To date, only a handful of studies have investigated the structure-activity relationships (SARs) and kinetic characterisation of pseudo-irreversible carbamate ChEIs, with earlier efforts focused on the isocyanate-derived monosubstituted aryl carbamates, such as physostigmine (1a) derivatives. The Decker group has repeatedly reported on a series of evodiaminederived BChEIs that among other interesting properties also feature a monosubstituted carbamate warhead. Following up on the representative n-heptyl carbamate 1b , Nmonosubstituted carbamates with a terminal basic heterocyclic or tertiary amine (e.g., 1d, Scheme 1) on a 2-10 carbon atom spacer had an extended duration of action (1-28 h), which was attributed to the interaction of the ω-moiety with the peripheral site. Groner et al. used two series of disubstituted N-alkyl-N-methyl carbamates with two different leaving group scaffolds to show that carbamoylation rates of hAChEs are mainly influenced by the size and number of degrees of freedom of the N-alkyl (R) substituent, while for hBChE, the rates were more dependent on the structure of the leaving group. Decarbamoylation rates were greater for hBChE than for hAChE, and also less dependent on the size of R. For hBChE, the dimethylcarbamoylated enzyme recovered the fastest. Interestingly, the bulkier N-cyclohexyl-N-methyl carbamate 1e was inactive. Temperature also had a substantial impact on the decarbamoylation rate. For AChE, Venkatasubban and co-workers reported on a series of monomethyl, dimethyl, ethyl methyl and diethyl carbamates with different leaving groups (phenols, fluoride). Due to long recovery times (up to several weeks), radiolabelling the hAChE and calculation of the final substrate turnover rate was necessary to accurately determine the decarbamoylation constants within a reasonable timeframe (up to 24 h). The increase in carbamate size by adding further N-methyl or N-ethyl groups had a significant impact on the decarbamoylation rates of 1f, with the approximate ratios H 2 N : MeNH : Me 2 N : EtMeN : Et 2 N= 8000 : 800 : 200 : 10 : 1. Moreover, AChE and BChE can be used as biomarkers for detection of Alzheimer's disease , Hirschsprung's disease and multiple sclerosis. To date, only a few AChE and BChE fluorescent probes and histochemical methods have enabled tissue ChE location and activity determination. Accordingly, the development of new chemical probes for investigating the roles of ChEs in physiological and pathophysiological processes in vitro and ex vivo is required by biologists and neurobiologists. In a previous publication we reported successful conversion of tryptophan-derived sp -rich selective hBChE inhibitors into pseudo-irreversible inhibitors through the introduction of a carbamate moiety that enabled covalent binding to the target and prolonged the inhibition (Scheme 2). In the present study, we follow up on this series of pseudo-irreversible covalent carbamate hBChE inhibitors and report on their SARs, selectivities, and potential to be used as clickable molecular probes for fluorescent labelling of ChEs. Scheme 2: Conversion of reversible selective hBChE inhibitor 1 scaffold to a pseudoirreversible covalent carbamate inhibitor 2 and the structure-activity relationship of the carbamate warhead (general structure 3).