, DMF)/n-methylpyrrolidone 605 (NMP) (1:1, v/v) with 1% Triton X-100) and coupling (DIC/ 606 HOBt chemistry; 3 equiv of protected amino acid derivatives were 607 used). A 10-fold molar excess of N-acetylimidazole in DMF was used 608 for acetylation of the N-terminus

, ) 2 was synthesized via coupling of the Fmoc-PEG 2 to the 610 amino group of terminal Pro residue. The N-terminal biotin group was 611 conjugated using a 5-fold molar excess of biotin and 1,3-612 diisopropylcarbidiimide (DIC) as the coupling agent in anhydrous 613 DMSO for 6 h at 30 °C. The synthesized peptides were

, Fully protected peptides were dissolved in DMF and their carboxyl 616 groups were activated with DIC and coupled with HBr×H 2 N-Ala P

, 4-Cl) 2 , or HBr×H 2 N-618 nVal P (O-C 6 H 4 -4-Cl) 2 in DMF in the presence of DIPEA. The mixture 619 was stirred for 6 h, and the DMF was removed under reduced 620 pressure. The resulting compounds were suspended in trifluoroacetic 621 acid (TFA)/phenol

, The crude peptides were purified by HPLC on a Beckman Gold

. System, Either isocratic 627 conditions or a linear gradient were applied (flow rate 3.0 mL/min, 628 monitored at 226 nm). The purity of the synthesized peptides was 629 verified on RP Kromasil 100, C8, 5 ?m column (4.6 mm × 250 mm) 630 (Knauer, Germany). The peptides were eluted with a linear gradient of 631 the above solvent system (10%?90% B) for 30 min, flow rate 1 mL/ 632 min, monitored at 226 nm. HPLC retention times and 1 H NMR t5 633 spectra of final phosphonate peptide inhibitors are shown in Table 5 634 and Supporting Information

, The specificity constants k cat /K m 640 for peptidyl-pNA substrates were determined under first-order the spectrophotometer (Versamax microplate reader, Molecular 644 Devices, Measurements were carried out at 37 645 °C in buffer 50 mM HEPES, 0.75 M NaCl, 0.05% NP40, pH 7.4. Final in PBS. FRET substrate ABZ-720 VADnVADYQ-EDDnp 15 (20 ?M final) was incubated with humR3 721 and macaque neutrophil lysate supernatant (10?500 nM) at 37 °C in 722 50 mM HEPES, 0.75 M NaCl, and 0.05% NP40, pH = 7.4. The acid (v/v), then 726 fractionated by Agilent Technology 1200 series HPLC system (Agilent 727 Technology

, Millipore) at a flow rate of 0.3 mL/min with a linear gradient

, v/v) of acetonitrile in 0.01% trifluoroacetic acid over 40 min

, Molecular docking was performed in order 732 to explain interactions of Ac-PYDA P (O-C 6 H 4 -4-Cl), Bt, vol.2

, Bt-nLeu(O-Bzl)YDA P (O-C 6 H 4 -4-Cl) 2 734 (8), and Bt-VYDnV P (O-C 6 H 4 -4-Cl) 2 (11) with humPR3 and 735 macPR3, vol.2, p.12

S. Server, 41 For the docking studies, inhibitor molecules 739 were used as a peptidyl phosphonic acids

, O-Bzl)YDA P (OH) 2 ] 741 instead of di(chlorophenyl)-phosphonate esters

, 753 (b) the terminal carbon of Ala/nVal side chain of ligand P1 position 754 and enzyme S1 binding pocket set at ?-carbon of Ile190 (distance 755 range: 5.5?6.5 Å for Ala and 2.2?5.0 Å for nVal), (c) Asp ?-carbon of 756 the inhibitor (P2 position) and PR3 ?-amine nitrogen of Lys99 757 (distance range 2.0?5.0 Å),and (d) ligand and Bt-Val4 762 fragment of inhibitor to explain the differences in activity of 11 toward 763 human and macaque enzyme. humPR3 or macPR3 binding site was 764 represented by all amino acid residues within 6 Å of inhibitor fragment 765 considered herein

,

, Spectroscopic data of synthesized inhibitors

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