As group-specific enzymes, purine NPs were therefore evolved to recognize multiple different purine nucleosides (and nucleobases) as substrates and catalyze transformations from and toward the natively occurring N9ribosyl purines with astonishing selectivity. Although the ability of purine NPs to promiscuously degrade "non-native" glycosylation regioisomers of purines is documented by a handful of examples, imperfect regioselectivity in glycosylation reactions with naturally occurring purines has not been reported yet for purine NPs. In addition, conceivable N7-ribosides such as N7X are not known as natural products. Providing an exception to the paradigm that natural purine nucleosides are uniformly N9-ribosides, we herein report on the synthesis of N7X by NPs as well as the unusual spectroscopic and physicochemical properties of this nucleoside. We initially discovered N7X as an unexpected byproduct of a reaction cascade involving a bacterial purine NP and a guanine deaminase (see Scheme S1). With the former enzyme cleaving guanosine into guanine and ribose 1phosphate (Rib1P) and the latter quantitatively hydrolyzing guanine into xanthine (X), the expected products of this sequence should be Rib1P and X (Fig. ). As N9xanthosine (N9X, the "native" isomer) is typically not converted by bacterial nucleoside phosphorylases to any significant extent (this process requires a specialized enzyme: xanthosine phosphorylase), the reverse reaction, glycosylation of X, similarly should not proceed. Thus, Rib1P and X should be terminal products of this reaction sequence. However, UV absorption spectra obtained during this transformation proved inconsistent with this assumption (Fig. ). Similar results were obtained when X and Rib1P were directly incubated with the purine NP. We consistently observed absorption at higher wavelengths than anticipated for X and HPLC analysis of the reaction mixture showed a single additional peak corresponding to an unknown product (Fig. ). HRMS clearly supported the formation of a riboside of X but the retention times and absorption spectra of the reaction product clearly did not match N9X (Figs. ). We nucleoside phosphorylases (with 500 µM X and 1 g L -1 enzyme). Please see the Supplementary Information for experimental details and the externally hosted supplementary material for all raw data. subsequently consulted DFT calculations to predict dominant tautomers and their UV spectra, which suggested N7X and O6-xanthosine (O6X) as sufficiently redshifted candidates, excluding O2-xanthosine (O2X, Fig. , please see the Supplementary Information and Chart S1 for details). A detailed NMR-spectroscopic analysis of a partially purified sample of the reaction product in D2O revealed N7X as the likely product. A weak HMBC correlation between the anomeric position on the ribosyl unit and C5 of the X unit, as well as a strong correlation to C8, make N7 the most likely ribosylation site. NOESY correlations between the ribosyl hydrogens (1′, 2′ and 3′) and H-8 support this assignment (Fig. ). Subsequent freeze-drying of the sample, trituration with DMSO-d6 and comparison to Bridson's 1998 NMR data (the only reported synthesis of N7X) unambiguously supported the formation of N7X (Table ).