The PUB module relies on a highly selective nucleoside phosphorolysis step, which can be leveraged to generate a UV spectroscopic read-out in the presence of phosphate. Given the inherent limitations of existing purine-based systems, which employ irreversible enzymatic phosphorolysis of a nucleoside precursor, we recognized that a conceptual redesign would be necessary to realize the versatility and scope required for many applications. Based on our previous forays into nucleoside phosphorylase chemistry and UV spectroscopic reaction monitoring, we hypothesized that we could employ in situ deprotonation of a pyrimidine nucleoside and its corresponding nucleobase, which then tautomerizes to a pyrimidinolate, to yield a spectral redshift (Fig. ). Thus, we envisioned that the phosphorolysis of a pyrimidine nucleoside could report on the presence of phosphate in solution through a characteristic UV spectroscopic signal reflecting conversion of the starting material. A systematic examination of commercially available uridine analogs identified 5-bromouridine (1a) as the ideal candidate. The nucleoside 1a ( = 7.95) can be deprotonated under mildly alkaline conditions in buffered solution and its corresponding anionic nucleobase 1b ( = 8.03) exhibits a 22 nm UV redshift, enabling their spectroscopic differentiation. Monitoring of the phosphorolysis of 1a to 1b at pH 9 yields highly overlapping absorption spectra with an isosbestic point at 288 nm (Fig. ) and a maximum difference of the extinction coefficients at 304 nm (∆ = 5.4 mM -1 cm -1 , Figs. and). Thus, monitoring at 304 nm coupled with isosbestic point normalization via parallel monitoring at 288 nm provides quantitative real-time conversion data for this transformation, given reference values for both compounds (Fig. ). As initial studies indicated that this reaction could be carried out with a variety of pyrimidine nucleoside phosphorylases, we elected the commercially available thermostable variant Y02 for further studies. An extensive evaluation of buffer systems revealed that the PUB module performs best in amino acid buffers such as asparagine ( = 8.8) or glycine ( = 9.6) or the amino sulfonate taurine ( = 9.1). Sulfonic acids from Good's series and the "better buffer" diethylpiperazine gave inferior results while other buffers including ammonia, borate or carbonate proved incompatible (see the SI for a detailed analysis and buffer recommendations; also see the SI section "Additional considerations"). Owing to the inherent selectivity of nucleoside phosphorlyases, the PUB module is highly selective to inorganic phosphate and exhibits no cross-reactivity with other phosphate species. Similarly, we found that the presence of other ions in solution has no significant impact on the performance of the module.