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β-Phenylalanine Ester Synthesis from Stable β-Keto Ester Substrate Using Engineered ω-Transaminases
(2018)
Amine transaminases are versatile biocatalysts for the production of pharmaceutically and agrochemically relevant chiral amines. They represent an environmentally benign alternative to waste intensive transition metal catalysed synthesis strategies, especially because of their high stereoselectivity and robustness. Therefore, they have been frequently used in the (chemo)enzymatic synthesis of amines and/or became attractive targets for enzyme engineering especially in the last decade, mainly in order to enlarge their substrate scope. Certainly, one of the most notable examples of amine transaminase engineering is the
manufacturing of the anti-diabetic drug Sitagliptin in large scale after several rounds of protein engineering. Thereby, the target amine was produced in asymmetric synthesis mode which is the most convenient and favored route to a target chiral amine, starting from the corresponding ketone. The choice of the amine donor is highly relevant for reaction design in terms of economical and thermodynamic considerations. For instance, the use of alanine as the natural amine donor is one of the most common strategies for the amination of target ketones but needs the involvement of auxiliary enzymes to shift the reaction equilibrium towards product formation. In fact, isopropylamine is probably one of the most favored donor molecules since it is cheap and achiral but it is supposed to be accepted only by a limited number of amine transaminases.
This thesis focusses on the optimization and application of amine transaminases for asymmetric synthesis reactions en route to novel target chiral amines using isopropylamine as the preferred amine donor.
G-Quadruplexe (G4) sind alternative Sekundärstrukturen, die von Guanosin-reichen DNA- oder RNA-Sequenzen ausgebildet werden können. In den letzten Jahren rückten diese tetrameren Konstrukte aufgrund ihres erst kürzlichen Nachweises in lebenden Humanzellen und ihrem Vorkommen in bestimmten funktionellen Genombereichen wie den Promotorregionen von Protoonkogenen oder den Telomeren zunehmend in den Fokus der Nukleinsäureforschung. Insbesondere ihre starke Korrelation mit Krebs macht Quadruplexstrukturen als Zielmotiv für die Entwicklung antikanzerogener Wirkstoffe höchst interessant. Um jedoch niedermolekulare Moleküle für therapeutische Zwecke nutzen zu können, muss zunächst auf molekularer Ebene ein grundlegendes Verständnis für die Ligand-Quadruplex-Interaktion geschaffen und anhand dieser Informationen Optimierungsmöglichkeiten für G4-bindende Liganden ergründet werden.
Ziel dieser Dissertation war die ausführliche Analyse der Bindung biologisch aktiver, Phenyl-substituierter Indolochinoline an Quadruplexstrukturen mittels diverser spektroskopischer und kalorimetrischer Methoden. Um simultan den Einfluss verschiedener Ligandstrukturelemente auf die G4-Anbindung für ein zukünftiges, rationales Wirkstoffdesign zu erforschen, wurde im Rahmen dieser Arbeit zunächst unterschiedlich modifizierte Indolochinolinderivate synthetisch hergestellt. In ersten spektroskopischen Experimenten sollten diese Moleküle anschließend nicht nur auf ihre generelle Interaktion mit verschiedenen G4-Topologien hin untersucht, sondern auch die Selektivität, die Bindungsaffinität und der Interaktionsmodus der Liganden ermittelt werden. Besonders die Indolochinoline mit basischen Seitenketten und einer N5-Methylierung zeigten eine hohe Affinität und Präferenz gegenüber parallelen Quadruplexstrukturen. Um detailliertere Informationen über diese Ligand-DNA-Wechselwirkung zu erhalten, erfolgten umfassende thermodynamische Bindungsstudien mittels isothermaler Titrationskalorimetrie. Anhand dieser Daten erfolgte eine Separation der Freien Bindungsenthalpie, die in dieser Form erstmalig für ein G4-bindendes Molekül beschrieben wurde. Hierbei zeigte sich, dass die Anbindung der Phenyl-substituierten Indolochinoline nicht nur durch hydrophobe Effekte, sondern vor allem durch spezifische molekulare Wechselwirkungen zwischen dem Ligand und der Quadruplex vorangetrieben wird.
In the 1940s cytochrome P450 monooxygenases have been discovered and have been the focus of many studies ever since. Although they catalyze very interesting reactions that might find applications in the production of fine chemicals or pharmaceuticals, their low activity and stability often reduces their economic value. Both properties, the activity and the stability, are influenced by the uncoupling of the catalytic cycle.
In this PhD thesis, an assay for the screening of activity and uncoupling of cytochrome P450 enzymes was successfully developed. After finding optimal conditions for the assay, concerning pH and enzyme concentration, the uncoupling of cytochrome P450 BM3 and five mutants (F87Y, R47L, Y51F, A82L and T268A) was investigated. With the results obtained, a comparison of data from literature was possible and revealed similarities. Additionally, through negative controls, the reliability of the assay could be further demonstrated. Although other methods have been described for the detection of hydrogen peroxide formation, the combination of NADPH consumption measurement and hydrogen peroxide formation in parallel was new and represents a very good basis for a pre-screening of large mutant libraries, followed by closer investigation of selected variants.
For the investigation of the activity of the CYP11A1 system, consisting of CYP11A1 and Adx and AdR as redox partner system, the expression and purification for all three proteins was investigated first. For the protein CYP11A1 and Adx, good expression levels were achieved, whereas for AdR the protein concentration obtained was very low. The purification of all three proteins was partially accomplished but left room for improvement. Therefore, in the Master thesis of Christopher Grimm, the pH and temperature stability of all three proteins was further investigated in order to improve conditions used for ion exchange chromatography and to investigate possible conditions for in vitro biocatalysis. As unfortunately even with further investigation of the expression of AdR, no improvement was achieved, a whole-cell system was further investigated. Here, the product formation could be increased 8-fold in comparison to the published data, from 0.27% conversion to 2.2% conversion over 24 h by using a different detergent for substrate solubilization, which might have led to a better substrate supply to the enzyme.
Due to the low activity and stability, a different P450 system, the CYP17A1 enzyme, was subsequently investigated, first by in vitro biocatalysis with the human CYP17A1 expressed in E. coli. Therefore, a suitable redox partner system needed to be found for efficient electron supply of the enzyme. In in vitro biocatalysis, in combination with the Pdx/PdR system of P. putida the CYP17A1 enzyme showed the highest conversion with 91% after 24 h. To investigate the activity of the enzyme further, all active site residues in 4 Å proximity to the bound substrate were exchanged with alanine. After expression of the variants, almost no correctly folded protein was obtained for the variants. Also, after investigating different buffers to possibly enhance the stability, no improvements were achieved. Therefore, a whole-cell approach with the bovine enzyme was chosen in order to investigate the activity of the alanine variants. Here the importance of positions N202, R239, G297, E305, and T306A, described in literature to be important for catalytic activity, was confirmed. Most importantly, three positions that alter the regioselectivity of the enzyme were identified. The reaction of the V483A mutant was therefore also further investigated by preparative biocatalysis. Afterwards the new product was separated by preparative HPLC and identified as 16α- hydroxyprogesterone as confirmed by NMR spectroscopy analysis.
In the last part of the thesis, another screening approach for possible high-throughput screening was investigated. In contrast to the other screening approach, here the investigation of the substrate conversion and the hydrogen peroxide formation were optimized for application in droplets. After finding that DCFH-DA was not sensitive enough towards hydrogen peroxide, the AmplifluTM Red probe was used. As both fluorescent products were found to stay in the aqueous phase above pH 7.4, the conditions investigated for the AmplifluTM Red assay were applied and only NADPH to substrate ratio was investigated by using an uncoupling variant, an active variant from literature and the cytochrome P450 BM3 wild-type enzyme. After finding a good ratio, the five variants used for the investigation of the AmplifluTM Red assay were investigated in the same concentration later on found in the droplets (1 cell per 4 pL), and one variant showed improved product formation compared to wild-type. This finding clearly shows the applicability of the assay for high-throughput screening in droplets.
Molybdopterin spielt in der Natur eine wesentliche Rolle, da es gebunden an Molybdän den Molybdän-Cofaktor bildet, der einer Reihe verschiedener Enzyme als katalytisches Zentrum dient. Der Molybdän-Cofaktor kann zwar aus dem Protein freigesetzt werden, erweist sich dann aber als instabil. Trotz langjähriger Bemühungen konnten der Molybdän-Cofaktor und seine biologischen Vorstufen bisher nicht auf chemischem Wege synthetisiert werden. Daher konnten die bisher gewonnenen Kenntnisse über diese Verbindung nicht anhand von Untersuchungen an dem freien Cofaktor gewonnen werden. Um den Cofaktor in seiner Chemie zu verstehen, beschäftigt sich diese Arbeit mit der chemischen Synthese von Modellverbindungen, die die Aufgaben des natürlichen Cofaktors nachbilden können. Um den Einfluss der verschiedenen Struktureinheiten auf die Stabilität oder die katalytische Aktivität zu verstehen und so ein tieferes Verständnis über Molybdopterin und mögliche Struktur-/Funktionsbeziehungen des natürlichen Cofaktors zu entwickeln, werden einzelne Strukturabschnitte untersucht. Im Rahmen dieser Arbeit war der Fokus das Verständnis der Chemie des Pyrazin-Pyran-Dithiolen-Strukturabschnittes und nach Möglichkeit die Entwicklung alternativer Modellverbindungen, die in der Lage sind Sauerstoff-Transport-Reaktionen zu katalysieren und/oder mit dem Apoenzym verbunden werden können. Im besten Falle kann so eine Modellverbindung als Behandlungsmöglichkeit der Molybdän-Cofaktor-Defizienz eingesetzt werden, bei Bindung mit dem Apoenzym für iSOD (isolierte Sulfitoxidase-Defizienz) oder bei Nichtbindung für MoCo-Defizienz Typ B. Für die Synthese der Pyrazin-Pyran-Dithiolen-Liganden sollten bereits literaturbekannte Syntheserouten insbesondere von Garner modifiziert und optimiert werden. Vergleichsweise sollten auch Ligandensysteme mit einer CH2-Gruppe anstelle der Sauerstofffunktion des Pyrans synthetisiert werden. Des Weiteren sollten neue Synthesewege zu strukturell und elektronisch ähnlichen Verbindungen entwickelt werden. Die so gewonnenen Ligandensysteme sollten anschließend mit vorzugsweise Molybdän, aber auch Wolfram komplexiert werden.
Unter Verwendung von rekombinanten Schweineleberesterasen wurden zwei Chemoenzymatische Prozesse sukkzessive etabliert, optmiert und im Maßstab vergößert. Es wurden zwei chirale Synthesebausteine beispielhaft hergestellt und charakterisiert.
Die Arbeit gibt einen Einblick in die Prozessoptimierung von chemoenzymatischen Syntheserouten unter ökonomischen Aspekten.
In an aerobic environment the occurrence of reactive oxygen species (ROS) is a common phenomenon. The diverse roles of ROS in cellular function and in diseases make them a target of interest in many research areas. Substances capable of directly or indirectly reducing the (harmful) effects of ROS are referred to as “antioxidants”. However, the term is applied miscellaneously in the chemical and the biological context to describe different attributes of a substance. In this work the potential of an electrochemical assay to detect different ROS in-vitro was explored. The method was optimized to investigate the radical scavenging activities (antioxidant potential) of trolox and different plant compounds (ascorbic acid, caffeic acid, epigallocatechin gallate, ferulic acid, kaempferol, quercetin, rutin, and Gynostemma pentaphyllum extract) in-vitro. The obtained data was compared to established antioxidant in-vitro assays. Further, the impact of the plant substances on cellular parameters was evaluated with the electrochemical assay and established cell assays.
The optimization of the electrochemical assay allowed the reproducible detection of ROS. The sensor electrode proved differently sensitive towards individual ROS species. The highest sensitivity was recorded for hydroxyl radicals while superoxide and hydrogen peroxide had little impact on the sensor. Extracellular ROS concentrations could be detected from cell lines releasing elevated ROS into the extracellular space. The antioxidant activity of the investigated plant substances could be demonstrated with all in-vitro assays applied. However, the absolute as well as the relative activity of the individual substances varied depending on the experimental parameters of the assays (pH, radical species, phase, detection method).
The plant compounds modified redox related intracellular parameters in different cell lines. However, a direct correlation between intracellular and extracellular effects of the plant compounds could not be established.
The work demonstrates the feasibility to use the electrochemical assay to sense ROS as well as to evaluate the radical scavenging activity of molecules. The in-vitro antioxidant activities demonstrated for the individual plant substances are not reliable to predict the cellular effects of the molecules.
The synthesis of pterin-dithiolene ligands was achieved by employing the radical nucleophilic substitution, i.e. the so-called “Minisci- Reaction”1. This protocol was used for the first time by Professor W. Pfleiderer on pterin substrates2 and proved a powerful method for the preparation of 6 acyl-pterins in course of this work. Subsequent construction of the dithiolene ring facilitates the synthesis of pterin-dithiolene ligands with completely unprotected pterin moieti.
The molybdenum cofactor is probably one of the most relevant discoveries in the recent history of pterin chemistry and biochemistry. Many efforts have been made for the preparation of compounds able to mimic the features of the Moco ligand system called "Molybdopterin". In fact, the study of MPT models enables a deeper understanding of the “mechanism of function” of this cofactor and most importantly, lays the foundation for a potential treatment for the Moco related diseases MoCOD and iSOD.
Humanity is constantly confronted with the emergence and reemergence of infectious diseases. Many of them produce large or devastating epidemics, like AIDS (HIV) and Ebola. Others have been long neglected, yet pose immediate threats to global public health as evidences the abrupt emergence of Zika virus in South America and its association with microcephaly in babies. The examples illustrate, that many of these diseases are provoked by RNA viruses. One of the first steps in understanding and eliminating those threats is the development of sensitive and rapid diagnostic methods. A general and relatively rapid method is the direct detection and examination of the agent’s genome. However, the nature of (re)emerging RNA viruses poses a series of very specific problems for the design of such methods. Therefore, a systematic approach was proposed for the design of DNA-hybridization-base methods to detect and characterize RNA viruses that will have both a high sensitivity and a specificity sufficiently broad to detect, per reaction, down to a single copy of any of the possible variants of the viral genome.
Following this approach a series of assays were designed, developed or adapted and put into use for detection and characterization of important RNA viruses. One of those viruses is West Nile virus (WNV), which after its explosive introduction into USA become the most widespread flavivirus throughout the world and, consequently, many countries began an intensive monitoring. While existing assay detected predominantly the Lineage 1, in Europa Lineage 2 was expected. Two new RT-qPCR for the detection of both lineages were developed, and reportedly used by independent laboratories. Due to more than 50000 associated deaths per year, the Hepatitis E virus also received an increasing attention to elucidate novel routes of transmission. This virus (especially genotype 3) has the zoonotic potential of transmission from pigs and wild boar to humans. RT-qPCR and nested qPCR for detection and characterization of this virus as well as a methodology for subtyping were developed and the first detected case of subtype 3b in a German wild animal was documented. In addition a novel assay for flaviviruses conformed by a RT-qPCR coupled with a low density DNA microarray was developed, which enabled the identification of WNV in mosquitoes from Greece. A RT-qPCR suitable for surveillance and diagnostic of all known variants of Venezuelan equine encephalitis virus was developed too. A causative agent of hemorrhagic infections, the Ngari virus, was detected and characterized in animal samples from Mauritania. These achievements were supported by the development of software applications for selection and visualization of primers and probes from aligned DNA sequences and for modeling of DNA hybridizations using unaligned sequences.
In conclusion a general methodology for rapid development of sensitive diagnostic methods based in DNA-hybridization technics (PCR, sequencing and microarray) was stablished and successful applications are reported.