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Chemoenzymatic Synthesis of Nucleoside Analogs As Potential Medicinal Agents

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Chemoenzymatic Synthesis of Nucleoside Analogs As Potential Medicinal Agents Chemoenzymatic synthesis of nucleoside analogs as potential medicinal agents vorgelegt von M. Sc. Heba Yehia Mohamed ORCID: 0000-0002-3238-0939 von der Fakultät III-Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktorin der Naturwissenschaften - Dr. rer. nat. – genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Dr. Roland Lauster, Medical Biotechnology, TU Berlin, Berlin Gutachter: Prof. Dr. Peter Neubauer, Bioprocess Engineering, TU Berlin, Berlin Gutachter: Prof. Dr. Jens Kurreck, Applied Biochemistry, TU Berlin, Berlin Gutachter: Prof. Dr. Vlada B. Urlacher, Biochemistry, Heinrich-Heine-Universität Düsseldorf, Düsseldorf Tag der wissenschaftlichen Aussprache: 16. Juli 2019 Berlin, 2019 Heba Y. Mohamed Synthesis of nucleoside analogs as potential medicinal agents Abstract Modified nucleosides are important drugs used to treat cancer, viral or bacterial infections. They also serve as precursors for the synthesis of modified oligonucleotides (antisense oligonucleotides (ASOs) or short interfering RNAs (siRNAs)), a novel and effective class of therapeutics. While the chemical synthesis of nucleoside analogs is challenging due to multi-step procedures and low selectivity, enzymatic synthesis offers an environmentally friendly alternative. However, current challenges for the enzymatic synthesis of nucleoside analogs are the availability of suitable enzymes or the high costs of enzymes production. To address these challenges, this work focuses on the application of thermostable purine and pyrimidine nucleoside phosphorylases for the chemo-enzymatic synthesis of nucleoside analogs. These enzymes catalyze the reversible phosphorolysis of nucleosides into the corresponding nucleobase and pentofuranose-1-phosphate and have already been successfully used for the synthesis of modified nucleosides in small scale. So far, the production of sugar-modified nucleosides has been a major challenge. In this study, it was shown that the synthesis of arabinose- or fluoroarabinose-containing nucleoside analogs by glycosylation starting from modified sugar-1-phosphates is possible with very high yields. This was not possible using transglycosylation reactions. One very evident example is the synthesis of 5-ethynyl-2´-deoxy-2´-fluorouridine arabinoside. It was not obtained by transglycosylation with fluoroarabinofuranosyl uracil as sugar donor whereas almost 50 % of the base was transformed by direct glycosylation with the corresponding 2-deoxy-2-fluoro-arabinofuransoe-1-phosphate. Halogenated ribo- and deoxynucleosides were produced at mg scale with a reagent grade purity of >95 %. The cytotoxic activity in different blood tumor cell lines (HL-60 and CCRF-CEM) was tested for these nucleoside analogs and it was shown that the enzymatically produced compounds showed similar IC50 values as their chemically produced counterparts. Compared to ribonucleosides, deoxyribonucleosides revealed a reduced non-specific cytotoxicity. In order to transfer the synthesis of modified nucleosides to a larger scale, the expression of thermostable nucleoside phosphorylases was first established by high cell density fed-batch cultivation in a laboratory bioreactor. Almost three times the volumetric yield of the recombinant protein PNP 03 was produced in the fed-batch cultivation in comparison to the shake flask cultures. The biocatalytic process was also optimized; exemplified by the synthesis of halogenated nucleoside analogs using a continuous enzyme membrane reactor. The results of the substrates conversion for nucleoside synthesis were very similar for discontinuous and continuous reactions. The enzymes were stable with natural substrates for several weeks and with modified substrates for up to 7 days. In addition, heat-treated cell lysate achieved a similar result to purified enzymes. Abstract I Synthesis of nucleoside analogs as potential medicinal agents Heba Y. Mohamed Zusammenfassung Modifizierte Nukleoside sind wichtige Medikamente zur Behandlung von Krebs, viralen oder bakteriellen Infektionen. Sie dienen auch als Vorläufer für die Synthese von modifizierten Oligonukleotiden (Antisense-Oligonukleotide (ASOs) oder short-interfering RNAs (siRNAs)), einer neuen und sehr effektiven Klasse von Therapeutika. Während die chemische Synthese von Nukleosidanaloga aufgrund von Mehrschrittverfahren und geringer Selektivität eine Herausforderung darstellt, bietet die enzymatische Synthese eine umweltfreundliche Alternative. Aktuelle Herausforderungen für die enzymatische Synthese von Nukleosidanaloga sind die Verfügbarkeit geeigneter Enzyme oder hohe Kosten für die Enzymproduktion. Um diese Herausforderungen zu bewältigen, konzentriert sich diese Arbeit auf die Anwendung von thermostabilen Purin- und Pyrimidinnukleosidphosphorylasen für die chemo-enzymatische Synthese von Nukleosidanaloga. Diese Enzyme katalysieren die reversible Phosphorolyse von Nukleosiden in die entsprechende Nukleobase und Pentofuranose-1-phosphat. Sie wurden bereits erfolgreich für die Synthese von modifizierten Nukleosiden im kleinen Maßstab eingesetzt. Bisher war die Herstellung von zuckermodifizierten Nukleosiden eine große Herausforderung. In dieser Studie wurde gezeigt, dass die Synthese von Arabinose- oder Fluoroarabinose-haltigen Nukleosidanaloga durch Glykosylierung ausgehend von modifizierten Zucker-1-Phosphaten mit sehr hohen Ausbeuten möglich ist. Dies war mit Transglykosylierungsreaktionen nicht möglich. Ein sehr anschauliches Beispiel ist die enzymatische Synthese von 5-Ethinyl-2´-deoxy-2´-fluorouridin arabinosid. Mittels Transglykosylierung mit Fluorarabinofuranosyluracil als Substrat war die Synthese nicht möglich. Hingegen wurden fast 50 % der Base durch direkte Glykosylierung umgesetzt. Halogenierte Ribo- und Desoxynukleoside wurden im mg-Maßstab mit einer Reinheit von >95 % hergestellt. Die zytotoxische Aktivität dieser Nukleosidanaloga wurde in verschiedenen Bluttumorzelllinien (HL-60 und CCRF-CEM) getestet und es wurde gezeigt, dass die enzymatisch hergestellten Verbindungen ähnliche IC50-Werte aufwiesen wie ihre chemisch hergestellten Pendants. Im Vergleich zu Ribonukleosiden zeigten Desoxyribonukleoside eine reduzierte unspezifische Zytotoxizität. Um die Synthese von modifizierten Nukleosiden in einen größeren Maßstab zu transferieren, wurde die Expression von thermostabilen Nukleosid-Phosphorylasen zunächst in einen Hochzelldichte-Fed- Batch-Prozess in einem Laborbioreaktor etabliert. In den Kultivierungen des Fed-Batch-Bioreaktors wurde im Vergleich zu Kulturen in Schüttelkolben fast die dreifache volumetrische Ausbeute des rekombinanten Proteins PNP 03 erzeugt. Der biokatalytische Prozess wurde am Beispiel der Synthese von halogenierten Nukleosidanaloga in einem kontinuierlichen Enzymmembranreaktor optimiert. Die Umsatzraten waren für diskontinuierliche und kontinuierliche Reaktionen sehr ähnlich. Die Enzyme waren bei natürlichen Substraten mehrere Wochen und bei modifizierten Substraten bis zu sieben Tage stabil. Mit Hitze-behandeltem Zelllysat wurden ähnliche Ergebnisse erzielt wie mit gereinigtem Enzym. II Zussamenfassung Heba Y. Mohamed Synthesis of nucleoside analogs as potential medicinal agents List of Contents Abstract .................................................................................................................................................... I Zusammenfassung ................................................................................................................................... II List of Contents ........................................................................................................................................ 1 Acknowledgements ................................................................................................................................. 3 List of Publications ................................................................................................................................... 5 List of Abbreviations ................................................................................................................................ 7 1. Introduction ...................................................................................................................................... 9 2. Scientific Background ...................................................................................................................... 11 2.1. Naturally occurring nucleoside analogs ...................................................................................... 12 2.2. Nucleoside analogs as therapeutics ............................................................................................ 13 2.3. Uptake and activation of nucleoside drugs in vivo ..................................................................... 13 2.4. Therapeutic applications of nucleoside analogs ......................................................................... 15 2.5. Chemical synthesis of nucleoside analogs .................................................................................. 17 2.6. Enzymatic synthesis of nucleoside analogs ................................................................................. 18 2.6.1. Nucleoside deoxyribosyltransferases (NDT)........................................................................ 20 2.6.2. Nucleoside phosphorylases (NPs) ....................................................................................... 20 3. Aim of the Project ..........................................................................................................................
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