7.13–7.19 (m, 5H), 7.23–7.26 (m, 2H), 7.30–7.33 (m, 3H), 7.55 (d, 1H, J = 8.0), 7.77 (d, 2H, J = 7.5), 8.25 (d, 1H, J = 7.0), 8.34 (d, 1H, J = 7.0), 8.95 (s, 1H), 10.70 (s, 1H), 10.77 (d, 1H, J = 2.5), 12.13 (broad s, 1H), MALDI-MS obsd 694.3321, calcd 694.3235 [(M + H)+ , M = C33H43N9O8].19b: 1H NMR (400 MHz, DMSO-d6): d 1.47–1.49 (m, 2H), 1.82–1.91 (m, 1H), 2.01–2.05 (m, 1H), 2.13–2.18 (m, 2H), 2.21–2.30 (m, 2H), 2.42–2.54 (m, 2H, overlap with DMSO), 2.69–2.80 (m, 3H), 2.89–2.94 (m, 1H), 3.04–3.07 (m, 2H), 3.16–3.20 (m, 2H), 3.79–3.84 (m, 1H), 4.09– 4.15 (m, 1H), 4.38–4.42 (m, 1H), 6.70 (broad, 1H), 6.88 (s, 1H), 6.97 (t, 1H, J = 7.6), 7.05 (t, 1H, J = 7.2), 7.14–7.19 (m, 2H), 7.30– 7.36 (m, 3H), 7.43–7.49 (m, 2H), 7.55 (d, 1H, J = 7.6), 7.65 (s, 1H), 7.77 (d, 1H, J = 7.6), 7.81–7.86 (m, 3H), 8.25 (d, 1H, J = 7.6), 8.39 (d, 1H, J = 6.4), 8.95 (s, 1H), 10.70 (s, 1H), 10.78 (s, 1H); MALDI-MS obsd 744.2910, calcd 744.3391 [(M + H)+ , M = C37H45N9O8]. Found at: doi:10.1371/journal.pone.0010504.s012 (164 KB TIF) Figure S9 Synthetic scheme for generation of diaster- eomerically pure conventional peptide hydroxamic acids. a) t-BuONH2 Aˆ?HCl, HBTU, DIPEA; b) LiOH then HCl. Note that these experiments were conducted to confirm the stereochemistry assignments made for each diastereomer 18a, 18b, 19a, and 19b (see Fig. S8). Found at: doi:10.1371/journal.pone.0010504.s013 (0.05 MB TIF) Figure S10 Alternate synthetic scheme for generation of conventional peptide hydroxamic acids. a) t-BuONH2?HCl, HBTU, DIPEA; b) LiOH then HCl; c) EtO2CCl, Et3N, 278uC then NH2-Arg(Pbf)-allyl?HCl. General Procedure for the Prepara- tion of Protected Hydroxamates 21 and 23 The protected hydroxamate 21 obtained as a colorless oil (290 mg, 88%) starting from (R)-acid 20 following the general procedure described for 6a and 6b. Also 23 was obtained as a colorless oil (485 mg, 94%) starting from (S)-acid 22. 21: 1H NMR (500 MHz, CDCl3): d 1.23 (s, 9H), 2.21–2.51 (m, 1H), 2.37–2.80 (m, 1H), 2.83–2.87 (m, 1H), 3.00–3.04 (m, 1H), 3.25 (broad, 1H), 3.65 (s, 3H), 7.14–7.15 (m, 2H), 7.20–7.23 (m, 1H), 7.27–7.30 (m, 2H), 7.75–7.90 (broad m, 1H). 23: 1H NMR (500 MHz, CDCl3): d 1.19–1.22 (m, 9H), 2.21– 2.84 (m, 1H), 2.40–2.84 (m, 1H), 2.99–3.04 (m, 1H), 3.18–3.24 (m, 1H), 3.34 (broad, 1H), 3.66 (s, 3H), 7.28–7.30 (m, 1H), 7.43–7.48 (m, 2H), 7.60 (broad s, 1H), 7.67 (broad, 1H), 7.77–7.82 (m, 3H). General Procedure for the Hydrolysis of Methyl Esters 21 and 23 to acids (R)-7a and (S)-7b A solution of the methyl ester 21 References 1. Zhao WQ, Alkon DL (2001) Role of insulin and insulin receptor in learning and memory. Mol Cell Endocrinol 177: 125–134. 2. Messier C, Teutenberg K (2005) The role of insulin, insulin growth factor, and insulin-degrading enzyme in brain aging and Alzheimer’s disease. Neural Plast 12: 311–328. 3. Duckworth WC, Kitabchi AE (1981) Insulin metabolism and degradation. Endocr Rev 2: 210–233. 4. Brange J, Langkjoer L (1993) Insulin structure and stability. Pharm Biotechnol 5: 315–350. 5. Farris W, Mansourian S, Chang Y, Lindsley L, Eckman EA, et al. (2003) Insulin- degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo. Proc Natl Acad Sci U S A 100: 4162–4167. 6. Duckworth WC, Bennett RG, Hamel FG (1998) Insulin degradation: progress and potential. Endocr Rev 19: 608–624. 7. Makarova KS, Grishin NV (1999) The Zn-peptidase superfamily: functional convergence after evolutionary divergence. J Mol Biol 292: 11–17. 8. Becker AB, Roth RA (1992) An unusual active site identified in a family of zinc metalloendopeptidases. Proc Natl Acad Sci U S A 89: 3835–3839. 9. Shen Y, Joachimiak A, Rosner MR, Tang W-J (2006) Structures of human insulin-degrading enzyme reveal a new substrate mechanism. Nature 443: 870–874. 10. Manolopoulou M, Guo Q, Malito E, Schilling AB, Tang WJ (2009) Molecular basis of catalytic chamber-assisted unfolding and cleavage of Inhibitors of IDE (335 mg, 1.14 mmole) in methanol (20 ml) and water (3 ml) was treated with LiOH monohydrate (96 mg, 2.28 mmole) in water (5 ml) at OuC for 6 h. The reaction mixture was diluted with water (10 ml), acidified with 1N HCl (to pH 2) and extracted with DCM (50 ml63). The organic layer was washed saturated sodium chloride (100 ml), dried over anhydrous sodium sulfate, filtered, and evaporated to give a waxy solid. The resultant solid was purified by silica gel column chromatography with 2% MeOH in DCM to give (R)-7a (waxy solid, 170 mg, 53%) and used without further purification. Acid (S)-7b was in prepared in a similar manner and a similar yield and used without further purification. Procedure for the Synthesis of 10b The protected dipeptide 10b was obtained as a white foam (80 mg, 73%) starting from (S)-acid 7b and Pbf-protected arginine allyl ester HCl salt following the general procedure described for 6a and 6b. The TLC profile and 1H NMR were the same for this material as those prepared by the separation of diastereomers. Procedure for the Synthesis of 18a The protected dipeptide 9a was obtained as a white foam (130 mg, 29%) starting from (R)-acid 7a and Pbf-protected arginine allyl ester HCl salt following the general procedure described for 6a and 6b. The TLC profile and 1H NMR were the same for this material as those prepared by the separation of diastereomers. The deprotected 18a was obtained from 9a as described above. In addition, this material demonstrated the same in vitro activity as the material prepared by the separation of diastereomers. Found at: doi:10.1371/journal.pone.0010504.s014 (0.07 MB TIF) Acknowledgments We thank Jake Ni and Li-An Yeh for assistance with compound screening, Chittaranjan Das and Michael J. Wolfe for advice on peptide synthesis, Camille Caclin for contributions to medicinal chemistry, Britney Popp for technical assistance, Dr. Michel Bernier for the generous gift of CHO-IR cells, and the staff of APS SBC and Biocars for help in data collection. Author Contributions Conceived and designed the experiments: MAL EM BET RLS GDC. Performed the experiments: MAL EM SH LR TS GMM MH PSM SC TPL BET MM. Analyzed the data: MAL EM SH SOAH SC BET WJT GDC DJS. Contributed reagents/materials/analysis tools: SC AHF SC LCC GDC. Wrote the paper: MAL EM SOAH WJT RLS GDC DJS. human insulin by human insulin-degrading enzyme. J Biol Chem 284: 14177–14188. 11. Malito E, Hulse RE, Tang WJ (2008) Amyloid beta-degrading cryptidases: insulin-degrading enzyme, presequence peptidase, and neprilysin. 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(2003) Kinetics of amyloid beta-protein degradation determined by novel fluorescence- and fluorescence polarization-based assays. J Biol Chem 278: 37314–37320. 18. Neant-Fery M, Garcia-Ordonez RD, Logan TP, Selkoe DJ, Li L, et al. (2008) Molecular basis for the thiol sensitivity of insulin-degrading enzyme. Proc Natl Acad Sci U S A 105: 9582–9587. PLoS ONE | www.plosone.org 12 May 2010 | Volume 5 | Issue 5 | e10504