Peptides were synthesized as described above at a 0.05 mmol scale. The peptide pellets were dissolved in 10 ml of water and directly purified by RP-HPLC on Waters HPLC system (Prep LC 2535 HPLC) using a semipreparative C18 column (Waters XBridge BEH300 prep C18, 5 μm, 10 mm by 250 mm), applying a linear gradient of 0 to 30% solvent B for more than 30 min [solvent A: 99.9% (v/v) H2O and 0.1% (v/v) TFA; solvent B: 99.9% (v/v) ACN and 0.1% (v/v) TFA] at a flow rate of 6 ml/min. Fractions containing the peptides were lyophilized and dissolved in H2O to prepare 5 mM stock solutions.

The peptides were cyclized with linkers in a combinatorial fashion in a 384-deep-well plate (3347, Corning) as follows. Seventy-eight microliters of 60 mM NH4HCO3 buffer (pH 8) was added to the wells by an automated dispenser (Multidrop Dispenser, Thermo Fisher Scientific) using eight lines and a small cassette. Two microliters of 5 mM purified linear peptides was transferred by a Biomek FXP laboratory automation workstation from a v-bottom 96-well plate (6018321, Ratiolab) containing different peptides in 88 wells and water in eight wells to eight quadrants of two 384-deep-well plates using a 96 pipetting head and 100-μl disposable plastic tips (AP96 P20, 717255, Beckman). Twenty microliters of 4 mM linker 1 to 7 (4 mM in ACN) was transferred from 96-well plates [v-bottom polypropylene (PP), 6018321, Ratiolab] to the two 384-deep-well plates using a 96 pipetting head and 100-μl disposable plastic tips (AP96 P20, 717255, Beckman) using the same workstation. In each transfer, one of the seven linkers placed in 88 wells of the 96-well plate was transferred along with ACN in eight wells. The plates were incubated at 37°C for 2 hours.

The reaction products were screened for inhibition of trypsin and thrombin in low volume flat bottom non-binding surface (NBS) 384-well plates (3820, Corning) as follows. The reaction mixture (1.5 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, Beckman). Human cationic trypsin (8.5 μl, 0.18 nM) or human α thrombin (3.53 nM) in assay buffer [10 mM tris-Cl (pH 7.4), 150 mM NaCl, 10 mM MgCl2, 1 mM CaCl2, 0.1% (w/v) bovine serum albumin (BSA), and 0.01% (v/v) Triton X-100] was dispensed with the BioTek MultiFlo dispenser to each well to reach final concentrations of 0.1 nM for trypsin and 2 nM for thrombin and incubated at RT for 15 min. Five microliters of fluorogenic protease substrates in assay buffer containing 3% dimethyl sulfoxide (DMSO) was dispensed to each well using the same dispenser but different cassettes for enzyme and substrate. For both enzymes, the substrate Z-Gly-Gly-Arg-AMC (150 μM) was added to reach a final concentration of 50 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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