Chemo-enzymatic synthesis of α-d-pentofuranose-1-phosphates using thermostable pyrimidine nucleoside phosphorylases

Kamel, Sarah; Weiß, Max; Klare, Hendrik F. T.; Mikhailopulo, Igor A.; Neubauer, Peter; Wagner, Anke

doi:10.1016/j.mcat.2018.07.028

Cited by 33 publications

(49 citation statements)

References 41 publications

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“…Profiting from the use of the thermostable NPases Y02 and N02, we were able to perform the phosphorolysis of 24 nucleosides, including 12 pyrimidine and 12 purine nucleosides, at temperatures of 40 to 70 °C. To prevent decomposition of the produced pentose‐1‐phosphates, which is known to happen rapidly at high temperatures and neutral pH values, we increased the reaction pH to 9. Under these conditions, both enzymes displayed excellent activity with all substrates 1 – 24 and allowed efficient reaction completion and monitoring.…”

Section: Resultsmentioning

confidence: 99%

Thermodynamic Reaction Control of Nucleoside Phosphorolysis

et al. 2020

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Nucleoside analogs represent a class of important drugs for cancer and antiviral treatments. Nucleoside phosphorylases (NPases) catalyze the phosphorolysis of nucleosides and are widely employed for the synthesis of pentose‐1‐phosphates and nucleoside analogs, which are difficult to access via conventional synthetic methods. However, for the vast majority of nucleosides, it has been observed that either no or incomplete conversion of the starting materials is achieved in NPase‐catalyzed reactions. For some substrates, it has been shown that these reactions are reversible equilibrium reactions that adhere to the law of mass action. In this contribution, we broadly demonstrate that nucleoside phosphorolysis is a thermodynamically controlled endothermic reaction that proceeds to a reaction equilibrium dictated by the substrate‐specific equilibrium constant of phosphorolysis, irrespective of the type or amount of NPase used, as shown by several examples. Furthermore, we explored the temperature‐dependency of nucleoside phosphorolysis equilibrium states and provide the apparent transformed reaction enthalpy and apparent transformed reaction entropy for 24 nucleosides, confirming that these conversions are thermodynamically controlled endothermic reactions. This data allows calculation of the Gibbs free energy and, consequently, the equilibrium constant of phosphorolysis at any given reaction temperature. Overall, our investigations revealed that pyrimidine nucleosides are generally more susceptible to phosphorolysis than purine nucleosides. The data disclosed in this work allow the accurate prediction of phosphorolysis or transglycosylation yields for a range of pyrimidine and purine nucleosides and thus serve to empower further research in the field of nucleoside biocatalysis.

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Section: Resultsmentioning

confidence: 99%

Thermodynamic Reaction Control of Nucleoside Phosphorolysis

et al. 2020

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“…Nucleoside phosphorylases (NPs), for instance, catalyze the reversible phosphorolytic cleavage of nucleosides into the corresponding free nucleobase and pentose-1-phosphate (Scheme 1) and are widely applied for the preparation of modified nucleosides. [1][2][3][4][5][6][7][8][9][10][11] Consequently, their kinetic and thermodynamic characterization has attracted increased interest and demanded the development of efficient analytical tools. [12,13] Recently, we reported a UV/Vis spectroscopy-based method for the monitoring of these reactions that largely eliminated the need for HPLC.…”

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confidence: 99%

Spectral Unmixing‐Based Reaction Monitoring of Transformations between Nucleosides and Nucleobases

et al. 2020

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The increased interest in (enzymatic) transformations between nucleosides and nucleobases has demanded the development of efficient analytical tools. In this report, we present an update and extension of our recently described method for monitoring these reactions by spectral unmixing. The presented method uses differences in the UV absorption spectra of nucleosides and nucleobases after alkaline quenching to derive their ratio based on spectral shape by fitting normalized reference spectra. It is applicable to a broad compound spectrum comprising more than 35 examples, offers HPLC‐like accuracy, ease of handling and significant reductions in both cost and data acquisition time compared to other methods. This contribution details the principle of monitoring reactions by spectral unmixing, gives recommendations regarding solutions to common problems and applications that necessitate special sample treatment. We provide software, workflows and reference spectra that facilitate the straightforward and versatile application of the method.

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“…The evaluation of enzymatic deoxyribose-1-phosphate forming reactions requires the fast detection of substrates and products. The detection of nucleoside and its corresponding nucleobase by HPLC, and thus the indirect determination of pentose-1-phosphate, has been the standard method to date (e.g., [8,34]). However, it is very time-consuming and laborious and therefore not suitable for use in high-throughput screenings.…”

Section: The Absorption Spectrum Of Thymine But Not Deoxythymidine Chmentioning

confidence: 99%

“…To date, our model does not include terms for the decay of enzyme activity or the degradation of any chemical species. We base these decisions on reports of the exceptional stability in alkaline conditions of deoxyribose-1-phosphate [34] and ribose-1-phosphate [42], as well as on the report of stable enzyme activity over days for thermophilic pyrimidine- [20] and purine-nucleoside phosphorylases [43] at even higher temperatures than those used in this study.…”

Section: Model Structurementioning

confidence: 99%

“…The stability of deoxyribose-1-phosphate and enzyme activity over the assay duration is key to correct conclusions from the experimental data. In addition to reports from literature on pentose-1-phosphate and enzyme stability [20,34,42,43], we performed a control experiment via a coupled read-out, providing evidence for fulfilling these preconditions ( Figure S2).…”

Section: Limitations and Domain Of Validity Of The Modelmentioning

confidence: 99%

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Dynamic Modelling of Phosphorolytic Cleavage Catalyzed by Pyrimidine-Nucleoside Phosphorylase

et al. 2019

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Pyrimidine-nucleoside phosphorylases (Py-NPases) have a significant potential to contribute to the economic and ecological production of modified nucleosides. These can be produced via pentose-1-phosphates, an interesting but mostly labile and expensive precursor. Thus far, no dynamic model exists for the production process of pentose-1-phosphates, which involves the equilibrium state of the Py-NPase catalyzed reversible reaction. Previously developed enzymological models are based on the understanding of the structural principles of the enzyme and focus on the description of initial rates only. The model generation is further complicated, as Py-NPases accept two substrates which they convert to two products. To create a well-balanced model from accurate experimental data, we utilized an improved high-throughput spectroscopic assay to monitor reactions over the whole time course until equilibrium was reached. We examined the conversion of deoxythymidine and phosphate to deoxyribose-1-phosphate and thymine by a thermophilic Py-NPase from Geobacillus thermoglucosidasius. The developed process model described the reactant concentrations in excellent agreement with the experimental data. Our model is built from ordinary differential equations and structured in such a way that integration with other models is possible in the future. These could be the kinetics of other enzymes for enzymatic cascade reactions or reactor descriptions to generate integrated process models.

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Chemo-enzymatic synthesis of α-d-pentofuranose-1-phosphates using thermostable pyrimidine nucleoside phosphorylases

Cited by 33 publications

References 41 publications

Thermodynamic Reaction Control of Nucleoside Phosphorolysis

Thermodynamic Reaction Control of Nucleoside Phosphorolysis

Spectral Unmixing‐Based Reaction Monitoring of Transformations between Nucleosides and Nucleobases

Dynamic Modelling of Phosphorolytic Cleavage Catalyzed by Pyrimidine-Nucleoside Phosphorylase

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