CA1318630C - Preparation of antibiotic chloropolysporin c - Google Patents

Abstract

ABSTRACT

The glycopeptide antibiotic chloropolysporin C is prepared from the related compound chloropolysporin B by enzymatic hydrolysis, using rhamnosidase.
Chloropolysporin C exhibits antibacterial activity and is useful in the treatment and prophylaxis of infections, and as a growth-promoting agent for animals.

Description

13~863~

PREPARATION OF ANTIBIOTIC CHLOROPOLYS~'ORIN C

This invention relates to a process for the preparation of the antibiotic chloropolysporin C from the related compound chloropolysporin B. Chloropolysporins B and C are glycopeptide antibiotics which exhibit antibacterial activity and are useful in the treatment and prophylaxis of infections, and as growth-promoting agents for animals.

As resistance to conventional antibiotics becomes increasingly established in common strains of pathogenic bacteria, the need for a wider variety of antibiotics for use in the fight against such bacteria becomes ever more crucial.
~oreover, various antibiotics, for example chloramphenicol, aureomycin, vancomycin and avoparcin, have been administered, or have been proposed for administration, to poultry and other farm animals, including the ruminants and pigs, for the prophylaxis of disease or to promote growth or milk production. However, an inherent disadvantage of the use of antibiotics in this way is that there is some risk that traces of the antibiotics or of metabolic products thereof may be found in animal products intended for human consumption (such as eggs, milk or meat); the alleged dangers of such residues are increasingly criticized by some sections of the community. There is, accordingly, a considerable desire amongst farmers for an antibiotic substance which will have the desired growth-promoting effect but which will leave no, or no significant, residues in animal products.
In European Patent Application No. 84304765.5 (published as EP-A-132349), assigned to the present assignees, there is disclosed an antibiotic, there referred to as "chloropolysporin", which was isolated from 2 ~31863~

~he culture medium of a microorgani6m identified as MicroPolV6~0ra 6p. SANX 60983. It was sub~equently discovered that the same microorganism, and hence others of the genu~ MicroPolY6pora, produce6 a further two new antibiotic 6ubstances that are highly effective against Gram-po6itive bacteria ~nd that show considerable promise for use as growth-promoting agents in animals, TheRe tWo antibiotic~, named chloropoly6porin B and chloropoly6porin C, their production by cultivation of microorganism~ of the genus MicroDolYsDora, and their therapeutic and veterinary use form the 6ubject of our European Patent Application No. 86300176.4 (publi6hed as ~P-A-la7722), When chloropoly6porin6 B and C are produced by microbial culture. the yield of chloropolysporin C in the resulting culture broth iB generally lower than that of chloropolysporin B. On the other hand, it has been establi6hed that chloropolysporin C exhibit6 higher antimicrobial activity against many organisms than doe6 chloropolysporin B. Accordingly, chloropolysporin C iB
potentially the more valuable component, for instance as a food additive, and there i8 a need for a procQ6s which can be used selectively to produce chloropolysporin C.

It has now been discovered, in accordance with the present invention, that chloropolysporin C can be produced by selective hydrolysis of chloropolysporin B, u~ing rhamno~ida~e.

It iS currently believed that chloropolysporins B and C may be represented by the planar 6tructural formula (I):

~31863~

Cl oR3 ~`1 R10 ,br ~ oa2 N ~ CH3 ~ OH
HO OH

in which:
for chloropolysporin B, Rl represents a l-L-ri6tosamine residue;
R2 represents a l-D-mannose residue:
R3 reprefien~s a l-D-glucoge residue: and R4 repre6ents a l-L-rhamno6e residue:
for chloropoly~porin C, R represents a l-L-ri6tosamine re6idue:
R2 represent6 a l-D-mannose residue:
R3 represents a l-D-glucose residue: and R repre6ents a hydrogen atom.
(Rigto6amine iB 3-amino-2,3,6-trideoxy-L-ribohexopyranose).

Accordingly, chloropolysporins B and C diPfer only in the substituent represented by -oR4 in the above formula.

It is probable that the various assymetric carbon atoms shown in the above formula (I) adopt, in chloropolysporins B and C, specific configurations, but these have not, to date, been elucidated.

`' 1318630 The proces6 of the invention involves an enzymatic hydroly~i6 and can be carried out under conditions such as reaction temperature, reaction time and pH which are generally well known Der se for this type of reaction.
For instance, it can be carried out in solution, with or without stirring, at a temperature of from 20 to 50OC, preferably 30O to ~0C, and mo6t preferably at about 37OC. A conventional aqueous solvent may be employed for thi6, for instance a 0.01 to o.lN phosphate buffer solution or a TRIS-HCl buffer 601ution. The pH of the reaction sslution will usually be wit~in the range of from 4 to 8, and preferably from 5 to 6. The reaction time will depend upon the other reaction conditions employed, such as the te~perature, pH, and concentration of the enzyme and of the chloropolysporin B 6ubstrate, but it will generally be within the range of from 5 hours to 3 days, and preferably from 15 to 24 hours.

The enzyme preparation used in the proce6s of the invention will usually have a rhamnosida6e activity of from 100 to 1,000 units/mg, and more preferably from 200 to 500 units/mg. The enzy~e i~ generally used iD an amount wich corre6ponds to a weight ratio of rhamnosîdase to chloropolysporin B within the range of from 1:0.01 to 1:50, and more preferably from 1:0.05 to 1:20.

There i~ no particular limitation on the type of rhamnosidase preparation used in the process of the invention, provided that it posse66e6 adequate rhamno6idase activity. In practice, an a-L-rhamno~idase (Enzyme Commis6ion No. ~.2.1.40) will normally be employed. The enzyme may be used in pure form, or in the form of a crude rhamno~idase preparation such as those produced commercially ~rom various microorganism6, and which may add~tionally contain some different enzymes and/or other materials besides the rhamnosidase itself, provided that such contaminant6 do not adversely affect the proce6s of the reaction to an unacceptable degree.

5 ~3186~0 Examples of 6uch rhamno~idase preparation6, which may be used in the proces6 of the inYention, include those produced by Sankyo Co Ltd , Japan, under the ~ ~
IlNaringinase'' and ~Sclase", and that produced by Tanabe Seiyaku Co Ltd,, aapan, under the trade name ~Kumitana6e~ Naringinase i6 an enzyme preparation in which the major enzyme component i~ a-rhamnosidase, with a minor amount of ~-gluco~idase, and i~ produced by the cultivation of the microorganism A6Derqillus usamii or of certain Penicillium species. Sclase is a pectinase-rich preparation containing naringinase, and i8 produced by cultivation of Coniothvrium diPlodiella~ A6Der~ ua usamii or AsDeraillu~ nicer. Kumitanase is a-rhamnosidase derived from AsDer~illus niaer.

AB an alternative to using a rhamnosidase preparation in soluble form, an immobilisQd enzyme preparation may also be used, either one in which the enzyme itself has been immobili~ed in a suitable carrier, or one containing immobili6ed cells of a rhamnosidase-producing microorganism - for example a naringinase-producing microorganism 6uch as AsDeraillus usamii, or a sclase-producing microorganism such as AsPeraillu6 niqer.
If such an immobilised form is used, the support material and techniques used for immobilisation of the en~yme or enzyme-producing microorganism may be cho~en from tho~e conventionally employed for this purpose in the enzyme art.

The process of the invention can be carried out by using as the 6tarting material a ~olution of previously i601ated chloropoly6porin B in either crude or purified form, and the concantration of chloropoly6porin B in the reaction solution i6 not particularly critical. However, in accordance with a preferred embodiment of the invention, the starting material may be a crude culture 6 i3~863~

broth containing chloropolysporin B, or a mixtuee of chloropolysporins B and C, a~ obtained by cul~ivation of a chloropolysporin-producing MicroPol~6Pora strain, by a proce6s such a6 already mentioned. In thi~ way, it is not nece6sary to isolate or purify the chloropoly~porin B
starting material before subjecting it to the proces6 of the invention, and this i6 advantageou6 especially for large-6cale commercial production. For example, the concentration of chloropoly~porin B starting material in 6uch a crude culture broth may be in the range of from 1,000 tO 10,000 y/ml. More generally, the concentration of chloropolysporin B in the reaction solution will usually be at least 1 to 5 y/ml, At the end of the reaction, the chloropolysporin C
formed by means of the enzymatic hydrolysis can be eecovered from the reaction mixture and purified by mean6 of variou~ techniques which are Der se well known in the production of biochemicals. For instance, it can be adsorbed onto an adsorbing resin such as I'Diaion"
(Registered Trade Mark) HP 20 (product of Mitsubi~hi Chemical Industries Ltd.) or "Amberlitel~ (Registered Trade Mark) XAD-2 or XAD-4 ~products of Rohm S Haas Co.), and eluted from the resin with a ~uitable eluent such as aqueous acetone or aqueou~ methanol. Another purification method which may be employed is rever6e phase chromatography, for example u~ing a silanizing 6ilica gel with a uniform particle size, preferably with a particle diameter within the range o from 0.06 to 0.2 mm. The eluent employed for thi~ technique may suitably be a mixture of aqueous ammonium formate and acetonitrile, a mixture of tri1uoroacetic acid and acetonitrile, or a mixture of phosphata buffer and acetonitrile; and the mo6t preferred eluent is a mixture containing about 85 par~s by volume sf 0.2% trifluoroacetic acid and about 15 parts by volume of acetonitrile.

7 131~63~

Thin layer chromatography and high performance liquid chromatography may be used to monitor the concen~ration of starting material and reaction product throughou~ the reaction, including the purification QtageS.

Since the ~tructure~ of chloropolysporin6 B and C have not been completely elucidated, they may be characterized by the properties 6et out below, in conjunction with the accompanying drawings. In ~he drawings:-Figures 1, 2 and 3 re~pectively show the ultra~ioletabsorption 6pectrum, the infrared abQorption spectrum and the nuclear magnetic re60nance spectrum of chloropoly6porin B;

Figures 4, 5 and 6 respectively 6how the ultraviolet ab60rption spectrum, the infrared absorption spectrum and the nuclear magnetic resonance spectrum of chloropolysporin C.

Chloropolysporin B, as its sulfate, is characterized by the properties:

(a) it takes the form of an amphoteric white powder, ~oluble in water:

(b) specific rotation: ~a]25-64.5 (C-1.04, O.lN
aqueous hydrochloric acid, ~odiu~ D-line);

(c) elemental analysi~:

C, 48.33%; H, 5.05%; N, 5.48%: Cl, 5.11%: S, 1.00~;

(d) on acid hydroly~i~ it yield6:

neutral ~accharides: glucose, mannose and rhamno~e;
amino acids: 3-chloro-s-hydroxyphenylglycine and N-methyl-~-hydroxyphenylglycine;

8 13~ 8~30 (e) ultraviolet absorption 6pectrum:

a~ illu~trated in Figure 1 of the accompanying drawings, having an ab~orption maximum ~max at Z80nm (Elcm=51) in a 0.1 N solution of hydrochloric acid, the absorbence, E, being measured at a concentration of 1% w~v;

(f) infrared absorption spectrum:

the infrared absorption spectrum (v cm 1) measured on a KBr disc is as shown in Figure 2 o~ the accompanying drawing6;

(g) nuclear magnetic resonance spectrum:

the nuclear magnetic resonance 6pectrum (~ ppm), measured at 270 MHz in deuterated dimethyl sulfoxide u6ing tetramethylsilane as the internal standard, is as illustrated in Figure 3 of the accompanying drawing~;

(h) 501ubility:

soluble in water, sparingly soluble in methanol and acetone, and in601uble in ethyl acetate, chloroform and benzene:

(i) color reactions:

positive in ninhydrin and Rydon-Smith reaction~;

(j) thin layer chromatography:

Rf value=0.65, using a cellulose sheet (Ea~tman~ as adsorbent and a 15:10:3:12 by volume mixture of butanol, pyridine, acetic acid and water as the developing solvent:

(k) high voltage paper electrophore~i6:

using Toyo~s filter paper No. 51A in a O.lM
TRIS-hydrochloric acid buffer 601ution of pH 7.5 (3300 ~olt/60cm, 1 hour), the migration distancè (detected by bioautography with Bacillus subtilis PCI 219) from the origin to the cathode wa6 4cm;

(1) molecular formula:

C83H8934N8~13 o.5H2S04. lOH20;
(m) molecular weight:

the molecular weight, measured by FAB-MS, was 1846 (~+, 18g7 ) .

"FAB-MS" is Fast Atom Bombardment Mas6 SpQctroscopy.

Chloropolysporin C, as its sulfate, may be characterized by the following propertie6:

(a) it takes the form of an amphoteric white powder, solubls in water;

(b) specific rotation: ~a] -64.4 (C=1.08, O.lN
aqueous hydrochloric acid, codium D-line);

lc) elemental analysis:

C, 50.53%; H, 4.69~; N, 6.14%; Cl, 5.62~; S, 1.12%;

10 13186~0 (d) on acid hydrolysis it yield6:

neutral saccharides: gluco6e and mannose:
amino acids: 3-chloro-4-hydro~yphenylglycine and ~-methyl-p-hydroxyphenylglycine:

(e) ultraviolet absorption 6pectrum:

as illustrated in Figure 4 of the accompanying drawings, having an absorption maximum ~max at 280nm (ElC~=57) in a 0.1 N solution of hydrochloric acid, the ab60rbence, E, being measured at a concentration of 1% wJv;

(f) infrared absorption 6pectrum:

the infrared absorption spectrum (~ cm 1) measured on a XBr di~c i~ as shown in Figure 5 of the accompanying drawings:

(g) nuclear magnetic resonance spectrum:

the nuclear magnetic resonance spectrum (~ ppm), measured at 400 MHz in deuterated dimethyl sulfoxide using tetramethyl6ilane as the internal standard, is aa illu~trated in Figure 6 of the accompanying drawings:

(h) solubility:

601uble in water, 6paringly soluble in methanol and acetone, and insoluble in ethyl acetate, chloroform and benzene:

(i) color reactions:

positive in ninhydrin and Rydon-Smith reactions:

(j) thin layer chromatography:

Rf value=0.65, u~ing a cellulo6e 6heet (Eastman) a~
ad60rbent and a 15:10:3:12 by volume mixture of butanol, pyridine, acetic acid and water a~ the developing solvent;

(k) molecular formula:

C77H7930N8C13. O.SH2S04. SH20;
(1) molecular weight:

the molecular weight, measured by FAB-MS, was 1700 (MH+, 1701).

The acute toxicity (LD50) value, a6 determined by intravenous admini6tration in mice (ICR, male, 5 weeks old) was 215 mgtkg for chloropolysporin B and 250 mg/kg for chloropolysporin C.

The minimal inhibitory concentrations (MICj of chloropolyspolins B and C against variou6 Gram-positive and Gram-negative bacteria were determined by the two-fold agar dilution method, using a Mueller-Hinton agar medium (protuced by Difco); the MIC against anaerobic bacteria wa6 determined using a GAM agar medium (produced by Nis6ui). The results are shown in Tables 1 and 2.

12 ~3~86~

Test strain MIC (~g/ml) Chloeopolysporin B C
_ _ . .
Staphylococcus aureus FDA Z09P JC-l 1.56 1.56 StaphYlococcu6 aureu~ SANK 70175 3.13 1.56 Staphylococcus aureus Smith 12.5 6.25 StaPhylococcu6 ePidermidis SANX 71575 3.13 3.13 Enterococcu6 faecalis SANK 71778 1.56 1.56 Bacillu8 subtili6 PCI 219 0.78 0.78 .
MYcobacterium ~meqmatis ATCC 607 25.0 12.5 Escherichia coli NIHJ JC-2 >100 >100 Klebsiella pneumonlae PCI 602 >100 >100 Pseudomonas aeruaino6a NCTC 10490 >100 >100 Serratia marcescens SANK 73060 >100 >100 Proteus mirabili_ SANK 70g61 >100 >100 . . _ . . _ . _ ~est strain MIC (~g/ml) Chloropoly6porin B C

Bacteroides fra~ilis >100 >100 Eubacterium cvlindroides 6.25 3.13 Fusobacterium necroDhorum >100 >100 PeDtococcus a6accharolvticu6 6.25 3.13 PeDtostreptocQccus Parvulus 0.78 0.39 Propionibacterium acnes 0.78 0.39 Clo~tridium ~Ymbiosum 1.56 0.39 Clostridium ramosum 1.56 1.56 Clos~ridium perfrinqens 0.20 0.10 Clostridium difficile 0.78 0.39 . _ . _ _ ~, ., __ ....... _ 13 ~318~

From the results given in the above Table6, it can be seen that chloropoly~porins B and C are effective against aerobic Gram-po6itive bacteria, such as Sta~hvlococcu~
aureus, StaphYlococcu6 eDidermidis, Enterococcus faecali6, Bacillu~ 6ubtili6 and MYcobacterium &meamatis, and against anaerobic Gram-positive bacteria, such as Eubacterium cYlindroides, PePtococcus asaccharolvticus, ProPionibacterium acnes, Clostridium 6vmbio6um, Cl06tridium Perfrinaen6 and Clostridium difficile.

CompariEon of the propertie6, chemical, physical and biological, given above of chloropoly6porin6 B and C with those of known antibiotic6 leads to the conclusion that they belong to the class of glycopeptide antibiotics containing chlorine, such a6 vancomycin, avoparcin a and ~, actinoidins A and B or A-35512 B. However, chloropolysporin6 B and C can be clearly di6tingui6hed from these ~nown antibiotics on the basis of the differences shown in the following Table 3. Specifically, they have different neutral saccharide components and different amino acids are produced on acid hydrolysis.
Moreover, they move a different distance on high voltage paper electrophoresi6 (HVPE 3300 volts~60 cm, 1 hour, pH
7.5, in 0.1 M TRIS-hydrochloric acid buffer solution), and they have different chlorine contents.

Antibiotic Neutral Amino acid Distance Chlorine saccha- content ride . (%) Vancomycin Glucose A~partic acid 4.89 N-methylleucine Avoparcin a Glucose, 4-Hydroxyphenyl- 9.4cm 1.85 Mannose, glycine, N-methyl-Rhamnose P-hydroxyphen qlYcine Avoparcin ~ Gluco6e, 3-Chloro-4-hydroxy- 9.4cm 3.65 Manno6e, phenylglycine, N-Rhamno~e methyl-~-hydroxy-_ phenYl~lvcine Actinoidin A Glucose, 4-Hydroxyphenyl- 2.02 Manno6e glycine, Phenyl-alanine ~ .
Actinoidin B Glucose, 2-Chloro-3-hydroxy- 3.96 Manno~e, phenylglycine, Phenvlalanine A-35512B Gluco8e, 1.82 Manno6e, Rhamnose, Fuco~e Actaplanin Glucose, 1.96 Mannose Rhamno6e Ri~tocetin A Gluco6e, 0 Manno6e Rhamnose Arabinose _ _ _ _ Ristocetin B Gluco~e, Mannose, Rhamnose _ 1 31~;3~

TABLE ~ (Cont'd) Chloro~oly- Glucose, 3-Chloro-4- 4 cm 5.11 spoein B Mannose, hydroxyphenyl-Rhamno~e glycine, N-methyl-~-hydroxyphenyl-qlvcine Chloropoly- Glucose, 3-chloro-s- 5.62 sporin C Mannose hydroxyphenyl-glycine, N-methyl-~-hydroxy-Dhenvl~lYcine The value reported above a6 ~'Di6tance~' is the di6tance of movement on high voltage paper electrophorasis, measured u6ing bioautography with Bacillus subtilis PCI
219 as the te6t organi6m.

From the above finding6, it can be seen that chloropolysporins B and C can be used as antibiotic6 against various diseases caused by bacterial infections.
The route of administration can vary widely and may be parenteral (e.g. by ~ubcutaneous, intravenou~ or intramu~cular in~ection or by suppository) or oral (in which case it may be in the form of a tablet, capsule, powder or granule). The do6e will, of course, vary with the nature of the disea6e to be treated, the age, condition and body weight of the patient and the route and time of administration; however, for an adult human patient, a daily dose of from 0.1 to 1.0 grams is preferred and thi6 may be administered in a single do~e or in divided dose6.

Moreover, in view of the strong activity of chloropolysporins B and C against infectious bacteria of the genus Cl06tridium, they can be expec~ed to be valuable growth-promoting agent~ for veterinary use. Bacteria of 16 13~

the genu~ Cl06tridium, particularly Clostridium perfrin~en6 and CloGtridium difficile, are often present in the intestine6 of farm animal6 and are the cause of diarrhoea. Since chloropolysporins B and C have a strong activity against 6uch microorganisms, they would 6uppre66 the growth of such microorgani6m6 in the intestines and thu6 impro~e the microbial balance of the intestines.
This, in turn, would improve feed efficiency, thus contributing to weight gain and improved milk production in variou6 farm animal6, including ruminants, pigs and poultry. Moreover, chloropoly6porins B and C, in common with the other glycopeptide antibiotics, are likely to have a low rate of absorption through the digastive organs and, as a result, where the chloropolysporin B or C i6 administered in the feed, little will remain in the animal body and hence in animal product6, such meat, milk or eggs. When the chloropolysporin B or C is used as a growth-promoting agent for animals, it is preferably administered orally. ~lthough it may be formulated into an edible compo6ition with any suitable carrier or diluent, it is particularly convenient to administer it in admixture with an animal feed or with drinking water.
When the chloropolysporin B or C i6 u6ed a6 a feed additive, it may be mixed alone with the feed or it may be mixed in combination with other non-toxic edible excipient6, nutrient 6upplement6 (e.g. vitamins, mineral6 or amino-acids), other antibiotic6, anticoccidial agents or enzyme6. For admini6tration to animal6 as a growth-promoting agent, the chloropoly6porin B or C need not nece66arily be in a completely purified form and it may be used in a crude or partially purified form, a6 obtained at any de6ired stage during the extraction and purification de6cribed above. For use as a growth-promoting agent, chloropoly6porin ~ or C is preferably employ~d in an amount of from 1 to 200, more preferably from 5 to 60, ppm by weight on the basis of the feed, drinking water or other carrier to which it is 1 3 ~

added; where an impure ~orm of chloropolysporin B or C is employed, a concentration having equivalent activity is used.

~ nimal~ to which chloropolyspo~in B or C can be administered include farm mammals (e.g. cattle, horse6, swine, ~heep and goat6), poultry (e.g. chickens, turkey~
and ducks) and pet animals (e.g. dogs, cats and birds).
Mo6t 6ignificantly, when chloropolysporin B or C i~
admini6tered orally to animal~, their growth is effectively promoted, but it i6 little ab~orbed from the gastro-inte6~inal tract and it exhibits low retention in animal tis6ues: thus, there iB an almos~ complete ab6ence of chloropoly6porin B or C residue6 in the products (e.g.
milk, meat or eggs) of animal6 to which it ha6 been administered, which is a great advan~age from the view point of food hygiene.

Chloropoly6porins B and C are produced by the cultivation of a MicroPolyspora strain herein identified as MicroDolvsPora 6p. SANK 60983, which was i~olated from a ~oil sample collected in Tochigi Prefecture, Japan.

The microorganism, MicroPolvspora sp. SANK 60983, has the characteristics de6cribed hereafter and i6 as described in Europsan Patent Application No. 8~304765.5 (publication ~P-A-132349), referred to above. These characteristics were determined by cultivation on various media prescribed by the ISP (International StreptomyceE
Project) or with the media recommended by S.A. Waksman in Volume 2 of ~The Actinomycete6", in all caRes at a temperature of 28C.

l. MorPholoaical Charac~eristics Strain SANK ~0983 grows relatively well on variou6 media. The aerial mycelium i6 hardly visible on almost 18 ~318~3~

all media but may occasionally be vi6ible on glycerol-asparagine agar or cn potato extract-carrot extract agar. The aerial and vege~ative mycelia bear, at the top and the middle, short chains of spore6, normallY
from 1 to 20, al~hough occasionally more than 20, ~pore&.
No distinct fragmentation of the hyphae i6 observed with the strain, although it may be ob6erved during later stages of the cul~ure.

2. Culture Characteri6tic~

Strain SANK 60983 can produce pale yellow, yellowish-brown or yellowish-gray colors. Aerial hyphae are not ob6erved on mo~t media, although white aerial hyphae are produced on ~ome media. No 601uble pigment iB
produced. Table 4 shows the resul~6 obtained after cultivation for 1~ day~ at Z8C on variou6 6tandard culture media. The color name6 and number6 u6ed were as6igned according to the ~Guide to Color Standard~, a manual published by Nippon Shiki6ai Kenkyusho, Tokyo, Japan.

lg 13~8630 _ ., ~edium Growth Aerial ~ever~e Soluble MYcelium Piqment Yea6t Abundant, None Yellowi~h- None extract- raised, brown malt wrinkled, (8-8-8 extract yellowish-agar brown (ISP 2) (8-8-8) Oatmeal Good, None Dull None agar smooth, yellow (ISP 3) dull (8-8-9) yellow (8-8-9) .
Inorganic Abundant, ~one Yellowish- None salt- smooth, gray (2-9-10) starch yellowi6h- to pale agar gray yellowi6h-(ISP g) (2-9-10) brown to pale . (6-8-9) yellowi6h-brown ~6-8-9) Glycerol- Good, Poor, Yellowi6h- Nohe a~paragine wrinkled, white brown agar yellowish- (2-9-10) (ISP 5) brown (2-9-10) ~31~63~

TABLE 4 (Cont~d) Medium Growth Aerial Reverse Soluble Mvcelium Piament Peptone- Moderate, None Pale None yeast smooth, yellowish-extract- pale brown iron agar yellowish-(ISP ~) brown (4-8-9) (2-8-9) , _ _ Tyrosine Abundant, None Dull None agar raised, yellow (ISP 7) wrinkled, (10-8-~) pale yellowi6h-brown (14-8-9) Sucrose Abundant, None Pale None nitrate raised, yellowi6h-agar wrinkled, brown pale (4-8-8) yellow (12-8-10) Glucose- Moderate None Yellowi6h- None asparagine smooth, gray agar yellowish- (2-9-10) gray (2-9-10) 6 3 ~

TABLE ~ ~Cont'd) _ _ _ _ .
Medium Growth Aerial Rever~e Soluble Mvcelium P ament Nutrient Modera~e None Pale None agar 6mooth, yellowi~h-(Difco) ~ale brown yellowi~h- (6-8-9) brown (6-8-9) Water Poor, None Yellowi~h- None agar 6mooth, gray yellowi6h- (1-9-10) gray (1-9-10) Potato Moderate Poor, Yellowish- None extract- gmooth, white gray carrot yellowi6h- (2-9-l0) extract gray agar (2-9-l0) ._ 3._ PHYSIOLOGICAL PROP~RTIES.

The ehy6iological eropertie~ of ~train SANK
60983 are shown in Table 5.

Decompo~ition: ~denine Ca6ein Xanthine Hypoxanthine +
Urea +

22 ~318630 TABLE S (Cont'd) Hydrolyfiis of starch +
Liquefac~ion of gelatin +
Coagulation of milk Peptonization of milk Reduc~ion of nitrate +
Secretion of deoxyribonuclea6e +

Melanin formation: ISP 1 Acid production from:
Sodium Acetate Sodium Succinate Sodium Citrate Sodium Pyruvate Sodium Tartarate D-Gluco6Q +
L-Arabinose +
D-Xylose +
Ino6itol +
D-Mannitol +
D-Fructo6e +
L-Rhamno6e +
Sucro6e Raffino6e +

23 13 1 8~3 0 TABLE 5 (Cont_~d) . ~
Utilization of D-Glucose +
carbon 60urce6: L-Ar~binose ~-~ylo~e +
Inositol +
D-Manrlitol +
D-Fructose L-Rhamnose +
Sucrose Raffinose +

Growth in NaCl: 3% w/v 5~ w/v +
7% w/~ +
10% w/v Range of growth 10C
temperature: 20C +
28C +
37C +

In the above Table, "+" means po6itive, ~'-" means negative and "+" means B lightly po 6 itive.

Although coagulation and peptonization of milk are both reported as negative, they may occasionally turn positive after long-term culture.

4. Whole Cell ComPOnent6 Acid hydrolyzates of bac~erial cells were as&ayed by paper chromatography, u~ing the me~hod of M.P. Lechevalier et al. ~I~The Actinomycetes Taxonomy~!, page 225 (1980)].
meso-Diaminopimelic acid, arabinose and galacto~e were 24 ~3 i 863 a found to be present in ~he cell walls, which are thus of Type IV, whilst the whole cell 6ugar pattern i6 of Type A The characteristic acyl group of the cell wall was also investigated by the method of Uchida et al. ~J. Gen.
Appl. Microbiol, 23,2~9 (1977)] and found to be ~he acetyl group.

None of the known genera of actinomycete~ has been reported to be capable of forming 6pores in the middla of the hyphae. However, from a comparison of other characteri6tics, the new 6train i~ clearly related to the genera ActinoDolv6Dora, SaccharoPolys~ora, Pseudonocardia and MicroDolv6~0ra. However, both ActinoPolY6Pora and Saccharop_lyspora allow spores to grow only on the tips of aerial hyphae, and the for~er is a highly halophilic genus, whilst the characteri6tic acyl group of the cell wall of the latter i6 the glycolyl group. For the6e reason6, the new strain SANK 60983 cannot be assigned to either of the6e genera. Although strains of the genu6 Pseudonocardia can grow spores on the aerial hyphae and on the vegetative mycelium, a6 does 6train SANK 60983, the 6ite of it6 growth takes place only at the tip of the hyphae and, moreover, lt6 hyphae characteri6tically grow by budding thus, strain SANK 60983 cannot be as6igned to the genus P6eudonocardia.

The only difference between the genus MicroDol~sPora and 6train SANK 60983 is that the site of growth of spores of MicroDoly~ora iB limited to the tips of the hyphae, whereas that of SANK 60983 iB at both the tip and the middle of the byphae.

At the present time, when there ha~ been virtu~lly no discu6sion in learned circles as to the implication~ for taxonomy of differences of thi6 type, it would seem inappropriate to differentiata between genera solely on the basis of difference6 in the si~e of growth of their 13~

spores. Accordingly, it seems most sati6factory to regard the strain SANK 60983 as repre6entative of a new species of the genu6 MicropolYs~ora and it h~s, accordingly, been named MicropolYspora 6p. SANK 60983. It should, however, be remembered that a~ignment of a strain of microorgani6m to any particular species, genus or even family i8 largely a matter of consen~us amongst tho~e experienced in the study of ~he particular class of microorgani~m involved and ~he original a6signment of a microorganism can be, and not infrequently i8, changed after wider discus6ion.

The strain SANK 60983 ha~ been depo~ited with the Fermentation Re6earch Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan, on 10th March 1983 under the acces6ion No. FER~ P-6985 and was re-deposited in accordance with the conditions stipulated by the Budapest Treaty with said Fermentation Research In6titute on 4th June 1984 under the acces6ion No. FER~ BP-538.

I~ has been established that strain SANK 60983 produces chloropoly6porin6 B and C. However, as i6 well known, the properties of microorganisms falling within the general category of the actinomycete6 can vary considerably and ~uch microorganisms can readily undergo mutation, both through natural cause6 and as the re~ult of induction by artificial means. Accordingly, other microorqanisms which can be clas~ified within the genus Micropolvspora and which 6hare with the 6train SANK 60983 the characteristic ability to produce chloropoly6porins B
and C can al60 be used to produce the 6tarting material for the process of the present invention.

The cultivation of microorganisms of the genus MicroPoly6pora to produce chloropoly6porin B and C can be performed under conditions conventionally employed for the cultivation of actinomycetes ~pecies, preferably in a 1318~3~

liquid culture, and de6irably with 6hakin~ or stirring and aeration. The nutrient medium u6ed for the cultivation i6 completely conventional and contain6 such confitituents as are commonly u6ed in the cultivation of the actinomyce~es. Specifically, the medium should contain an assimilable carbon source, sui~able example of which include gluco~e, maltose, sucro6e, mannitol, molasses, glycerol, dextein, starch, soybean oil and cottonseed oil;
an assimilable nitrogen ource, suitable examples of which include soybean meal, peanut meal, cottonseed meal, fish meal, corn steep liquor, peptone, meat extract, pres6ed yeast, yeas~ extract, sodium nitrate, ammonium ni~rate or ammonium sulfate: and one or more inorganic salts, ~uch a6 sodium chloride, pho6phates, calcium carbonate and ~race metal 6alt6. Where cultivation is effected in a liquid medium, it i8 generally desirable to incorporate an anti-foaming agen~ (for example silicone oil, vegetable oil or a suitable surfactant) in the medium, The cultivation is suitably performed a~ a sub~tantially neutral pH value and at a temperature of from 2~ to 30C, more preferably at about 28C.

The production of chloropoly6porin6 B and C as cultivation proceed6 may be monitored by a variety of conventional microbiological assay techniques for monitoring the production of antibiotic~ (when they are produced by microbial culture) and which require little or no elaboration here. A suitable technique might be the paper disc-agar diffusion a66ay (using, for example, a paper di6c of diameter about 8mm produced by Toyo Kagaku Sangyo Co.f Ltd) and using, for example, Bacillu~ subtili~
PCI 219 or StaPhylococcus aureu6 FDA 209P JC-l as tbe ~e~t organism.

The amount of chloropolysporins B and C produced normally reaches a maximum after cultivation has proceeded 27 ~ ~1 8~ ~Q

for 55-70 hour6 and i~ is clearly desirable to separate the chloropolysporin~ from the culture medium no later than the time when this maximum ha6 been reached.
However, this period may vary, depending upon the cultivation conditiong and technique~, and a shorter or longer period may be appropriate, depending upon the circum6tance~. The correct cultivation time may readily be a~6es6ed for every ca6e by routine experiment, u6ing suitable monitoring techniques, e.g. as described above.

Chloropolysporin6 B and C are mainly relea6ed into the liquid portion of the cultured broth and can thu6 be eecovered by removing solid matter, including the mycelium, for example by filtration (preferably u6ing a filter aid such a6 diatomaceou6 earth) or by centrifugation. It can then be recovered from the separated liquid portion by conventional technique6 and, if desired, then purified.

Chloropolysporin6 B and C are preferably 6eparated from other products in said liquid portion by mean6 of an ad60rbent, either by ad60rbing the impuritie6 or by adsorbing the chloropoly6porins or by ad60rbing both separately or together and then eluting the chloropolycporins. A wide range of ad60rbQnt6 may be u6ed; examples which we have found to be particularly sati6factory include: activated carbon; and adsorbing re6ins such a~ Amberlite (registered trade mark) XAD-2, XAD-4 or XAD-7 (products of Rohm and Haa6), Diaion (regi~tered trade mark) ~P 10, HP 20, CHP 20P or HP 50 (products of Mitsubishi Chemical Industries Co., Ltd.) and Polyamide gels (a product of Woelm Pharma, ~est Germany~.
The impuritie~ pre6ent in the liquid por~ion may be removed by pa6~ir.g the solution containing the chloropolysporin~ through a layer or column of one or more of the aforemen~ioned adsorbent6 or by ad~or~ing the chloropolysporin~ on one or more of the adsorbents and 2~ 13~8~30 then eluting the chloropolysporin6, either separately or together, with a 6uitable eluent. Suitable eluents include mixture6 of methanol, acetone or bu~anol with water.

The chloropoly6porins B and C thu~ obtained may be further purified by variou~ means. Suitable method~
include: partition column chromatography u~ing a cellulo~e product, such a6 Avicel (a regi6tered trade mark for a ~roduct of A~ahi Chemical Industry Co., Ltd.) or Sephadex LH-20 (a regi6tered trade mark for a product of Pharmacia, Sweden); reverse pha~e column chroma~o~raphy u~ing a carrier for the rever6e pha~e; extraction based on the di~ferences in di~tribution in solvents between chloropolysporins B and C and their contaminating impurities; or the counter-current distribution method.
These purification techniques may be used singly or in combination and may, if needed, be repeated one or more times.

If de~ired, chloropolysporin~ B and C can be separated from each other by chromatography. A preferred 6y6tem for this purpoBe i8 Sy6tem 500 (a product of Waters Co.), using the Preppack C18~cartri~ é. A suitable eluent is a buffered mixture containing acetonitrile and maintained at a slightly acidic pH value.

Depending upon the culture condition~, chloropolysporins B and C can exist in the mycelium from the cul~ure broth and can be extracted therefrom by conventional techniques. For example, ~hey can be ex~racted with a hydrophilic organic solvent (such a6 an alcohol or acetone), and then the solvent removed from the exteact to leave a re6idue, which i6 dis601ved in an aqueous medium. The chloropQlysporins can be extracted from ~he resulting 601ution and purifie~ as de6cribed above.

Chloropoly~porin~ B and C thu6 obtained have, a~ ~heir sulfates, the phy~ical and chemical propertie6 described above. They are normally and preferably 6eparated from the culture bro~h in the form of a water-601uble sal~ and are most conveniently charac~eri~ed in the form of such a salt, i.e., as herein, in the form of the sulfate, since chloropolysporin~ B and C them6elves (i.e. the free ba6es) are insoluble in water.

Where the chloropolysporin B or C i6 isolated in the f~rm of a salt, it may be converted to the free base by conventional mean~, such a6 the use of ion-exchange resins or of adsorbents for reverse phase chromatography. An aqueous solution of a salt will normally have an acidic pH
value: adjustment of this pH value to approximate neutrality will result in mainly preciei~ation of the free ba~e, which may then be collected by suitable means, e.g.
filtration or centrifugation. This product will, however, normally be contaminated by impurities, including minor proportion6 of the relevant salt , and will, therefore, normally require further purification. Accordingly, a more preferred method is by using, for example, a suitable ion-exchange resin or an adsorbent for reverse phase chromatography. These compounds, however, ~hare wi~h known glycopeptide antibiotics, such as avoparcin, the property of being very difficult to isolate in the form of the ~ree base [see e.g. W.~. McGahren et al., Journal of Antibiotics, XXXVI, 12, 1671 (1983)] and they are, accordingly, preferably isolated and employed in salt form.

The invention is further illustrated by the following non-limiting Examples, which describe the production of chloropolysporin C by enzymatic hydrolysi6 of chloropolysporin B, and by the following Preparation which describes ~he production of the starting material by microbial culture.

~3~86~

3.8 g of ~he enzyme naringina6e (product of Sankyo Co.
Ltd.) were added to a solution of 10.5 g of chloropolysporin B in 2 litres of 0.06M aqueous pho~phate buffer (pH 5.8), and the mixture was allowed to react for 21 hours at 37C under gentle stirring, until the chloropolyspor}n B ~tar~ing material could no longer be detected by HPLC.

After completion of the reacton, the reaction mixture wa~ pas6ed through a column containing 370 ml of Diaion HP20 ad~orbent resin (product of Mitsubi~hi Chemical Indu6trie6 Ltd.), and the column was then wa6hed with water and eluted with 700 ml of 50~ v/v aqueous acetone.
The solvent was evaporated off from the eluate, under reduced pre6sure, and the residue was lyophilized giving 7.8 q of crude product, which was then purified by the following procedure.

The whole of the crude product was dissolved in 30 ml of 50% v/v aqueous methanol, and the solution was ad60rbed r c,~;/e".~, rfc ,~ " GI
on a 3 x 50 cm column of Toyopearl HW-~OF resln ta~product of Toyo Soda Co. Ltd.~. The column was developed and eluted with 50% v/v aqueous methanol. The eluate was collected in fractions of 20 ml each, and the fraction~ in which chloropolysporin C was detected by HPLC were selected. These fractions were combined and the solvent was distilled off from them under reduced pres6ure. The re6idue was acidified to pH 4.0 with hydrochloric acid and lyophilized, yielding 5.6 g of chloropolysporin C
hydrochloride.

200 mg of the enzyme 6clase (product of Sankyo Co.
Ltd.) were added to a solution of 10 mg of 31 ~318~3~

chlo~opoly~po~in B in 5 ml of o.lM ~queou~ phosphate buffer, and the mixture was allowed to react for about 24 hour~ at room t~mperature, under gentle stirring After completion of the reaction, the mixtu~e wa6 ad60rbed on~o a column containing 5 ml of Diaion HP 20 re6in ~product of Mit6ubishi Chemical Indu6trie6 Ltd.), and the column wa6 then wa6hed with water and eluted with 50~ v/v aqueou6 acetone. The fractions of eluate containing chloropoly6porin C were conden~ed under reduced pres6ure and the re~idue was lyophilized, giving 5 mg of crude product. The crude product may be purified by the following procedure.

8 g of crude product containing chloropoly6porin C was dis601ved in 40 ml of water and the 601ution adju6ted to pH 3.0 with dilute hydrochloric acid. Thi6 solution was ad60rbed onto a 4 x 40 cm column of Polyamide gel (product of Woelm GmbH ~ Co.), which wa6 developed with water, and fractions of 20 ml each were collected. The fraction6 containing chloropoly6porin C (as detected by HPLC) were combined and conden6ed under reduced pre66ure. The re6idue wa6 acidified to pH 4.0 with hydrochloric acid and then lyophilized, giving 4.6 g of chloropoly6porin C
hydrochloride.

PRE~PARAT I ON

one loopful growth of MicroDolY~Dora 6p. SANK 60983 wa6 inoculated into a 500 ml Erlenmeyer flask containiag 80 ml of medium A, which ha6 the following compo6ition (percen~age6 are by weight):

MEDIUM A
Glucose 3%
Pressed yeast 1%
Soybean meal 3~
Calcium carbonate 0.4%
Magnesium sulfate 0.2%
Anti-foaming agent (supplied under the trademark Nissan CB-442) 0.01%
Water the balance (adjusted to pH 7.0~

The microorganism was then cultured for 84 hours at 28C, using a rotary shaker at 220 r.p.m.

25 ml of the resulting seed culture were inoculated into each of four 2 litre Erlenmeyer flasks, each containing 500 ml of medium B, which has the following composition (percentages are by weight):

MEDIUM B
Glucose 5%
Yeast extract 0.1%
Soybean meal 1%
Polypepton (a trademark for a product of Daigo Eiyo Co. Ltd., Japan) 0.4%
Beef extract 0.4%
Sodium chloride 0.25%
Calcium carbonate 0.5%
Anti-foaming agent (supplied under the trademark Nissan CB-442) 0.01%
Water the balance (adjusted to pH 7.2) The microorganism was then cultured at 28C for 24 hours, using a rotary shaker at 220 r.p.m.

~1863~

The resulting cultured broth~ were combined. 750 ml of thiS broth were then inoculated into each o~ two 30 litre jar fermentors, each containing 15 litres of medium B, and the microorganism was then cultured at 28C for 69 hours, whilst aerating at the ra~e of 15 li~res per minute and stirring.

At the and of this time, batches of cultured broth separately cultivated as described above were combined to give a total of 30 li~res of cul~ured broth. Celite 5g5 (a registered trade mark for a product of Johns-Manville Products Corp, New Jersey, U.S.A.) filter aid was added to the cultured broth and the mix~ure wa6 filtered, to give 30 litres o~ a filtrate. This filtrate was adsorbed on 3 litre~ of Diaion HP 20 (a product of Mitsubishi Chemical Industrie~ Co., Ltd.), and the ad60rbent was wa~hed with water and then eluted with 50~ ~/v aqueous acetone. Acetone wa6 evaporated from the combined active fraction6 by evaporation under reduced pre6~ure; and the concentrate thus obtained was lyophilized, giving 44 g of a crude powder.

41 g of this powder were dissolved in water and adsorbed onto 1.8 litres of Diaion HP 20, wa~hed with 5 litre6 of water and 2 litres of 10~ v/v aqueous acetone, and ehen eluted with 4 litres of 50~ v/v aqueou6 acetone. The active fraction~ from the elution were collected and condensed to a volume of 1 litre by evaporation under reduced pressure. The condensate was centrifuged at 5000 r.p.m. and the resulting precipitate was dried, to give 9.6 g of crude powder containing chloropolysporin~ B and C.

This crude powder was dis601ved in 1 litre of 50 v/v aqueous methanol and then adsorbed on~o Z00 ml of acidic alumina (a product of Woelm Pharma, West Germany), which had previously been equilibrated with 34 ~ 63 ~

50% v/v aqueous methanol. The ad~orbed product was then eluted with the same solvent, and ~:he active fraction6, a total of 1.1 litres, were collected. The combined active fractions were pas6ed through 60 ml of Dowex 21 K
(OH ~, and eluted with water. The active fractions from this elution, a ~o~al ~olume of 1.2 li~res, were collected and ~hen condensed by evaporation under reduced pressure to a volume of 30 ml, This condensa~e was lyophilized, to give 1.23 g of powder. The powder wa~ dissolved in aqueous hydrochloric acid of pH 4.0 and then adsorbed onto 56 g of Polyamide filled wi~h water (a product of Woelm Pharma, ~est Germany). This was subjected to gradient elution with 400 ~1 of water and 1.2 litres of methanol, in 20 ml fractions, up to raction 80. Fractions 30-60 were collected and combined. The methanol was distilled off under reduced pres6ure and the resulting concentrate was lyophilized, to give 738 mg of a white powder containing chloropoly6porins B and C.

4.4 g of this crude white powder containing chloropolysporins B and C were dissolved in 80 ml of a mixed solvent consi~tinq of 15 parts of acetonitrile and 85 parts of a buffer solution (containing 0.2~ wtv sodium heptanesulfonate, 2.5~ w/v acetic aeid and 0.5%
w/v concentrated aqueous ammonia): the solution was then ad60rbed on a Syste~ 500 chromatography system (a product of Waters Co), using a Preppack C18 cartridge. This was developed and eluted with the same mixed solvent as mentioned above at a flow rate of 100-150 ml per minu~e. Chloropolysporin ~ was elu~ed in the solvent after between 800 ml and 1700 ml of the eluant had passed ~hrough the cartridge, whils~
chloropolysporin C was eluted after between 1700 and 4700 ml of the eluant had passed.

~3~3~

The active fractions containing chloropoly6porin B
were collected and adju6ted to a pH value o~ 7Ø They were then concentrated by evaporation under reduced pres6ure, to di6till o~f the acetonitrile. The re~ulting concentrated 601ution was ad~orbed on a Diaion HP 20 column (100 ml), washed with water. and then eluted with 500 ml of 70~ v/v aqueous ace~one. The eluate wa~ condensed by evaporation under reduced pre6sure, and the residue wag lyophilized to afford chloeopolysporin B heptane6ulfonate as a powder.

200 mg of this powder were di~olved in S ml of water, and then 1 ml of a 10~ w/v aqueou6 601ution of sodium dodecyl~ulfate was dropped into the re6ulting solution. The precipitate which formed was collected by centrifugation at 3000 rpm for 10 minute6. Thi6 precipitate was su~pended in water and the su6pen6ion was again centri~uged at 3000 rpm for 10 minute6 to wash the precipitate. Thi6 operation was reeeated a further three times to wash the precipitate. The precipitate was then dissolved in 3 ml of methanol and the insoluble residue was filtered off. 2 ml of a 0.5M methanolic solution of teiethylamine 6ulfate were added dropwise and the re6ulting precipitate wa6 collected by centrifugation at 3000 rpm for 10 minute~. This precipitate was su6pended in a small amount of methanol and again centrifuged at 3000 rpm for 10 minute6. This wa6 repeated a further three times to wa6h the precipitate. The precipitate wa6 then dis601ved in 1.5 ml of water and the in601uble residue wa~ filtered off.
Lyophilization of the fil~rate gave 65 mg of chloropolysporin B ~ulfate.

Claims (8)

1. A process for preparing the antibiotic chloropolysporin C, represented by the planar structural formula:

in which R1 represents a 1-L-ristosamine residue, R2 represent6 a 1-D-mannose residue, R3 repre6ents a 1-D-glucose residue, and R4 represents a hydrogen atom, and which compound as its sulfate is characterized by the properties:

(a) it takes the form of an amphoteric white powder, soluble in water;

(b) specific rotation: [.alpha.]25-64.4° (C=1.08, 0.1N
aqueous hydrochloric acid, sodium D-line);

(c) elemental analysis: .

C, 50.53%; H, 4.69%; N, 6.14%; Cl, 5.62%; S, 1.12%;

(d) on acid hydrolysis it yields:

neutral sacchasides: glucose and mannose;
amino acids: 3-chloro-4-hydroxyphenylglycine and N-methyl-p-hydroxyphenylglycine;

(e) ultraviolet absorption spectrum:

as illustrated in Figure 4 of the accompanying drawings, having an absorption maximum .lambda.max at 280nm (ElCm=57) in a 0.1 N solution of hydrochloric acid, the absorbence, E, being measured at a concentration of 1% wtv:

(f) infrared absorption spectrum:

the infrared absorption spectrum (? cm-1) measured on a KBr disc is as shown in Figure 5 of the accompanying drawings;

(g) nuclear magnetic resonance spectrum:

the nuclear magnetic resonance spectrum (.delta. ppm), measured at 400 MHz in deuterated dimethyl sulfoxide using tetramethylsilane as the internal standard, is as illustrated in Figure 6 of the accompanying drawings;

(h) solubility:

soluble in water, sparingly soluble in methanol and acetone, and insoluble in ethyl acetate, chloroform and benzene;

(i) color reactions:

positive in ninhydrin and Rydon-Smith reactions;

(j) thin layer chromatography:

Rf value=0.65, using a cellulose sheet (Eastman) as adsorbent and a 15:10:3:12 by volume mixture of butanol, pyridine, acetic acid and water as the developing solvent;

(k) molecular formula:

C77H79O3ON8Cl3, O.5H2SO4, 5H2O;
(1) molecular weight:

the molecular weight, measured by FAB-MS, is 1700 (MH+, 1701);

which proces6 is characterized by subjecting to enzymatic hydrolysis, using rhamnosidase, the antibiotic chloropolysporin B, which has the said planar structural foemula (I) in which R1 represents a 1-L-ristosamine residue, R2 resresents a 1-D-mannose residue, R3 represents a 2-D-glucose residue, and R4 represents a

2-L-rhamnose residue, and which as its sulfate is characterized by the properties:

(a) it takes the form of an amphoteric white powder, soluble in water;

(b) specific rotation: [.alpha.]25-64.5° (C=1.04, O.lN
aqueous hydrochloric acid, sodium D-line);

(c) elemental analysis:

C, 48.33%; H, 5.05%; N, 5.48%; Cl, 5.11%; S, 1.00%;

(d) on acid hydrolysis it yields:

neutral saccharides: glucose, mannose and rhamnose, amino acids: 3-chloro-4-hydroxyphenylglycine and N-methyl-p-hydroxyphenylglycine;

(e) ultraviolet absorption spectrum:

as illustrated in Figure 1 of the accompanying drawings, having an absorption maximum .lambda.max at 280nm (Elcm=5l) in a 0.1 N solution of hydrochloric acid, the absorbence, E, being measured at a concentration of 1% w/v:

(f) infrared absorption spectrum:

the infrared absorption spectrum (? cm-1) measured on a KBr disc is as shown in Figure 2 of the accompanying drawings;

(g) nuclear magnetic resonance spectrum:

the nuclear magnetic resonance spectrum (.delta. ppm), measured at 270 MHz in deuterated dimethyl sulfoxide using tetramethylsilane as the internal standard, is as illustrated in Figure 3 of the accompanying drawings;

(h) solubility:

soluble in water, sparingly soluble in methanol and acetone, and insoluble in ethyl acetate, chloroform and benzene;

(i) color reactions:

positive in ninhydrin and Rydon-Smith reactions;

(j) thin layer chromatography:

Rf value=0.65, using a cellulose sheet (Eastman) as adsorbent and a 15:10:3:12 by volume mixture of butanol, pyridine, acetic acid and water as the developing solvent;

(k) high voltage paper electrophoresis:

using Toyo's filter paper No. 51A in a 0.1M
TRIS-hydrochloric acid buffer solution of pH 7.5 (3300 volt/60cm, 1 hour); the migration distance (detected by bioautography with Bacillus subtilis PCI 219) from the origin to the cathode is 4cm;

(l) molecular formula:

C83H89O34N8Cl3. 0.5H2SO4. 1OH2O;
(m) molecular weight:

the molecular weight, measured by FAB-MS, is 1846 (MH+, 1847).

2. A process as claimed in claim 1, in which the hydrolysis is performed at a pH in the range of from 5 to 6.

3. A process as claimed in claim 1, in which the hydrolysis is performed at a temperature of from 30°C to 40°C.

4. A process as claimed in claim 1, in which the hydrolysis is performed with an enzyme preparation having an .alpha.-rhamnosidase activity in the range of from 200 units/mg to 500 units/mg.

5. A process as claimed in claim 1, in which the hydrolysis is performed with an enzyme concentration which corresponds to a weight ratio of rhamnosidase to chloropolysporin B within the range of from 1Ø05 to 1:20.

6. A process as claimed in claim 1, in which the hydrolysis is performed in a solution containing from 1,000 to 10,000 .gamma./ml of chloropolysporin B.

7. A process as claimed in claim 1, in which the hydrolysis is performed in a culture broth containing chloropolysporin B as obtained by cultivation of a chloropolysporin-producing Micropolyspora strain.

8. A process as claimed in claim 1, in which the hydrolysis is performed for a period of time in the range of from is to 24 hours.