NO851655L - MICROBIOLOGICAL PROCEDURE FOR THE PREPARATION OF ANTRACYCLINE DERIVATIVES. - Google Patents

Description

The invention relates to a microbiological method for producing the compound of formula I

in which R means hydrogen or hydroxy and R1 and R2 are the same or different, and means sugar combinations of the following composition:

-Roa-dF-CinA

-Roa-dF-Acu

-Roa-dF=CinB

-Roa-Rod-CinA or

-Roa-Rod-Acu

or R2 has the above-mentioned meanings and R^ means the sugar combination of the following composition:

-Roa-dF-Rod

-Roa-dF-CinB

-Roa-dF-Acu

-Roa-Rod-Acy

-Roa-dF-CinA

-Roa-Rod-Rod or

-Roa-Rod-CinA

where Roa means rhodosamine, dF means deoxyfucose, Rod means rhodinose, Acu means aculose, ConA means cinerulose, A and CinB means cinerulose B, and dF means CinB, that the sugar building blocks deoxyfucose and cinerulose B are joined by an additional ether bridge next to the usual glycosidic bonds, the method being characterized by a compound of formula II

wherein R has the meaning mentioned under formula I, and R^ and R^

are the same or different and mean sugar combinations with the following composition:

-Roa-dF-Rod

-Roa-Rod-Rod

-Roa-dF-CinA or

-Roa-Rod-CinA

or only R^ or only R^ have the meanings mentioned and the second substituent resp. means

-Roa-dF-Rod

-Roa-dF-CinB

-Roa-dF-Acu

-Roa-Rod-Acu

-Roa-dF-CinA

-Roa-Rod-Rod or

-Roa-Rod-CinA

react with a specific oxidoreductase and the compound formed is isolated from the reaction medium in a manner known per se by extraction and purified.

The oxidation of terminal sugar molecules anthracycline triglycosides by reaction with an oxidoreductase or already known (A. Yoshimoto et al., The Journal of Antibiotics Vol. XXXII, 472 (1979)). This is, however, an anthracycline derivative that is only unilaterally substituted with triglycosides.

Surprisingly, however, by applying this method to anthracycline derivatives with two triglycoside chains on the molecule, a selective oxidation of terminal sugar moles is possible. Thus, for example, by reacting a cytorhodin of formula II with an oxidoreductase preparation, prepared from the culture filtrate of Streptomyces galilaeus (ATCC 31133), only the lower of the triglycoside side chain (-R3) is oxidized.

As oxidoreductases, oxidizing enzymes and enzyme preparations such as e.g. cytoplasmic membrane suspensions, which are isolated in the usual way from the culture filtrate of microorganisms, preferably of Streptomyces purpurascens (DSM 2658) or of Streptomyces galilaeus (ATCC 31133).

The enzymatic method according to the invention is carried out at temperatures of 20-45°C, preferably 37°C and a pH of 4-7, preferably 5-6, the reaction time can be a few hours to 3 days. To increase yields, further enzyme can be dosed. After completion of the reaction, the desired compounds are isolated from the reaction medium by extraction and possibly by column chromatography, preferably on silica gel or on chemically bound reversed phases ("reversed-phase"-Adsorbent), or by drop resistance chromatography, separated and purified.

With the method according to the invention, e.g. the following compounds: Cytorhodin U (compound of formula I, in which R.^= -Roa-Rod-Rod

and R2 means -Roa-dF=CinB).

Cytorhodin V (compound of formula I, in which R 1 is -Roa-Rod-Rod and R 2 is -Roa-Rod-Acu).

Cytorhodin P (compound of formula I, wherein R means -Roa-Rod-Acu and R2 means -Roa-dF-CinA).

Cytorhodin D (compound of formula I, in which R 1 means -Roa-Rod-Acu, and R 2 means -Roa-dF-CinB).

1-Hydroxy-cytorhodin V

1-Hydroxy-cytorhodin P

1-Hydroxy-cytorhodin D

1-Hydroxy-cytorhodin B (compound of - formula I, wherein Roa-Rod-Rod, and R2 means Roa-dF-CinA).

The cytorhodins obtained according to the invention are distinguished by a high cytostatic effect.

The starting compounds of formula II with R=H can be prepared according to German Offenlegungsschrift DE 33 23 025 by fermentation of the micro-organism •Streptomyces purpurascens~ (DSM 2658) and subsequent isolation.

According to this publication, the strain Streptomyces purpurascens (DSM 2658) is fermented in a nutrient medium in the usual way, at temperatures of approx. 24-40°C at a pH of 6.5-8.5, and aerobic conditions are observed. From the separated mycelium, anthracycline preparations (cytorhodins) are extracted by extraction, for example, with aqueous acetone at a pH of 3.5, the acetone is removed, and the aqueous phase is extracted at a pH of 7.5 with ethyl acetate. The culture filtrate is extracted at a pH value of 7.5, preferably with ethyl acetate. The ethyl acetate extracts from the mycelium and the culture filtrate are combined and evaporated to dryness gives a raw residue. Preferably, the residue is further processed as possibly mentioned in the aforementioned German patent application.

Accordingly, crude residue is dissolved in toluene and extracted with an acetate buffer (pH 3.5), obtaining a toluene phase and an aqueous phase. The aqueous phase is further processed as follows: After setting the pH to 7.5, methyl acetate is extracted again

and the ethyl acetate phase is concentrated, obtaining a so-called cytorhodin crude mixture, from which the anthracycline derivatives (cytorhodins) of formula II are recovered chromatographically.

The starting compounds of formula II with R = OH (according to German application P 34 25 357.2) are prepared analogously to the above-mentioned method with the difference that the fermentation is carried out at an increased degree of aeration from 0.8 to 1.2 vvm over a time of 70 to 130, preferably 90 to 110 hours.

The invention shall be explained in more detail by means of the following examples:

Example 1

The isolation of oxidoreductase from Streptomyces galilaeus ATCC 31133.

Streptomyces galilaeus - ATCC 31133 was inoculated as a spore suspension in a 2000 ml Erlenmeyer flask with 500 ml of the following nutrient solution:

and incubated for 48 hours at 28°C and 200 rpm. my. on a rotary shaker. 900 ml of this pre-culture served as inoculum for a 12-litre fermenter which was filled with 9 liters of culture solution: Trace element solution

After a fermentation duration of 3-4 days at 28°C, 400 revolutions per my. and an aeration degree of 0.5 vvm, the culture solution is harvested, centrifuged and the culture filtrate is concentrated ten times. This concentrate was brought to saturation with ammonium sulphate

of 50%, the supernatant discarded, the precipitate taken up in tris-HCl buffer, pH 7.4 (10 mM), and dialyzed against 100 times the volume of the same buffer. The enzyme solution was then stirred with deionized DE 52 (Whatman) in a batch mixture, unbound protein removed with the above buffers adding NaCl (20 mM) by washing, then specific oxidoreductase eluted with the same buffer adding NaCl (250 mM) and concentrated by means of a precipitate of ammonium sulphate (80%). The resulting enzyme solution with 130 mU/mg protein was lyophilized

cert and stored at -18°C. The determination of the oxidoreductase activity took place by measuring the ^C^, SOm formed with the substrate cytorhodin B is transferred in a coupled reaction by means of peroxidase to 6-phenylenediamine. The concentration of the yellow dye formed is directly proportional to the amount Yi^ O^ formed by the enzyme. 1 int. unit of enzyme activity corresponds to the turnover of 1 ymol of substrate/minute.

Example 2

Preparation of an oxidoreductase preparation from Streptomyces purpurascens - DSM 2658.

Streptomyces purpurascens - DSM 2658 was grown on yeast-malt agar with the following composition:

In addition, the medium was distributed on Roux bottles and sterilized for 30 minutes at 121°C, cooled, inoculated with a spore suspension and incubated for 7-14 days at 25°C. Subsequently, good growth and good spore formation could be determined. From this stock culture, a spore suspension was prepared with 10 7 spores/ml in sterile NaCl (0.8% strength). 2 ml served as inoculum of 50 ml of the following pre-culture medium: Each time 50 ml of the above-mentioned "medium" was distributed in 300 ml Erlenmeyer flasks and sterilized for 30 minutes at 121°C. The inoculated flasks were incubated for 2-3 days at 25°C and 200 revolutions per minute on a rotary shaker. 10 ml of the aqueous pre-culture was used as inoculum for

- following - main culture:

Each time 500 ml of the above-mentioned medium was divided into 2000 ml Erlenmeyer flasks and sterilized for 30 minutes at 121°C. The inoculated flasks were incubated for 4-5 days at 25°C, and 200 revolutions per minute on a rotary shaker.

The washed cultures were harvested, centrifuged, and the cells washed with NaCl.

For the enzymatic reactions, either the whole cells or membrane fractions of these cells were used.

Example 3:

Preparation of cytorhodin U.

35 mg cytorodin B (compound of formula II in which R3 means

-Roa-Rod-Rod and R4 means -Roa-df-CinA) was dissolved in 5 ml of 96% ethanol supplemented with 30 ml of citrate-NaOH buffer pH 5.5, and by adding 1.4 ml of oxidoreductase from Streptomyces galileus (1.6 U/ml) stirred at 37°C with 150 revolutions per minute. The reaction was followed by HPLC (Lichrosorb, Si 6 0 7 y (Merck), chloroform: methanol: acetic acid (96 %): water: tri-ethylamine in the ratio 80:10:10:2:0.01, flow 0.5-1 .5 ml. detection by flow photometer at 495 nm). After 48 hours, the output compounds could no longer be detected.

The reaction solution was mixed with 200 µl of 0.1 mol EDTA solution, adjusted with 1-N NaOH to pH 7.5, extracted 3 times with each time 10 ml of ethyl acetate. The acetic ester phase was dried with Na 2 SO 4 , freed from desiccant and carefully evaporated in vacuo.

The residue (25 mg) was dissolved in the above-mentioned mobile phase and chromatographed on 35 g silica gel "Lichrosorb" Si 60, 7 y (Merck)

in a steel column 16 x 250 mm, at a flow rate of 10 ml/minute. The fractions 60-70 (each 2 ml) which according to analytical HPLC contain the compound Cytorodin U are combined, evaporated in vacuo, the residue taken up with 4 ml of Na-acetate solution, pH 3.5. The solution was mixed with 0.1 ml of 1 mM EDTA solution, and washed with toluene fat and softener free, adjusted with IN NaOH to pH 7.5, and the crude product extracted with methylene chloride. After the usual drying over Na2S04 and evaporation in vacuum, pure cytorhodin U (1 mg) is obtained.

Cytorhodin U:

red amorphous substance, easily soluble in methanol, ethyl acetate, chloroform and toluene, insoluble in water and hexane, soluble in 1%

aqueous D-glucinic acid or D-lactobionic acid.

Thin layer chromatography:

Kieselgel 60 F254~finished plates "Merck", system chloroform:methanol:acetic acid (96%):water 80:10:10:2, RH = 0.25.

<1>H-NMR: fig. 1.

Absorption spectrum:

a) 234 (4.67) 253 (SCH) 295 (3.86) 495 (4.10 b) 235 (4.68) 253 (SCH) 295 (3.92) 495 (4.18) c) 244 (4.70) - 290 (SCH3.84) 580, 610 (4.18

(FAB-MS confirmed)

<C>60<H>84<N>2°21 M ber'1168

Cytorhodin. U have. formula. I, wherein ;.R1 means -Roa-Rod-Rod

and R2 is -Roa-df-CinB.

Example. 4

Preparation of cytorhodin' V.. -

6? mg Cytorhodin A (compound of formula II, in which R^ and R^ each means -Roa-Rod-Rod) as D-gluconate (equivalent to 54 mg cytorhodin A free base), dissolve in 45 ml citrate NaOH buffer pH 5.5 , and stirred after adding 2 ml of a solution of oxidoreductase of Streptomyces galilaeus at 37°C with 150 revolutions per minute. minute. The turnover was followed by HPLC. After 48 hours, 35% of the starting compound had been converted. The reaction solution was kept frozen. After thawing, 0.2 ml of a 0.1 mM EDTA solution was added, pH adjusted with 1-N NaOH to pH 7.5, and extracted with ethyl acetate. (3 times each 30 ml). The separated organic phase was dried with Na 2 SO 4 , and carefully evaporated under reduced pressure.

As in example 3, the residue (33 mg) was dissolved in the mobile phase, separated by preparative HPLC.

Fractions 30 - 45 contained the turnover product,

fractions 90 - 120 contained unreacted starting compound.

As in example 3, the reaction product was washed free of fat and plasticizer and isolated as an amorphous red powder (1.0 mg).

Cytorhodin V:

red, amorphous substance, easily soluble in methanol, ethyl acetate, chloroform and toluene, insoluble in water and hexane, soluble in 1% aqueous D-gluconic acid or D-lactodioic acid.

Thin layer chromatography as in example 4: RF = 0.21.

<1>H-NMR; fig. 2

Absorption spectrum

Cytorhodin V has formula I, wherein -Roa-Rod-Rod and R2 means Roa-Rod-Acu.

Example 5

Preparation of cytorhodin P and D.

2 5 ml cytorhodin B (compound of formula II in which R^ means

-Roa-Rod-Rod and R4 means -Roa-df-CinA) is dissolved in 3 ml of 90% ethanol, mixed with 20 ml of citrate-NaOH buffer pH 5.5, and oxidized at 37°C with 2.5 ml suspension of cell membranes (0.5 g/ml) from Streptomyces purpurascens - DSM 2658 (ex. 2) under stirring (150 revolutions/min). According to HPLC, after 24

hours still 21% of the starting compound present and the compounds Cytorhodin P and Cytorhodin D formed in a ratio of approx. 5 : 1.

As in examples 3 and 4, the mixture of the product was isolated from the reaction solution, separated by preparative HPLC, and the product isolated in pure form.

It was formed from pure compounds Cytorhodin D (0.7 mg), Cytorhodin P (1.9 mg) and unreacted Cytorhodin B (1.2 mg).

Cytorhodin P:

red, amorphous substance, easily soluble in methanol, ethyl acetate, chloroform and toluene, insoluble in water and hexane, soluble in 1% aqueous D-gluconic acid or D-lactobionic acid.

Thin layer chromatography can in example 4: RF - 0.27.

3 H-NMR: fig. 3'

Absorption spectrum

Cytorhodin P of formula I, wherein -Roa-Rod-Acu and

R2 is -Roa-dF-CinA.

Cytorhodin D has the formula I, wherein R 1 means -Roa-Rod-Acu and

R2 means -Roa-dF=CinB.

Example 6

Preparation of 1-hydroxy-cytorhodin U and 1-hydroxy-cytorhodin B

50 mg of a 1:1 mixture of 1-hydroxy-cytorhodin N (compound of II, in which R^ means Roa-dF-Rod and R^ means Roa-Rod-Rod) and 1-hydroxy-cytorhodin 0 (compound of formula II in which R^ means Roa-Rod-Rod, and R^ means Roa-dF-Rod), was dissolved with 3 ml of 96% ethanol, supplemented with 30 ml of citrate-NaOH buffer, pH 5.5, and by adding 2 ml of oxidoreductase from Str. galilaeus (1.6/ml) stirred at 37°C at 150 rpm. The turnover was monitored with HPLC (Lichrosorb Si 60

7y (Merck), chloroform:methanol:acetic acid (96%):water:triethylamine in the ratio 80:10:10:2:0.01, flow 0.5 - 1.5 ml, detection by flow photometer at 495 nm). After 17 hours, the starting compound was no longer detectable, and the compounds 1-hydroxy-cytorhodin B and 1-hydroxy-cytorhodin 0 were newly formed in a ratio of 3:6.

The reaction solution was mixed with 200 µl, 0.1 mol EDTA solution, with 1N NaOH adjusted to pH 7.5, and extra-

hardened three times with each time 10 ml acetic acid ethyl ester. Vinegar-

the ester phase was dried with Na 2 SO 4 , freed from desiccant and carefully evaporated in vacuo.

The residue (45 mg) was dissolved in the above-mentioned phase, chromatographed on 35 g of silica gel "Lichrosorb" Si 60, 7y (Merck) in a steel column 16 x 250 mm at a flow rate of 10 ml/min. The fractions (each 2 ml) which according to analytical HPLC contained the new compounds were combined, evaporated in vacuo, the residue taken up with 4 ml of Na-acetate solution pH 3.5. The solution was mixed with 0.1 ml of 1 mM EDTA solution, and washed fat- and softener-free with toluene, 1-N NaOH, adjusted to pH 7.5, and the crude product extracted with methyl chloride. After the usual drying over Na2SO4 and evaporation in a vacuum, pure 1-hydroxy-cytorhodin U (3.5 mg) and 1-hydroxy-cytorhodin B (2 mg) are obtained.

1-Hydroxy-cythorhodin U and 1-Hydroxy-cythorhodin B: red violet, amorphous substances, easily soluble in methanol, ethyl acetate, chloroform and toluene, insoluble in water and hexane, soluble in 1% aqueous D-gluconic acid or lactobionic acid .

Thin layer chromatography: silica gel 60 F254~^erdi9Plate "Merck" system chloroform: methanol: acetic acid (96%) in water, 80:10:10: 2, RH = 0.2 5 (1-hydroxy-cytorhodin U) and RH is 0 ,18 (1-hydroxy-cytorhodin B).

1- hydroxy- cytorhodin U:

Absorption spectrum:

1-Hydroxy-cytorhodin U of formula I, wherein R 1 is -Roa-Rod-Rod and R 2 is -Roa-dF=CinB.

1- hydroxy- cytorhodin B

Absorption spectrum:

1-Hydroxy-cytorhodin B of formula I, wherein Roa-Rod^Rod and R2 means Roa-df-CinA. Example 7 Preparation of 1-hydroxy-cytorhodin P and 1-hydroxy-cytorhodin D 25 mg of 1-hydroxy-cytorhodin B (compound of formula II, in which R3 means -Roa-Rod-Rod and R4 means -Roa-dF-CinA) is dissolved in 3 ml 90% ethanol, mixed with 20 ml citrate-NaOH buffer pH 5.5, oxidized with 2.5 ml suspension of the cell membrane (0.5 g/ml) from Streptomyces purpurascens - DSM 26 58 (example 2 ) while stirring (15 0 revolutions/min). According to HPLC, after 24 hours there was also approx. 22% of the starting compound present, and the compounds cytorhodin P and cytorhodin D newly formed in a ratio of approx. 4.5:1.

The mixture of products was isolated as indicated in examples 3-6 from the reaction solution, separated by preparative HPLC and the products isolated in pure form.

It was formed from pure compounds 1-hydroxy-cytorhodin D (0.9 mg), cytorhodin P (3.2 mg), and unreacted cytorhodin B (1.5 mg).

1- hydroxy- cytorhodin P and - D

violet, amorphous substances, easily soluble in methanol, ethyl acetate, chloroform and toluene, insoluble in water and hexane, soluble in 1% aqueous D-gluconic acid and D-lactobionic acid.

Thin layer chromatography as in Example 4: RF = 0.27 (1-hydroxy-cytorhodin P) and 0.35 (1-hydroxy-cytorhodin D).

1-OH-P;

Absorption spectrum:

Example 8

Preparation of 1-hydroxycytorhodin V:

50 mg of 1-hydroxy-cytorhodin A (compound of formula II in which R^ and R^ each mean -Roa-Rod-Rod) as D-gluconate (equivalent to 37 mg of 1-OH-cytorhodin A free base) were dissolved in 40 ml citrate-NaOH buffer, pH 5.5, and by adding 2 ml of a solution of oxidoreductase from Streptomyces galilaeus stirred at 37°C with 150 revolutions per minute. The turnover was followed by HPLC. After 48 hours, approx. 35% of outgoing connections converted. The reaction solution was mixed with 0.2 ml of a 0.1 mM EDTA solution, pH adjusted with 1N NaOH

at pH 7.5, and extracted with ethyl acetate (3 times 30 ml). The separated organic phase was dried with Na^SO^ and carefully evaporated under reduced pressure.

As in example 3, the residue (35 g) was dissolved in the mobile phase and separated by preparative HPLC. Fractions 32-47 contained the reaction product, fractions 88-110 did not react with starting compounds.

As in example 3, the reaction product was washed free of fat and plasticizer and isolated as an amorphous red powder (2.0 mg).

1- hydroxy- cytorhodine V:

red violet, amorphous substance, easily soluble in methanol, ethyl acetate, chloroform and toluene, insoluble in water and hexane, soluble in 1 aqueous G-gluconic acid or D-lactobionic acid.

Thin layer chromatography as in example 4: RF = 0.21.

Absorption spectrum:

The identification of the components in the above-mentioned examples took place under the following measurement conditions: Proton resonance spectra (^H-NMR spectra) were measured on a HX-270 Bruker Fourier-transform nuclear resonance spectrometer at 270 MHZ. The concentration is 2-4 mg/0.5 ml 99.8% CDCl 3 , the solutions were shaken immediately after preparation with 0.1 ml 5% Na 2 CO 3 in 99.5% D 2 O.

The signals marked with a star in the figures originate from low molecular weight impurities in the % 0 range and from solvent residues.

The mass spectra were measured on MS-902 S,AEI mass spectrometer using a FAB (solid atom bombardment) ion source. The substances were introduced into a matrix, and thioglycerol into the ion source, partly with the addition of ammonium chloride.

The absorption spectra were measured in the range 200-700 nm.

a) water-methanol 1 : 9

b) 10% 1 n HCl in methanol

c) 10% 1 n NaOH in methanol.

The substance concentrations were 10 - 30 mg/l, indicated by absorption maxima in nm and the molar extinction coefficients (log e).

Determination of the cytotoxic activity:

The determination of the cytostatic effect of the compounds mentioned here took place on L1210 leukemia cells in mice. IN

in detail the following test systems are used:

a) Proliferation equipment.

This method is determined in vitro after incubation of the cells

with different concentrations of the test substance, the extent to which the cells can incorporate radioactive DNA precursors (e.g.<14>C labeled Thymidine). Untreated L1210 cells were subjected to the same experimental conditions and serve as a control. In the following, the method is briefly described: L1210 cells in exponential growth phase (5 x 10<3>/ml in RPMI 1640) are incubated in a microtiter plate for 72 hours with different concentrations of the test substance (37°C, 5% C02, 95% relative humidity ). The control consists of cells that are only incubated with fresh medium. All provisions are carried out as 4 times

14

decision. After 65 hours, 50 µl of C-1-thymidine (1.5 µc/ml) are added to radioactively mark the cells' DNA. After 7 hours of incubation, the cells are aspirated, the DNA is separated with 5% trichloroacetic acid and washed in sequence with water or methanol. After drying at 50°C, the DNA incorporated radioactivity is determined after adding 5 ml of scintillation liquid.

The results are given in terms of the scintillation index after incubation with the test substance as a percentage of untreated controls. From the measured values determined in this way, the sintering effect curve is determined and IC5g', i.e., is determined graphically. the concentration which, under the test conditions, lowers the incorporation of radioactive thymidine by around 5% compared to the controls. the IC^^ value

of the compounds mentioned here compared to adriamycin (ADM) are summarized in table 1. b) Colony formation of L1210 leukemia cells in soft agar. This method serves to demonstrate an impact of the test substance

on the weight ratio of the cells over several generations (at a cell cycle time of 10-12 hours, observed during the test period of 7 days for approx. 14 consecutive generations).

In this sample, cytostatic active substances cause a reduction in the observed colony numbers compared to untreated controls. In detail, the test is carried out as follows: 500 leukemia cells per plate is incubated with different concentrations of test substance for 1 hour at approx. 30°C. Then washed twice with McCoy 5A medium, and poured into Petri dishes after adding 0.3% agar. Controls are incubated in fresh medium only. Instead of 1 hour's incubation, in many cases different concentrations and test substance are mixed with the upper agar layer in order to achieve a continuous exposure of the cells over the total incubation time. After the agar solidifies, the plates are incubated in a culture cabinet for 7 days at 37°C (5% CC>2, 95% relative humidity). The number of colonies formed with a diameter of 60 y is then counted. The results are given as colony numbers in treated agar plates in % of untreated controls. From the thus obtained dose-effect curve, IC5 rn 0 is determined as a measure of the effect of the substance. The results for the compounds mentioned here compared to adriamycin are summarized in table 1.

Claims (4)

1. Method for preparing compound of formula IN in which R means hydrogen or hydroxy and R.^ and R.sub.2 are the same or different and mean sugar combinations of the following composition: -Roa-dF-CinA -Roa-dF-Acu -Roa-dF=CinB -Roa-Rod-CinA or -Roa-Rod-Acu or R2 has the above meaning, and means sugar combinations of the following composition: -Roa-dF-Rod -Roa-dF-CinB -Roa-dF-Acu -Roa-Rod-Acu -Roa-dF-CinA -Roa-Rod-Rod or -Roa-Rod-CinA where Roa means rhodosamine, dF means deoxyfucose, Rod means Rhodinose, Acu means aculose, CinA cinerulose A and CinB means cinerulose B and dF means C inB, that the two sugar building blocks deoxyfucose and cinerulose B are linked by means of an additional ether bridge next to the usual glycosidic bond, characterized in that a compound of formula II in which R means hydrogen or hydroxy and R^ and R^ are the same or different and mean sugar combinations of the following composition: -Roa-dF-Rod -Roa-Rod-Rod -Roa-dF-CinA or -Roa-Rod-CinA or only R^ or only R^ have the aforementioned meanings and the second substituent resp. means -Roa-dF-Rod -Roa-dF=CinB -Roa-dF-Acu -Roa-Rod-Acu -Roa-dF-CinA -Roa-Rod-Rod or -Roa-Rod-CinA is reacted with a specific oxidoreductase and the compound or compounds formed are isolated from the reaction medium in a manner known per se by extraction and purified. 2. Method according to claim 1, characterized in that the enzymatic reaction is carried out at a temperature of 20-45°C, a pH of 4-7 and a reaction duration of a few hours up to 3 days. 3. Method according to claims 1 and 2, characterized in that the oxidoreductase extracted from the culture filtrate of Streptomyces galilaeus (ATCC 31133) or Streptomyces purpurascens (DSM 2658) is used. 4. Process according to claims 1 and 2, characterized in that in the preparation of a compound of formula I in which one of the sugar combinations means -Roa-dF-Rod -Roa-dF-CinB -Roa-dF-Acu -Roa-dF-CinA -Roa-Rod-Acu -Roa-Rod-Rod or -Roa-Rod-CinA and R_ means one of the sugar combinas ions -Roa-dF -CinA -Roa-dF-Acu -Roa-dF=CinB -Roa-Rod-CinA or -Roa-Rod-Acu is reacted with a compound of formula II, in which R^ has the meanings mentioned above for R^ and R4 means one of the sugar combinations -Roa-dF-Rod -Roa-dF-CinA -Roa-Rod-Rod or -Roa-Rod-CinA with an oxidoreductase extracted from the culture filtrate of Streptomyces galilaeus (ATCC 31133).