Characterization of 2-deoxyribose-5-phosphate aldolase in the reaction of statin side chain synthesis

Statini su heterogena skupina lijekova koja se koristi pri snižavanju kolesterola te sprečavanju pojave kardiovaskularnih bolesti. Strukturno svi statini posjeduju 3,5-dihidroksi kiselinski bočni lanac s dva kiralna centra kojeg je teško sintetizirati zbog potrebe za visokom kemijskom i stereokemijskom čistoćom. U radu je provedena sinteza (3R,5R)-6-kloro-3,5-dihidroksiheksanala (laktola), prekursora za dobivanje statina. Sinteza se provela dvostrukom aldolnom adicijom acetaldehida i kloroacetaldehida kataliziranom enzimom 2-deoksiriboza-5-fosfat aldolaza (DERA). Ispitani su utjecaji različitih pufera i pH vrijednosti na stabilnost i aktivnost enzima DERA. Kao najoptimalniji uvjeti (pufer i pH) pokazali su se 100 mM fosfatni pufer i pH 6 pri kojim je enzim DERA kinetički okarakteriziran u svim stupnjevima reakcije. U reakciji acetaldehida i kloroacetaldehida te u reakciji acetaldehida i 4-kloro-3-hidroksibutanala kinetika je opisana dvosupstratnom Michaelis-Menteničinom jednadžbom dok je kinetika enzima u reakciji trimerizacije acetaldehida opisana kinetikom prvog reda, te je postavljen matematički model procesa u različitim tipovima reaktora. Ispitana je deaktivacija enzima u prisutnosti različitih koncentracija acetaldehida, kloroacetaldehida i međuprodukta, 4-kloro-3-hidroksibutanala, te je opisana kinetikom drugog reda. Sinteza laktola i validacija modela je provedena u tri različita tipa reaktora (kotlasti reaktor, ponovljivi kotlasti reaktor i kotlasti reaktor s dotokom) kako bi se dobio uvid i pronašlo najbolje rješenje za prevladavanje nedostataka stabilnosti korištenog enzima.Statins are a heterogeneous group of drugs used to reduce cholesterol and prevent the phenomenon of cardiovascular disease. Structurally, all statins possess a 3,5-dihydroxy acid side chain with two chiral centers which is difficult to synthesize due to the need for high chemical and stereochemical purity. In this study the synthesis of (3R,5R)-6-chloro-3,5-dihydroxyhexanal (lactol), a precursor for the preparation of statin was investigated. The synthesis was performed in the reaction of double aldol addition of acetaldehyde and chloroacetaldehyde catalyzed by the enzyme 2-deoxyribose-5-phosphate aldolase (DERA). The influence of different buffers and pH values on the stability and activity of the DERA enzyme was tested. 100 mM phosphate buffer and pH 6 were chosen as the optimal conditions (buffer and pH). Enzyme DERA was kinetically characterized in all steps of the investigated reactions. In the reaction of acetaldehyde and chloroacetaldehyde and in reaction of acetaldehyde and 4-chloro-3-hydroxybutane kinetics was described by the two-substrate Michaelis - Menten equation, while the kinetics of the enzyme in the reaction of acetaldehyde trimerization is described by the first order kinetics. Mathematical model of the process was proposed in different types of reactor. Enzyme deactivation was tested in the presence of different concentrations of acetaldehyde, chloroacetaldehyde and intermediate, 4-chloro-3-hydroxybutanal, and was described by second order kinetics. The lactol synthesis and validation of the model were carried out in three different types of reactors to gain insight and find the best solution to overcome the stability deficiencies of used enzyme