Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Patent
1997-08-23
1999-11-23
Witz, Jean C.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
435170, 549292, 560256, C12P 718, C12P 104, C07D30930, C07C 6702
Patent
active
059898771
DESCRIPTION:
BRIEF SUMMARY
FIELD AND BACKGROUND OF THE INVENTION
The present invention provides an improvement for the recovery of lovastatin, compactin or pravastatin from fermentation broths.
Lovastatin for instance is produced as a secondary metabolite by various microorganisms such as Aspergillus terreus (U.S. Pat. No. 4,231,938) or Monascus ruber (U.S. Pat. No. 4,323,648). During the fermentation also lovastatin related byproducts such as 4-acetyl lovastatin are produced.
Lovastatin, usually in the acid form, can be isolated from the fermentation broth in different ways. The first stage is formed by purification yielding crude crystals. These crude crystals still comprise related compounds like 4-acetyl lovastatin. As lovastatin is a pharmaceutical compound that has to meet high purity requirements, additional purification in order to remove the lovastatin related impurities is necessary. The lovastatin-related impurities are generally removed by multiple recrystallizations, by column chromatography as described in U.S. Pat. No. 4,231,938 or preparative HPLC (WO 92/16276), decreasing the yield significantly.
BRIEF DESCRIPTION OF THE INVENTION
By the process of the present invention impurities present in the broth filtrate are removed, thus preventing the need for their removal via additional recrystallizations and resulting in an increased yield.
During the application of the process of the present invention to a broth filtrate of a microorganism producing statins as for instance Aspergillus terreus, surprisingly 4-acetyl lovastatin is selectively converted into lovastatin instead of being converted into dehydro lovastatin via dehydration which occurs for pure 4-acetyl lovastatin (see FIG. I). Another surprising fact is that the 2-methyl butanoate group is not removed during the application of the invention.
The process of the present invention has neither been described nor suggested in the prior art.
BRIEF DESCRIPTION OF THE FIGURES
FIG. I. Reaction scheme of the deacylation of 4-acetyl lovastatinic acid to lovastatinic acid (a) and the dehydratation of 4-acetyl lovastatinic acid to dehydro lovastatinic acid (b)
FIG. II. Thin layer chromatogram (TLC) showing reduction of impurities in crude lovastatin crystals after applying the process of the invention. Eluent: chloroform/methanol=9/1; detection: iodine staining; run product: 2 .mu.l of a solution, consisting of crude lovastatin crystal in toluene, concentration 50 g/l.
Right side: crude crystal from untreated broth filtrate; left side: crude crystal from broth filtrate which has been stirred for 2 hours at 50.degree. C. and pH 12.5.
DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns a process to improve the recovery of lovastatin, pravastatin or compactin from a broth filtrate. This process comprises: consisting of lovastatin, compactin or pravastatin resulting in a product medium filtrate about pH 10. Preferably, said method also comprises heating the clarified broth filtrate above approximately 50.degree. C.
The process of the present invention offers a simple and selective method of the deacylation of 4-acylated statins in broth filtrates, resulting in an improved yield and purity of the crystals. During the treatment at high pH the 4-acylated statin is converted into the related statin. The conversion rate of, for instance, 4-acetyl lovastatin into lovastatin in broth filtrate is dependent on the pH and the reaction temperature. In a preferred embodiment of the present invention, the treatment is carried out at pH-values above pH=10, more preferably between pH=10 and pH=13, most preferably between pH=11 and pH=12.5. Also temperatures between 60.degree. C. and 95.degree. C. are preferred. By applying higher pH-values and/or higher temperatures the reaction time for complete deacylation decreases.
The process of increasing the pH can be advantageously be applied to filtrates of fermentation broths from any microorganism that is capable of producing a member of the group consisting of lovastatin, pravastatin or compactin. Microorganisms capable
REFERENCES:
patent: 5451688 (1995-09-01), Kogen et al.
De Pater Robertus M.
Sibeyn Mieke
Gist-Brocades B.V.
Hanley Susan
Witz Jean C.
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