Abu, Rohana

Biokatalyse tiltrækker i stigende grad opmærksomhed fra både akademiske og industrielle forskere på grund af dets evne til at katalysere meget selektive reaktioner. Der er for nyligt udvist stor interesse for anvendelsen af enzymatiske kaskader som værktøj i organisk syntese af værdifulde kemiske forbindelser (f.eks. chirale molekyler) specielt som farmaceutiske mellemprodukter og for at levere komplekse reaktioner der udviser bemærkelsesværdige fordele i forhold til ét-trins systemer. På trods af denne interesse er evalueringer af enzymatiske kaskader ud fra et procesmæssigt synspunkt kun sjældent blevet rapporteret i den videnskabelige litteratur. Målet med denne afhandling er derfor at evaluere proceskoncepterne indenfor enzymatiske kaskader ud fra en systematisk fremgangsmåde ved hjælp af værktøjer som termodynamisk og kinetisk analyse. Der er inddraget tre relevante case studier for at eksemplificere fremgangsmåden. I det første case studie blev termodynamiske og kinetiske studier brugt til at evaluere fordelagtigheden af en redox neutral kaskade til asymmetrisk amination af alkoholer til de tilsvarende chirale aminer. Denne syntetiske kaskade er potentielt attraktiv fordi den syntetiserer værdifulde chirale molekyler fra relativt billige råmaterialer og ligeledes maksimerer atomøkonomien. Reaktionsskemaet består af to primære enzymer (alkoholdehydrogenase og ω-transaminase) der er direkte involveret i hovedsyntesen. Alanindehydrogenase blev introduceret som sekundært enzym for at regenerere co-faktor NAD og co-substrat alanin in situ og for at forskyde ligevægtpositionerne i hovedsyntesen. Denne strategi kunne i princippet opnå høj grad af konvertering hvor ammoniak, som eneste reagent, anvendes i overskud for at drive konverteringen. Resultaterne indikerer kvantitativt at mulighederne for at forbedre termodynamisk begrænsede reaktioner ikke kun er via anvendelsen af enzymkoblede reaktioner (at koble den ufordelagtige reaktion med en energimæssigt foretrukken reaktion) men også ved at matche de relative reaktionshastigheder mellem de forbindende enzymer. Når reaktionstrinnene i en kaskade er uafhængige kan kinetikken kontrolleres yderst effektivt for at opnå en tilstrækkelig omdannelse til et givent slutprodukt. Dette eftervises i det andet case studie involverende kinetiskmodellering af dannelsen af 2-ketoglutarat fra glukoronat. Denne kaskade bestående af fire enzymer (uronatdehydrogenase, glucaratdehydratase, keto-deoxy-d-galactaratdehydratase og α-ketoglutaric-semialdehyde-dehydrogenase), brugt i nævnte rækkefølge, succesfuldt opnåede høj grad af omdannelse.  Endelig blev det tredje case studie brugt til at undersøge effekten af aktivitetskoefficienter i enzymkatalyserede reaktioner. Ofte er den ‘oplagte’ koncentrationsbaserede ligevægtskonstant (K’) brugt i stedet for en aktivitetsbaseret ligevægtskonstant til at beskrive reaktionsligevægte i biologiske systemer. Det blev antaget at den kemiske aktivitet for reaktanterne er ækvivalent med den respektive reaktants molære koncentration ved ligevægt, fordi reaktionen foregår i en fortyndet vandig opløsning hvorved effekten af aktivitetskoefficienten kan negligeres. På trods af dette, bliver der ofte argumenteret for at effekten burde undersøges. Derfor blev amineringen af cyclohexanon med (S)-1-phenyletylamin katalyseret af ω-transaminase enzymet brugt som en reaktionsmodel til at undersøge betydningen af aktivitetskoefficienterne. Resultaterne viser at værdier af aktivitetskoefficienterne for komponenterne afviger signifikant fra et, hvilket viser at reaktionssystemet ikke kan antages af opføre sig ideelt. Det er vist at termodynamiske og kinetiske analyser er effektfulde værktøjer til af evaluere og opnå virksomme kaskader for ikke-naturlige stofskifteveje. Dog skal det siges at mere meningsfulde ligevægtsdata fra enzymkatalyserede reaktioner vil være brugbare for at fastslå effektiviteten af sådan en strategi.Biocatalysis is attracting significant attention from both academic and industrial scientists due to the excellent capability of enzyme to catalyse selective reactions. Recently, much interest has been shown in the application of enzymatic cascades as a useful tool in organic synthesis to synthesize valuable compounds (e.g. chiral molecules) especially as pharmaceutical intermediates and to assist complex reactions that otherwise has problems as single step system. Despite this interest, process evaluation of many enzymatic cascades has only rarely been reported in the search for process improvement and implementation. Hence, the goal of this thesis is to evaluate the process concepts in enzymatic cascades in a systematic manner, using tools such as thermodynamic and kinetic analysis. Three relevant case studies have been used to exemplify the approach. In the first case study, thermodynamic and kinetic studies were used to evaluate the favourability of a redox neutral cascade for the asymmetric amination of alcohols to their corresponding chiral amines. This synthetic cascade is potentially attractive since it synthesizes valuable chiral molecule from cheap raw materials as well as maximising the atom economy. The scheme consists of two primary enzymes (alcohol dehydrogenase and ω-transaminase) that are directly involved in the main synthesis. Alanine dehydrogenase was introduced as a secondary enzyme to regenerate the co-factor NAD+ and co-substrate alanine in situ as well as to shift the equilibrium positions in the main syntheses. In principle, this strategy could successfully achieve high conversion, using ammonia as the sole reagent used in excess to drive the conversion. The findings herein indicate that quantitatively the possibilities for improving the conversion of thermodynamically limited reactions are not only via application of enzyme coupling reactions (coupling the unfavourable reaction with an energetically favourable reaction) but also by matching the relative reaction rates between the interconnecting enzymes. When the reaction steps are independent in a cascade, the kinetics can be controlled in a highly efficient way to achieve a sufficiently favourable conversion to a given target product. This is exemplified in the second case study, in the kinetic modelling of the formation of 2-ketoglutarate from glucoronate, the second case study. This cascade consists of 4 enzymes (uronate dehydrogenase, glucarate dehydratase, keto-deoxy-d-galactarate dehydratase and α-ketoglutaric semialdehyde dehydrogenase) run in that order to successfully achieve high conversion. Finally, a third case study was used to explore the effect of activity-coefficients in enzyme-catalysed reactions. Frequently, the ‘apparent’ or (concentration-based) equilibrium constant (Κ′), instead of activity-based equilibrium constant, was used to describe reaction equilibria of biological systems. It is assumed that the reactant activity is equal to the respective reactant molar concentration at equilibrium since many reactions operate in dilute aqueous solutions and thus neglect the activity coefficient effect. The effect of such assumption was therefore tested with the cyclohexanone amination with (S)-1-phenylethylamine catalysed by ω-transaminase. The findings showed that the activity coefficients of the components significantly deviate from unity, indicating its non-ideal behaviour in the reaction medium. Hence, thermodynamic and kinetic analyses are powerful tools to evaluate and to achieve workable cascades for non-natural pathways. Additionally, more meaningful equilibrium data from enzyme-catalysed reactions can be a useful way to determine the effectiveness of a given cascade strategy

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Process Evaluation Tools for Enzymatic Cascades Welcome Message

http://orbit.dtu.dk/ws/files/139518947/Process_evaluation_tools_for_enzy_atic_cascades.pdf

Process Evaluation Tools for Enzymatic Cascades Welcome Message

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Online Research Database In Technology