label peptides and proteins with a label capable of detection using the light emission of firefly luciferin-luminescent beetle luciferase luminescence system, and can adjust the amount of label to obtain a suitable amount of luminescence Another object of the present invention is to provide a novel ε-N-luciferyl lysine compound that is promising as a peptide labeling agent.

  Another object of the present invention is to provide a peptide labeling agent that can adjust the labeling amount of a peptide or protein and thereby detect light emission under suitable image conditions.

  In addition, the present invention can detect an appropriate amount of light by adjusting the introduction amount of a labeling agent when performing image monitoring by luminescence detection using an amino acid bound to a luminescent substrate of a firefly luciferin-luminescent beetle luciferase luminescence system as a labeling agent. It is another object of the present invention to provide a peptide labeling method capable of performing image monitoring.

  As a result of diligent studies to solve the above problems, a compound in which lysine having two amino groups is used as an amino acid to be bound to the luminescent substrate and the luminescent substrate is bound to the ε-amino group is used as a labeling agent. By using it, a part or all of the lysine contained in the peptide becomes a labeled peptide, so that it is possible to obtain a peptide with an increased or decreased amount of the labeling agent introduced as necessary, and there is a limit on the amount of emitted light. It was found that it was relaxed, and the present invention was achieved.

According to one embodiment of the present invention, the ε-N-luciferyl lysine compound is represented by the following general formula.

(R in the general formula is a methyl group, and Pv is a hydrogen atom or a fluorenylmethoxycarbonyl group.)
Moreover, according to one aspect of the present invention, a peptide labeling agent has the ε-N-luciferyl lysine compound.

  Moreover, according to one aspect of the present invention, the peptide labeling method comprises dissociating luciferin from the peptide by preparing a peptide labeled with luciferin by peptide synthesis using the ε-N-luciferyl lysine compound. Allows detection of luminescence in the presence of activity.

  The luminescence detection is detection of luminescence by the action of the luminescent beetle luciferase enzyme, and the amount of the ε-N-luciferyl lysine compound incorporated into the peptide can be adjusted in order to adjust the amount of detectable luminescence.

  In the peptide labeling method, the luciferin undergoes an oxidation reaction in the presence of pyrophosphate and Mg ions, thereby stabilizing the luminescence behavior and enabling quantitative analysis of the luminescence intensity by the luminescent beetle luciferase.

  The oxidation reaction can be performed using a luminescent beetle luciferase, an oxidase, or an oxidizing agent.

  According to the present invention, there is provided a novel compound that can be introduced into a peptide chain by reaction of an α-amino group of lysine by binding a luminescent substrate of a luminescent system by luminescent beetle luciferase and an ε-amino group of lysine. Thus, a promising compound as a peptide labeling agent capable of arbitrarily controlling the amount of light detected by adjusting the amount of this compound introduced into the peptide is provided. The setting of the amount introduced into the peptide makes it possible to realize a light emission intensity suitable for image monitoring, and therefore it is possible to adjust and optimize the light emission amount in image monitoring.

  With the progress of synthetic reaction technology and the development of peptide synthesizers, it is now possible to synthesize peptides from amino acids according to a preset amino acid sequence, and any natural or artificial peptide (a protein that is a high molecular weight peptide) Including) can be prepared. Therefore, a labeled peptide can be obtained by performing peptide synthesis using a labeled amino acid.

  That is, if a compound in which a luminescent substrate, that is, firefly luciferin (hereinafter referred to as luciferin) is bound to an amino acid is prepared and a peptide is synthesized using this compound, a peptide labeled with the luminescent substrate is obtained, and the luminescent substrate is obtained from this peptide. By detecting bioluminescence by the luminescent beetle luciferase (hereinafter referred to as luciferase) when the label is dissociated, the presence of the peptide and the enzyme activity causing the label dissociation is monitored. In other words, the amino acid to which the luminescent substrate is bound functions as an amino acid type peptide labeling agent that can label the peptide by being introduced into the peptide.

  The amino acid type peptide labeling agent is prepared by condensing a luminescent substrate using an amino acid having a functional group such as a hydroxyl group, an amino group, a thiol group, and a carboxyl group in addition to the carboxyl group and the α-position amino group. Can do. Examples of such amino acids include asparagine, aspartic acid, cysteine, glutamine, glutamic acid, lysine, arginine, histidine, serine, threonine, tryptophan, and tyrosine, but they have reactivity with luminescent substrates and applicability as labeling agents. Therefore, in the present invention, an amino acid type peptide labeling agent employing lysine and condensed with luciferin which is a luminescent substrate is proposed.

  Lysine is an amino acid having two amino groups. Regarding the condensation site with luciferin, there are amidation with an α-position or ε-position amino group of lysine and esterification with a carboxyl group, but the amino group or carboxyl group of lysine binds to luciferin. In the case of the synthesized compound, only one of the remaining α-amino group or carboxyl group can be incorporated into the peptide chain from the compound when synthesizing the peptide, so that it is limited to introduction into the N-terminus or C-terminus of the peptide. The That is, the application of the labeling agent is limited to labeling at the peptide end, and only one molecule of the labeling agent can be incorporated into one peptide chain. In this case, when the molecular weight of the molecule to be labeled is large or when detection is performed in a low concentration state, the presence of the labeling agent is small, so that detection is difficult due to a lack of the amount of emitted light, or a clear luminescent image is obtained. The possibility that it is not possible arises.

  In comparison, ε-N-luciferyl lysine in which the amino group at the ε-position of lysine is bound to luciferin has an α-amino group and a carboxyl group incorporated into the peptide chain in the same manner as lysine when introduced into the peptide chain. Therefore, it can be incorporated not only at the terminal of the peptide but also at any position in the peptide. Therefore, a peptide obtained by substituting a part or all of lysine constituting the peptide with ε-N-luciferyl lysine according to the amino acid sequence of the target peptide can be obtained. That is, a peptide in which part or all of the lysine site in the peptide is labeled with luciferin can be obtained. In addition, since an arbitrary amount of ε-N-luciferyl lysine can be introduced into the peptide, the frequency of labeling can be adjusted. Therefore, even a high molecular weight peptide or a peptide in a low concentration condition can be a labeling method capable of providing a light amount suitable for luminescence detection and image monitoring by increasing the labeling amount.

  Hereinafter, the novel ε-N-luciferyl lysine compound according to the present invention and a peptide labeling method using the compound as a peptide labeling agent will be described in detail.

The present invention proposes an ε-N-luciferyl lysine compound represented by the following formula as a promising new compound as a peptide labeling agent.

(R in the above general formula is a methyl group, and Pv is a hydrogen atom or a fluorenylmethoxycarbonyl group.)
The condensation of the carboxyl group of firefly luciferin and the amino group at the ε-position of the lysine ester is carried out by gently activating the carboxyl group of luciferin in the coexistence state of luciferin and lysine ester. Obtained by condensation. The ε-amino group of the lysine ester is more reactive than the α-amino group. However, when the activity of the carboxyl group of luciferin is high, a compound in which both amino groups of the lysine ester are condensed with luciferin is produced. Prepare by activation.

  The ε-N-luciferyl lysine compound can be used as a substitute for lysine when performing peptide synthesis, whereby a peptide in which lysine in the peptide is labeled with luciferin can be obtained. That is, the ε-N-luciferyl lysine compound functions as a peptide labeling agent. The amount of labeling can be adjusted by the amount of ε-N-luciferyl lysine compound used. The peptide synthesis method may be performed by arbitrarily selecting and using various methods and equipment based on existing organic synthetic chemistry, or may be automatically synthesized by a peptide synthesizer. Peptide synthesis methods include peptide formation using a condensing agent, active esterification method, mixed acid anhydride method, ligation, solid phase synthesis method and the like.

  When an ε-N-luciferyl lysine compound is used as a peptide labeling agent in organic synthetic chemical peptide synthesis by a conventional method, the α-amino group or carboxyl group needs to be protected. In particular, since an amino acid having a free α-amino group is easily racemized, it is always necessary to protect the amino group when the α-amino group is not reacted (the carboxyl group is condensed to form a peptide). Therefore, as a compound that is actually supplied to peptide synthesis as a peptide labeling agent, a compound in which a carboxyl group is protected with a methyl group as a protecting group R, or an α-amino group with a fluorenylmethoxycarbonyl group as a protecting group Pv. A protected compound is included, and an ε-N-luciferyl lysine compound having such a protecting group can be appropriately selected according to the use mode in peptide synthesis. These ε-N-luciferyl lysine compounds can be introduced and desorbed by a known method. Specifically, the protecting group R of the carboxyl group is alkoxylated by treatment with an alcohol solution of hydrochloric acid or a reaction product of alcohol and thionyl chloride, and removed by a hydrogenation reaction or treatment with trifluoroacetic acid. Release. For the protecting group Pv of the α-amino group, a fluorenylmethoxycarbonyl group is introduced by reacting with N- (fluorenylmethoxycarbonyloxy) succinimide in the presence of an amine, and is eliminated by treatment with piperidine. .

  Peptides synthesized using ε-N-luciferyl lysine compounds as described above behave in the same manner as peptides having the same amino acid sequence without labeling, and are similarly subjected to chemical and biochemical effects from the surroundings. . As a result, if there is a chemical environment that promotes enzymatic activity or similar hydrolysis, such as acylase, which is a kind of amidase, in the environment from which the peptide was obtained, the amide bond between the luciferin moiety and the lysine moiety is degraded. Luciferin dissociates from the peptide. Therefore, by detecting luminescence of the dissociated luciferin using luciferase, the behavior, movement and arrangement of the peptide are visualized and imaged. By utilizing this, for example, it is possible to check the function of synthetic peptides and to trace behavior and movement, such as movement inside and outside the boundary through cell membranes, localization / dispersion tendency, reaction / interaction with other substances, etc. Investigation is possible. In addition, when a gene encoding the amino acid sequence of an enzyme having a hydrolytic ability to release luciferin from a labeled peptide is incorporated into a vector and expressed in a cell, the peptide synthesized using the amino acid type labeling agent of the present invention acts. Then, since the release of labeled luciferin occurs in the cell having the vector, the gene can be traced by detecting the luminescence, and the introduction state of the gene and the behavior of the cell can be monitored and imaged.

  Since the bioluminescence detection of the released luciferin is enabled by the presence of the luminescent enzyme, oxygen and ATP, a luciferase composition containing these and appropriately adjusting the pH can be used as the luminescence kit. The pH of the luminescent system is not particularly limited, but is preferably 4 to 10, more preferably 6 to 8. If necessary, potassium phosphate, Tris-HCl, glycine, HEPES, etc. are used to stabilize the pH. These buffering agents can be used as appropriate. Although the light emitting system is an aqueous system, the presence of a hydrophilic organic compound is allowed, and therefore, for example, tetrafluoroacetic acid, acetic acid, formic acid and the like may be contained.

  Luciferin can also be detected by chemiluminescence. Chemiluminescence occurs by oxidizing the luciferin to produce a peroxide, and the decomposition product of the peroxide becomes an excited luminescent species. Oxidation proceeds by air oxidation with t-butoxypotassium in DMSO. Therefore, when such an oxidation system is allowed, detection and visualization by chemiluminescence are possible.

  Therefore, when the peptide labeling agent of the present invention is used, protein processing enzyme expression, protein amount assay, and protein localization bioimaging can be performed based on the above-described luminescence form. It is also possible to bioimage a sugar chain addition process necessary for protein ripening and to observe protein / protein interactions.

  Hereinafter, the present invention will be described in detail with reference to examples. The present invention is not limited in any way by these examples. In the present application, “%” indicates “% by mass” unless otherwise specified.

[Preparation of ε-N-luciferyl lysine methyl ester]
(D) -Luciferin (103.8 mg, 0.37 mmol), (L) -lysine methyl ester dihydrochloride (103.7 mg, 0.42 mmol) and 1-hydroxybenzotriazole (62.3 mg, 0.46 mmol) N, N-diisopropylethylamine (150 μl, 1.56 mmol) was added to 1.5 ml of anhydrous DMF solution containing While cooling this D-luciferin solution in an ice bath, 1 ml of a chloroform solution containing 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (99 mg, 0.52 mmol) was added dropwise over 1 minute. After stirring for 3 hours, the reaction was carried out by stirring for 14 hours at room temperature. The contents during the reaction were traced by analyzing by HPLC. The analysis conditions were as follows, and the methyl ester of ε-N- (D) -luciferyl- (L) -lysine eluted at around 9.0 minutes.

  Distilled water (30 ml) was added to the reaction solution, and extracted with 30 ml of ethyl acetate three times. The ethyl acetate extracts were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude ε-N- (D) -luciferyl- (L) -lysine methyl ester (purity 60-70%). This was separated and purified by a column using the following analysis conditions, and confirmed by mass spectrometry and NMR analysis. The obtained compound data is as follows.

<Analysis conditions>
Analytical column: Kanto Chemical Mightysil PR-18 GP (H), 250 × 4.6 (5 μm)
Solvent: 0.05% TFAaq: CH3CN = 1: 9 → 9: 1 (20 minutes), 1: 9 (20-30 minutes)
Flow rate: 1 ml / min, column temperature: 20 ° C
Detection: 254, 330 nm (DAD), 510 nm (exp. = 330 nm, FD)
<Compound data of ε-N- (D) -luciferyl- (L) -lysine methyl ester>
MS (ESI) [M + H]; m / z 423.1213
1H-NMR (270 MHz, CD3OD): δ1.46-1.60 (2H, complex), 1.65-1.77 (2H, complex), 1.88-2.00 (2H, complex), 2. 19 (2H, t, J = 7.5 Hz), 3.69 (1H, d, J = 9.7 Hz), 3.75 (1H, d, J = 9.7 Hz), 3.82 (3H, s ), 4.03 (1H, dt, J = 6.5, 2.3 Hz), 5.34 (1H, t, J = 9.7 Hz), 7.07 (1H, dd, J = 8.9, 2.3 Hz), 7.34 (1 H, d, J = 2.3 Hz), 7.90 (1 H, d, J = 8.9 Hz)

[Preparation of α-N-fluorenylmethoxycarbonyl-ε-N- (D) -luciferyl- (L) -lysine methyl ester]
Ε-N- (D) -Luciferyl- (L) -lysine methyl ester obtained in Example 2 (50 mg, 0.12 mmol), 1 ml of dichloromethane and 9-fluorenylmethyl chloroformate (151.2 mg, 0.58 mmol) ) Were mixed and dissolved. Then, N, N-diisopropylethylamine (200 μl, 2.08 mmol) was added dropwise and stirred at room temperature for 3 hours. After confirming the disappearance of the raw material by HPLC under the same analysis conditions as in Example 1, the contents during the reaction were
30 ml of distilled water was added and extracted three times with 30 ml of ethyl acetate. The ethyl acetate extracts were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude α-N-fluorenylmethoxycarbonyl-ε-N- (D) -luciferyl- (L) -lysine. This was separated and purified by a column using the same analysis conditions and confirmed by mass spectrometry. The obtained compound data is as follows.

<Compound data of α- (N-fluorenylmethoxycarbonyl) -ε- (D) -luciferyl- (L) -lysine methyl ester>
MS (ESI) [M + Na ⁺ ]; m / z 667.0852
MS (ESI) [M + K ⁺ ]; m / z 683.0740

Claims (4)

An ε-N-luciferyl lysine compound represented by the following general formula.

(R in the general formula is a methyl group, and Pv is a hydrogen atom or a fluorenylmethoxycarbonyl group.)   A peptide labeling agent having the ε-N-luciferyl lysine compound according to claim 1.   By preparing a peptide labeled with luciferin by peptide synthesis using the ε-N-luciferyl lysine compound according to claim 1, it is possible to detect luminescence in the presence of an activity to dissociate luciferin from the peptide. Peptide labeling method.   The luminescence detection is detection of luminescence by the action of a luminescent beetle luciferase enzyme, and the amount of ε-N-luciferyl lysine compound incorporated into a peptide is adjusted in order to adjust the amount of detectable luminescence. Peptide labeling method.