Figure S6 Lack of toxicity of IDE inhibitors used in cell- based assays. A, B, Potential cytotoxicity of Ii1 and ML3-XF evaluated by lactose-dehydrogenase release (A) and (3-(4,5-di- methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium (MTS) (B) conversion assays. Assays were conducted on confluent CHO-IR cells using the CytoTox 96TM and the CellTiter 96H AQueous Non-Radioactive Cell Proliferation Assay (ProMega) according to manufacturer’s recommendations. Essentially identical results were obtained with HeLa cells (not shown). Found at: doi:10.1371/journal.pone.0010504.s010 (0.12 MB TIF) Figure S7 Confirmation that FITC-labeled species se- creted by FITC-insulin-loaded CHO-IR cells are pre- dominantly breakdown products. A, Relative fluorescence intensity of conditioned medium (CM) from FITC-insulin-loaded cells, unconditioned medium, CM from unloaded cells, and-as a key control-the latter medium supplemented with intact FITC- insulin such that the fluorescence is equivalent to that in the CM from FITC-insulin-loaded cells. B, Levels of intact insulin present in the latter samples quantified using a homogeneous time- resolved fluorescence-based assay (CIS-Bio). Note that levels of intact insulin are greatly reduced in the CM from FITC-insulin- loaded cells as compared to that in CM from unloaded cells supplemented with a fluorescent equivalent of intact insulin. C, Surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) spectra from CM supplemented with intact FITC-insulin (upper panel) or CM from FITC-insulin-loaded cells (lower panel). Note that intact FITC-insulin (expected mass = 6197.4; observed mass 6196.37) is detectable in the former sample, but only fragments thereof are detected in the latter. Found at: doi:10.1371/journal.pone.0010504.s011 (0.35 MB TIF) Figure S8 Synthetic scheme for inhibitor Ii1. a) 10% Pd/C, H2 (1 atm), aqueous NaOH, EtOH, rt, 48 h; b) BnBr, K2CO3, acetone, reflux, 16 h; c) LDA, THF, 278uC, 1 h then BrCH2CO2t-Bu; d) TFA - CH2Cl2 (1:1), rt, 1 h; e) t-BuONH2?HCl, DMF, HBTU, DIPEA; f) 10% Pd/C, H2 (1 atm), EtOH; g) EtO(C = O)Cl, Et3N, THF/DMF, 220uC, 30 min then NH2-Arg(Pbf)-allyl?HCl, Et3N, 220uC for 1 h then rt for 16 h; h) chromatography on silica gel; i) Pd(PPh3)4, morpholine; j) HBTU, DIPEA; k) 10% Pd/C, H2 (1 atm), EtOH/MeOH; l) 5% anisole in TFA, 40uC, 4.5 h; m) reverse HPLC purification. Hydrocinnamic acid, 2a, was purchased from Sigma-Aldrich. 2-naphthalenepropanoic acid (2b): A mixture of 3-(2- naphthyl)acrylic acid (1, 1 g, 0.54 mmol) and 10% Pd-C (100 mg) in ethanol (20 mL) was treated with 20% aqueous NaOH (5 mL) and stirred under hydrogen (1 atm) for 48 h. The mixture was filtered and the filtrate was acidified with 1N HCl and the extracted with EtOAc (250 mL). The organic extract was dried over anhydrous magnesium sulfate, filtered and concentrated to afford 2b (710 mg, 70%), which was used for the next step without further purification: 1H NMR (500 MHz, DMSO-d6): d 2.64 (t, 2H, J = 7.8), 2.99 (t, 2H, J = 7.8), 7.41–7.49 (m, 3H), 7.72 (s, 1H), 7.82–7.87 (m, 3H), 12.16 (br s, 1H). Procedure for the Preparation of Esters 3a and 3b A mixture of the acid (2a, 1.5 g, 10 mmole) and potassium carbonate (1.4 g, 10 mmole) in acetone ( 30 ml) was treated with benzyl bromide (1.3 ml, 11 mmole) while stirring at room temperature and heated at reflux overnight. The reaction mixture was cooled, filtered and evaporated to an oil. The resultant oil was purified by silica gel column chromatography with 5% EtOAc in hexane to give 3a as a colorless oil (1.92 g, 80%):1H NMR (500 MHz, CDCl3): d 2.69 (t, 2H, J = 8.0), 2.97 (t, 2H, J = 7.8), 5.11(s, 2H), 7.18–7.21 (m, 3H), 7.26–7.37 (m, 7H). Inhibitors of IDE 3b (oil, 1.26 g, 87%):1H NMR (500 MHz, CDCl3): d 2.79 (t, 2H, J = 7.5), 3.15 (t, 2H, J = 7.8), 5.12 (s, 2H), 7.27–7.34 (m, 6H), 7.41–7.48 (m, 2H), 7.63 (s, 1H), 7.75–7.82 (m, 3H). Procedure for the Alkylation of Esters 3a and 3b To a stirred solution of LDA (2.0 M in THF, 2.7 ml, 5.4 mmole) and anhydrous THF (5 ml) at 278uC was dropwise added ester 3a (1.28 g, 5.3 mmole) in THF (6 ml). After 1 h at 278uC, tert-butyl bromoacetate (0.87 ml, 5.9 mmole) was added neat and the reaction mixture was slowly allowed to warm to room temperature overnight. A saturated aqueous ammonium chloride solution (2 ml) was added to the reaction mixture followed by EtOAc (100 ml). The organic layer was washed with saturated sodium chloride (100 ml), dried over anhydrous sodium sulfate, filtered, evaporated to give an oil. The resultant oil was purified by silica gel column chromatography with 7% EtOAc in hexane to give crude 4a (1.2 g, 63% mixture of mono- and dialkylated ester) as a colorless oil and used for the next step without further purification. 4b (oil, 830 mg, 40%):1H NMR (500 MHz, CDCl3): d 1.38 (s, 9H), 2.39 (dd, 1H, J = 5.0, 16.5), 2.65 (dd, 1H, J = 8.8, 17.3), 2.94 (dd, 1H, J = 7.5, 13.0), 3.17–3.26 (m, 2H), 5.05 (d, 1H, J = 12.0), 5.11 (d, 1H, J = 12.5), 7.16–7.19 (m, 2H), 7.22–7.29 (m, 4H), 7.42–7.48 (m, 2H), 7.58 (s, 1H), 7.73– 7.76 (m, 2H), 7.78–7.81 (m, 1H). Procedure for the Deprotection of Alkylated Esters 4a and 4b to acids 5a and 5b A mixture of the ester (4a, 1.2 g, 3.78 mmole, crude mixture (see above)) and TFA (10 ml) in dichloromethane (20 ml) was stirred at room temper- ature for 4 h. The reaction mixture was evaporated to give an oil, which was purified by silica gel column chromatography with 30% EtOAc in hexane to give 5a as a colorless oil, which solidified under house vacuum (530 mg, 53% from crude starting material):1H NMR (500 MHz, CDCl3): d 2.47 (dd, 1H, J = 4.5, 17.5), 2.75 (dd, 1H, J = 9.5, 17.0), 2.79 (dd, 1H, J = 8.5, 13.5), 3.08 (dd, 1H, J = 6.3, 13.8), 3.15–3.20 (m, 1H), 5.11 (s, 2H), 7.12–7.13 (m, 2H), 7.19–7.35 (m, 8H). 5b (waxy oil, 770 mg, 95%):1H NMR (500 MHz, CDCl3): d 2.50 (dd, 1H, J = 4.8, 17.3), 2.78 (dd, 1H, J = 9.0, 17.5), 2.96 (dd, 1H, J = 7.5, 13.0), 3.21–3.30 (m, 2H), 5.09 (dd, 2H, J = 12.3, 15.3), 7.17–7.19 (m, 2H), 7.22–7.28 (m, 4H), 7.42–7.48 (m, 2H), 7.58 (s, 1H), 7.73–7.76 (m, 2H), 7.78–7.81 (m, 1H). Procedure for the Preparation of Protected Hydroxamates 6a and 6b A mixture of the acid (5a, 600 mg, 2 mmole) and HBTU (770 mg, 2.02 mmole) in anhydrous DMF (20 ml) was stirred at room temperature for 1 h. Then O-tert-butylhydroxylamine HCl (265 mg, 2.1 mmole) and DIPEA (0.74 ml, 2.1 mmole) were added and the resulting reaction mixture was stirred overnight. The reaction mixture was diluted with EtOAc (100 ml), washed with saturated sodium chloride (100 ml), dried over anhydrous sodium sulfate, filtered, evaporated to give an oil. The resultant oil was purified by silica gel column chromatography with 20% EtOAc in hexane to give 6a (oil, 700 mg, 95%): 1H NMR (500 MHz, DMSO-d6): d 1.11 (s, 9H), 2.20 (dd, 1H, J = 5.5, 15.0), 2.36 (dd, 1H, J = 9.0, 15.5), 2.79–2.80 (m, 2H), 3.07–3.13 (m, 1H), 4.99 (s, 2H), 7.11–7.12 (m, 2H), 7.20–7.35 (m, 8H), 10.36 (s, 1H). 6b (oil, partially purified, mixture of product and acid-starting material). Procedure for the Preparation of Acids 7a and 7b A mixture of hydroxamate (6a, 700 mg, 1.89 mmol) and 10% Pd-C (200 mg) in ethanol (20 mL) was stirred under hydrogen (1 atm) for 16 h. The mixture was filtered and concentrated to afford 7a (white solid, 480 mg, 90%), which was used for the next step without further purification: 1H NMR (500 MHz, DMSO-d6): d 1.12 (s, 9H), 2.06 (dd, 1H, J = 5.8, 15.0), 2.28 (dd, 1H, J = 8.8, 15.3), 2.73 (dd, 1H, J = 6.8, 13.3), 2.86 (dd, 1H, J = 7.3, 13.3), 2.93–2.98 (m, 1H), 7.16–7.22 (m, 2H), 7.27–7.30 (m, 3H), 10.31 (s, 1H), 12.24 (br s, 1H). 7b (purified by silica gel column chromatography with 5% MeOH in DCM, white foam, 220 mg, 80%): 1H NMR (500 MHz, DMSO-d6): d 1.12 (s, 9H), PLoS ONE | www.plosone.org 10 May 2010 | Volume 5 | Issue 5 | e10504