CS197288B2 - Process for preparing aminoderivatives of sugar - Google Patents

Description

The present invention relates to a process for the preparation of novel aurnosugar derivatives. Further, reference is made to pharmaceutical compositions containing these am'nucaric derivatives as active ingredients, as well as to controlling carbohydrate exchange in humans and animals by suppressing glycoside hydrolase with these aminosugars, such as for the treatment of Diabetes, obesity and increased blood fat levels. .

A number of microorganisms are known to class

Actinomycetes suppresses glycosylation ^d hydrolases, wherein depending on the conditions obtained in the cultivation Inhib 'tors with - -potlačujícím effect mainly amylase or sucrase (see, e.g., U.S. - writings 3,876,766, 3,855,066 and 3,879,546).

A process for the preparation of the novel aminosugar derivatives of general formula I has now been discovered

wherein n 1 is a number from 1 to 7 and n 2 is a number from 1 to 7, wherein the sum of ni and na is 2 to 8.

Depending on the sum of ni-j-nz, these aminosugar derivatives predominantly have the effect of inhibiting the action of amylase or sucrase. Thus, the aminosugar derivatives which are directed to suppressing the action of amylase are derivatives with n1 -n2 = 4 to 8, in particular p. ni---.ni = 4 and 5. Aminosugar derivatives which are directed to suppress the action of sucrose are derivatives with ni - n.2 = 1 to 3, suitably with ni-Jn? = = - 2 to 3 and preferably with nn = 2. ;

In addition, it has been discovered that - surprisingly - in - vivo - a - strong - suppression of starch degradation by amino-sugar derivatives with m4-n = 1-3, in particular ni = 2, is achieved.

The process for producing the amino sugar derivatives of the invention is carried out by culturing strains SE 50 (ATCC 31 042), SB 18 [ATCC 31041], SE 82 (ATCC 31045), SE 50/13 (ATCC 31 043) and SE 50 / 110 (ATCC 31 044) of the genus Actinoplanaceae aerob in a solid or liquid medium containing a source of carbon and nitrogen, as well as salts and antifoams, at a temperature of 15 to 45 ° C and a pH of 5.0 to 8.5 and required the compounds are isolated in a manner known per se.

Thus, strains SE 50 (ATCC 31 042, CBS 961.70), SB 18 (ATCC 31041, CBS 957.70) and SE 82 (ATCC 31 045, CBS 615.71) as well as mutants or variants of these strains are suitable for the production of the aminosugar derivatives of the invention. . The yields of the amino-sugar derivatives obtained are particularly good in this regard with the strains SE 50/13 (ATCC 31 043, CBS 614.71) and SE 50/110 (ATCC 31 044, CBS 674.73). Both strains correspond as far as possible to the original strain SE 50 (ATCC 31 042, CBS 971.60) from which they were obtained by natural selection without inducing changes.

For. cultivation - microorganisms. solid or liquid, but especially liquid, aqueous nutrient media are used which contain, in a conventional concentration, the known starting materials for carbon and nitrogen, salts and antifoaming agents at conventional concentrations.

The starting materials for carbon are predominantly carbohydrates, in particular starches, maltose, glucose, as well as complex mixtures of substances, such as commercially available malt extract.

Suitable starting materials for nitrogen are the complex mixtures known per se, such as casein hydrolyzate, yeast extract, peptone, fish meal, meat extract, mixtures of these starting materials, as well as individual amino acids and / or ammonium salts as nitrogen sources. .

The microorganisms are grown aerobically in aerated shake cultures or in conventional culture containers.

The type of concentration of the carbonaceous substance used as starting material affects, in conjunction with the strain used for fermentation, the type of predominantly end product.

Thus, for example, in nutrient solutions containing 2% by weight of starches, a compound with ni-n2 = 4 to 8 is formed. The SE 50/13 strain (CBS 614.71) is particularly suitable for the preparation of these amino-sugar derivatives with ni-m = 4 to 8. ).

However, if the nutrient solution contains sufficient glucose (about 3.5% by weight), 0.1 to 3% by weight of starches are sufficient to obtain predominantly mixtures of the aminosugar derivatives with ni-j-n2 = 4-8. may be used as starting compounds for the production of lower homologues by a chemical or enzymatic hydrolysis process.

Aminosugar derivatives with ni-J ns = 1 to 4 are largely obtained when working in a starch-free nutrient solution. The addition of maltose to the nutrient solution is particularly favorable. Thus, for example, with the addition of maltose and using strain SE 50 (CBS 961.70), a mixture of amniugar derivatives with predominantly ni-j-n2 = 2 to 3 is obtained. carbon only uses glucose. It should be noted here that excess glucose during long-term fermentation also produces high molecular weight amino-sugar derivatives.

If a nutrient solution that does not contain glucose is used and maltose is added as the sole carbon starting material, it is predominantly an aminosugar with ni - | - nz = 2. In a technically preferred embodiment, pure maltose can be replaced by a cheaper product , such as malzine, it is a natural commercially available malt extract.

The strain SE 50/10 (CBS 674.73) has proven to be particularly suitable for the production of amino-sugar derivatives which predominantly contain derivatives thereof with n = 1 to 3.

This strain provides yields of low molecular weight amino sugar derivatives that. are about twice that obtained with strain SE 50/13 (CBS 614.71).

The cultivation of microorganisms is generally carried out at incubation temperatures between 15 and 45 ° C, preferably between 24 and 32 ° C. The use of strains SE 50 (CBS 961.70) or SE 50/13 (CBS 614.71) yields amino-sugar derivatives with ni-j-Π2 = 4 'to 8 at a higher temperature, e.g. 28 ° C, while at a lower temperature, e.g. ° C, for example the strains SE 50 (CBS 961.70) and SE 50/110 (CBS 674.73) are predominantly amino-sugar derivatives with ni + n 2 - = 1 to 3.

The cultivation period is generally 1 to 8 days, preferably 2 to 6 days. Longer cultivation times, especially in connection with excess carbohydrates in the culture medium, support the formation of amino-sugar derivatives with higher ni-j-nz values.

The pH in the culture medium varies generally from 5.0 to 8.5, with pH values of 6.0 to 7.0 being preferred.

The end of the fermentation is generally determined by determining the suppressing effect in the enzyme inhibition assay, as well as determining the composition of the fermentation broth by thin layer chromatography.

The separation and isolation of the individual amino-sugar derivatives according to the invention is based either on microbiological culture broths or hydrolysates or incubation mixtures, in which enzymatic and / or microbiological degradation is carried out by salting out (.alpha.) .Alpha.-amino-sugar derivatives.

The processing is carried out in different ways depending on the value for Ц- nz. Thus, the production of pure aminosugar derivatives with ni + пг = 1 to 4 is generally carried out by optionally treating the culture broth or hydrolyzate with activated charcoal at neutral pH, after prior separation of the mycel, and then desorbing the activated charcoal aminosugar derivatives by treatment of activated carbon with an aqueous alcohol or acetone, preferably 50 to 80% acetone. The desorption is particularly quantitatively evaporated at a pH in the acid range of 1.5 to 4, preferably 2 to 3.

In the case where the separate culture broths or hydrolysates are very dark colored, it has proven advantageous to first treat the activated carbon or absorbent resins, such as the commercially available Laxapol Ca 9221 (grain size 0) prior to the above-described activated carbon treatment at acidic pH. 35 mm, manufactured by Bayer AG) at a pH of from 2 to 6, preferably from 2 to 3, in order to obtain discoloration. In this case, the activated carbon preferably binds only the dye and not the aminosugar derivatives.

Separation of the individual aminosugar derivatives of the invention from the activated carbon de-sorbate obtained as described above is then carried out. is generally carried out by passing the desorbate through a known acid cation exchanger, such as Dowex 50 W (manufactured by Dow Chemicals) (pH 1 to 8 preferably 2 to 4; at a lower ionic strength, a conductivity of <10 mS.cnr ^1, preferably <2 mS.cnr ^1]. Individual aminosugar derivative of the same time bind to the resin. for such cation can be bound particularly advantageous here to aminosugar derivatives of the acetone solution (50 to 80% acetone, pH 1-5, preferably 2 to 4).

The selective desorption of the aminosugar derivatives of the invention from the cation exchangers is then generally carried out using aqueous acids or bases as eluting agents, preferably ammonia or hydrochloric acid at concentrations of 0.01 to 1 val / liter.

From the individual desorbates, the corresponding aminosugar derivative is then obtained after neutralization of the desorbates with a weakly acidic or basic ion exchange resin or after removal of the base or acid under vacuum, in the case of volatile bases or acids, evaporation of the solution by lyophilization or precipitation with organic solvents, preferably acetone.

Furthermore, low molecular weight aminosugar derivatives derived from inert saccharides can be separated by chromatography on a cellulose-based ion exchange resin, preferably based on phosphorus-cellulose (manufactured by Serva, Heidelberg). Buffers, preferably phosphate buffer, of low ionic strength, preferably 2 to 100 mM, in particular 5 to 10 mM, in the pH range of 2.5 to 8, preferably 5 to 6, are used as the eluent. and, if possible, a low salt content in fractionating preparations whose coloring substances are less disturbing.

To prepare pure individual aminosugar derivatives, the pre-purified preparations are then chromatographed over a suitable molecular sieve, for example, Bio-Gel P-2 (manufactured by Bio-Rad, Munich), and the eluate fractions subjected to thin layer chromatography. The fractions containing the pure compounds of the invention are pooled, re-chromatographed and then, after evaporation, lyophilized or precipitated with an organic solvent as described above.

All amino-sugar derivatives are characterized in that they form, in acid total hydrolysis, the "component I" of the formula C 13 H 21 O 7 N, as well as glucose. For “structural component

I ', the following formula was derived:

(Component. LJ member of the Amino Sugar Row of the Initial Rivals of the Invention has a single glucose unit. This compound is a colorless amorphous substance with good solubility in water, dimethylformamide, dimethylsulfoxide, methanol and hot ethanol. water = 10: 6: 4, this compound has an R-value _of 0.46 (= 0.50 maltose and glucose 0.65) on the silica film F 1500 [Schleicher and Schüll] 0.47 (maltose = 0 54 and glucose = 0.66) on F 254 silica gel plates [Merck, Darmstadt].

When using the spraying agent silver nitrate and sodium hydroxide, a brown-black color is obtained at room temperature or after slight heating.

The compound is silylated together with D-glucose as standards in a mixture of pyridine (1), trimethylchlorosilane (0.5) and N-methyl-trimethylsilyltrifluoroacetamide (1) and then subjected to 1.8 m glass column chromatography packed with 3% SE 30 silicone elastomer (Packard) on WAW chromosorb.

The injection and detector temperatures are 300 ° C. The temperature of the furnace is 220 ° C, isothermally until the elution of α- and β-D-glucose standards. The temperature is then raised at a rate of 15 ° C per minute to 300 ° C.

A flame ionization detector is used, introducing nitrogen as carrier gas at a rate of 40 ml / minute, air as combustion gas at a rate of 80 ml / minute and hydrogen at a rate of 20 ml / minute. The compound has a retention time of 16 to 17 minutes (α-D-glucose = 3 minutes, β-D-glucose = 4 minutes and sucrose = 12 to 13 minutes).

The non-crystallized sample of the compound obtained by evaporation of the methanolic solution is dissolved in water and the specific optical rotation is determined [α] _D = + 134.3 °.

The NMR spectrum in CD3OD at 220 MHz is shown at rt. 1 (first coordinate axis = δ ppm).

The predominant features of the NMR spectrum are shown in Table 1.

Table 1

in ppm Multiplicity Relative intensity 1.3 doublet, J = 6.5 Hz 3 H 2.3 Triplet, Ji and J2 8 to 10 Hz 1 H 3.15 Triplet, Ji and J2 7 to 9 Hz 1 H 3,3-3,9 signals are not merely assignable 12 H 4.13 AB-system, J = 12 Hz 2 H 4.48 and 1 doublet, J = 7 Hz) 1 H 5.1 J 4.9 doublet, J = 2.5 Hz J singlet -L И 11 H 5.0 doublet, J = 2-3 Hz for deuterium exchanged protons IH 5.8 (hardly recognizable) doublet, J - 3 to 4 Hz 1 H (hard to discern) 33 H

When this compound is reacted in a 1: 1 mixture of acetic anhydride and pyridine at room temperature, a decaacetyl derivative (molecular weight: 903) is obtained. The NMR spectrum of the decaacetyl derivative is shown in Figure 2.

If the reaction is carried out in ethyl acetate / acetic anhydride in a ratio of 1: 1 with a catalytic amount of sulfuric acid, mass spectroscopy, besides the decaacetyl derivative, also shows the formation of an undeacetyl derivative (molecular weight: 945).

The mass spectrum of the decaacetyl derivative has a molecular peak at 903 (2.5% relative intensity) and a baseline peak at 843. The important partial peaks in the above mass spectrum range are 844 (55% relative intensity [II.a.])

784 (36% r. I.)

783 (34% I),

759 (34% I),

556 (36% r. I.)

496 (37% y.) A

436 (29% g. I.).

Methylation with CH 3 I / NaH in dimethylsulfoxide according to Hakomori yields the decamethyl derivative as the main product and also the undecamethyl derivative in small amounts.

The molecular spectrum has a molecular peak at 623 (6.1% relative intensity) and a baseline peak at 535. A second molecular peak was observed at 637 (0.1% relative intensity).

Spectroscopic data and chemical properties show that the compound has the structure of Formula IIa

OH OH CHt У-0 CHpM (l · нон У-N ⁰⁾ OH \ ^ OH CH-OH H OH OH H (+ (1 / aJ

In particular, the NMR spectrum shows that the compound of formula (IIa) has the structure O- / 4,6-bisdesoxy-4- [1-S- (1,4,6 / 5) -4,5,6-tri hydroxy-3- hydroxymethylcyclohex-2-en-1-ylamino] -? D-glucopyranosyl] - (1-4) -D-glucopyranose consistent with Formula IIb

The following board member (ш + П2 = 2) has a total composition of C25H43O18N and is a well water soluble product. When thin layer chromatography has an R value _of 0.35 on silica-gel film F 1500 and 0.33 on silica gel plates F 254, using the above described systems.

The optical rotation in water [α] _D is - | -167 ° / o.

The NMR spectrum in D2O at 220 MHz is shown in Figures 3 and ^13, and the C-NMR spectrum in Fig. 4.

Methylation as described above provides a 13-fold methylated compound and, to a small extent, a 14-fold methylated product.

The mass spectrum of the methylated product had a molecular peak of 827 (1.5% relative intensity) and was consistent with the formula C38H69NO18. The second molecular peak with 0.1% relative intensity is at 841.

Important partial peaks are:

739 (27% г. I.) 592 (3.7% г. I.) 535 (30% I) 388 (9% y. 1.) 386 (13% г. I.) 284 (13% г. I.) 187 (12% г. I.) 171 (40/ _0% I.) 101 (34% г. I.) 88 (25% I) 75 basic peak.

It follows from the chemical and spectroscopic properties that the compound has the structure of formula IIIa:

wherein the second member of the aminosugar derivatives of the invention belongs to the O- / 4,6-bisdesoxy-4- [1S- (1,4,5,6 / 5) -4,5,6-trihydroxy-3-hydroxymethylcyclo-hexyl- 2-en-1-ylamino] -α-D-glycopyranosyl- (1-> 4) -Oα-D-glucopyranose of 1- (1-> 4) -D-glucopyranose conforms to formula IIIb

The nearest higher member of the series (n1-j2 = 3) is obtained in two isomeric forms, one of the two isomeric forms predominantly entering.

In acid partial hydrolysis, both isomeric compounds can be resolved into the first row member (m + иг = 1) and glucose at a molar ratio of 1: 2.

The product present in minor amounts is compound O74,6-bisdesoxy-4- [1,5- (1,4,6 / S) -4,5,6-trihydroxy-3-hydroxymethylcyclohex-2-enylamino] -aD -glucopyranosyl / - (1-4) -OaD-glucopyranosyl- (1-4) OaD-glucopyranosyl- (1-4) -D-glucopyranose according to formula IV:

197268 ^{но г} ноон

О ZE

Isomeric product present in larger quantities ^: is the compound O- / 4,6-bisclesoxy-4 - [: L S- (1,4,6 / 5) [4,5,6-trihydroxy-3-hydroxymethyl] -4- O-α-D-glucopyranosyl 1- (1-4) -cyclo-14-hex-2-en-1-ylamino] -α-D-glucopyranosyl] - (L-> 4) -Oxa-D-glucopyranosyl- (4? 4) - D-glucopyranose according to formula V

О α:

1S

These isomers are F plates 1500 using n-butanol, ethanol and water in a ratio of 50:30:20 and the value of R _{g ÚKOZ} 0.41 to 0.46. The presence of isomers with the structure represented by formula V can be demonstrated when the hydrogenation degradation product (palladium on activated carbon) is analyzed. These include degradation products: 3-hydroxymethyl-4,5,6-trihydroxy- [4-Cba-D-glucopyranosyl- (1)] -cyclohexane, O- (4-amino-4,6-bisdesoxy-α-D-glucol) pyranosyl) - (1- + 4) -O-β-D-glucopyranosyl- (1- + 4) -D-glucopyranose and glucose.

Since, in this degradation of the C-N-bond at the alkyl position, it is cleaved and hydrogenated with hydrogen, it is clear that this compound, which is isomeric to the compound of formula IV, is converted to a compound of formula III and / or IIIb, however an additional glucopyranosyl group is present at the 4-position of the cyclohexene ring.

PdíCJH ^

CH.OH CH.OH γ-ο но- <У-оч>

^{+ H} Z ^N \) ~ O \ / ° \ / ⁽ но OH OH OH OH н о OH OH OH

The presence of the isomers of the structure shown in Formula IV can be demonstrated by the formation of validatol and O- (4-amino-4,6-bisdesoxy-α-D-glucopyranosyh).

- (T> 4) -Cha-D-glucopyranose 1- (1-> 4) -O-α-D-glucopyranosyl- (1-> 4) -D-glucopyranose.

_: MOH! сн ₃ снрн CHOH снрн% \ · 'Η Λ Г ^0th SMELL

HO-C \\ N ohoh \ ohoh он он он он OH OH

он он он он он OH

In further tests, isomers with ni + P 2 = 3 are methylated according to known methods, hydrolyzed and reduced with sodium borohydride, acetylated and analyzed by gas chromatography. While the compound of formula IV only provides 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-D-glucitol, the compound of formula V provides 1,4,5-tri-O- acetyl-2,3,6-tri-O-methyl-D-glucitol and 1,5-di-Oacetyl-2,3,4,6-tetra-O-methyl-D-glucitol in a 2: 1 molar ratio. Isomers with ni + P 2 = 3 of formula IV and formula V are not degraded by β-amylase.

The isomeric compounds of formulas IV and V are separated by chromatography on an acidic ion-exchange resin with 0.025 N hydrochloric acid as the eluent.

The product with ni + P 2 = 4 is hydrogenated on palladium on carbon catalyst. The non-basic hydrogenolysis product was separated over an acid ion exchanger and separated by preparative thin layer chromatography. Following acylation, the following two pseudotrisaccharides are identified by mass spectrometry as the main degradation products:

HO OH OH OH OH OH (as decaacetate with molecular peak at m / e = 906) a

МО

Higher members of the series that contain 4 to 8 glucose units with a molecular weight of 969 to 1617 cannot suppress sucrose so much.

TLC using n-butanol, ethanol and water in a ratio of 50:30:20 provided on Kieselgel Plate F 1500 R value of _{gluc In:}

(as nonaacetate with molecular peak at m / e = 848).

Thus, the largely predominant major component of component 5 is the isomer corresponding to formula VI with a glucosidically linked maltose unit attached to the cyclitol moiety.

This structure is also the result of degradation with β-amylase in which the major part of the component with ni + P 2 = 4 degrades to the compound of formula III and maltose.

The component with ni + nz = 5 behaves as follows when degrading with β-amylase:

Approximately 90% degrades to the compound of formula V and the corresponding amount to maltose, suggesting that the isomeric maltose unit linked glycosidically to the cyclitol residue is the major component of the product; about 10% is not degraded by β-amylase.

0.30 to 0.34

0.21 to 0.23

0.14 to 0.16

0.09 to 0.11 glucose units, glucose units, glucose units and glucose units.

As with the lower members of this series, it provides total acid hydrolysis, "component I" and glucose, and mutually different molar ratios, namely 1: 4, 1: 5, 1: 6, 1: 7 and 1: 8 (with the percentage glucose content being 74.4%, 79.7%,

83.3%, 86.5% and 89.1%).

When thin layer chromatography using silica gel plates 1500 F with n-butanol, ethanol and water in a ratio of 45:35: 20, the solvent is observed in triplicate developing these _Rf values:

Ratio Rf Glucose: Component I

glucose 0.77 maltose 0.65 maltotriosis 0.51 maltotetraosis 0.39 maltopentaosis 0.27 That made the score 4 - 1 0.25 maltohexaosis 0.21 It was 5 - 1 at the break 0.18 maltoheptaosis 0.15 6: 1 0.13 maltooctaosis 0.11 7: 1 0.09 8: 1 0.07

The catalytic hydrogenation described above also shows that the individual, but not all, glucose units of these higher members are in the 4-position of the cyclohexene group.

Since these compounds contain linear oligoglucoside chains having a 1-4 bond, they can be used as substrates for certain enzymatic degradation of carbohydrates. Obviously, the range of enzymes is limited to those enzymes that are not substantially suppressed by these compounds.

Bacterial α-amylases and fungal α-amylases can be used to break down an oligoglucoside chain that contains 3 or more glucoside units to low molecular weight compounds and inert carbohydrate fragments such as maltose and maltotriose. This degradation method is applicable to compounds with ш + иг? 3 and the concept is further evidence for α, 1-4 binding. Compounds of the invention with 4 to 7 glucoside units are partially degradable by β-amylase. Since β-amylase cleaves maltose units from the irreducible end of the glucose chain with α, 1-4 linkages, careful analysis of the β-amylase degradation product provides valuable information about each of the oligoglucoside substituents bonded at the 4-position of the cyclohexene ring, especially according to its chain length and number. glucose 'in the chain bonded to bisdesoxyglucose, i.e. at the reducible end of the compound. However, compounds containing 4,5,6,7 or 8 glucose units cannot be completely degraded by β-amylase. The resistance of some of the compounds appears to be attributed to the structural conditions for β-amylase attack.

The results of β-amylase degradation can be summarized as follows:

197288 20 May Removal of the maltose unit 19 Dec Number of glucose units in the starting material Number of glucose units in the degradation product 4 4 0 2 1 5 5 0 3 : 1 6 6 0 4 1 2 2 7 7 0 5 1 3 2 8 8 0 6 1 4 2 2 3

Optionally, some of the starting material is recovered.

With regard to the β-arnylolytic degradation, it should be emphasized once again that β-amylase makes it possible to remove only maltose units and only from the non-reducible end of the oligosaccharides. It should be avoided that any theory leads to some limitations. Although the exact structure of these higher compounds is not fully elucidated, it must be concluded from the above observations that compounds with 4, 5, 6, 7 and 8 glucose units belong to derivatives of lower compounds of formula IIIa containing chains of 2, 3, 4, 5 and 6 glucose units linked to each other by α, 1-> 4 bonds, wherein the oligosaccharide chain in the ε-conformation is linked to the 4-position of the cyclohexene ring.

Methylation of compounds with ethyl iodide and sodium hydride in dimethyl sulfoxide, total hydrolysis, derivative preparation and finally gas chromatographic analysis provide

A 2,3,6-trimethylglucose derivative such that glucose units are necessarily 1 > 4 bound in an attached linear structure.

One of the two methylation products which, depending on the methylation compound, is present at different scales or is not present at all under certain circumstances, · is 2,3,4,

6-tetramethyl derivative. The occurrence of this derivative and its molar ratio to the trimethyl derivative is dependent on the substituents attached to the cyclohexene ring.

Animals and humans are known to ingest blood and carbohydrate-containing foods and beverages (such as cereal and potato starches, fruit, fruit juices, beer, chocolate) to increase their blood sugar levels due to rapid degradation of carbohydrates by glycoside hydrolases (e.g. salivary and pancreatic amylases, maltase, sucrose) proceed according to the following scheme:

amylase starches, optionally, glycogenes

maltase

--'----- ► maltose '----—> glucose sucrose sucrose ----—> glucose +

This hyperglycemia is particularly severe in diabetic patients and is manifested continuously. In obese individuals, hyperglycaemia due to food often causes particularly strong insulin secretion, which in turn leads to increased fat production and a reduction in its degradation. In conjunction with such an increase in the amount of sugar in the blood occurs. people with good metabolism and in obese people due to insulin secretion often lower their blood sugar. It is known that both hypoglycemia and the stomach present in the stomach promote the formation of gastric juice, which itself aids or promotes the development of gastric ulcer, gastric ulcer or duodenal inflammation.

It has now been discovered that. The amino-sugar derivatives of the invention greatly reduce food-induced hyperglycemia, hyperinsulinemia and hypoglycemia, and in particular also the food-reactive increase in blood sugar.

Furthermore, it is known that carbohydrates, particularly sucrose, are broken down in the oral cavity by the action of microorganisms, thereby promoting tooth decay.

The amino-sugar derivatives of the invention can be used to prevent or reduce such dental caries.

Amylase test in vitro

An amylase · inhibitor unit (1 AIE) is defined as the amount of inhibitor that suppresses two amylase units by 50 percent.

Amylase unit (AE) is the amount of enzyme that cleaves 1 equivalent of starch glucosidic bonds per minute under the test conditions below. The cleavage bond is determined colorimetrically as μνβΐ of the reducing sugars with dinitrosalicylic acid and reported using a maltose calibration curve as When performing the test, 0.1 ml of amylase solution (20 to 22 AE / ml) is mixed with 0 to 10 µg inhibitor or 0 to 20 µΐ of the test solution in 0.4 ml - 0.02 M sodium glycerophosphate buffer and 0.001 M calcium chloride CaCl 2 [pH 6.9] and equilibrated in a water bath at 35 ° C for about 10 to 20 minutes, then incubated for 5 minutes with 0.5 ml of 1% starch solution (soluble starch # 1252 from Merek Darmastadt) heated to 35 ° C at 35 ° C and then 1 ml of dinitrosalicylic acid reagent (according to P. Bernfeld in Colowick-Kaplan, Meth. Enzymol., Vol. - 1, p. 149) is added. The mixture was heated in a boiling water bath for 5 minutes, then cooled and 10 ml of distilled water were added. The extinction at 540 nm is measured against the corresponding determined blank value without amylase. For the evaluation, the effect of the amylase after the inhibitor was added is subtracted from the pre-constructed amylase calibration curve and the percent suppression of the amylase employed is calculated from this. Percent suppression is expressed as a function of μξ inhibitor quotient + *

AE + + 'where + is based on dry matter and ++ is in an uninhibited batch of the same series, the 50% suppression point is subtracted from the curve and recalculated to the AIE / mg inhibitor.

Sucrose assay in vitro

A sucrose inhibitor unit (1SIE) is defined as the amount of inhibitor that suppresses two sucrose units by 50%. The sucrose unit (SE) is the amount of enzyme that cleaves 1 μπιο · 1 sucrose into glucose and fructose per minute under the test conditions below. The number of μΐηοΐύ of glucose produced is determined quantitatively by the glucosoxidase reaction under conditions in which there is no further cleavage of sucrose by the action of sucrose. To carry out the test, 0,05 ml of sucrose ¹ 'solution, adjusted to 0,12 SE, is mixed with 0 to 20 µg inhibitor or 0 to 20 µΐ of the solution to be tested and supplemented with 0,1 M sodium maleate buffer [pH 6,0] to 0.1 ml. It is equilibrated at 35 ° C for 10 minutes and then 0.1 ml is added

A 0.05 M sucrose solution previously heated to 35 ° C in 0.1 M sodium maleate buffer (pH 6.0). Incubate for 20 minutes at 35 ° C and stop the sucrose reaction by adding 1 ml of glucosoxidase reagent 2 ² 'and incubate for an additional 30 minutes at 35 ° C. 1 ml of 50% sulfuric acid is then added and measured at 545 nm against the corresponding blank. For evaluation, the percentage suppression of the sucrose activity used is calculated and SIE / g and SIE / liter, respectively, are calculated from a 50 ·% glucose-suppression curve.

1) Soluble saccharase from the small intestine mucosa according to B. Borgstrom, A. Dahlquista, Acta Chem. Scand. 12, (1958), p. 1997, with 0.1 sodium maleate buffer [pH 6.0], diluted to the corresponding SE content.

2) Glucosoxidase reagent was prepared by dissolving 2 mg of glucosoxidase (Boehringer grade I) in 100 ml 0.565 M Tris-HCl buffer [pH 7.0] and then adding 1 ml detergent solution (2 g tritone X 100 + 8) g 95% ethanol p.a.), 1 ml dianisidine solution (260 mg o-dianisidine dihydrochloride in 20 ml water) and 0.5 ml 0.1% aqueous peroxidase solution (Boehringer, lyophilisate, grade II).

Maltase test

A maltase inhibitor unit (1MIE) is defined as the amount of inhibitor that suppresses two maltase units by 50%. Maltase Unit (ME) is the amount of enzyme that cleaves 1 μΐηοΐ maltose to 2 μΐηοΐγ glucose per minute under the test conditions below. The number of μΐύηοΐύ of glucose formed is determined quantitatively by the glucosoxidase reaction under conditions in which maltose is no longer cleaved by maltase. To perform the test, mix 0,05 ml of ¹ 'maltase solution, adjusted to 0,060 to 0,070 ME, with 0 to 20 μ £ inhibitor - or 0 to 20 μ - of the solution to be tested and make up with 0,1 M sodium maleate buffer [pH 6,0 ] to 0.1 ml. Equilibrate for 10 minutes at 35 ° C and then add 0.1 ml of a 0.05 M maltose solution previously heated to 35 ° C in 0.1 M sodium maleate buffer (pH 6.0) and incubate for 20 minutes at 35 ° C and terminating the maltase reaction by adding 1 ml of glucosoxidase reagent ² 'and incubating for an additional 30 minutes at 35 ° C. Then 1 ml of 50% sulfuric acid is added and measured at 545 nm against the corresponding blank.

For evaluation, the percent suppression of the maltase treatment used is calculated and the MIE / g and MIE / liter, respectively, are calculated from the 50% suppression using the glucose calibration curve.

1) Soluble maltose from the small intestine of the pig according to B. - Borgstrom, A. Dahl197288 quista, Acta Chem. Scand. 12, (1958) p. 1997, with 0.1 M sodium maleate buffer [pH 6.0], diluted to the corresponding ME content.

2) Preparation of the glucosoxidase reagent: j as described in the sucrose assay in Note 2).

The results of an in vitro enzyme suppression test for the individual amino sugar derivatives of the invention are shown in Table 2.

Table 2

Formula Number of glucose units Suppression of AIE / g α-amylase action Suppression of SIE / g sucrose effect Suppression of MIE / g maltase activity II в 1 300 000 30 000 5 000 III в 2 300 000 68 000 15 000 in 3 1 400 000 21000 5 000 4 to 6 17 500 000 8 500 - 5 to 7 30 000 000 2 500 -

As can be seen from the table, the suppressing effect in in vitro experiments against pancreas-α-amylase with increasing molecular weight increases strongly; thus, compounds with 5 to 7 glucose units exhibit 100 times more potent enzyme suppression in vitro than compounds with 1 or 2 units. The suppression of the effect of sucrose is most potent in a two-unit derivative, the 1-glucose unit derivative is only half as strongly suppressed, and the higher member has a further decrease in the specific effect of suppressing the effect of sucrose.

In vivo, the efficacy of suppressing the action of sucrose is approximately parallel to that of a specifically observed suppressing effect. In contrast, it has surprisingly been found that in an in vivo starch loading test for compounds with 1, 2 or 3 glucose units compared to in vitro amylase suppression, a 10 to 40-fold potency is found.

To achieve diet-related hyperglycemia and hyperinsulinemia, groups each receive 6 un-fed rats:

(a) 2,5 g of sucrose

(b) 2,5 g of maltose; or

(c) 1 g of cooked starch per os in. aqueous solution or as a suspension. Six other rats receive said carbohydrates in equal amounts and additionally a glucoside hydrolase inhibitor at said dosage.

In addition, 6 additional rats receive the corresponding amount of salt solution.

Blood glucose and serum insulin are measured at short time intervals in blood from the retroorbital Venenplexus. Blood glucose determination is carried out in an autoanalyzer (Technicon (§) according to Hoffman: J. Biol. Chem. 120, 51 (1937) or enzymatically with glucosoxidase and o-dianisidine hydrochloride), serumin insulin determination according to the method of Hales and Randle [Biochem . J. 88: 137 (1963)].

The results of the in vivo inhibition of sucrose (sucrose processing) in fasted rats are shown in Table 3 below:

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Table 4

Formula

Glucose units

In vitro Inhibition of Sucrose Action (SIE / mg) (ED 50 mg / kg)

lib (1) 30 3.0 IIIb. (2) 68 0.21 in (3) 21 2.65 - (4 to 6) 8.5 -15,30 - (5 to 7) 2.5 -52.00

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From the above data, it is apparent that while the degree of suppression of the action of sucrose is demonstrated both in vitro and. also in vivo, as a distinct function of molecular weight, suppression of amylase activity in vitro depends on molecular weight, while suppression of starch degradation in vivo does not decrease with a decrease in molecular weight, but remains surprisingly constant.

Thus, although the effect of amylase suppression in vivo decreases with molecular weight, the ED 50 values of suppression of starch diet in the gastrointestinal tract are substantially the same for all compounds of the invention. These data are summarized in Table 6.

Table 6

Formula Glucose units Suppression of α-amylase action in vitro AIE / mg Suppression of starch intake in the gastrointestinal tract in vivo (ED 50 mg / kg) lib (1) 300 0.76 illb (2) 300 1.60 V, (3) 1 400 1.61 =. '· (4 to 6) 17 500 ’1.42 = - '- ·'. (5 to 7) 30000 1.00

• Surprise. it is also possible with compounds of formulas IIb, illb and V to simultaneously suppress the action of sucrose and starch eating in the gastrointestinal tract at a precisely predicted and characteristic dose.

In addition, the compounds have a beneficial effect with respect to glucose absorption. This is apparent from the data presented in Table 7 (hungry rat experiments) for the compound of formula illb (ni = 2).

Table 7

Dose

Blood glucose in mg% (mean + bp) minutes 30 minutes 45 minutes salt solution 72 + 4.6 glucose (control) 142 + 12 glucose-j-30 mg 135 + 13 glucose + 60 mg 12 µm: + ^ 1 ^ 3 *

78 + 1.3

142 + 12

128 + 5.5

118 + 2.9 **

87 + 6.8

158 + 19

132 + 7.3

130 + 9.0 * = Probability against glucose control group = 0.05 * = Probability against glucose control group = 0.01

Unexpectedly, purification of the mixture of higher members of this series and use in pure form results in even greater effect and specificity. This can be demonstrated by comparing the in vitro suppression of α-amylase and sucrose activity shown in Table 2 and that shown in Table 8 for pure compounds.

Number of glucose units

Table 8

Suppression of AiE / g 5-amylase treatment

Suppression of the action of sucrose SiE / g

4 67,000,000 7000 5 57,000,000 3500 6 42,000,000 1200 7 24 000 000 60 8 5 000 000 10

Previous results show a high effect of a compound with 4 glucose units as an α-amylase inhibitor. Also, compounds with 5 and 6 units have a significantly higher specific suppressing effect than has been found with the prior art formulations. The decrease in the suppressing effect occurs with an increase in molecular weight, as is evident in the 7 and 8 glucose unit compounds, although these compounds still have a substantial suppressing effect. In contrast, molecular weight dependence is evident when suppressing the effect of sucrose in vitro. A compound with 4 glucose units has about 1/10 of a specific effect as a compound with 2 glucose units, against which the suppressing effect of a compound with 8 glucose units is negligible. ·

The compounds may be used without dilution, for example, as a powder or in a gelatin shell, or in combination with a carrier in pharmaceutical compositions.

The pharmaceutical preparations may contain a greater or lesser amount of inhibitor, for example 0.1 to 99.5% in combination with a pharmaceutically acceptable non-toxic inert carrier, the carrier may comprise at least one solid, semi-solid or liquid diluent, filler and / or non-toxic inert pharmaceutically compatible formulation aid. Such pharmaceutical preparations are preferably in the form of dosage units, i.e., physically discrete units comprising determining the amount of inhibitor that corresponds to a fraction or 'multiple of the dose required to achieve the desired suppressing effect. Dosage units may contain 1, 2, - 3, 4 or more individual doses or 1/2, 1/3 or 1/4 of this unit dose. Preferably, the unit dose contains a sufficient amount of the active ingredient to achieve one or more dosage units of the desired suppressing effect when administered according to a predetermined dosage regimen, with the entire, half, third or quarter of the daily dose being administered usually once, twice, three or four times. per day. Other therapeutic agents may also be used. Although in any case careful consideration should be given to dosing and dosing regimens, using 'thorough' professional judgment and taking into account the age, weight and condition of the patient, the type and severity of the disease is generally within a range of about 30 to about 3. 105 AIE / kg and about 1 to about 1. 104 SIE / kg body weight per day. In many cases, a sufficient therapeutic effect is obtained with a smaller dose, while in other cases a larger dose is needed.

Oral administration is performed using solid and liquid dosage units such as powders, tablets, dragees, capsules, granules, suspensions, solutions and the like.

Powders are made by comminuting the substance to a suitable size and mixing with a comminuted pharmaceutical carrier. Although edible carbohydrates such as starches, lactose, sucrose or glucose are normally used for this purpose and can also be used here, it is desirable to use non-metabolizable carbohydrates such as cellulose derivatives.

Sweetening, flavoring, preservative, dispersing and coloring agents can also be used with them.

Capsules can be prepared by mixing the powder mixture described above and filling it into ready-made gelatin containers. A glidant such as diatomaceous earth, talc, magnesium stearate, calcium stearate or solid polyethylene glycols may be added to the powder mixture prior to filling. The composition may also contain disintegrants or co-solvents such as agar-agar, calcium carbonate or sodium carbonate to facilitate the availability of the inhibitor when using the capsule.

Tableting is accomplished, for example, by making a coarse or fine grain powder mixture and adding a glidant - and a disintegrant. Tablets are formed from this mixture. The powder mixture is obtained by mixing the substances which have been comminuted and suitably diluted with diluents or other carriers as described above. Optionally, a binder such as carboxymethylcellulose, alginate, gelatin or polyvinylpyrrolidone, a dissolution retarder such as paraffin, a resorption accelerator such as a quaternary salt and / or an adsorbent such as bentonite, kaolin or middle calcium phosphate are added. The powder blend may be granulated together with a binder such as syrup, starch grease, acacia grease, or solutions based on cellulosic or polymeric materials. The product is then passed through a coarse sieve. Alternatively, the powder mixture may be passed through a tabletting machine and the non-uniformly formed pieces obtained may be comminuted to a grain size. A lubricant such as, for example, stearic acid or a salt, talc or mineral oil may be added to prevent the resulting grains from sticking in the tabletting nozzle. These glidant compositions are then compressed into tablets. The active ingredient may also be combined with free-flowing inert carriers and directly used in the manufacture of tablets by omitting the granulation and grinding steps. The product may be provided with a clear or opalescent protective coating, such as a shellac, sugar or polymer coating, and the coating may be polished with a wax. Dyestuffs may be added to the coating to differentiate between different dosage units.

Forms of oral administration, such as solutions, syrups or elixirs, may be prepared in dosage units such that a specified amount of preparation contains a specified amount of active ingredient. A syrup may be prepared by dissolving the active ingredient in an aqueous solution containing a suitable flavoring agent, the elixirs being prepared using non-toxic alcoholic carriers. Suspensions may be dispersions of the compound in a non-toxic carrier. Co-solvents and emulsifiers such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitester, preservatives and flavor enhancers such as essential oil, carbohydrate and the like can also be added.

Dosage prescriptions - may also - be provided for capsules. In addition, the dosage can be provided in such a way that the active substance is administered in a delayed manner, for example when retention of the active substance in polymeric substances, waxes and the like.

The toxicity of these compounds is very low. Also without final purification, for example, the crude product of Example 8 with 26,000 SIE g is tolerated without side effects when 340,000 SIE / kg is administered orally in mice and rats. Also with intravenous injection, mice tolerate 10,000 SIE / kg without side effects.

Pharmaceutical. .přípravky invention may contain '(Ake. Other pharmaceutically ^Prior: variables compounds, particularly other oral antidiabetic agents, such as β-cytotropní sulfonylureas and biguanides lowering blood sugar.

In addition to the aforementioned pharmaceutical preparations, foodstuffs containing these active substances, such as sugar, bread, potato products, fruit juices, beer, chocolate and other types of confectionery and canned foodstuffs, can also be produced. such as marmalade, wherein therapeutically effective amounts of at least one inhibitor of the invention are added to these products.

Lighting of some used reagents and excipients

The following may be used as ion exchangers. commercially available products:

Amber lit IRA 410 Cl ~ (anion exchanger), Amberlit (ЫСОзЧогта) (anion exchanger,

Amberlite IRC 120 (H + form), (strongly acid cation exchanger),

Amberlit IRA 410 OH ^- (strong basic anion exchange resin), Amberlit IRC 50 H + (weakly acidic cation exchanger) (manufactured by Rohm and Haas' Company), Dowex 50 WX 4H + (strongly acidic cation exchanger) (branded by Dow Chemical Co. Midland , Michigan, USA).

As the cellulose anion exchanger, for example, EAE-cellulose from Schleicher and Schull can be used.

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

Malcin is a natural malt extract of the form of Diamalt AG, Munich (Germany).

Levapol (g) is a non-specific adsorption resin from Bayer AG, Leverkusen (Germany).

Klarcel is auxiliary. filter medium from CECA, Velizy-Viilacoublay (France).

SE 50 is a silicone elastomer from Hewlett Packard.

The micro-organisms used are stored in the American Culture Types Collection (ATCC) under the following numbers:

ATCC strain —- Nr.

SE 50 (CBS 961.70) 31042

SE 18 (CBS 957.70) 31041

SE 82 (CBS 615.71) 31045

SE 50/13 (CBS 614.71)

SE 50/110 (CBS 674.73) 31044

Example 1

A glass fermenter with 8 'liters of nutrient solution containing 5.0% starch, 1% yeast extract, 0.2% K2HPO4 is inoculated with a shake flask with strain SE 50/13 (CBS 614.-71) for 3 days and incubated with vigorous stirring and aeration for 3 days ^: at 28 degrees Celsius to obtain a culture broth with 105,000 AIE / ml.

liters of this fermentation broth, after cooling to 20 [deg.] C., are adjusted to pH 2.5 with semi-concentrated nitric acid, 30 g of carborafine activated carbon are added and stirred for 10 minutes. It is then centrifuged at 10,000 rpm for 15 minutes and the clear pale yellow residue is neutralized with ammonia to 500 ml. 500 ml of the concentrate was stirred with 200 g of anion exchange resin Amberlit IRA 410 'Cl for 45 minutes, separated on a. and add 4/5 of the volume, i.e. 400 milliliters of methanol, to precipitate the major part of the high molecular weight starch degradation product (along with the remaining activated carbon still present). It is then centrifuged at 5000 rpm for 5 minutes. 850 ml of the residue are added dropwise with vigorous stirring to 4 'liters of dried ethanol. The white flaky precipitate was collected on suction, washed three times with dried ethanol and twice with ether, and dried under vacuum at 50 ° C. Yield: 36 g of a white powder with 10.10 ⁶ AIE / g. In some other examples, the substance is used.

Suppression of enzyme action on thin-layer plates

To assess the final product and the composition of the preparation by thin-layer chromatography, 1 μΐ of fermentation broth or 1 µg of the preparation is applied to the finished silica gel plates (Schleicher und Schüll, Dassel; type F 1500) and developed twice with n-butanol, ethanolO1 and water at a ratio of 50: 30: 20.

For coloring to suppress the sucrose action, developed and well dried enzyme plates (20 ml per 20 x 20 cm plate) were sprayed and the gel allowed to solidify. It is then incubated for 5 minutes in a humid chamber at room temperature and finally sprayed with a substrate gel. After this second gel layer has solidified, the plates are placed in a humid chamber and incubated at 40 ° C. The inhibitor stained (light spots, red-brown background) developed after 60 to 90 minutes. . At the moment of optimum color development, the process is interrupted and the agar-coated plates on them are dried by flowing hot air.

Preparation of gels

Enzymgel: 1.5 g agarose (L‘Industrie Biologique Francais) is suspended in 100 ml

0.2 M sodium maleate buffer (pH 6.0) and then dissolved by boiling. Cool the clear agarose solution to 50 ° C and add 250 μϊ of Triton X-100 (2 g Triton X 100 -) - 8 g ethanol pa) and 0,5 ml dianisidine solution (20 mg dianisidine in 1 ml acetone), Immediately before using the gel, 1 ml of GOD / POD reagent (12.5 mg glucose oxidase, Boehringer grade I) and 2.5 mg peroxidase, grade II, Boehringer lyophilisate dissolved in 5 ml maleate buffer are added. ) and 4 to 5 units of small intestine sucrose: The gel must be maintained at 50 ° C until spraying, otherwise it will solidify in the nozzle during spraying.

Substrate gel: 0.5 g of agarose is suspended in 100 ml sodium maleate buffer (pH 6.0) and dissolved at boiling. It is then cooled to 50 ° C, 100 µl of Triton (2 g of Triton X-100-1-8 g of ethanol p.a.) are added and 1 gram of sucrose is added (Serva No. 35,579). After dissolving sucrose, the gel is ready for use.

To color the acylase inhibition, the developed and dried thin plates for amylase-gel chromatography (20 ml per 20 X X 20 cm plate) are sprayed and allowed to solidify. After 5 minutes of incubation at room temperature, a layer of 0.5% starch solution (1 g Merek No. 1252 starch, in 200 ml of 0.2 M glycerophosphate buffer 0.01 M CaCl2 (pH 6.9), dissolved) is applied to the plates. boiling) and left there for 2 minutes at 40 ° C with shaking. The plates are then rinsed well with distilled water and wetted with dilute iodine solution (4 ml iodine base solution to 500 ml water, iodine base solution: 2.2 g iodine | -4.4 g potassium iodide, dissolved in undiluted starches). 100 ml of water). After about 1 minute the color is optimal. They take pictures immediately, because the blue spots quickly fade.

Preparation of amylose gel g agarose is dissolved in 100 ml of buffer 0.2 M natriumglycerofosfátu 0.01M calcium chloride (pH 6.9) at 100 ° C and, after cooling to 50 ° C was added 100 _(l Triton X-100 (2 g Triton X-100- | -8 g ethanol pa) 100 µΐ of a suspension of crystalline amylase (10 µg of pancreatic amylase per ml of saturated ammonium sulphate solution, supplied by Boehringer) is added immediately before spraying.

Example 2

If a 1 liter Erlenmeyer flask is inoculated with 120 ml of nutrient solution containing 4% starch, 2,4% glucose, 0,9% casein hydrolyzate and 0,9% yeast extract, adjust the pH to 7,6 with sodium hydroxide, add 0,4% of calcium carbonate and sterilized for 30 minutes at 121 ° C, with 3 ml of the initial culture of strain SE 82 (CBS 615.71) growing in a nutrient solution consisting of 2% starch, 1% glucose, 0.5% casein hydrolyzate and 1% yeast extract, pH adjusted to 7.2 with NaOH, added 0.4% calcium carbonate, sterilized at 121 ° C for 30 minutes and incubated for 5 days at. 28 ° C on a bender shaker, to obtain a culture solution of 122,000 AIE / ml. During processing, the mycelium is separated from the combined culture solutions by centrifugation at 12,000 rpm. 300 ml of the culture filtrate are adjusted to pH 2.5 with partially concentrated nitric acid and stirred for 10 minutes with 2.4 g of activated carbon. by centrifugation at 12,000 rpm. 300 ml of methanol are added to the solution neutralized to pH 6 with 10N potassium hydroxide, allowed to stand briefly at 12 000 rpm. removes the precipitate. Now, the residue is added dropwise to 3 liters of ethanol and, after standing for a short time, the precipitate is collected by centrifugation at 12,000 rpm. The precipitate was washed twice with absolute ethanol and once with ether and dried in vacuo. This gave 2.23 g of a product with 7.45 * ¹⁰ &lt; ⁶ &gt; AIE / g, which contained 95% of compounds having a n-n = 4 or more glucose units.

Example 3

When a 1 liter Erlenmeyer flask is inoculated with a 120 ml nutrient solution containing 3.5% glucose, 2% starch, 0.5% casein hydrolyzate, 1.3% yeast extract, 0.3% calcium carbonate and 0.3% medium potassium phosphate, prior to sterilization, adjusted to pH 7.8, sterilized for 30 minutes at 121 ° C, with 6 ml of SE 50/110 strain in a nutrient solution containing 3% soy flour, 3% glycerin and 0.2% calcium carbonate, and Incubate for 3-4 days on a rotary shaker at 24 ° C to obtain a culture solution containing 153,000 AIE / ml and 12,200 SIE / liter.

liter of culture solution is adjusted to pH 2.5 with nitric acid, stirred for 10 minutes with 5 g of activated carbon and then centrifuged at 5000 rpm for 30 minutes. Finally neutralized by adding 25 g · l ^ A ^ i ^ rlitu IRA 410 (OH ^_ form). The neutral residue is adjusted to 100 ml in a rotary instrument, 100 ml of methanol are added and filtered. The filtrate was stirred in 2 L of absolute ethanol, the precipitate was removed to dryness and washed three times with acetone / ether and dried in vacuo.

Yield: 14 g of white powder with 5.10 ⁶ AIE / g; predominantly compounds with at least ni + rn = 4 glucose units.

Example 4

When working according to Example 3 but with 0.5% starch, a culture broth of 40,000 AIE and 184 SIE / ml is obtained after a four-day fermentation. The culture broth comprises a mixture of compounds of the invention having at least one glucose unit.

Example 5

If a 1 liter Erlenmeyer flask is inoculated, containing 120 ml of nutrient solution consisting of 3% glucose 197288, 0,6% casein hydrolyzate, 1,6% yeast extract, 0,3% calcium carbonate, 0,3% medium potassium phosphate, The pH is adjusted to 7.8 with potassium hydroxide prior to sterilization, with an initial culture of SE 50/110 (CBS 674.73) according to Example 3 and incubated for 4 days at 24 ° C on a bender shaker to obtain a culture broth of 10,800 SIE / liter. containing predominantly a compound of the invention with a single glucose unit.

liters of the culture filtrate are separated from the mycelium at 13,000 rpm, adjusted to pH 2.5 with half-concentrated nitric acid and stirred with 55 g of activated carbon and 200 g of clarcelium for 15 minutes. After removal of the solid by suction, it is neutralized with concentrated ammonia to pH 7, the solution is evaporated to 1.5 liters and precipitated with a 5-fold amount of ethanol.

The flocculent precipitate obtained is collected on a continuous rotor centrifuge at 12,000 rpm. and evaporate the yellowish residue to 150 ml and centrifuge on a slow-running centrifuge to separate a small portion of insoluble material. 50 ml of this solution is loaded onto a strongly acidic cation exchanger Amberlite IR-120 (H ⁺ form) packed column (30 XX 300 mm; 30 ml water per hour). After collecting a total of 300 ml of eluate containing inert carbohydrates as well as a proportion of unadsorbed inhibitory active ingredients, the cation exchanger with about 400 ml of water is transferred to a beaker and concentrated ammonia is added with stirring until the pH is adjusted to 11.5. After 30 minutes of further stirring, the cation exchanger was removed, evaporated to 1/20 volume, filtered through a column (20 X 150 mm) of an Amberlit IRA-410 ion exchanger (НСОз ^- form) and collected at a flow rate of 30 ml / h about 500 ml eluate. which was evaporated to give 1.3 g of crude product after lyophilization.

For further purification, the crude preparation was fractionated on Bio-Gel P-2, 100-200 mesh polyacrylamide gel (Bi-Rad, Munich). A 50 mm diameter and 450 mm length column is used to treat the water at a flow rate of 40 ml / h, collecting 10 ml fractions each. All fractions were assayed by anthrone carbohydrate assay as well as a sucrose inhibition assay on inhibitory active ingredients. The sucrase inhibitor-containing fractions were further tested by thin layer chromatography according to Example 1 for their individual components. The fractions containing the compound with 1 glucose unit were pooled, evaporated and lyophilized. 35 mg of a substance with 0.3.10 ⁶ AIE / g and 30,000 SIE / g are obtained.

Example 6

If a 1 liter Erlenmeyer flask is inoculated with 120 ml of a nutrient solution containing 5% starch, 1% yeast extract, 0.2% medium potassium phosphate each with 2 ml of the starting culture according to Example 3, a solution is obtained after a three-day incubation at 28 ° C. cultures with amylase inhibitor yield:

strain AIE / ml

SE 50 (CBS 861.70) 37,000

SE 50/13 (CBS 614.71) 109,000

SE 50/110 (CBS 674.73) 53 500 which is predominantly a mixture of compounds with 4 or more glucose units.

Example 1 a d 7

If a 1 liter Erlenmeyr flask is inoculated with 120 ml of nutrient solution containing 1,3% maltose, 3,5% glucose, 0,5% casein hydrolyzate,

1.3% yeast extract, 0.3% calcium carbonate and 0.3% medium potassium phosphate, each with 2 ml of the starting culture according to Example 3, the following yields are obtained after a four-day incubation on a bending shaker at 24 ° C with different strains:

strain SIE / ml AIE / ml

SE 50 (CBS 961.70) 25580

SE 50/13 (CBS 614.71) 14.81460

SE 50/110 (CBS 674.73) 57,9755 which is predominantly a mixture of compounds with 4 or less glucose units.

Example 1 a d 8

When seeded in a fermenter with 100 liters of nutrient solution containing 3.5% glucose, 2.5% malt extract maleate, 0.5% casein hydrolyzate, 1.3% yeast extract, 0.3% calcium carbonate, 0.3% medium potassium phosphate and 0.1% antifoam, with 5 liters of the culture starting material of Example 3 and incubated with stirring at aeration for 5 days at 24 ° C to obtain a culture solution of 73,000 SIE / liter containing mainly compound with ni + P 2 = 2.

liters of fermentation batch with mycelium are adjusted to pH 2.5 with concentrated nitric acid while monitoring with a pH meter and 900 g (= 1%) of activated carbon are added under stirring to adsorb the major portion of the resulting colorants. Stir for 15 minutes, then separate on a centrifuge at 3000 rpm. from the mycelium and the major part of the coal, and the residue is then filtered with the addition of 3 kg of clarcel through pressure suction. 61 liters of a light brown clear filtrate were obtained, containing 60,000 SIE / l.

The filtrate is adjusted to pH 7 with concentrated ammonia and the active ingredient is mixed with 1300 g (2%) of activated carbon for 30 minutes for adsorption. It is filtered through a suction filter and the activated carbon sediment is well compressed to dryness and stirred three times with 4 liters of 50% acetone at pH 2.5 for 15 minutes each time in order to desorb the active ingredient from the coal. Acetone desorbates were combined by filtration to separate the coal. The combined desorbates were evaporated on a rotary evaporator to 250 ml, an equal (250 ml) volume of methanol was added and filtered through a pleated filter. The filtrate (480 ml) was added dropwise to 5 liters of acetone with vigorous stirring. The precipitate was collected on suction and washed three times with acetone and ether. It is then dried under vacuum at. Deň: 32 ° C. Yield: 230 · g with 8500 SIE / g.

g of the above crude product is dissolved in 1 liter of water and stirred with 300 g of strongly acidic cation exchanger Dowex ^R 50WX 4 H + (200 to 400 mesh) for 30 minutes. The resin was filtered off and washed three times with 2 liters of 0.001 N hydrochloric acid. The washed Dowex cation exchanger is then suspended in 500 ml of water and adjusted to pH 9.0 by addition of 25% ammonia while monitoring with a pH meter. Finally, desorbate twice with 500 ml of 0.6% ammonia, combine the desorbates and evaporate to 100 ml on a rotary evaporator. To decolour this concentrate, 2 g of DEAE-cellulose anion exchanger (Schleicher und Schull, No. 02035, 0.6 mval / g) is stirred for 5 minutes and then centrifuged. The light yellow residue was added to an equal volume (100 mL) of methanol and then dropped into 2 L of acetone with vigorous stirring. The precipitate is suction filtered, washed with acetone and ether and dried under vacuum at 35 ° C. Yield 4.2 g with 26,000 SIE / g.

For proper purification, 4.0 g inhibitor was gel filtered in 0.5 g aliquots through a Bio-Gel P-2 polyacrylamide gel. 0.5 g of the preparation is then dissolved in 10 ml of water and applied to a column of Bio-Gel P-2 (200-400 mesh, Bio-Rad) of 5 cm diameter and 95 cm length. Develop with water at a flow rate of 80 ml / h. 12 ml fractions were collected. The total carbohydrate content (in the form of anthrone assay, as an extension at E6O0) as well as the content of sucrose inhibitor and amylase inhibitor were examined in all fractions. In addition, fractions were tested by thin layer chromatography (enzyme inhibitor staining according to Example 1).

Fractions containing 4-6 glucose units were combined, evaporated to 10 ml in vacuo and precipitated by dropwise addition to 200 ml dry ethanol. The precipitate was collected by centrifugation, washed with acetone and ether, and dried in vacuo. Yield from 4.0 g crude inhibitor: 0.2 g of 4-6 glucose units with 17.5 x ^{10 6} AIE / g and 8500 SIE / g.

Fractions containing the 3-unit compound were treated in the same manner, with precipitation of 200 ml of acetone. Yield from 4.0 g crude inhibitor: 0.1 g of compound with n1-n2 = 3, with 1.4 x 10 6 AIE / g and 21,000 SIE / g.

It is isolated from the fractions containing the compound with n1 + .02 = 2 (precipitated with acetone)

0.9 g of compound with m + n2 = 2 units with 0.3.106 AIE / g and 68,000 SIE / g.

Example 1: 9

If 3 small fermenters are inoculated with 8 liters of nutrient solution each

7.5% malt extract maleate, 0.3% casein hydrolyzate, 0.7% yeast extract, 0.3% calcium carbonate, 0.3% medium potassium phosphate, with 5% of the initial culture of strain SE 50/110 (CBS 674.73) ). (obtained according to Example 3), a culture broth of 73 SIE / ml is obtained after a five-day incubation at 24 ° C, which predominantly contains a compound with ni-J-nz = 2. After centrifugation [30 minutes, 3000 rpm] after separation of the mycelium, 20.5 liters of dark brown culture solution are obtained with 67,000 SIE / liter. It is adjusted to pH 3.5 with nitric acid and 60 g of Lewapol adsorption resin (Ca 9221, 0.35 mm grain, Bayer) per liter, i.e. 1.23 kg of Lewapol, are added to decolorize. After stirring for 20 minutes, it was collected on a suction filter (Seitz K3). The decolorized culture solution was neutralized with ammonia (18.5 L, 67,000 SIE / L). Finally . 20 g / liter of activated carbon, i.e. 370 g, is stirred for 20 minutes for adsorption of the active compound. It is then filtered through a filter (Seitz K3), which is coated with a clarifier filter aid. The filtrate (17.5 L, 3600 SIE / L) is discarded. The residue on carbon was washed three times with 2 liters of distilled water. For desorption of the active substance from coal, three times. with 1 liter of 80% acetone. 15 minutes, adjusting the pH to 2.5 with concentrated hydrochloric acid. The desorbates were combined (2.4 L, 371,000 SIE / L). 20 g / liter of Dowex H + cation exchanger (Dowex 50 WX 4, H + form from Serva, Heidelberg), i.e. 46 g of Dowex, is introduced into the desorbate and stirred for 20 minutes. The resin was then filtered off (Dowex fraction I) and washed with about 75% acetone. The filtrate and washings (3 l = 215,000 SIU / liter) was stirred with 60 g / liter of a strongly basic anion exchange resin Amberlite IRA 410 (OH ^_ -form, Serva, Heidelberg) and adjusted to pH 7. It was then filtered and the filtrate ( 2.8 liters, 219,000 SIE / liter) add 72 grams of Dowex H + cation exchanger and stir for 20 minutes. The pH 3.0 is maintained by hanging a porous nylon bag filled with Amberlit IRA 410 OH- anion exchange resin. It is then filtered from Dowex (Dowex Fraction II) and the filtrate (2.6 L, 27,000 SIE / L) discarded.

The dowex fractions I and II are washed separately three times each with 75% acetone at pH 3.5 and then desorbed three times with 100 ml each (dowex fraction I) or 150 ml (dowex fraction II) of 0.6% ammonia. In the first desorption, in which the amount of · ammonia to neutralize the resin · Dowex is adjusted by adding concentrated. ammonia pH to 9 when monitored by pH meter. The 3 dowex fractions I and II are combined, evaporated to near dryness on a rotary evaporator, taken up in 50 ml of water, adjusted to pH 3-4 with hydrochloric acid and added with 50 ml of methanol. The solution is dropped into 1.5 L of absolute acetone with stirring, the precipitate is suction filtered and washed three times with acetone as well as once with ether and dried under vacuum.

Yield:

fraction I 6.5 g 25,000 SIE / g fraction II 12.3 g 66,000 SIE / g Yield Volume SIEy / lttr (liter) 1) Culture solution 20.5 67 000 2) After discoloration Lewapol 19.5 67 000 3] After adsorption to activated carbon 18.5 3 600 4) 1. desorbate 0.7 742 000 5) 2. desorbate 0.9 329 000 6) 3. desorbate 0.8 85 000 7). Mixed desor- worry (4—6) 2.4 371 000 8) After the 1st dowex adsorption 3.0 215 000 9) After neutralization with IRA OH ' 2.8 219 000 10) After the 2nd dowex adsorption 2.5 27 000 11) United NI-I3 de- sorbates of dowe- X fractions 0.29 682 000 12) connected NH3 de- sorbates of dowe- X fractions II 0.45 1 419 000 13] Precipitation fraction I 6,5 g 25,000 / g Precipitation, fraction II 12,3 g 36,000 / g

Example 10

When 2 g of the preparation as described in Example 1 are incubated in 60 ml of 20 mM sodium glycerophosphate buffer [pH 6.9] and 1 mM calcium chloride with 1 g of α-amylase from Aspergillus spec. (Servos No. 13,418) with continuous stirring at 37 ° C for 120 hours, then heated at 100 ° C for 5 minutes and centrifuged at 4000 rpm. from the insoluble fraction, 1.9 g of product with 3500 SIE / g and 2.106 AIE / g are obtained after lyophilization of the solution. When tested by TLC and sucrase inhibitor staining as described in Example 1, it appears that the compounds with suppressive activity are essentially compounds of the invention having n-j = 1, 2 and 3 .

Example 11

When 2 g of the formulation as described in Example 1 are inhibited in 30 ml of 20 mM acetate buffer [pH 4.8] with 1.25 mg / Boehringer bathylalase under constant agitation Fractions I and II contain as suppressant component mainly a compound of the invention with ni-j-n = 2, in addition to a small proportion of the compound with ni-j-β 2 = 3.

The following table shows the sucrose-induced suppression in successive preparations:

Total SIE SIE '% yield 1,373,500 100 ALIGN! 1,306,500 95 66 600 (4.8 postponed) 519 400) 37.8] AND 296 100} 883 500 21.6 64.4 68,000 J 5,0] 890 400 64.8 645 000 613 200 67 500 (4.9 postponed) 197 780 14.4 ’ 60.9 638 550 46,5 ' 162 500 11,8] 1 and (· 44.0 442 800 32,2 J

It is allowed to stand at 37 ° C for 120 hours, heated to 100 ° C for 5 minutes and centrifuged at 4000 rpm. thus, after freeze-drying a solution of 1.5 g of product with 1800 SIE / g and 3.8.10 ⁶ AIE / g is obtained. When this product is tested by thin layer chromatography and the coloring of a sucrose inhibitor as described in Example 1, it turns out that the suppressing compounds are essentially compounds of the invention with n 5 = 2 and 3.

Example 12

If a 200 ml Erlenmeyer flask is inoculated with 25 ml of nutrient solution containing 0,1% potassium potassium phosphate, 0,2% ammonium sulphate, 0,05% magnesium sulphate, 0,05% potassium chloride,

0.01% ferrous sulfate, 2% of the preparation as described in Example 1, with an Asp spore suspension. niger ATCC 11 3-4 and incubates at 28 ° C with the baking shaker, so that after 6 days, the Al 2 O - 2 - liter decreases from 210 300 AIE / ml to 53 000 AIE / ml and after 10 days. days on

21300 AIE / ml. At the same time, the SIE / ml content increased from 7.0 to 72 SIE / ml.

ml of the solution incubated for 10 days with the spore suspension is centrifuged to separate the mycelium at 3000 rpm for 30 minutes. 15 ml of the residue (72,000 SIE / liter) is desalted by stirring for 30 minutes with 2 g of Amberlit IRC 50 H ⁺ cation exchanger and 1 g of Amberlit IRA 410 OH anion exchanger (conductivity less than 2 mS.cm ¹ ). The ion exchangers are filtered off and the filtrate is allowed to flow at a rate of 5 ml / h through a Dowex H ⁺ cation exchanger in a column (diameter 1 cm, length 10 cm) equilibrated with 0.001 N hydrochloric acid. It is then washed with 200 ml of 0.001 N hydrochloric acid. For desorption, 10 ml / h of a 0.6% ammonia solution was pumped through the column and 5 ml fractions were collected. The fractions showing sucrose suppression activity were pooled, rotovapped to 2 ml and methanol (2 ml) was added. The solution is adjusted to pH 3-4 and precipitated by the dropwise addition of 100 ml of acetone. The precipitate was collected on suction, washed with acetone and ether and dried in vacuo. The yield is 26 mg with 28,000 SIE / g, where the substance consists of compounds with ni + P 2 = 2 and 3. The pure compound with ni + P 2 = 2 is obtained from this product as described in Example 8 by gel filtration column of Bio-Gel P-2. 7 mg of the compound with ni + пг = 2 of 60,000 SIE / g are obtained.

Example 13 liters of culture filtrate obtained from the fermentation broth as described in Example 5 by removing mycelium at 13,000 rpm. and having an activity of 13,000 SIE / g is mixed to reduce the salt content (conductivity of the culture filtrate about 10 mS.cnr ¹ ) with 500 g of an ion exchange mixture of 2.5 parts Amberlite IRC-50 (HMorma) and 1 part Amberlite IRA-410 (OH-form) 1 hour. The ion exchangers are separated, the solution is evaporated to a somewhat less than 100 ml volume and to remove insoluble matter at 20,000 rpm. spin for 15 minutes. The residue was made up to 100 ml (conductivity has nearly 3.5 mS.cnr ¹⁾ P-cellulose (Serva no. 45130, prepared according to known methods and in 5 mM ammonium phosphate buffer [pH 5.5] equilibrated). The buffer is the eluent. The flow rate is 90 ml / h and 18 ml fractions are collected.

The eluate fractions are then assayed for their carbohydrate content [using the anthrone assay] and sucrose inhibiting agents (using the sucrose inhibition assay). Combine the fractions which according to the anthrone test, nearly free of carbohydrates, while also in the test of inhibition of sucrase specific activity (fractions 60 to 170), evaporated to 150 ml and filtered through a column (50 XX 300 mm) anion exchange resin Amberlite IRA-410 (HCO 3 ^_ -form). For better control of desionization, the eluate is collected in fractions (10 ml fraction in 20 minutes) and tested for carbohydrate (using anthrone test: still almost negative), phosphate (using ascorbic-molybdate: completely negative) reagent and suppressing the effect of sucrose (by enzyme inhibition assay). The suppressive fractions (3 to 30) were pooled, evaporated, lyophilized, redissolved and lyophilized. 280 mg of crude inhibitor are obtained.

Further purification of the crude inhibitor on Bio-Gel P-2 is performed as described in Example 6 by fractionation. From the fractions containing the pure compound with n 2 P 2 = 1, 30 mg of the product with 0.3 is isolated after lyophilization. .10 ⁶ AIE / g and 35 000 SIE / g.

Example 1 a d 1 4

For example, to obtain aminocyclic derivatives with 5 to 7 glucose units, starting from a preparation as described in Example 1.

g of the preparation of Example 1 is dissolved in 250 ml of water. The conductivity of this solution is 10 mS.cm ⁴ and pH 5.5. For desalting, 60 g of a slightly acidic cation exchanger Amberlite IRC 50 H + (cation exchanger, which binds the amino-sugar derivatives only in traces from an aqueous solution) and 20 g of anion exchanger Amberlit IRA 410 OH are added to the solution and stirred for 20 minutes. The filtrate (δ conductivity 0.5 mS.cm ¹ , pH 3.5) is adjusted to pH 3.0 (conductivity 0.6 mScm ¹ ) with 1 N hydrochloric acid. This solution was pumped at a rate of 42 ml / h with a Dowex 50 W (H ⁺ form) cation exchange column packed in a 2.5 cm diameter and 40 cm height column (equilibrated in 0.001 N hydrochloric acid and finally washed with 2 liters of 0.001 N hydrochloric acid). After washing the column, eluting with 1.2% aqueous ammonia and collecting 10 ml fractions, the suppressing fractions were combined, the ammonia was drawn off in vacuo and the solution was then evaporated to 30 ml in vacuo. The solid was collected by suction filtration and dried in vacuo after washing with ethanol and ether to yield 4.4 g with 26.5 * ¹⁰ &lt; ⁵ &gt; AIE / g.

0.5 g of the substance was applied to a Bio-Gel P-2 preparative column as described in Example 8 and developed for gentle purification. Fractions containing by thin layer chromatography (stained with an amylase inhibitor) of compound with n1 + 112 = 5-7 were combined, evaporated in vacuo and precipitated with dry ethanol as described above. The yield from 0.5 g of crude product is 0.2 g of the aminosugar derivative with ni + 112 = 5-7, with 30 x ^{10 6} AIE / g and 2500 SIE / g.

Example 1 a d 1 5

This example shows how compounds can be eluted from cation exchangers under acidic conditions.

A 1.5 cm diameter column was packed with 30 g (wet weight) Dowex ^R 50 WXX 4 (H ⁺ form) cation exchanger, 200-400 mesh in 0.001 N hydrochloric acid. 500 milliliters of mixed desorbate (400,000 SIE / liter, pH 2.5, 60% acetone) is then pumped through the column obtained for Example 9 (Table, Run 7) for about 1 hour and then washed with 500 ml of 0.001 N acid. hydrochloric acid. Under these conditions, the compound elutes only with traces of activity. It is then desorbed with 0.0125 N hydrochloric acid, the conductivity or refractive index of the eluate exiting the column being registered. In addition, the eluate is tested for SIE content. The active fractions 74 to 100 are combined and, after addition of the Amberlit IRA 410 OH enex, neutralized, then evaporated to 5 ml, treated with 5 ml of methanol and precipitated by addition to 200 ml of acetone. After washing with acetone and ether, it is dried under vacuum.

The yield was 1 g of compound with n1 + P2 = 2 with 65,000 SIE / g.

Compounds with ni-112-3 and 4 glucose units can be obtained from the active front fractions.

This method of acid desorption also allows, on the contrary, to be alkaline. desorption fractionation to obtain individual annocarbons of this series.

When the preparation with 4-8 glucose units as described above is subjected to this procedure, while the neutralized eluate is lyophilized, the following single larger fractions are obtained:

ni + nn = 4 = 67,000 AIE / mg ni + P2 = 5 = 57,000 AIE / mg ni -j- · nn = 6 = 42,000 AIE / mg ni -j- P2 = 7 = 24,000 AIE / mg ni + nn = 8 = 5,000 AIE / injg

Example 16

The β-amylase degradation method described above is carried out as follows:

100 mg of the compound was dissolved in 1.9 ml of 20 mM sodium acetate buffer (pH 4.75) and 0.1 ml of β-butylase from Butate (Boehringer, 5 mg / ml, 500 U / mg) was added. The mixture is kept at 37 ° C for 48 hours and heated to 100 ° C for 5 minutes and finally centrifuged at 4500 rpm to remove the precipitated protein and other impurities. The whole mixture was then loaded onto a Bio-Gel P-2 column (22 mm diameter, 100 cm length, thermostat maintained at 65 ° C) and eluted with water at a flow rate of 25 rpm / h. The eluate is passed through a conductometer and a highly sensitive reflectometer with flow cells. Fractions are always collected by

2.5 ml. Fractions can be tested for amylase or sucrose inhibiting activity or by means of an antimony assay. Buffer-derived electrolytes and enzyme preparation elute with the exclusion volume and degradation products according to decreasing molecular weight. Complete separation of compounds whose molecular weight differs only slightly is carried out by repeated chromatography under the conditions described above. The fractions containing the product to be isolated were pooled and lyophilized.

Example 17

To separate the isomeric compounds with three glucose units, 10 g of the isomeric mixture dissolved in water is added to a column packed with Dowex.RTM. 50 WX 4 (H @ ⁺ form) cation exchange resin. The column is first washed with water until the eluate is neutral and then eluted with 0.025 N hydrochloric acid. Fractions were collected in 3 ml increments and assayed by thin layer chromatography. Thin layer chromatography is performed on silanized silica gel plates with a mixture of ethyl acetate, methanol, water and 25% ammonia in a ratio. 100: 60: 40: 2 with triple development. The compound of formula IV proceeds greater than the starting point. the fractions 215-272 containing 6 g of the isomer of formula V and the fractions 288-294 containing 600 milligrams of the isomer of formula IV were combined, neutralized with an anion exchange resin Amberlit IRA-410 (OH-form) and evaporated.

In vitro suppression of sucrose action of an isomer of Formula IV isolated. this procedure is 19,000 SIE / g.

Example 18

Purified preparations of compounds of the formula I may be obtained by repeated gel permeation chromatography according to this example.

6.0 g of the crude preparation of the compound with ni + -П2 = 4 and 5 obtained according to Example 8 or 17 are freed from the admixtures with salts by dissolving the material in 1 nM ammonia and this solution is applied to a column (50 mm diameter and 100 cm long) ) filled with Sephadex G-10 ^R. The column is eluted with 1 nM ammonia and the eluate is monitored using a flow refractometer and a flow cell conductometer. The eluate can also be tested by thin layer chromatography on silica gel plates with n-butanol, ethanol and water in a ratio of 45:35:20. Fractions containing pure material were pooled and lyophilized. Yield: 4.4 g.

2.0 g of this material was loaded onto a column (diameter 50 mm, length 100 cm) packed with Bio-Gel P-2R, 200-400 mesh, for further separation. The column is maintained at 65 ° C by a thermostat and eluted with water at a flow rate of 80 ml / h.

The unusable fraction and the salt-containing fraction which may still be present after chromatography on Sephadex are discarded. The system consisting of the column, circulation pump, flow refractometer and flow conductometer is sealed and the solution is circulated through the gel. As progress has been made, separation is tested by constantly monitoring the refractive index and conductivity. Sufficient separation is achieved after 5 circulations. The column is then reconnected to the solvent reservoir and eluted with 16 ml fractions each. Fractions were assayed by thin layer chromatography as described above and fractions containing pure compounds and intermediate fractions were pooled and lyophilized. Yield 300 mg of compound with m + P 2 = 4; 420 mg of compound with ø n2 = 5 and 100 mg of less separated substances as an intermediate.

Example 19 One liter Erlenmeyer flasks each containing 200 ml of nutrient solution containing 2% starch, 1% glucose, 0.5% NZ amines, 1% yeast extract and 0.4 percent calcium carbonate (sterilized for 30 minutes at 121 ° C) (PH adjusted to 7.2 before sterilization) are inoculated with 2 ml of the starting culture (obtained in the same nutrient solution, inoculated with oatmeal agar flasks from slanted tube cultures). These flasks were cultured at 28 ° C on a bender shaker. After a three-day cultivation period, the contents of all three flasks were pooled and the mycelium separated on a centrifuge. 425 ml of a residue are obtained which contains 1100 AIE / ml, 0.17 MIE / ml and 1.0 SIE / ml.

Claims (1)

A process for the preparation of amino-sugar derivatives of the general formula I wherein m is the number 1 to 7 and П2 stands for numbers 1 to 7, the sum of ni а П2 stands for numbers 2 to 8, characterized in that strains SE 50 (ATCC 31 042), SB 18 (ATCC 31 041), SE 82 (ATCC 31 045) are cultivated, SE 50/13 (ATCC 31,043) a SE 50/110 (ATCC 31 044) of the genus Actinoplanaceae aerob in a solid or liquid medium containing a source of carbon and nitrogen, as well as salts and antifoams, at a temperature of 15 to 45 ^° C and a pH of 5.0 to 8.5 and the desired compounds are isolated in a manner known per se.