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.

The Peculiar Case of the Hyperthermostable Pyrimidine Nucleoside Phosphorylase from Thermus Thermophilus

“…Reaction monitoring was performed as described previously. [7,33,34] Typically, samples of 50 µL were withdrawn from reaction mixtures containing 1 mM UVactive compounds and pipetted into 450 µL 100 mM aqueous NaOH to stop the reaction. To record the UV spectra of these alkaline samples, 200 µL of the quenched samples were transferred to a UV/Vis-transparent 96-well plate (UV star, GreinerBioOne, Kremsmünster, Austria) and UV absorption spectra were recorded from 250 to 350 nm in steps of 1 nm with a high-throughput plate reader (BioTek Instruments, Winooski, USA).…”

“…[20] Likewise, reference spectra and software for spectral unmixing are available from the same repository [35,36] and described in previous works. [33,34] Author Information…”

Kinetic Analysis of the Hydrolysis of Pentose-1-Phosphates Through Apparent Nucleoside Phosphorolysis Equilibrium Shifts

“…The respective degree of conversion of the nucleoside to the nucleobase was then obtained via deconvolution of the experimental UV absorption spectra using suitable reference spectra. [15,16] All raw data presented in this report, along with metadata and calculations are freely available from an external online repository. [18] Likewise, reference spectra and software for spectral unmixing are available from the same repository [19,20] and detailed in previous works.…”

“…[18] Likewise, reference spectra and software for spectral unmixing are available from the same repository [19,20] and detailed in previous works. [15,16]…”

“…Monitoring of the phosphorolysis reaction can be achieved via the two UVactive components (nucleoside and nucleobase). [15,16] The graphs shown in B only serve an illustrative purpose and do not represent real data.…”