SUPPORTING INFORMATION Backbone modification of a parathyroid hormone receptor-1 antagonist/inverse agonist Ross W. Cheloha, Tomoyuki Watanabe, Thomas Dean, Samuel H. Gellman*, Thomas J. Gardella* *Corresponding authors:
Supporting Information Protease Susceptibility Assays. Peptide concentration was determined by UV Vis spectroscopy (calculated from the UVvis absorption at 280 nm, based on an extinction coefficient for the tryptophan sidechain chromophore of 5,690 M 1 cm 1 ). Peptide stock solutions were prepared in degassed water to a concentration of 200 µm. For trypsin proteolysis, sequencing-grade trypsin from bovine pancreas was purchased from Sigma Aldrich and prepared to a stock concentration of 100 µg/ml in 1 mm HCl. The protease reaction was carried out in 0.6 ml Eppendorf tubes at room temperature. The reaction solution was prepared by combining 40 µl of 200 µm peptide (final concentration 40 µm), 20 µl of 10X TBS ph 8.5 (final concentration 15 mm Tris, 150 mm NaCl, or 1x TBS), 130 µl of water, and 0.5 µl of 100 µg/ml protease (added last, final concentration 0.25 µg/ml, in a total volume of 200 µl). Each proteolysis experiment was run in duplicate. Following addition of protease, the reaction was timed and quenched by combining a 50 µl aliquot of the proteolysis mixture with 50 µl of 0.1% trifluoroacetic acid in acetonitrile. A portion (75 µl) of the quenched reaction mixture was injected onto an analytical RP-HPLC, and peaks were analyzed. The time course of peptide degradation was experimentally determined by integrating the area of the peak corresponding to the intact peptide in a series of HPLC traces, with duplicate proteolysis reactions being used to generate error bars corresponding to the standard deviation. The final 25 µl of the reaction solution was used to acquire MALDI TOF mass spectrometry data for identification of peptide fragments resulting from proteolysis. Proteolysis was observed at all predicted trypsin cut sites for peptide 1. Exponential decay curves and half-life values were generated using GraphPad Prism. The proteolysis assay using proteinase K was run under identical conditions to the assay run with trypsin with the following exceptions. Proteinase K was dissolved at a stock concentration of 250 µg/ml in water, and a peptide stock was prepared at a concentration of 100 µm in degassed water. The protease reaction was prepared by combining 100 µl degassed water, 20 µl 10x TBS (final concentration 15 mm Tris, 150 mm NaCl), 40 µl of 100 µm peptide stock (final concentration 20 µm), and 40 µl of proteinase K was added last. Protease activity was quenched at the indicated time point by transferring 50 µl of the protease mixture into 100 µl of 0.1% trifluoroacetic acid in water (volume/volume). Peptide Synthesis and purification. Protected amino acids were activated with 2-(1H-benzotriazole-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate (HBTU) and N-hydroxybenzotriazole (HOBt) in the presence of N,N-diisopropylethylamine (DIEA). The growing peptide chain was deprotected using 20% piperidine in DMF. Protected β 3 homoamino acids were purchased from PepTech or ChemImpex. After synthesis, the peptides were cleaved from the resin and side chains were deprotected using reagent K (82.5% TFA, 5% phenol, 5% H2O, 5% thioanisole, 2.5% ethanedithiol) for two hours. The TFA solution was dripped into cold diethyl ether to precipitate the deprotected peptide. Peptides were purified on a prep C18 column using reverse phase HPLC. Purity was assessed by analytical RP HPLC (solvent A: 0.1% TFA in water, solvent B:
0.1% TFA in acetonitrile, C18 analytical column (4.6 X 250 mm), flow rate 1 ml/min, gradient 10 60% B solvent over 50 minutes). See the supporting table 1 for information regarding analytical HPLC retention times, purity data and a list of observed masses from MALDI TOF MS (average [M+H] +, m/z). See supporting table S1 for a summary of HPLC and MALDI-TOF data and supporting figure S6 for HPLC chromatograms of peptide purity checks and MALDI- TOF-MS of purified materials.
Supporting figure S6: HPLC and MALDI-TOF mass spectra data Peptide 1
Peptide 2