JP2001114797A - Calix[4]resorcarene derivative having sugar cluster - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new type of sugar component-containing synthetic compound which can be applied under conditions approximating to those of a biosystem. SOLUTION: This calix[4]resorcarene derivative is represented by the general formula (I), wherein R is a hydrophobic group; X is CH2CH2NHCOCH2S-Sugar; Sugar is the residue of a sugar constituting a sugar chain.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a synthetic compound having a sugar component, and more particularly to a compound having a cluster of saccharide components such as sialic acid and having a strong interaction with a sugar-specific substance. The present invention relates to a macrocyclic compound exhibiting a function.

[0002]

2. Description of the Related Art A sugar chain composed of sialic acid and other saccharides is present on the surface of a cell membrane, and is used for adhesion and information exchange between cells, cell proliferation and differentiation, infection of cells with viruses and bacteria, and the like. It is involved in many important phenomena in the body.

As one of the means for studying such cellular phenomena, various models designed to simulate the functions of sugar chains or sugars in order to develop useful drugs and reagents, etc. Compounds have also been synthesized, but few are able to create an environment similar to real biological systems.

[0004] For example, since sugar is extremely water-soluble, it is not possible to grasp functions such as sugar bond by hydrogen bond in water in a normal state. Therefore, many conventional model systems for examining the functions of sugars or sugar chains have been limited to those applied in nonpolar organic solvents, not in water as in biological systems. Further, conventional model compounds relating to sugars or sugar chains are exclusively aimed at a single function, and there are few synthetic compounds that have multifunctions that can be applied to complex biological systems.

[0005] It is an object of the present invention to provide a new type of sugar component-containing synthetic compound which can be applied under conditions similar to a biological system and exhibits multiple functions.

[0006]

Means for Solving the Problems The present inventor has used calixresorcarene, a kind of macrocyclic compound, to derive a novel compound which can achieve the above-mentioned object. That is, the present invention provides a calix [4] resorcarene derivative represented by the following general formula (I).

[0007]

Embedded image

However, in the formula (I), R represents a hydrophobic group,
X represents _{CH 2 CH 2 NHCOCH 2 S-} Sugar, Sugar represents a residue of a sugar constituting a sugar chain.

In a preferred embodiment of the present invention,
In the above, Sugar is a sialic acid residue, and in a particularly preferred embodiment, X contains sialic acid represented by the following structural formula (II).

[0010]

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[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The calixresorcalene from which the compound of the present invention is derived is also called a resorcinol cyclic tetramer, a calixresorcinalene, a resorsialen, a calixresorcinol, or the like. It is a cyclic compound having eight phenolic hydroxyl groups that are H (hydrogen atoms). Calix of the present invention [4]
The resorcalene derivative is formed by introducing a sugar constituting a sugar chain such as sialic acid in the form of an S-glycoside bond into a site of the calixresorcalene (resorcinol cyclic tetramer) where eight hydroxyl groups are present. The formed synthetic compound.

The hydrophobic group represented by R in the general formula (I) representing the calix [4] resoralene derivative of the present invention is not particularly limited, but is generally a linear alkyl group having 1 to 18 carbon atoms. For example, an undecyl group (C ₁₁ H ₂₃ ) is preferred.

Further, C in the formula (I) represented by X
Sugar of H ₂ CH ₂ NHCOCH ₂ S-Sugar
Examples of the sugar residues constituting the sugar chain include sialic acid, N-acetylglucosamine, galactose, mannose, fucose and other sugar residues. These different sugars are 2
Also included are sugar residues that are linked together. As described later, the present invention is based on the fact that clusters of various sugars can be efficiently introduced into a calixresoralene structure by S-glycoside bonds.

In order to produce the calix [4] resorcalene derivative of the present invention, in the above formula (I), X is H
(Hydrogen atom) calixresorcarene (resorcinol cyclic tetramer) as a basic skeleton, and a chloroacetamide group at the terminal of the eight phenolic hydroxyl groups, that is, X = CH ₂ CH ₂ NHCOCH in the formula (I) _A material into which ₂ Cl has been introduced is used as a starting material. As is well known, calixresoralene (resorcinol cyclic tetramer) serving as a basic skeleton is obtained by using resorcinol and an appropriate aldehyde [an appropriate aliphatic or aromatic aldehyde depending on R of the formula (I)]. Can be easily obtained by heating in an acidic alcohol solution (for example, F. Weinelt et al., J. Am.
Org. Chem., 56 , 5527 (1991).

On the other hand, the sugar to be introduced into the calixresorarene raw material is obtained by protecting a hydroxyl group other than the anomeric position of the saccharide with an appropriate protecting group (eg, acetyl group) and introducing a halogen at the anomeric position. Are synthesized.
This is treated with potassium thioacetate, and only the anomeric position is selectively hydrolyzed to obtain a sugar derivative having an SH residue (S
ugar-SH).

Next, the thus obtained saccharide derivative is used as a starting material [in the formula (I), X = CH ₂ CH ₂ N
Halogenated with a calixresoralene which is HCOCH ₂ Cl] to introduce eight sugar residues, and by removing a protecting group (acetyl group), calix [ 4]
A resorcalene derivative is obtained. According to the present invention, by reacting a calixresorcarene raw material as a sugar derivative having an anomeric position of a sugar as an SH group, the coupling reaction proceeds extremely efficiently, and eight phenols having a calixresorcalene structure are formed. S on all of the hydroxyl groups
-A calix [4] resorcarene derivative in which various sugars (sugar residues) are introduced via glycoside bonds to form sugar clusters can be obtained.

The calix [4] resorcarene derivative of the present invention synthesized as described above has the following unique properties and exhibits multiple functions. (1) The cyclic tetramer structure of resorcinol in the calix [4] resorcarene derivative of the present invention forms aromatic ring vacancies with high electron density, and captures (inclusions) various molecules as guests in the vacancies. can do. The guest molecule may be anionic or cationic.

(2) The calix [4] resorcarene derivative of the present invention can be easily adsorbed on a hydrophobic surface via a lower hydrophobic chain [a tail represented by R in the formula (I)].

(3) The calix [4] resorcarene derivative of the present invention has a sugar cluster close to the cell surface at the upper part of the eight phenolic hydroxyl groups, and contains a substance specific to the sugar. Perform strong interactions. For example, the calix [4] resorcarene derivative of the present invention having a sialic acid cluster strongly interacts with sialic acid-specific lectins and influenza virus (human, type A).

(4) The calix [4] resorcarene derivative of the present invention has extremely high water solubility and does not aggregate at a low concentration (generally about 1 mM or less).

By combining the above properties, calix [4] resorcarene derivative of the present invention is
It is expected that it can be applied to various uses. For example, the calix [4] resorcarene of the present invention can be transported to a receptor site (sugar-specific site) in a biological fluid (aqueous solution) with a medicinal component or a probe component captured as a guest molecule in an aromatic ring vacancy. It can be used as such a drug or reagent.

The calix [4] resorcarene derivative of the present invention examines various physiological functions and the like based on the interaction between a sugar (sugar chain) and a substance specific thereto in an aqueous solution similar to a biological fluid. It can also be used as a model compound. Further, the calix [4] resorcarene derivative of the present invention is adsorbed (immobilized) on a hydrophobic support (carrier) to collect or remove, detect (analyze), or remove sugar-specific substances or viruses. It can also be applied as a functional material to be purified.

[0023]

EXAMPLES Next, examples will be shown to further clarify the features of the present invention, but the present invention is not limited to these examples. In the following description, Me represents a methyl group (CH ₃ ) and Et represents an ethyl group (C ₂ H ₅ ).

[0024]Example 1 Calix [4] Resorcarene
Derivative synthesis  In the general formula (I) described above, when R is C₁₁H₂₃And
X is the sialic acid cluster-containing potassium of the formula (II) described above.
[4] resorcalene derivative was synthesized as follows.
(See FIG. 1).

Octa (chloroacetamide) compound (compound 1b in FIG. 1) 597 in which a chloroacetamide group is introduced into eight phenolic hydroxyl groups of resorcinol cyclic tetramer
mg (0.290 mmol) was dissolved in a mixed solvent of Et ₃ N / DMSO to obtain a 1% solution (25 mL).

On the other hand, hydroxyl groups other than the anomeric position of sialic acid are protected with acetyl, and a base atom is introduced into the anomeric position, which is treated with potassium thiosulfate, and further hydrolyzed, whereby the anomeric position is protected. Glycosyl thiol (compound 2 in FIG. 1) was prepared.

1469 mg of this glycosyl thiol
(2.9 mmol) was added to the above octa (chloroacetamide) compound 1b in Et ₃ N / DMSO,
The coupling reaction was performed at room temperature for 12 hours under a nitrogen atmosphere. After completion of the reaction, the solvent was removed by freeze-drying, and the residue was dissolved in saline and extracted with CHCl ₃ . The extract was evaporated to dryness, and the residue was reprecipitated with Et ₂ O to obtain octo (thioglycoside) of calix [4] resorcarene (compound 1c in FIG. 1). Yield 88% (1
493 mg). 127 ° C.

The obtained octa (thioglycoside) compound (1148 mg, 0.203 mmol) was dissolved in MeOH / DM
Dissolved in SO to form a 50% solution to which Na
A solution of OMe in MeOH (28%, 6 mL) was added and reacted with stirring for 8 hours. After completion of the reaction, MeOH was removed, 0.05N NaOH (20 ml) was added to the residue, and the obtained mixture was ion-exchanged with a resin (Dowex).
50) to remove the base and freeze-dry. After the residue was purely dissolved, inorganic salts were removed using a dialysis membrane with a transport critical molecular weight of 1000 (Spectra / Por membrane MWCO 1000). After dialysis, the solvent was removed by freeze-drying to obtain the desired calix [4] resorcarene derivative (compound 1d in FIG. 1). Yield 95% (845 mg).

Identification data: mp 250 ° C. (dec); IR (KBr) 3425, 2923, 2852, 1651, 1
590, 1378, 1294, 1193,1058, 597 cm ^-1 ; ¹ H NMR (DMS
Od ⁶ ) δ0.83 (t, 12H), 1.20 (m, 72H), 1.63-1.69 (m, 1
6H), 1.88 (s, 24H), 2.60 (d, 8H), 3.16-5.17 (m, 148
H), 6.40 (br s, 4H), 6.77 (br s, 4H), 7.93 (m, 8H),
8.18 (m, 8H),; ¹³ C NMR (DMSO-d ⁶ ) δ 14.17, 22.36,
22.90, 29.03, 31.60, 52.29, 63.06, 66.58, 66.96, 6
8.64, 71.36, 76.02, 82.11, 93.88, 125.66, 154.50, 1
67.91, 171.40, 172.01; Elemental analysis, calculated values (C ₁₉₂
H ₃₀₄ N ₁₆ O ₈₀ S ₈ ): C (51.05), H
(7.14), N (4.96), measured values: C (50.88), H (7.0
7), N (4.78).

[0030]Example 2: Guest capture ability experiment  In this example, calix [4] synthesized in Example 1 was used.
The resorcalene derivative (compound 1d in FIG. 1) is used as a host.
To capture various guest molecules in aqueous solution
It is an example.

As an anionic guest, rose bengal (compound 3a in FIG. 1) which is a well-known singlet oxygen generating compound was used (at a concentration of 1.0 × 10 ⁻⁵ M), and the pH was 7.
8 (buffer: 25 mM Tris-HCl)
The complex forming ability was examined while changing the concentration of calix [4] resorcarene derivative 1d in the range of 0 to 5.0 × 10 ⁻⁴ M. FIG. 2 shows a chart of the electronic spectrum. Iso-absorption point of the spectrum and Job
From the plot analysis, a 1: 1 complex was formed between 3a and 1d, and the coupling constant was K = 2.1 × 10 ⁴ M
It was found to be ^-1 .

As examples of other guests, some halogen-substituted fluorescein compounds (compounds 3b to 3d in FIG. 1)
When a similar experiment was conducted with respect to
The affinity for d was determined for phloxin B (compound 3b, K =
1740M- ¹ )> erythrosin B (compounds 3c, 80
0)> eosin Y (compound 3d, <50).

Further, similar experiments were carried out for ethyl violet, methylene blue and rhodamine 6G as cationic guests.
It shows that the calix [4] resorcarene derivative of the present invention sufficiently captures cationic guests. In the case of these cationic guests, formation of a complex having a guest / host ratio of 2 or more was also observed.

[0034]Example 3: Lectin binding test  This example shows the calix of the present invention synthesized in Example 1.
[4] Resorcalene is fixed on the hydrophobic support below.
And the resorcinol cyclic tetramer above it
Sialic acid clusters introduced into the phenolic hydroxyl groups of the body
Indicates binding of lectin in aqueous solution.
You. In experiments, the binding (adsorption) phenomenon can be detected directly
SPR (surface plasmon resonance: surface
(Plasmon resonance) method. The equipment used was Pharmaci
a BIAcore X manufactured by Biotech.

The sensor chip of this SPR device (a glass substrate on which a gold thin film coated with a long-chain alkenylthiol is adhered) is calix [4] in a flow cell.
Aqueous solution of resorcalene derivative 1d (pH 7.0, 150
50 mM containing 50 mM CaCl ₂ and 50 mM CaCl ₂
By treating with 1 mM (Tris-HCl), 1d was immobilized on the sensor chip from the aqueous solution. Resonance signal RU (resonance unit) at the time of saturation
Was 1280. This value corresponds to a packing density of 0.18 molecules / nm ² , that is, an occupied area per molecule of the compound 1d of 5.7 nm ² . This occupied area is the sialic acid cluster site (diameter 0.2 to 0.2) in a state where calix [4] resorcarene 1d is folded.
0.25 nm). From these results, calix [4] resorcarene derivative 1d forms a densely packed monolayer, and its hydrophobic chain (C ₁₁ H)
₂₃ ) is adsorbed on the hydrophobic surface of the sensor chip via
On the other hand, it is considered that the sialic acid cluster site is exposed in the bulk aqueous solution.

In fact, the sensor chip on which calix [4] resorcarene is immobilized is attached to TTA (tachyple
When treated with an aqueous solution (0.1 mg / ml) of S. us tridentatas: a sialic acid-specific lectin from horseshoe crab, the increase in RU (resonance unit) at saturation was about 2%.
000 and Calix [4] resorcalene derivative 1
d was found to be strongly bound.

[0037]Example 4: Influenza virus red blood
Sphere aggregation inhibition experiment  Influenza virus has a sialic acid residue at the oligosaccharide end
Has the property of specifically recognizing groups, and sialic acid on the surface of erythrocytes.
It is known that it recognizes acid residues and causes red blood cell aggregation.
I have. In this example, the sialic acid class synthesized in Example 1 was used.
[4] resole allene attraction of the present invention
The conductor 1d interacts with the influenza virus and
Effect of inhibiting hemagglutination by influenza virus
Is played.

As an experimental method, a solution (1 mg / ml) of calix [4] resoleallene derivative 1d having a sialic acid cluster was prepared, and this was used as a mother liquor in a 96-well microtiter plate to prepare 0.01% gelatin. A solution was prepared by diluting with a phosphate buffer solution (PBS) twice. To this was added influenza A virus and 0.5% human erythrocytes fixed amount (2 ⁴ HA units) at 4 ° C., when it disappeared aggregation inhibition, i.e. detecting the thinnest concentration as inhibitory effect ability did.

A / PR / of influenza A virus
8/34 (H1N1) strain, A / Memphis / 1/71 (H
The values for the 3N2) strain and A / Aichi / 2/68 (H3N2) were 44 μg / ml (10 μM), 2
2 μg / ml (5 μM) and 44 μg / ml (10 μM)
M), and the calix [4] resorcarene derivative of the present invention has an ability to inhibit hemagglutination caused by influenza A virus of various strains.

[0040]Example 5: Neutralization of influenza virus
Titration experiment  Kidney cells are infected by influenza virus
And release lactate dehydrogenase. This
In Example 1, utilizing this phenomenon, prepared in Example 1.
The calix of the present invention having a sialic acid cluster [4]
Influenza virus infection with resorcalene derivative 1d
It shows that it has a neutralizing ability.

In the experiments, MDCK (Madi) was used as kidney cells.
ne-Darby canine kidney) cells were used. MDCK cells maintained in Eagle's minimal essential medium containing 5% fetal calf serum were treated with influenza virus (TCID _50).
100 μl) and calix [4] resorcarene derivative (0.01-2000 μg / ml).
Incubated at 4.5 ° C. for 20 hours. The released lactate dehydrogenase was quantified by a colorimetric method, and the concentration of 1d when the release of lactate dehydrogenase stopped was taken as the neutralization point.
₅₀ (concentration required for 50% neutralization) was determined, and A / PR / 8/34 (H1N
1) 200 μg / ml (46 μM) and 100 μg / m for the A, Memphis / 1/71 (H3N2) and A / Aichi / 2/68 (H3N2) strains, respectively.
1 (23 μM), and 200 μg / ml (46 μM)
Met.

[Brief description of the drawings]

FIG. 1 summarizes the structural formulas of the compounds used in the examples illustrating the features of the present invention.

FIG. 2 is an electronic spectrum diagram of a complex formed from calix [4] resorcarene derivative of the present invention and rose bengal.

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Claims (3)

[Claims] 1. A calix [4] resorcalene derivative represented by the following general formula (I). Embedded image [However, in the formula (I), R represents a hydrophobic group, and X represents CH ₂
CH ₂ NHCOCH ₂ S-Sugar, Sug
ar represents a sugar residue constituting a sugar chain. ] 2. The calix [4] resorcarene derivative according to claim 1, wherein the sugar is sialic acid. 3. The calix [4] resorcarene derivative according to claim 2, wherein X is represented by the following structural formula (II). Embedded image