AU619034B2 - A new microorganism for breaking down moenomycins, a process for the breakdown, and the use of the breakdown products - Google Patents

A new microorganism for breaking down moenomycins, a process for the breakdown, and the use of the breakdown products Download PDF

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AU619034B2
AU619034B2 AU39985/89A AU3998589A AU619034B2 AU 619034 B2 AU619034 B2 AU 619034B2 AU 39985/89 A AU39985/89 A AU 39985/89A AU 3998589 A AU3998589 A AU 3998589A AU 619034 B2 AU619034 B2 AU 619034B2
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Werner Aretz
Dirk Bottger
Kurt Hobert
Gerhard Seibert
Alois Tumulka
Peter Welzel
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Hoechst AG
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Abstract

It is possible with the aid of a Bacillus species or the appropriate enzymes obtained therefrom to degrade phosphoglycolipid antibiotics. The degradation products of the moenomycins have antibiotic activity or can be used as building block in the synthetic preparation of transglycosylase inhibitors.

Description

ture of Applicant (s) Sealsof Company and SIgnatures of Its Officers as 44b 1 e 619034 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION
(ORIGINAL)
Class Application Number: Lodged: Complete Specification Lodged: *e 0 Accepted: Published: "tiltitv: 0* A S S 'Related Art; Form Int. Class S Name of Applicant: 0* 0 Address of Applicant Actual Inventor: 5* HOECHST AKTIENGESELLSCHAFT 50 Bruningstrasse, D-6230 Frankfurt/Main 80, Federal Republic of Germany WERNER ARETZ, DIRKBOTTGER, GERHARD SEIBERT, ALOIS TUMULKA, PETER WELZEL and KURT HOBERT &WD-Wkf W =---eatermark Patent Trademark Attorneys 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.
Address for Service Complete Specification for the invention entitled: A NEW MICROORGANISM FOR BREAKING DOWN MOENOMYCINS, A PROCESS FOR THE BREAKDOWN, AND THE USE OF THE BREAKDOWN PRODUCTS The following statement is a full description of this invention, including the best method of performing it known to us HOECHST AKTIENGESELLSCHAFT Dr.KH/gm HOE 88/F 225 Description A new microorganism for breaking down moenomycins, a process for the breakdown, and the use of the breakdown products Moenomycin A (Fig. 1) is the main component of Flavomycin* which is used in livestock nutrition. Like other known phosphoglycolipid antibiotics it inhibits the biosynthesis of the peptidoglycan framework of the bacterial cell wall. Closer investigations showed that the transglycosylation reaction of the penicillin-binding protein lb of E. coli is inhibited by these substances [Huber Antibiotics, V-l, pages 135 to 153 (1979)]. 0. 0 L.o* Attempts at specific enzymatic or microbial breakdown of 15 phosphoglycolipid antibiotics have hitherto failed.
Surprisingly, a new Bacillus species which is able to cleave the phosphoglycolipid antibiotics to defined end products has now been isolated from a contaminated fermenter for the preparation of flavomycin using *d0 Streptomyces ghanaensis. Thee end products have anti- S biotic activity or can be used as building blocks in the synthesis of new transglycosylase inhibitors.
I 10 bcerHice t le inv ailn Cletos es 1. Bacillus spec. DSM 4675 and the variants an anits thereof.
2. The= l ge product of moenomycin A with the Sformula_ m a I lb Hence the invention relates to: 1. Bacillus spec. DSM 4675 and the variants and mutants thereof. The term variants and mutants of DSM 4675 means DSM 4675 derivatives which have the ability to cleave phosphoglycolipid antibiotics.
2. The cleavage product of moenornycin A with the formula I when produced by the Bacillus of claim 1.
H
2 N 0 CONH 2 0 OH Ho- J/ NHA H o 0 OH -0 H 0 CO HAC
HO
ao60 OH
MC
6 o* o o o o oe o* o *o S- 2
I
I i 4H iC 3. The cleavage product of the phosphoglycolipid antibiotics with the general formula Ro-CH 2
-CH-OR
2 COOH
(II)
in which R 1 is hydrogen or a phosphono group
[-PO(OH)
2 and R 2 is a (C 5 to C 55 )-alkyl group which can be branched or unbranched, saturated or unsaturated.
S 4. The enzymes with whose aid the phosphoglycolipid antibiotics can be cleaved at the phosphoglycosidic linkage, or the cleavage products specified under 3.
can be cleaved at the monophosphate ester linkage.
5. A process for the preparation of the breakdown products specified under 2. and which comprises I incubating phosphoglycolipid antibiotics with Bacillus spec. DSM 4675.
6. The use of the substances specified under 2. and 3.
as building block for the synthetic preparation of transglycosylase inhibitors or as substance having antibiotic activity.
The invention will be described in detail hereinafter, especially in the preferred embodiments. It is furthermore defined in the claims.
I 15500 -3 Bacillus spec. was deposited with the number DSM 4675 under the provisions of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturt2_ GmbH (German Microorganism and Cell Culture Collection) in Braunschweig, FRG, on June 23, 1988. The characteris tics of the strain may be said to be the following: 1. Taxonomic properties of Bacillus sp. DSM 4675 A) Morphology motile rods; up to 5 pm long; some in short 'i 0 chains see terminal spore; sporangium swollen e* Gram-positive.
*I go.
o B) Growth on various media (28°C; 48 hours) 1. Antibiotic medium 3 (Difco) rough, lobed colonies of diameter 1-2 mm 2. Luria broth (Bacto tryptone 10 g/l; Bacto yeast 5 g/l; NaCl 5 g/l smooth, glossy round colonies of diameter 2-3 mm; :I *C opaque 3. Nutrient broth (Difco) white, glossy colonies with irregular margin 4. Christensen urea agar (Difco) i C growth positive McConkey agar (Difco) growth positive 6. BROLAC agar (lactose) (Difco) growth positive 7. Simmons citrate agar (Difco) growth negative -4- 8. No growth in the presence of 7 or 10 NaCi in a peptone/meat, extract medium (Difco) C) Physiological properties 1.Oxidase 2. Catalase+ 3. Hemolysis L4. Aminopeptidase Nitrate reduction 6. Phenylalanine deaxninase 7. Growth at 30 0 C fI x:40 0 C 1 8. Anaerobic growth U solid liquid 9. Gas formation from glucose 110. Indole formation 411. Arginine dihydrase C SS'..12. Urea breakdown 9 920 13. Esculin hydrolysis 1 Gelatin breakdown fl-Galactosidase+ 16. Lysine decarboxylase 17. Ornithine decarboxylase i~ 18. H 2 S production .19. Tryptophan deaminase Alkal. phosphatase 21. Voges-Proskauer reaction D) Fermentation of carbohydrates C source Assimilation Acid formation Adipate Adonitol Arabinose++ Caprate Citrate I
I-
5 C source Assimilation Acid formation Dulcitol Fructose Galactose Gluconate Glucose Inositol Lactose Malate Malonate Maltose Mannitol Mannose Melibiose Phenylacetate Raffinose Rhamnose Sucrose Salicin SSorbitol Trehalose Xylitol Xylose N-Acetyl-glucosamine Taking account of taxonomic features and with the aid of "Bergey's Manual of Systematic Bacteriology" (Vol. 2, Williams and Wilkins publ., Baltimore, 1986) the strain can be assigned to the genus Bacillus. To determine the species, parallel comparative examinations of type cultures of Bacillus macerans, circulans, lentus, alcalophilus, stearothermophilus, licheniformis, polymyxa and fastidiosus were carried out. All the comparison strains showed distinct differences from Bacillus spec. DSM 4675.
Nor were any of these strains able to break down phosphoglycolipid antibiotics, especially moenomycin A. The conclusion to be drawn from this is that the strain DSM
A
I
C
~1 1 e t
I
I
6 4675 is a new species.
The invention also relates in each case to the mutants and variants which, as is known, may arise spontaneously or be generated by treatment with physical agents, for example irradiation, such as ultraviolet or X-rays, or with chemical mutaLgens such as, for example, ethyl methanesulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 2-hydroxy-4-methoxy-benzophenone
(MOB).
Suitable and preferred as carbon source for the aerobic fermentation of Bacillus spec. DSM 4675 are assimilable carbohydrates and sugar alcohols, such as glucose, 2* lactose or mannitol, as well as carbohydrate-containing natural products such as malt extract. Suitable and preferred nitrogen-containing nutrients are: amino acids, .15 peptides and proteins, as well as the breakdown products thereof, such as peptone or tryptone, furthermore meat extracts, milled seeds, for example of corn, beans, soybean or the cotton plant, disillation residues from the production of alcohol, meat meals or yeast extracts, as well as ammonium salts and nitrates. The nutrient solution may additionally contain, for example, chlo- .rides, carbonates, sulfates or phosphates of the alkali metals or alkaline earth metals, iron, zinc and manganese as additional inorganic salts.
The growth of the microorganism and the formation of the enzymes necessary for the breakdown reactions according to the invention is particularly good in a nutrient medium containing corn starch, soybean meal, sucrose, glycerol, peptone and/or corn steep as carbon and nitrogen sources.
The fermentation is carried out aerobically, that is to say, for example, submerged with shaking or stirring in shaken flasks or fermenters, where appropriate introducing air or oxygen. The fermentation can take place in a temperature range from approximately room temperature to 7 preferably at about 35 to 37"C. The culturing time is generally 24 to 48 hours.
The cultures of Bacillus DSM 4675 obtained in this way, or preparations thereof, can be used to cleave the phosphoglycolipid antibiotics. These include, in particular, the antibiotics of the moenomycin group, for example pholipomycin, the prasinomycins 2 the diumycins (macarbomycins) esanchomycin, prenomycin and teichomycin, and other structurally related substances which have a correspondingly functionalized phosphoglyceric acid S. Takahashi et al., Tetrahedron Lett. 1983, 499 2) F.L. Weisenborn et al., Nature 213, 1092 (1967) 3) S. Takahashi et al., J. Antibiot. 26, 542 (1973)].
The enzymes are particularly preferably used to break 1..5 down the moenomycins, for example Flavomycin.
When Bacillus cells are used it is advantageous to permeabilize the latter, for example with cetyltrimethylammonium, salts. It is likewise possible to use protein Sisolates from the Bacillus cells, or enzyme extracts 20 which have been partially enriched, for example, by salting-out or chromatography or, of course, the purified enzyme. It is furthermore possible to employ the enzyme and cells in free or immobilized form.
S 0 The enzymatic breakdown is depicted in the following diagram using moenomycin A as an example.
25 diagram using moenomycin A as an example.
i. Enzymatic breakdown of moenomycin A MOENOMYCIN A a a Moenomycinase
HOOC
HO S HO MB MBAse
HO
MA
9 It is evident from this diagram that two enzymes are needed to prepare the cleavage products. An enzyme, which the inventors have called moenomycinase, is required to cleave the phosphoglycosidic linkage. Moenomycinase is associated with the cytoplasmic membrane of Bacillus spec. DSM 4675 and can be obtained from the microorganism by enzyme isolation methods known per se.
Moenomycinase has a pH optimum of about 8.0-8.5, in p,-rticular 8.2-8.3. The temperature optimum of the enzyme is 45-55 0 C, in particular 49-51 0 C. Moenomycinase has a Km value for moenomycin A of 4-10 ill l i u ramo icXromo\C Two cleavage products are obtained. Cleavage product I comprises the sugar component of the phosphoglycolipid antibiotics. Also obtained is the cleavage product with e .,15 the general formula II
R
1 -CH2-CH-OR 2
COOH
•i in which R 1 is a phosphono group and R 2 is a (C 5 to C 55 4 alkyl group, preferably a (Co 1 to C 30 )-alkyl group, in particular a (C 20 to C 25 )-alkyl, each of which can be &20 branched or unbranched, saturated or unsaturated, prefer- 6 ably branched and unsaturated.
Moenomycins are preferably employed as substrates, so that the resulting cleavage products are the substances S* corresponding to the compound MC, as well as the compounds MB and MA (see formula diagram).
Another enzyme is required for the dephosphorylation of the phosphoglyceric acid lipid, which is obtained by incubation with moenomycinase, of the general formula II in which R 1 is hydrogen, and can also be obtained from the microorganism according to the invention. The inventors have called this enzyme MBase. MBase can likewise be isolated from the microorganism by methods known per se.
For example, the cells are disrupted with ultrasound, and 10 the resulting crude extract is further enriched either by ammonium sulfate fractionation (25-55 saturation) or ultracentrifugation. This is followed by dialysis. The moenomycinase and MBase are finally separated by chromatography.
The MBase is increasingly inactivated at temperatures above 37"C as well as when the pH falls in the acid pH range below pH The cleavage of the moenomycins, as well as of the phosphoglycolipid antibiotics can, as already mentioned, be carried out with whole cells or enzyme isolates.
seeo The reaction is generally carried out in aqueous medium at a pH of about 5.5-8.5, preferably pH 7-8. The reaction time is generally 5-48 hours, preferably about 24 hours.
The reaction temperature can be from 4 to 60 0 C, preferably 30 to 37"C. The substrate concentration ought to be in the range from 0.1 to 5 preferably 1 to 2 It is still possible to carry out the reaction at temperatures or pH values which are higher or lower than stated. However, the moenomycinase is then less active.
The reaction products resulting from moenomycin A are the substances MB and MC depicted in the diagram. The product SMA can be obtained by incubation of MB with MBase at to 37"C, preferably at 35 to 37°C, and pH 5.5 to preferably pH 6 to 8, over a period of about 24 hours.
The said reaction products can be used as antibiotic (for example MB) or as building block for the synthesis of transglycosylase inhibitors (for example MA and MC).
The invention is described in more detail by means of examples. Unless stated otherwise, percentage data relate to weight.
11 Example 1 Maintenance of the Bacillus spec. DSM 4675 strain Bacillus spec. DSM 4675 is maintained on the following solid nutrient medium (medium 1): Bacto tryptone (Difco) Yeast extract (Difco) NaCl Agar 10 g/1 5 g/l 5 g/l 15 g/1 •o°°o pH 7.2 The medium is distributed over test tubes and sterilized at 121 0 C for 30 minutes, then cooled, inoculated with the culture and incubated at 37"C for 2-3 days.
The grown culture is rinsed off to provide the inoculum for the following, moenomycin-containing main culture (medium 2): 15 *5 20 0
S
Corn starch Soybean meal Sucrose CaCO3 Corn steep CoSO 4 ®Genapol (alkyl polyglycol ester Moenomycin A 40 g/l 35 g/l 10 g/l 8 g/1 4 g/l 20 mg/l 5 ml/l 3 g/l (sterile filtered) pH 7.6 300 ml Erlenmeyer flasks each containing 30 ml of this medium are inoculated and then incubated at 37 0 C and 190 rpm for 8-48 hours. Analysis of the culture filtrate by thin-layer chromatography shows that the compounds MA, MB and MC are detectable as cleavage products of moenomycin, and that towards the end of the reaction there has been complete disappearance of the moenomycin employed.
m~ ~UW 12 Example 2 Preparation of cell-free extracts To prepare cell-free extracts, Bacillus spec. DSM 4675 is cultured in a fermenter. For this, cells are rinsed off the aga: plate to provide a 10 ml inoculum for a preculture (500 ml of medium 2 without Flavomycin in a 2 1 Erlenmeyer flask) which is then incubated at 37 0 C and 190 rpm for 24 hours.
A 12 1 laboratory fermenter containing 9 1 of medium 3 is I used for the main culture stage: *o 0 Peptone 12.5 g/l Glycerol 20.0 tg/l Citrate 2.0 g/1l K2HPO 4 1.5 g/l MgSO 4 x 7H 2 0 0.5 g/l FeCl 3 x 6H 2 0 0.04 g/l So** Desmophen (propylene glycol) 5.0 ml/l pH 6.8 S This is inoculated with 500 ml of preculture and incubated at 37 0 C, 300 rpm and an aeration rate of 0.5 vvm for 24 hours.
The grown culture is centrifuged, and the cell paste is resuspended in potassium phosphate buffer (pH 7.0) 50 mM (1 g of wet cells 2 ml of buffer). The cells are then disrupted with ultrasound, a French Press® or Dyno Mill®, and the resulting crude extract is used for the conversion.
In a test mixture containing 100 pl of crude extract, 12 mg of moenymycin and 900 pl of potassium phosphate buffer (pH 8.0) 50 mM there is within 7-24 hours at 37°C breakdown of the substrate employed. The reaction products found are MA, MB and MC.
i 13 Example 3 Preparative conversion of moenomycin A Preparative conversions are carried out in a 12 1 fermenter containing 8.2 1 of potassium phosphate buffer (pH 8.0) 50 mM, 800 ml of crude enzyme extract and 100 g of moenomycin A at 37 0 C and 100 rpm. The progress of the conversion is followed by TLC. The entire reaction mixture is freeze-dried after 8-48 hours. The moenomycin breakdown is generally 40-60 (UV analysis from the 10 TLC). The resulting reaction products are MA, MB and MC.
Example 4 *o S Preparation of the breakdown products 100 g of freeze-dried mixture from the enzymatic conversion were taken up in 2 1 of water and extracted twice O.15 with 2 1 of ethyl acetate each time. Centrifugation was g necessary for complete phase separation in this case. The combined organic extracts were dried over sodium sulfate, o filtered and evaporated to dryness. 0.4 g (0.4 of a dark brown oil was obtained, and thin-layer chromatography in the system n-butanol/acetic acid/water 3/1/2 on silica gel (Merck 60 F254 aluminum TLC sheet-), spraying with molybdatophosphoric acid/cerium(IV) sulfate color reagent (abbreviated to PMS reagent hereinafter), showed that it comprised mainly component MA (Rf value 0.85).
The remaining aqueous phase was then extracted twice with 2 1 of n-butanol each time. Once again, centrifugation was necessary for phase separation. The combined butanol phases were then concentrated as far as possible, and the residue was taken up in a little water and finally freeze-dried. 7.4 g (7.4 of a yellow powder were obtained and were found on examination by thin-layer chromatography (using the abovementioned conditions and I 14 the same detection) to comprise mainly the component MB (Rf 0.52).
The aqueous phase from which non-polar substances had been removed in this way was freeze-dried. The amount of residue resulting from this was 76.9 g (76.9 with a total amount of 85 Thin-layer chromatography showed that this pale yellow powder was composed of a polar main substance, called MC (Rf remaining moenomycin A and by-products.
JO The crude products of components MA, MB and MC obtained in this way were further purified as follows.
0 Example S* a) Purification of the breakdown product MA 400 mg of MA crude product were chromatographed on 120 g I 15 of silica gel (Merck 60, 15 40 mcm) which had been i* adjusted to a pH of 7.5. (The column material had been pretreated in the following way for this purpose: the silica gel was stirred in 500 ml of 2N HC1 for one hour, then filtered off with suction and washed to neutrality.
The pH was then adjusted to 7.5 with IN NaOH, and finally washing with 2 1 of water and 500 ml of methanol was carried out. The material pretreated in this way was dried and activated at 120 0 C overnight.) Chloroform/ethanol 1/1 was used as eluent. The substance was loaded onto the coltunn in 3 ml of solvent mixture, and 216 fractions each of 2.5 ml were collected. Using TLC analysis (TLC plates and detection as in Example 1, solvent system as for column eluent), fractions 115-175 were combined, dried on sodium sulfate and finally evaporated to dryness. 27 mg of spectroscopically pure MA were obtained.
0
I-
15 b) Purification of the breakdown product MB 3.1 g of MB-containing crude product from the extraction were chromatographed on 500 g of silica gel which had been adjusted to a pH of 7.5 using the process explained in Example 2. Using a medium-pressure chromatography system (MPLC), chloroform/ethanol/water 4/7/1.5 was used for the elution at a flow rate of 10 ml/min and a pressure of 2-5 bar. After a fore-run of 600 ml, 220 fractions each of 10 ml were collected, combining on the 10 basis of the TLC. Besides mixed fractions containing MB, o.
fractions 90-170 yielded 1.28 g of pure MB after evaporation, taking up in water and freeze-drying.
c) Purification of the breakdown product MC 2.2 g of polar crude product from the aqueous phase of the extraction were likewise chromatographed under pressure (MPLC). 500 g of silica gel with a pH of were employed (process in Example and the eluent used S was ethyl acetate/i-propanol/water 4/5/5. The amount to be loaded was suspended in methanol with 15 g of silica ,20 gel, the solvent was evaporated off, and the support 4 treated in this way was introduced into a precolumn, and then elution was carried out at a flow rate of 5 ml/min under a pressure of 2-4 bar. A fore-run of 940 ml was followed by fractionation in 250 fractions each of 10 ml.
The fractions were tested by thin-layer chromatography in the system ethyl acetate/i-propanol/water 1/1/1 using PMS color reagent and detection of the UV absorption at 254 nm, and were combined. Besides mixed fractions, concentration of fractions 120-190 to the aqueous phase and subsequent freeze-drying revealed 1.3 g of pure MC, which was investigated by spectroscopy.
Example 6 Elucidation of the structures of the products MA and MB obtained from moenomycin A by enzymatic breakdown.
16 (The numbers of the structures relate to the formula diagram on page 19) The structure deduced for MA was la. The assignment of the structure is based on the 3C NMR spectrum of la.
Reaction of la with diazomethane yielded the methyl ester lb which is characterized by a 1 H NMR and an El mass spectrum.
The structure deduced for MB on the basis of "C and FAB mass spectrum was 2. Hydrogenation of 2 yielded the decahydro derivative 3a which reacted with diazomethane to give 3b, which had already been obtained previously fro,. moenomycin A by another route. It is consistent with I the proposed structures that it was possible to convert 2 (MB) enzymatically into la (MA).
Description of the experiments la: 'C NMR (100.6 MHz, CD30D): moenocinol moiety: 6 67.5 123.5 141.6 and 141.8 (C-3 and 32.3 and 32.5 (C-4 and 126.7 35.9 40.9 (C- 30.7 151.1 33.4 122.7 (C-13), 137.3 36.4 27.7 125.3 (C-17), 132.2 25.9 17.8 16.1 (C-21), 109.2 27.3 (C-23 and C-24), 23.8 Glyceric acid moiety: 6 175.9 80.6 64.1 '25 28
H
4 60, (446.6) Ib: la was converted into the H' form in aqueous solution using ®Dowex 50 form). la (H form, 18.5 mg, 0.04 mmol) was dissolved in methanol (3 ml) and, at OOC, excess ethereal diazomethane solution was added. The reaction mixture was maintained at 0°C for 2 h and at for 12 h and then evaporated to dryness. Column chromatography (5 g of SiO 2 petroleum ether/ethyl acetate 2:1) yielded Ib (3 mg).
-17 1 H NMR (80 MHz, CDC1 3 6 0.96 6H, CH 3 -23 and CH 3 24), 1.61 6H), 1.68 3H), 1.73 3H) (4xCH.), 1.90-2.12 (allyl 2.62 (broad d, J 7Hz, CH 2 -12), 3.78 OCH 3 3.60-4.30 (OCH 2 and OCH signals), 4.62 (broad s, CH 2 4.87-5.47 (olefinic Hs). C 2 9H 4 8
O
4 (460.7), MS: m/z =460 271 230 (19), 199 43 (100).
2: 13C NMR (100.6 MHz, D 2 moenocinol moiety: 6 =68.6 (C- 124.5 142.9 34.6 34.0 C- 10), 128.1 143.6 37.8 44.1 151.4 37.2 123.5 138.3 (C-14), Goo#'e 42.2 29.1 126.9 133.1 (C-18), a 4 I~ '28.0 19.9 18.2 111.2 29.7 0& 105 (C-23, 24), 25.9 Glyceric acid moiety: 6 179.0 81.8 68.6 -C 2 8
H
4 7 0 7 P (526.7), FAB-MS (matrix: DMSO/glycerol): m/z =615 (M-3H+4Na)+, 593 (M- 2H+3Na)+, 571 492, 267, 231, 185, .165, 143, 3a: I 2 (14.9 mg, 28.3 pmol) and PtO 2 (4 mg) were stirred in 04s methanol (3 ml) and acetic acid (50 pl) in an H 2 atmosphere under normal conditions for 3 days. The catalyst was filtered off, and evaporation yielded 3a (13.5 mg).
625 M-3+4N)+,603(M-2H+3Na)+, 581 558 (11- 3b: 111 3a was treated in aqueous solution with the ion exchanger (Dowex 50, H+ form) in order to liberate all acidic groups. The resin was filtered off and then the solution was freeze-dried. 8.5 mg (15.9 pmol) of the sample treated in this way were dissolved in methanol (2 u7, An excess of ethereal diazomethane solution was added at 0 0 C. The mixture was left to stand at 0 0 C for 2 h and at -18 0 C for 12 h. Evaporation and column chromatography (5 g of S'O 2 1 petroleum ether/ethyl acetate 1:1) yielded 3b 0 mg) 'H NMR (80 20 MHz, CDC1 3 6 3.75 OCH 3 and 2 d, 3 1jip =10 and 12 Hz, P(OCH 3 2 3.20 4.40 (OCH 2 and OCH MUltiplets), C1 3
H
63 0 7 P (578.8), MS: m/z ()=563 (0.1, Ir M-C1 4
H
2 9 1, breakage of the link between C-8 and C-9 of the 25 perhydromoenocinol moiety), 229 212 (6, 127' 57 (100).
0 4* *1 0*e 19
M
C
ROO~H
RO en zyme Ioenomycin A 0O
S
5@ S S
OS
*5SS
S.
SS S
SOSS
S S S. S SS 55
S
S
00*@ S S 55
*S
S
SS
S.
5 S 5* enzyme
HOOCH
H0 *~H O- O,,p H 2 /Pt R0% ROOC
H
RO 0 0S S S 55 5.5.5.
S
3 R a H b2 CD 3
H
3 CO.. 5. OH 04aM P
)(C
Hzl 0O 2 i -j r 4 1 P 20 Example 7 Antibiotic activities of the cleavage products An agar dilution test with Mueller-Hinton agar was carried out to determine the antibacterial activities (Antibiotics in Laboratory Medicine, V. Lorian, Ed., Baltimore 1986, pages 1-10).
Minimum inhibitory concentration (pg/ml) Cleavage products Str.
pyogenes Staph.
aureus 503 E.coli DC2 .o eec e e
C
O
see.
e e SO Sees 0 >100 3.125 >100 >100 25 >100 >100 >100 >100 Example 8 0 e
S
SII
C
Transglycosylase assay 00 The inhibition of the polymerization of the peptidoglycan-sugar chains by the cleavage products was carried 0" out by the assay described by Izaki Biol. Chem. 243, 3180-3192, 1968) using lipid intermediates from the cell membrane of E. coli K 12.
S: It emerges from this that moenomycin A (20 pg/ml) inhibits the transglycosylase reaction by 52.7 and the cleavage product MB inhibits the enzyme by 32.9 e 94~

Claims (8)

1. Bacillus spec. DSM 4675 and the variants and mutants thereof said variants and mutants having the ability to cleave phosphoglycolipid antibiotics.
2. The compound of the formula I when produced by the Bacillus of claim 1. H 2 N O CONH 2 0 OH HO-H 0 .NHA HO L o o MC I. oo 4 U 0 1 4 CI A 21 1. Bacillus spec. DSM 4675 and th, -thereof. 2. The compound of the formula I Ltants I A 0* 0 S 000 0 0* 0 00 0000 S. 0060 0e 0 00 .0 *0 a 0 0 0* S0 S 4@ 0 S@ 0 0 0* 0* 0* 00 0 *00000 6
3. .The compound of the formula II L-j'n oc~C~X'L R 1 0-CH 2 -CH-OR 2 II b OCH in which R' is hydrogen or a phosphono group and R 2 is a branched or unbranched, saturated or unsatur- ated (C 5 to -alkyl1 group.
4. The compound as claimed in claim 3, wherein the alkyl group has a chain length of 10 to 30 carbon atoms The compound as claimed in claim 3 or 4, wherein the alkyl group has a chain length of 20 to 25 carbon atoms.
6. Moenomycinase having the following characteristics cleavage of phosphoglycolipid antibiotics at the phosphoglycosidic linkage a pH optimum of 8.0 to a temperature optimum of 45 to 550C and _1 22 a K, value of 4 to 10 pmolar based on moenomycin A as substrate.
7. An enzyme from Bacillus spec. DSM 4675, which dephosphorylates the compound as claimed in claim 3, and is inactivated above 37"C and when the pH decreases at pH below
8. A process for the preparation of compounds of the formula I as claimed in claim 2, which comprises incubating moenomycin A with Bacillus spec. DSM 4675 or an enzyme isolate obtained therefrom.
9. A process for the preparation of the compound of the formula II as claimed in claim 3, which comprises incubating phosphoglycolipid antibiotics with Bacillus spec. DSM 4675 or an enzyme isolate ob- tained therefrom. The process as claimed in claim 8 or 9, wherein incubation is carried out at a pH of 5.5 to Soso% DATED this 17th day of August 1989. .HOECHST AKTIENGESELLSCHAFT S WATERMARK PATENT TRADEMARK ATTORNEYS 50 QUEEN STREET MELBOURNE. VIC. 3000. I
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EP0652205A3 (en) * 1993-11-04 1995-08-30 Hoechst Ag Moenomycin degradation products containing hydroxylated or oxidized lateral lipid chain and moenomycin analogs, process for preparing and their use.
DE19709897A1 (en) * 1997-03-11 1998-09-17 Hoechst Ag Bismuth salts of antibiotics of the moenomycin group, process for their preparation, their use and medicaments containing such salts
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EP1069130B1 (en) * 1999-07-15 2004-08-11 Hoechst Marion Roussel Moenomycin A Derivatives, their preparation, and use as antibacterial products

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