FI56698C - PROCEDURE FOR INSULATION AND MICROBIOLOGICAL PROCESSING OF MICROBIOLOGICAL AMINOSCOCKETS - Google Patents

Description

VJ * r »l rq-i ANNOUNCEMENT γγλΛλ

JeST} (11) UTLÄGGNINGSSKRIFT 5 86 98 C (45) Patent granted on 10 03 1930

Patent neddelnt (5l) Ky.lk. * / Lnt.CI. · Q; «| 2 D 13/04-C 07 H 15/20 FINLAND — FINLAND (2.1) Ptunttlhikamus — Paunttntdkning 27 ^ 0/7 ^ + (22) Haktmlspllvi — Ansökningadag 19. 09 · 7 4

(23) The beginning of the cloud — Glltighettdig 19.09 · 7 U

(41) Became Public — Blivit offantllg oo no 7t

National Board of Patents and Registration 3 · ί; «« K .. «ί.4..Μ« · νΜΙ. * Η (44) Date of issue of Nlhtivlkiipanon |

Patent * och registerstyrelsen Antökan utlagd och utl.ikrlften publicarad 30.11.79 (32) (33) (31) Privilege requested — Begird prlorltet 22.09.73 26.10.73, 2i.ll.73 Federal Republic of Germany-Förbundsrepubliken Tyskland (DE) P 23 ^ + 7782.9, p 231 + 7782.9, p 23 ^ 7782.9 (Tl) Bayer Aktiengesellschaft, Leverkusen, Federal Republic of Germany Förbunds-republiken Tyskland (DE) (72) Werner Frommer, Wuppertal-Elberfeld, Bodo Junge, Wuppertal-Barmen,

Uwe Keup, Wuppertal-Elberfeld, Lutz Miiller, Wuppertal-Elberf eld,

Walter Puls, Wuppertal-Elberfeld, Delf Schmidt, Wuppertal-Vohvinkel,

Federal Republic of Germany-Förbundsrepubliken Tyskland (DE) (7 * +) Oy Kolster Ab (5I +) Method for isolation and purification of microbiologically prepared amino sugar derivatives - Förfarande för isolering och rening av mikrobiologiskt framställda aminosockerderivat

It is already known that some actinomycetes, in particular microorganisms of the family Actinoplanaceae, develop inhibitors of glycoside hydrolases, mainly enzymes that break down carbohydrates in the gastrointestinal tract. One such group of inhibitors is relatively heat resistant and resistant to acids and alkalis at room temperature. These inhibitors are amino sugars in chemical structure.

Most of the inhibitors in this group are very potent amylase inhibitors, but as sucrase inhibitors only moderate or weak (see Finnish Patent Publication No. 1 + 9 77 * +) ·

The invention relates to a process for the isolation and purification of microbiologically prepared amino sugar derivatives of the general formula 2 06698 OH_ .HH CH3

HO - / \ - N- / R

ch2oh h oh oh where R is a saccharide chain with 1-7 hexose units.

The process according to the invention is characterized in that microorganisms belonging to the genus Actinoplanes under aerobic conditions and in an aqueous nutrient medium after separation of the mycelium obtained by culturing and optionally culturing the obtained filtrate, e.g. the bound substances are selectively desorbed with a mixture of water and alcohol or water and acetone adjusted to pH 1.5, preferably 50-80% acetone, c) the desorbate obtained is applied to a cation exchanger, which is preferably strongly acidic, and d) to a cation exchanger the bound amino sugars are either completely desorbed with ammonia and then fractionated in a manner known per se using gel filtration or a cellulose column as individual components, or the amino sugars are selectively eluted with dilute acid and the eluates thus obtained are isolated individual amino sugars.

As mentioned above, these new amino sugar derivatives are potent inhibitors of gastrointestinal glycoside hydrolases. In this case, the lower members, where R corresponds to an oligosaccharide chain having 1 or 2 hexose units, have a predominantly strong sucrase inhibitory effect, while the higher members strongly inhibit predominantly amylase.

The amino sugar derivatives prepared by the process of the invention have a strong specific sucrase and / or amylase inhibitory effect. Some new amino sugar derivatives have a much greater in vivo inhibitory effect than their in vitro inhibitory effect.

It is also particularly surprising that the lower members of the homologous series according to the invention inhibit glucose resorption in vivo.

Based on these surprising properties, the preparation of new amino sugar derivatives is important pharmacologically.

3,06698

Strains of the genus Actinoplanaceae belonging to the family Actinoplanaceae belonging to the actinomycetes are used to carry out the method according to the invention. Examples are the strains Actinoplanes spec. SE 50 (CBS 961 70), SB 18 (CBS 957 70) and SE 82 (CBS 615 70). These microorganisms have already been described in German Application Publication No. 2 06U 092 and have been deposited with numbers in parentheses at Centralbureau voor Schimmelcultures (Baara / The Netherlands).

Strains SE 50/13 (CBS 611 * 71) and SE 50/110 (CBS 67-73) proved to be particularly suitable for the overall yield of the amino-sugar derivatives prepared according to the invention and / or for the size of the residue R of the amino-sugar derivatives formed as a mixture during fermentation. The descriptions of both strains are quite similar to the parent strain SE 50. These strains were obtained without the use of mutagens by natural selection from strain SE 50.

The culture to be treated in the process according to the invention has been obtained by culturing microorganisms belonging to the order Actinonycetales in an aqueous nutrient medium which, in addition to the carbon source, contains a nitrogen source, salts and antifoams in conventional concentrations which can vary within wide limits.

Carbon sources are mainly carbohydrates, in particular starch, maltose, glucose and mixtures of two or three such substances, and even more complex mixtures, such as malt extract.

As the nitrogen sources, the usual complex mixtures can be used in microbiology, such as, for example, casein hydrolyzate, yeast extract, peptone, fish powder, fish extracts, corn steep liquor extract and also mixtures thereof as well as amino acids and / or ammonium salts.

The cultivation is performed with aerobically conditioned vibration cultures or tank cultures. The quality and concentration of the carbon source and the strain used for fermentation affect the size of the residue R in the final product to such an extent that individual members of the homologous series or at least members of the very narrow region of the homology series can be almost selectively reduced. In this case, it turns out that. in nutrient solutions containing more than 2% of starch, amino sugar derivatives with U-7 hexose units are primarily formed, and that. strain SE 50/13 (CBS 61U 71) is particularly suitable for this production. In some cases, however, it is sufficient to add 0.1-3%, mainly 0.5-2% starch, to nutrient solutions containing e.g. 3-5% glucose as the basic carbon source, in order to obtain mixtures of amino sugar derivatives containing the h-J hexose unit. It has further been found that in sugar-free nutrient solutions, and in particular by adding maltose to the nutrient solution, in particular strain SE 50 (CBS 961 70), amino sugar derivatives containing mainly 2 to 3 hexose units can be obtained. Particularly suitable in the process according to the invention for the preparation of an amino sugar derivative in which R is glucose are nutrient solutions in which only glucose is the carbon source. If there is an excess of glucose in the nutrient solution, long juicers also form during long fermentation. amino sugar derivatives. With certain limitations, this can be attributed to the fact that during fermentation, the depletion of nitrogen sources occurs simultaneously with the depletion of glucose. If only glucose is used in nutrient solutions and maltose α is added as the carbon source, a predominantly amino sugar derivative with 2 hexose units is obtained. Pure maltose can then also be replaced by cheaper mixtures, such as Meltzin, with a naturally occurring malt extract which. rely on Diamalt AG, Munich. Corresponding to the maltotriose content, the following higher homology is also formed. Strain SE 50/110 proved to be particularly suitable for the preparation of lower homologues, which in optimal nutrient solutions gives lower homologues in approximately twice as high yields as strain SE 50/13.

The other composition of the nutrient solution, in particular the concentrations of nitrogen sources and the salt composition, can vary widely during fermentation.

The nutrient solutions are sterilized in the usual way. The pH ervos of the nutrient solution are between 5 * 0-8.5 »mainly between 6.0-7.8. Incubation temperatures are between 15 and ^ 5 ° C, mainly between 2U-32 ° C. When preparing higher homologues using strains SE 50 and SE 50/13, a higher temperature, e.g. 28 ° C, is more preferred; when preparing lower homologues using strains SE 50 and SE 50/110, a lower temperature, e.g. 2 U ° C, is more preferred. The cultivation time is 1-8 days, mainly 2-6 days. Longer culture times favor the formation of higher homologues, especially when carbohydrates are present in excess. The end point of fermentation is determined by determining the amount of inhibitory activity in the enzymatic inhibition assay and the composition by plate chromatography.

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The resulting amino sugar derivatives are isolated and purified from microbiological culture broths.

The compounds prepared by the process of the invention are recovered by adsorbing them on activated carbon at neutral pH and then extracting them with aqueous alcohols or acetone, mainly 50-80% acetone. Removal is particularly complete with a pH in the acid range of 1.5 to 1+, mainly in the range of 2-3.

If the starting solutions are very dark colored, they are subjected to a decolorization treatment with activated carbon at acidic pH (pH = 1-3) or an adsorption resin, e.g. Levapol ® Ca 9221 (non-specific polystyrene-based resin), before adsorption of the substances according to the invention. (Bayer AG), at a pH between 2-7, mainly between pH 2-3. Activated carbon primarily binds dyes in the acidic region, whereas Lewapol does not adsorb the amino sugar derivatives of the invention in a neutral or acidic environment.

To isolate amino sugar derivatives having an inhibitory effect from inactive saccharides and other inactive compounds, the weak basicity of the amino sugar components is exploited. Under suitable conditions (pH = 1 to 8, mainly 2-1; low ionic strength, corresponding to a conductivity <10 mS, mainly <2 mS), homologous amino sugar derivatives bind strongly to acidic cation exchangers such as Dowex 50 W in H + form (Fa. Dow Chemicals). Aminosugar derivatives having an inhibitory effect bind particularly well from an acetone solution (acetone 50-80%, pH 1-5, mainly 2-1) to a cation exchanger having a substantially higher capacity for said substances under these conditions. Provided that the solution contains more than 50% γ in acetone, the binding to even weakly acidic ion exchangers, such as Amberlite IRC-50 (H + form), is quite good.

According to the invention, aqueous solutions of bases or acids, mainly ammonia or hydrochloric acid at concentrations of 0.01 to 1 Val / liter are used for the extraction of amino sugar derivatives from cation exchangers. Inhibitively active amino sugar derivatives are recovered from the extracts - after neutralization with the corresponding weakly acidic or basic ion exchangers, or after removal of the base or acid in vacuo using suction in the case of volatile bases or acids - by concentration of the solvent by lyophilization or by lyophilization 10-20 parts by volume of acetone.

Separation of small molecule amino sugar derivatives with inhibitory activity from neutral saccharides is also possible by chromatography on cellulose-based ion exchangers, mainly phosphocellulose 6 G66S8 (Phospho-Cellulose, Serv., Heidelberg). Buffers are used as eluents, in particular phosphate buffers with low ionic strength, in particular 2-100 mM, in particular 5-10 mM, at a pH between 2.5-8, mainly between pH -6. A prerequisite for efficient fractionation is that the salt contents of the preparations to be fractionated are as low as possible, while the dyes then interfere less.

To prepare individual members of the homologous series of inhibitory amino sugar derivatives of the invention as pure products, the pre-purified preparations are chromatographed on a suitable molecular sieve, e.g.

(S) Bio-Gel ^ P-2 (polyacrylamide, Fa. Bio-Rad, Munich) and the eluate fractions are examined by plate chromatography. Fractions containing pure amino sugar derivatives are combined, rechromatographed and lyophilized after concentration or precipitated with organic solvents as described above.

According to their chemical nature, the substances prepared by the process of the invention belong to carbohydrates. They form sets of homologues, the basic backbone of which is to be taken from the following amino sugar derivative (C 1 H 2 O 2 N) (R = hexose).

OH OH CH3

CH 2 OH h OH XOH

The higher members thus differ from the base body in that they each have one monosaccharide unit, mainly a hexose unit, especially a glucose unit, more than the previous member of the series.

1 06698

An example of the structural principle of a homologous series is the case where the saccharide chain is formed by 1 to 7 glucose units. (The formula then represents an oligosaccharide chain with n = 1 to 7 glucose units): OH OH CH3 / CH2OH \ CH2OH h OH OH \ 0H OH / n

The basic body of the series corresponds to the case where n = 1 and is referred to as "Component II". Each member of the series differs from the next lower or next higher member in that it has one more or less glucose unit. A compound greater than one unit of glucose "Component II" is then referred to as "Component III", a compound greater than 2 units of glucose is referred to as "Component IV", and so on.

All these compounds are known from the fact that in complete acid hydrolysis they form glucose and a more undefined so-called "Component I".

"Component II" can be characterized in terms of its physicochemical properties, structure and reactivity as follows:

Component II is a colorless, amorphous solid that is highly soluble in water, dimethylformamide, dimethyl sulfoxide, and methanol. When heated, it also dissolves in ethanol.

a) Plate chromatography:

Propellant: ethyl acetate / MeOH / H 2 O 10: 6: ** (DC-Fertigfolien = ready-made membranes for plate chromatography).

I. DC-Fertigfolien F 1500 Kieselgel, Schleicher & Schtlll

Rf values: II 0, ** 6 maltose: 0.50 glucose: 0.65 II. DC-Fertigplatten F 25 ** Kieselgel, Merck

Rf values: II 0, ** 7 maltose: 0.5 ** glucose: 0.66 $ 6698

Component II is visualized on silica gel plates, most preferably with AgNO 2 / NaOH spray reagent. The brownish-black color develops even at room temperature or when slightly warmed · b) Gas chromatography »

For gas chromatographic study, component II is silylated with α- and β-D-glucose or also using sucrose as an internal standard in an ee of pyridine (1), trimethylchlorosilane (0.5) and N-methyl-trimethylsilyl-trifluoroacetamide (1).

Column »180 cm, glass, packing 3 56 SE 50 / Chromosorb WAW

Temperatures »Nozzle body 3 ° 0 ° C

Detectors J00 ° C

Oven 220 ° C isothermal until α- and β-D-glucoBi peaks are eluted; then temperature program 15 ° C / min. Up to 300 ° C.

Detectors: F I D (Flammen Ionisations Detektor)

Gases: Carrier gas: 40 ml / million

Fuel gas: Air 80 ml / min.

20 ml / min.

Retention: o-D-glucose 3 min · β-D-glucose 4 min.

Sucrose 12 - 13 min.

II 16 - 17 min.

o) Rotation angle »

The non-crystallized sample II obtained by concentrating the methanolic solution of II was dissolved in water and the specific rotation was determined: [<x] D - + 134.3 °.

d) IR spectrum »Slightly worded, poorly structured IR spectrum when using Oria.

Main absorption bands at the valence oscillation sites of 0-H »and C-0.

e) RMR spectrum »CD ^ QD / 220 MHz» (Figure 1) 6, ppm Multiplicity relative intensity

1.3 doublets; J-6, 3Hz 3 H

2.3 "triplets"; JjU.Jg 8-10 Hz 1 H

3.15 "triplet"; J ^. 7-8 Hz 1H

3.3 ~ 3.9 signals cannot be specified in 12 H

4.13 AB system; J-12 Hz 2 H

4.48 * 7 doublets; J »7 Hz” 7

and 5.1 f doublets; J-2.5 HzJ

^ protons exchanged for deuterium 4.9 against the singlet 11 H 9 500698

5.0 doublets; J-2-3 Hz 1 H

(weakly distinguished)

5.8 doublets; J »3-4 Hz 1 H

(weakly distinguished) _

35 H

(f) Component II armament:

Component II is reacted in a 1: 1 mixture of acetic anhydride and pyridine at room temperature to give the decaacetyl derivative (molecular weight 903). it can be shown that in addition to the decaacetyl derivative, an undecaacetyl derivative has also been formed (molecular weight: 943) · HMR spectrum of the decaacetyl derivative: (Fig. 2)

Deca-acetyl. MS spectrum of the derivative:

Molecular peak: 903 (relative intensity 2.5 ^)

Base peak: 843

Important fragment peaks in the above range: 844 (55 # rel. Intensity) 784 ($ 36 rel. Intensity) 783 (34 i rel. Intensity) 759 ($ 35 rel. Intensity) 556 (36 i rel. Intensity) 496 (37 96 rel. Intensity) 436 (29 i »rel. Intensity) g) Methylation of component II:

Methylation of component II with a CH 2 Cl 2 / NaH combination in dimethylsulfoxide according to Hakomori gives the decamethyl derivative of component II as a major product, also a small amount of the undecamethyl derivative. Mass Spectrum:

Molecular peak: 623 (relative intensity 6.1 #)

Base peak: 535 2nd molecular peak: 637 (relative intensity 0.2 j6)

Based on the spectroscopic values and chemical properties, the following structural formula is obtained for component II:

OH OH CH- vCH „0H

w Vo y-o

HO - / \ N 0 · / \ - OH

/ = \ H r ~ \ / “Λ

CHgOH H OH OH OH OH

10 56698

Component II is a mixture of the X and β forms, as shown in particular by NMR spectra.

The following member of the homologous series, "Component III" ^ 25H43 ° 18N ^ 0n structural formula

OH OH CH3 CH2OH CH2OH

-Q- ° -Q- ° -Q- "

CH 2 OH H OH OH OH OH OH OH

Component III has the following physical and chemical properties:

It is a highly water-soluble amorphous solid product.

a) Plate chromatography:

Propellant: Ethic ester / MeOH / H 2 O 10: 6: I. DC-Fertigfolien F 1500 Kieselgel (Schleicher & Schtlll):

Rf values: III 0.35 maltose: 0.50 II. DC-Fertigplatten F 254 Kieselgel (Merck):

Rf values: III 0.33 maltose: 0.54

Component III is obtained with AgNOg / NaOH spray reagent already at room temperature or with a slight heating of the plates to a brownish-black color spot.

b) IR spectrum: Slightly telling, poorly separated IR spectrum when using KBr, main absorption bands in the valence vibration range of 0-H and C-0 (3700 to 3100 cm -1 or 1180 to 950 cm -1).

c) Rotation angle: Δ7d in H 2 O: + 147.2 ° d) NMR spectrum of III in D 2 O / 220 MHz: (Figure 3) e) Methylation according to Hakomori *:

Methylation of III with CH 2 Cl 2 and NaH in DMSO as the main product also gives a 14-fold methylated product.

(f) Mass spectrum of the methylation product:

The molecular peak at 827 (1.5% rel. Intensity) corresponds to the gross formula CggHggNO ^ g. (At a relative intensity of 0.1, 841 has a second molecular peak).

$ 6698 11 The main fragment peaks are: 739 (27% rel. Intensity) 592 (3.7% rel. Intensity) 535 (30% rel. Intensity) 388 (9% rel. Intensity) 386 (13% rel. Intensity) 284 ( 13% rel. Intensity) 187 (12% rel. Intensity) 171 (% rel. Intensity) 101 (34% rel. Intensity) 88 (25% rel. Intensity) 7 5 base peaks.

Component IV is the next higher member of the homologous series. The compound decomposes in acid into partial hydrolysis components II and glucose in a molar ratio of 1: 2.

Plate chromatography:

Propellant: n-butanol / ethanol / water = 50:30:20.

DC-Ferfigplatten F 1500 Kieselgel (Schleicher & SchUll):

Rglukoosi'arvot! IV: ° »H1 · ° · 1'6

The inhibitory effect of higher homologues, in particular components V to VIII, on sucrase is very much lower and on pancreatic o (amylase) very much stronger than that of the lower members of the series (components II to IV).

When hydrolyzing higher components, lower homologues formed as intermediates can always be shown among the products; cleavage products also include glucose and maltose. Thin layer chromatography:

Driving Este; n-butanol / ethanol / water * 50:30:20 DC-Fertigplatten F 1500 Kieselgel (Schleicher & SchUll):

Rglucose ”values: V: °» 30 “°» 3I + VI: 0.21 - 0.23 VII: 0.14 - 0.16 VIII: 0.09 - 0.11

The specific inhibitory activities of inhibitory amino sugar derivatives are determined by enzyme inhibition assays in vitro.

amylase

An amylase inhibitor unit (1 AIY) is defined as the amount of inhibitor capable of inhibiting two amylase units by 50%. One amylase unit (AY) is the amount of enzyme which cleaves 1 μm equivalent of glucosidic bonds in starch in one minute under the experimental conditions specified below. The iVal of the cleaved 12,56698 bonds is determined as the iVal value of the reducing sugars formed with dinitrosalicylic acid colorimetrically and is expressed as pVal-maltose equivalents using the maltose reference curve. To perform the experiment, to 0.1 ml of amylase solution (20 to 22 AY / ml), add 0-10 μg of inhibitor or 0-20 μΐ of test solution in 0.4 ml of a 0.02 molar mixture of sodium glycerophosphate buffer / 0.001 mol of CaCl2, pH at 6.9, and the mixture is allowed to equilibrate in a water bath at 35 ° C for about 10-20 minutes. It is then incubated for 5 minutes with 0,5 ml of a 1% starch solution preheated to 35 ° C (Stärke, löslich, Firma Merck, Darmstadt,

No. 1252) at 35 ° C and then 1 ml of dinitrosalicylic acid reagent (according to P. Bernfeld, Colowick-Kaplan, Meth. Enzymol., Band 1, page 149) is added. To develop the color, heat the batch in a boiling water bath for 5 minutes, then cool and add 10 ml of distilled water. The extinction at 540 nm is measured against an amylase-free blank prepared in a similar manner. To interpret the test result, the amylase reference curve prepared previously is read after the addition of inhibitor, the amylase activity still effective, and from this the percentage inhibitory effect of the amylase used is calculated. The percentage inhibition is given by the equation jjg inhibitor + AY + + + calculated on a dry matter basis + ♦ as a function of the AY of the uninhibited batch of the same series, read from the curve the 50% inhibition point and convert to AIY / mg inhibitor.

Sakkaraasikoe

The sucrase inhibitor unit (SIY) is defined as the amount of inhibitor that inhibits two acacase units by 50%.

One unit of sucrase (SY) is the amount of enzyme which, under the experimental conditions specified below, breaks down 1 μmol of sucrose into glucose and fructose in one minute. The molar amount μ of glucose formed is determined by the glucose oxidase reaction under conditions in which no further cleavage of sucrose by sucrase occurs. To perform the experiment, 0-20 μg of inhibitor or 0-20 μΐ of test solution and 0.1 molar sodium maleate buffer pH 6.0 are added to 0.05 ml of precipitate-rasase solution with SY adjusted to 0.12. until the volume is 0.1 ml. Equilibrate for 10 minutes at 35 ° C and then add 0.1 ml of a 0.05 molar solution of sucrase preheated to 3 ° C in 0.1 molar sodium maleate buffer pH 6. Incubate for 20 minutes at 35 ° C and stop the sucrase reaction by adding 1 ml of glucose oxidase reagent and 56698 incubation is continued for 30 minutes at 35 ° C. 1 ml of 50% HgSO4 is then added and the measurement is carried out at 5 to 5 nm against the corresponding blank value. To interpret the test results, the percentage inhibitory capacity of the sucrase used is calculated and the value obtained from the $ 50 inhibitory site is converted to SIY / g or SIY / liter using the glucose reference curve.

1) Sucrase dissolved from porcine small intestinal mucosa (B. Borgström, A. Dahlquist. Acta Chem. Scand. 12, (1958), page 199T) · Diluted with 0.1 molar sodium maleate buffer to a pH of 6.0 corresponding to SY.

2) The glucose oxidase reagent is prepared by dissolving 2 mg of glucose oxidase (Boehringer No. 15 ^ 23) in 100 ml of 0.565 moles of Tris-HCl buffer pH 7.0 and then adding 1 ml of detergent solution (2 g of Triton X 100 + 8 g of 95 # ethanol pa), 1 ml of dianisidine solution (260 mg of o-dianisidine. HCl in 20 ml of H 2 O) and 0.5 ml of 0.1% aqueous peroxidase solution (Fa. Boehringer No. 15302 ).

Maltaasikoe

The Maltase Inhibitor Unit (MIY) is defined as the amount of inhibitor that inhibits two maltase units by 50%. One unit of maltase (MY) is the amount of enzyme which, under the experimental conditions specified below, degrades 1 μmol of maltose to 2 μmol of glucose in one minute. The μ molar amount of glucose formed is quantified by the glucose oxidase reaction under conditions in which maltose is no longer degraded by maltase. To perform the experiment, 0 to 20 mg of inhibitor or 0 to 20 ml of test solution are added to 0.05 ml of malt solution adjusted to 0.060 to 0.070 MY, and then 0.1 mol of sodium maleate buffer pH 6.0 is added until the volume is 0, 1 ml. Equilibrate for 10 minutes at 35 ° C and then add 0.1 ml of a 0.05 molar maltose solution preheated to 35 ° C in 0.1 molar sodium maleate buffer (pH 6.0). Incubate for 20 minutes at 35 ° C and stop the malting reaction. „. ...

by adding 1 ml of glucose oxidase reagent, and germination is continued for 30 minutes at 35 ° C. Then 1 ml of 50% H 2 SO 4 is added and the measurement is performed at 5 ^ + 5 nm against the corresponding blank value.

To interpret the test results, the inhibitory effect of the maltase used is calculated as a percentage and the value obtained from the 50% inhibitory site is converted to MIY / g or MIY / liter by means of a glucose comparison curve.

1) Maltase dissolved from porcine small intestinal mucosa (B. Borgström, A. Dahlquist, Acta Chem. Scand 12, (1958), page 1997). Diluted with 0.1 mol of sodium maleate buffer (pH 6.0) to a corresponding MY concentration.

2) Preparation of glucose oxidase reagent: as described in the sucrase test (p. 19, footnote 2).

111 S66S8

The results of in vitro enzyme inhibition experiments using private homologues of the amino sugar derivatives of the invention and certain homoloft mixtures are summarized in the following table, in which the hexose units represent glucose units: n - number of hexose units ΑΓΥ / g SIY / g MIY / g

Amino sugars ohdan-woman with n - 1,300,000 30,000 5,000 n - 2,300,000 68,000 15,000 n - 3 1,400,000 21,000 5,000 n - 4-6 17,500,000 8,500 n - 5-7 30,000,000 2,500

As can be seen from the table, as the molecular weight of the homologous series increases, the specific inhibitory activity against pancreatic α-amylase increases sharply; thus, compounds with n = 5-7 »inhibition of enzyme activity in vitro are 100 times more potent than an amino sugar derivative with n - 1 or n - 2« On the other hand, sucrase inhibition is most pronounced for a derivative with n - 2. , the prophylactic effect is only half as effective, and the specific sucrose-inhibitory effect of higher derivatives drops further.

In an in vivo sucrose loading assay, the activity has been found to be approximately parallel to the specific inhibitory activity observed in the in vitro assays. In contrast, in vivo starch loading experiments showed an 10-40-fold increase in the activity of amino sugar derivatives with n 1, 2, and 3 compared to amylase inhibition in in vitro experiments. (See Table 1 for Tables 2, 3, and 5).

It is known that animals and humans experience hyperglycemia after ingestion of carbohydrate-rich nutrients and nutrients (e.g., grain, potato starch, fruit, fruit juices, beer, chocolate) due to the rapid breakdown of carbohydrates by glucose hydrolases (e.g., glucose hydrolases). -amylase, maltases, saccharases) according to the following diagram * anvlaasl3 Pitaase \ Starch or glycogen maltose r glucose

Sucrose glucose + fructose 15 5Ö6S8 This hyperglycemia is particularly pronounced and persistent in diabetics. In obese people, dietary hyperglycemia often results in particularly high insulin secretion, which in turn increases fat production and reduces fat breakdown. In the presence of such hyperglycaemia, hypoglycaemia is frequently observed in insulin-dependent and obese individuals. It is known that both hyperglycemia and ingestion in the stomach promote the formation of gastric fluid, which in turn causes or promotes the formation of gastric ulcer, gastric ulcer or duodenal ulcer.

It has now been found that the glucoside hydrolase inhibitors obtained and isolated by the process according to the invention significantly reduce dietary hyperglycemia, hyperinsulinemia, hypoglycaemia after the metabolism of rats and / or humans has been affected by wheat starch or sucrose and maltose or maltose. through the stomach. In addition to this, they prevent the absorption of glucose from the intestine. Furthermore, the conversion of carbohydrates into adipose tissue lipids and the storage of dietary fat in adipose tissue is reduced or slowed.

It is further known that microorganisms break down carbohydrates in the oral cavity, especially sucrose, and thus promote the formation of tooth decay.

Glycoside hydrolase inhibitors are therefore suitable as therapeutic agents for the following diseases: obesity, blood obesity (arterial embrittlement), diabetes, pre-diabetes, gastric ulcer, gastric ulcer, duodenal ulcer and tooth decay.

Clarifications of some of the reagents and excipients used: Amberlite IRA 410 Cl "(anion exchanger); Amberlite IRC 120 (H + form) strongly acidic cation exchanger; Amberlite (HCO 2 'form) (Anion exchanger); Amberlite IRA 410 OH” (strongly basic anion exchanger), Amberlite IRC 50 H * (strongly acidic cation exchanger), trademarks of Rohm and Haas Company, and Dowex 50 WX 4H + (strong acid cation exchanger), a trademark of Dow Chemical Co. Midland, Michigan, USA.

16 56698

As the cellulose-based anion exchanger, for example, DE AE-Zellulose, Schleicher und Schull.

As the polyacrylamide gel, for example, Biogel-P-2 (trademark of Bio-Rad Laboratories, Richmond, California, USA) can be used.

Maltzin is a natural malt extract from Diamalt AG, Munich (BRD).

(S)

Levapol® is an unspecified polystyrene-based adsorption resin from Bayer AG, Leverkusen (BRD).

. Clarcel is a filter aid (diatomaceous earth / cellulose) from CECA, Velizy-Villacoublay, France.

SE 30 is a silicone elastomer from Hewlett Packard.

The hexose units in the following examples are glucose units.

Plate Chromatography Study To evaluate the composition of the final fermentation product and preparations by plate chromatography, 1 μl of fermentation broth or 1 μg of preparations are placed on silica gel ready-made membrane plates (Schleicher & Schull, Dassel. Typ F 1,500) and developed twice with n-butanol / ethanol / ethanol. / 30/20.

To obtain a sucrase inhibition dye, an enzyme gel (20 ml / 20 x 20 cm plate) is injected onto a developed and well-dried plate and the gel is allowed to solidify. It is then pre-incubated for 5 min. in a humidity chamber at room temperature and then spraying sufficient substrate gel. After this second gel layer has solidified, the plate is placed in a humid chamber and germinated at 60 ° C. Inhibition color (light spots, reddish brown background) develops in 60-90 minutes. The development of color formation is stopped at the optimum moment, and the plate with its agar layers is dried in a ventilation dryer with warm air.

Preparation of gels

Enzyme gel: 1.5 g of Agarose (L'Industrie Biologique Frangais) is suspended in 100 ml of 0.2 M Na-maleate buffer, pH

6.0, and then dissolved by boiling. Cool the clear Agarose solution to 50 ° C and add 250 μl of Triton X-100 solution (2 g of Triton X-100 + 8 g of ethanol pa) and 0,5 ml of dianisidine solution (20 mg dianisidine / 1 ml acetone) by swinging the vessel. . Just before using the gel, add 1 ml of G0D / POD reagent (12.5 mg Glucoseoxidase, Reinheitsgrad I, Boehringer, Best. Nr.

15 ^ 23 and 2.5 mg Peroxidase, Reinheitsgrad II, Fa. Boehringer, Best. Nr. 15302, dissolved in 5 ml of maleate buffer) and 1+ to 5 units of sucrose obtained from porcine small intestine Ί ^. The gel must be kept at 50 ° C until spraying, otherwise it will solidify in the nozzles during spraying.

17 56698

Substrate gel: 0.5 g of agarose is suspended in 100 ml of Na-maleate buffer, pH 6.0 and dissolved by boiling. The enzyme preparation prepared as described in footnote 1) is then cooled to 50 ° C, 100 Triton (2 g Triton X-100 + 8 g ethanol, pa) and 1 g sucrose (Serva No. 35579) are added. · After the sucrose has dissolved, the gel is ready to use.

To provide an amylase inhibitory dye, an amylase gel (20 ml / 20 x 20 cm plate) is sprayed onto the developed and dried chromatographic plate and allowed to solidify. After pre-incubation for 5 minutes at room temperature, the plate with gel membranes is placed in a 0.5% starch solution (1 g of starch, Merck Kr. 1252, dissolved by boiling in 200 ml of a buffer solution of 0.2 m glycerophosphate / 0.01 m CaCl 2, pH 6 , 9) and kept there for 2 min. at + 0 ° C and invert the solution. The plate is then rinsed with well-distilled water and immersed in a dilute Jg solution (U ml Jg stock solution in 500 ml Hg0: or Jg stock solution: 2,2 g Jg + i +, U g KJ dissolved in 100 ml Hg0 O). The optimum color is formed after about 1 minute. The disc is photographed immediately as the blue spots fade soot.

Preparation of amylase gel 1 g of agarose is dissolved in 100 ml of buffer solution at 100 ° C 0.2 m sodium glycerophosphate / 0.01 m CaCl 2, pH 6.9, and after cooling to 50 ° C, 100 μl of amylase crystal suspension (10 mg porcine pancreatic amylase / ml saturated NH 4 sulphate solution, Boehringer, No. 15017).

Example 1 per 100 liters of nutrient solution containing 3.5% glucose, 2.5 μl malt extract dry powder, 0.5% casein hydrolyzate, 1.3% yeast extract, 0.3% CaCO 2, 0.3% KgHPO 4 and 0, 1% antifoam, inoculated with 5 liters of a pre-culture of strain SE 50/110 in a nutrient solution containing 3% soybean meal, 3% glycerol and 0.2% CaCO 3. The culture was incubated with stirring and conditioning for 5 days at 2U ° C to obtain a culture solution containing 73,000 SIY / l and containing mainly .. an amino sugar derivative with n = 2.

The pH of a 90 liter batch, with mycelium, was adjusted to 2.5 with concentrated HNO 2 using a pH meter, and 900 g (= 1%) of activated carbon (Merck) was added with stirring to adsorb most of the dye formed. The mixture was stirred for 15 minutes, and the mycelium and most of the carbon were isolated in a centrifuge at 3,000 rpm, and the supernatant was finally filtered through a suction filter with the first addition of 3 kg of Clarcel. 65 liters of amber, clear filtrate with a SIY content of 60,000 SIY / l were obtained.

18 56698

The pH of the filtrate was adjusted to 7 with concentrated NH 4 and the filtrate was mixed with 1300 g (2 ¢) of activated carbon (Merck) for 30 minutes to adsorb the active substance. It was filtered through a suction filter and the activated carbon sediment was washed 3 times with 10 liters of distilled water. The carbon was then compressed to dryness and mixed to extract the active substance from the carbon three times with 1 liter of 50% acetone at pH 2.5, stirring for 15 minutes each time. The acetone extracts separated by filtration from carbon were combined. The combined extract was concentrated on a rotary evaporator to 250 mL, the same volume (250 mL) of methanol was added and filtered through a pleated filter. The filtrate (U80 ml) was added dropwise with vigorous stirring to 5 liters of acetone. The separating precipitate was filtered off with suction and washed 3 times with acetone and ether. It was then dried in vacuo at 35 ° C.

Yield 230 g of product with 8,500 SIY / g.

25 g of the above crude product was dissolved in 1 liter of H 2 O and the solution was stirred for 30 min. With 300 g of Dovex® 50WX 1+ H (0.07-0.037 mm). The resin was filtered off and then washed 3 times with 2 liters of 0.001 N HCl. The washed Dcwex was then suspended in 500 ml of HgO and the pH of the suspension was adjusted to 9.0 with 25% NH 4 using a pH meter. It was then extracted twice more with 500 ml of 0.6% NH 4 (each time), the extracts were combined and concentrated on a rotary evaporator to 100 ml. To remove color from this concentrate, it was stirred for 5 min. With 2 g of DEAE cellulose (Fa.

Schleicher und Schiill Nr. 02035, 0.6 mVal / g), and then centrifuged. To the pale yellow overlay was added the same volume (100 mL) of methanol, and then this was added dropwise with vigorous stirring to 2 liters of acetone. The precipitate was filtered off with suction, washed with acetone and ether and dried in vacuo at 35 ° C. Yield 1+, 2 g of product with 26,000 SIY / g. For further purification, 0.0 g of inhibitor was gel filtered in 0.5 g portions through Biogel P-2. To this end, each 0.5 g preparation was dissolved in 10 any HgO and applied to a Biogel P-2 column (0.07-0.037 mm, Bio-Rad) with a diameter of 5 cm. Development was performed with water at a flow rate of 80 ml / h. 12 ml fractions were collected. Total carbohydrate content (anthrone test, extinction E 620) as well as sucrase inhibitor and anylase inhibitor concentrations were determined from all fractions. In addition, fractions were examined by plate chromatography (Enzyme inhibition color: see "Plate Chromatography Study").

.9 S6698

Fractions found to contain amino sugar derivatives with n = U-6 were combined, concentrated in vacuo to 10 mL and mixed by dropwise addition to 200 mL of dry alcohol. The precipitate was centrifuged off, washed with acetone and ether and dried in vacuo; yield U, from 0 g of crude inhibitor: 0.2 g of an amino sugar derivative with n = 1 + -6 and having 17.5 x 10 4 AlI / g and 8,500 SIY / g. Fractions containing an amino sugar derivative with n = 3 were treated in the same manner with precipitation with 200 ml of acetone, yield U, from 0 g of crude inhibitor: 0.1 g of amino sugar derivative with n = 3 and 1, U x 10 ^ AIY / g and 21,000 SIY / g.

From the fractions containing the amino sugar derivative with n = 2 (precipitation with acetone), 0.9 g of the amino sugar derivative with n = 2 and 0.3 x 10 6 Al / g and 68,000 SIY / g were isolated.

Example 2

Three small fermentors, each containing 8 L of nutrient solution containing 7> 5% malt extract dry powder, 0.3% casein hydrolyzate, 0.7% yeast extract, 0.3% CaCO 2, 0.3% KgHPO 4, were seeded 5% preculture of strain SE 50/110 in a nutrient solution containing 3% soybean meal, 3% glycerol and 0.2% CaCO 3 to give, after 5 days incubation at 21 ° C, a culture broth of 73 SIY / ml , and containing mainly an amino sugar derivative with n = 2. After centrifugation to separate the mycelium (30 min, 3,000 rpm), 20.5 liters of a deep brown culture solution with 67,000 SIY / l was obtained. The pH was adjusted to 3.5 with HNO 2 and 60 g of Lewapol (Ca 9221, grain size 0.35 mm, Bayer) / 1 = 1.23 kg of Lewapol were added to remove the color. 20 min. after mixing, it was suction filtered through a Seitz K 3 filter. The decolorized culture solution was neutralized with NH 4 (18.5 L, 67,000 SIY / L). The effect of adsorbing the substance was mixed with 20 g of activated carbon / 1 = 370 g and stirring was continued for 30 minutes. It was then filtered through a Seitz K 3 filter with a layer of Clarcel filter aid on top. The filtrate (17 »5 1, 3 600 SIY / l) was discarded. The residual carbon was washed 3 times with 2 L of distilled water. To extract the active ingredient from the carbon, this was stirred 3 times in succession for 15 minutes with 1 liter of 80% acetone (each time), the pH being adjusted with conc. With HCl to 2.5. The extracts were combined (2.1+ 1.371,000 SIY / l). To this extract was added 20 g of Dowex H / 1 (Dowex 50W X 1 », H form, Fa. Serva, Heidelberg) = U6 g of Dowex and mixed for 20 minutes. The resin was then filtered off (Dowex fraction I) and washed with a small amount of 75% acetone. The filtrate and washings (31 = 215,000 SIY / l) were mixed with 60 g of Aniberlite IRA 1 * 10 (OH form) (Serva Heidelberg) / l until the pH was 7. It was then filtered and the filtrate (2.8 1, 219,000 SIY / l) 72 g of Dowex H + resin were added and mixed for 20 minutes.

During this time, the pH was maintained at 3 ° 0 by hanging a porous nylon bag filled with Amberlite IRA 1 * 10 OH. The Dovrex was then filtered off (Dowex fraction II), the filtrate (2.6 L, 27,000 SIY / l) was discarded.

Dovex fractions I and II were each washed 3 times separately with 75% acetone at pH 3.5 and then extracted 3 times with 100 ml (each time) (Dowex fraction I) or 150 ml (Dowex fraction II) 0.6% NH 4. In the first extraction, when the amount of ammonia was not sufficient to neutralize the Dowex resin, the pH was adjusted to conc. NH 4 by adding a pH meter to 9. The three extracts of Dowex fractions I and II were each combined, concentrated to near dryness on a rotary evaporator, dissolved in 50 mL of H 2 O, adjusted to pH 3-1 + with a pH meter and 50 mL of methanol was added. The solutions were added dropwise with stirring to 1.5 liters of absolute acetone, the precipitate formed was filtered off with suction and washed 3 times with acetone and once with ether. Drying took place in vacuo.

Yield: Fraction I 6.5 g 25,000 SIY / g

Fraction II 12.3 g 36,000 SIY / g.

Fractions I and II contain, as inhibitory components, mainly an aminosugar derivative with n = 2, and in addition small amounts of mainly higher homologues (n = 3).

Yield Volume (l) SIY / 1 SIY total SIY yield,% 1) Culture solution 20.5 67,000 1,373,500 100 2) After decolorization with Lewapol 19.5 67,000 1,306,500 95 3) After adsorption on activated carbon 18.5 3 600 66 600 (1 *, 8 rejected) 1+) 1st extract 0,7 7 ** 2 000 519 1 * 00) 37,8) ^ 5) 2nd extract 0,9 329 000 296 100) 883 500 21.6) 61 * - 6) 3. new 0.8 85,000 68,000) 5.0) 7) New mixture (U-6) 2.1 * 371,000 89Ο 1 * 00 61 *, 8 8) 1. After Dowex adsorption 3.0 215 000 61 * 5 000 9) After neutralization with IRA OH 2.8 219 000 613 200 10) 2. After Dowex adsorption 2.5 27 000 67 500 (* +, 9 rejected) 11) Combined NH_ extract

From the Dowex fraction Γ 0.29 6 82 000 19 7 7 80 11 *, 1 *) 12) Combined NH 2 extract ^ 60-

From the Dowex fraction "ΊΙ 0.1 * 5 1 1 * 19,000 63Ö 550 1 * 6.5) 13) Precipitation Fraction I 6.5 g 25,000 / g 162,500 11.8)) 1 * 1 * -

Precipitation Fraction II 12.3 g 36,000 / g 1 + 1 * 2,800 32.2) 56698

Example 3 200 g of a preparation obtained by inoculating 8 liters of a nutrient solution containing 5.0% starch, 1.0% yeast extract and 0.2% Κ ,, ΗΡΟ ^, 3 days old strain SE 50/13 (CBS 611+ , 71) shaking culture and incubating the culture for 3 days at 28 ° C with vigorous stirring, were added to 9 * + 0 ml of distilled water and 60 ml of concentrated H 2 SO 4 and, the solution was refluxed for 1+ hours (internal temperature 98 -100 ° Cv oil bath temperature: 11 + 0 ° C). To the cooled, black-brown solution was added 10 g of activated carbon (Merck Art. 2186) and stirred for one hour. The activated carbon was then filtered off, washed with water and the pH of the filtrate was adjusted to 7-8 with about 250 ml of 10N KOH. The solution was stirred for tann with 50 g of activated carbon. The carbon was filtered off, washed with 2 L of water and the filtrate was discarded. To extract the product from charcoal, this was digested overnight with 2 liters of 30% alcohol. Finally, the carbon was filtered off and the alcoholic solution was evaporated to dryness on a rotary evaporator. Residue: 6.2 g. This crude product (6.2 g) was dissolved in 500 ml of water and the solution was gently stirred for 1 hour with 30 g of Amberlite IR 120 (H + form). The ion exchanger was filtered off and washed with distilled water until the filtrate was neutral and glucose-free. The ion exchanger was then stirred overnight in 1000 ml of water with 15 ml of 25% NH 4, separated and discarded. The filtrate was evaporated to dryness on a rotary evaporator. Residue: 3.7 g.

For further purification, chromatography was performed using cellulose. U, 5 g of the material extracted from the ion exchanger was applied to a column packed with cellulose with a length of 011 1 m and a diameter of 2.5 cm. Ethanol / H 2 O 5: 1 was first used as the eluent, ethanol / H 2 O 3 was first used to elute the amino sugar derivative with n = 1. At a drip rate of 20 drops per minute, 1 * + ml fractions were taken at a time. Each separate fraction was examined by plate chromatography. Fractions U7-85 were evaporated to dryness to give 1.6 g of a slightly brownish-colored amino sugar derivative with n = 1. Quantitative coloring impurities are not significant. The amino sugar derivative with n 58 l is obtained as a colorless ✓ resin if the purification step with a strongly acidic ion exchanger is performed on a column instead of the batch method.

22 56698

Example ^ (Example of extraction of compounds of the invention with an acidic solution) ... ®

A column with a diameter of 1.5 cm was charged with 30 g of moist Dowex 50 W x lt, (H +) 0.07 ^ -0.037 mm, 0.001 norm. HCI. 500 ml of the mixed extract (U00,000 SIY / l, pH 2.5 → 60% acetone) obtained according to Example 2 (Table, item 7) was then pumped through the column for about 1 hour and then the column was rinsed with 500 ml. 0.001 N hydrochloric acid. HCl. Under these conditions, only a small amount eluted from the activity. The 0.0125 norm was then extracted. HCl to record the conductivity of the column eluate. In addition, the SIY content of the eluate was examined. The active fractions 7-100 were combined and neutralized by adding the prepared Amberlite IRA bVS OH, then concentrated to 5 ml, 5 ml of methanol was added and precipitated by dropwise addition of the solution to 200 ml of acetone. After washing with acetone and ether, it was dried in vacuo.

Yield 1 g of an amino sugar derivative with n = 2 and 65,000 SIY / g.

Amino sugar derivatives with n 5 3 and n = · (can be recovered from the active pre-fractions.

This acid extraction method thus makes it possible, in contrast to the alkaline extraction method, to fractionate amino sugar derivative homologues.

Therapeutic experiments I Experimental arrangements for the action of glucoside hydrolase inhibitors and glucose resorption. in rats and humans

To induce dietary hyperglycemia and hyperinsulinemia, fasting rats (6) or fasted subjects are orally administered either 2.5 g of sucrose or 2.5 g of maltose or 1 g of cooked starch or 2.5 g of glucose per kilogram, or 50 g of sucrose or 50 g of maltose or 50 g of cooked starch or 50 g of glucose per person orally as an aqueous solution or suspension. The other 6 rats receive the same amounts of said carbohydrates and in addition one of the active ingredients mentioned in the examples at the indicated doses. Healthy subjects are administered alternately one of said carbohydrates every other day or less frequently with or without any of said active ingredients. Blood glucose and serum insulin are determined both before and shortly after administration of the active ingredient carbohydrate 5 minutes to 3 hours after administration from blood taken from the retro-orbital venous plexus of rats or from blood taken from the elbow vein or capillaries of 23,56698 subjects. blood glucose

(f D

assays are performed on an Auto-Analyzer (Technicon w, Hoffman: J · Biol. Chem. 120, 51 (1937) or enzymatically with glucose oxidase and o-dianisidine hydrochloride), serum insulin assays by the method of Hales and Rande: Biochem. J. 88, 137 (19 ^ 3) - II Experimental set-up to show inhibition of carbohydrate conversion of adipose tissue lipids

Rats (6) receive by gavage one of said carbohydrates in inactive form together with a suitable dose of the same carbohydrate, „but labeled with radioactive carbon (^ C), - the active ingredient mentioned in the above-mentioned examples. At short intervals from the start of the experiment, the animals are sacrificed and the adipose and / or kidney surrounding adipose tissue is sampled, the lipids are extracted with appropriate fat solvents, e.g. chloroform / methanol (2: 1), and the lipid-free metabolic metabolites are washed away. Lipids isolated in this way are assayed for radioactivity on a Liquid Scintillation Counter. Activity is expressed in dpm / 1 g adipose tissue.

In the tables, P denotes the probability of error and s denotes the standard deviation.

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Claims (1)

A process for the isolation and purification of microbiologically prepared amino sugar derivatives of the general formula OH ° H CH 3 h ° \ / 1r ch 2oh h 0H 0H wherein R is a saccharide chain having 1 to 7 hexose units, characterized in that microorganisms belonging to the genus Aetinoplanes -organisms under aerobic conditions and in an aqueous nutrient medium after separation of the mycelium obtained by culturing and possibly decolorization of the obtained culture filtrate, for example with activated carbon at pH 1-3 a) with a controlled mixture of water and alcohol or water and acetone, preferably 50-80 to # # acetone, c) the desorbate obtained is applied to a cation exchanger, which is preferably strongly acidic, and d) the amino sugars bound to the cation exchanger are either completely desorbed with ammonia and then fractionated in a manner known per se using gel filtration or a cellulose column as individual components, or the amino sugars are selectively eluted with a dilute acid, and the individual amino sugars are isolated from the eluates thus obtained.