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

Abstract

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.

Keywords: UV/Vis spectroscopy; nucleobases; nucleoside phosphorylase; nucleosides; spectral unmixing.

Scheme 1

Nucleoside/nucleotide phosphorolysis of pyrimidine or purine species. With the exceptions of cytosine and 1,2,4‐triazole‐3‐carboxamide, all nucleobases featured in this report are described.

Figure 1

The principle of spectral unmixing‐based reaction monitoring. A) Enzymatic phosphorolysis of thymidine (1) into 2‐deoxyribose‐1‐phosphate (2) and the free nucleobase 3 as well as deprotonation after alkaline quenching. Representative resonance structures are shown. B) The substrate 1 and product 3 of the reaction have markedly different UV absorption spectra under alkaline conditions. C) The spectra of 1 (blue) and 3 (red) can be fitted to an experimental spectrum (black line) obtained during a reaction to derive the individual contributions of both species to the observed spectrum (hashed areas). D) Unmixing of multiple experimental spectra obtained during a reaction (left) enables reaction monitoring by deriving the degree of conversion at every sampled timepoint (right). Spectral unmixing of nucleoside transformations generally includes background correction, normalization to the isosbestic point of base cleavage, and fitting of the respective reference spectra. The spectra and conversions presented in this figure serve an illustrative purpose and were generated from the reference spectra of 1 and 3, as described in the externally hosted Supporting Information.36 Typical reaction conditions include a nucleoside concentration of 2 mM, 10 mM phosphate, 50 mM buffer of choice and 10 μg⋅mL⁻¹ NP in a total volume of 500 μL.12

Figure 2

Common background signals. A) Examplary UV‐active reactants 1 and 3. B) Standard background observed from the absorption of the 96‐well plate filled with water or aqueous NaOH. Signals for 1 and 3 represent typical signal intensities observed for reactions with 2 mM nucleoside substrate and a dilution factor of 15 during sampling. C) Baseline shift observed in approximately 1–2 % of measurements. D) Background observed in reactions with significant protein content. Purified Escherichia coli thymidine phosphorylase was used to recreate typical protein backgrounds by 15‐fold dilution in 100 mM NaOH. Note that some proteins can cause significantly more or less background. E) Representative background observed with some organic solvents. DMF was diluted tenfold in 100 mM NaOH to record the background signals.