The SNAP-<i>tag</i> technology revised: an effective <i>chemo-enzymatic approach</i> by using a universal azide-based substrate

SNAP-<i>tag</i>^® is a powerful technology for the labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. Although commercially available or ad hoc produced, their synthesis and purification are necessary, increasing time and costs. To address this limitation, here we suggest a revision of this methodology, by performing a <i>chemo-enzymatic approach</i>, by using a BG-substrate containing an azide group appropriately distanced by a spacer from the benzyl ring. The SNAP-<i>tag</i>^® and its relative thermostable version (<i>Ss</i>OGT-<i>H⁵</i>) proved to be very active on this substrate. The stability of these <i>tags</i> upon enzymatic reaction makes possible the exposition to the solvent of the azide-moiety linked to the catalytic cysteine, compatible for the subsequent conjugation with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. Our studies propose a strengthening and an improvement in terms of biotechnological applications for this self-labelling <i>protein-tag</i>.