טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentDerry Netta-Lee
SubjectBiocatalytic Synthesis of Chiral Sulfoxides and Epoxides
Using Toluene Monooxygenases
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Ayelet Fishman
Full Thesis textFull thesis text - English Version


Abstract

The production of enantiomerically pure compounds is of steadily increasing importance to the chemical and pharmaceutical industries since the world market for chiral fine chemicals, pharmaceuticals, agrochemicals, and flavor compounds rapidly expands. Chiral sulfoxides have major importance in the pharmaceutical industry. For example, omeprazaole is a chiral sulfoxide, and its (S)-enantiomer, esomeprazole, is the leading drug used for treatment of ulcer. Chiral epoxides are important intermediates in the chemical synthesis of optically active pharmaceuticals, polymers and agrochemicals. Enantiopure styrene oxide is an epoxide mainly used to synthesize enantiopure pharmaceuticals such as anti-diabetic agents. The objective of the present research was to develop novel biocatalytic processes for the production of esomeprazole and chiral styrene oxide based on the selective oxidation of omeprazole-sulfide and styrene, respectively, with toluene monooxygenases (TMOs).

TMOs are soluble, non-heme diiron multi component enzymes which utilize oxygen to catalyze the hydroxylation of hydrocarbon substrates. A high throughput method for measuring omeprazole-sulfide oxidation was developed, based on the fluorescence signal obtained from acid activation of omeprazole. Unfortunately, screening of thousands of TMO variants did not reveal positive hits. It is concluded therefore, that omeprazole-sulfide is not a potential substrate for toluene monooxygenases in the present whole cell system, and that a biocatalytic process based on these enzymes is not feasible. On the other hand, we show for the first time, that TMOs are capable of performing the enantioselective epoxidation reaction of styrene. Furthermore, screening of random libraries and saturation mutagenesis libraries at key positions in the α-hydroxylase subunit of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 and toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, revealed variants whose catalytic activity was altered, so that the rate and enantioselectivity were improved. Variant TOM TomA3 V106C was found to oxidize styrene 7.5 times faster than wild-type enzyme. The enantiomeric excess increased from almost no selectivity for the wild-type to 36% (pro-R) for the mutant. T4MO variant TmoA I100L did not change the wild-type oxidation rate, but the enantiomeric excess rose from 23% to 58% (pro-S).

In silico modeling suggested that residues Leu at position I100 of T4MO and Cys at position V106 of TOM, control the size of the hydrophobic gate and thus, restrict the entrance of the substrate and influence the reaction rate. In addition, these positions regulate proper alignment of the substrate near the diiron atoms, consequently, controlling the enantioselectivity of the product.