Evaluacija fluoriranih derivata stiren oksida kao supstrata halogenhidrin-dehalogenaze

master's thesis

• Title (english): Evaluation of fluorinated derivatives od styrene oxide as substrates of halohydrin dehalogenase
• Author: Marko Pranić
• Mentor: Zvjezdana Findrik Blažević (mentor)
• Committee member: Zvjezdana Findrik Blažević (predsjednik povjerenstva)
• Committee member: Martina Sudar (član povjerenstva)
• Committee member: Marija Vuković Domanovac (član povjerenstva)
• Granter: University of Zagreb; Faculty of Chemical Engineering and Technology; Zagreb
• Defense date and country: 2020-12-21, Croatia
• Scientific / art field, discipline and subdiscipline: TECHNICAL SCIENCES; Chemical Engineering

Abstract

Fluorirani aromatski spojevi su važni kiralni građevni blokovi u farmaceutskoj industriji. Jedna od metoda njihove sinteze je i biokatalitički put korištenjem enzima halogenhidrin-dehalogenaze (HHDH). U ovom radu proučavana je reakcija otvaranja epoksidnog prstena, fluoriranog derivata stiren oksida uz azid kao nukleofil. Reakcija je katalizirana s HHDH iz divljeg soja mikroorganizma poznata i pod nazivom HheC. Provedene su preliminarne reakcije u vodenom mediju s 5 % organskog otapala. U njima je evaluirano šest različitih epoksida s atomima fluora kao supstituentima na stiren oksidu. Metodom početnih brzina određeni su kinetički modeli i parametri za svaku od reakcija. Utjecaj koncentracije epoksida (cepox) na brzinu enzimske reakcije odgovara Michaelis-Meteničinoj enzimskoj kinetici, dok utjecaj koncentracije azida odgovara kinetičkom modelu s inhibicijom supstrata. Enzimsku kinetiku u vodenom mediju bilo je moguće mjeriti samo do koncentracije epoksida od 20 mM, zbog njihove ograničene topljivosti. Kako bi se povećala topljivost epoksida, reakcije je potrebno provoditi pri većim udjelima organskog otapala. Usporedbom procijenjenih kinetičkih parametara i pregledom literature određeno je da položaj supstituenta ima presudnu ulogu na aktivnost enzima i afinitet supstrata prema enzimu. Od ispitivanih stiren oksida najbolji supstrat za HheC je onaj koji ima najudaljenije supstituente od epoksidnog prstena, odnosno u para položaju.

Abstract (english)

Fluorinated aromatic compounds are important chiral building blocks in the pharmaceutical industry. One of the methods for their synthesis is a biocatalytic path with enzyme halohydrin dehalogenase (HHDH). In this work, the ring-opening reaction of epoxides was studied. Substrates were fluorinated derivates of styrene oxide with azide as a nucleophile. The reaction was catalyzed by wild-type HHDH, also known as HheC. Preliminary reactions were ran in a buffered medium with 5 % of organic solvent. Six different epoxides with fluorine atom on different positions of the phenyl ring were evaluated. By using the initial reaction rate method, kinetic models and parameters for each reaction were determinend. The influence of the concentration of epoxides (cepox) on the rate of enzymatic reactions fits Michaelis-Menten kinetics, while the influence of azide concentration fits the Michaelis-Menten kinetics with substrate inhibition. Because of low epoxide solubility, enzymatic kinetic in the water medium can be determined up to the epoxide concentration of 20 mM. To increase epoxide solubility reactions need to be carried out at higher concentration of organic solvent. By comparing the estimated kinetic parameters and literature search, it was found that the substituent position has a deterministic role in enzyme activity and affinity of the substrate towards the enzyme. From the tested styrene oxides, the best substrate for HheC is the one that has substituents on the opposite side from the epoxide ring.

• Keywords: epoksid; halogenhidrin-dehalogenaza; kinetika enzimske reakcije; evaluacija supstrata
• Keywords (english): epoxide; halohydrin-dehalogenase; enzyme reaction kinetics; substrate evaluation
• Language: croatian
• URN:NBN: urn:nbn:hr:149:888012
• Project: Number: IP-2018-01-4493; Title: Enzimska sinteza fluoriranih kiralnih građevnih blokova; Acronym: EnzyFluor; Leader: Maja Majerić-Elenkov; Jurisdiction: Croatia; Funder: HRZZ; Funding stream: IP
• Study programme: Title: Environmental Engineering - Graduate study; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka ekoinženjerstva (magistar/magistra inženjer/inženjerka ekoinženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2022-11-30
• Created on: 2022-07-19 13:01:28