Isolera instrument

SG-17, an MMP-sensitive lipopeptide (SKK(stearate)SGPLGIAGQSK(stearate)KS), was prepared by microwave (MW)-assisted Fmoc solid-phase peptide synthesis (Fmoc-SPPS) on Wang resin (loading between 0.2–0.4 mmol/g), using a CEM Liberty Blue synthesizer. Coupling reactions were performed with amino acids (5 eqs., 0.2 M in DMF), using diisopropyl carbodiimide (DIC, 0.5 M in DMF) and Oxyma Pure (1 M in DMF) as coupling reagents. The MW synthesis cycle entailed 15 s at 75 °C–170 W, followed by 110 s at 90 °C–40 W. N-Fmoc deprotection was performed using 20% piperidine in DMF with a MW cycle entailing 15 s at 75 °C–155 W, followed by 60 s at 90 °C–50 W. Final cleavage in solution entailed shaking the resin for 3 h in a 90:2.5:2.5:5 TFA/TIPS/H₂O/phenol mixture. The lipopeptide was then purified in reverse-phase chromatography with a Biotage Isolera instrument equipped with a C18 column. 0.1% TFA in water was used as phase A and 1% TFA in ACN as phase B, going from 20 to 100% phase B in 18 min (Rt = 16.6 min for MSLP-1). Electrospray ionization high-resolution time-of-flight mass spectrometry (ESI-HR TOF-MS) on a Q-TOF Synapt G2-Si yielded m/z 1503.7206 [M+2H]²⁺, 702.8170 [M+3H]³⁺ values for the pure lipopeptide.

For most general experimental techniques and procedures, see reference [80 (link)]; ¹H-NMR and ¹³C-NMR chemical shifts (δ, ppm) are relative to signals of residual CHD₂OD in CD₃OD δ_H 3.27 (central line of a quintuplet), ¹³CD₃OD [δ_C 49.0 (central line of a septuplet)], and ¹³C-4 of C₅D₅N [δ_C 134.3 (central line of a triplet)], respectively. All the NMR experiments were performed on a Bruker AV300 spectrometer, at 300 (¹H) and 75.47 MHz (¹³C), respectively. Deuterated solvents (purity 99.8%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). ESI-MS experiments were carried out on a Thermo-TSQ mass spectrometer, by flow injection analysis (FIA), with the electron-spray ionization source (ESI) at 5 kV on TIP capillary. Spectroscopy grade solvents (Sigma-Aldrich) were used. Preparative medium-pressure liquid chromatographic (MPLC) separations were carried out on a Biotage Isolera instrument (Biotage, Uppsala, Sweden).