Biokatalitička azidoliza fluoriranih derivata stiren oksida

master's thesis

• Title (english): Biocatalytic azidolysis of fluoro-substituted stiren oxide derivatives
• Author: Petra Marin
• Mentor: Marina Cvjetko Bubalo (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Cell Technology, Application and Biotransformations); Zagreb
• Defense date and country: 2020-06-09, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Halogenhidrin-dehalogenaze (HHDH) su enzimi koji kataliziraju nukleofilno otvaranje epoksida koristeći niz malih aniona kao što su azid, cijanid, cijanat i dr. Upotrebom ovih enzima moguće je pripraviti enantiomerno čiste fluorirane građevne blokove čija je dostupnost od velikog značaja pri razvoju lijekova u farmaceutskoj industriji. U okviru ovog rada, provedena je prekomjerna ekspresija 7 različitih HHDH, divljih tipova (HheA, HheB i HheC) i genetski modificiranih varijanti (HheA-N178A, HheC-T134V, HheC-W249P i HheC-W4). Uspješnost prekomjerne ekspresije proteina potvrđena je SDS-poliakrilamidnom elektroforezom. Ispitana je katalitička aktivnost genetski modificiranog enzima HheA-N178A u reakcijama azidolize 5 fluoriranih derivata stiren oksida različitog položaja supstitucije (1a-1e). HheA-N178A je pokazala nisku do vrlo visoku S-enantioselektivnost prema ispitanim supstratima (E = 2 – 165). Za visoku enantioselektivnost reakcije presudan je supstituent na para-položaju, a izrazito nepovoljan utjecaj na enantioselektivnost ima metilna skupina kao drugi supstituent na kiralnom centru supstrata.

Abstract (english)

Halohydrin dehalogenases (HHDH) are enzymes that catalyze the nucleophilic opening of epoxides using a number of anionic nucleophiles, including azide, cyanide, nitrite, cyanate, etc. The use of HHDH gives possibility to prepare enantiomerically pure fluorinated building blocks whose availability is of great importance in pharmaceutical industry. In this thesis, overexpression of 7 different HHDH, wild-type (HheA, HheB and HheC) and genetically modified variants (HheA-N178A, HheC-T134V, HheC-W249P and HheC-W4) was performed. The success of protein overexpression was determined by SDS-polyacrylamide electrophoresis. Catalytic activity of genetically modified halohydrin dehalogenase HheA-N178A in reactions of azidolysis of 5 fluorinated styrene oxide derivatives of different substitution positions (1a-1e) was explored. HheA-N178A showed low to very high S-enantioselectivity towards the tested substrates (E = 2 - 165). Para-position of substituent is crucial for the high enantioselectivity of the reaction, and the methyl group has an extremely unfavorable effect on the enantioselectivity as a second substituent at the chiral center of substrate.

• Keywords: azidoliza; epoksidi; halogenhidrin-dehalogenaze; kinetička rezolucija; β-azidoalkoholi
• Keywords (english): azidolysis; epoxides; halohydrin-dehalogenases; kinetic resolution; β-azidoalcohols
• Language: croatian
• URN:NBN: urn:nbn:hr:159:923960
• Study programme: Title: Bioprocess Engineering; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva (magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2020-06-19 09:25:40