Design And Synthesis Of Substrate-Based Fluorescent Sensors Targeting Serine Hydrolases

Butyrylcholinesterase(BChE)and Chymotrypsin(CTR)are two important enzymes in the serine hydrolase family.While BChE is mainly responsible for the degradation of chemicals related to neurological diseases including acetylcholine,butyrylcholine,succinylcholine,etc.For instance,its abnormality can induce nervous system diseases such as Alzheimer’s disease.As for CTR,it is mainly responsible for the digestion of useless peptides in the body.As a metabolizing enzyme,the catalytic reaction of CTR often occurs in the pancreas of the organism,and its abnormality usually leads to a variety of pancreatic diseases.Developing corresponding enzyme-activity monitoring methods is necessarily indispensable for the diagnosis,treatment and drug development of related diseases.At present,the detection of BChE activity often relies on multi-stage mass spectrometry or the indirect Ellman’s analysis.Meanwhile,the tools of CTR-activity measurement mainly focus on peptide-based fluorescent or UV substrate.But it cannot be ignored that the two aforementioned methods bear several drawbacks such as high costs and cumbersome experimental process.Owing to its high temporal-spatial resolution and remarkable signal-noise ratio,nonpeptide-based small-molecule probes have an advantage over traditional analyses in terms of economic costing and operational inconveniences when they are applied in monitoring enzymatic activity.In this thesis,a series of "Turn-ON" small-molecule fluorescent probes for targeting BChE and CTR were synthesized,individually.Compound Ⅱ-12 was screened as the best sensor with the highest selectivity towards BChE,and the probe Ⅲ-2&Ⅲ-11 were selected as ideal probes with considerable selectivity and specificity for CTR.Especially,the probe Ⅱ-12,using cyclopropenyl group as the recognition unit for BChE,showed more than 60-fold enhancement of fluorescent intensity when incubated with BChE and nearly 31-fold of the discrimination between BChE and AChE,and this sensor played the advantage of near-infrared fluorescent dye in imaging the endogenous BChE including SH-SY5Y cells,zebrafish,and AD-model mouse.While compound III-2(with 4-bromobutryl group)and Ⅲ-11(with 2-thiophene acetyl group)showed 66-fold and 262-fold enhancement of fluorescent intensity when incubated with CTR,respectively.At the same time,they showed 14.5-fold and 14.5-fold of the discrimination between CTR and trypsin.Besides,both probes were comparable to the commercial substrate(AMC-FPAA-Suc)in inhibitor screening and kinetic studies.It was noteworthy that probe Ⅲ-11 had successfully tracked CTR in zebrafish for the first time,indicating its potential to act as a new tool for CTR-related drug discovery or mechanism research in the future.