Mannose azide 4 was synthesised via a BF3•OEt2 mediated glycosylation of D-mannose pentaacetate 1 with 4nitrophenol, to afford a-aryl mannoside 2 (Figure ). Subsequent deacetylation of this protected sugar was followed by the hydrogenation of the nitro group to an amine group, which was followed by squarate ester coupling to afford mannoside 3. As the installation of a sugar into an OPAL probe had not previously been attempted, we chose to make our α-mannose presenting squarate mannoside amenable to derivatisation through azide-alkyne 'click' reactions via the addition of 1-amino-2-azidoethane to afford the novel azide containing mannoside squarate 4. Two OPAL probe precursor peptides with different serine-glycine Gly-Ser linker lengths (n= 1 or 3) were then synthesised via solid-phase peptide synthesis (SPPS). These precursor peptides featured an L-propargylglycine residue at their Ctermini, which we selectively conjugated to our versatile mannose azide 4 prior to resin cleavage in an adaption of the on-resin Cu-catalysed 'click' method of Sewald and coworkers. Following cleavage from the resin, a stoichiometric quantity of NaIO4 was then used to cleanly and selectively convert the more reactive 1,2-amino alcohol motif present in the OPAL probe precursor peptides S8 and S9 to an aldehyde motif without oxidising the 1,2-diol of the mannose motif, affording two activated α-mannosepresenting OPAL probes 5 and 6 for bioconjugation (Figure ). After freshly installing N-terminal α-oxo aldehyde functionality into colicin E9, the OPAL probes 5 and 6 were used to construct two α-mannose-presenting glyco-colicins (Gly-Ser)1 and (Gly-Ser)3 with different length linkers (Figure ). The success of OPAL conjugation was confirmed through analysis by SDS-PAGE and lectin blotting with concanavalin A (a lectin which binds to terminal α-mannose units) (Figure ). OPAL bioconjugation of probes 5 and 6 to colicin E9 was evident in the lectin blots, with no lectin binding to unmodified colicin E9 observed.