Modelling of the α-chloroaldehyde oxidation catalyzed by oxidoreductases

Statini su skupina lijekova koja se koristi kako bi se snizila razina kolesterola u krvotoku čovjeka s ciljem sprječavanja razvoja kardiovaskularnih bolesti. Statini posjeduju 3,5-dihidroksi kiselinski bočni lanac s dva kiralna središta koji su zaslužni za ostvarivanje glavne funkcije lijeka. 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 katalizirana oksidoreduktazama. Kao oksidoreduktaze korišteni su enzimi aldehid dehidrogenaza (AlDH) i ketoreduktaza (KRED). Obzirom da navedeni enzimi za svoju aktivnost trebaju prisutnost koenzima NAD(P)+ provedena je njegova regeneracija enzimom NAD(P)H oksidazom (NOX). Ispitana je kinetika svih enzima. Oksidacija laktola uz AlDH opisana je dvosupstratnom Michaelis-Menteničinom kinetikom s uključenom inhibicijom supstratom, NAD+, te kompetitivnom inhibicijom produktom, NADH. Oksidacija laktola uz KRED opisana je dvosupstratnom Michaelis-Menteničinom kinetikom s uključenom inhibicijom supstratom, NADP+, te kompetitivnom inhibicijom produktom, NADPH. Kinetika enzima NOX u reakciji oksidacije NADPH opisana je Michaelis-Menteničinom kinetikom s kompetitivnom inhibicijom produktom (NADP+). Pored navedenih kinetičkih mjerenja ispitan je i utjecaj aldehida na stabilnost enzima AlDH, KRED i NOX-a. Razvijen je matematički model procesa te je validiran provedbom reakcije u kotlastom reaktoru. Provedeni su eksperimenti oksidacije pročišćenog laktola s navedenim enzimima uz regeneraciju koenzima, te sirovog laktola dobivenog aldolnom adicijom acetaldehida i kloroacetaldehida katalizirane enzimom 2-deoksiriboza-5-fosfat aldolazom (DERA).Statins are group of drugs used for lowering cholesterol level in human bloodstream in order to prevent cardiovascular diseases. Statins possess 3,5-dihydroxy acid side chain with two chiral canters which are responsible for achieving the main function of the drug. Synthesis of statin 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 catalyzed by oxidoreductases in order to produce the statin side chain. For this purpose, enzymes aldehyde dehydrogenase (AlDH) and ketoreductase (KRED) were tested. Since the oxidoreductases for its activity demand the use of coenzyme NAD(P)+ which need to be regenerated, the enzyme NAD(P)H oxidase (NOX) was used for its regeneration. The kinetics of all used enzymes was examined. Oxidation of lactol catalysed by AlDH was described by double-substrate Michaelis-Menten model with include substrate (NAD+) inhibition and with competitive inhibition by product (NADH). The kinetics of the same reaction catalysed by KRED was described by double-substrate Michaelis-Menten model with substrate (NADP+) inhibition and with competitive inhibition by product (NADPH). In the reaction of coenzyme regeneration, the kinetics of NOX was described by Michaelis-Menten model with competitive product (NADP+) inhibition. Beside kinetic measurements, the influence of the aldehydes on the stability of the AlDH, KRED and NOX was examined. Mathematical model of the process was developed and was validated by carrying out the reaction in a batch reactor. Experiments of lactol oxidation catalyzed by enzyme AlDH and KRED was carried out with coenzyme regeneration. As the substrates purified lactol as well the crude lactol, produced in the reaction of double aldol addition of acetaldehyde and chloroacetaldehyde catalyzed by 2-deoxyribose-5-phosphate aldolase (DERA) were used