Due to the brightness and excellent photostability, rhodamine B was chosen as a basis of the clickable fluorophore. Secondary amides of rhodamine B exist in a non-fluorescent spirocyclic form at neutral pH and transform into ring-opened fluorescent forms at lower pH. To remove this impact of pH on the fluorescence, a ω-azido tertiary amide 26 was prepared with 1-(2azidoethyl)piperazine, which is not prone to spirolactamisation and subsequent loss of fluorescence. Human plasma is reported to contain 3.5-9.3 mg/L (41-109 nM) BChE 2 and is therefore an excellent and readily available biological sample for labelling experiments. Three time-dependent carbamate inhibitors were chosen based on their potencies, selectivity ratios and decarbamoylation rates. The stability of carbamates in aqueous solution was also checked , and were shown to be stable for weeks under physiologically similar conditions (Table ). The compounds were incubated with plasma, the whole samples were labelled using in situ click reaction with Cu + and 26, and the mixture was resolved using SDS-PAGE, as described in the Experimental Section. The proteins were transferred to a nitrocellulose membrane using Western blotting to improve fluorescent detection. Clearly visible fluorescent bands were present only in the plasma sample treated with inhibitor 17 (Figure left, lane 4) and in the recombinant hBChE and hAChE samples labelled with 17 (Figure left, lanes 9 and 10, respectively). The labelling was abolished by preincubation with BChE-selective inhibitors ethopropazine and tetraisopropyl pyrophosphoramide (iso-OMPA; lanes 5-7) and also with AChE-selective BW284c51 (1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide; lanes 8, 11). The latter is somewhat surprising, as the reported IC 50 for hBChE inhibition by BW284c51 is 354 µM. Additional immunostaining with anti-hBChE antibodies was performed (Figure , right), and the fluorescently labelled bands in lanes 4 and 9 were also detected by antibodies. The expected mass of fully glycosylated native plasma hBChE is 85 kDa , which agrees with the signal below the 98 kDa marker. The origins of the other fluorescent and immunostained bands are unknown; some possibilities include carbamate also binding to other serine hydrolases in plasma , degradation of hBChE upon storagethe socalled "storage bands" , and unspecific binding to human serum albumin (67 kDa). This requires further exploration, but for this proof-of-concept study, the fluorescent labelling of hBChE with clickable carbamates was confirmed. The successful labelling only in the case of the carbamate 17 with the terminal alkyne on the longest chain (i.e., 11 carbon atoms vs. 3 and 5 carbon atoms for 18 and 16, respectively) would suggest that a sufficiently long linker is required on both the side of the alkyne partner as well as the azide fluorophore, to successfully span the active site gorge and participate in the click reaction. However, this was not supported by computational studies (vide infra); therefore, factors other than steric limitation might be at work here. Perhaps the click reaction deeper within the active site gorge is hindered, or the leaving group 5 remains bound in the active site (as in the crystal structure; see below), to prohibit the fluorophore terminal azide from entering deep enough into the active site to reach the carbamate alkyne and for the click reaction to occur.