WO2000055349A1

WO2000055349A1 - 3,3'-diketotrehalose

Info

Publication number: WO2000055349A1
Application number: PCT/JP2000/001702
Authority: WO
Inventors: Koji Sode
Original assignee: Koji Sode
Priority date: 1999-03-18
Filing date: 2000-03-21
Publication date: 2000-09-21
Also published as: AU3196000A; JP4468592B2

Abstract

A novel trehalose derivative represented by formula (I). This compound is useful as a trehalose inhibitor and a stabilizer in freeze-drying proteins.

Description

Specification

3, 3'—Dikelet trehalose technical field

The present invention relates to a novel trehalose derivative, a method for producing the same, and a trehalase inhibitor and a lyophilized stabilizer containing the trehalose derivative of the present invention. Background art

Trehalose (Hi-D-darcoviranosyl Hi-D-darcopyranoside) is a non-reducing disaccharide in which two molecules of D-glucose are in a 1,1-linked form. Although there are three isomers, only the α-isomer exists in nature and is widely distributed in many organisms. In insects, it is present in hemolymph and not only exists as main blood sugar, but also acts as an antifreeze to maintain cold resistance.

Trehalase is an enzyme specific to trehalose, which hydrolyzes trehalose into two molecules of glucose. Trehalase is widely distributed in the animal and plant kingdoms, including bacteria, yeast, fungi, insects, and mammals.

Intestinal trehalase in mammals is localized in epithelial cells of the small intestine and has the function of hydrolyzing ingested trehalose. However, the physiological function of renal trehalase has not yet been elucidated due to the absence of trehalose in the blood. So far, trehalase derived from pig kidney has been reported to have affinity for various trehalose derivatives (Figures 1 and 2). a, Trehalose is a substrate for trehalase, but its isomer, 3,3 / trehalose, hi,) 3-trehalose has no affinity. Also, as in the case of D, D-mannopyranosyl, D-mannopyranoside and D, D-Darcopyranosyl a, D-mannopyranoside, the affinity changes greatly with only a slight change in the hydroxyl group of the substrate. The various properties of trehalase are described in Eu r. J. Biochem. 240, 692-69 8, 1996: Bioch imi. Biopros. Acta 1244, 291-294, 1995: Biooc. H imi. Biophys. A cta 791, 45-49, 1984. In many insects, trehalose is present in hemolymph and is the major blood sugar. It is also a major storage sugar in yeasts and fungi. Trehalase, which is present in insects, yeast, and fungi, is considered to be a regulatory enzyme that acts to supply glucose to each organ. Therefore, trehalase inhibitors are expected to be useful as fungicides or insecticides.

Examples of trehalase inhibitors that have been reported to date include, for example, S-GI (deoxyno jiri my cin)> trehalostatin (treha 1 ostatin), validamycin A (va 1 id amy cin A), noridoxy Lumin A (va 1 i doxy l ami ne A), MDL 25637, kasuno no spermine (cast ano s pe rmi ne), treno V-noline (trehazo 1 in), sudatrestin (suidatrestin) (Figure 3). Of these, burrs Doxil § Min A, Torehazorin, scan one Datoresu Chin, inhibition constant K i values, respectively ^{2. 4X 10- 9 M, 1. 9X} 10_ 8 M, be a 5. 0 X 10- ⁸ M It is reported to be a strong trehalase inhibitor. Trehazolin and sodatrestin are inhibitors specific to trehalase because they do not inhibit dalcosidases other than trehalase. In fact, rhodamycin A has already been used as a fungicide against the phytopathogenic fungus Rhizoct on iaso 1 ani to damage the rice sheath. Validoxylamine A is also thought to have an insecticidal effect on tobacco pests.

For various trehalase inhibitors, see Comp. Biochem. Phy siol. B120, 639-646, 1998: Arch. Biochem. Biophys., 316 (2), 821-826. , 1995: Agric. Bio 1. Chem., 55 (3), 895-897, 1991: J. Antibiotics 44 (10), 1165-1168, 1991: J. Antibiotics 40 (4), 563—565, 1987: Comp. Biochem. Phy siol. 61 B, 111—114, 1978.

Trehalose is also known to have the ability to protect proteins when they are lyophilized, and is used as a cryoprotectant for biological molecules such as enzymes, membranes, vaccines, and animal and plant cells and organs. (Biotec hno l. Annu. Re v., 2: 293-314, 196). For example, it has been found that when trehalose is used as a lyoprotectant, the stability of glucose dehydrogenase during lyophilization is significantly increased. However, trehalose is hydrolyzed by trehalase present in the blood to produce glucose, and is used as a substrate for G3DH. Therefore, glucose assay enzyme and 1,5-anhydro-D-dalsitol analysis It could not be used as a freeze-drying stabilizer for enzymes for use.

Therefore, an object of the present invention is to provide a novel trehalose derivative useful as a trehalase inhibitor and a protein freeze-drying stabilizer. Disclosure of the invention

The present invention provides the following formula:

A novel compound represented by the formula: 3,3'-Diketotrehalose. It has been found that the compounds of the present invention are not degraded by trehalase and have high trehalose inhibitory activity. Furthermore, the compounds of the present invention have been found to be useful as stabilizers for freeze-drying proteins. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows various trehalose derivatives.

FIG. 2 shows various trehalose derivatives. FIG. 3 shows various trehalase inhibitors.

Figure 4 shows the principle of the enzyme electrode method.

FIG. 5 shows the ¹³ C NMR structural analysis of the compound of the present invention.

FIG. 6 shows the ¹³ C NMR structural analysis of the compound of the present invention and a comparative compound. FIG. 7 shows the hydrolysis products of the compounds of the present invention.

FIG. 8 shows the change over time when the compound of the present invention is hydrolyzed.

FIG. 9 shows a Dickson plot for the trehalase inhibitory effect of the compound of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

The 3,3′-diketotrehalose of the present invention can be easily synthesized by oxidizing trehalose with glucose 3 _ dehydrogenase. Although it can be chemically synthesized, saccharides have a large number of hydroxyl groups, so that a multi-step reaction is required to modify a specific site.

Glucose 3-dehydrogenase (glucose 3-dehydrogenase; G3DH) is an enzyme that specifically dehydrogenates the C-13 hydroxyl group of saccharides to produce 3-ketosugar. G3DH has been so far 4 types (H1 omo nass p., Cytophag a ma ri no fl av a> Ag r ob acteri um t ume faciens> F l avoba cteri um sacchar oph i lm G3DH) Have been reported.

G3DH can be prepared, for example, using the method described in Enzyme Microchrome. Techno 1.22: 269-274, 1998. Briefly, te, Halomon assp. Hiichi 15 strain (De leyasp. A-15 strain) is cultured according to a conventional method, and the collected cells are disrupted to obtain a water-soluble fraction. This is purified by a method such as dialysis, gel filtration, or ion exchange chromatography. The 3,3′-diketotrehalose of the present invention can be produced by oxidizing trehalose using the G 3 DH enzyme electrode method. Figure 4 shows the principle of the enzyme electrode method. Shield the electrode cell from light and add trehalose, G3DH, and an electron mediator to lO OmM phosphate buffer (pH 7.5). Electronic mediator Examples thereof include potassium ferricyanide, phlegmene, osmium derivatives, phenazine methosulfate, and the like. Perform constant-potential electrolysis of 370 mVVsAg / AgC1 using platinum for the counter electrode, platinum mesh for the working electrode, and AgC1 for the reference electrode. Monitor the reaction by thin layer chromatography and observe the aging of the trehalose spot. After completion of the reaction, the reaction product can be purified using silica gel column chromatography, ion exchange chromatography, or the like. Alternatively, for example, by using potassium ferricyanide at a concentration of 10 OmM or more, the enzymatic reaction can be advanced without using an electrode.

The invention also features a trehalase inhibitor comprising a 3,3'-diketotrehalose of the invention. 3, 3 'of the present invention - diketo trehalose, that the inhibition constant ^{K i = 2. 42X 10_ 6 M} , to have a high Torehara Ichize inhibitory activity was s Mii. Therefore, the trehalase inhibitor of the present invention is useful as a research reagent for elucidating the effects of trehalose and trehalase, and as a fungicide or insecticide.

Further, the present invention is characterized by a protein freeze-drying stabilizer containing 3,3′-diketotrehalose. In particular, when the enzyme is lyophilized, a high residual enzyme activity can be obtained after thawing the enzyme by adding the treoctylose derivative of the present invention. Furthermore, since the trehalose derivative of the present invention is not decomposed by trehalase present in blood to produce glucose, it is very useful as a freeze-drying stabilizer for enzymes such as GOD and GDH for glucose assay. is there.

The entire contents of the specification of Japanese Patent Application No. 11-073568, which is the application on which the priority claim of the present application is based, are incorporated herein as a part of the present specification.

Hereinafter, the present invention will be described more specifically with reference to Examples, but these Examples do not limit the scope of the present invention. Example

Preparation of G3DH

Halomon ass p. Α- 15 strains, 10 g per liter Tons, lg yeast extract, 30 gNaC l, ₂ gK 2 HP_〇 _4, 10 ga- medium containing methyl -D- Darukoshido (pH7. 0) in, for 24 hours aerobically cultured at 30 ° C. The collected cells were suspended in a 2 OmM phosphate buffer (pH 6.0) and disrupted by a French press. The supernatant was separated by ultracentrifugation (40000 rpm, 1.5 h), and ammonium sulfate was added at a rate of 243 gZ1 under ice cooling to carry out salting out. The supernatant was separated by centrifugation (10000 g, 20 minutes), and dialyzed overnight in 20 mM phosphate buffer (pH 6.0) to obtain crude purified G3DH. The crude purified G3DH was subjected to hydrophobic chromatography by high performance liquid chromatography (8020 series: Tosoh Corporation) to purify G3DH. Enzyme activity was measured according to a conventional method, and the amount of enzyme that oxidizes 1πι1 of glucose per minute was defined as 1 unit (U). Example 2

Oxidation of Torayachi-Rose Using Enzyme Electrode Method

The cell for the electrode was shielded from light, and trehalose (Hayashibara Co., Ltd .; 0.13 g: final concentration of 2 OmM), G3DH (10 U) derived from human fifteen obtained in Example 1 and an electronic medium—potassium ferricyanide as an evening meal (Final concentration: 10 OmM), and the total volume was adjusted to 15 ml with 10 OmM phosphate buffer (PH7.5). The reaction temperature was maintained at 26 ° C. using a water bath, and constant potential electrolysis of 37 OmVvs AgZAgC 1 was performed using a platinum counter electrode, a working electrode platinum mesh, and a reference electrode Ag / Ag C 1. To observe changes over time, samples were sampled at 200/21 intervals every 2 hours, and each sample was dried, dissolved in methanol, and centrifuged (15000 rpm, 5 minutes) to remove insoluble substances. Similarly, another methanol extraction was performed to completely remove the residual protein. The obtained sample was dried to remove methanol, redissolved in distilled water, and the obtained sample was observed by thin-layer chromatography. As a developing solvent, a mixed solution of acetonitrile: water = 7: 3 was used.

As a result of thin layer chromatography, the spot of trehalose (R f = 0.45) faded with time, and a spot appeared at the position of R f = 0.57. The trehalose derivative was synthesized in about 12 hours by the electrode reaction. Example 3

Purification of reaction products

The reaction solution after the electrode reaction was placed in an eggplant-shaped flask, concentrated using a rotary evaporator, dried, and then dissolved in methanol. The insolubles were filtered by suction, the obtained filtrate was concentrated again, dried and dissolved in distilled water, and unreacted trehalose and the product were separated by an open column filled with silica gel. Acetonitrile: water = 7: 3 was used as the eluent. The solution separated in this way was concentrated, dried and dissolved in distilled water, and the cations present in the reaction solution were adsorbed and removed by an open column filled with Amberlite (cation exchange resin). Water was used as the eluent. The eluted aqueous solution was further concentrated, dried and dissolved in distilled water, and anions contained in the reaction solution were adsorbed and removed by a column filled with Amberlite (anion exchange resin). Water was used as the eluent. The eluted aqueous solution was concentrated and dried to collect a reaction product. Example 4

Analysis of the reaction product by ^{1 3} CNMR

The reaction product obtained in Example 3 was dissolved in heavy water (D ₂ 〇), and the structure was analyzed by ¹³ C NMR. Trehalose was similarly used for comparison.

The structure of the purified sample was analyzed by ¹³ CNMR, and the result shown in FIG. 5 was obtained. This was compared with Trehachi-Rose and 3-ketotrehalose (Figure 6). When the peak of the reaction product was compared with the position of the trehalose peak, the C-13 peak among the product peaks was shifted downfield. This shift is the same as C-3 of 3-keto trehalose. This indicated that the reaction product was 3,3'-diketotrehalose, which has keto groups at the 3-position on both glucose residues. Example 5

Analysis of reaction products by acid hydrolysis

The purified reaction product is dissolved in water 201, and 4N sulfuric acid 200 / il (final concentration 2 N) was added, and heat treatment was performed at 99 ° C for 5 hours to hydrolyze. While checking the pH of the obtained solution with pH test paper, 1 ON NaOH was added little by little until the solution became neutral. The sample thus obtained was confirmed by thin-layer chromatography. Acetonitrile: water = 6: 4 was used as a developing solvent. For comparison, trehalose was similarly hydrolyzed and confirmed.

FIG. 7 shows the results of thin-layer chromatography. The hydrolysis produces two molecules of glucose from trehalose. In contrast, no glucose was produced from the reaction product. If the reaction product is 3-ketotrehalose with a keto group at the C-3 position of one of the two glucose residues of trehalose, a spot should appear at the same position as glucose after hydrolysis. It is. Therefore, it was clarified that the reaction product was 3,3′-diketotrehalose, in which a keto group was inserted at the C-13 position of both glucose residues of two molecules of trehalose. Example 6

Effect of trehalase on 3, 3'_diketotrehalose

Trehalose, 3, 3'—Diketotrehalose, 17 mg each (final concentration 5 OmM), weigh, trehalase from pig kidney (SI GMA) 300 ^ 1 (0.08 U), 1 OmM phosphate buffer (pH 6.0) 700 1 was added to make the total volume 1 ml, and the reaction was started with stirring at room temperature. At 0, 5, 10, 23, and 33 hours from the start of the reaction, 200 1 samples were taken, and the change over time in hydrolysis was observed. Each of these samples was concentrated under vacuum, dissolved in methanol 2001, and extracted with methanol by precipitating the protein by centrifugation. The supernatant was concentrated, and the remaining protein was completely removed by performing another methanol extraction. The sample thus obtained was dried under vacuum overnight to completely remove the metal. The residue was dissolved in 200 × 1 of water, and observed by high performance liquid chromatography (CCPS, CO-8020, RI_8020, Chromatocorder 21: Tosoichi Co., Ltd.). The column was a sugar analysis column, the flow rate was 0.65 m 1 Zmin, and the eluent was water.

The results of HPLC are shown in the graph of FIG. According to the graph, It was observed that halos was converted to glucose. In contrast,

3'-diketotrehalose did not decrease under the same degradation conditions as trehalose, indicating that hydrolysis by trehalase did not occur. Therefore, it was shown that trehalase does not use 3,3'-diketotrehalose as a substrate. Example 7 '

Trehalase inhibitory activity

3, 3'-Diketotrehalose solution 100 1 (The final concentration in the total amount of trehalase reaction system 200 / X 1 is 0, 0.1, 0.5, 1.0, 5.0, 10.0 M And trehalase 26 1 (0.007U) derived from pig kidney were stirred at 37 ° C for 5 minutes. Next, trehalose (final concentration: 0, 5, 10, 20 mM) was added to bring the total amount to 2001. The buffer used was a 50 mM sodium phosphate buffer (PH 6.5). The trehalase reaction was allowed to proceed by stirring this solution at 37 ° C for 30 minutes, and then inactivated by heat treatment at 95 ° C for 5 minutes to stop the reaction. The glucose concentration in the reaction solution was measured, and the trehalase activity was calculated from the amount of glucose produced. 1 U of trehalase activity was defined as an amount that produced 2 mol of glucose from 1 mol of trehalose per minute.

From the relationship between trehalose concentration and trehalase activity, the Michaelis constant Km of trehalase for trehalose was determined to be 6.6 mM. In addition, the reaction rate decreases as the 3,3'-diketotrehalose concentration increases, indicating that trehalase is inhibited by 3,3'-diketotrehalose. Figure 9 shows a Dickson plot. In the Dickson plot, the inhibitor constant is determined from the inhibitor concentration at the crossing point when two different substrate concentrations are plotted on the same graph on the graph of the inhibitor concentration Vs1 / reaction rate. As a result, the inhibition constant K i of 3,3′-diketotrehalose for trehalase is 2.

It was determined to 42X 10 one ⁶ M.

The above results indicate that 3,3′-diketotrehalose has an inhibitory effect on trehalase. Example 8

Protein freeze-drying stabilization

The stabilizing effect of the compound of the present invention on freeze-drying of protein was examined using glucose dehydrogenase (GDH) having pyromouth quinoline quinone (PQQ) as a coenzyme. PQQGDH derived from Ac inetoba ctercalco aceticus was produced in recombinant E. coli based on the gene sequence described in Mo 1. Gen. Genet. (1989), 217: 430-436. The water-soluble fraction obtained by disrupting the cells was purified by cation chromatography. This enzyme, final concentration 5 M PQQ, was holoenzyme by one base 1 hour incubator Bok at room temperature in the presence of IMMC aC 1 _2. Next, the compound of the present invention was added to a final concentration of 5 OmM, and the sample was dispensed at 60 / xL and frozen at -80 ° C. As a control, the enzyme was frozen without adding the compound of the present invention. Next, the frozen enzyme was dried in a desiccator at 10 Pa and 0 ° C. for 12 hours. After the lyophilized enzyme was heat-treated at 4, 28, 45 or 70 ° C for 1 hour, the residual activity of the enzyme was measured. The enzymatic activity was measured using PMS (phenazine methosulfate) -DCIP (2,6-dichlorophenolindophenol) in 10 mM phosphate buffer pH 7.0. The change in absorbance at 600 nm was tracked using a spectrophotometer, and the rate of decrease in the absorbance was defined as the reaction rate of the enzyme. At this time, one unit was defined as the enzyme activity for reducing DC IP of 1; mo 1 per minute. The molar extinction coefficient of DC IP at pH 7.0 was 16.3 mM- ¹ .

At any of the measured temperatures, the sample to which the compound of the present invention was added had higher residual activity of the enzyme than the control. That is, it was found that the compound of the present invention has a freeze-drying stabilizing effect on the enzyme. Industrial applicability

The trehalose derivative of the present invention is useful as a trehalase inhibitor and a protein freeze-drying stabilizer.

Claims

The scope of the claims

1. The following formula:

3, 3 'One diketo trehalose represented by

2. The method for producing 3,3′-diketotrehalose according to claim 1, wherein trehalose is oxidized by glucose 3 dehydrogenase.

3. Trehalase inhibitor containing 3,3'-diketotrehalose according to claim 1 c

4. A freeze-dried protein stabilizer containing 3,3′-diketotrehalose according to claim 1.