Methods for the preparation, isolation and purification of epothilone B, and X-ray crystal structures of epothilone B

Abstract

The present invention relates to improved methods for the production, isolation and purification of epothilone B. These methods include, for example, a fermentation process for the production of epothilone B, isolation via adsorption onto a resin, and subsequent purification.

1. A process for isolation of epothilone B from an epothilone-producing microorganism comprising:
 (a) fermenting a strain of epothilone-producing microorganism in the presence of a resin that adsorbs epothilone B by hydrophobic interaction;  
 (b) collecting the resin in a water-based medium;  
 (c) extracting the resin with a solvent selected to extract epothilone B and to separate it from the water-based medium; and  
 (d) crystallizing epothilone B from the extraction phase; wherein said fermentation step further comprises feeding an additive capable of improving the amount of epothilone B produced as compared with the amount of epothilone A produced  that increases the ratio of epothilone B to epothilone A produced from the fermentation, as compared with when the fermentation is performed without feeding the additive.  
 
     
     
       2. The process of  claim 1  wherein the crystallized epothilone B from step (d) is substantially pure. 
     
     
       3. The process of  claim 1  wherein the resin is extracted with a polar solvent. 
     
     
       4. The process of  claim 1  wherein said fermentation step further comprises fermenting said epothilone-producing microorganism in the presence of skim milk, soy flour, yeast extract, maltrin starch, and/or glycerol. 
     
     
       5. The process of  claim 1  wherein said fermentation step comprises continuously feeding said additive capable of improving the ratio of epothilone B to epothilone A. 
     
     
       6. The process of  claim 1  wherein said additive is a propionic acid salt or ester. 
     
     
       7. The process of  claim 6  wherein said additive is sodium propionate, propionic acid methyl ester or propionic acid ethyl ester. 
     
     
       8. The process of  claim 1  wherein the crystallization is conducted to reduce the amount of epothilone A to about 55% or less of the amount of epothilone A present after extraction step (c). 
     
     
       9. The process of  claim 8  further comprising
 (e) at least a second crystallization step effective to reduce the amount of epothilone A to about 55% or less of the amount of epothilone A present after crystallization step (d).  
 
     
     
       10. The process of  claim 1  wherein the epothilone-producing microorganism is a strain of Sorangium cellulosum. 
     
     
       11. The process of claim  10    1 , wherein said epothilone- producing  microorganism is Sorangium cellulosum strain ATCC No. PTA 3880. 
     
     
       12. The process of claim  10    1 , wherein said epothilone- producing  microorganism is Sorangium cellulosum strain ATCC No. PTA 3881. 
     
     
       13. The process of  claim 1  wherein the resin is a styrene/divinylbenzene-based polymer. 
     
     
       14. The process of  claim 13  wherein the resin is present in a range of from about 0.2 w/v % to about 5.0 w/v %. 
     
     
       15. The process of  claim 1  wherein said step (d) comprises:
 (i) adding a second solvent in which epothilone B is either not soluble or sparingly soluble;  
 (ii) removing at least a portion of the extraction solvent; and  
 (iii) transitioning the resultant solvent or solvent mixture to a temperature at which epothilone B crystallizes.  
 
     
     
       16. The process of  claim 15  wherein the extraction solvent is ethyl acetate or MTBE, and the second solvent is toluene. 
     
     
       17. The process of  claim 1  further comprising:
 (f) prior to step (c), washing the resin with aqueous acetonitrile, or aqueous methanol, or an aqueous medium comprising a detergent and an amine reagent added in base form, the aqueous medium selected to not elute epothilone B.  
 
     
     
       18. The process of  claim 1 , wherein step (c) further comprises polish filtering the epothilone B containing solvent. 
     
     
       19. The process of  claim 1 , wherein epothilone B and epothilone A are produced in an epothilone B/A ratio of at least one. 
     
     
       20. The process of  claim 1 , wherein epothilone B and epothilone A are produced in an epothilone B/A ratio of at least 1.5. 
     
     
       21. The process of  claim 1 , wherein epothilone B and epothilone A are produced in an epothilone B/A ratio in the range of 1.5 to 4.0.