Aim:To develop probes for detecting the binding specificity between β-secretaseand substrate,and provide reliable biological activity data for further researchingencircling substrate-based inhibitors.Methods:To prepare the inhibitors,thehydroxyethylene(HE)segment including P_1 and P_1′ was synthesized after multi-step reactions;the combination of all segments was then completed through solidphase synthesis.Recombinant human β-secretase ectodomain(amino acid resi-dues 1-460)was expressed as a secreted protein with a C-terminal His tag in insectcells using baculovirus infection,and all compounds were evaluated in thisβ-secretase enzyme assay.In order to understand the interaction in detail,the theoretical methods,namely molecular dynamics(MD)simulation and mo-lecular mechanics-generalized-born surface area(MM-GBSA)analysis,were per-formed on the complex of β-secretase and OM99-2 to obtain the geometrical andenergetical information.Results:We designed and constructed a positional scan-ning combinatorial library including 16 compounds;all members of the librarywere synthesized based on HE dipeptide isostere.Structure-activity relationshipstudies at the P_4-P_1 and P_1′-P_4′ positions led to the discoveries of P and P’sidesbinding specificity and potent inhibitors 14,18,and 22.The binding free energyon the whole system and every residue were compared to the biological assayresult.Conclusion:The removal of P_4′ yielded inhibitor 22(A_3*B_2)with highpotency;further truncation of P_3′ gave inhibitor 18(A_3*B_1)with equal activity,implying that the right side of the inhibitors play a less important role and could beeasily simplified,while change on the P side may cause substantial results. Aim: To develop probes for detecting the binding specificity between β-secretase and substrate, and provide reliable biological activity data for further researchingencircling substrate-based inhibitors. Methods: To prepare the inhibitors, thehydroxyethylene (HE) segment including P_1 and P_1 ’was synthesized after multi-step reactions; the combination of all segments was then completed through solid phase synthesis. Recombinant human β-secretase ectodomain (amino acid resi-dues 1-460) was expressed as a secreted protein with a C-terminal His tag in insect cells using baculovirus infection, and all compounds were evaluated in this β-secretase enzyme assay. In order to understand the interaction in detail, the theoretical methods, namely molecular dynamics (MD) simulation and mo-lecular mechanics-generalized-born surface area (MM-GBSA) analysis, were per-formed on the complex of β-secretase and OM99-2 to obtain the geometrical andenergetical information. Results: We designed and constructed a positional sc an-ning combinatorial library including 16 compounds; all members of the library article synthesized based on HE dipeptide isostere. Structure-activity relationship studies at the P_4-P_1 and P_1’-P_4 ’positions led to the discoveries of P and P’sidesbinding specificity and potent inhibitors 14,18, and 22. The binding free energyon the whole system and every residue were compared to the biological assayresult. Conlusion: The removal of P_4 ’yielded inhibitor 22 (A_3 * B_2) with high potency; further truncation of P_3’ 18 (A_3 * B_1) with equal activity, implying that the right side of the inhibitors play a less important role and could be beeily simplified, while changing on the P side may cause substantial results.