Visual Abstract

A new fluorescent ribonucleoside alphabet (mthN) consisting of pyrimidine and purine analogues, all derived from methylthieno[3,4‐d]pyrimidine as the heterocyclic core, is described. Large bathochromic shifts and high microenvironmental susceptibility of their emission relative to previous alphabets derived from thieno[3,4‐d]pyrimidine (thN) and isothiazole[4,3‐d]pyrimidine (tzN) scaffolds are observed. Subjecting the purine analogues to adenosine deaminase, guanine deaminase and T7 RNA polymerase indicate that, while varying, all but one enzyme tolerate the corresponding mthN/mthNTP substrates. The robust emission quantum yields, high photophysical responsiveness and enzymatic accommodation suggest that the mthN alphabet is a biophysically viable tool and can be used to probe the tolerance of nucleoside/tide‐processing enzymes to structural perturbations of their substrates.

Guanine deaminase (GDA) deaminates guanine to xanthine. Despite its significance, the study of human GDA remains limited compared to other metabolic deaminases. As a result, its substrate and inhibitor repertoire are limited, and effective real-time activity, inhibitory, and discovery assays are missing. Herein, we explore two emissive heterocyclic cores, based on thieno­[3,4-d]­pyrimidine ( thN) and isothiazole­[4,3-d]­pyrimidine ( tzN), as surrogate GDA substrates. We demonstrate that, unlike the thieno analog, thG N , the isothiazolo guanine surrogate, tzG N , does undergo effective enzymatic deamination by GDA and yields the spectroscopically distinct xanthine analog, tzX N . Further, we showcase the potential of this fluorescent nucleobase surrogate to provide a visible spectral window for a real-time study of GDA and its inhibition.

No abstract