Peptides were synthesized by Fmoc chemistry on Rink amide MBHA resin (resin, 0.3 mmol/g) at a scale of 5 μmol in 96-well reaction plates (32.410, Intavis) using an automated peptide synthesizer (Intavis MultiPep RSi). The coupling was carried out twice for each amino acid (4 equiv) using HATU (4 equiv) and NMM (10 equiv) in 130 μl of DMF without shaking for 45 min. The resin was washed twice with 225 μl of DMF, and unreacted amino groups were capped by incubation with 5% acetic anhydride and 6% 2,6-lutidine in 100 μl of DMF without shaking for 5 min. The resin was washed six times with 225 μl of DMF, and Fmoc groups were removed by incubation twice with 120 μl of 20% (v/v) piperidine in DMF without shaking for 5 min. The resin was washed eight times with 200 μl of DMF. At the end of the synthesis, the resin was washed twice with 200 μl of DCM. In all washing steps, the resin was incubated for 1 min.

Peptides were cleaved from the resin, and the protecting groups were removed under reducing conditions as follows. Volumes of 300, 100, and 100 μl of cleavage solution (90% TFA, 2.5% H2O, 2.5% thioanisole, 2.5% phenol, and 2.5% EDT) were added sequentially to wells of the reaction plate and incubated each time for 30 min to pass the resin by gravity flow. The flow-through was collected in a 96-deep-well plate (32.296, Intavis). Another 100 μl of cleavage solution was added, vacuum was applied, and the volume of the reaction solution was reduced by evaporation to 0.2 ml. Peptides were precipitated by addition of 1.5 ml of cold diethyl ether to each well and incubation for 1 hour at −20°C and pelleted by centrifugation at 4000g for 30 min. The peptide pellets were dissolved in ACN/H2O (1:1) and lyophilized, and the peptide was redissolved in H2O. The concentration was determined by measuring absorption at 280 nM using a nanodrop 8000 device (Thermo Scientific). The concentration was adjusted by addition of H2O to obtain stock solutions of 5 mM in v-shaped PP 96-well plates. The peptides were cyclized with linkers in a combinatorial fashion in 384-deep-well plates, as described above for the pilot-scale macrocycle library.

The reaction products were screened for inhibition of thrombin and KLK5 in low volume flat bottom NBS 384-well plates (Corning 3820) as follows. Reaction mixture (2 μl) was transferred from the reaction plate to the assay plate using the Biomek FXP laboratory automation workstation and a 384 pipetting head using 50-μl disposable plastic tips (AP384 P30XL, A22288). Eight microliters of human α thrombin (3.75 nM) or KLK5 (3.38 nM) in assay buffer [10 mM tris-Cl (pH 7.4), 150 mM NaCl, 10 mM MgCl2, 1 mM CaCl2, 0.1% (w/v) BSA, and 0.01% (v/v) Triton X-100] were dispensed with the BioTek MultiFlo dispenser to each well to reach final concentrations of 2 nM for thrombin and 5 nM for KLK5 and incubated at RT for 15 min. Five microliters of fluorogenic protease substrates in assay buffer containing 3% DMSO were dispensed to each well using the same dispenser. For thrombin, the substrate Z-Gly-Gly-Arg-AMC (150 μM) was added to reach a final concentration of 50 μM, and for KLK5, Val-Pro-Arg-AMC (300 μM) was added to reach 100 μM.

The residual protease activity was measured by monitoring the change in fluorescence intensity over time. The fluorescence intensity was measured with a fluorescence plate reader (excitation, 360 nm; emission, 465 nm; Tecan Infinite F500) at 25°C for 30 min with a reading every 3 min. The extent of inhibition (%) was calculated by comparing the residual activity to a control without macrocycle.

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