Synthesis and Characterization of Cyclic Dipeptides

Molecular biology reagents and chemicals were purchased from Fisher Scientific, Sigma-Aldrich, or New England Biolabs, Inc if not specifically indicated. Primers were ordered from Sigma-Aldrich. 4-Me-d,l-tryptophan was from MP Biomedical (Santa Ana, CA), while NOC-5 (3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene) was purchased from EMD Millipore. Nine cyclic dipeptides used in this study were purchased from Chem-Impex Int’l, Inc. Brevianamide F was purchased from Ark Pharm, Inc. Escherichia coli BL21-GOLD (DE3) (Agilent) and BAP1 were used for routine molecular biology studies (Table S4) and protein expression, respectively, and were grown in Luria-Bertani broth or Terrific broth. DNA sequencing was performed at Eurofins. Primers used in this study were listed in Table S5. TB14 used in the study was reported in our previous study.^{[32 (link)]} 1D and 2D NMR spectra of compounds were recorded in CD₃OD on a Bruker 600 MHz spectrometer using 1.5mm High Temperature Superconducting Cryogenic Probe at the University of Florida, Gainesville, FL, USA. Spectroscopy data were collected using Topspin 3.5 software. All ¹H NMR were water suppressed to get rid of high background water peak. HRMS data were obtained using a Thermo Fisher Q Exactive Focus mass spectrometer equipped with an electrospray probe on Universal Ion Max API source.