DE2550110A1 - PROCESS FOR THE CONVERSION OF CEPHALOSPORINE COMPOUNDS - Google Patents

Description

Dr. F. Zumstein Sr. - Dr. E. Assmann - Dr. R. Koenigsberger Dipl.-Phys. R. Holzbauer - Dipl.-Ing. F. Klingseisen - Dr. F. Zurnstein jun.

PATENT LAWYERS

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Case Ceph 204
10 / DE

GLAXO LABORATORIES LIMITED, Greenford, Middlesex / England

Process for converting τοη qephalosporin compounds

The invention relates to the conversion of cephalosporin compounds and in particular the enzymatically catalyzed hydrolysis of 3-acyloxymethylcephalosporins.

The cephalosporin compounds mentioned herein are named generally with reference to the term "Cepham" (J.Am.Chem.Soc. 1962 , 84 , 3400). The term "oephem" refers to the cepham structure with a double bond.

The 3-hydroxymethylcephalosporin compounds are valuable intermediates in the synthesis of a number of cephalosporin antibiotics with substituted methyl groups in 3-Stellur ± g due to the chemical reactivity of the hydroxyl group, which is why the / Jiydroxv; _x

fmet'Eylgruppe easily into the desired 3- (substit.-methyl) group can be transferred. In addition, the 7-acylanido ~ 3-

80982Q / 1U8

hydroxymethylceph-3-em-4-carboxylic acids antibiotic properties. The production of 3-hydroxymethylcephalosporin compounds by hydrolysis of 3-acyloxymethylcephalosporins, in particular naturally occurring 3-acetoxymethylcephalosporin compounds produced by fermentation, such as cephalosporin C ß, 6R, 7R) -3-acetoxyme thy 1-7- (D-5-amino-5 -carboxypentanamido) -ceph-3-em-4-carboxylic acid_7 and derivatives thereof, for example H-protected derivatives and compounds in which the DS-amino-S-carboxypentanoyl group has otherwise been converted or removed, and if necessary has been replaced by another acyl group , is therefore of considerable interest.

The hydrolysis of 3-acyloxymethyl-ceph-3-em-4-carboxylic acids too their 3-hydroxymethyl analogs has been developed by chemical methods generally found to be quite useless since such reactions are of a rapid and essentially irreversible nature Lactonization are accompanied, a reaction of 3-hydroxymethylund 4-carboxy-groups is included and / or by destruction of the ß-lactam ring system.

However, it has been found that it is possible to use 3-acyloxymethylceph-3-em-4-carboxylic acids by enzymatically catalyzed methods to hydrolyze under conditions whereby the lactonization and the degradation of the ß-lactam is essentially or completely avoided. Although esterases come from a number of sources, e.g. from plants, in which these enzymatically catalyzed methods can be applied, however, difficulties can arise in the isolation of sufficient quantities of esterase from some of these sources; the esterases, which are derived from microorganisms are therefore most expedient ^ in view of the relative ease with the microorganisms can be grown on a large scale using standard fermentation techniques to allow easy digestion of the esterase to offer.

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It has now been found that esterases from certain microorganisms of the Basidiomycetes class cause hydrolysis
of 3-acyloxymethyl-ceph-3-em-4 - carboxylic acids to their 3-hydroxymethyl analogs.

According to the invention, therefore, a process for converting ^ -Acylox ^ ethyl-ceph-S-em- ^ carboxylic acid to a 3-hydroxymethyl analog thereof by hydrolysis is created, which is characterized in that the hydrolysis is catalyzed by an esterase, which by cultivating a microorganism
of the Basidiomycetes class, which is capable of producing the required esterase activity.

The 3-Acyloxymethyleeph-3-em-4 - carboxylic acids, which according to the
Invention hydrolyzed include compounds of the general formula

ι ^{r2 R}

TtTL

J Nv _x ^ -CH ₂ O. CO.

3 (D

COOH
wherein R is an amino group or blocked amino group, for example a ^ _20 " ^car ^ ^ox y ^ ^sc ^ ^e acylamido group;
R is hydrogen or a lower alkyl, lower alkoxy, lower alkylthio, or lower alkanoy! Group (the term
As used herein, "low" denotes groups with
no more than 8, preferably no more than 6 carbon atoms);

R .CO is a C ₂ ^ ₂₀ carboxylic acyl group; and B is ~ S or (α- or ß-).

Acylamido groups R, which in the compounds of formula I

may be present are, for example, the following: the D-5-Am: üio-5-carbo: xypentanamido group, which occurs in naturally occurring, compounds produced by fermentation such as cephalosporin C are found; F-protected derivatives of the D-5-amino-5-carboxypentanamido group, e.g. wherein the amino group is substituted by a protecting group of the type described in British Patents 1,041,985, 1,302,015 or 1,313,207 is, for example a lower alkyl group, an aryl-lower alkyl ! group, an ary! group (e.g. 2,4-dinitrophenyl) or a Acy! Group, especially a lower alkanoyl group (e.g. acetyl, Propionyl or butyryl), an α-halo or α, cc-dihalo-lower alkanoy! Group (e.g. chloroacetyl or dichloroacetyl), an Aroy! group (e.g. benzoyl, chlorobenzoyl, mtrobenzoyl or tosyl), a lower alkoxycarbonyl group (e.g. t-butoxycarbonyl), a Ary 1-lower alkoxycarbony! Group (e.g. benzyloxycarbonyl) or a diacyl group such as phthaloyl; an acylamido group by converting the D-S-amino-S-carboxypentanamido group, e.g. a 4-carboxybutamido group, which is derived therefrom e.g. by enzymatic Oxidation is preserved; Formamido; a group of the formula

* R (CH ₀ ) _. CONS-

where R is a carbocyclic or heterocyclic aryl group (e.g. phenyl; phenyl substituted by one or more Halogens, hydroxy, lower alkyl, nitro, amino, - lower alkanoyl, lower alkoxy or lower alkylthio; Thienyl or furyl) or an aryloxy, arylthio, aryl-lower alkoxy or aryl-lower alkylthio group (e.g. phenoxy, phenylthio, 5-methy1-1,3,4-thiadiazol-2-ylthio or benzylthio) and η is a number from 1 to 4; a group of the formula

R ^a -OH-CONH-

60982GMU6

Where R ^{a is} an ary! Alkoxy or lower-alkylthio) and X is amino, protected amino (e.g. containing any of the N-protective groups mentioned in connection with the 1-5-amino-5-carbo2ypentanamido group, e.g. a t.-butoxycarbony group), Carboxy, carbalkoxy or hydroxy ;, a group of the formula

R ^a -C-CO1IH- ■

ff

H.

OR ^b

where E ^{a has} the meaning given above (e.g. where R ^{a is} phenyl, substituted phenyl, haphthyl, thienyl, furyl or pyridyl), and R is hydrogen, acyl (e.g. lower alkanoyl), lower alkyl (e.g. methyl, ethyl, Propyl or butyl), cycloalkyl (e.g. with 5 to 7 carbon atoms, such as cyclopentyl or cyclohexyl), aryl (e.g. carbocyclic aryl, such as phenyl), or aryl-lower alkyl (e.g. benzyl or phenethyl). Examples of groups R which come under the above general formulas which may be present in the compounds of the formula I include pheny / acetamido, chienylacetamido, 2-hydroxy-2-phenylacetamido, 2-t.-butoxycarbonylamino-2-pheny! acetamido and syn-2-furyl-2-methoxyiminoacetamido.

Acyl groups R. CO, which are contained in the compounds of formula I. include a number of aliphatic, araliphatic and aromatic groups, for example lower alkanoyl groups, such as acetyl, propionyl and butyryl; low-Alkenoy! groups, such as crotonoyl; Aryl-lower alkanoyl groups, such as Phenylacetyl; and aroyl groups such as benaoyl. As stated above, the method according to the invention finds particular application the hydrolysis of cephalosporin C and derivatives thereof, i.e.

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Compounds of the formula I in which R .CO is an acetyl group.

The groups R and B in formula I are preferably hydrogen or ^ S.

The level of the desired esterase activity produced by a particular microorganism of the Basidiomyeetes class can easily be determined, for example by some preliminary experiments on a small scale using a suitable 3-acyloxymethylceph-3-em-4-carboxylic acid substrate, e.g. cephalosporin C. , the reaction system then being checked, for example by thin layer chromatography, to determine the amount of 3-hydroxymethyl-eeph-3-em-4-carboxylic acid formed. The following method can be used for example for yeasts: the test organism can be subcultured in a usual Hährmedium such as agar slant with 2-DG $ lucose, \ fo yeast extract, 1 fo peptone, 0.5 ° f potassium dihydrogen phosphate and

2 fo agar powder, and the cultivation is carried out 2 to 4 days at 25 ⁰ C.

An inoculum is transferred from the incline by means of a sterile loop into a miniature flask which contains 3 ml of medium containing 2.7 fo D-glucose, 1.3 fo yeast extract, 1.3 fo oxoid peptone and 0.7 fo potassium dihydrogen phosphate and furthermore 1 ml of an 8- fold solution of cephalosporin C potassium salt. The flask, along with an uninoculated flask, is used as a blank

Shaken for 3 days at 25 ^° C. and the contents then analyzed by thin layer chromatography.

Samples of the contents of the flask (1 μl) and standard samples of cephalosporin C and its 3-hydroxymethyl analogues (1 μg of 20 mg / ml) v / ground on cellulose plates, which are buffered to pH 5.8 to 6.0 with M / 15 phosphate buffer, spotted and the plates are developed in 70% aqueous n-propanol. After drying, the plates are examined under ultraviolet light and the

'609 * 20/1146

Degree of deacetylation of cephalosporin C becomes qualitative determined by the intensity of the spots of the starting material and the 3-hydroxymethyl product from the Eolbenin content.

For non-yeasts, the following slightly modified procedure can be used.

The! Least organism is grown on an agar medium comprising 4 io maltose, one fo peptone, 2.4 $> malt extract agar powder and 2.5 $ and is set prior to sterilization to pH 7 »5th

The slants are incubated for 7 days at 25 ⁰ C and an inoculum is from each slope in 40 ml of a liquid medium transferred, the 2 fo glucose, 1 fo yeast extract, 1 fo peptone and 0.5 io potassium dihydrogen phosphate at pH 5.8. The culture is incubated for 10 days at 25 ⁰ C on a shaker and used a second liquid stage of the same medium with the addition of 0.1 io cephalosporin C to oculieren. After a further incubation period of 3 to 8 days, depending on the rate of growth, the cultures will. harvested.

Homogeneous suspensions of each culture are made by vigorous mixing made in a blender.

There are prepared reaction mixtures containing 2 ml of the homogeneous ■ NEN cell suspension, 2 ml of 0.5 M potassium phosphate buffer pH 6.0 and 6 ml of a 3.33 $ solution of cephalosporin C and are included for 5 days at 25 ⁰ C. incubated with shaking.

Each sample is buffered by thin layer chromatography using cellulose beBchichtetenPlatten to pH 5.8 to 6.0, and using a 70 io v / v aqueous n-Propanollösungsmittelsystems tested. The presence of 3-hydroxymethylephalosporin product is determined by UV irradiation of the thin layer chromatography plate.

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Zones corresponding to authentic samples of (6R, 7R) -7- (D-5-Aiaino-5-carboxypentanamido) -3-hydroxymethyl-ceph-3-em-4-carboxylic acid (DAC) are eluted and the amount of DAC is determined by determining the optical density at 260 nm.

Organisms. which have no esterase activity do not generate a spot corresponding to the 3-hydroxymethyl compound.

Microorganisms of the Basidiomycetes class, which are used as a source of the desired esterase activity are useful include: yeast microorganisms belonging to the order TJstilagenales or belong to the family Sporobolomycetaceae. Yeast microorganism

aG ejms Y men in the Ustilagenale3 include organisms of the species sporidium and Bhodosporidium, for example strains of Iieueo-sporidium scottii, Rhodosporidium toruloides and Rhodosporidium sphaerocarpum. Yeast microorganisms in the Sporobolomycetaceaen

XG enus X /
include organisms of the species V Bullera, Sporidiobolus and Sporobolomyces, for example strains of Bullera alba, Bullera tsugae, Sporidiobolus johnsonii, Sporidiobolus ruinenii, Sporobolomyces roseus and Sporobolomyces salmonicolor. Non-yeast microorganisms of the Basidiomycetes class which produce the desired esterase activity can also be used. Examples of such microorganisms are smut fungi of the order Ustilagenales, for example strains of Ustilago maydis; Microorganisms of the order Agaricales, for example strains of Schizophyllum commune and Coprinus comatus; and microorganisms of the order Polyporales, for example strains of Polyporus dichrous, Polyporus versicolor, Poria monticola. If IT leaky yeast microorganisms are to be used, it may be advantageous to select such a microorganism that can be grown in submerged culture.

Yeast microorganisms of the genus Rhodosporidium, for example Organiasn of the species Rhodosporidium toruloides, such as Rhodosporidium toruloideG CBS 14 and Rhodosporidium toruloides CBS 349 and mutants thereof, are a particularly useful source of

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the esterase activity due to the high pus of the desired End product they produce.

In general, the microorganism used as the source for the Esterase activity used can be conveniently preserved, either by freeze-drying a suspension of the microorganism in 2 S & -iger skimmed milk in sealed glass ampoules or by ordinary subculture of the microorganism on solid medium, e.g., yeast extract / peptone / agar medium. The freeze-dried Culture can be restored by adding sterile water. Freeze drying is the preferred method for the storage of yeast cultures.

The wiedersuspendierten microorganisms may be grown, for example by Aufstreieh vaccination on a solid Hährmedium, for example an aqueous medium containing 2 fo glucose, 1 fo Hefeexfcrakt, 1 fo peptone and 0.5 ^ potassium dihydrogen phosphate, solidified with 2 fo Agar (all percentages are by weight / Volume) at pH 5.6. The surface cultures are grown, for example, is covered at 25 ⁰ C until the agar and can then several months at a low temperature, for example, are kept 5 ° C. " The organism can be grown submerged, expediently at 25 ° C., by using, for example, a liquid culture medium that contains a source of carbon, nitrogen and trace elements

inoculating a sample of the surface culture; an example of a suitable culture medium for this purpose is that described above for surface growth without agar is described. It is often convenient to keep a liquid culture of the organism, which are used for inoculation of subsequent liquid cultures,., e.g.

i5Cgrvienae ^ b_weraen can, _f

the production of breeds on a large scale because this makes the The inconvenience of having a large surface area for liquid inoculations is avoided, and a larger amount of inoculation can be used. The subsequent incubation of such a liquid culture for a suitable time, for example about 3 Sage, appropriately obtained at 25 ° 0 microorganism suspension can then be used as a source for the esterase, which for

6Ö982Ö / 1US

Deacylation of the 3-acyloxymethyl-eeph-3-em-4-carboxylic acid used as starting material is used. In the case of some organisms, for example Leucosporidium scottii is a lower one Incubation temperature, e.g. around 17 ° C »more appropriate.

The formation of the esterase can in certain cases be accelerated by adding an inducing agent to the liquid growth medium. Suitable such agents include cephalosporin C, cephalotin Z ^ 6R, 7R) -3-aceto2q5rmethyl-7- (thien-2-yl) -acetamidoceph-3-em-4-carboxylic acid7 and its salts (e.g. the alkali salts such as the potassium salt ), wherein the means, such as potassium cephalosporin C, have proven effective in amounts of less than 0.1 $> weight / volume. The inducing agent is expediently added to the liquid culture medium after sterilization. The agent can be sterilized by passing it through a sterile filter, while the culture medium can be sterilized by, for example, heating in an autoclave at about 121 ^° C. and 10 500 kg / m 2, for example for 15 minutes.

The esterase can be used in several different forms for hydrolysis of 3-acyloxymethylceph-3-em-4-carboxylic acid can be used. For example, a sample of the liquid culture medium itself can be used as the esterase source, if desired after the microorganism cells have been torn apart, e.g. by conventional methods such as ultrasound treatment or treatment with lytic enzymes. An aqueous extract of the suspension resulting from disruption of the cells can also be used will. Alternatively, the whole cells filtered from the liquid culture medium can be used, likewise the corresponding filtrate; if desired, the fiI-stepped To use, the cells can be broken up again before filtering * for example as described above.

The use of the whole cells is a special part of the gate

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this slightly, for example as a cell suspension, from the liquid Growing medium can be separated, they can also be easily stored, e.g. as dried or frozen Paste, which can be added directly to the reaction mixture for hydrolysis and they are light, for example by filtration, can be removed from the reaction mixture after the hydrolysis has ended. The cells separated in this way can again, for example after washing with water, for the hydrolysis of further samples of 3-acylo3gnae "fcbylceph-3-em-4-carboxylic acid starting material be used; enough esterase activity remains to produce a hydrolyzed cephalosporin product of satisfactory quality in constant yield, during several recyclings of the cells.

If desired, the whole cells in or on an inert Matrix, e.g. A polymer or a membrane, can be immobilized or fixed, e.g. by covalent bonding to an inorganic or organic polymer or by sticking in or on a laser, ^ .B * cellulose triacetate or

t bullets.)

in a wrapper, such as pearls or before going to the hydrolysis reaction system can be added to keep the cells protected and losses during their return for another operation stay to a minimum. A preferred matrix for use in immobilizing the cells is polyacrylamide gel. The esterase can also be used in a cell-free form, for example obtained by precipitation from a piltrate or cell extract obtained from the liquid culture medium as described above using a suitable protein precipitating agent such as a salt or solvent. For example, the precipitated, cell-free esterase can be added directly to a hydrolysis reaction system or it can dissolved in water and added as an aqueous solutioni

Alternatively, the esterase can be in a fixed, that is, immobilized Porm, can be used, e.g. by solubilization or

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Attachment to or in an inert matrix; suitable immobilized Shapes are those described in British Patent 1,224,947 and Belgian Patent 782,646. For example, an esterase obtained from an extract of the liquid culture medium or from the redissolution of precipitated esterase, be covalently bonded to an otherwise inert inorganic or organic polymer or attached to or in a fiber, for example a fibrous polymer such as cellulose triacetate, or on or in a membrane or to a polymer such as polyacrylamide gel an ion exchange resin are absorbed or in an envelope, such as pearls, globules or granules can be occluded. the Immobilized, i.e. immobilized, esterases of this type can advantageously be used in batch processes where the esterase is to be reused and in the hydrolysis of a continuous flow of 3 — acyloxy— methylceph ^ -em ^ -carboxylic acid by, for example, passage through a column containing the immobilized esterase.

The hydrolysis reaction is expediently initiated by contacting the esterase with an aqueous medium which contains the 3-acyl oxymethylceph-3-em-4-carboxylic acid or a salt thereof, for example an alkali metal salt such as the sodium or potassium salt, the medium suitably contains 0.5 to 10 fa weight / volume of the cephalosporin compound. The medium can, if desired, be sterilized before the hydrolysis, but it is not necessary that sterile conditions be maintained during the reaction and in such cases the hydrolysis with the gate part can also be carried out under non-sterile conditions.

the 3-acyloxymethyl-ceph-3-em-4-carboxylic acid by fermentation produced cephalosporin is, as is the case with cephalosporin C, so can that of the cultivation of the cephalosporin producing organism (e.g. an organism of the genus Cephalosporium) obtained fermentation broth itself with the

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Esterase treated - so the need for interim Separation of the cephal ο sp orin connection is bypassed. The mycelium can if desired from the broth before treatment but in many cases it is more convenient to use all of the broth straight away.

The hydrolysis can be carried out at a pH between 4 and 8, for example. The pH can be kept at a value of, for example, 6.0 during the entire reaction by using a buffer, for example phosphate buffer, but it may also be necessary to add smaller amounts of a strong base. The hydrolysis is expediently carried out at ambient temperature, ie about 25 ^° C., and advantageously with stirring and / or aeration of the reaction mixture.

The required amount of esterase or esterase-containing material for a given reaction can be determined by preliminary tests in can be determined on a small scale. The period of time for complete deacylation of the 3-acylo: zymeth.yl-ceph-3-em-4-carboxylic acid becomes on the nature of the starting material and the esterase and the applied Depending on the reaction conditions. The course of the reaction can be conveniently followed by the product by thin layer or paper chromatography using a suitable The carrier / solvent system combination separated and densitoinetrically is determined.

\ Jerm. the 3-acylo2: yinethylceph-3-em-4-carboxylic acid used as starting material has a D-5-amino-5-carboxypentanamido group in the 7-position, as is the case with cephalosporin C of the Pail, the hydrolytic deacylation process according to the invention can be carried out with a enzyme-catalyzed oxidative deamination of the group in the 7-position, e.g. to a 4-carboxybutanamido group, combined with a fungal oxidase, especially an oxide from the yeast Trigonopsis variabilis, as described in British patent 1 272 769 or the Belgian patent

£ 09820/1145

-H-

782 393 is described. The enzymatic ^ conversion of the groupings in 3- and 7-position can either be consecutive or at the same time.

That for the isolation of the 3-Hydrozymethyl-ceph. ~ 3-em-4-carboxylic acid product The method used depends on the nature of the product and the reaction system, but the usual ones are generally used Techniques applied. If, for example, whole cells are used as a source of ester, they can be filtered or centrifugation and reused if desired, and the solution is purified by filtration, e.g. a kieselguhr bed, further clarified. When the 3-hydroxymethylceph-3-em-4-carboxylic acid that is Desacety! -Cephalosporin C, so can this can be isolated, for example by desalination of the solution by absorption on charcoal, then by elution with Acetone and water, further purification of the eluate by absorption on an anion exchange resin (e.g. Amberlite IRA-68 in acetate form), Elute the desacety 1 — cephalosporin from the resin with potassium acetate solution and precipitate the product with acetone «Others Techniques Used in Isolating the Various Cephalosporin Products Solvent extraction »acid precipitation and precipitation at the isoelectric point can be used (in the case of zwitterion products such as (6R, 7R) -7 ~ amino-3 ~ hydrosymethylceph-3-em-4-carboxylic acid).

The following examples illustrate the invention. All temperatures are given in ⁰ C and the nature and purity of the products were determined using the following techniques.

nLlSLj & ^ LJ ο ο, 1

f Thin layer clock chromatography (DSC), ultraviolet spectrophotometry (U.T.), infrared spectrophotometry (IR), nuclear magnetic resonance (IT.M.R.) and high pressure

. - Liquid chromatography A ^ h.ρ.1.c.). ["high pressure liquid chromatography)

example 1

3-eia-4-carboxylic acid

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A solution of 4 g (6R, 7R) -3 ~ acetox; 7methyl-7- (D-5-amino-5-carbo2cypentanamido) -eeph-3-em-4-carboxylic acid, as the potassium salt in 80 ml of demineralized water "were mixed with 20 ml of a suspension of cells obtained by culturing Rhodosporidium toruloides CBS 14 (40 ml) in 0.5 m pH 6.0-Ealium-

cCli issi phosphate buffer he treated. (The microorganism was grown on the yeast extract-peptone medium as described above for 72 hours, after which the cells were harvested by centrifugation and washed with water). The resulting mixture was kept at 25 ⁰ C and shaken on a rotary shaker, the Yerlauf the reaction was monitored by thin layer chromatography. After 42 hours, when complete deaetylation was indicated by thin layer chromatography, the reaction mixture was centrifuged to remove the solids and the resulting solution was treated with acetone (5 volumes). The precipitate formed was collected by centrifugation and dried under vacuum at room temperature to give the title compound (4 »56 g). This was determined by thin layer chromatography on cellulose-coated plates using a 70 $ yol / vol aqueous n-propanol solvent system in comparison with an authentic sample and densitometrically shown to have a purity of about 53.5 $ what corresponds to a total yield of 100 io.

Example 2

(6R, 7R) -7- (D-5-amino-5-carbo3: ypentanamido) -3-hydro3-cymeth.yl-ceph-3-em-4-carboxylic acid

The procedure of Example 1 was repeated except that a suspension of cells obtained by culturing Sporidiobolus johnsonii CBS 5470 was used, and the enzyme-catalyzed deacetylation was carried out for 114 hours. 4.03 g of the title compound was obtained, this material was about 37.5 # purity, indicating an overall yield of 63 r> .

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Example 3 ,

4 ~ carboxylic acid

A solution of 4 g of sodium (6R, 7R) -3-acetoxymethyl-7- (thien-2-yl-acetamido) -ceph ~ 3 ~ e: m-4 - carboxylate in 80 ml of demineralized water was mixed with a suspension treated by cells obtained by culturing Rhodosporidium toruloides CBS 14 in the same manner as described in Example 1. The deacetylation was determined by thin layer chromatography to be complete after 42 hours, after which the reaction mixture was centrifuged to remove the solids and the remaining solution was mixed vigorously with 200 ml of butyl acetate. The pH of the mixture was adjusted to 2.2-2.5 by dropwise addition of concentrated phosphoric acid * The aqueous layer was separated and extracted with a further 20 ml of butyl acetate, adding a few drops of * Gemex "detergent to break the emulsion formed The butyl acetate fractions were combined and a 12.5% w / t tolumene solution of potassium acetate in denatured alcohol (approximately 8 ml) was slowly added until no further precipitation occurred, and the precipitate was isolated by suction, with a small volume Washed acetone and dried under vacuum at room temperature to give the title compound (2.87 g). This was compared by thin layer chromatography on cellulose coated αϊ plates using an ethyl acetate-n-butanol-sodium acetate solvent system with an authentic sample and densitometrically it was found that the compound was about 75% pure $>' was present, indicating an overall yield of 65 #.

Example 4

(6R, 7R) -3-1 ^ dro3cyi ethyl-7 - (thien-2-yl-acetamido) -ceph-3-em-4-carbonic acid

The procedure of Example 3 was repeated with the exception

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that a suspension of cells "obtained by culturing Sporidiobolus johnsonii CBS 5470" was used and the enzymatic-catalyzed deacetylation proceeded for 114 hours. 1.99 g of the title compound were obtained; this material had a purity of about 77 fi, v / as indicated a total yield of 46 fo.

Examples 5 to 9 illustrate methods which are applied can be adjusted to the degree of the desired esterase activity produced by a particular microorganism testing.

Example 5

(a) A liquid yeast extract / peptone nutrient medium, as described above, but containing an additional 2 fo weight / to lumen (6R, 7R) -3-Ac et oxymethyl-7- (D-5 ~ amino-5-carboxypentanamido ) ceph - ^ - EMH - carboxylic acid contained as KJaliumsalz, was inoculated with Rhodosporidium toruloides CBS 14, which had previously been cultured on agar slant with yeast extract / peptone medium for 4 days at 25 ⁰ C. The liquid culture was incubated on a rotary shaker for 3 days at 25 °. Samples of culture medium were then subjected to thin layer chromatography on cellulose coating ended plates buffered at pH 5.8 to 6.0 with 0.067 M phosphate buffer, and using a 70 <fo Yol / Tol aqueous n-propanol solvent system. TJT irradiation of each plate revealed the integrity of a spot corresponding to the authentic (6R, 7R) -7- (D-5 - amino-5-carboxypentanamido) -3-hydroxymethyl-ceph-3_em-4-carboxylic acid.

(b) The procedure of (a) was repeated with the exception that (6R, 7R) -3-acetoxymethy1-7- / 2- (fur-2-y1) -2-methoxyiminoacetamideq7-ceph-3-em-4 -carboxylic acid (syn-isomer) instead of (6R, 7R) -3-acetoxymethyl-7 ~ (D ~ 5-amino-5-carboxypentanamido) -ceph-3-em-4 ~ carboxylic acid, Potassium salt was used. The exam of the liquid culture medium after incubation

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Thin-layer chromatography showed the presence of (6R, 7E.) - 7 - ^ - (l \ ir-2-yl) -2-methoxyiminoacetamiao7-3-hydroxymethyl-ceph.-3-em-4-carboxylic acid (syn isomer).

Example 6

The procedures of Examples 5 (a) and (b) were repeated with with the exception that the Ehodosporidium toruloides replaces GBS 14 was caused by one of the following microorganisms:

Ehodosporidium toruloides CBS 349

Ehodosporidium sphaerocarpum OBS 5939

Leucosporidium scottii CBS 5930

Sporidiobolus johnsonii CBS 5470

Sporobolomyces roseus CBS 486

Bullera alba CBS 501

Sporobolomyces salmonicolor CBS 496

In all cases either (6R, 7R) -7- (1) -5-amino-5-carboxypentanamido) -3-hydroxymethyl-ceph-3-em-4-carboxylic acid was used or (6E, 7R) -7- / 2 -]? ur-2-yl) -2 ~ methoxyim: uioace tamidQL7-3-hydroxymethy 1-ceph-3-em-4-carboxylic acid (syn-isomeres) found suitable.

Example 7

An inoculum of Sporidiobolus ruinenii CBS 5001, which had previously been cultured for 4 days at 25 ° on agar slants with yeast extract / peptone medium as described above, was incubated for 3 days at 25 ° in 40 ml of an aqueous medium containing 0, 3 i ° w / v yeast extract, containing 0.3 $ w / v malt extract, 0.5 $ w / Yolumen peptone and 1.0 $ 6 weight / Yolumen D-glucose. A portion of this liquid culture (1 ml) was then used to inoculate 53 ml of an aqueous medium containing $ 0.23 w / volume yeast extract, 0.23 fo w / volume malt extract, 0.38 fo w / volume peptone, 0 , 75 # weight / ^{volume D-glucose and 1.96 c} / o weight / volume (6R, 7R) -3-acetoxymethyl-7- (D-5 ~ amino-5 ~ carboxypentanamido) -ceph-3-em- 4-carboxylic acid as the potassium salt

603820/1141

held. The resulting liquid culture was used for 5 days Incubated 25 ° on a rotary shaker, after which the culture medium by thin layer chromatography using the method in Example 5 was tested and the presence of (6R, 7R) -7- (D-5-amino-5-carboxypentanamido) -3-hydroxymethylceph-3 ~ em-4-carboxylic acid was established.

Example 8

The procedure of Example 7 was repeated except that the Sporidiobolus ruinenii CBS 5001 was replaced by Bullera tsugae CBS 5038 was replaced. Thin layer chromatography confirmed the generation of (6R, 7R) -7- (D-5-amino-5-carboxypentanamido) -3-hydroxymethyl-ceph-3-em-4-carboxylic acid confirmed.

Example 9

A liquid yeast extract / peptone storage medium as described above was inoculated with Ustilago maydis IMI 103761, which had previously been grown on inclined plates of modified Sabouraud agar for 72 hours at 25 °. The liquid culture was incubated for 72 hours at 25 ° on a rotary shaker, after which a portion of the culture medium (1 ml) was mixed with 8 ml of 0.1 M phosphate juff and 1 ml of 10 5 weight / volume (6R, 7R) - 3-acetoxymethyl-7- (D-5-amino-5-earboxypentanamido) -ceph-3-em-4-carboxylic acid was incubated for 4-8 hours at 25 ° on a rotary shaker. The samples of the culture medium were then examined by thin layer chromatography on cellulose-coated plates using a 70 tfo v / v aqueous n-propanol solvent system. UV irradiation of each plate revealed the presence of a stain corresponding to an authentic sample of (6R, 7R) -7- (D-5-amino-5-carboxypentanamido) -3-hydroxymethyl-ceph-3-em-4 -carboxylic acid. The concentration of this material was 1.7 mg / ml compared to a concentration of 0.5 mg / ml obtained in a control experiment where no microorganism was used.

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Example.1O

ceph-3-em-4-carboxylic acid (syn-isomeres)

(a) A medium containing 2.2 $> G-lucosemonohydrat, 1 fo Oxoid yeast extract, 1 <fo Oxoid peptone, 1.5 $> potassium dihydrogen phosphate, 0.1 io polypropylene glycol / white mineral oil (1: 1) uolo , -1 # Cephalosporin C potassium salt, v / urde inoculated with a liquid culture of Rhodosporidium toruloides CBS H (100 ml) and fermented for 32 hours with stirring and aeration (3 l / min.). The broth was harvested by centrifugation, the supernatant liquid was decanted and a mobile slurry was obtained by adding demineralized water.

(b) a reaction mixture of 8.0 g of (6R, 7R) -7 ~ Z2- (I \ ir-2-yl) -2-metho2yiminoacetamido7-3-acetoxymethylceph-3-em-4-carboxylic acid (syn-Isomeres), 2.6 g sodium hydroxide, 120 ml demineralized Water, 2.2 ml of orthophosphoric acid and 18 ml of a slurry of Rhodosporidium toruloides CBS 14-, as indicated under (a) was incubated at 25 ° on a rotating shaker. After 20 hours, thin layer chromatography (Silica gel plates with 0.5 m sodium chloride as solvent) the reaction can be shown to be complete. The reaction mixture was centrifuged and the supernatant decanted off and filtered through a block of asbestos. 14 ml of methyl isobutyl ketone were added to the clear piltrate, the mixture was magnetically stirred and a 20 <£ solution of orthophosphoric acid was added dropwise until the pH was 2.0. The mixture was then allowed to settle, the lower aqueous layer was pipetted off and the solvent layer, which contained an abundant white precipitate was filtered in vacuo. The precipitate was mixed with 10 ml of methyl isobutyl ketone and washed with 2 × 10 ml of ice-cold water. The product was then filtered off with suction and dried in vacuo at room temperature and gave 5> 01 g of the title compound. The IR and NMR spectra of the product consistently showed that it was the

Title compound acted. The purity was found to be 92.2 fo by high pressure liquid chromatography.

Example 11

(6R _f 7R) -7-Amino-3-hydroxymethyl-ceph-3-eia-4-carboxylic acid

A reaction mixture consisting of 6.4 g (6R, 7R) -7-amino-3-acetoxymethyl-ceph-3-em-4-carboxylic acid toluene-p-sulfonate, 120 ml demineralized water and sufficient ammonia solution (specific Weight 0.880) to dissolve the cephalosporin; and 12 ml of a slurry of Rhodosporidium toruloides OBS 14, prepared as in Example 10 (a), was adjusted to pH 6.5 with 20% orthophosphoric acid. The mixture was shaken on a moving in a circle shaker at 25 ⁰ C and after 20 hours, the analysis by thin layer chromatography (silica gel with 0.5 m-Fatriumchloridlösung) indicates that the reaction was complete.

The reaction mixture was centrifuged, the supernatant decanted and filtered through a layer of asbestos. The pH of the solution was then lowered to 3.6 by adding dropwise with stirring 20% orthophosphoric acid and the resulting precipitate was filtered off and washed with 5 ml of ice-cold water and 10 ml of acetone. A second crop was obtained from the Ultrat by adding an equal volume of acetone. The resulting precipitate was filtered off and washed with 10 ml v / ater and 20 ml acetone. The two pesticides were dried separately in a vacuum oven at room temperature and combined to give 1.99 g of the title compound. The MER and IR spectra of the product were consistent with the title compound. The purity of the product was determined by high pressure liquid chromatography to be 98.1 fo.

SG982G / 1US

Example 12

Deacetylation of (6R, 7R) -7- (D-5-benzoylamino-5-carboxypentanamido) -3-acetoxymethyl-ceph-3 ~ em-4-carboxylic acid

The cell residue from the centrifugation of the reaction mixture of Example 10 (b) was gev / ash and a mobile slurry was obtained by adding demineralized water. 18 ml of this slurry were added to a reaction mixture consisting of 8 g of (6R, 7R) -7- (D-5-benzoylamino-5-carboxypentanamido) -3-acetoxymethyl-ceph-3-em-4-carboxylic acid, 2, 5 g sodium hydroxide, 120 ml demineralized water and 2.2 ml orthophosphoric acid (specific weight 1.75) and was adjusted to pH 6.0. The mixture was incubated for 21 hours on a rotating shaker in a circle at 25 ⁰ C, followed by thin layer chromatography (SiIicagel plates with chloroform / methanol / formic acid 48/8/2) was determined that the reaction was complete. After centrifuging the mixture and decanting the supernatant, the product was characterized by reaction with diphenyldiazomethane, whereby diphenylmethyl ~ (6R, 7R) -7 ~ (D-5-benzoylamino-5-diphenylmethoxycarbonylpentanamido) -3-hydroxymethyl-ceph-3 -em-4-carboxy-lat (8.5 g) which was found to be identical to an authentic sample by IR and NMR spectroscopy.

Example 13

(6R, 7R) -7-Z2- (Eur-2-.yl) -2-methoxyiminoacetamido7-3-h.ydroxymethyl-ceph-3-em-4-carboxylic acid (syn-isomeres)

A culture (60 ml) of Rhodosporidium toruloides OBS 349, prepared by fermentation on a Gluco se / yeast extract / Pep ton medium ^ T for 3 days in the presence of 0.1 fo cephalosporin C potassium salt, was centrifuged and the cells in 25 ml of water resuspended. This suspension was added to a solution of 4.0 g of (6R, 7R) -7-Z? - (Pur-2-yl) -2-methoxyiminoacetamido7-3-acetoxymethyl-ceph-3-em-4-carboxylic acid (syn- Isomeres), 1.3 g sodium hydroxide and 1.1 ml orthophosphoric acid in 30 ml demineralized water. The reaction mixture was on a chute! - y in the same way as described in Example 10a,

609820 / 1US

device at 25 ⁰ C 23 hours incubated, followed detected by thin layer chromatography that the reaction was complete. The reaction mixture was then worked up in the same manner as in Example 10 and gave 2.5 g of the title compound. The identity of the product was confirmed by thin layer chromatography and IR and NMR spectroscopy.

Example 14

(6R, 7R) -7- (1H? ~ Amino-5-carboxypentanamido) -3-hydroxymethyl-ceph-3-em-4-carboxylic acid

A suspension of cells from Rhodosporium toruloides CBS 349 was prepared as in Example 13 with the cells suspended in 0.25 M phosphate buffer, pH 6.0. A solution of 6.7 g of cephalosporin C potassium salt in 60 ml of demineralized water was added to this yeast suspension (40 ml). The mixture was incubated for 20 hours on a shaker at 25 ^° C., after which it was determined by thin layer chromatography that the reaction had ended. The reaction mixture was worked up in approximately the same manner as in Example 1 and gave 4.27 g of the title compound with A ","., At 260 µm. The product was found to be identical to an authentic sample of the title compound on two thin layer chromatography systems. The UY absorption indicated a purity of $ 45.

Example 15

(6R, 7R) -3-Hydroxymethy l-7-Z2-methoxy imino-2- (fur-2-vlV acetal id oj -ceph-3'-em-4-carbpnic acid (syn-isomer)

16.0 g of sodium hydroxide and 13.5 ml of orthophosphoric acid were in 750 ml of distilled water / water dissolved. 50 g (6R, 7R) -3-acetoxymethyl-7- {2-meihoxy-jnrino - 2 - (fur-2-yl) -acetamido7-ceph-3-em-4-carboxylic acid (syn-isomer) were added and this dissolved so that gave a solution with a pH of 6.4. The pH was adjusted to 6.3 by adding 20% orthophosphoric acid and a suspension of Rhodosporidium toruloides CBS 14 cells

609820MUS

(150 g) was added. The mixture was stirred and aerated for 8 hours at 25 °, during which time 105 ml of an m-sodium hydroxide solution was added to keep the mixture in the pH range of 5.8 to 6.3. The conversion was shown to be complete by thin layer chromatography. The mixture was stirred and vented for an additional 16 hours, then the yeast was removed by centrifugation. The yeast was washed by suspending it in 200 ml of water and centrifuging again. The main fraction was stirred with 2 g of charcoal for 30 minutes and then filtered through a Hyflo-Supercel bed. The bed was washed with the yeast washes. 250 g of sodium chloride and 500 ml of 4-methylpentan-2-one were added to the filtrate, and the pH was adjusted to 2.0 by adding 20% orthophosphoric acid over 20 minutes. The mixture was cooled in an ice bath while acidifying. The stirring was stopped and the layers allowed to settle. The clear aqueous layer was discarded and the precipitated solid was filtered off and washed with 50 ml of 4-methylpentan-2-one and with three times 40 ml of water. The product was dried in vacuo at 40 ° for 24 hours to give 38.39 g of the title compound as an off-white solid. The purity was found to be 99.1 <fo by high-performance liquid chromatography, and the total impurities were found to be 2.5 $> by thin layer chromatography.

Example 16

(a) (6R, 7S) -3-acetoxymethyl-7-methoxy-7-phenyl-acetamido-ceph-3-em-4-carboxylic acid

A solution, cooled to -78 °, of 1.5 g of t-butyl (6R, 7R) -3- & oetoxymethyl-7-phenylacetamido-ceph-3-em-4-carboxylate in 10 ml of dry tetrahydrofuran was stirred for 3 minutes and cooled to -78 ° solution of 358 mg of lithium methoxide placed in 8.8 ml of dry methanol and 50 ml of tetrahydrofuran under nitrogen. After 3 minutes, 0.69 ml of t-butyl hypo-

609820 / 1U5

chlorite was added and stirring continued for a further 5 minutes as the yellow solution in a likewise stirred mixture of 150 ml of ethyl acetate and 150 ml of water containing 1 g of ammonium chloride and 1 g of sodium metabisulfite was poured. The watery Phase was separated and extracted with 50 ml of ethyl acetate and the organic phases were combined and washed with 50 ml of brine washed, dried and evaporated to dryness in vacuo to give 1.6 g of t-butyl (6R, 7S) -3-acetoxymethyl-7-methoxy-7-phenylacetamido-ceph ^ -em - ^ - carboxylate.

A stirred solution of 1.6 g of this product in 6.8 ml of trifluoroacetic acid and 1.7 ml of anisole was kept at about 23 ° and the remaining trifluoroacetic acid was removed by evaporation and then by distillation with toluene (2 × 15 ml). A solution of the brown residue in 25 ml of sodium bicarbonate solution was stirred with 25 ml of ethyl acetate for 10 minutes. The aqueous layer was separated, adjusted to pH 2 with 2N hydrochloric acid and extracted with 3 × 25 ml of ethyl acetate. The extract was washed with brine, dried and evaporated to give 1.3 g of a yellow solid. A solution of this contaminant in 50 ml of acetone was treated with charcoal, the mixture was filtered and the filtrate was concentrated to dryness in vacuo. Trituration of the residue with 25 ml of ether gave the title compound (500 mg), P = 167-170 °, fqj ^ + 190 ° (c 0.28, DMSO), λ ___ (0.1 M-pH 6 phosphate buffer) 265 mn (έ \ * 196)

(b) Sodium (6R, 7S) -3-hydroxymethyl-7-iiiethoxy-7-phenylacetamifloceph-3-em-4-carboxylate

A suspension of 200 mg (6R, 7S) -3-ethoxymethyl-7-methoxy-7-phenylacetamido-ceph-3-em-4-carboxylic acid in 3 ml of water was made up by adding saturated aqueous sodium bicarbonate pH 6 adjusted. 3 ml of 0.4 M pH 6 phosphate buffer were added and the resulting solution with Rhodosporidium toruloides CBS 14- (1 g of the frozen homogenized product) at about Stirred at 20 °. After 16 hours, the thin-film test showed

6Q982Ö / 1U5

Chromatography (chloroform: methanol acetic acid = 90: 16: 5) that the substrate was completely converted. The mixture was clarified by centrifugation and the supernatant liquid decanted and freeze-dried. Trituration of the residue with 10 ml of ether resulted in 400 mg of a colorless pesticide, which was identified as containing the title compound by NMR spectroscopy.

Example 17

(a) The organisms listed in Table I were grown on an agar medium containing 4 fo maltose, 1 f> peptone, 2.4% malt extract and 2.5 f> agar powder contained and was adjusted before sterilization to pH 7 ». 5

The inclined plates were incubated for 7 days at 25 ⁰ C and an inoculum was transferred from each plate in a liquid medium (40 ml) containing 2 fo G-lucose, 1 f> yeast extract, peptone and 0.5 1 fo fo Ealiumdihydrogenphosphat at pH 5.8. The culture was incubated for 10 days at 25 ⁰ C on a shaker and used to inoculate the same to a second liquid medium level unter'Zugabe of cephalosporin G (0.1 fo). After a further incubation period of 3 to 8 days, depending on the rate of growth, the cultures were harvested. Homogeneous suspensions of each culture were prepared by mixing vigorously using a mixer. Reaction mixtures were prepared which contained 2 ml of the homogeneous cell suspension, 2 ml of 0.5 M potassium phosphate buffer pH 6.0 and 6 ml of a 3.33% cephalosporin C solution and it was stirred for 5 days at 25 ⁰ C incubated.

Each sample was examined by thin layer chromatography under Using the method described in Example 5.

9820/1145

The zones correspond to the authentic samples of (6R, 7R) -7- (D-5-amino ~ 5-carboxypentanamido) -3-hydroxymethyl-ceph-3-em-4-carboxylic acid (DAC) were eluted and the amount of DAC by optical Density determination determined at 260 mn.

The results obtained are shown in Table I.

(b) The above procedure (a) was repeated with (6R, 7R) -7-ZJ2- (Eur-2-yl) -2-methoxyiminoacetamido7-3-acetoxymethyl-ceph-3-em-4-carboxylic acid instead of cephalosporin C in the reaction mixture. The 3-hydroxymethyl compound obtained was determined by thin layer chromatography checked using the method in the example. The results obtained are shown in Table I.

Table I.

Concentration of the 3-hydroxymethyl 1 compound in the reaction mixture (mg / ml)

organism DAC (6R, 7R) -7- / 2- (l ^ ir-2-yl) -2-me-
thoxyiminoacetainiuoj? —3 — lyräXü—
xymethy1-ceph-3-em-4-carbon-
acid Polyporus dichrous CBS 446. ΐ> υ
Polyporus versicolor 7.7 8.5 CBS 471.72
Poria monticola 7.7 9.2 CBS 336.49
Coprinus comatus 10.3 7.0 CBS 150.59
SchisoTJhyllum commune 10.3 9.5 CBS 103.20
Control attempt without
organism 9.1
4.0 7.3
4.6

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Claims (19)

Claims Ί »J Process for converting a 3-acyloxymethyl-ceph-3-em-4-carboxylic acid to a 3-hydroxymethyl-eeph-3-eiii-4-carboxylic acid by hydrolysis, characterized in that the hydrolysis is catalyzed by an esterase produced by cultivating a microorganism from the Basidiomycetes class capable of producing the required esterase activity. 2. The method according to claim 1, characterized in that the microorganism belongs to the order Ustilagenales, family Sporobolomycetaceae or belongs to the order Polyporales. 3. The method according to claim 1 or 2, characterized in that the microorganism belongs to the genus Rhodosporidium. 4. The method according to claim 1 or 2, characterized in that the micro-organism belongs to the genus Leucosporidium, TJstilago, BuI-lera, Belongs to Sporidiobolus and Sporobolomyces. 5. The method according to any one of claims 1 to 3j, characterized in that that the microorganism is a strain of Rhodosporidium toruloides. 6. The method according to any one of claims 1 to 4, characterized in that that the microorganism is a strain of Leucosporidium scottii, Rhodosporidium sphaerocarpum, Bullera alba, Bullera tsugae, Sporidiobolus johnsonii, Sporidiobolus ruinenii, Sporobolomyces roseus, Sporobolomyces salmonicolor, Ustiligo maydis, Schizophyllum commune, Coprinus comatus, Polyporus dichrous, Polyporus ver3icolor and Poria monticola is. 7. The method according to claim 5, characterized in that the Microorganism Rhodosporidium toruloides CBS 14 or a Mutant of it is. 809820 / 1US 8. The method according to claim 5, characterized in that the microorganism Rhodosporidium toruloides CBS 349 or a Mutant of it is. 9. The method according to any one of the preceding claims, characterized in that as 3 ~ acylozymethyl-ceph-3-em-4 - carboxylic acid a compound of the general formula CH ₂ O.CO.R ³ COOH uses v / ird, -wherein R is an amino group or blocked Is amino group; R is a carbon atom or a lower alkyl, lower alkoxy, lower alkylthio or lower alkanoyl group; R .CO is an earboxylic acyl group with 2 to 20 carbon atoms; and B is T ^ -S or ^ S - ^ 0. 10. The method according to any one of the preceding claims, characterized characterized in that as 3-acyloxymethyl-ceph-3-em-4-carboxylic acid a 3-aceto2ymethyl-ceph-3-em-4 ~ carboxylic acid is used will. 11. The method according to claim 9 or 10, characterized in that ρ
that R is a hydrogen atom and B r = S. 12. The method according to any one of claims 9 to 11, characterized in, that R is selected from the group D-5-amino-5-carbozypentanamido and IT-protected derivatives thereof; 4-carboxybutanate nido; Pornaiaido; a group of ponies S09820 / 1U5 R. (CH ₂ ) _n . COHH- wherein R is a carbocyclic or heterocyclic aryl group or an aryloxy, arylthio, aryl-lower-alkoxy or Aryl-lower-alkylthio group and η is an integer of Is 1 to 4; a group of the formula R ^a -CH-CONH-X where R is an ary! group and Ί. the radical is amino, protected amino, carboxy, carbalkoxy or hydroxy; and from a group of the formula R ^a _ c - COEH- away wherein R is as defined above and R is a hydrogen atom or an acyl, lower-alkyl, cycloalkyl, aryl or Ary is 1-lower-alkyl! Group. 13. The method according to claim 12, characterized in that R is selected from the groups D-5-benzoylamino-5-carboxypentanamido, Thienylacetamido, 2-Bydrosy-2-pheny! Acetamido, 2-t-butoxycarbonylamino-2-phenylacetamido and syn-2-3Turyl-2-aiethoxyiminoacetamido. 14. The method according to any one of claims 9 to 13, wherein R .CO is an Acy! group selected from the low-Alka groups lower-alkenoyl, aryl-lower-alkanoyl and aroyl. 15. The method according to claims 9 to 14, characterized in that that the group R ^ .CO- is an "Acety! group. 609820/1145 16. The method according to claim 10, characterized in that as 3-aceto2ymethyl-ceph ~ 3-em-4-carboxylic acid the cephalosporin C. is used. 17. The method according to claim 16, characterized in that the whole broth from a cephalosporin C fermentation with the Esterase treated v / ird. 18. The method according to any one of the preceding claims, characterized characterized in that the S-acyloxymethyl-ceph-S-em - ^ - carboxylic acid is brought into contact with a source of esterase, which whole cells that are obtained from a culture broth of the microorganism contains. 19. The method according to any one of claims 1 to 17, characterized in, that the 3-acyloxymethyl-ceph-3-em-4 ~ carboxylic acid with cell-free esterase is brought into contact, which was immobilized or fixed in or on an inert matrix. 609820 / 1U5