GB2185480A - Production of avermectins - Google Patents

Abstract

Avermectins are more efficiently produced by fermentation of novel microorganisms which do not also produce significant levels of oligomycins. The microorganism may be a mutant strain of streptomyces avermitilis.

Description

SPECIFICATION Macrolide production This invention concerns a new process for making avermectins.

Background of the Invention Avermectins are macrolide compounds which exhibit potent anthelmintic, insecticidal and acaricidal activities. Typically they are produced by fermentation of a microorganism belonging to the genus Streptomyces, as described in British Patent 1,573,955.

Avermectins are compounds of the following general structural formula:

where the group R1 is an 4'-(a-L-oleandrosyl)-x-L-oleandrose disaccharide and the groups R2, R3 and X distinguish the different individual avermectins, as shown in Table TABLE I -R2 -R3 Avermectin A18 -CH3 -C2H5 -CH=CH Avermectin Alb -CH3 -CH3 -CH=CH Avermectin B18 -H -C2Hs -CH=CH Avermectin Bib -H -CH3 -CH=CH Avermectin A2 -CH3 -C2H5 -CH2-CH(OH)- Avermectin A2b -CH3 -CH3 -CH,-CH(OH)- Avermectin B28 -H -C2H5 -CH2-CH(OH) Avermectin B2b -H -CH3 -CH2-CH(OH) The compounds are of use in veterinary, agriculture and human medicine.

The production of avermectins using microorganisms, such as Streptomyces avermitilis ATCC 31267, ATCC 31271 or ATCC 31272 described in the British Patent 1,573,955, additionally gives in high concentration a compound which will be called the CFA-compound. This substance is insoluble in water but it is soluble in the solvents normally used in avermectin extraction.

Although the particular choice of microorganism and fermentation conditions will affect the ratio of CFA-compound to avermectins, the range is typically for example from 5:1 to 1:5.

Object of the Invention The object of the present invention is to facilitate the extraction of avermectins, in preference to the CFA-compound.

Summary of the Invention To this end, the present invention provides an avermectin-producing microorganism which on fermentation to produce avermectin compounds gives a fermentation broth which contains substantially no CFA-compound.

Preferred Embodiments For example, the ratio of CFA-compound to avermectins using the present microorganism is preferably less than 1:20, better still less than 1:50. The level of the CFA-compound is below that at which it can be detected by TLC or HPLC.

For assaying for CFA-compound and avermectins by HPLC or TLC, the sample shall be prepared as follows: mycelium from 10 ml of whole broth is mixed with 10 ml of methanol, the mixture is shaken, and the resultant extract is filtered and it is ready for assay. Sample quantification is made using 96% avermectin B18 and 98% CFA-compound as standards.

With such an assay when using the previously availabe avermectin-producing microorganism, it is found on TLC plates, that the CFA-compound shows a different retention factor (Rf) than avermectins. For instance the Rf is 0.52 in a ethyl acetate-dichloromethane (1:1, v/v) development system and 0.46 in a chloroformethyl acetate-methanol-dichloromethane (9:9:1:2, v/v/v/v) system when Kieselgel-60-F254 (20x20 cm, 0.25 thick, E. Merck, Darmstadt) plates are used.

When TLC plates are visualized with p-anisaldehyde-sulphuric acid, the CFA-compound is coloured brown, different to the violet colour produced by avermectins.

We have found that the CFA-compound can be quantitatively analyzed by high performance liquid chromatorgraphy (HPLC) using the following conditions: Waters chromatograph (pumps models 600 and M-45); injector, U6K; Detector Lambda Max 480; Philips Recorder PM-8220; Waters radial compression C18 reverse phase column (10x0.8 cm); mobile phase, methanol-water (85:15,v/v); sample volume, 50 ,ul; flow rate, 2ml/min; chart speed, 3 mm/min; wave-length, 246 nm; and chromatogram time, 10 min. Under these conditions, the CFA-compound retention time is 4.0 minutes.

Such a CFA-compound can be extracted and purified from methanolic extracts of Streptomyces avermitilis ATCC 31267 or ATCC 31272 fermentation broths by silica-gel column chromatography and crystallization in a methanol-water mixture.

The CFA-compound exhibits the following characteristics: specific rotation [ai25'CD=73.6 (c=0.5 9/100 ml methanol), melting point mp=166 C, and two absorption maxima at 225 nm (e=19800) and at 231 nm (e=17600). By comparison of these date the IR, H1-NMR and C13-NMR spectra with published data, the CFA-compound was identified as oligomycin A, and verified by comparison with an authentic sample.

To suppress the CFA-compound from the avermectin fermentation broth, mutants can be prepared in accordance with this invention from an avermectin-producing strain to give strains which do not yield the CFA-compound.

Thus, while the ATCC 31267 parental strain gives a CFA concentration higher than 100 Ag/ml, the mutant of this invention normally does not produce a detectable amount of the CFAcompound (using HPLC or TLC).

Avermectin-producing strains of this invention can readily be obtained using available mutation agents, such as radiation or chemical agents. A simple screening procedure will enable detection of desired mutants.

An example of the microroganism of this invention is the strain of Streptomyces avermitilis known by our designation AV-5/4 and has been deposited for patent purposes in accordance with the Budapest Treaty at the National Collection of Industrial Bacteria on 20 December 1985, and given the number NCIB 12197.

A characteristic of the particular strain AV-5/4 that differentiates it from its parental strain is the aerial mycelium colouration, which in ISP No. 2 culture medium is green-coloured instead of the brownish-gray original colour.

The present invention is not restricted to the particular strain AV-5/4. It extends not only to other mutant strains derived from other avermectin-producers, especially of S. avermitilis, but also to mutants of the strain AV-5/4.

The microorganisms of this invention may be employed to produce avermectins in the substantial absence of the CFA-compound. To this end, the present invention provides a process for producing avermectins which involves fermentative cultivation of a microorganism of this invention.

The fermentation media to make avermectins by the present process can be those usually employed for actinomycetes fermentations of secondary metabolites. Such media include an organic nitrogen source, for example, one or more defatted meals from oily seeds, aminoacids, protein extracts and hydrolized proteins, and a carbon source for example, one or more assimilable sugars, polyols or vegetable and/or animal oils. Mineral salts can be added to the culture medium to provide the microorganism with ions necessary for its metabolism, and antifoam agents can be employed when needed. To develop the fermentation, the culture medium pH should normally be controlled between 5.0 and 8.5, preferably between 5.5 and 8.0. Examples of suitable pH regulators are the biological buffers (MOPS, TES, HEPES, etc,), which may be used in concentrations ranging for instance between 0.1 and 1.0%.

To carry out a fermentation with a strain of this invention, a fresh spore suspension from slant tubes, or frozen or lyophilized spores or mycelium can be used. With such spore suspension, a vegetative phase is obtained by culturing it in a liquid medium. This culture can be used as inoculum for fermentors. This step can be repeated several times depending on the fermentor volume.

The suitable fermentation vessels for this process can be conical flasks and the conventional aerobic fermentation tanks, fitted with means for agitation and aeration. The volume of such recipients preferably ranges between 200 ml and 250 m3, and the working volume between 10 and 75% of their total capacity. Before inoculation, the fermentation medium needs to be sterilized and the fermentation has to be conducted in conditions that prevent external combination with undesirable microorganisms. If during the fermentation process nutrients and/or other necessary substances are added, they and the addition system shall be previously sterilized.

The batch fermentation can suitably take from 3 to 15, mor usually 5 to 10 days at 20 to 35, more usually 25 to 30"C.

The avermectins are insoluble in water, and therefore they are in the solid part of the fermentation broth. The broth solids are preferably separated either by filtration or by centrifugation, and subsequently washed with water. Afterwards they can be extracted with a watermiscible solvent, such as methanol, ethanol or acetone. The extract is then usually concentrated 10 to 20 times until a precipitate is obtained, which is separated by centrifugation.

In the case of extraction with acetone, a dense oily phase is obtained. In the case of extraction with methanol, a solid is precipitated. The solid or the oily phase can be taken into a water non-miscible solvent, such as chloroform or methylene chloride, and the solution filtered.

The yield in this step is ordinarily higher than 96%.

The extract can be evaporated to dryness, dissolved in methanol and adsorbed on a hydrophobic resin of the XAD type (trade mark of the Company Rohm & Haas), such as XAD-2, XAD-4 or XAD-8. The whole process is conveniently performed at room temperature.

For best results, the adsorption of the methanolic extract on the resin takes place simultaneously with the addition of a 55% methanolic solution, whereby a slight precipitation occurs of the avermectins and other substances present in the extract. This precipitation does not provoke any flow decrease, and avoids the elution of the avermectins before the washing step. Once the adsorption is over, the resin can be washed with a 55% methanolic solution and treated with methanol. The methanolic eluate contains practically 100% of the avermectin content of the adsorbed extract, while the purity of these macrolides in the eluate is three times higher.

The methanolic eluate can be evaporated to dryness, and dissolved in a solvent mixture (such as chloroform-ethyl acetate-methylene chloride) used to equilibrate a silica-gel column, where the avermectins will be separated and purified. The avermectin solution is typically adsorbed in a silica-gel column, which is eluted with the same solvent mixture used for the equilibration of the column. The eluate is collected in fractions, which are analyzed for instance by HPLC and TLC.

Similar samples are then combined and evaporated. the residue can be dissolved in a methanolwater mixture and crystallized at 4-8"C. Four crystalline products are obtained, with the spectroscopic and physical characteristics of the avermectins A18, A2a, B18 and B2a, respectively. Their structures can be confirmed by H'-NMR, C13-NMR and MS.

The avermectins B18 and/or B28 can be transformed to the commercially important 22,23-dihydroderivative of avermectin Bla. In particular, the avermectin B18 can be transformed to the corresponding 22,23-dihydroderivative by hydrogenation with homogenous catalysis, such as by use of the Wilkinson catalysts.

Examples of the Invention The present invention is illustrated in non-limiting manner by the following Examples: EXAMPLE 1 A 250 ml conical flask containing 40 ml of the following culture medium was inoculated with a spore suspension from a slant of ISP No. 2 medium from incubation of the AV-5/4 strain for 14 days at 28"C.

beef extract 0.3 g tryptone 0.5 g soybean meal 0.5 g giucose 0.1 g soluble starch 2.4 g yeast extract 0.5 g calcium carbonate 0.4 g distilled water to 100 ml The pH was not adjusted and the conical flasks were sterilized at 121"C for 20 minutes. After inoculation, the flasks were incubated at 28"C for 48 hours in an orbital shaker with 5 cm eccentricity at 250 rpm.

This culture was used to seed 250 ml conical flasks containing 30 ml of the following culture medium: glycerol 2.0 g corn dextrin 2.0 9 dry distiller's solubles 0.7 g MOPS 0.5 g autolyzed yeast 0.75 g glucose 0.4 g PPG-2000 0.25 g distilled water to 100 ml The initial pH was adjusted to 7.3 with sodium hydroxide and sterilized at 121"C during 20 minutes. The incubation was performed at 28"C in an orbital shaker with 5 cm eccentricity at 200 rpm during 6 to 7 days. When fermentation was finished, the conical flasks were collected and the fermentation broths analyzed by HPLC, verifying the final concentration, which expressed as avermectin B1a, was higher than 20 ,ug/ml. The CFA-compound final concentration was not detectable.TLC analysis revealed the existence of the four major components of avermectins A1a, A2a, B1a, B28) but not the presence of CFA-compound. The four minor avermectin components were not present at a sufficiently high level for detection by TLC.

EXAMPE 2 and COMPARATIVE EXAMPLE 1 Following the protocol described in Example 1, a comparison was made of the production of avermectins and CFA-compound by ATCC 31267 and AV-5/4 strains, verifying by HPLC analysis that the final concentration of avermectins, expressed as avermectin B1a, was higher than 20 ,ltg/ml for the former strain and was not detectable for the later. In the fermentation broth of ATCC 31267, the presence of a predominant spot corresponding to CFA-compound was observed also by TLC analysis while such spot was not detectable in the case of the AV-5/4 broth.

EXAMPLE 3 34 litre of the fermentation broth obtained according to the process of the Example 1 were filtered under vacuum with a Bcichner apparatus. The solid phase on the paper filter was also washed with water under vacuum and 7.9 kg of solids were obtained. The solids were extracted twice with 17 litre of acetone for 3 hours under agitation. The extract was concentrated at reduced presure and at 400C to a volume of 1900 ml, whereby a solid phase was produced, which was separated by centrifugation. This was the avermectin rich phase.

The rich phase was dissolved in 250 ml of chloroform and then it was filtered. The filtrate was evaporated to dryness, and was then dissolved again in methanol. The sample solution was applied on a XAD-2 column, previously equilibrated with a 55% methanolic solution. During the adsorption of the sample, a 55% methanolic solution was also added to the resins, in order to retain the avermectins in the resin. Once this adsorption was finished, the resin was washed with the methanolic solution, previously mentioned, until the column effluent was colourless. The avermectins were eluted from the resin with methanol.

The dry eluate was dissolved in 35 ml of a solvent mixture chloroform-ethyl acetate-methanol-dichloromethane (9:9:1:2, v/v/v/v). The solution was applied to a silica-gel column (70-280 mesh) with a volume of 600 ml (90x30 cm), equilibrated with the same solvent mixture. The colume was eluted with the same solvent mixture at a flow of 3 ml/min. Fractions with similar contents were combined, evaporated to dryness and crystallized in a mixture of methanol-water at 4"C during 48 hours, giving four compounds.

The compounds obtained by this process were identified through their spectroscopic and physical characteristics as the avermectins A18, A2a, B1a and B2a, respectively.

EXAMPLE 4 Five hundred milligram of avermectin B18 and 200 mg of triphenylphosphinerhodium chloride were dissolved in 50 ml benzene and shaken under a nitrogen atmosphere at 2.7 kg/cm2. The hydrogenation was followed by HPLC analysis.

After 8 hours, the reaction product was filtered and evaporated to dryness at reduced pressure, purified by column chromatography on silica-gel, and 350 mg of a product were obtained. This product was shown by spectroscopical and physical studies to be the 22,23-dihydroderivative of the avermectin B1a. The yield was 70%.

Claims (10)

1. A process for producing avermectin compounds which process comprises fermentative cultivation of an avermectin-producing microorganism which on fermentation to produce avermectin compounds gives a fermentation broth which contains substantially no CFA-compound.

2. A process according to claim 1, wherein the ratio in the fermentation broth of the CFA compound to avermectin compounds is less than 1:20.

3. A process according to claim 1, wherein the level in the fermentation broth of the CFAcompound to avermectin compounds is below that at which it can be detected by TLC.

4. A process according to claim 1, 2 or 3, wherein the fermentative cultivation takes 5 to 10 days at 25 to 30"C at pH 5.5 to 8.0.

5. A process according to any preceding claim, wherein at least one of the avermectin compounds is extracted from the fermentation broth.

6. A process according to claim 5, wherein avermectin B1. is extracted from the fermentation broth.

7. A process according to claim 6, wherein the extracted avermectin B,, is subjected to 22,23-dehydrogenation.

8. A process according to claim 7, wherein the 22,23-dehydrogenation is effected using a Wilkinson catalyst.

9. An avermectin-producing microorganism which on fermentation to produce avermectin compounds gives a fermentation broth which contains substantially no CFA-compound.

10. Streptomyces avermitilis AV-5/4 (NCIB 12197).