Peptide synthesis and purification

SHA was synthesized by Wuxi AppTech. All peptides were synthesized using standard Fmoc solid phase peptide synthesis protocols using a CEM Liberty Blue peptide synthesizer with microwave-assisted coupling and deprotection steps. Peptides containing a l-aspartate or l-glutamate were synthesized on a preloaded Fmoc-L-Asp(Wang resin LL)-ODmab or Fmoc-L-Glu(Wang resin LL)-ODmab resin, where the acidic side-chain of the amino acid is tethered to the resin, and were cyclized on-bead by a standard coupling reaction following deprotection of the C-terminal -ODmab protecting group with 2% (v/v) hydrazine monohydrate in dimethylformamide (DMF). All other peptides were synthesized with the C-terminus tethered to Cl-TCP(Cl) ProTide resin from CEM, cleaved from the resin with 1% (v/v) TFA in dichloromethane (DCM), and cyclized by a solution-phase coupling reaction prior to the final total deprotection. Crude peptides were purified based on mass via reverse phase HPLC using a Waters AutoPurify HPLC/MS system in line with a SQD2 mass spectrometer. Peptides were typically purified via a water (0.1% formic acid) and acetonitrile (0.085% formic acid) gradient at 2%/min on an XBridge Prep C18 10 um, 19 × 150 mm column. Masses and purities were assessed via electrospray ionization mass spectrometry during and subsequent to purification on an SQD2 mass spectrometer. Due to epimerization during cyclization, for some peptides two major peaks with correct mass was obtained. For such peptides, both peaks were collected and tested. LC/MS data for the peptides for which HDAC inhibition assays were performed are shown in Supplementary Fig. ¹². Source Data are provided as Supplementary Data ². Sequences of all peptides tested are provided in the Supplementary Data file ².