JPS617288A - Alkylthioglucoside derivative - Google Patents

Abstract

NEW MATERIAL:The compound of formula I (R is 6-10C alkyl). EXAMPLE:n-Octylthioglucoside. USE:Solubilizing agent for biological substance. It can be produced at a low cost, and can be reconstituted to an artificial membrane. PREPARATION:For example, the acetyl-protected glucosethiourea compound of formula II is made to react with the compound of formula RX (X is halogen) (e.g. n-octyl bromide) e.g. in a 1:1 mixture of acetone and water, in the presence of potassium carbonate and sodium sulfite, etc. for 15hr under stirring, extracting the reaction product with methylene chloride, etc., and eliminating the protecting group by hydrolyzing with an alkali.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel alkylthioglucoside which has a surfactant action and is useful as a solubilizer for biological membranes and white matter. Biological membranes are composed of a double layer of polar lipids and various types of white matter that are responsible for the metabolism, transport, and information transmission of biological substances.These membrane proteins were extracted.

Attempts to understand life phenomena by examining their properties in detail have become widespread. In this case, alkyl glucoside derivatives represented by the general formula (R in the formula is an alkyl group having 6 to 10 carbon atoms), especially n-octyl glucoside, are known as membrane protein solubilizing agents, but in the synthesis process, large amounts of Since it uses a silver catalyst, it has the drawback of being expensive to produce and difficult to use as a general-purpose surfactant. Another disadvantage is that they are degraded by glucosidases that coexist when solubilizing biological components, resulting in loss of surface activity. Furthermore, in order to analyze the properties and functions of the solubilized and separated biomembrane, it is incorporated into an artificial membrane and the membrane is reconstituted, but the conventionally used octyl glucoside has problems with the reproducibility of diluted reconstitution. .

The present inventors have conducted extensive research to develop a powerful compound using this type of alkyl glucoside derivative, which has excellent ability to solubilize and reconstitute membranous white matter. It was discovered that derivatives were suitable for the purpose, and based on this knowledge, the present invention was made.In other words, this invention is based on the general formula (where R is a linear chain having 6 to 10 carbon atoms). The present invention provides an alkylthioglucoside derivative represented by (which is an alkyl group).

The alkyl group in the general formula (1) has 6 to 10 carbon atoms.
an alkyl group such as n-hexyl group, n-heptyl group, or n-octyl group.

Examples include n-nonyl group and n-decyl group. The compounds of this invention are all novel compounds that have not been described in any literature, and are represented by the formula (1), for example.

OO Acetyl-binding glucose thiophores, general formula % formula % () (in the formula, R has the same meaning as above, and X is a halogen atom) approximately equimolar of h-substituted alkyl halides. Let it react.

It can then be produced by performing deprotection by alkaline hydrolysis. Acetyl-protected glucose thio I (base structure) represented by general formula (1) is first brominated by adding 25-thihydrobromic acid/acetic acid to pentaacetyl glucose. It can be synthesized by reaction.

Specifically, the thiopretrogen (1) and the W-substituted alkyl halide (1) are dissolved in an acetone-water (1;1) mixed solvent,
Potassium carbonate-sulfite) was added, and the reaction was allowed to proceed with stirring for 15 hours. The product was extracted with methylene chloride, and after evaporation of the solvent, deacetylation was performed to obtain the final product, alkyl thioglucoside (+). obtain.

The compound of this invention has the ability to solubilize biological materials without altering their properties. It is suitable as a biological material solubilizer because it is difficult to be degraded even by degrading enzymes (glucosidase). Another advantage is that it can be efficiently reproduced when solubilized and separated biomembrane substances are artificially reconstituted.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 n-octyl thioglucoside is produced by bromination of pentaacetyl glucose, production of a thiominato compound, and octylation with n-octyl bromide.

Finally, the acetyl group can be deprotected for synthesis. That is, first, 133 g of pentaacetyl glucose
140 m of 25-thihydrobromic acid/acetic acid was added to bromate the mixture. One hour after pentaacetylglucose was completely dissolved, pour the reaction solution into water in Step 11.

Extract with 550 ml of chloroform. Separate the chloroform phase.

The solvent is distilled off under reduced pressure to obtain a prohuman body. Brome body 140
Dissolve 1-, thio f (element 30
Add FK and heat to reflux.

The precipitated white crystals of thio-L element (1) are collected as P. 10 g of the obtained thio vapor and 45 g of n-octyl bromide were added to 500 MI of acetone-water mixed solvent (191), and 3 g of potassium carbonate and 2 g of sodium sulfite were added as catalysts.
The reaction is allowed to proceed for 5 hours with stirring. 2 with 200d of methylene chloride
Extract times. After washing the methylene chloride phase with water, it is dried over dichloromethane, and the resulting octyl compound is purified by silica gel column treatment using benzene-acetone. Deacetylation can be performed using alkaline methanol.

Add 100ml of 5mM sodium hydroxide-methanol solution to 7g of octyl compound and react for 2 hours.

Neutralize with acetic acid 12. Evaporate to dryness. The obtained white waxy solid is dissolved in dichloromethane and purified by silica gel column chromatography to obtain the target pure n-octylthioglucoside 4.5B.

Calculated value (regret) 54.52 9.15 Actual value (
H) 54.41 898 The infrared absorption spectrum of this product is shown in Figure 1.

Example 2 In place of n-octyl bromide in Example 1, the chain length is changed by 1. As the substituted alkyl bromide, n-hexyl bromide, n-heptyl bromide, n-nonyl bromide, n-nonyl bromide By performing the same operation using n-decyl, corresponding alkylthioglucoside compounds were obtained. The following table shows the results of elemental analysis of the compound obtained.

Table 1. Elemental analysis results of n-alkylthioglucoside 1, B-hexylthioglucoside OizHtiO
g8 51.40 B, 63 50.98 8,42
2, n-heptylthioglucoside O+sHnOg
8 53.03 8.90 52.88 8,903,
n-nonylthioglucoside 0m5I (saoi
s 55.87 9.3B 55.66 9.33
4 n-decylthioglucoside 0m6H*tOs8
57.11 9.59 56.89 9.51 Example 3 The solubilizing ability of octyl thioglucoside and octyl glucoside, which is known as a superior membrane self-solubilizing agent, was compared. E. coli membrane vesicles were prepared by the French press method. In 50mM potassium phosphate buffer (pm 7.5)
Membrane vesicles (1,51F', il/ml), 1
M dithiothreitol was mixed with various concentrations of octylthioglucoside or octylglucoside in the presence of lipid (4"?/g#) and incubated at 6°C for 10 minutes.

Ultracentrifuge (170,000Xg, 1 hour) K,
The solubilized component (supernatant) and the insolubilized component (precipitate) were separated. Both proteins were subjected to constant protein infusion to examine their solubilizing ability. 70 at 35mM or more of octylthioglucoside
It can solubilize membrane proteins of chi. A similar solubilization IC50mM or higher of octyl glucoside is required. Figure 2 shows a soluble comparison. Denaturation of the solubilized protein was barely observed in either case.

Example 4 An artificial membrane experiment is conducted to analyze the properties and functions of biological membranes and to study the functions of biological membranes. In this case, a problem arises as to how to incorporate the neural tissue extracted from the biological membrane into the artificial membrane.

The easiest to use artificial membrane is Lipocy J.
The most relaxed, simple and rapid reconstitution method is the surfactant dilution method. Dilute reconstitution methods using octyl glucoside have already been reported (e.g. Tsuchiya et al. J, B.
jol, chem, 257.5125 (198
2) ) (However, this method has a narrow range of 1113 octylglucosides that can be used for reconstitution, and there are problems with reproducibility. Good results were obtained.Escherichia coli membrane proteins were solubilized according to the method described in Example 3.Solubilized white matter (150)Le) and lipids (50)Ll.

25w9) and further various concentrations (30-75mM
) of octylthioglucoside. 15 at 22°C
After incubating for a minute, the mixture is diluted with 30 times phosphate buffer. Put this in an ultracentrifuge (Zoo, OOOXg, 1 hour)
and collect the reconstituted proteoribosomes. The melibiose transport activity of this reconstituted proteolibosome was investigated (pressure reconstitution is possible when using 45rnM to 70mM octylthioglucoside;
It was found that the best value was around mM. The results are shown in Figure 3. The results obtained by the octyl thioglucoside dilution method (up to about 70 nanomoles, melibiose/machi protein) are lower than the results obtained by the octyl glucoside dilution method (up to about 40 nanomoles melibiose/■ long protein). It's expensive. Reproducibility is also higher when using octylthioglucoside.

Example 5 The sensitivity of surfactants to ρ-glucosidase was investigated.

In 50 mM acetate buffer, in the presence of 43 mM octylthioglucoside or octyl glucoside, p-glucosidase (Sigma, lj; mul
sin) at 37°C, and sampling was performed at each time interval to examine the decomposition of both glucosides. As a result, significant decomposition was observed in octyl glucoside;
No degradation was observed with octylthioglucoside. A comparison of decomposition rates is shown in Figure 4.

[Brief explanation of the drawing]

Figure 1 is an infrared absorption spectrum of off-thioglucoside, Figure 2 is a comparison of the solubilizing ability of surfactants, Figure 3 is the influence of octylthioglucoside concentration on the reconstitution of melibiose transport carrier into liposomes, and Figure 4 is The figure shows a comparison of stability against C-glucosidase action. -0-0- in the figure is octylthioglucoside. -41-t represents octyl glucoside. Above FJUA, (hr) flute now

Claims (1)

[Claims] 1. An alkylthioglucoside derivative represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼ (R in the formula is an alkyl group having 6 to 10 carbon atoms). 2. A method for solubilizing a substance using the alkylthioglucoside derivative according to claim 1; 3. A method for reconstituting a solubilized substance into an artificial membrane using the alkylthioglucoside derivative according to claim 1.