Chemo-enzymatic synthesis of selenium-modified nucleosides, nucleotides and nucleic acids
Final Report Abstract
The aim of the joint project was to synthesize Se-modified nucleic acids by a chemoenzymatic approach for crystallography. The German partner was mainly focusing on the enzymatic synthesis of the building blocks: 2-Se-modified nucleosides and nucleotides. Despite the high similarity of Se-modified bases to natural bases, the synthesis of the Senucleosides was very challenging. Se-modified bases and -nucleosides show only low solubility and are instable under oxidizing conditions. Although the production of single Senucleosides in a transglycosylation reaction has been demonstrated in advance of this project, the observed product yields were very low. In order to improve the product yields in a rational approach, studies on thermodynamic characteristics of the reaction were first carried out. Here it was shown that the thermodynamic equilibrium is very unfavorable for the preparation of the Se-nucleosides. Based on this knowledge, conditions (very low phosphate concentration, 10-fold excess of sugar donor) could be defined that allowed an improved synthesis of the Se nucleosides in a transglycosylation reaction. Since the high sugar donor excess had a negative impact on downstream processing, the purification process had to be optimized first. However, a combination of silica chromatography and HPLC finally enabled the purification of the target molecules. However, since maximum product yields of 50% at most were observed for the 2-seleniummodified nucleosides even after optimization of the transglycosylation, an alternative approach for the preparation of the Se nucleosides was sought. Here, direct glycosylation was first validated, and product yields could be slightly increased. An analysis of existing methods for increasing product yields for modified nucleosides led us to test the use of a sucrose phosphorylase in combination with direct glycosylation. This approach showed that product yields >90% was achieved with a small excess of substrate. Another advantage of this method is that downstream processing is simplified, and the E-factor was significantly improved compared to chemical synthesis or transglycosylation. For the synthesis of the Se-modified nucleotides, a generalizable method for the synthesis of NTPs was first developed using selected natural and modified nucleoside substrates. The influence of an integrated ATP regeneration system was evaluated and found to have a very positive impact on the enzymatic cascade synthesis of NTPs. After the development of a high throughput enabling method for substrate screening of nucleoside kinases, it was possible to identify suitable enzyme candidates for Se-NMP synthesis. Based on these results, a thinlayer chromatography-based screening for the preparation of Se-modified NTPs was performed. Focusing on thermostable enzymes, cascades were identified that successfully allowed the enzymatic synthesis of the targeted Se-NTPs.
Publications
- Human Deoxycytidine Kinase Is a Valuable Biocatalyst for the Synthesis of Nucleotide Analogues. Catalysts 2019, 9, 997
Hellendahl, K.F.; Kamel, S.; Wetterwald, A.; Neubauer, P.; Wagner, A.
(See online at https://doi.org/10.3390/catal9120997) - General Principles for Yield Optimization of Nucleoside Phosphorylase-Catalyzed Transglycosylations. ChemBioChem 2020, 21, 1428
Kaspar, F.; Giessmann, R. T.; Hellendahl, K.F.; Neubauer, P.; Wagner, A.; Gimpel, M.
(See online at https://doi.org/10.1002/cbic.201900740) - Modular Enzymatic Cascade Synthesis of Nucleotides Using a (d)ATP Regeneration System. Front. Bioeng. Biotechnol. 2020. 8, 854
Fehlau, M.; Kaspar, F.; Hellendahl, K.F.; Schollmeyer, J.; Neubauer, P.; Wagner, A.
(See online at https://doi.org/10.3389/fbioe.2020.606584) - Spectral Unmixing-Based Reaction Monitoring of Transformations between Nucleosides and Nucleobases. ChemBioChem 2020, 21, 2604
Kaspar, F.; Giessmann, R.T.; Westarp, S.; Hellendahl, K.F.; Krausch, N.; Thiele, I.; Walczak, M.C.; Neubauer, P.; Wagner, A.
(See online at https://doi.org/10.1002/cbic.202000204) - Optimized Biocatalytic Synthesis of 2-Selenopyrimidine Nucleosides by Transglycosylation. ChemBioChem 2021, 22, 2002
Hellendahl, K.F.; Kaspar, F.; Zhou, X.; Yang, Z.; Huang, Z.; Neubauer, P.; Kurreck, A.
(See online at https://doi.org/10.1002/cbic.202100067) - Semi-Automated High-Throughput Substrate Screening Assay for Nucleoside Kinases. Int J Mol Sci. 2021, 22(21), 11558
Hellendahl, K.F.; Fehlau, M.; Hans, S.; Neubauer, P.; Kurreck, A.
(See online at https://doi.org/10.3390/ijms222111558)
