Journal of Medicinal Chemistry raphy (heptane/ethyl acetate 1:1, Rf = 0.14) gave (R)-8 as a transparent oil (690 g, 88% yield over two steps). Analytical chiral HPLC (hexane/ethanol 70:30) tR 9.2 min, 100% purity. 1H NMR (CD3OD) δ 7.43−7.25 (m, 5H), 5.20−5.02 (m, 2H), 5.00−4.85 (m, 1H), 4.65−4.51 (m, 1H), 4.50−4.38 (m, 1H), 4.21−3.80 (m, 8H), 3.21−3.03 (m, 1H), 3.02−2.89 (m, 1H), 2.79−2.61 (m, 1H), 2.52− 2.38 (m, 1H), 1.98−1.35 (m, 13H), 1.34−1.18 (m, 2H), 1.16−1.09 (m, 3H) 1.04−0.81 (m, 12H). 13C NMR (CD3OD) δ 174.9, 172.3, and 172.1 (rot), 171.6 and 171.4 (rot), 157.5, 157.2, 157.0, and 156.7 (rot), 137.6, 129.4, 129.0, 128.8, 128.7, 80.2, 68.4, and 68.3 (rot), 62.2 and 62.1 (rot), 54.2, 54.5, 49.6, 46.5, 45.9, and 45.6 (rot), 42.8, 42.2, and 41.9 (rot), 28.7, 26.4, and 25.7 (rot), 25.6, 25.4, 23.7, 23.6, 23.5, 22.2, 22.0, 21.4, 14.5; [α]D20 −43.5 (c 1.0, CH3OH). IR (KBr) v 3318, 2957, 1754, 1709, 1640, 1445, 1206 cm−1. HRMS (FT-ICR-MS) calcd for C34H54N4O8 [M + H]+ 647.4014; found 647.4015. (−)-Ethyl 2-[(Benzyloxycarbonyl)(1-((tert-butoxycarbonyl-L- leucinyl)-L-leucinyl)-(S)-piperidin-3-yl)amino]acetate ((S)-8). Deprotection of (S)-5 (535 mg, 1.0 mmol) with TFA (1.0 mL, 13 mmol) and dichloromethane (6 mL) were performed as described for the synthesis of (S)-5 to give the Boc-deprotected intermediate. The intermediate and the second Boc-L-Leu × H2O (275 mg, 1.10 mmol) were coupled as described for the synthesis of (S)-5 with EDC (212 mg, 1.10 mmol), HOBt (149 mg, 1.10 mmol), Et3N (406 mg, 4.02 mmol), and molecular sieves (240 mg) in dichloromethane (11 mL). Purification by flash chromatography (heptane/ethyl acetate 1:1, Rf = 0.24) gave (S)-8 as a transparent oil (590 g, 91% yield over two steps). Analytical chiral HPLC (hexane/ethanol 70:30) tR 5.6 min, 100% purity. 1H NMR (CD3OD) δ 7.47−7.23 (m, 5H), 5.28−5.02 (m, 2H), 5.00−4.86 (m, 1H), 4.67−4.50 (m, 1H), 4.48−4.36 (m, 1H), 4.23− 3.97 (m, 6H), 3.96−3.81 (m, 1H), 3.74−3.62 (m, 1H), 3.49−3.36 (m, 1H), 3.27−3.17 (m, 1H), 3.05−2.90 (m, 1H), 2.73−2.60 (m, 1H), 2.54−2.35 (m, 1H), 2.14−1.08 (m, 18H), 1.00−0.74 (m, 12H). 13C NMR (CD3OD) δ 174.8, 172.6, and 172.5 (rot), 171.7 and 171.5 (rot), 157.5, 156.8, and 156.5 (rot), 137.6 and 137.5 (rot), 129.3, 129.1, and 128.9 (rot), 128.7, 128.6, 80.2, 68.5, 68.3, and 68.1 (rot), 62.1, 54.3, 50.2, 46.3, 46.2, 45.7, 43.4, 42.7, 41.9, 30.0, 28.8, 26.2, 25.8, 23.8, 23.6, 22.4, 22.2, 22.0, 14.5; [α]D20 −9.2 (c 0.5, CH3OH). IR (KBr) v 3319, 2957, 1753, 1709, 1641, 1447, 1205 cm−1. HRMS (FT- ICR-MS) calcd for C34H54N4O8 [M + H]+ 647.4014; found 647.4014. (−)-2-[(Benzyloxycarbonyl)(1-((tert-butoxycarbonyl-L-leucin- yl)-L-leucinyl)-(R)-piperidin-3-yl)amino]acetamide ((R)-9). Com- pound (R)-9 was synthesized from (R)-8 (213 mg, 0.33 mmol) and NH3 in methanol (7 N) (2.0 mL) as described for (S)-2, but the reaction mixture was stirred for 168 h. Purification by flash chromatography (dichloromethane/methanol 95:5, Rf = 0.05) gave the product as a white solid (119 mg, 63%). Analytical chiral HPLC (hexane/ethanol 70:30) tR 13.7 min, 100% purity. 1H NMR (CD3OD) δ 7.42−7.24 (m, 5H), 5.20−5.05 (m, 2H), 4.96−4.87 (m, 1H), 4.66− 4.52 (m, 1H), 4.48−4.37 (m, 1H), 4.14−3.84 (m, 6H), 3.17−2.92 (m, 2H), 2.73−2.59 (m, 1H), 2.54−2.40 (m, 1H), 2.03−1.91 (m, 1H), 1.87−1.34 (m, 14H), 1.00−0.84 (m, 12H). 13C NMR (CD3OD, 50 °C) δ 175.0, 174.4, 172.6, and 172.4 (rot), 157.7, 157.5, 137.8, 129.5, 129.0, 128.7, 80.6, 68.7, 55.2, 54.8, 54.6, 47.6, 46.7, 46.1, 43.0, 42.3, 42.1, 29.1, 28.7, 26.6, 26.0, 25.7, 25.3, 23.6, 23.3, 22.2, 21.9; [α]D20 −50.0 (c 0.67, CH3OH). IR (KBr) v 3408, 2956, 1704, 1638, 1449 cm−1. HRMS (FT-ICR-MS) calcd for C32H51N5O7 [M + H]+ 618.3861; found 618.3863. (−)-2-[(Benzyloxycarbonyl)(1-((tert-butoxycarbonyl-L-leucin- yl)-L-leucinyl)-(S)-piperidin-3-yl)amino]acetamide ((S)-9). Com- pound (S)-9 was synthesized from (S)-8 (394 mg, 0.61 mmol) and NH3 in methanol (7 N) (4.0 mL) as described for (R)-7. Purification by flash chromatography (dichloromethane/methanol 95:5, Rf = 0.13) gave (S)-7 as a white solid (325 mg, 86%). Analytical chiral HPLC (hexane/ethanol 70:30) tR 8.4 min, 100% purity. 1H NMR (CD3OD) δ 7.51−7.22 (m, 5H), 5.27−5.06 (m, 2H), 4.98−4.86 (m, 1H), 4.66− 4.50 (m, 1H), 4.47−4.37 (m, 1H), 4.23−3.79 (m, 6H), 3.28−3.11 (m, 1H), 3.06−2.91 (m, 1H), 2.72−2.58 (m, 1H), 2.54−2.38 (m, 1H), 2.04−1.81 (m, 2H), 1.80−1.25 (m, 13H), 0.99−0.75 (m, 12H). 13C NMR (CD3OD, 50 °C) δ 174.9, 174.3, and 174.2 (rot), 172.7 and 172.3 (rot), 157.5, 137.8, and 137.7 (rot), 129.4, 128.9, 128.7, 80.5, Article 68.6, 56.4, 54.7, 47.3, 46.4, 46.1, 43.5, 42.8, 42.1, 42.0, 29.1, 28.8, 26.2, 25.8, 25.7, 23.6, 23.4, 22.2, 22.0; [α]D20 −9.5 (c 0.67, CH3OH). IR (KBr) v 3407, 2957, 1707, 1638, 1450 cm−1. HRMS (FT-ICR-MS) calcd for C32H51N5O7 [M + H]+ 618.3861; found 618.3861. (−)-2-[(L-Leucinyl-(1-L-leucinyl-(R)-piperidin-3-yl))amino]- acetamide × 2TFA ((R)-LLpipG) ((R)-10). Compound (R)-9 (282 mg, 0.46 mmol) was deprotected as described for (S)-LpipGP ((S)- 18) but with stirring for 3 h during the first deprotection step. Purification by flash chromatography (ethyl acetate/methanol 20:3) gave the Cbz-deprotected intermediate (189 mg). Further depro- tection (180 mg) was performed as described for (S)-LpipGP ((S)- 18) with TFA/dichloromethane (1:10) (3 mL) and with stirring for 14 h to give the white TFA salt of (R)-LLpipG ((R)-10) (251 mg, 90%). Analytical reversed phase HPLC (the mobile phase was 10.8− 37.2% CH3CN in H2O for 30 min with 0.1% TFA throughout) tR 8.3 min, 100% purity. 1H NMR (CD3OD) δ 4.93−4.82 (m, 1H), 4.67 (app d, 1H), 4.09 (app d, 1H), 4.04−3.83 (m, 3H), 3.82−3.64 (m, 2H), 3.47−3.38 (m, 1H), 3.26−3.09 (m, 2H), 2.84 (app t, 1H), 2.32− 2.11 (m, 1H), 1.99−1.41 (m, 6H), 1.04−0.76 (m, 12H). 13C NMR (CD3OD) δ 172.8, 170.7, 168.6, 55.6, 54.8, 52.7, 46.7, 46.4, 44.5, 41.8, 41.3, 28.0, 25.7, 25.1, 25.0, 23.5, 23.1, 21.9, 21.7; [α]D20 −13.6 (c 1.0, CH3OH). IR (KBr) v 3422, 2964, 1676, 1202, 1135 cm−1. HRMS (FT-ICR-MS) calcd for C19H37N5O3 [M + H]+ 384.2969; found 384.2967. For NMR spectral assignment, see the Supporting Information. (+)-2-[(1-(L-Leucinyl-L-leucinyl)-(S)-piperidin-3-yl)amino]- acetamide × 2TFA ((S)-LLpipG) ((S)-10). Compound (S)-9 (230 mg, 0.37 mmol) was deprotected as described for (R)-LLpipG ((R)- 10) to give the white TFA salt of (S)-LLpipG ((S)-10) (209 mg, 92%). Analytical reversed phase HPLC (the mobile phase was 10.8− 37.2% CH3CN in H2O for 30 min) tR 8.60 min, 100% purity. 1H NMR (CD3OD) δ 4.83−4.77 (m, 1H), 4.53−4.39 (m, 1H), 4.13−3.86 (m, 3H), 3.75−3.65 (m, 1H), 3.63−3.45 (m, 2H), 3.39−3.32 (m, 1H) 2.60 (app t, 1H), 2.31−2.14 (m, 1H), 2.09−1.97 (m, 1H), 1.94−1.81 (m, 1H), 1.80−1.56 (m, 5H), 1.55−1.39 (m, 1H), 1.04−0.86 (m, 12H). 13C NMR (CD3OD) δ 173.0, 171.3, 168.7, 56.1, 55.2, 52.7, 47.1, 46.9, 44.1, 41.6, 41.3, 27.5, 25.8, 25.2, 24.4, 23.5, 23.2, 22.1, 21.8; [α]D20 +14.3 (c 1.0, CH3OH). IR (KBr) v 3419, 2966, 1674, 1202, 1137 cm−1. HRMS (FT-ICR-MS) calcd for C19H37N5O3 [M + H]+ 384.2969; found 384.2966. Biological Studies. Purification of IdeS. For recombinant IdeS expression an ideS allele was PCR-amplified from genomic streptococcal DNA using primers 5′-TGTTACCATGGA- TAGTTTTTCTGCTAAT-3′ introducing a NcoI restriction site and 5′-GCTAGGTACCTTAATTGGTCTGATTCCAAC- TAT-3′ introducing an Acc65I restriction site for subsequent cloning into plasmid pETtrx_1a.29 IdeS was expressed in Escherichia coli strain BL21 to obtain a His-tagged Trx-IdeS protein. The fusion protein was purified on Ni2+-resin using standard protocols. The His-Trx tag was removed by enzymatic cleavage with Tev-protease in His-trap binding buffer (20 mM Tris-HCl pH8.0, 150 mM NaCl, 10% glycerol) at 30 °C for up to 20 h followed by a second round of purification on Ni2+-resin to remove uncleaved fusion protein.29 The collected flow through, containing IdeS without tag, was purified by size exclusion chromatography on a HiPrep 16/60 Sephacryl S-300 HR column (GE Healthcare). Purified protein was kept in PBS at −20 °C until use. IdeS Activity Assay and Inhibitor Screening. IdeS (10 μL) (1.83 μM in PBS) was mixed with peptide or peptide analogue (3 μL) (30 mM in 50% DMSO, 50 mM phosphate buffer pH 7.4) and incubated for 15 min at room temperature prior to the addition of IgG1 (10 μL) (Sigma I5154, 8.07 μM in 20 mM TRIS-buffered saline pH 8.0) as substrate. The reactions were incubated for 20 min at 37 °C before termination by the addition of iodoacetamide (10 μL, 16.5 mM) (Sigma I1149). Samples were run in triplicates. Controls where IdeS had been replaced with PBS were included in duplicates for each peptide/peptide analogue. Samples were analyzed by SDS-PAGE and surface plasmon resonance spectroscopy. 2558 dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560