Optimization of reaction conditions of enzyme-catalyzed α-chloroaldehyde oxidation

Statini su heterogena skupina lijekova koja se primjenjuje za snižavanje razine kolesterola u krvotoku čovjeka s ciljem prevencije kardiovaskularnih bolesti. Statini posjeduju 3,5-dihidroksi kiselinski bočni lanac s dva kiralna središta. Radi velikih zahtijeva kemijske i stereokemijske čistoće, njihova sinteza predstavlja izazov. U ovom radu provedena je biokatalitička oksidacija α-kloroaldehida (laktola) u aktivni kiselinski oblik bočnog lanca statina. Reakcija je katalizirana enzimom aldehid dehidrogenazom (AlDH) uz regeneraciju koenzima NAD+ enzimom NADH oksidazom (NOX). Ispitan je utjecaj različitih pH na aktivnost i stabilnost enzima AlDH i NOX. Kao optimalni uvjet za provedbu spomenute reakcije pokazala se vrijednost pH 8. Enzimi su u optimalnom mediju kinetički okarakterizirani u svim stupnjevima reakcije. Kinetika enzima AlDH u reakciji oksidacije laktola opisana je dvosupstratnom Michaelis-Menteničinom kinetikom s kompetitivnom inhibicijom supstratom (NAD+) i kompetitivnom inhibicijom produktom (NADH). Za kinetiku povratne reakcije pretpostavljeno je da se radi o kinetici drugog reda, a kako bi se procijenio kinetički parametar, provedena je oksidacija laktola bez regeneracije koenzima. Kinetika enzima NOX u reakciji oksidacije NADH opisana je Michaelis-Menteničinom kinetikom s kompetitivnom inhibicijom produktom (NAD+) te je nadograđena nekompetitivnom inhibicijom laktolom. Razvijen je matematički model procesa te je validiran provedbom reakcije u kotlastom reaktoru. Usporedno su provedena tri eksperimenta s različitim početnim koncentracijama koenzima. Proveden je i eksperiment u kojem je kao supstrat poslužio sirovi produkt (nepročišćeni laktol) dvostruke aldolne adicije acetaldehida i kloroacetaldehida katalizirane enzimom 2-deoksiriboza-5-fosfat aldolazom (DERA). Kako bi se mogla pratiti inaktivacija enzima tijekom provedbe eksperimenata, razvijeni su testovi za spektrofotometrijsko određivanje aktivnosti svakog pojedinog enzima. Kao potencijalno bolja metoda regeneracije koenzima, ispitan je postupak s lawsone kinonom umjesto enzima NOX.Statins are heterogeneous group of drugs used for lowering cholesterol level in human bloodstream preventing cardiovascular diseases. Structurally, they possess 3,5-dihydroxy acid side chain with two chiral centers. Synthesis of statine side chains is a challenge due to high demands for chemical and stereochemical purity. In this work a biocatalytical oxidation of α-chloroaldehyde (lactol) was carried out. Reaction is catalyzed by aldehyde dehydrogenase (AlDH) with coenzyme regeneration by NADH oxidase (NOX). The effect of various pH on activity and stability of both enzymes was examined. pH 8 was chosen as the optimal for the implementation of investigated reaction. Enzymes were kinetically characterized in all steps of the investigated reactions. Kinetics of AlDH in reaction of lactol oxidation was described by double-substrate Michaelis-Menten model with competitive inhibition by substrate (NAD+) and with competitive inhibition by product (NADH). Kinetics of the reverse reaction was assumed to be of the second order and to assess kinetic constant, lactol oxidation without coenzyme regeneration was performed. In reaction of coenzyme regeneration the kinetics of NOX was described by Michaelis-Menten model with competitive inhibition by product (NAD+) and was upgraded by non-competitive lactol inhibition. Mathematical model of the process was developed and was validated by carrying out the reaction in a batch reactor. Three parallel experiments with different initial coenzyme concentrations were conducted. Oxidation of lactol was also carried out with the crude product (lactol) using reaction mixture from double aldol addition of acetaldehyde and chloroacetaldehyde catalyzed by 2-deoxyribose-5-phosphate aldoase (DERA). In order to monitor enzyme inactivation during the experiments, tests for spectrophotometric AlDH and NOX activity determination were developed. As potential alternative for enzyme regeneration, procedure with lawsone quinone instead NOX was investigated