| Abstract | Biocatalysis is an emerging and important scientific filed for the asymmetric synthesis of pharmaceuticals and fine chemicals that has accomplished astonishing growth in industrial sector in recent years. For individual biocatalytic synthesis to reach its full potential, the process must be explored from the aspect of various scientific fields, including protein engineering, organic chemistry, reaction engineering, and more. The focus of this PhD thesis are halohydrin dehalogenases (HHDHs), promising and yet quite unexplored group of enzymes, and their potential for industrial-scale synthesis. Through extensive research of the existing literature, a serious lack of kinetic data in biotransformations with enzymes from HHDH group was determined. Therefore, one of the objectives of the thesis was to gain insight into kinetic characteristics of HHDH from Agrobacterium radiobacter AD1 (HheC) and develop a mathematical model in synthesis of important building blocks, since this approach may lead to the discovery of enzyme kinetic limitations and process bottlenecks, and, more importantly, enable the enhancement of process outcome through model-based simulations. The investigated reactions were kinetic resolutions for the synthesis of optically pure, fluorinated β-substituted alcohols and epoxides that represent valuable building blocks in pharmaceutical and fine chemicals industries. Ring-opening reactions of fluorinated styrene oxide derivatives, catalyzed by wild-type HheC and its variants W249P and ISM-4, were explored. Fluorinated derivatives of styrene oxide with substituents in para-position were found to be most convenient substrates for HheC based on the activity, enantioselectivity and hydrolytic stability, whereby 2-[4-(trifluoromethyl)phenyl]oxirane stood out as the best option. Enzyme variant W249P, with exchanged Trp and Pro amino acids on position 249, displayed higher substrate affinity in comparison to the wild type. Hence, it was selected for further kinetic investigation. The synthesis of (R)-2-azido-1-[4-(trifluoromethyl)phenyl]ethanol was described by double substrate Michaelis-Menten kinetics, with the presence of enzyme inhibitions with reacting substrate (R)-2-[4-(trifluoromethyl)phenyl]oxirane,
opposite enantiomer (S)-2-[4-(trifluoromethyl)phenyl]oxirane,
product (R)-2-azido-1-[4-(trifluoromethyl)phenyl]ethanol,
by-product rac-2-[4-(trifluoromethyl)phenyl]-1,2-ethanediol, and co-solvent DMSO.
Apart from numerous inhibitions and substantial hydrolysis effect, operational stability decay was found to contribute greatly to the synthesis outcome on higher concentration scale, since deactivation constant is directly correlated to the initial concentration of the substrate, 2-[4-(trifluoromethyl)phenyl]oxirane. The mathematical model was developed, and process simulations were employed in process optimization. Significant improvements in process metrics were achieved by modifying reactor set-up and selecting suitable initial conditions. The optimized biocatalytic system in repetitive batch reactor led to high reaction yield and optical purity (Y = 95%, ee > 99 %). However, process should be optimized further in order to increase reaction productivity that meets the target for industrial synthesis. The existence of various enzyme inhibitions and concentration-dependent enzyme deactivation, as well as low solubility and hydrolytic instability of the substrate, makes the system convenient for the switch from aqueous to alternative media. In the second part of the thesis, the focus was on the investigation of the influences of organic solvents (OSs) on HheC catalytic and structural performances. The stability of HheC in presence of dimethyl sulfoxide (DMSO), the most used co-solvent in HHDH-catalyzed biotransformations, was found to be preserved in cases when DMSO volume ratio does not exceed 30% (v/v). In higher DMSO content, HheC is not able to retain native structure and is completely and rapidly inactivated at 50% (v/v)
co-solvent. This was confirmed by the combination of experimental studies, including monitoring enzyme stability and protein size distributions during incubation with different DMSO content, together with molecular dynamic studies (MD). DMSO also proved to be a mixed-type inhibitor of HheC in the reactions of para-nitro-2-bromo-1-phenylethanol (PNSHH) dehalogenation and para-nitro styrene oxide (PNSO) ring-opening with bromide ions. The inhibitory behavior was detected by kinetic Lineweaver-Burk analysis and confirmed by MD. Likewise, DMSO was found to be inhibitor of W249P variant in
(R)-2-[4-(trifluoromethyl)phenyl]oxirane azidolysis during kinetic investigation. Wild-type HheC also displayed inadequate catalytic properties in presence of other tested
water-miscible co-solvents, specifically dimethylformamide, methanol, isopropanol, acetonitrile, and tetrahydrofuran. In case of hydrophobic solvents, a direct correlation between HheC activity and logP value was found in PNSHH ring-closure reaction. Knowledge of such a relationship makes biocatalysis with organic solvents more predictable, which may reduce the need to experiment with a variety of solvents in the future. However, this trend was not reported for PNSO ring-opening reaction. From hydrophobic OSs, tested alkanes (cyclohexane, n-hexane, n-heptane) were found to be compatible with HheC activity and stability during incubation, indicating the preservation of high enzyme structural integrity in these biphasic systems. Chloroform and toluene displayed inhibitory properties, especially in ring-opening reaction, which is more valuable from the synthetic point of view. In comparison to the wild type HheC, thermostable variant ISM-4 performed better in presence of OSs in terms of activity, stability, and enantioselectivity. In other words, the link between thermal stability and resistance to the action of OSs was established. These results revealed that ISM-4 has excellent potential for biotransformations in organic media, and as such should be explored for future implementations. |
| Abstract (croatian) | Biokataliza je važno znanstveno područje u razvoju za asimetričnu sintezu lijekova i finih kemikalija koje je postiglo zapanjujući rast u industrijskom sektoru posljednjih godina. Kako bi pojedinačna biokatalitička sinteza dosegla svoj puni potencijal, proces se mora istražiti s aspekta različitih znanstvenih područja, uključujući proteinsko inženjerstvo, organsku kemiju, reakcijsko inženjerstvo itd. U fokusu ovog doktorskog rada su halogenhidrin-dehalogenaze (HHDH), obećavajuća ali još nedovoljno istražena skupina enzima, te njihov potencijal za industrijsku sintezu. Opsežnim istraživanjem postojeće literature utvrđen je ozbiljan nedostatak kinetičkih podataka u biotransformacijama s enzimima iz skupine HHDH. Stoga je jedan od ciljeva doktorskog rada bio dobiti uvid u kinetičke karakteristike HHDH iz Agrobacterium radiobacter AD1 (HheC) i razviti matematički model za sintezu važnih građevnih blokova, budući da ovaj pristup može dovesti do otkrića kinetičkih ograničenja enzima i uskih grla procesa te, što je još važnije, omogućiti poboljšanje ishoda procesa kroz simulacije temeljene na modelu. Istraživane reakcije bile su kinetičke rezolucije za sintezu optički čistih, fluoriranih β-supstituiranih alkohola i epoksida koji predstavljaju vrijedne gradivne elemente u farmaceutskoj industriji i industriji finih kemikalija. Istražene su reakcije otvaranja prstena fluoriranih derivata stiren-oksida, katalizirane divljim tipom HheC i njegovim mutantima W249P i ISM-4. Utvrđeno je da su fluorirani derivati stiren-oksida sa supstituentima u parapoložaju najprikladniji supstrati za HheC na temelju aktivnosti, enantioselektivnosti i hidrolitičke stabilnosti, pri čemu se 2-[4-(trifluormetil)fenil]oksiran istaknuo kao najbolja opcija. Enzim W249P, sa zamijenjenim Trp i Pro aminokiselinama na poziciji 249, pokazao je veći afinitet prema supstratu u usporedbi s divljim tipom, stoga je odabran za daljnje kinetičko istraživanje. Sinteza (R)-2-azido-1-[4-(trifluorometil)fenil]etanola opisana je dvosupstratnom Michaelis-Menteničinom kinetikom, uz prisutnost inhibicija enzima sa supstratom (R)-2-[4-(trifluormetil)fenil]oksiranom,
suprotnim enantiomerom (S)-2-[4-(trifluormetil)fenil]oksiranom,
produktom (R)-2-azido-1-[4-(trifluormetil)fenil]etanolom,
nusproduktom rac-2-[4-(trifluorometil)fenil]-1,2-etandiolom, i ko-otapalom DMSO.
Osim brojnih inhibicija i značajnog učinka hidrolize, utvrđeno je da pad operacijske stabilnosti enzima uvelike pridonosi ishodu sinteze pri višim koncentracijama, budući da je konstanta deaktivacije enzima izravno ovisna o početnoj koncentraciji supstrata, 2-[4-(trifluorometil)fenil]oksirana. Razvijen je matematički model, a pri optimizaciji procesa korištene su simulacije procesa. Značajna poboljšanja u ishodu sinteze postignuta su modificiranjem tipa reaktora i odabirom odgovarajućih početnih uvjeta. Optimizirani biokatalitički sustav u repetitivnom šaržnom reaktoru doveo je do visokog iskorištenja na produktu i optičke čistoće (Y = 95%, ee > 99 %). Međutim, proces treba dodatno optimizirati kako bi se povećala produktivnost reakcije koja ispunjava ciljeve za industrijsku sintezu. Postojanje različitih inhibicija i deaktivacije enzima ovisne o koncentraciji, kao i niska topljivost i hidrolitička nestabilnost supstrata, čine sustav pogodnim za prelazak s vodenog na alternativni medij. U drugom dijelu doktorskog rada fokus je bio na istraživanju utjecaja organskih otapala (engl. organic solvent – OS) na katalitička i strukturna svojstva HheC. Utvrđeno je da je stabilnost HheC u prisutnosti dimetil-sulfoksida (DMSO), najčešće korištenog ko-otapala u HHDH-biotransformacijama, očuvana u slučajevima kada volumni udio DMSO ne prelazi 30% (v/v). Pri većem udjelu DMSO, HheC nije u stanju zadržati prirodnu strukturu te se potpuno i brzo inaktivira pri 50% (v/v) ko-otapala. To je potvrđeno kombinacijom eksperimentalnih istraživanja, uključujući praćenje stabilnosti enzima i raspodjele veličine proteina tijekom inkubacije s različitim sadržajem DMSO, zajedno s molekulsko-dinamičkim studijama (engl. molecular dynamics – MD). Prema HheC, DMSO se također pokazao kao inhibitor miješanog tipa u reakcijama dehalogenacije para-nitro-2-bromo-1-feniletanola (PNSHH) i otvaranja prstena para-nitro stiren oksida (PNSO) s bromidnim ionima. Inhibicijsko ponašanje otkriveno je kinetičkom Lineweaver-Burk analizom i potvrđeno MD analizom. Isto tako, tijekom kinetičkih ispitivanja, otkriveno je da je DMSO inhibitor W249P mutanta tijekom azidolize (R)-2-[4-(trifluorometil)fenil]oksirana. Divlji tip HheC također je pokazao neadekvatna katalitička svojstva u prisutnosti drugih testiranih ko-otapala koja se miješaju s vodom, a posebice dimetilformamida, metanola, izopropanola, acetonitrila i tetrahidrofurana. U slučaju hidrofobnih otapala, izravna korelacija između aktivnosti HheC enzima i logP vrijednosti pronađena je u reakciji zatvaranja prstena PNSHH. Saznanje o takvoj ovisnosti čini biokatalizu s organskim otapalima predvidljivijom, što može smanjiti potrebu za eksperimentiranjem s različitim otapalima u budućnosti. Međutim, ovaj trend nije pronađen u reakciji otvaranja PNSO prstena. Od hidrofobnih OS, utvrđeno je da su alkani (cikloheksan, nheksan, n-heptan) kompatibilni s HheC aktivnošću i stabilnošću tijekom inkubacije, što ukazuje na očuvanje visokog strukturnog integriteta enzima u ovim dvofaznim sustavima. Kloroform i toluen pokazali su inhibicijska svojstva, posebno u reakciji otvaranja prstena koja je vrjednija sa sintetičkog gledišta. U usporedbi s divljim tipom HheC, termostabilni mutant ISM-4 demonstrirao je bolje rezultate u prisutnosti OS u smislu aktivnosti, stabilnosti i enantioselektivnosti. Drugim riječima, za slučaj ovog enzima utvrđena je poveznica između termičke stabilnosti i otpornosti na djelovanje otapala. Ova istraživanja otkrila su da ISM-4 mutant enzima ima izvrstan potencijal za biotransformacije u organskim medijima, te bi ga kao takvog trebalo istražiti za buduće primjene. |
