N7X has several unusual physicochemical properties. First, N7X is the only known natural nucleoside whose UV absorption spectra are redshifted compared to those of the free nucleobase (Fig. ). 20 N7X's sweeping absorption spectra (particularly those of the anion, Fig. ) are more reminiscent of free pyrimidine nucleobases than purine nucleosides. Secondly, N7X has a pKa of around 8.5 (Figs. and), as determined by UV spectroscopy, which is notably higher than free X (ca. 7.3) and N9X (ca. 5.3). Both of these properties may originate from the unique electronic situation in N7X's nucleobase. Although DFT calculations suggest that the negative charge in the anions of both N7X and N9X predominantly resides at O2 (Chart S1), only N7X can form an intramolecular H-bond between O6 and the 2′-OH (Fig. ). This grants N7X's nucleobase more aromatic character and leads to a pronounced redshift in its absorption spectra, in line with the trends exhibited by pyrimidine nucleosides and -bases. Thirdly, N7X displays an equilibrium constant of phosphorolysis of ca. 0.5 at pH 10, which we determined by continuously monitoring the glycosylation of X by UV spectroscopy using principles of spectral unmixing (Figs. and). This unusually high value could be qualitatively confirmed by reactions monitored discontinuously by HPLC (Table ). As purine nucleosides typically have equilibrium constants of phosphorolysis of 0.01-0.1 (irrespective of the sugar moiety), this makes N7X's phosphorolysis the most exothermic one known among purines. The next closest equilibrium constants of phosphorolysis of purines are those of N9X (0.2, determined by HPLC, Shugar reported 0.05) and inosine (0.1, determined by discontinuous spectral unmixing). To our surprise, non-selective formation of N7X is a common reactivity across the entire family of NPs (Fig. ). We made our initial discovery using the purine NP from Geobacillus thermoglucosidasius (GtPNP), which almost exclusively furnishes N7X under alkaline conditions. Compared to its native reactions (e.g. the phosphorolysis/glycosylation of adenosine or guanosine), which proceed with rate constants of >1 s -1 , the formation of N7X is a slow process. Under alkaline conditions, this transformation happens well over an order of magnitude slower and with a KM value exceeding the solubility limit of free X, as determined from kinetic experiments assessed by UV spectroscopy (Fig. ). Interestingly, at neutral pH values, this enzyme also yields N9X as a product of the same reaction and slowly degrades N7X (likely via hydrolysis, Fig. ). This process is pH-dependent (it does not happen at pH 10 to a relevant degree, Fig. ) and enzyme-catalyzed (purified N7X is stable, Fig. ). Other purine NPs, such as the ones from Thermus thermophilus (TtPNP), Escherichia coli and human, also make both N7X and N9X in different ratios, as do the xanthosine phosphorylase from E. coli (EcXP) and the pyrimidine NP from G. thermoglucosidasius (GtPyNP, albeit much slower, Fig. and Table ). Although the elucidation of the exact mechanisms underpinning the observed selectivity preferences are beyond the scope of this work, we hypothesize that differences in H-bonding behavior may be responsible. PNPs preferentially yielding N7X possess a nucleobasecoordinating aspartate, while those preferentially making N9X have an asparagine at the analogous position (Fig. ).