Mathematical model of dynamic kinetic resolution catalyzed by halohydrin dehalogenase

Optički čiste tvari su vrijedni prekursori u farmaceutskoj industriji i industriji finih kemikalija, stoga pretvorba racemičnih smjesa u enantiomerno čiste produkte predstavlja izazov. Jedna od metoda deracemizacije je dinamička kinetička rezolucija (DKR) koja se zasniva na kinetičkoj rezoluciji u kombinaciji s in situ racemizacijom sporije reagirajućeg enantiomera supstrata. Halogenhidrin dehalogenaze (HHDH) u metaboličkim putovima kataliziraju reverzibilnu dehalogenaciju vicinalnih halogenhidrina do epoksida. U radu je razvijen matematički model DKR rac-2-bromometil-2-metil-oksirana i dobivanja (S)-5-bromometil-5-metiloksazolidin-2-ona uz enzim HheC iz Agrobacterium radiobacter AD1. Ispitivana DKR se sastoji od tri reakcijska stupnja: i. favorizirano nastajanje (S)-oksazolidinona reakcijom (S)-epoksida i cijanatnih iona, ii. nastajanje 1,3-dibromo-2-metil-2-propanola iz (R)-epoksida i bromidnih iona, te iii. nastajanje rac-epoksida iz dibromoalkohola. Matematički model sastoji se od kinetičkog modela i bilanci tvari u reaktoru. Određivanjem kinetike reakcije DKR utvrđen je različit afinitet enzima prema enantiomerima. U i. stupnju reakcije za (S)-epoksid utvrđeno je da supstrati prate Michaelis-Menteničinu kinetiku uz inhibicijski učinak (S)-epoksida na enzim pri višim koncentracijama. Za (R)-epoksid utvrđeno je da prati kinetiku 1. reda, a cijanatni ioni Michaelis-Menteničinu kinetiku. Bromidni ioni te suprotni enantiomeri epoksida u oba slučaja imaju inhibicijski učinak na enzim. U ii. stupnju DKR oba epoksida prate kinetiku 1. reda, a bromidni ioni Michaelis-Menteničinu kinetiku uz inhibiciju u slučaju (S)-epoksida. U slučaju (S)-epoksida također postoji inhibicijski učinak suprotnim enantiomerom i cijanatnim ionima. Otkriveno je da je reakcija zatvaranja dibromoalkohola odnosno iii. stupanj DKR spontan kemijski proces pri kojem rac-epoksid nastaje prema kinetici 1. reda. Optički čisti (S)-5-bromometil-5-metiloksazolidin-2-on može se dobiti uz pažljivo odabrane reakcijske uvjete. DKR je potrebno provoditi pri niskim koncentracijama rac-epoksida, radi inhibicije supstratom (S)-epoksidom u i. stupnju, te niskim koncentracijama bromidnih iona, koje su dovoljne za provođenje ii. stupnja i ne inhibiraju enzim u i. stupnju. Optically pure compounds are valuable precursors in pharmaceutical and fine chemicals industry, so the conversion of racemic mixtures to enantiomerically pure products represents a challenge. One of the deracemization methods is the dynamic kinetic resolution (DKR) which couples a kinetic resolution with in situ racemization of the slower reacting substrate enantiomer. Halohydrin dehalogenases (HHDH) in metabolic pathways catalyze the reversible dehalogenation of vicinal halohydrin to epoxide. In this paper, a mathematical model of rac-2-bromomethyl-2-methyl-oxirane DKR has been developed to obtain (S)-5-bromomethyl-5-methyloxazolidin-2-one with the HheC enzyme from Agrobacterium radiobacter AD1. The examined DKR consists of three reaction steps: i. favored formation of (S)-oxazolidinone in (S)-epoxide and cyanate ions reaction; ii. the formation of 1,3-dibromo-2-methyl-2-propanol in (R)-epoxide and bromide ions reaction; and iii. the formation of rac-epoxide from dibromo-alcohol. The mathematical model consists of the kinetic model and the mole balance in the reactor. By determining the DKR reaction kinetics, different affinity of the enzyme towards the enantiomers has been observed. In the i. reaction step for (S)-epoxide, it has been determined that both substrates follow Michaelis-Menten kinetics with the inhibitory effect on the enzyme at higher concentrations of the (S)-epoxide. The (R)-epoxide follows the first-order kinetics and the cyanate ion Michaelis-Menten kinetics. Bromide ions and the opposite epoxide enantiomers in both cases have an inhibitory effect on the enzyme. In the ii. reaction step both epoxides follow first-order kinetics and the bromide ions follow Michaelis-Menten kinetics with the inhibitory effect in the case of (S)-epoxide. In the case of (S)-epoxide enzyme inhibition with another enantiomer and cyanate ion is present as well. It has been found that the reaction of dibromo-alcohol closure, i.e. iii. reaction step, is a spontaneous chemical process in which rac-epoxide is formed by first-order kinetics. Optically pure (S)-oxazolidinone can be obtained with carefully selected reaction conditions. DKR should be carried out at low concentrations of the rac-epoxide, because of the (S)-epoxide substrate inhibition in the i. reaction step, and low concentrations of bromide ions, which should be sufficient enough to perform the ii. reaction step and which do not inhibit the enzyme in the i. step.