KR800001434B1 - Process for preparing amino-sugar derivatives - Google Patents

Abstract

Amino-sugar derivs. (I) inhibiting amylase and saccharase were prepd. by aerobic culture of actionoplanaceae SE 50 and SE 82 or their variants. The microorganisms were cultured in the media contg. starch or glucose as carbon source, and caseine, enzyme ext., peptone, fish pellet, cone flake soln. and meat ext. as nitrogen source in aerobic state with stirring at 24-32≰C and ph 6.0-7.0 for 2-6 days. I(n1+n2 = 3-8) is useful for the therapy of diabetes mellitus, obesity and adiposis.

Description

Method for preparing amino-sugar derivatives

Figure 1: NMR Spectrum (220MHz out of CD ₃ OD)

Figure 2: N.M.R. of decaacetyl derivatives. spectrum

Figure 3: NMR Spectrum (220 MHz in D ₂ O)

Figure 4: 13C-N.M.R Spectrum

The present invention relates to a method for preparing an amino-sugar derivative of the following structural formula (I) effective in treating diabetes, obesity and hyperlipidemia by having an action of inhibiting the hydrolytic action of amylase or saccharase enzyme will be.

In the above structural formula

n ₁ is an integer from ₁ to 8

n ₂ is an integer 0 or 1-8

n ₁ + n ₂ is 3-8 and the derivative is substantially free of structural isomers when n ₁ + n ₂ is 3 or 4 and homologues of compounds of formula (I) when n ₁ + n ₂ is 5 or more There is no.

Since the amino-sugar derivative according to the present invention regulates carbohydrate metabolism in humans and animals by inhibiting the hydrolase of glycosides, it is an active substance effective for treating diseases such as diabetes, obesity and hyperlipidemia.

Various microorganisms of Actinomycetales produce inhibitors that inhibit the hydrolytic enzymes of glycosides depending on the culture conditions, and these inhibitors are known to have mainly inhibitory effects on amylase or saccharase enzymes. have. [Reference: U.S. Patent Nos. 3,876,766, 3,855,066 and 3,879,546]

Our old German publication No. 2,347,782 describes a process for the preparation of amino-sugar derivatives having the structure (A).

R is an oligosaccharide containing 1-7 monosaccharide units.

The compound of formula (A) is prepared by culturing microorganisms belonging to Actinoplanaceae and Actinomycetales according to our previous specification on nutrient medium, and in preferred strains to actinoplanes. SE 50 (CBS961,70), SB 18 (CBS 957,70), SE 82 (CBS615,71), SE 50/13 (CBR641,71) or SE 50/110 (CBS 674,73).

Depending on the conditions for culturing the microorganism, the compound of formula (I) containing 1,2,3 or 4 monosaccharide units per molecule can be prepared without substantially producing a higher molecular weight homologue.

We now find that surprisingly the preparation of the compound of formula (A) containing at least three monosaccharide rings per molecule according to the recipe of our previous specification is accompanied by the production of isomers of formula (A). Furthermore, we can now separate the isomers of compounds containing 3 or 4 saccharide rings relative to each other per molecule and to prepare amino-sugar derivatives containing 3-8 monosaccharide rings without homologue compounds.

Depending on the sum of n ₁ + n ₂ , this amino-sugar derivative has the function of mainly inhibiting saccharase or mainly amylase enzymes. For example, amino-sugar derivatives with n ₁ + n ₂ of 4-8, preferably n ₁ + n ₂ of 4 or 5, are effective as inhibitors of subenzymes, whereas amino- with n ₁ + n ₂ of 3 Sugar derivatives are effective as saccharase enzyme inhibitors.

Amino-sugar derivatives with n ₁ + n ₂ 3 surprisingly have a strong inhibitory effect on starch in vivo. The preparation of the amino-sugar derivative according to the present invention is carried out according to a known method by culturing the strain belonging to the actinomycetales neck, preferably the strain belonging to the actinoplanacea family according to a known method and then separating The present invention relates to a method of isolating each compound.

An amino-sugar derivative having a total value of n ₁ + n ₂ of 1-4 can also be obtained by chemically or enzymatically hydrolyzing an amino-sugar derivative having a high molecular weight.

In order to prepare the compounds according to the invention actinoplanes SE 50 (CBS 961.70), SB 18 (CBS 957.70) belonging to the Actinoplanaceaaceae and Actinomycetales, preferably belonging to the Actinoplanes And SE 82 (CBS 615.71) or mutants or variants thereof are incubated according to known methods. The strains SE 50/13 (CBS 164.71) and SE 50/110 (CBS 674.73) are particularly suitable in relation to the total production. The properties of both strains correspond to those of the parent strain SE50 (CBS 971.60) obtained by natural selection without the use of mutagenic substances.

A solid or liquid, in particular a nutrient medium aqueous solution containing a starting carbon source and a nitrogen source, salts and antifoaming agents at a usual concentration which is liquid and known, is used for the cultivation of microorganisms. As the carbon source, carbohydrates, in particular starch, reverse sugar, glucose and complex mixtures such as commercial malt extract are used as starting materials.

As the nitrogen source, complex mixtures such as known hydrolyzed caseins, enzyme extracts, peptones, fish meal, corn slices, meat extracts and mixtures of these substances are used as starting materials, and individual amino acids and / or ammonium salts can also be used as nitrogen sources. Can be. The microorganisms are introduced into the air and incubated aerobically in a shaker incubator or in a conventional culture vessel.

As can be seen, the properties of the final product produced as the main product are influenced by the nature and concentration of the starting material used as the carbon source in relation to the specific strain used in the fermentation. Thus, for example, in a nutrient solution containing at least 2% (by weight) starch, a compound having, in particular, n ₁ + n ₂ of 4-8 is produced.

The SE 50/13 (CBS 614.71) strain is suitable for the preparation of amino-sugar derivatives with n ₁ + n ₂ of 4-8. However, if the nutrient solution contains a sufficient amount of glucose (about 3.5% by weight), starch of 0.1-3% (by weight) is sufficient to obtain a mixture of amino-sugar derivatives with n ₁ + n ₂ of 4-8. All amino-sugar derivatives thus obtained can be used as starting materials for preparing lower homologues by chemical or enzymatic hydrolysis.

On the other hand, starch-free nutrients, in particular SE 50 (CBS 961.70) strains, are used to produce a mixture of compounds with mainly n ₁ + n ₂ 3 and compounds with R having two monosaccharide units. (I.e., homologues with small molecular weight of the compound of formula (I) wherein n ₁ + n ₂ is 2 and n ₂ is 0)

Excess glucose is present and compounds with longer chains are also produced when the fermentation period is extended. Using a nutrient solution in which glucose is completely suspended and maltose as a starting material as a carbon source, the main product obtained is an amino-sugar with n ₁ + n ₂ = 2. Instead of pure maltose, cheaper products such as Maltzin, a natural malt extract that is commercially available, can be used, and higher molecular weight oligosaccharides are also produced depending on the nature of maltotriose. . Monarch SE 50/110 (CBS 674.73) has been found to be particularly suitable for preparing amino-sugar derivatives, in particular containing derivatives whose predominantly n ₁ + n ₂ is 1-3. This strain produces lower amino-sugar derivatives in twice the yield than none using SE 50/13 (CBS 614.71). In general, the microorganism is incubated at a culture temperature of 15-45 ℃, preferably 24-32 ℃. Thus, when using SE 50 strains (CBS 961.70) or SE 50/13 (CBS 614.71), amino-sugars with n ₁ + n ₂ 1-8 are produced at higher temperatures, such as 28 ° C, whereas 24 ° C At lower temperatures, such as the amino-sugar derivatives of n ₁ + n ₂ 1-3, strains SE 50 (CBS 961.70) and SE50 / 110 (CBS 674.73) are produced.

Incubation period is generally 1-8 days, preferably 2-6 days. Particularly when excess carbohydrates are added in the nutrient medium, long-term culturing is good for producing higher amino-sugar derivatives of n ₁ + n ₂ . In general, the pH of the medium is 5.0-8.5 and 6.0-7.0 is preferred. The final product of fermentation is generally detected by measuring the degree of inhibition by enzyme inhibition test as well as by measuring the composition of the fermentation vegetation by thin layer chromatography.

As mentioned above, when chemically or enzymatically hydrolyzing a relatively high molecular weight amino-sugar derivative to a monosaccharide to prepare a lower molecular weight derivative, it is generally obtained from a relatively high molecular weight amino-sugar derivative. Chemical hydrolysis is treated for 10-180 minutes with an aqueous 1-5N inorganic acid solution at a temperature of 50-100 ° C., especially at a temperature of 90-100 ° C., especially at 90-100 ° C. Also possible enzymatic hydrolysis is generally in combination with hydrolysis, in particular β-amylase or amylase or α-amylase enzymes obtained from B. Subtilis that cannot be inhibited by the compounds according to the invention or Incubate with amyloglucosides enzyme. Or 1-10% (by weight) relatively high molecular weight amino-sugar derivatives, preferably with Aspergillus niger (ATCC11,394) suitable for culturing in a nutrient medium containing amino-sugar as the only carbon source. Incubate the same strain.

Thus, the isolation and isolation of the amino-sugar derivatives according to the present invention can be carried out by enzymatic and / or microorganisms of broths or cultures of hydrolyzed products or microorganisms or amino-sugar derivatives having a relatively high molecular weight. Start with the incubated mixture digested by. High molecular weight substances containing 4-8 glucose units are first bleached, concentrated in solution and initially separated by direct precipitation. This material is also prepared according to the preparation described later.

Short chain compounds containing 3 or 4 glucose units are first separated by adsorption with activated charcoal at neutral pH, followed by separation with an aqueous alcohol solution or acetone, especially acetone of 50-80% strength. This separation can proceed completely in the acidic pH range of pH 1.5-4, preferably pH 2-3.

If the starting material solution is very dark in color, it is activated carbon or adsorptive resin, initially at acidic pH (pH 1-3), ie commercially available Lewapol CA 9221 (particle size: 0.35 mm, manufactured by Bayer). Is decolorized by treatment at pH 2-7, preferably at pH 2-3 before treatment with the above-mentioned activated carbon. Within the acid range, activated carbon only adsorbs the dye selectively, while amino-sugar derivatives do not adsorb within the neutral or acid range.

In order to isolate the pure compound according to the present invention, its weak basic properties can be used. Suitable conditions, i.e. pH 1-8, preferably pH 2-4, and an example of a compound from a small low ionic Doha Kang than 2mS and cm ^-1 to preferred, corresponding to a small conductivity than 10mS cm ^-1 and which contains hydrogenated It is bound to strong acidic cation ion exchangers such as the Dow X 50W (available from Dow Chemical). The compounds are particularly successfully bound to cation exchangers from acetone solutions (50% -80% acetone, pH 1-5, preferably 2-4) and under these conditions actually increase the adsorption rate for these compounds. can see.

If the solution contains more than 50% acetone, it is also possible for the compound to bind to a weakly acidic exchanger such as Amberlite IRC-50 (in the hydrogenated form). The separation of the amino-sugar derivatives according to the invention from this cationic resin is generally carried out using acidic or basic aqueous solutions, preferably ammonia or hydrochloric acid at a concentration of 0.01-1 equivalents / l as eluent. It is best to let them.

The separated product is neutralized with a weak acid or weak base exchanger, or the basic acid is concentrated by volatilization in vacuo and the product is freeze-dried and concentrated or precipitated with an organic solvent, preferably acetone.

In addition, a small molecular weight amino-sugar derivative can be separated by inert sugars by chromatography with an exchanger made of cerose, preferably phosphorus-cerose (manufactured by Sherpa Co., Heidelberg). As the eluent, a buffer solution having a low ionic strength, preferably 2-10 mM, particularly 5-10 mM and having a pH range of 2.5-8, preferably 5-6, preferably a phosphate buffer solution is used. What is needed in advance for effective fractionation is that the salt content in the formulations used under fractionation is as low as possible.

In order to prepare each compound according to the present invention in pure form, the pre-purified preparation is chromatographed with a suitable molecular sieve such as Bio-Gel P-2 (manufactured by Bio-Rad, Munich). This eluted fraction is subjected to caterpillar chromatography and subjected to pure fraction containing the compound according to the present invention, again chromatographed, concentrated to the end and freeze-dried or settling the organic solvent as described above. . Hydrolyzed to total acid, all amino-sugar derivatives consist of “Component I” (C ₁₃ H ₁₉ O ₇ N) and glucose.

The structural formula of “component I” is as follows:

ll 회사제품임)상에서는 Rf치가 0.46(엿당은 0.50이고 포도당은 0.95임)이며 F 254 실라카겔판(메르크회사 제품임, 다름슈타트)상에서 Rf치가 0.47(엿당은 0.54이고 포도당은 0.66임)이다, 질산은/수산화나트륨 분무용시약을 사용하면 실은 또는 약간 가온한 상태에서 흑갈색 점을 얻는다.The conformation of homologues with a low molecular weight of the compound according to the present invention is as follows (ie, a compound of formula (I) wherein n is 0 and n ₁ is 1 or 2) The compound having n ₁ + n ₁ is 1 is water, dimethylform It is a colorless amorphous material with good solubility in amide, dimethyl sulfoxide, methanol and hot ethanol. Thin layer chromatography using ethyl acetate / methanol / water (10: 6: 4) gave compound F 1500 silica gel membrane (Schleicher & Sch).

ll company product) has an Rf value of 0.46 (yield sugar is 0.50 and glucose is 0.95) and an Rf value of 0.47 (yield sugar is 0.54 and glucose is 0.66) on F 254 silica gel gel (manufactured by Merck, Darmstadt). The use of silver nitrate / sodium hydroxide spray reagents results in dark brown spots with either real or slightly warm conditions.

The compound is silylated together with a standard D-glucose or sucrose in a mixture of pyridine (1), tritylchlorosilane (0.5) and N-methyl-trimethyl-silyl-trifluoro acetamide (1), followed by chromosomal. Gas chromatography is performed on an 8 foot glass tube filled with 3% SE 30 silicone elastomer (available from Pakart) on Chromosorb WAW. Injection temperature and probe temperature are 300 ℃. The oven temperature is 220 ° C and the temperature is kept constant until the α- and β-D-glucose standards are eluted. The temperature is then raised to 300 ° C. at a rate of 15 ° C./min. The flame ionization gun is used to supply nitrogen to the carrier gas at a rate of 40 ml / min. The combustion gas supplies air at a rate of 80 ml / min and hydrogen at a rate of 20 ml / min. Compounds appear after 16-17 minutes (α-D-glucose after 3 minutes, β-D-glucose after 4 minutes and sucrose after 12-13 minutes)

는 +134.3°이다. 220MHz인 N.M.R로 CD₃OD중에서 스펙트럼을 찍어보면 제1도와 같은 결과를 보인다. (좌표축=δppm) N.M.R. 스펙트럼의 주 특징은 다음 표 I에서 보인 바와같다 :A sample of amorphous compound obtained by concentrating a methanolic solution is dissolved in water and the specific optical density is measured.

Is + 134.3 °. The spectrum of CD ₃ OD with NMR of 220MHz shows the same result as in FIG. (Coordinate axis = δppm) The main features of the NMR spectrum are shown in Table I below:

TABLE I

If the compound is reacted in acetic anhydride / phytinine (1: 1) mixture at room temperature, a large caracetyl derivative (molecular weight: 903) is obtained. The N.M.R spectrum of the dacaacetyl derivative is as shown in FIG.

The reaction can be added to the catalytic amount of sulfuric acid and advanced seeker decaacetyl derivatives in glacial acetic acid / acetic anhydride (1: 1) to determine the production of undeacetyl derivatives (molecular weight: 945) by mass spectrometry. The mass spectrometry of the decaacetyl derivative shows molecular perc (2.5% relative strength) at 903 and reference perc at 843. Peaks for the critical areas in the upper mass region are 844 (55% relative strength), 784 (36% relative strength), 784 (34% relative strength, 759 (34% relative strength), 556 (36% relative strength) 496 (37% relative strength) and 436 (29% relative strength).

Examination of the spectroscopic data and chemical properties revealed that the compound has the following structural formula (IIA).

In particular, the NMR spectrum is 0- {4,6-bis-deoxy-4- [1S- (1,4,6 / 5) -4,5 in which the compound of formula (IIB) has a steric structure as follows: , 6-trihydroxy-3-hydroxymethylcyclohex-2-en-1-yl-amino] -α-D-glucopyranosyl}-(1 → 4) -D-glucopyranose.

The next lower molecular weight homologue of the present invention is an amorphous solid product having a general composition of C ₂₅ H ₄₃ O ₁₈ N and easily soluble in water. Thin layer chromatography with the above developing agent gave an Rf value of 0.35 on an F1,500 silica gel film and an Rf value of 0.33 on an F254 silica gel plate.

The compounds have the IR spectrum did not less crucial has a main absorption band of OH and CO vibration (3700-3100cm ^-1 and 1180-950cm ^-1, respectively) did not have good resolution. The degree of beneficiation [α] _D in water is +167.0. In D ₂ O, the spectrum was taken by NMR at 220 MHz, and the result is shown in FIG. 3 and FIG. 4 of the 13C-NMR spectrum.

The methylation reactions described above give compounds with 13-methyl groups and also compounds with small amounts of 14-methyl groups. The mass spectrometry of the methylated product shows a molecular peak at 872 (1.5% relative strength) and the corresponding empirical formula is C ₃₈ H ₆ ⁶ NO ₁₈ . The second molecular peak with a relative strength of 0.1% is at 841. The most important partial peaks are: 739 (27% relative strength), 592 (3.7% relative strength), 535 (30% relative strength), 388 (9% relative strength), 386 (13% relative strength), 284 (13% Relative strength), 187 (12% relative strength), 171 (40% relative strength), 101 (34% relative strength), 88 (25% relative strength), and the reference peak is 75. This compound has the following structural formula (III) as determined by chemical properties and spectroscopic analysis.

This compound has 0- {4,6-bis-deoxy-4- [1S- (1,46 / 5) -4,5,6-trihydroxy-3 having a stereostructure as shown in Formula (IIIB) -Hydroxy methylcyclohex-2 -en-1-ylamino] -α-D-glycopyranosyl}-(1 → 4) -0-α-D-glucopyranosyl (1 → 4) -D- Glucopyranose.

Compounds in which n ₁ = + n ₂ is 3 are obtained in two isomeric forms and can be partially acid hydrolyzed to separate the two isomeric compounds with the compound of formula (IIA) and glucose in a 1: 2 molar ratio.

The product present in small amounts is 0- {4,6-bis-deoxy-4- [1S- (1,4,6 / 5) -4,5,6- having a conformation as in formula (IV) Trihydroxy-3-hydroxymethylcyclohex-3-en-1-ylamino] -α-D-glucopyranosyl- (1 → 4) -0-α-D-glucopyranosyl- (1 → 4 ) -0-α-D-glucopyranosyl- (1 → 4) -D-glucopyranose.

The large amount of isomer products present is 0- {4,6-bis-deoxy-4- [1S- (1,4,6 / 5) -4,5,6-trihydroxy- having the following conformation 3-hydroxymethyl-4-0-α-D-glucopyranosyl- (1 → 4) -cyclohex-2-en-1-ylamino] -α-D-glucopyranosyl]-(1 → 4 ) -0-α-D-glucopyranosyl- (1 → 4) -D-glucopyranose.

The R glucose value of this isomer on an F1500 plate chromatographed using n-butanol / ethanol / water (50:30:20) is 0.41-0.46. The presence of isomers having the structure of formula (V) can be determined by analyzing the product obtained by hydrocracking (palladium / activated carbon). This degradation product is 3-hydroxymethyl-4,5,6-trihydroxy-4-0-α-D-glucopyranosyl-1-hexane, 0- (4-amino-4,6-bis-dec Oxy-α-D-glucopyranosyl)-(1-4) -0-α-D-glucopyranosyl- (1 → 4) -D-glucopyranoose and glucose.

Since the double bond of the hydrogenated hexene group was reduced and the amino bond was decomposed, it is clear that the compound of formula (VI) and the isomer are structurally related to the compound of formula (IIIA) or (IIIB), but cyclohexene ring The presence of a glucopyranosyl group at position 4 of is also characteristic.

The presence of the isomer of Structural Formula (IV) and 0- (4-amino-4,6-bis-deoxy-α-D-glucopyranosyl)-(1 → 4) -O-α- Confirmed by producing D-glucopyranosyl (1 → 4) -0-D-glucopyranose. See S, Horii et. al Jouvnal of Antibiotics, xxlv, 59 (1971).

In the second test, isomers with three glucose units are methylated hydrolysis, reduced to sodium borohydride, acetylated and analyzed by gas chromatography according to known methods. If the compound of formula (IV) is only 1,4,5-tri-0-acetyl-2,3,6-tri-0-methyl-D-glucitol, then the compound of formula (V) is 1,4,5-tri -0-acetyl-2,3,6-tri-0-methyl-D-glucitol and 1,5-di-0-acetyl-2,3,4,6-tetra-0-methyl-D-glu Sytol is contained in a 2: 1 molar ratio.

Isomer compounds of the formulas (IV) and (V) are not degraded by -amylase.

The product with n ₁ -n ₂ of 4 is hydrogenated using palladium / charcoal as catalyst. Hydrocracked, degraded non-basic products are separated by acidic ion exchange and isolated by preparative thin layer chromatography. Acetylation confirms the next principal component by mass spectrometry.

(Decaacetyl derivatives have a molecular peak where m / e is 906)

(Nonaacetyl derivative has molecular peak where m / e is 848)

The main component of the substance of n ₁ + n ₂ is 4 having the following three-dimensional structure.

This result is confirmed by decomposing most components having n ₁ + n ₂ of 4 with the compound of formula (IIIB) by β-amylase.

Isomer compounds of structural formulas (IV) and (V) can be separated by chromatography with an acidic ion exchange resin using 0.025 hydrochloric acid as the eluent.

Digestion with [beta] -amylase yields a product with n ₁ -n ₂ of 5 corresponding to 90% of the compound of formula (V). The glycoside linkage with maltoztriose units leads to the product bound to the cyclitol residue being the main product. About 10% is not degraded by the β-amylase enzyme.

It constantly has the following three-dimensional structure:

Higher-value compounds of this family of glucose units with a molecular weight of 969-1,617, 4-8, have less inhibitory activity of the saccharase enzyme, although in vivo tests have greater inhibition of α-amylase. Acid hydrolysis of these higher levels of the compound allows the lower levels of the constituents to be detected as intermediates with glucose and maltose in each case. Chromatography on an F 1,500 silica gel plate with n-butanol / ethanol / water (50:30:20) showed an R glucose value of 0.30-0.34 (4 glucose units); 0.21-0.23 (5 glucose units); 0.14-0.16 (6 glucose units) and 0.09-0.11 (7 glucose units).

Total hydrolysis of the lower compounds in this series resulted in discrete molar ratios of “component I” and glucose, namely 1: 4, 1: 5, 1: 6, 1: 7 and 1: 8 (percentage of glucose 74.4%, 79.7%, 83.3%, 86.5%, and 89.1%, respectively.

Chromatography with F 1500 silica gel plate using n-butanol / ethanol / mole (45:35:20) as a solvent gave the following Rf:

Catalytic hydrogenation as described above also shows that some, but not all, of the high levels of this class of compounds bind to position 4 of the cyclohexene group.

Since this compound contains a linear oligoglucoside chain with 1 to 4 bonds, it can be used as a substrate for any enzyme that degrades carbohydrates. The scope of the enzyme is clearly limited to what is substantially not inhibited by the compound. The α-amylase of bacteria and fungi can be used for the degradation of all oligoglucosides containing three or more glucose units, which break down to produce compounds of lower molecular weight and inert sugars such as maltose and maltotriose. This decomposition process is also used to identify 1 → 4 bonds. Compounds according to the invention with glucose units of 4-8 can also be degraded by β-amylase. Since β-amylase breaks down maltose from non-reducing ends of glucose containing 1 → 4 bonds, carefully analyzing the degradation products obtained from β-amylase results in each oligo bound to position 4 of the cyclohexene ring. Valuable information about glucoside substituents, particularly the length of the chain and the number of glucose in the chain bound to bis-deoxyglucose, ie the reducing end of the compound. However, compounds with 4,5,6,7 or 8 glucose units are not completely degraded by β-amylase. The resistance of this part of the compound is apparently due to the inadequate structural conditions that β-amylase can attack.

The decomposition results by β-amylase are summarized as follows.

In some cases, small amounts of starting material, if possible, isomers are recovered. Regarding degradation by the β-amylase enzyme, it should be again emphasized that β-amylase can only remove units of maltose continuously and only remove them from non-reducing ends in oligosaccharides. As a result, although not related to all theories, the compounds with 4,5,6,7 and 8 glucose units found above have 2,3,4,5 and 6 glucose units of structural formula (IIIA) and It is known that α, 1 → 4 bonds are formed, and finally, a derivative is bound to the α-position at position 4 of the cyclohexene ring.

The compound is methylated in methyldimethylide / sodium hydride in dimethylsulfoxide, total hydrolysis, and the derivatives are produced and then analyzed by gas chromatography to obtain 2,3,6-trimethylglucose derivatives and thus the glucose unit is necessarily 1 to 4 on the linear structure.

Depending on the type of methylated compound, the second methylated product, which is present in various amounts or which is not present under any conditions, is a 2,3,4,6-tetramethyl derivative. The presence of this derivative and the relative molar ratio of 2 to the trimethyl derivative depend on the type of substituents attached to the cyclohexene ring.

The following isomeric compounds of the formula (I) with n ₁ + n ₂ of 6 and 7 are mainly related to actinoplanacea under the conditions described above to facilitate the production of compounds with larger molecular weights of the formula (I). It is believed to be produced by culturing microorganisms belonging to:

After ingesting foods and drinks containing large amounts of carbohydrates (e.g. grain starch, sensed starch, fruits, fruit juices, beer and chocolate), glucoside hydrolase (e.g. amylase and salivary glands and maltase and It is known that carbohydrates are rapidly decomposed by Saccharase, which leads to hyperglycemia in animals and humans.

This hyperglycemia is particularly pronounced and persistent in diabetics. In fatty patients, dietary hyperglycemia often affects insulin oversecretion, increasing the production of fatty substances and reducing fat metabolism. In people with healthy metabolism and fatty people, this form of hyperglycemia results in insulin secretion and often hypoglycemia. Both hypoglycemia and milk juice in the stomach promote secretion of gastric juice, which is known to cause or promote gastritis or gastric ulcer or duodenal ulcer.

It has now been found that glycoside hydrolase inhibitors obtained according to the present invention can prevent hyperglycemia, hyperinsulinemia, hyperglycemia due to dietary aspects. This can be seen by observing the sucrose and maltose in the stomach after feeding the rats and / or humans. This compound not only promotes the passage of carbohydrates in the stomach but also inhibits glucose absorption in the intestine. The conversion of carbohydrates into fat tissue and incorporation of dietary fat into fat storage tissue is thus reduced or delayed.

Or carbohydrates, especially sucrose, are broken down by microorganisms in the mouth, thereby promoting bone formation.

The amino-sugar derivatives according to the invention can be used for the prevention or inhibition of this type of osteodermogenesis.

The following describes the cross section of the inhibitory action of the compounds according to the invention.

Amylase Test (In Vitro Test)

Amylase Inhibitor Unit (1AIU) defines two amylase units by the amount of inhibitor up to 50%. The amylase unit (AU) is the amount of enzyme that degrades 1 μ equivalent of glycoside bonds in starch for 1 minute under the following test conditions. The μ equivalent of the cleaved bond is measured colorimetrically with dinitrosalicylic acid as the μ equivalent of the reducing sugar produced and expressed in μ equivalent of maltose with the aid of the standard sugar curve. To test, add 0.10 μg of inhibitor or 0-20 mL of test solution in 0.4 ml of 0.02 M sodium glycerophosphate buffer / 0.001 M CaCl _{2 at} pH 6.9 to 0.1 ml of amylase solution (20-22 AUml) The mixed solution is left for 10-20 minutes in a 35 ° C water bath. After 5 minutes of incubation at 35 ° C. with 0.5 ml of 1% starch solution (Merck's Soluble Starch, Darmstadt, No.1,252), which was previously heated to 35 ° C., 1 ml of dinitrosyl acid reagent [( (See P. Bernfeld in Colowick-KCplan, Metho Enzymo), vol. 1, p. 149). To express color, batch is heated in a water bath boiled for 5 minutes, cooled, and 10 ml of distilled water is added. Absorbance at 540 mm is measured against a correspondingly prepared amylase-free blan k value. For evaluation, the percentage of inhibition of amylase used is calculated from the value, intertwined from the previously prepared amylase quantitative curve, which is still effective after adding the inhibitor. The inhibitor percentage is plotted in numbers and the inhibitor is converted to AIU / mg units by intertwining the 50% inhibition point from the curve.

+: For dry matter.

++: AU in batches of the same series not inhibited

[Saka Raise examination (test of living)]

Saccharase Inhibitor Unit (SIU) is defined as the amount of inhibitor that inhibits two Saccharase units by 50%. Saccharase unit (SU) is the amount of enzyme that breaks down 1 μmol of sucrose into glucose and fructose in one minute under the following test conditions. Μmol of glucose produced is quantitatively determined with the help of glucose oxidase under which saccharose is no longer degraded.

m and A. Dahlguist, Fctachem. Scand 12, (1958), 1997페이지에 따라 돼지의 소장 점막으로부터 얻은 가용성인 사카레이즈임]을 pH 6.0인 0.1M 소듐 말레에이트로 희석하여 0.12SU로 한것을 0-20㎍의 저해제 또는 0-20㎕의 시험용액과 혼합하고 이를 pH 6인 0.1M 소듐 말레에이트 완충용액으로 0.1ml로 해준다. 혼액을 35℃에서 10분간 방치하고 미리 35℃로 가온시킨 pH 6.0인 0.1M의 소듐 말레에이트완충용액중의 0.1ml의 0.05M 서당용액을 그 후에 가해준다. 혼액을 20분간 35℃에서 배양해주고 사카레이즈 반응을 1ml의 포도당 산화효소 반응물질[참조 : 포도당산화효소 반응물질은 pH 7.0인 100ml의 0.565M 트리스-염산 완충용액에 2mg의 포도당 산화효소(베링거 존회사제품, No. 15,423)를 용해시키고 이어서 1ml의 세척액(2g의 트리론x100+8g의 95% 분석용 에탄올), 1ml의 디아니시린용액(20ml의 물중의 260mg의 0-디아니시딘 2HCl임)과 0.5ml의 0.1% 강도인 과산화효소(베링거존회사제품임 No.15,302) 수용액을 가해주어 제조한 것임]을 가하여 멈추게하고 혼액을 또 30분간 35℃에서 배양시킨다. 그 후에 1ml의 50% 황산을 가하고 혼액을 상응하는 비교치에 대해 545nm에서 측정해 본다. 평가키 위해서 사용된 사카레이즈의 저해 백분율을 계산해내어서 이를 포도당 표준곡선의 도움으로 50% 저해점으로부터 SIU/g 또는 SIU/ℓ로 전환시킨다.To perform this test, 0.05 ml of a soluble saccharose solution containing 0-20 µg of inhibitor or 0-20 µl of test solution in 0.12SU [B. Borgst

m and A. Dahlguist, Fctachem. According to Scand 12, (1958), p. 1997, the soluble saccharose obtained from the small intestine mucosa of pigs was diluted to 0.12 SU with 0.1 M sodium maleate at pH 6.0, 0-20 μg inhibitors or 0-20 Mix with μl of test solution and make 0.1ml with 0.1M sodium maleate buffer at pH 6. The mixture is left at 35 ° C. for 10 minutes and 0.1 ml of 0.05 M Sucrose solution in 0.1 M sodium maleate buffer solution, pH 6.0, previously warmed to 35 ° C. is then added. The mixture was incubated at 35 ° C. for 20 minutes and the saccharase reaction was carried out with 1 ml of glucose oxidase reactant. Company, No. 15,423), followed by 1 ml of wash solution (2 g of Triron x 100 + 8 g of 95% analytical ethanol), 1 ml of dianicillin solution (260 mg of 0-diisinidine 2HCl in 20 ml of water). ) And 0.5 ml of 0.1% strength peroxide enzyme (manufactured by adding an aqueous solution of Bering Co., Ltd. No. 15,302)] to stop the mixture, and the mixture is incubated at 35 ° C for 30 minutes. Then 1 ml of 50% sulfuric acid is added and the mixture is measured at 545 nm for the corresponding comparison. Calculate the percentage inhibition of saccharose used for evaluation and convert it from the 50% inhibition point to SIU / g or SIU / l with the help of the glucose standard curve.

[Malayse test (test of living)]

m과 A. Dahlguist, Acta Chem. Scand. 12,(1958), 1997페이지에 따른 돼지소장의 점막으로부터 얻는 가용성 말레이즈로 pH 6.0인 0.1M 소듐 말레에이트에 의해 적당한 MU 함량을 갖도록 희석시킨 것임]에 가해주고 혼액을 pH가 6.0인 0.1M 소듐 말레에이트를 가하여 0.1ml로 해준다. 혼액을 10분간 35℃에서 방치하고 미리 35℃로 가온된 pH 6.0인 0.1M의 소듐 말레에이트 완충액중의 0.1ml의 0.05M 엿당용액을 그 후에 가해준다. 혼액을 35℃에서 20분간 배양해주고 말레이즈반응을 1ml의 포도당 산화효소물질[참조 : 포도당 산화효소물질은 상기 참조의 사카레이즈 시험부문에서 기술한 바와 같은 것임]을 가해주어 중지시키고 혼액을 또 30분간 35℃에서 배양시킨다. 그 후에 1ml의 50% 황산을 가하고 혼액을 상응하는 비교치에 대해 545nm에서 측정해 본다.Malays inhibitor unit (MIU) is defined as the amount of inhibitor that inhibits two Malays units by 50%. Malays unit (MU) is the amount of enzyme capable of breaking down 1 μmol of maltose into 2 μmol of glucose in 1 minute under the following test conditions. The amount of μmol produced is quantitatively determined by glucose oxidase reaction under the condition that maltose no longer degrades by malease. To perform the test, 0.05 ml of Malays solution containing 0-20 μg of inhibitor or 0-20 μl of test solution containing 0.060-0.070 MU [see B. Borgstr

m and A. Dahlguist, Acta Chem. Scand. 12, (1958), soluble malees obtained from the mucosa of the swine small intestine according to page 1997, diluted to a suitable MU content by 0.1 M sodium maleate at pH 6.0] and the mixture was mixed with 0.1 M at pH 6.0. Sodium maleate is added to make 0.1 ml. The mixture is left at 35 ° C. for 10 minutes and 0.1 ml of 0.05 M maltose solution in 0.1 M sodium maleate buffer, pH 6.0, previously warmed to 35 ° C. is then added. Incubate the mixture for 20 minutes at 35 ° C, and stop the Malase reaction by adding 1 ml of glucose oxidase (see below). Incubate at 35 ° C for min. Then 1 ml of 50% sulfuric acid is added and the mixture is measured at 545 nm for the corresponding comparison.

To assess, calculate the percentage of inhibition used and convert from 50% inhibition to MIU / g or MIU / l with the aid of the glucose standard curve.

Table II summarizes the results of the enzymatic inhibition test of the living body for the specific compounds of the present invention and the discontinuous ones of the compounds of higher molecular weight according to the present invention.

As shown in the table above, the inhibitory effect of certain organisms on the α-amylase of the pancreas greatly increases as the molecular weight of the same series increases, such that a compound having 5-7 glucose units has 1 or 2 units. In vitro tests have more than 100 times stronger enzyme inhibition than compounds.

Saccharase inhibition is much stronger than derivatives with two units, and enzyme inhibition by derivatives with one glucose unit has only half the action, and higher levels of compounds have no specific action against Saccharase inhibition. Further decreases.

In vivo testing shows that the inhibition of saccharose is nearly parallel to the specific inhibitory activity found in biopsy. On the other hand, surprisingly, a 10-40-fold increase in the effect of in vivo starch loading compared to the amylase inhibition test in vivo was found in compounds with 1,2 or 3 glucose units.

To prepare dietary hyperglycemia and hyperinsulinemia, in each case six groups of hungry rats a) 2.5 g of sucrose, b) 2.5 g of maltose, or c) 1 g of boiled starch, may be an aqueous solution or suspension. To administer. Six other mice are given the same amount of carbohydrate as mentioned and in addition dosed glycoside hydros inhibitors.

In addition, up to six different groups of rats are given a positive salt solution.

으로 함] 또는 포도당 산화효소와 0-디아니시딘 하이드로클로라이드로 효소적으로 실시하고 혈중 인슐린의 측정은 참조의 방법에 따라 실시한다[참조 : Hales or Randle; Biochem. J. 88, 137(1963)].Blood glucose levels and blood insulin levels are measured at short intervals of fatigue in the posterior ocular vein. Measurement of blood glucose levels was performed with an automated analyzer [Hoffman, J. Biol. Chem. Technican according to 120 51 (1937)

Or enzymatically with glucose oxidase and 0- dianisidine hydrochloride, and the measurement of insulin in the blood is carried out according to the reference method [Hales or Randle; Biochem. J. 88, 137 (1963).

The results of the in vivo test (sustained dose) of saccharase in starved rats are shown in Table III.

TABLE III

As can be seen from the above data, the in vivo action on the inhibitory action of saccharase is parallel to the inhibition action found in the test of the living body. Thus the ED ₅₀ increases as the sugar decay units decrease. The results are summarized in Table IV.

TABLE IV

Surprisingly, the in vivo starch degradation by the compounds of formulas (IIB), (IIIB) and (V) is much better than expected based on the data found in the in vitro test for amylase inhibition and thus the expected sample. Does not correspond to

Table V below shows in vivo test data obtained after administration of starch to coarse mice as above:

TABLE V

From the above table, the degree of saccharase inhibition showed a characteristic function of molecular weight in both in vitro and in vivo tests, whereas in amylase inhibition, molecular weight had a direct function in the in vivo test, but starch in the in vivo test Digestion inhibition was observed to be surprisingly constant, not decreasing as the molecular weight decreased. Thus, even if in vitro testing the amylase dropping decreases with molecular weight, the ED ₅₀ for this compound is substantially the same as for higher molecular weight compounds. This is summarized in Table VI below.

Table VI

Thus, purely low molecular weight compounds can simultaneously inhibit both digestion of sucrose and starch, and at such precise and predictable characteristic dose levels.

The compound also has a useful effect on glucose uptake, as can be seen in Table VII, which summarizes the results of the formula (IIIB) compound (n = 2) for starved mice.

TABLE VII

Purification of mixtures of higher molecular weight compounds of this class results in an unexpected increase in activity and specificity. This can be seen by comparing the in vitro inhibitory activity of α-amylase and saccharase given in Table II and the data in Table VIII below for purified compounds.

TABLE VIII

정도의 작용을 가지는 반면 8개의 포도당 단위를 갖는 화합물의 저해 작용은 겨우 최소 작용을 갖을 뿐이다.The results show that compounds with four glucose units have high activity as α-amylase inhibitors. Compounds with 5 and 6 units also show a much higher specific inhibitor than all previous formulations. Of course, as the molecular weight increases, it is obvious that the inhibition activity is gradually inhibited, as shown in the compound having 7 and 8 glucose units, although still showing a relatively high inhibitory action. Saccharase inhibition was shown to be inversely proportional to molecular weight in vitro. Compounds with four glucose units can be used to determine the specificity of the compounds with two glucose units.

While having a degree of action, the inhibitory action of a compound with eight glucose units is only minimal.

The compounds can be administered in powder or gelatin capsules without dilution, and can also be administered as pharmaceutical compositions in admixture with a carrier.

Pharmaceutical compositions comprising the compound according to the invention as an active ingredient (ie, the compound of formula (I) is substantially free of its isomers) may be mixed with a solid or liquefied gaseous excipient or with a solvent having a molecular weight of 200 or less excluding surfactants. It is prepared by mixing with a non-liquid diluent.

Pharmaceutical compositions containing sterile or isotonic aqueous solutions containing the compounds according to the invention as active ingredients can also be prepared.

In the present invention, the compound of the present invention may be used alone or in combination with a diluent to form a unit liquid.

Pharmaceuticals containing the compounds according to the invention are prepared by tablets (which contain confectionary tablets and granules), dragees, capsules, pills, ampoules or suppositories, alone or in combination with diluents.

까지)을 담체와 혼합하여 함유한 물리적으로 3분되어 응집시킨 단위를 의미한다. 약제가 일일용량 또는 일일용량의 반,

또는

용량인가는 약물을 각각 하루에 한번, 두번, 세번 또는 네번 투여하느냐에 따라 정해진다.As used herein, “pharmaceutical” means a physically distinct aggregated segment suitable for medical administration. As used herein, the term "medical agent in unit dosage form" is the amount divided by the daily dose or the multiple dose (up to four times the dose) or the daily dose (

Refers to a unit that is physically 3 minutes agglomerated and mixed with a carrier. The medicament is a daily dose or half the daily dose,

or

Dosage depends on whether the drug is administered once, twice, three or four times a day, respectively.

Pharmaceutical compositions containing the compounds according to the invention as active ingredients include ointments, gels, pastes, creams, sprays (with aerosols), lotions, suspensions, water-soluble or water-insoluble diluents, emulsions, syrups , Granules or powders.

Diluents used in pharmaceutical compositions (eg granules) suitable for the manufacture of tablets, dragees, capsules and pills are:

a) fillers and weights: starch, sugar, mannitol, silicic acid;

b) binders: carboxymethyl, cerose and other cerose derivatives, alginates, gelatin, polyvinyl pyrrolidone;

c) humectant: glycerol;

d) disintegrants: Hanchin-agar, calcium carbonate, sodium bicarbonate;

e) dissolution: paraffin;

f) Promote absorption: Quaternary ammonium compound

g) surfactants: cetyl alcohol, glycerol monostearate;

h) adsorbents: kaolin, bentonite

i) Suspending agents: talc, calcium and magnesium stearate, solid polyethylene glycols

Tablets, dragees, capsules and pills prepared from the pharmaceutical compositions containing the compounds according to the invention may typically be coated, encapsulated or protected with a mold and may contain milky agents.

It may also be configured to release only the active ingredient as long as possible, preferably at a particular site of the intestine. Coatings, encapsulations or protective molds can be made, for example, of polymeric materials or waxes.

The active ingredient may also be microencapsulated with one or several diluents mentioned above.

Diluents used in pharmaceutical compositions suitable for suppositories include, for example, polyethylene glycol and fats (nose; cocoa oil and higher esters [e.g., esters of C ₁₄ -alcohols and C ₁₆ fatty acids]) or mixtures of these diluents. There is a diluent that is soluble or insoluble in water.

Pharmaceutical compositions such as ointments, pastes, creams and gels include animal and vegetable fats, waxes, paraffins, starches, tragacanth, cerose derivatives, polyethylene glycols, silicones, bentonite, salicylic acid, talc and zinc oxide And conventional diluents, such as mixtures of these substances.

Pharmaceutical compositions such as powders and sprays may contain conventional diluents such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder or mixtures of these materials. The aerosol may contain conventional propellants such as chlorofluorohydrocarbons.

Pharmaceutical compositions such as solutions and emulsions may contain conventional diluents such as solvents, solubilizers and emulsifiers (excluding solvents having a molecular weight of 200 or less, excluding surfactants, as mentioned above, of course). Examples include water, ethyl alcohol, isopropyl, alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (e.g., dropping oil) , Glycerol, tetrahydrofurfuryl alcohol, fatty acid esters of polyethylene glycol and sorbitol, or mixtures thereof. For parenteral administration, liquids and emulsions must be sterilized with appropriate blood and isotonic solutions.

Pharmaceutical compositions such as suspensions include water, ethyl alcohol, propylene glycol, surfactants (e.g., ethoxylated isostearyl alcohol, polyoxyethylene sorbite and sorbitan esters), microcrystalline cellulose, aluminum metahydroxy Side, bentonite, hanchin-agar and tragacanth or mixtures thereof.

All pharmaceutical compositions according to the invention may contain flavoring and flavoring agents (eg peppermint oil and camphor oil) and sweetening agents (eg saccharin) as well as colorants and preservatives.

The pharmaceutical composition according to the present invention preferably contains 0.1-99.5%, more preferably 0.5-95%, of the active ingredient relative to the total composition.

In addition to the compounds according to the invention, the compositions and medicaments containing them may also contain other pharmaceutically active compounds. It may also contain various kinds of compounds according to the invention.

In the drug containing the compound according to the invention all diluents can be all the diluents mentioned above. Such agents may contain a solvent having a molecular weight less than 200 as the sole diluent.

The distinct agglomerating moieties constituting the medicament containing the compound according to the invention can generally be suitably applied by shape or packaging for administration and include, for example, tablets (sweets and granules), pills, dragees, capsules, It can be the same as suppositories and ampoules. Some of these can be made to release the active ingredient slowly. Such as capsules may contain a protective seal that allows some of the medicament to be physically distinguished and aggregated.

The preparation of the above-mentioned pharmaceutical compositions and medicaments is by the method of mixing the active ingredient with a diluent according to known methods into a pharmaceutical composition (e.g. granules) and subsequently preparing the composition as a medicament (e.g. tablets). .

A medicament in which the compound according to the invention is used alone or in combination with a diluent can be administered to humans or animals for use in the prevention and treatment of the abovementioned diseases.

The compound may be administered orally, parenterally (eg, intramuscularly, intraperitoneally or intravenously), rectally or locally, preferably orally. Preferred pharmaceutical compositions and drugs are therefore suitable for oral administration.

Although the dose is carefully adjusted in each case according to the diagnosis, age, weight of the patient, and the nature and gravity of the patient's condition and clinical condition, the typical dose is 30-3x10 ⁵ AIU / kg and 1-1x10 ⁴ SIU per day. / kg. In some cases, sufficient therapeutic effects can be obtained at lower doses, and in some cases, higher doses may be required.

The toxicity of this compound is very low. The crude product of Example 8, with an activity of 26,000 SIU / g, even without final purification, can tolerate the administered subject without side effects. This is true because the compound according to the present invention is tolerated without side effects when administered to mice and rats in a case of 340,000 SIU / kg. Intravenously, mice withstand 10,000 SIU / kg without side effects.

In addition to the compositions and medicaments containing the compounds according to the invention, the compounds according to the invention may be included with nutrients in human food or animal feed. The diet includes sugars, potato bread products, fruit juices, beer, chocolate and other dairy products, which can be stored in the form of jams, to which at least one inhibitor of the therapeutic amount according to the invention is added. Nutrients suitable for incorporation into animal food include oil confectionery, cereals (eg barley), fishmeal, soybean meal, sugar beets, raw vegetables and hay flakes.

The following examples describe the present invention in detail. An example of a typical ion-exchange resin used ambeo light (AMBERITE) IRA 410 Cl ^- (anion exchanger); Amberlite IRC 120 (H ⁺ form) (strong acid ion exchanger); Ambeo light (HCO ₃ ^- form) (anion exchanger); Ambeo Light IRA 410 OH ^- (strongly basic ion exchanger); Amberlite IRC 50H ⁺ (weak acid cation exchanger) and Dow Xa 50 WX4 H ⁺ (strong acidic ion exchanger).

The microbial strains described and used herein are stored in the American Type Culture Collection as having the following serial numbers.

Example 1

A fermentor containing 8 liters of nutrient solution with 5.0% starch, 1.0% yeast extract and 0.2% K ₂ HPO ₄ was inoculated with a flask shaken with SE 50/13 (CBS 614.71) for 3 days. The mixture is incubated with intensive stirring for 3 days at 28 ° C. with air. This gives a broth for culture containing 105,000 AIU / ml.

After cooling to 20 ° C., 6 L of medium broth was adjusted to pH 2.5 by diluting the concentrated nitric acid in half, 30 g of Carboraffin activated carbon was added, and the mixture was stirred for 10 minutes. After centrifugation at 10,000 rpm for 15 minutes, the clear pale yellow supernatant was neutralized with ammonia and concentrated to 500 ml. 500 ml of the concentrate is stirred with 200 g of Amberlite IRA 410 Cl ⁻ for 45 min and the latter is filtered off.

4/5 of 400 ml of methanol is then added to the filtrate to precipitate the starch decomposition product of the polymer (along with the remainder of the activated carbon). The compound is centrifuged at 5000 rpm for 5 minutes. 850 ml of the supernatant is added dropwise to 4 L of anhydrous alcohol with intensive stirring. The white, soft precipitate was filtered, washed three times with anhydrous alcohol and two times with ether and dried at 50 ° C. in vacuo.

Yield: 36 g of white powder containing 10 × 10 6 AIU / g, this precipitate is used in several of the following examples.

[Inhibition of Enzymes in Bacterial Plates]

In order to evaluate the composition of the preparation by TLC and the final product of fermentation, 1 ml of fermentation broth or 1 mg of preparation was applied to a silica gel film prepared in advance (Messrs. Schleicher and Schiill, Dassel, Type F 1500). -Develop twice in the developing solution with butanol / ethanol / water = 50/3020.

For Saccharase Inhibition Staining, the developed, well-dried plate is sprayed with enzyme gel (20 ml / 20 × 20 cm plate) and the gel is hardened. Subsequently, the plate is preincubated for 5 minutes in a humid container at room temperature, followed by saturation by sufficiently spraying with a substrate gel.

After hardening the second layer of this gel, the plate is placed in a humid container and incubated at 40 ° C. Inhibition staining (faint, spot, reddish brown background) is developed for 60-90 minutes. The test is stopped at the best coloration time and the agar layer applied thereon is dried with warm air using a blower.

[Production of Gel]

Enzyme Gel: 1.5g of agarose (L'Industrie Bioloigue Francais) is suspended in 100ml of 0.2M sodium maleate buffer solution at pH 6.0 and heated to dissolve. The clear agarose solution is cooled to 50 ° C. and 250 ml of Triton X-100 solution (combined with Triton X-1002g and 8g analytical ethanol) is added with shaking. Immediately before using the gel, 1 ml of GOD / POD solution (12.5 mg of glucose oxidase for tablets 1.Messrs. Boehringer, Order, No. 15,423 and 2.5 mg of peroxide for tablets II, Messrs, Boehringer, Add No. 15,302 in 5 ml of maleate buffer) and 4-5 units of saccha raise from the small intestine of pigs. The gel must remain at 50 ° as it solidifies in the nozzle during spraying.

Substrate gel: 0.5 g of agarose is suspended in 100 ml of sodium maleate buffer, pH 6.0, heated to dissolve. The solution is then cooled to 50 ° C. and 100 ml of Triton (2 g of Triton X-100 combined with 8 g of analytical ethanol) are added.

After sacrose is dissolved, the gel is prepared. The dried and dried TLC plates are sprayed with amylase gel (20 ml / 20 × 20 cm plates) for dyeing to inhibit amylase and the gel is solidified. After pre-incubation for 5 minutes at room temperature, 1 g of starch dissolved in a gel-covered plate was heated in 200 ml of starch solution 0.2 M glycerophosphate buffer / pH6.9, 200 ml of 0.01 M calcium chloride, Merch No. 1,252) and allowed to stand at 40 ° C for 2 minutes with shaking. Then wash the plate well with distilled water. To stain undecomposed starch, it is dipped in lean iodine solution. (4 liters of 500 ml of water iodine stock solution; iodine stock solution; 2.2 g of iodine dissolved in 100 ml of water and 4.4 g of calory iodide). Dyeing is best after about 1 minute. The blue spot will fade out quickly, so take a picture of the plate immediately.

Preparation of Amylase Gel

1 g of agarose is dissolved in 100 ml of 0.01 M calcium chloride buffer containing 0.2 M sodium and glycerophosphate / pH 6.9 at 100 ° C., cooled to 50 ° C., and 2 ml of 100 ml of Triton X-100 and 8 g of ethanol for analysis are combined. ) 100 ml of amylase crystalline suspension is added immediately before spraying.

Example 2

Nutritional solution containing 4% starch, 2.4% glucose, 0.9% hydrolyzed casein and 0.9% yeast extract, adjusted to pH 7.6 with sodium hydroxide, 0.4% calcium carbonate, sterilized for 30 minutes at 121 ° A 1 L conical flask containing 120 ml contains 2% starch, 1% glucose, 0.5% hydrolyzed casein and 1% yeast extract, adjusted to pH 7.2 with sodium hydroxide and 0.4% carbonic acid Calcium was added to inoculate strain SE 82 (CBS615.71) previously incubated in nutrient solution sterilized at 121 ° C. for 30 minutes. The mixture is then incubated in a rotary shaker at 28 ° C. for 5 days to obtain a solution for the medium containing 122,000 AIU / ml. To develop the eggs little by little, the mycelia were centrifuged at 12,000 rpm, separated from the bound medium solution, and 300 ml of the medium filtrate was diluted to half with concentrated nitric acid to pH 2.5 and 2.5 g in the mixture. Put analytical activated carbon of and stir for 10 minutes. After separating activated carbon at 12,000 rpm, 300 ml of methanol was added to the solution, neutralized to pH 6 with 10NKOH, and the mixture was allowed to stand for a short time and removed from the precipitate at 12,000 rpm.

After the mixture was allowed to stand for a short time, the supernatant was added dropwise to 3 L of ethanol, and the precipitate was separated by centrifugation at 12,000 rpm, and the precipitate was washed twice with anhydrous ethanol, once with ether, and dried under vacuum. 2.23 g of product containing at least 95% of a compound having 7.445 × 10 ₆ AIU / g and n ₁ + n ₂ = 4 and more glucose units are obtained.

Example 3

Containing 3.5% grapevine, 2% starch, 0.5% hydrolyzed casein, 0.3% yeast extract, 0.3% calcium carbonate and 0.3% potassium monohydrogen phosphate, to pH 7.8 before sterilization, Strain SE 50/110 (CBS 674.73), incubated in a 1 l conical flask containing 120 ml of nutrient solution sterilized at 121 ° C. for 30 minutes in a nutrient solution containing 3% soy flour, 3% glycerol and 0.2% calcium carbonate. After 6 ml inoculation, the mixture is incubated in a rotary vibrating shaker at 24 ° C. for 3-4 days to obtain a solution for medium with 153,000 AIU / ml and 12,200 SIU / l.

1 L of the medium solution is adjusted to pH 2.5 with nitric acid, 5 g of activated carbon is added thereto, the mixture is stirred for 10 minutes, and the mixture is centrifuged at 5000 rpm for 30 minutes. The supernatant is then neutralized by adding 25 g of Amberlite IRA 410 (OH ⁻ type). The neutral supernatant is concentrated to 100 ml on a rotary evaporator, mixed with 100 ml of methanol and the mixture filtered. The filtrate is poured into 2 l of anhydrous alcohol. The separated precipitate is filtered, washed with acetone 3 and ether 3 and dried in vacuo.

Yield: 14 g of a white powder containing 5 × 10 ⁶ AIu / g and containing a compound of glucose unit with n ₁ + n ₂ = at least 4.

Example 4

In addition to the use of 0.5% starch, according to the method of Example 3, after fermentation for 4 days, a culture broth containing 40,000 AIU and 184 SIU / ml is obtained. Culture broth comprises a mixture of compounds according to the invention with at least 1 glucose unit.

Example 5

Contains 3% glucose, 0.6% hydrolyzed casein, 1.6% yeast extract, 0.3% calcium carbonate and 0.3% potassium monohydrogen phosphate, adjusted to pH7.8 with potassium hydroxide and then sterile nutrient solution 120ml Into a 1 l conical flask was inoculated with strain SE50 / 110 (CBS 674.73) previously cultured according to Example 3. After culturing the mixture at 24 ° C. in a rotary vibration shaker for 4 days, a broth for medium containing the compound according to the present invention having 10,800 SIU / L and 1 glucose unit is obtained.

Five liters of media filtrate separated from the mycelium at 13,000 rpm was diluted to half the concentration of nitric acid to pH2.5, and 55 g of activated carbon (“Merck”) and 220 g of clacel were added and stirred for 15 minutes. After filtration to remove the solids, the filtrate was neutralized to pH 7 with concentrated ammonia, the solution was concentrated to 1.5 L, and 5 times ethanol was added to form a precipitate. The soft precipitate is separated at 12,000 rpm by a continuous centrifuge, the light yellow supernatant is concentrated to 150 ml and centrifuged by low rotation to separate small amounts of insoluble material. 50 ml of this solution was injected into a column (30 × 300 mm; 30 ml of water per hour) filled with Amberlite IRA-120 (H ⁺ type), and 300 ml of eluate containing inert sugars and composition having a non-absorbing action was taken. The exchanger is then transferred to a glass baker with 400 ml of water, followed by addition of ammonia and stirring until the pH reaches 11.5. After at least 30 minutes of exchange, the exchanger is separated and the solution is concentrated to 1/20 of its capacity and then filtered through a column (20x150 mm) with Amberlite IRA-410 (HCO ₃ ⁻ type). About 500 ml of the eluate is collected at a flow rate of 30 ml / hour, the eluate is concentrated and lyophilized to obtain 1.3 g of crude product.

The crude formulation is fractionated in Bio-Gel P-2,100-200 mesh (M essrs. Bio-Rad, munich) for further purification. Using a column of 500 mm diameter and 450 mm length treated with water at a flow rate of 40 ml / hour, 10 ml of fractions are obtained. All fractions are used for carbohydrate tests by the Antron test and compositional tests with retardation action by the Saccharase Retardation Test. Fractions with saccharose inhibitors are further examined by TLC to determine the content of their individual composition according to Example 1. Fractions comprising a compound with one glucose unit are combined, concentrated and lyophilized. 35 mg of material with 0.3 × 10 ⁶ AIU / g and 30,000 SIU / g are obtained.

Example 6

1 ml of conical flasks each containing 120 ml of 5% starch, 1% yeast extract and 120% nutrient solution with 0.2% potassium monohydrogen phosphate were inoculated with 2 ml of the pre-cultured strain according to Example 3, respectively, and the following yields were obtained. The solution for the medium containing the compound having four or more glucose units in the mixture with the amylase blocking material having it is incubated at 28 ° C. for three days.

Example 7

In a 1 l conical flask each containing 120 ml of lactose with 1.3% lactose, 3.5% glucose, 0.5% hydrolyzed casein, 1.3% yeast extract, 0.3% calcium carbonate and 0.3% potassium monohydrogen phosphate 2 ml of the strains previously cultured according to Example 3 were each inoculated, and the other strains having the following yields and the compound having 4 or more glucose units were incubated for 4 days in a rotary vibration shaker at 24 ° C.

Example 8

A fermentor containing 100 liters of nutrient solution with 3.5% glucose, 2.5% maltzin, 0.5% casein hydrolysate 1.3% yeast extract, 0.3% calcium carbonate, 0.3% potassium monohydrogen phosphate and 0.1% antifoam 5 L of the culture medium previously inoculated according to Example 3 was inoculated, and the mixture was cultured by feeding air with stirring at 24 ° C. for 5 days. A solution for the medium of 73,000 SIU / l and a compound according to the invention n = 2 is obtained.

90 l of fermentation batch with mycelium is adjusted to pH 2.5 with concentrated nitric acid using a pH meter, and 900 g (= 1%) of activated carbon (Merck) is added while stirring to absorb the dyes formed. The mixture is stirred for 15 minutes, the mycelium and activated charcoal are separated at 3,000 rpm using a centrifuge, and the supernatant to which 3 kg of Klasel is added is filtered through a pressure filter. 6 L of a pale brown clear filtrate with a SIU content of 60,000 SIU / L is obtained.

The filtrate is adjusted to pH 7 with concentrated ammonia, and 1300 g (2%) of activated carbon (Merck) is added and stirred for 30 minutes to absorb the active compound. The mixture is filtered through a pressure filter and the activated carbon precipitate is washed three times with 10 liters of distilled water. Compress the activated carbon until it is dry and add 3x4 L of 50% acetone in each case to separate the active substance from the activated carbon and stir to pH 2.5. The activated carbon is separated by filtration, and the product separated from acetone is combined.

The separated and combined product is concentrated to 250 ml on a rotary evaporator, the same amount (250 ml) of methanol is added and the mixture is filtered through a slotted filter. The filtrate (480 ml) is added dropwise to 5 L of acetone under vigorous and stirring. The separated precipitate is filtered off, washed three times with acetone and ether, and dried in vacuo at 35 ° C. Yield: 230g of 8,500SIU / g

) 50Wx4H⁺(200-400메쉬)를 넣고 30분간 교반한다. 수지를 여과하고 0.001N 염산 2ℓ로 3번 세척한다. 그후 세척한 도웩스를 물 500ml에 현탁시키고 pH를 25% 암모니아를 넣어 pH 미터를 사용하여 9.0으로 맞춘다. 그 후 생성물에 각각 0.6% 암모니아 500ml를 넣고 2번이상 분리시키고, 떼어낸 생성물을 조합하여 회전증발기로 100ml까지 농축시킨다. 이 농축물을 탈색시키기위해 2g의 DEAE 셀루로오즈(Messrs,Schleicherand Sch

ll, No.02,035,0.6밀리당량/g)를 넣고 5분간 교반한 후 혼합물을 원심 분리한다. 동량(100ml)의 메탄올을 미황색의 상등액에 가하고 혼합물을 그 후 2ℓ의 아세톤에 적가시키며 격렬하게 교반한다. 침전물을 여과시키고, 아세톤, 에테르로 세척한후 35°에서 진공으로 건조한다.25 g of the crude product was dissolved in 1 L of water and 300 g of Doex was added to the solution.

) Add 50Wx4H ⁺ (200-400 mesh) and stir for 30 minutes. The resin is filtered and washed three times with 2 L of 0.001 N hydrochloric acid. The washed dopses are then suspended in 500 ml of water and the pH is adjusted to 9.0 using a pH meter with 25% ammonia. Thereafter, 500 ml of 0.6% ammonia was added to the product, and the mixture was separated two or more times, and the separated product was combined and concentrated to 100 ml by a rotary evaporator. 2 g of DEAE cellulose (Messrs, Schleicherand Sch) was used to discolor this concentrate.

ll, No.02,035,0.6 milliliter equivalents / g) and stirred for 5 minutes, followed by centrifugation of the mixture. Equivalent amount (100 ml) of methanol is added to the pale yellow supernatant and the mixture is then added dropwise to 2 L of acetone and stirred vigorously. The precipitate is filtered off, washed with acetone, ether and dried in vacuo at 35 °.

Yield: 4.2 g of product containing 26,000 SIU / g, 4.0 g of blocking material was gel filtered through Biogel P-2 in 0.5 g increments for better purification. For this purpose 0.5 g of each formulation is dissolved in 10 ml of water and injected into a Biogel P-2 column (200-400 mesh, Bio-Rad) 5 cm in diameter and 95 cm in length. The column is developed in water at a flow rate of 80 ml / hour. 12 ml of fractions are obtained. The total amount of carbohydrates (due to the absorption of E 620 from the Antron test), the amount of saccharose and amylase was determined.

In addition, fractions are tested by TLC. (Staining to Block Enzyme According to Example 1)

Fractions containing compound with 4-6 glucose units are collected, concentrated in vacuo to 10 ml, and precipitated by dropwise addition of the concentrate to 200 ml of dry alcohol. The precipitate is centrifuged, washed with acetone and ether and dried under vacuum; Yield from 4.0 g of crude material; Fractions containing 4 g of glucose units, 0.2 g of compounds containing 17.5 × 10 ⁶ AIU / g and 8,500 SIU / g, and compounds containing 3 units are developed in the same manner. Precipitation rate Precipitate with 200 ml acetone; Yield from 4.0 g of crude material; n ₁ + n _{2 = 3} and 1.4x10 ⁶ AIU / g and containing 21,000SIU / g compound 0.1g, and n ₁ + n _{2 = 2} units of 0.3x10 ⁶ AIU / g and 68.000SIU / g containing 0.9 g of compound is separated from a fraction containing a compound having n ₁ + n _{2 = 2} .

Example 9

In each of the three small fermentors containing 7.5 liters of maltzin, 0.3 percent hydrolyzed casein, 0.7 percent yeast extract, 8 liters of nutrient solution with 0.3 percent calcium carbonate and 0.3 percent potassium monohydrogen phosphate, Inoculate 5% of the precultured strain SE 50/110 (CBS 674.73) obtained according to 3 and incubate at 24 ° C. for 5 days to obtain a broth containing a compound with 73 SIU / ml and n ₁ + n ₂ = 2. Centrifugation (30 min, 3,000 rpm) to separate the mycelium yields 20.5 L of a deep brown medium solution containing 67,000 SIU / L.

The pH is adjusted to 3.7 with nitric acid and added to decolorize Lewapol (Lewapol, Ca 9221, 0.35 mm grain siye, Messrs.Bayer) / L 60 g = 1.23 kg. After stirring for 20 minutes, the mixture is filtered through a Sitz K3 filter. The decolorized solution for medium is neutralized with ammonia. (18.5 liters, 67,000 SIU / L). 20 g = 370 g of activated carbon per liter are added and adsorbed to adsorb the active material and the mixture is stirred for 30 minutes. Thereafter, the filtrate was filtered with a Sights K3 filter, which was covered with a layer of the subcell of the filter. Discard the filtrate (17.5 liters, 3,600 SU / L). The residue of activated charcoal is washed three times with 2 liters of distilled water. In order to remove the active substance from the activated carbon, the latter was added three times in succession with 1 L of acetone having 80% strength, respectively, and the pH was adjusted to 2.5 by adding concentrated hydrochloric acid. Collect the removed product. (2.5 liters, 371,000 SIU / L) The removed product is charged with dopants H ⁺ 20 g (Dopps 50Wx4, H ⁺ type, Messrs, Serva, Heidelbery) = 46 g dopants per liter and the mixture is stirred for 20 minutes. The resin is then filtered (Doxess fraction 1) and washed with some acetone at 75% strength. Add filtrate and washed liquor (3 liters = 215,000 SIU / l) to 60 g of Amberlite IRA 410 (OH ^- type) (Messrs. Serva, Heidelberg / l) and stir until pH 7. The mixture is then filtered and Add 72 grams of Dow's H ⁺ 72 g to the filtrate (2.8 liters, 219,000 SIU / l) Stir the mixture for 20 minutes, hanging the porous nylon bag filled with Amberlite IRA 410OH ^- to pH3.0 Dodds is then filtered (Dodds Fraction II) and the filtrate (2.6 L, 27,000 SIU / L) is discarded.

Dodds fractions I and II are each washed three times independently with 75% acetone to pH 3.5 and desorbed three times with 100 ml of 0.6% ammonia (Dawx fraction I) or 150 ml (Dawx fraction II), respectively. Ammonia is adjusted to pH 9 using ammonia during the first desorption that is not sufficient to neutralize the Dox resin. The products desorbed three times from Dow's Fraction I and Dow's Fraction III are then assembled and concentrated on a rotary evaporator until they are almost dry. Add the residue to 50 ml of water, adjust the pH to 3-4 with hydrochloric acid, and add 50 ml of methanol. The solution is added dropwise to 1.5 L of anhydrous acetone with stirring, and the collected precipitate is filtered off and washed three times with acetone and once with ether. The product is dried in vacuo.

Yield: 6.5 g of fraction I, 25,000 SIU / g

Fraction II 12.3 g, 36,000 SIU / g

As a composite material having a blocking action, the fractions I and II comprises n ₁ + n ₂ = 3 the small amount of the addition of compound n ₁ + n ₂ = ₂ the compounds according to the invention.

The following table shows the saccharase retardation action of the formulations continuously.

[Yield]

Example 10

200 g of the formulation of the type described in Example 1 are dissolved in 940 ml of distilled water and 60 ml of concentrated sulfuric acid, and the solution is heated with reflux for 4 hours. (Internal temperature; 98-100 ° C; Oil bath temperature; 140 ° C) 10 g of activated carbon (Merck, Type 2186) was added to the cooled dark brown solution and the mixture was stirred for 1 hour. The activated carbon is then filtered and washed with water, and the filtrate is adjusted to 7-8 by adding 250 ml of 10N KOH. Add 50 g of activated carbon to the solution and stir thoroughly for 1 hour. Activated charcoal is filtered, washed with 2 liters of water and the filtrate is removed. In order to desorption, add 30 liters of alcohol with 2 L of activated carbon and leave overnight. Finally activated charcoal is filtered and the alcoholic solution is concentrated on a rotary evaporator. Residue: 6.2 g This crude product (6.2 g) was dissolved in 500 ml of water, and 30 g of Amberlite IR 120 (H ⁺ type) was added to the solution and stirred with care for 1 hour. The exchanger is filtered and the filtrate is washed with distilled water until neutral and free of glucose. Thereafter, the exchanger was put in a solution containing 15 ml of 25% strength ammonia in 1,000 ml of water, stirred overnight and separated. The filtrate is concentrated in a rotary evaporator. Residue: 3.7g

Chromatography is used for further purification. 4.5 g of desorbed material from the exchanger are placed in a column of 1 L length and 2.5 cm width filled with cellulose. The developing agent used is initially used in an ethanol / water ratio of 5: 1, and 3: 1 ethanol / water is used for the first time to elute the compound according to the invention where n = 1. 14 ml fractions are obtained at 20 drops per minute. Individual fractions are examined by TLC. Fraction 47-85 is obtained after concentrating 1.6 g of the compound according to the invention with 1 glucose unit which inhibits the off-brown discoloration. Decolored impurities are not quantitatively important. Compounds having one glucose unit are obtained as colorless resins if the purification step of the strong acid ion exchange resin is carried out in a column rather than by a batch process.

Example 11

As described in Example 1, 200 g of the precipitate was dissolved in 940 ml of distilled water and 60 ml of concentrated sulfuric acid, and the solution was heated with reflux for 1/4 hour (internal temperature; 98-100 ° C., oil bath temperature; 140 ° C.) 10 g of activated carbon (Merck, Art. 2186) is added to the cooled dark brown solution and the mixture is stirred for 1 hour. After that, the activated carbon is filtered and washed with water, and the filtrate is adjusted to pH 7-8 by adding 250 ml of 10N KOH. 50 g of activated carbon was added to the solution and stirred for 1 hour. Activated charcoal is filtered off, washed with 2 liters of water and discarded the filtrate. To desorb, add 2 liters of 30% strength alcohol to activated carbon and stir overnight. Finally, the activated carbon is filtered off and the alcoholic solution is concentrated in a rotary evaporator. Residue; 8.0g

The residue is added to 15 ml of water and placed in a column (20 cm high, 2.4 cm in diameter) filled with 50 g of Amberlite IR 120 (H ⁺ type). The solution is absorbed at three drops per minute and the column is washed with water until all nonbasic composition is removed. (12 drops / min) The basic product is eluted from the column with ammonia at 0.5% strength. (12 drops / min) The aqueous solution is evaporated to dryness in a rotary evaporator. Residue: 4.1g

2 g of this residue are dissolved in a little water and placed in a column filled with Sephadex G-15 (height: 200 cm, diameter: 3.0 cm). The column is eluted with water. Each 2 ml fraction is collected at a flow rate of 8 ml / hour. Individual fractions are examined by TLC. In fractions 85-94 you get 280 mg of a compound with two glucose units and a special action of 50,000 SIU / g.

Example 12

60 g of 20 mM sodium glycerophosphate buffer, pH 6.9 with 1 mM calcium chloride, 2 g of the same formulation as described in Example 1, 1 g of alpha-amylase obtained from the genus Aspergillus (SERVA No. 12,418) was added for 120 hours. After incubation with constant stirring at 37 ℃ heated to 100 ℃ for 5 minutes. The undissolved material is centrifuged at 4,000 rpm and the solution is lyophilized to give 1.9 g of product with 3,500 SIU / g and 2 × 10 ⁶ AIU / g. When this product is tested by TLC and tested with saccharose deterrent bleaching as described in Example 1, it is confirmed that the compound with existing blocking action is essentially a compound according to the invention having 1,2,3 glucose units. Will know.

Example 13

As described in Example 1, 1.25 mg of beta: amylase (Boehringer 15,471) obtained from sweet potato was added to 2 g of the formulation contained in 30 ml of 20 mM acetic acid buffer solution having a pH of 4.8, and incubated with constant stirring at 37 ° C. for 120 hours. . And finally, heated to 100 ° C. for 5 minutes, centrifuged the insoluble material at 4,000 rpm, and freeze-dried the solution to obtain 1.5 g of product with 1,800 SIU / g and 3.8 × 10 ⁶ AIU / g. If this product is tested with TLC and a saccharose stopper bleach as described in Example 1, it will be appreciated that the compounds with existing stop action are essentially compounds according to the invention with 2.3 glucose units.

Example 14

200 ml triangle containing 0.1 ml potassium monohydrogen phosphate, 0.2% ammonium sulfate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.01% iron sulfate and 25 ml of nutrient solution with 2% formulation as described in Example 1 The flask is inoculated with spore suspension of strain Asper. Niger ATCC 11,394 and incubated at 28 ° C. on a rotary shaker. The AIU concentration drops from 210,000 AIU / ml to 53,000 AIU / ml after 6 days and to 21,300 SIU / ml after 10 days. At the same time the SIU / ml content rises from 7.0 to 72 SIU / ml.

20 ml of the solution incubated with suspended spores for 10 days is centrifuged to separate the mycelium at 3,000 rpm for 30 minutes. 2 g of Amberlite IRC 50H ⁺ and 1 g Amberlite IRA 410OH (conductivity of 2 mScm ^-1 or less) are added to 15 ml of supernatant (72,000 SIU / l) and desalted with stirring for 30 minutes.

The mixture is filtered and the filtrate is developed at a rate of 5 ml per hour through a column of Dow's H ⁺ (1 cm x 10 cm) equilibrated with 0.001 N hydrochloric acid. The column is then washed with 200 ml of 0.001 N hydrochloric acid. To desorb, 0.6% strength ammonia solution is injected into the column (10 ml / hour) and 5 ml fractions are collected.

Fractions with saccharose retardation are collected, concentrated to 2 ml on a rotary evaporator and mixed with 2 ml of methanol. The solution is adjusted to pH 3-4 and precipitated by dropwise addition of 100 ml of acetone. The precipitate is filtered off, washed with acetone and ether and dried in vacuo.

Yield; 26 mg containing a compound with 28,000 SIU / g and 2,3 glucose units

Separation of the pure compound with two glucose units from this product is accomplished by gel filtration through a column containing Biogel P-2 as described in Example 8.

7 mg of compound having 60,000 SIU / g of 2 glucose units are obtained.

Example 15

Centrifuge the mycelium at 13,0000 rpm and have an activity of 13,00SIU / g Amber for 1 hour to reduce salt content in 2 liters of filtrate for culture media obtained from fermentation batch as described in Example 5. Add 2.5 g of Lit IRC-50 (H ⁺ type) and 1 part of Amberlite IRA-410 (OH ^- type) and stir. (Conductivity of the filtrate for the medium; about 10 ms. Cm ^-1 ) The exchanger is separated and the solution is concentrated to slightly less than 100 ml and centrifuged to remove insoluble composition at 20,000 rpm for 15 minutes. Make the supernatant up to 100 ml; The conductivity is 3.5 mS.cm ^-1 and it is placed in a column (55x400 mm) of P-cellulose (balanced with 5 mM ammonium phosphate buffer to pH 5.5 pretreated according to the method of SERVA No. 45,130) and depurified. The above-mentioned phosphate buffer is used as a developer. The flow rate is 90 ml / hour and 18 ml of fraction are obtained.

The effluent was tested for carbohydrate content (by antimony test) and its saccharose jersey composition content (by saccharase jersey test) and then almost free of carbohydrates in the anthone test, and similarly in the saccaine jersey test. The active fractions are combined (fractions 60-170), concentrated to 150 ml and filtered through a column (50x300 mm) containing Amberlite IRA-410 (HCO- ³ form).

Eluates are collected in fractions to better control deionization. (10 ml per fraction in 20 minutes) and carbohydrates (by minus in each case by the anthone test) and phosphoric acid (by minus in each case by the iscorbic acid-molybdate reagent) test. The saccharase jersey test is performed. Fractions with inhibition (3-30) are pooled, concentrated, lyophilized, redissolved and lyophilized to yield 280 mg of crude material.

The crude material is fractionated with Biogel P-2 as described in Example 5 for further purification. From a fraction containing a compound with pure glucose units, 30 mg of a product of 0.3 × 10 ⁶ AIU / g and 35,000 SiH / g are obtained after lyophilization and separation.

Example 16

The starting material used to separate the compound having 5-7 glucose units may be, for example, the preparation as described in Example 1. For this purpose 30 g of the formulation according to example 1 are dissolved in 250 ml of water. The conductivity of the solution is then 10 mS.cm ^-1 and the pH is 5.5. To the solution is added 60 g of Amberlite IRC 50H ⁺ (a weak acid cation exchange resin that only binds an amino sugar derivative from an aqueous solution) and Amberlite IRA 410OH ^- 20 and desalted with stirring for 20 minutes. The filtrate (conductivity 0.5mScm ^-1 , pH 3.5) is adjusted to pH 3.0 with 1N hydrochloric acid. The solution is pumped at a rate of 42 ml / hour to a column filled with Dowex 50 W × 4,200-400 mesh (H ⁺ , 2.5 cm in diameter, 40 cm in height, equilibrated with 0.001 N hydrochloric acid). The dodec is then washed with 2 l of 0.001 N hydrochloric acid. After washing the column the elution is with 1.2% strength aqueous ammonia and 10 ml fractions are collected. Fractions with blocking action are collected, and ammonia is removed in vacuo.

The solution is then eluted to 30 ml in vacuo. The product is precipitated by dropwise addition of 600 ml of anhydrous alcohol, and the precipitate is filtered, washed with alcohol and ether, and dried under vacuum. The yield containing 26.5 × 10 ⁶ AIU / g is 4.4 g. A portion of 0.5 g is applied to the biogel P-2 column prepared as described in Example 5 and used for better tableting. Fractions containing 5-7 glucose units are collected according to TLC (amylase low color), concentrated in vacuo and precipitated with anhydrous alcohol as described above. Yield obtained from 0.5 g of crude product; 0.2 g amino sugar derivative of 5-7 glucose units containing 30 × 10 ⁶ AIU / g and 2,500 SIU / g

Example 17

This example illustrates how the compounds according to the invention can be eluted from cation exchange resins under acidic conditions.

A column 1.5 cm in diameter is filled with 30 g of Dow's 50w × 4 (H ⁺ ) 200-400 mesh. 500 ml of a mixed desorbent (400,000 AIU / l) at pH 2.5 obtained according to Example 9 (Table, run No. 7) were pumped into the column in about 1 hour and finally washed with 500 ml of 0.001 N hydrochloric acid. Under these conditions only a small amount of active substance is eluted. From there, 0.0125N hydrochloric acid is added to the column eluate, which is dependent on conductivity or refractive index, and finally desorption occurs.

Therefore the SIU content of the eluate is tested. The precipitate formed by the addition concentrated to 5ml and then neutralized and added dropwise to the reaction and then in 5ml methanol 200ml ^{acetone-combining} the active fractions 74-100, and cancer suberic discrete IRA 140OH. Washed with acetone and ether and dried in vacuo.

Yield; Compound with 1 Glucose Unit of 25,000 SIU / g A compound having 3-4 glucose units can be obtained from the first active fraction.

Therefore, this process of acid desorption is possible compared to alkaline desorption, fractionation, and fractionation of individual amino sugar derivatives. In this preparation, the dependent, individual higher fractions prepared as described above with 4-8 units, except that only the neutralized eluate is lyophilized, are obtained as follows.

4 glucose units = 67,000 AIU / mg

5 glucose units = 57,000 AIU / mg

6 glucose units = 42,000 AIU / mg

7 glucose units = 24,000 AIU / mg

8 glucose units = 5,000 AIU / mg

Example 18

The beta-amylase digestion process described above is carried out as follows.

Dissolve 100 mg of compound in 1.9 ml of 20 mM sodium acetate buffer (pH 4.75), add 0.1 ml of beta-amylase (Boehr.inger No. 15471; 5 mg / ml; 500 u / mg) from sweet potatoes and mix the mixture at 48 ° C. Incubate for hours, heat at 100 ° C. for 5 minutes, and centrifuge at 4500 rpm to remove precipitated protein and other impurities. The whole mixture is then poured into a column (diameter 22 mm; length 1000 mm, kept constant at 65 ° C.) and eluted with water at a rate of 25 ml per hour. The eluate is controlled by a conductivity meter and a highly sensitive refractometer arranged to pass through the cell. 2.5 ml of fractions are collected and these fractions can be used for carbohydrate content testing by amylase or saccharase retardation or anthrone reactions. Electrolytes and aerosols made from buffers are eluted with invalid substances, and the degradation products are eluted with decreasing molecular weight.

To completely separate a compound having a slight difference in molecular weight, it is accomplished by recycling the chromatography using the same conditions as described above. Fractions containing the isolated product are collected and lyophilized.

Example 19

In order to separate the isomers with glucose content, 10 g of a mixture of isomers dissolved in water is placed in a column filled with Dow's-50Wx4 (H ⁺ ). The column is first washed with water until the eluent is neutral and then eluted with 0.025 N hydrochloric acid.

Take 3 ml fractions of each and test by TLC. It is carried out on silanized silica gel plates (Merck, Germany) in a three-star developing solvent with ethyl acetate + methanol + water + 25% ammonia ratio of 100: 60: 40: 2. Compounds having the structure IV pass a wider distance from the original than compounds having the structure V.

Fractions 215-272, which contain 6 g of the isomer having Structural Formula V and fractions 215-288, which contain 600 mg of the isomer having Structural IV, are collected, neutralized with Amberlite IRA-140 (OH ⁻ ) and evaporated.

In vitro saccharase inhibition of isomers having Structural Formula IV isolated by this preparation method is 19,000 SIE / g.

Example 20

Purification of the compound of n ₁ + n ₂ = 4 is obtained by circulating gel permeation chromatography according to the following examples; A preparation of 6.0 g of a compound having n ₁ + n ₂ = 4 and 5 obtained as described in Example 8 or 17 was dissolved with 1 mM ammonium hydroxide and filled with Sephadex G-10 ^(R) . The solution is placed in a column (500 nm in diameter; 100 cm long) to separate it from the salt contaminants. The column is eluted with 1 mM ammonium hydroxide.

Thereafter, the eluate flows through the conductivity meter and the refractometer attached to the container. TLC (n-butanol; ethanol: water = 45: 35: 20) with a silica gel plate is therefore a suitable method for testing the eluate. The fractions containing the material to be purified are combined and lyophilized. Yield: 4.4 g: This material is further separated by adding 2.0 g of this material to a column filled with Biogel P-2 ^(R) , 200-400 mesh. The column is heated at 65 ° C. and water is used as the eluent at a rate of 80 ml per hour.

Fractions that occupy the invalid material are discarded together with fractions containing salts which may still be present after Sephadex chromatography. The columnar system, the motion pump, the refractometer and the conduction system are closed and the solution is pivoted through the gel. The separation process is checked by continuously adjusting the refractive index and the conductivity system. Sufficient separation occurs after five rotations. The column is then reconnected to a solvent reservoir and the column is eluted to yield 16 ml volumes of each.

Fractions are tested by TLC, as described above, and the fractions containing pure compounds as well as intermediate fractions are collected and lyophilized.

Yield; 600 mg of a compound having n ₁ + n ₂ = 4; 420 mg of a compound having n ₁ + n ₂ = 5; 100 mg of material collected from intermediate fractions

Claims (1)

A method for preparing an amino-sugar derivative of the following structural formula (I), which is characterized by culturing a strain belonging to Actinomycetales, isolating it, and isolating it with each compound.

Where n ₁ is an integer of 1-8, n ₂ is an integer of 0 or 1-8, in which case n ₁ + n ₂ is 3-8 and n ₁ + n ₂ is 3 or 4 And when n ₁ + n ₂ is 5 or more, there is virtually no homology with the compound of formula (I).

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