2-alkenyloxy-3-(phosphono)oxypropanoic acids derived from the cleavage of phosphoglycolipid antibiotics

A new Bacillus species and the appropriate enzymes obtained therefrom can be used to break down phosphoglycolipid antibiotics. The breakdown products of moenomycins display antibiotic activity or can be used as building blocks for the synthetic preparation of transglycosylase inhibitors.

Moenomycin A (FIG. 1) is the main component of Flavomycin.RTM. 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 G., Antibiotics, V-1, pages 
135 to 153 (1979) ]. Attempts at specific enzymatic or microbial breakdown 
of 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 Streptomyces ghanaensis. These end products have antibiotic activity 
or can be used as building blocks in the synthesis of new transglycosylase 
inhibitors. 
Hence the invention relates to: 
1. Bacillus spec. DSM 4675 and the variants and mutants thereof. 
2. The cleavage product of moenomycin A with the formula I 
##STR1## 
3. The cleavage product of the phosphoglycolipid antibiotics with the 
general formula 
##STR2## 
in which R.sup.1 is hydrogen or a phosphono group [--PO(OH).sub.2 ]and 
R.sup.2 is a (C.sub.5 to C.sub.55)-alkyl group which can be branched or 
unbranched, saturated or unsaturated. 
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 3., which comprises 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. 
Bacillus spec. was deposited with the number DSM 4675 under the provisions 
of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und 
Zellkulturen GmbH (German Microorganism and Cell Culture Collection) in 
Braunschweig, FRG, on Jun. 23, 1988. The characteristics 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 .mu.m long; some in short 
chains 
terminal spore; sporangium swollen 
Gram-positive. 
B) Growth on various media (28.degree. 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; 
opaque 
3. Nutrient broth (Difco) 
white, glossy colonies with irregular margin 
4. Christensen urea agar (Difco) 
growth positive 
5. McConkey agar (Difco) 
growth positive 
6. BROLAC agar (lactose) (Difco) 
growth positive 
7. Simmons citrate agar (Difco) 
growth negative 
8. No growth in the presence of 7 or 10% NaCl in a 
peptone/meat extract medium (Difco) 
C) Physiological properties 
1. Oxidase + 
2. Catalase + 
3. Hemolysis - 
4. Aminopeptidase - 
5. Nitrate reduction - 
6. Phenylalanine deaminase 
- 
7. Growth at 30.degree. C. 
+ 
40.degree. C. + 
50.degree. C. + 
8. Anaerobic growth 
solid - 
liquid - 
9. Gas formation from glucose 
- 
10. Indole formation - 
11. Arginine dihydrase 
- 
12. Urea breakdown - 
13. Esculin hydrolysis 
+ 
14. Gelatin breakdown - 
15. .beta.-Galactosidase 
+ 
16. Lysine decarboxylase 
- 
17. Ornithine decarboxylase 
- 
18. H.sub.2 S production 
- 
19. Tryptophan deaminase 
- 
20. Alkal. phosphatase 
- 
21. Voges-Proskauer reaction 
- 
______________________________________ 
D) Fermentation of carbohydrates 
Acid 
C source Assimilation 
formation 
______________________________________ 
Adipate - 
Adonitol - 
Arabinose + + 
Caprate - 
Citrate - 
Dulcitol - 
Fructose + + 
Galactose - 
Gluconate + 
Glucose + + 
Inositol - 
Lactose + + 
Malate - 
Malonate - 
Maltose + + 
Mannitol + + 
Mannose + + 
Melibiose + + 
Phenylacetate - 
Raffinose + + 
Rhamnose - 
Sucrose + + 
Salicin - 
Sorbitol - 
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 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 mutagens 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, lactose or mannitol, as well as carbohydrate-containing 
natural products such as malt extract. Suitable and preferred 
nitrogen-containing nutrients are: amino acids, 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, 
chlorides, 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 50.degree. C., 
preferably at about 35.degree. to 37.degree. 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.sup.1), the prasinomycins.sup.2), the diumycins 
(macarbomycins).sup.3) esanchomycin, prenomycin and teichomycin, and other 
structurally related substances which have a correspondingly 
functionalized phosphoglyceric acid. 
FNT 1) S. Takahashi et al., Tetrahedron Lett. 1983, 499 
FNT 2) F. L. Weisenborn et al., Nature 213, 1092 (1967) 
FNT 3) S. Takahashi et al., J. Antibiot. 26, 542 (1973) 
The enzymes are particularly preferably used to break 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 isolates from the Bacillus cells, or enzyme extracts 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. 
The enzymatic breakdown is depicted in the following diagram using 
moenomycin A as an example. 
##STR3## 
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 particular 8.2-8.3. The 
temperature optimum of the enzyme is 45.degree.-55.degree. C., in 
particular 49.degree.-51.degree. C. Moenomycinase has a K.sub.m value for 
moenomycin A of 4-10 millimolar. 
Two cleavage products are obtained. Cleavage product I comprises the sugar 
component of the phosphoglycolipid antibiotics. Also obtained is the 
cleavage product with the general formula II 
##STR4## 
in which R.sup.1 is a phosphono group and R.sup.2 is a (C.sub.5 to 
C.sub.55)-alkyl group, preferably a (C.sub.10 to C.sub.30)-alkyl group, in 
particular a (C.sub.20 to C.sub.25)-alkyl, each of which can be branched 
or unbranched, saturated or unsaturated, preferably branched and 
unsaturated. 
Moenomycins are preferably employed as substrates, so that the resulting 
cleavage products are the substances 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.sup.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 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.degree. C. 
as well as when the pH falls in the acid pH range below pH 5. 
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. 
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.degree. 
to 60.degree. C., preferably 30.degree. to 37.degree. 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 MA can be obtained by incubation 
of MB with MBase at 30.degree. to 37.degree. C., preferably at 35.degree. 
to 37.degree. C., and pH 5.5 to 8.5, 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. 
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) 10 g/l 
Yeast extract (Difco) 5 g/l 
NaCl 5 g/l 
Agar 15 g/l 
pH 7.2 
______________________________________ 
The medium is distributed over test tubes and sterilized at 121.degree. C. 
for 30 minutes, then cooled, inoculated with the culture and incubated at 
37.degree. C. for 2-3 days. 
The grown culture is rinsed off to provide the inoculum for the following, 
moenomycin-containing main culture (medium 2): 
______________________________________ 
Corn starch 40 g/l 
Soybean meal 35 g/l 
Sucrose 10 g/l 
CaCO.sub.3 8 g/l 
Corn steep 4 g/l 
CoSO.sub.4 20 mg/l 
.RTM. Genapol (alkyl polyglycol ester) 
5 ml/l 
Moenomycin A 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.degree. 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. 
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 agar plate to provide a 10 
ml inoculum for a preculture (500 ml of medium 2 without Flavomycin in a 2 
l Erlenmeyer flask) which is then incubated at 37.degree. C. and 190 rpm 
for 24 hours. 
A 12 l laboratory fermenter containing 9 l of medium 3 is used for the main 
culture stage: 
______________________________________ 
Peptone 12.5 g/l 
Glycerol 20.0 tg/l 
Citrate 2.0 g/l 
K.sub.2 HPO.sub.4 1.5 g/l 
MgSO.sub.4 .times. 7H.sub.2 O 
0.5 g/l 
FeCl.sub.3 .times. 6H.sub.2 O 
0.04 g/l 
Desmophen (propylene glycol) 
5.0 ml/l 
pH 6.8 
______________________________________ 
This is inoculated with 500 ml of preculture and incubated at 37.degree. 
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.RTM. 
or Dyno Mill.RTM., and the resulting crude extract is used for the 
conversion. 
In a test mixture containing 100 .mu.l of crude extract, 12 mg of 
moenymycin and 900 .mu.l of potassium phosphate buffer (pH 8.0) 50 mM 
there is within 7-24 hours at 37.degree. C. 50% breakdown of the substrate 
employed. The reaction products found are MA, MB and MC. 
EXAMPLE 3 
Preparative Conversion of Moenomycin A 
Preparative conversions are carried out in a 12 l fermenter containing 8.2 
l of potassium phosphate buffer (pH 8.0) 50 mM, 800 ml of crude enzyme 
extract and 100 g of moenomycin A at 37.degree. 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 TLC). The resulting reaction products are MA, 
MB and MC. 
EXAMPLE 4 
Preparation of the Breakdown Products 
100 g of freeze-dried mixture from the enzymatic conversion were taken up 
in 2 l of water and extracted twice with 2 l of ethyl acetate each time. 
Centrifugation was necessary for complete phase separation in this case. 
The combined organic extracts were dried over sodium sulfate, 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 sheets), 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 l 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 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=0.1), remaining moenomycin A and by-products. 
The crude products of components MA, MB and MC obtained in this way were 
further purified as follows. 
EXAMPLE 5 
A) Purification of the Breakdown Product MA 
400 mg of MA crude product were chromatographed on 120 g of silica gel 
(Merck 60, 15-40 mcm) which had been 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 HCl for one hour, then filtered off 
with suction and washed to neutrality. The pH was then adjusted to 7.5 
with 1N NaOH, and finally washing with 2 l of water and 500 ml of methanol 
was carried out. The material pretreated in this way was dried and 
activated at 120.degree. C. overnight.) Chloroform/ethanol=1/l was used as 
eluent. The substance was loaded onto the column 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. 
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 basis of the TLC. Besides mixed fractions containing MB, 
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 7.5 were employed (process in Example 2), and the eluent used was 
ethyl acetate/i-propanol/water=4/5/5. The amount to be loaded was 
suspended in methanol with 15 g of silica gel, the solvent was evaporated 
off, and the support 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 
(The numbers of the structures relate to the formula diagram on page 19) 
The structure deduced for MA was 1a. The assignment of the structure is 
based on the .sup.13 C NMR spectrum of 1a. Reaction of 1a with 
diazomethane yielded the methyl ester 1b which is characterized by a 
.sup.1 H NMR and an EI mass spectrum. 
The structure deduced for MB on the basis of .sup.13 C and FAB mass 
spectrum was 2. Hydrogenation of 2 yielded the decahydro derivative 3a 
which reacted with diazomethane to give 3, which had already been obtained 
previously from moenomycin A by another route. It is consistent with the 
proposed structures that it was possible to convert 2 (MB) enzymatically 
into 1a (MA). 
Description of the Experiments 
1a: 
.sup.13 C NMR (100.6 MHz, CD.sub.3 OD): moenocinol moiety: .delta.=67.5 
(C-1), 123.5 (C-2), 141.6 and 141.8 (C-3 and C-7), 32.3 and 32.5 (C-4 and 
C-5), 126.7 (c-6), 35.9 (C-8), 40.9 (C-9), 30.7 (C-10), 151.1 (C-11), 33.4 
(C-12), 122.7 (C-13), 137.3 (C-14), 36.4 (C-15), 27.7 (C-16), 125.3 
(C-17), 132.2 (C-18), 25.9 (C-19), 17.8 (C-20), 16.1 (C-21), 109.2 (C-22), 
27.3 (C-23 and C-24), 23.8 (C-25). 
Glyceric acid moiety: .delta.=175.9 (C-1), 80.6 (C-2), 64.1 (C-3). 
C.sub.28 H.sub.46 O.sub.4 (446.6) 
1b: 
1a was converted into the H.sup.+ form in aqueous solution using .RTM.Dowex 
50 (H.sup.+ form). 1a (H.sup.+ form, 18.5 mg, 0.04 mmol) was dissolved in 
methanol (3 ml) and, at 0.degree. C., excess ethereal diazomethane 
solution was added. The reaction mixture was maintained at 0.degree. C. 
for 2 h and at 20.degree. C. for 12 h and then evaporated to dryness. 
Column chromatography (5 g of SiO.sub.2, petroleum ether/ethyl acetate 
2:1) yielded 1b (3 mg). 
.sup.1 H NMR (80 MHz, CDCl.sub.3): .delta.=0.96 (s, 6H, CH.sub.3 -23 and 
CH.sub.3 -24), 1.61 (s, 6H), 1.68 (s, 3H), 1.73 (s, 3H) 
(4.times.CH.sub.3), 1.90-2.12 (allyl Hs), 2.62 (broad d, J=7 Hz, CH.sub.2 
-12), 3.78 (s, OCH.sub.3), 3.60-4.30 (OCH.sub.2 and OCH signals), 4.62 
(broad s, CH.sub.2 -22), 4.87-5.47 (olefinic Hs). --C.sub.29 H.sub.48 
O.sub.4 (460.7), MS: m/z (%)=460 (0.03), 271 (10), 230 (19), 199 (68), 43 
(100). 
2: 
.sup.13 C NMR (100.6 MHz, D.sub.2 O): moenocinol moiety: .delta.=68.6 
(C-1), 124.5 (C-2), 142.9 (C-3), 34.6 (C-4), 34.0 (C-5, C-10), 128.1 
(C-6), 143.6 (C-7), 37.8 (C-8), 44.1 (C-9), 151.4 (C-11), 37.2 (C-12), 
123.5 (C-13), 138.3 (C-14), 42.2 (C-15), 29.1 (C-16), 126.9 (C-17), 133.1 
(C-18), 28.0 (C-19), 19.9 (C-20), 18.2 (C-21), 111.2 (C-22), 29.7 (C-23, 
24), 25.9 (C-25). Glyceric acid moiety: .delta.=179.0 (C-1), 81.8 (C-2), 
68.6 (C-3). --C.sub.28 H.sub.47 O.sub.7 P (526.7), FAB-MS (matrix: 
DMSO/glycerol): m/z=615 (M- 3H+4Na).sup.+, 593 (M-2H+3Na).sup.+, 571 
(M-H+2Na).sup.+, 492, 267, 231, 185, 165, 143, 115. 
3a: 
2 (14.9 mg, 28.3 .mu.mol) and PtO.sub.2 (4 mg) were stirred in methanol (3 
ml) and acetic acid (50 .mu.l) in an H.sub.2 atmosphere under normal 
conditions for 3 days. The catalyst was filtered off, and evaporation 
yielded 3a (13.5 mg). --C.sub.28 H.sub.57 O.sub.7 P (536.7), FAB-MS 
(matrix: DMSO/glycerol: m/z=625 (M-3H+4Na).sup.+ 603 (M-2H+3Na).sup.+, 581 
(M-H+2Na).sup.+, 558 (M-H+Na)+, 514, 500, 498, 432, 404, 362, 340, 298, 
288, 266, 186, 164, 142, 115, 93. 
3b: 
3a was treated in aqueous solution with the ion exchanger (Dowex 50, 
H.sup.+ 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 .mu.mol) 
of the sample treated in this way were dissolved in methanol (2 ml). An 
excess of ethereal diazomethane solution was added at 0.degree. C. The 
mixture was left to stand at 0.degree. C. for 2 h and at 20.degree. C. for 
12 h. Evaporation and column chromatography (5 g of SiO.sub.2, petroleum 
ether/ethyl acetate 1:1) yielded 3b (4.0 mg). 
.sup.1 H NMR (80 20 MHz, CDCL.sub.3): .delta.=3.75 (s, OCH.sub.3 and 2 d, 
3J.sub.H,P =10 and 12 Hz, P(OCH.sub.3).sub.2), 3.20-4.40 (OCH.sub.2 and 
OCH multiplets), --C.sub.13 H.sub.63 O.sub.7 P (578.8), MS: m/z (%)=563 
(0.1, M-CH.sub.3), 519 (1, M-COOCH.sub.3), 452 (1, M-(CH.sub.3 O).sub.2 
P(O)OH), 381 (3, M-C.sub.14 H.sub.29, breakage of the link between C-8 and 
C-9 of the 25 perhydromoenocinol moiety), 229 (8, a), 212 (6, b), 127 
(20), 57 (100). 
##STR5## 
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 (.mu.g/ml) 
Str. Staph. 
pyogenes aureus E. coli 
Cleavage products 
77 503 DC2 
______________________________________ 
MA &gt;100 &gt;100 &gt;100 
MB 3.125 25 &gt;100 
MC &gt;100 &gt;100 &gt;100 
______________________________________ 
EXAMPLE 8 
Transglycosylase Assay 
The inhibition of the polymerization of the peptidoglycan-sugar chains by 
the cleavage products was carried out by the assay described by Izaki (J. 
Biol. Chem. 243, 3180-3192, 1968) using lipid intermediates from the cell 
membrane of E. coli K 12. 
It emerges from this that moenomycin A (20 .mu.g/ml) inhibits the 
transglycosylase reaction by 52.7% and the cleavage product MB inhibits 
the enzyme by 32.9%.