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

• Published in: Chembiochem : a European journal of chemical biology Vol. 21; no. 18; pp. 2604 - 2610
• Main Authors: Kaspar, Felix, Giessmann, Robert T., Westarp, Sarah, Hellendahl, Katja F., Krausch, Niels, Thiele, Isabel, Walczak, Miriam C., Neubauer, Peter, Wagner, Anke
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 14.09.2020; John Wiley and Sons Inc
• Subjects: Absorption spectra; Bases (nucleic acids); Chromatography, High Pressure Liquid; Communication; Communications; Data acquisition; High-performance liquid chromatography; Liquid chromatography; Monitoring; Nucleic Acid Conformation; nucleobases; nucleoside phosphorylase; Nucleosides; Nucleosides - chemistry; Software; spectral unmixing; Spectrophotometry, Ultraviolet; UV/Vis spectroscopy; nucleosides; nucleoside phosphorylase; spectral unmixing; nucleobases; UV/Vis spectroscopy
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202000204

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 bases of life and their analogues display UV absorption spectra that differ from those of their respective nucleosides under alkaline conditions. This enables robust, versatile and fast analysis of reactions between these species.