JP2003246776A - Cross-linkable photochromic molecule and driving reagent for biofunctional molecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cross-linkable photochromic molecule which can make a biofunctional molecule change the structure reversibly and a driving reagent for the biofunctional molecule which can generate mechanical energy by photo- irradiation. <P>SOLUTION: The cross-linkable photochromic molecule is indicated in formula (8a) or (8b). The driving reagent for the biofunctional molecule is bonded to two thiol radicals in the biofunctional molecule via the maleinimide radicals in the formula (8a) or (8b) to cross-link the thiol radicals. The distance between the thiol radicals can be changed within a constant range by irradiation of an ultraviolet ray or a visible ray. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a driving reagent for a crosslinkable photochromic molecule and a biofunctional molecule.

[0002]

2. Description of the Related Art Photochromic molecules are compounds that reversibly change their structure by absorbing light of a specific wavelength, such as azobenzene derivatives, metacyclophane derivatives, fulgide derivatives, dithienylethene, spiropyran derivatives, anthracene dimers, stilbene derivatives. Etc. are known. These photochromic molecules are used in optical storage materials, light control materials, display materials, optical switch elements and the like.

The azobenzene derivative is a typical photochromic molecule and undergoes a cis-trans structure change upon irradiation with light. When the azobenzene derivative is irradiated with ultraviolet rays, it becomes a cis type (5a), and when it is irradiated with visible light, it becomes a trans type (5b).

[0004]

[Chemical 5]

Such properties of the azobenzene derivative are
For example, it can be used for drive control of a molecular shuttle. Figure 3
As shown in (a), a molecule in which a ring-shaped molecule 2 is fitted into a dumbbell-shaped molecule 1 is called a rotaxane. Figure 3
As shown in (b), the portions (stations) 3a, 3 where the interaction with the ring-shaped molecule 2 is relatively strong at two locations on the shaft portion.
If b is provided, the ring-shaped molecule 2 is station 3a,
Reciprocates between 3b (Molecular shuttle).

In recent years, nanotechnology for constructing a molecular assembly such as a micro-nanomachine or a molecular device by using an intermolecular interaction with a molecule as a part has been attracting attention. In a molecular assembly such as a micro-nano machine, intermolecular interaction is used for driving and controlling.

Formula (6) shows an example of rotaxane. In the rotaxane of the formula (6), an azobenzene derivative is introduced into the shaft portion, and cyclodextrin is used as a ring-shaped molecule.

[0008]

[Chemical 6]

In the rotaxane of the formula (6), the shaft portion 4 bends and stretches as schematically shown in FIG. 4 in response to the cis-trans structure change of azobenzene. When the shaft portion 4 bends, the cyclodextrin 5 cannot move and the molecular shuttle stops. Since the structural change of azobenzene is controlled by light irradiation, the molecular shuttle can also be controlled by light irradiation.

Further, metacyclophane-ene becomes a ring-opening structure (7a) by irradiation with visible light, and becomes a ring-closing structure (7b) by irradiation of ultraviolet rays. By utilizing this structural change, molecular recognition and metal recognition can be switched on / off by light irradiation (optical switching). A molecular recognition site S such as calixarene is introduced into metacyclophane-ene. Alternatively, a metal recognition site such as a ligand may be introduced.

[0011]

[Chemical 7]

In the ring-closed structure (7b), since the molecular recognition sites S are fixed at positions distant from each other, the molecule (guest molecule) to be recognized cannot be captured (OFF state). On the other hand, when the ring-opening structure (7a) is taken, the movable range of the molecular recognition site S is expanded (on state), and the pair of molecular recognition sites S can be brought close to each other (7c). Therefore, the guest molecule can be incorporated into the molecular recognition site S (7d). Thus, the metacyclophane-ene derivative can control the recognition or capture of molecules and metals by light irradiation.

Further, the fulgide derivative reversibly changes color in accordance with the structural change caused by light irradiation. The fulgide derivative has a ring-closed structure upon irradiation with ultraviolet light and changes to red. When it is irradiated with visible light, it becomes a ring-opened structure and becomes colorless. A material obtained by dispersing or copolymerizing a fulgide derivative in a polymer liquid crystal can reversibly change the alignment state and birefringence of the liquid crystal by utilizing the structural change of the photochromic molecule. As described above, photochromic molecules such as azobenzene have various uses, and their applications to micro-nanomachines and functional materials are being actively researched.

[0014]

However, the above-mentioned conventional photochromic molecule is merely used for the change of color of the molecule itself due to light irradiation, or is merely used for switching, and has a polymer structure. Not designed as a source of driving force to change. On the other hand, proteins, which are macromolecular biofunctional molecules, are mostly used as enzymes that perform chemical reactions. Artificial micro-nanomachines and the like that utilize changes in the higher-order structure of proteins as mechanical energy have not been put to practical use.

Conventionally, various reagents for chemically modifying a protein have been developed, but basically, these do not affect the higher-order structure of the protein as much as possible, and are localized at a specific site of the protein. Designed to cause structural changes. In addition, proteins generally have temperature, salt concentration, and pH.
Structural changes occur depending on the environmental conditions such as or due to the presence of specific metal ions or coenzymes, but the reversible structural changes are limited.

Even when a protein undergoes a reversible structural change, it is easy to change the conditions under which such a structural change occurs in a short time or to change the structure to a different structure alternately at arbitrary time intervals. is not. Therefore, in order to construct a micro-nanomachine etc. using proteins,
A means for changing the higher-order structure of the protein is required without changing the environment of the protein (solution composition or temperature).

The present invention has been made in view of the above problems, and therefore an object of the present invention is to provide a crosslinkable photochromic molecule capable of reversibly causing a structural change in a biofunctional molecule. . Further, the present invention is
It is an object of the present invention to provide a biofunctional molecule driving reagent capable of producing minute mechanical energy in a biofunctional molecule by light irradiation.

[0018]

In order to achieve the above object, the crosslinkable photochromic molecule of the present invention has the formula (1)
Alternatively, it has a structure represented by (2). Thereby, for example, it becomes possible to crosslink two thiol groups in a polymer such as a protein and change the distance between the thiol groups within a predetermined range according to the wavelength of irradiation light. Further, in the crosslinkable photochromic molecule of the present invention, since the binding portion with the thiol group is an N-substituted maleimide, the reaction with the thiol group proceeds under mild conditions.

Alternatively, the crosslinkable photochromic molecule of the present invention may be one in which a group that binds to a specific amino acid residue is introduced, as shown in formulas (3) and (4). Also in this case, the length of the crosslinked portion can be changed by irradiation with light.

In order to achieve the above object, the driving reagent for a biofunctional molecule of the present invention is characterized by having a structure represented by the formula (1) or (2). This makes it possible to reversibly change the structure of biofunctional molecules such as proteins within a certain range by light irradiation. Therefore,
It becomes possible to construct molecular motors, micro-nanomachines, etc. using biofunctional molecules.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the crosslinkable photochromic molecule and the driving reagent for biofunctional molecules of the present invention will be described below with reference to the drawings. (Embodiment 1) Crosslinkable photochromic molecule 4,4'-azobenzenedimaleimide (4,4'-azo-benzene) of the present embodiment
-di-maleimide (ABDM)) is represented by the following formula (8a) or (8b).

[0022]

[Chemical 8]

ABDM is composed of two maleimide groups that bind to proteins and azobenzene that is photo-driven. The two benzene rings have a cis type and a trans type depending on the wavelength of the irradiated light. Cis type (8
a) and becomes a trans type (8b) by irradiation with visible light.

The maleimide group binds specifically to cysteine residues in proteins. Generally, the N-substituted maleimide (9a) is mild to the thiol (9b) (room temperature, pH).
It reacts within a few minutes at about 5 to 9 neutral) to form the thioether (9c).

[0025]

[Chemical 9]

Therefore, according to ABDM, two cysteine residues in the protein molecule are cross-linked. The cross-linking span (cross-link span) changes according to the cis-trans structure change of ABDM. Cross-link span is 5-6Å with cis type ABDM, transformer type ABDM
It becomes 15-16Å in DM.

Cis type ABDM and trans type ABDM
, The absorption maximum wavelength in the absorption spectrum is different, and cis type and trans type can be distinguished based on this. ABD
When M is irradiated with ultraviolet rays to form a cis type, the maximum absorption wavelength is 325 nm. Further, when the ABDM is irradiated with visible light to form a trans type, the absorption maximum wavelength becomes 345 nm.

FIG. 1 shows a change in absorption spectrum when a trans ABDM is irradiated with ultraviolet rays to be changed to a cis type. A mercury lamp (115V, 0.
Ultraviolet light having a wavelength of 366 nm was irradiated using 16A) as a light source. 70% ethanol was used as a solvent, and ultraviolet irradiation was performed at room temperature. As shown in FIG. 1, a transformer-type ABDM
The absorption maximum wavelength at (0 min) is 345 nm,
The absorption maximum wavelength shifts to 325 nm on the short wavelength side with the passage of time after the start of ultraviolet irradiation.

FIG. 2 shows a change in absorption spectrum when a cis type ABDM is irradiated with visible light and changed to a trans type. The ABDM was illuminated with a fluorescent lamp (27 W). 70% ethanol was used as a solvent, and irradiation with visible light was performed at room temperature. As shown in Fig. 2, cis-type ABDM
The absorption maximum wavelength at (0 min) is 325 nm,
The absorption maximum wavelength shifts to 345 nm on the long wavelength side with the passage of time after the start of visible light irradiation. The rate at which the ABDM changes from the trans type to the cis type or from the cis type to the trans type changes depending on the sample concentration and the output of the light source.

Two cysteine residues in the protein molecule have been cross-linked by ABDM, and the ABD is exposed to light.
When M is structurally changed between cis type and trans type, a structural change can be caused in the protein molecule. That is, the structural change of ABDM due to light irradiation can be used as a driving force for the structural change of protein. Further, by using the laser pulse light, it is possible to cause the cis-trans type change of the ABDM alternately at high speed.

Next, a method for synthesizing ABDM will be described. (I) Coupling of 4,4′-azoaniline (4,4′-azoaniline) and maleic anhydride As shown in the formula (10), 236 μmol of 4,4′-azoaniline (a) and 472 μmol of maleic anhydride are used. Was reacted in 2.6 ml of tetrahydrofuran (THF) at 4 ° C. for 22 hours. The suspended reaction solution was centrifuged at low speed to collect the precipitate. 2.6 ml of T was added to the recovered precipitate.
After adding HF and suspending, centrifugation was performed to obtain the compound (b).
Got

[0032]

[Chemical 10]

(II) Formation of Maleimide Group by Dehydration Condensation As shown in formula (11), the precipitation of compound (b) is 2.6.
Suspend in 0.5 ml of THF, add 0.5 ml of acetic anhydride and 5
It was transferred to a small test tube together with 0 mg of anhydrous sodium acetate, vacuum-sealed, and heated at 100 ° C. for 22 hours. 10 ml of water was added to the reaction solution in which the product (ABDM) was solubilized, and the mixture was stirred at 4 ° C for 10 minutes. Insolubilized ABDM
Was collected by low speed centrifugation. 1 again for the collected ABDM
It was washed by adding 0 ml of water, and was collected by low speed centrifugation. Water was dried using a rotary evaporator and a high vacuum pump to obtain a compound (c).

[0034]

[Chemical 11]

Compound (c) is a single layer having an Rf value of 0.82 when the mobile phase is 1-butanol: acetic acid: water = 5: 2: 3 in thin layer chromatography (TLC) using a silica gel plate. Spotted. Further, in the mass spectrometry, the compound (c) showed a molecular weight corresponding to ABDM.

ABDM manufactured by the above manufacturing method
It is also possible to cyclically drive the biofunctional molecule by binding the biofunctional molecule to the biofunctional molecule and alternately irradiating it with ultraviolet rays and visible light at a constant cycle using, for example, a pulse laser. Instead of using a pulsed laser, a continuous wave laser or a light source for stationary light other than a laser may be used, and a shutter or the like may be provided on the optical path to alternately irradiate ultraviolet rays and visible light.

(Embodiment 2) The protein-driven reagent of this embodiment is represented by the following formula (12a) or (12b):
At least one of the maleimide groups of BDM is replaced with another group that covalently bonds to a specific amino acid residue or a compound that strongly interacts with a specific protein.

[0038]

[Chemical 12]

Here, X and Y represent the same or different groups. Examples of groups that can be introduced into X and / or Y include succinimidyl ester (13a) and isothiocyanate (13b) (-N =
C = S) (2), sulfonyl chloride (-SO ₂ C
l) (13c).

The aldehyde (13d) reacts with the amino group to form a Schiff base. Reduction of the Schiff base gives stable compounds. Further, phthalaldehyde (13e) reacts with an amino group in the presence of a reducing agent such as 2-mercaptoethanol. These (13a) ~
(13e) binds to the N-terminal of the protein or the ε-amino group of the lysine residue.

[0041]

[Chemical 13]

Examples of the group which selectively bonds to the thiol group include benzofurazan (benzooxadiazole) (14a), iodoacetamide (14b) and aziridine (14c) in addition to the above maleimide group. . Also, 5,5'-dithiobis (2-nitrobenzoic acid) that reacts with thiol groups (Ellman's reagent or DTNB)
(14d) and 2,2'-dipyridyl disulfide (14e)
A symmetric disulfide compound, such as, may be introduced instead of the maleimide group.

[0043]

[Chemical 14]

Alternatively, the maleimide group of ABDM may be replaced with one of the systems known to specifically bind strongly, such as avidin and biotin, antigen and antibody, and receptor and ligand. . For example, if a low molecular weight compound such as biotin is introduced into the ABDM instead of the maleimide group, a protein such as avidin can be crosslinked.

In addition to the above, a precursor of nitrene or carbene such as azide or diazoacetyl may be introduced into X and / or Y of the formula (12). Azide (15a) and diazoacetyl (16a) are commonly used as a reactive group of a reagent for photoaffinity labeling.

In photoaffinity labeling, a reactive group activated by light irradiation is introduced into a ligand in advance,
It is a method of binding a ligand to a protein by light irradiation in a state where the ligand is adsorbed to a specific site of the protein by utilizing specific binding.

The nitrene (15b) produced by irradiating the azide (15a) with light and the carbene (16b) produced by irradiating the diazoacetyl (16a) with each other include an insertion into a C—H bond and an aromatic ring. Since various reactions such as addition to double bond and hydrogen abstraction reaction occur, the binding site selectivity of the reactive group itself is poor. However, by utilizing photoaffinity labeling, it is also possible to crosslink a specific site of a biofunctional molecule with a photochromic molecule.

[0048]

[Chemical 15] (In the formula, R ^{1 to} R ⁶ represent hydrogen or hydrocarbon. A substituent may be introduced into the hydrocarbon.)

[0049]

[Chemical 16] (In the formula, R ^{1 to} R ⁶ represent hydrogen or hydrocarbon. A substituent may be introduced into the hydrocarbon.)

In the ABDM and its derivatives, a spacer having a specific length may be provided between the benzene ring and the maleimide group or its substituent (or compound). However, in order to accurately control the change in the crosslink span of the ABDM derivative, the spacer is preferably a group having high rigidity.

According to the above-described driving reagent for the crosslinkable photochromic molecule and the biofunctional molecule of the present invention,
It becomes possible to reversibly cause a specific structural change in a biofunctional molecule. Therefore, the crosslinkable photochromic molecule of the present embodiment is useful as a reagent for protein research.

By cross-linking different sites of the protein using the biofunctional molecule driving reagent of the present embodiment, a molecular motor or micro-nanomachine having a size of several tens of nanometers that can be driven and controlled by light irradiation can be provided. Can be built. Such a micro-nano machine etc.
The length of the cross-linked portion can be changed within a fixed range of 5 to 17Å, and can be applied to, for example, the medical field.

The embodiments of the crosslinkable photochromic molecule and the driving agent for the biofunctional molecule of the present invention are not limited to the above description. For example, a biofunctional molecule is not limited to a naturally occurring protein, and if it includes a group or site that specifically binds to both ends of ABDM or a derivative thereof, a subunit of the protein or a mutant prepared using an expression system is used. Or a complex of the protein with a coenzyme or another material. Besides, various modifications can be made without departing from the scope of the present invention.

[0054]

According to the crosslinkable photochromic molecule of the present invention, it becomes possible to reversibly cause a structural change in a biofunctional molecule. According to the biofunctional molecule driving reagent of the present invention, it becomes possible to continuously generate minute mechanical energy by light irradiation.

[Brief description of drawings]

FIG. 1 shows an absorption spectrum when the crosslinkable photochromic molecule of the present invention is irradiated with ultraviolet rays to change from a trans type to a cis type.

FIG. 2 shows an absorption spectrum when the crosslinkable photochromic molecule of the present invention is irradiated with visible light to change from cis type to trans type.

3 (a) and 3 (b) are schematic views of a molecular shuttle.

FIG. 4 is a schematic diagram showing control of a molecular shuttle using a conventional crosslinkable photochromic molecule.

[Explanation of symbols]

1 ... Dumbbell-shaped molecule, 2 ... Ring-shaped molecule, 3a, 3b ...
Station, 4 ... Shaft part, 5 ... Cyclodextrin.

Claims (3)

[Claims] 1. A crosslinkable photochromic molecule represented by the following formula (1) or (2). [Chemical 1] [Chemical 2] 2. A crosslinkable photochromic molecule represented by the following formula (3) or (4). [Chemical 3] [Chemical 4] (However, in each formula, X and Y represent the same or different groups, X and / or Y are succinimidyl ester,
Isothiocyanate, sulfonyl chloride, aldehyde, phthalaldehyde, maleimide, benzofurazan,
It shall be selected from iodoacetamide, aziridine, disulfide compounds, biotin, azide, diazoacetyl. ) 3. A compound represented by the formula (1) or (2) according to claim 1, which bonds with two thiol groups in a biofunctional molecule through a maleimide group to crosslink between the thiol groups, A reagent for driving a biofunctional molecule, which changes the distance between the thiol groups within a certain range by irradiation with ultraviolet rays or visible rays.