To investigate the effect of aryl-OH functionalities, we prepared a set of three symmetric bis-hydroxyaryl-Tz (with 'aryl' referring to phenyl and pyridyl). Applying standard procedures for tetrazine synthesis we obtained the hydroxylated bis-phenyl-Tz 4 (hPh2) and the two bis-hydroxypyridyl-tetrazines 5 (h2P2) and 6 (h4P2) starting from the respective nitriles (Fig. ). Unexpectedly, buffered aqueous solutions (pH 7.4) of 5 and 6 did not show the pink color as usually characteristic to 1,2,4,5-Tz. The observed color ranged from yellow at µM concentrations to orange at higher concentrations (e.g., 1 mM; Fig. ) and was first mistaken for immediate degradation, but later confirmed to be due to the spectroscopic properties of this new class of Tz in aqueous solution. Consistent with the yellow color, UV-Vis spectroscopy revealed an increased absorption between 400 nm and 450 nm (see Supporting Information) that disappears upon click reaction (e.g., with rTCO-sarcosine ), as clearly visible at higher concentrations (Fig. ).