CA2062446A1 - Process for the preparation of ma - Google Patents

Abstract

Abstract of the Disclosure A process for the preparation of MA

The invention relates to a process for the preparation of MA, which ensures MA production by enzymatic catalysis in glycine/NaOH buffer, and by the use of a phosphate-containing culture medium for Bacillus sp. DSM 4675 and by extraction of the biomass with acetone.

Description

20~244~

HOECHST AKTIENGESELLSCHAFT HOE 91~F 069 Dr. SIJfe Description A pro~ess for the preparation of MA

Moenomycin A is the main component of Flavomycin- which is used in livestock nutrition. Like other known phospho-glycolipid antibiotics, it inhibits the bio~ynthesis of the peptidoglycan framework of the bacterial cell wall.
More detailed investigations have found that the trans-glycosylation reaction of the penicillin-binding protein lb of E. coli is inhibited by these substances ~Huber G., Antibiotics, V-1, pp. 135 - 153, (1979)]. Attempts at specific enzymatic or microbial degradation of phosoho-glycolipid antibiotics initially ~ailed.

European Application EP 0 355 679 describes a process for the degradation of moenomycins (= phosphoglycolipid antibiotic) to MA, MB and MC catalyzed by the enzymes moenomycinase and MBase from Bacillus sp. DSM 4675.
Moenomyfins Moenomycinase MC~MB
J! MBase MA

Examples of antibiotics in the moenomycin group are pholipomycin", the prasinomycins2), the diumycins (macarbomycins)3 esanchomycin, prenomycin and teichi-mycin, and other structurally related substances which have a corresponding functionalized phosphoglyceric acid ~ 1) S. Takahashi et al., Tetrahedron Lett. 1983, 499 2) F.L. Weisenborn et al., Nature 213, 1092 (1967) 3) S. Takahashi et alO, J. Antibiot. 26, 542 (1973)].

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2~2~46 In addition, EP 0 355 679 describes the aerobic fermen-tation of ~acillus spec. DSM 4675, the cleavage products resulting from the degradation o~ the moenomycins, the enxymes catalyzing the degradation, and the use of the degradation products as synthetic building blocks for the preparation of transglycosylase inhibitors tMA~ or as substance with antibiotic activity (MB).

The process in the abovementioned application gives a 1%
yield of MA because it is directed at the biologically active, i.e. antibiotically active, MB.

However, there is a distinct need to optimize processes for the preparation of MA because MA is a valuable building block for novel MA analogs, i.e. for novel transglycosylase inhibitors.

The invention thus relates to:
1. A process for the preparation of MA of the formula I

~0 ~OOC H ~ ~ I

by enzymatic degradation of phosphoglycolipids, wherein the enzymatic catalysis takes place in a glycine/NaOH bu~fer.

2. A process as claimed in claim 1, wherein the culture medium for 3acillus sp. DSM 4675 i8 optimized with respect to the titer~ of moenomycinase and MBase by addition of phosphate.
5 3. A process as claimed in claim 1, wherein acid or alkaline phosphatase is employed as substitute for the enzyme MBase.

4. A process as claimed in claim 1, wherein the fil-trate of the biomass i9 extracted with ethyl acetate ' 20624~

and then with acetone, and the biomass itself is extracted by stirring with acetone.

The invention is described in detail hereinafter, especi-ally in the preferred embodiments. It is furthermore defined in the claims.

Unless otherwise indicated, percentage data relate to weight.

Bacillus sp. was deposited with the number DSM 4675 under the conditions of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH in Braunschweig, Germany, on June 23, 1988.

The growth of the microorganism Bacillus sp. DSM 4675 and the production of the enzymes necessary for the degradation reactions according to the invention is particularly ~ood in a nutrient medium with the main components: citric acid, sodium gluconate, glycerol, peptone, phosphate and a vitamin solution. The concen-tration of the phosphate, for example potassium phos-phate, is preferably 50-100 mM. The nutrient medium can, however, also be employed without phosphate or with phosphate in any desired physiological concentration. The content of gluconic acid or salt thereof i9 1-2%, prefer-ably 2%.

The fermentation is carried out aerobically, that is to say, for example, submerged with shaking or stirring in shaken flasks or fermenters, where appropriate introdua-ing air or oxygen. The fermentation can be carried out in a temperature range from about room temperature to 50C, preferably at about 35 to 37C. The culture time is generally 8 to 48 bours, preferably 16-18 hours.

As described in EP 0 355 679, when Bacillus cells are used it is advantagsous for them to be permeabilized, for example with cetyltrimethylammonium salts, or to be .

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2~2446 lyophilized. It is likewise possible to operate with protein isolates from the Bacillus cells or with enzyme extracts which have been partially concentrated by salting out or chromatography, or naturally with the purified enzyme. It is furthermore possible to employ the enzyme in free or immobilized form.

Lyophilized cells are preferably employed as source of enzyme for the enzymatic cleavage of the moenomycins to MA in the process according to the invention.

It is evident from the diagram on page 1 that two enzymes are necessary for the preparation of MA. One enzyme is needed for the cleavage of the phosphoglycosidic linkage of moenomycin A, and this was called moenomycinase by the inventors. Moenomycinase is associated with the cyto-plasmic membrane of Bacillus 9p. DSM 4675 and can be obtained from the microorganism by methods known per se for enzyme isolation.

MBase can likewise be isolated from the microorganism byknown methods. For example, the cells are disrupted with ultrasound, and the resulting crude extract is further concentrated either by ammonium sulfate fractionation (~5-5S% saturation) or ultracentrifugation. This i9 followed by dialysis. The moenomycinase and MBase are finally separated by chromatography.

In the process according to the invention the enzymatic cleavage, i.e. the conversion of the moenomycins into MA, preferably takes place in one mixture (Example 2).

The cleavage of the moenomycins is carried out with lyophilized cells or enzyme isolates, but preferably with lyophilized cells.

The reaction takes place in glycine~NaOH buffer. The pH
of the buffer is preferably pH 8.0-8.5, otherwise in the range pH 7.5-10. The reaction takes place at 34-39C, .
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2~624~6 preferably at 37~C. The pH of the en~yme reaction i~ in the range pH 7.0-9.0, preferably 7.8. The reaction time is generally 5-48 hours, preferably about 24 hours. The substrate concentration ought to be in the range from 0.1 to 5%, preferably 1 to 2%.

It is still possible likewise to carry out the reaction at higher or lower temperatures or pH values than stated.
However, the enzyme activity is then lower.

It is possible to employ for the degradation of MB to MA
besides MBase, as described in EP 0 355 679, also phosphatase. Preferably used are the acid phosphatase from potatoes and the alkaline phosphatase from calf intestine. The enzymes are commercially available (Sigma). Both enzymes can be employed in immobilized and non-immobilized form.

The MA of the formula I obtained by the cleavage reaction is subsequently isolated and purified. This is carried out by extraction of the filtrate of the biomass or of the biomass itself with organic solvents. Preferably employed as solvent is acetone in a ratio of 0.2-1, preferably 0.3, by volume. The purification by chroma-tography takes place by using a petroleum ether/acetone or petroleum ether/ethyl acetate mixture as washing liquid. Methanol is employed as MA eluent.

The resulting reaction product MA can be used as synthetic building block ~or transglycosylase inhibitors.

The invention is described further by means of examples.

Example 1 Maintenance of the Bacillus sp. DSM 4675 strain The maintenance of the strain and the culturing of the preculture are described in European Patent Application .. . .

~ - 6 - 20~2~6 0 355 679 (Examples 1 and 2~.
a) A 12 1 laboratory fermenter containing 9 1 of medium of the following composition serves as main culture stage:

Peptone 12.5 g/l Glycerol 20.0 g/l Citrate 2.0 g/l Na gluconate 10.0 g/l K2HPO4 10.0 g/l MgSO4 x 7H2O 0.5 g/l FeCl3 x 6H2O 0.04 g/l Vitamin solution 1 ml Vitamin solution:
Nicotinic acid 0.35 g/l Thiamine HCl 0.30 g/l D-Biotin 0.01 g/l p-Aminobenzoic acid 0.20 g/l Pyridoxal HCl 0.10 g/l Ca pantothenate 0.10 g/l Vitamin B12 0.05 g/l This ls incubated with 500 ml of preculture at 37C, 300 rpm and an aeration rate of 0.5 w m for 16-18 hours.

The fully grown culture is centrifuged and then lyophilized.

~he novel medLum and the shortened ~ermentation tlme result in a doubling of the biomass yield. The biomas~ i9 characterized by mean~ of the optical den~ity. ~he measured OD = 7. In addition, the resulting cells degrade moenomycins to MA with a high yield.

In a test mixture with 100 ~1 of crude extract, 12 mg of moenomycin A and 900 ~1 of potassium phosphate buffer (pH 8.0) 50 mM, 50% of the substrate employed is degraded within 7-24 hours at 37C. The reaction products found ` - 7 - 2062~46 are MA, Ms and MC.

b) If the medium described under a) is used but without the addition of phosphate there is a distinct reduction in biomass. The measured OD = 3.
Example 2 Conversion of the moenomycins into MA (enzymatic eleavage) Lyophilized cells of Bacillus sp. DSM 4675 are used for the conversion.

a) 90-200 g of lyophilisate are suspended in 9 1 of glycine/NaOH buffer, 100 mM, pH 8.5, and, after addition of 135 g of moenomycin mixture or after addition of MB, 1.8 g of Na azide and 214 mg of CoCl2 ~ incubated at 37C
and 190 rpm for 6-48 hours.

The course of the reaction, which is followed by TLC
analysis, shows that up to 80% of the substrate is degraded to MB and MA. Of the cleavage produets, about 10-20% comprises MB and about 80-90% eomprises MA.

This way of earrying out the reaetion makes large-seale produetion of MA possible.

b) A yield of 60% MA is obtained when a buffer of identieal eomposition but with pH 9.0 i8 employed.

e) The yield of MA is likewise redueed to 60% when a tris-HCl buffer tl00 mM tris, pH 7.8, otherwise the eomposition eorresponds to the buffer stated under a)] is used.

``` 206244~ -- 8 --Example 3 Enæymatlc cleavage of MB using phosphatases The use of acid phosphatase from potatoes and alkaline phosphatase from calf intestine for the preparation of MA
from MB was investigated.

The conversion of MB (5 mg/ml) into MA is about 50% with acid phosphatase (10 U/ml of mixture) at pH 4.8 and room temperature, and more than 90% with alkaline phosphatase (50 U/ml) at pH 8.0 within 144 hour~. The conversion rate of the alkaline phosphatase i~ distinctly increased (c 24 h) in the presence of 0.1 mM ZnCl2 and MgCl2 at pH 10.5 (glycine/NaOH buffer, 100 mM) and 37C.

It is likewise possible to use immobilized alkaline phosphatase. In this case 35 U/ml under the above-mentioned conditions convert more than 90% of the sub-strate within 28 h.

Example 4 Isola,tion of the Flavomycin degradation product MA

The solids present in the suspension resulting from the enzymatic conversion are removed by centrifugation.

The resulting biomass is extracted by stirring several times with the same volume of acetone at room temperature until MA i9 no longer detectable in the organic phase by thin-layer chromatography. The MA-containing extracts are combined.

The filtrate obtained after removal of the biomass is initially extracted once with 1/3 of its volume of ethyl acetate. The aqueous-organic, colloidal solution i~ then extracted with 1/3 of its volume of acetone until MA is no longer detectable by TLC in the organic phase which `` 9 20~2~
can be separated off in each case.

The MA-containing extracts are combined with the acetone extracts of the biomass, and the solvent is removed in vacuo.

The Flavomycin component MB can be isolated from the remaining aqueous reaction solution by extraction several times with n-butanol. The crude MB obtained after removal of the solvent by distillation in vacuo can be used anew for the enzymatic reaction for preparing MA.

On average, about 110 g of crude M~ and about 85 g of crude M~ are obtained by the extraction process described above from about 500 g of the Flavomycin A/C complex.

Example 5 Purification of MA by chromatography The MA obtained after evaporation of the solvent is subsequently purified by column chromatography on silica gel.

For this, about 20 g of crude MA are dissolved in the minimum amounts of a 1:1 petroleum ether/acetone mixture and loaded under 7-10 bar at a flow rate of 5 l/h onto a steel column containing about 2.1 kg of silica gel 60 (pH
- 7.5) as stationary pha~e. Washing i~ then carried out with about 10 l of a petroleum ether/acetone ~6:4~
mixture under the same conditions. The washing liquid i9 collected in a single fraction. Elution i9 then carried out with about 5 l of pure methanol.

The methanol eluate is collected in fractions each of 0.1 l. The MA-active pure fractions detected by TLC are combined, and the solvent i9 removed in vacuo. MA is obtained as a pale yellow highly viscous oil. Marginal fractions can ~e rechromatographed. About 13 g of pure `` 2~62~46 substance are obtained from about 20 g of crude MA.

Precoated silica gel plates are used for the detection of MA by thin-layer chromatography. The mobile phase used is a solvent mixture composed of chloroform/methanol/ acetic acid t80: 10: 1). Detection is effected by staining the developed plates with PMS and subsequently drying them at 130C. Also applied as comparison substances are MA, MB
and the Flavomycin A/C complex.

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Claims (9)

1. A process for the preparation of the compound of the formula I

I

by enzymatic degradation of phosphoglycolipids, wherein the enzymatic catalysis takes place in a glycine/NaOH buffer.

2. The process as claimed in claim 1, wherein the glycine/NaOH buffer has the pH 8.0-8.5.

3. The process as claimed in claim 1, wherein the culture medium for Bacillus sp. DSM 4675 is opti-mized with respect to the titers of moenomycinase and MBase by addition of phosphate.

4. The process as claimed in claim 3, wherein the phosphate is added in a concentration of 50-100 mM
to the buffer.

5. The process as claimed in claim 1, wherein acid or alkaline phosphatase is employed as substitute for the enzyme MBase.

6. The process as claimed in claim 5, wherein the acid phosphatase from potato is employed.

7. The process as claimed in claim 5, wherein the alkaline phosphatase from calf intestine is employed.

8. The process as claimed in claim 1, wherein the fil-trate or the biomass is extracted with ethyl acetate and then with acetone, and the biomass itself is extracted by stirring with acetone.

9. The process as claimed in claim 8, wherein the filtrate of the biomass and the biomass itself are mixed with acetone in a ratio of 1:0.3 by volume.