Introduction

A number of sulphur compounds are known in which the sulphur is bonded to three substituents and also retain a lone pair of electrons.  These types of compounds can in suitable cases exhibit chirality. Sulphoxides also have a structure with three different substituents and a lone pair of electrons around a sulphur atom and so can exhibit chirality¹.

Sulphoxides have been obtained in optically active forms by resolution of the racemates. However, optically active sulphoxide have also been prepared by asymmetric synthesis, which are carried out by oxidation of unsymmetrical substituted sulphide.  Sulphoxidation in chiral environments is either limited to specific substituents or gives only moderate to low enantiomeric excess of product and use of chiral oxidants can be similarly unrewarding².

The asymmetric oxidation of a prochiral sulphide is undoubtedly a more direct and commercial method for the synthesis of sulphoxide than the kinetic resolution of a reacemic sulphoxide. The transformations with metal catalyst or isolated enzymes are tedious and expensive.  Asymmetric sulfoxidation by whole cells are much cheaper and more convenient, as it does not require expensive co-factors³.

One of the earliest methods of preparing chiral sulphoxides, microbial biotransformation, has not been extensively adopted for synthetic purposes.  Despite the proven success of this method in performing stereospecific asymmetric oxidation of certain prochiral sulphides and the associated developments in the application of isolated oxidase enzyme methodology, further work is still necessary to identify new substrates and  specific microorganisms which will prove useful for the production of chiral sulphoxides.

The use of enzymes as reagents in organic synthesis provides exciting opportunities for the exercise of regio and stereoselectivity.  In case of oxidative reactions catalyzed by oxygenase enzymes, whole microbial cells in an actively growing or resting stage are commonly employed^4,5,6,7. In general, organic sulfides, along with other xenobiotic organic compounds are oxidized both in vivo and in vitro by either one or both of two types of mono oxygenases enzymes, those dependent upon cytochrome P-450 for activation and transfer and other using a flavin molecule for the purpose². It has generally been assumed that the fungal enzymes responsible for oxidation of sulphide to sulphoxide are cytochrome P-450 containing monooxygenases.

some selected  fungi were screened for  their capacity to carry out sulphoxidation of  5- Methoxy-2-[[(4-methoxy –3, 5-dimethyl-2-pyridinyl) methyl]-sulphinyl]-1 H benzimidazole to omeprazole.

The sulphide selected for screening is an intermediate in the synthesis of omeprazole, a potent proton pump  inhibitor. It acts as an inhibitor of gastric H+, K+ ATPase, the gastric acid pump. This being sulphoxide, has an asymmetric center in the sulphur atom and exists in two optical isomers.  The S isomer is the desirable sulphoxide for the observed clinical activity.

Fungi selected for screening are Aspergillus niger MTCC 961, Pichia farinosa MTCC 246, Saccharomyces cerevisiae MTCC 174, Candida vishwanathii MTCC 1629, Rhizopus stolonifer MTCC 2198, Aspergillus niger,Aspergillus flavus, Aspergillus oryzae, pencillium species, (All isolated from soil) and bakers yeast.

Materials and Methods    

Microorganisms

Saccharomyces cerevisiae: MTCC 174 was obtained from MTCC, Chandigarh. The organism was maintained on YEPD media containing, yeast

extract 3.0 g, peptone 10.0 g, dextrose 20.0 g, agar 20.0 g and distilled water 1000 ml.

Pichia farinosa MTCC 246 was obtained from MTCC, Chandigarh. The organism was maintained on MYA media containing malt extract 3.0 g, yeast

extract 3.0 g, peptone 5.0 g, dextrose 10.0 g,  agar 20.0 g and distilled water 1000 ml.

Aspergillus niger (soil isolate) and Aspergillus niger MTCC 961. The organisms were maintained on MRBA media containing, dextrose 10.0 g, peptone 5.0 g,

potassium dihydrogen phosphate 1.0 g, Magnesium sulphate 0.5 g, Rose Bengal

0.0035 g, Agar 20.0 g, distilled water 1000 ml and streptomycin 0.03 g.

Candida viswanathii MTCC 1629: The organism was obtained from MTCC, Chandigarh and maintained on YEPD medium.

Pencillium species (soil isolate): The organism was maintained on MRBA medium.

Rhizopus stolonifer MTCC 2198: The organism was maintained on MRBA Medium.

Chemicals:  All chemicals and solvents were from local suppliers and of analytical  grade.

Omeprazole standard was obtained from the industry.

Cultivation of Saccharomyces cerevisiae, Pichia farinosa and Candida viswanathii

The organisms from the slant culture was subcultured  into 300ml YEPD medium containing Yeast extract 0.3 g, peptone 1.0 g, dextrose 2.0 g and distilled water 100 ml, pH was adjusted to 7.0 and was sterilized at 121ºC for 15 min. The cultures were grown at 30ºC, 160 rpm for 24 h.

10% volume of the fermentation medium was used for inoculation of 2.5 L of YEPD medium. The inoculated medium was incubated at 30ºC, 160 rpm for 48 h. After 48 h of growth, the cells were separated by filtration using buchner  funnel and the biomass was washed with phosphate buffer twice.

Cultivation of Aspergillus niger, Rhizopus stolonifer and Pencillium.

The spores from the maintenance culture were inoculated to, 2 L of potato dextrose medium containing potato 400.0 g, dextrose 20.0 g and distilled water 2000ml.

The pH of the medium was adjusted to 6.0.  The medium was sterilized at 121 ºC for 15 min. The inoculated medium was incubated at 25ºC for 5 days to get sufficient biomass. The mycelial biomass was separated by filtration and washed with phosphate buffer twice.

Procedure for Bio-oxidation

10 g of the biomass was taken in 20 ml phosphate buffer of  pH 7.6, 20 mg of omeprazole intermediate  dissolved in 2 ml alcohol was added to the above suspension and incubated at 30˚C, 160 rpm for 48 h.

The reaction mixture was filtered to remove the biomass. Filtrate was extracted with alkaline methylene dichloride (20 ml x 3), washed with 20  ml of brine twice and dried over anhydrous sodium sulphate. The organic extract was then concentrated by evaporation.

HPLC Analysis

The oxidized product of omeprazole was quantified by HPLC.

Chromatographic condition

Mobile phase : phosphate buffer (pH 7.6)  : methanol ( 25:75)

Column:                C18 phenomenex

250 x4.6 mm, .5 μm,

Flow rate:              0.8 ml/min.

Wave length:         280 nm

Injection volume:   20 μl.

Standard Preparation

25 mg of the Standard was dissolved in 25 ml of methanol and diluted to get concentration of 2.5 to 10.0 μg.

Sample preparation

The concentrated sample was made up to 10 ml with methanol.

Results and Discussion

Totally ten organisms were selected for carrying out sulphoxidation of omeprazole intermediate (Table – 1), out of which one species of Aspergillus niger showed negligible conversion while Rhizopus stolonifer was found to be effective in bringing about sulphoxidation of the selected substrate. Even though Aspergillus species are known to have good sulphoxidation acitiviy, the above selected species were inactive in general on the omeprazole intermediate.

Table 1

The reaction was monitored by TLC (Benzene: ethyl acetate: methanol:: 5:3:1), and the product was confirmed by HPLC and LCMS. The standard for the analysis was obtained from industry.

Maintainence of alkalinity during reaction and extraction process is critical in sulphoxidation of omperazole intermediate as omperazole is unstable in acidic or neutral medium.

References

1. Michael North, Principles and Applications of Stereochemistry, Stanely Thomas (publishing) Ltd., United Kingdom, 56 (1998).
2. Herbert Leslie Holland, Chiral Sulfoxidation by Biotransformation of Organic Sulfides, Chem.Rev., 88, 473(1988).
3. Ai-Tao Li, Jian-Dong Zhang, Jian-He Xu, Wen-Ya Lu and Guo-Qiang Lin, Appl. Environ. Microbiol., 75, 551 (2009).
4. European Patent EP0795024
5. Herbert Leslie Holland, Frances M. Brown and Brett G. Larsen, Tetrahedron Asymmetry, 6, 1561(1995).
6. Kurt Faber, Biotransformations in Organic Chemistry, Springer, New York, 177 (2004).
7. Derek R. Boyd, Narain D. Sharma,Simon A. Haughey,Martina A. Kennedy, Brian T. McMurray, Gary N. Sheldrake,Christopher C. R. Allen,Howard Dalton and Kenneth Sproule,  J. Chem. Soc., 1, 1929(1998).