Recombinant Macrocyclic Lanthipeptides Incorporating Non-Canonical Amino Acids (original) (raw)

2017, Journal of the American Chemical Society

Materials: E. coli DH10B was used for general molecular biology purposes. LB and TB medium were purchased from Corning, LB agar purchased from Fisher Scientific, Isopropyl-ß-thiogalactoside (IPTG) was purchased from Anatrace, 4-12% (wt/vol) Bis-Tris gels for SDS-PAGE were purchased from Invitrogen, Q5 DNA Site-Directed Mutagenesis Kit, restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (NEB) and oligonucleotides were purchased from Integrated DNA Technologies (San Diego, CA). Plasmid DNA preparation was carried out with QIAprep Spin Miniprep Kit (Quiagen). Unless otherwise mentioned, all chemicals and solvents were purchased from Sigma-Aldrich and used without further purification. Peptide concentrations were measured using BCA assay from Pierce. LCMS conditions for the analysis of Nisin analogs: Agilent 6520 accurate-mass quadrupole-time-of-flight (QTOF) instrument was used to carry out high-resolution mass spectrometry for all peptide samples, which was equipped with reverse phase liquid chromatography and an electrospray ionization (ESI) source. Samples in distilled deionized water were injected (10-15 µL) at a concentration of 0.1 mg/mL and separated on a 150 mm reverse phase C8 wide pore column heated to 70 °C to improve peak resolution (Phenomenex, Aeris WIDEPORE XBC8, LC column 150 x 2.1 mm). Peptides were eluted in a gradient of H 2 O + 0.1% formic acid (solvent A) and acetonitrile + 0.1% formic acid (solvent B) using the following method: 5% B for 2 min, 5-60% B for 10 min, 60-80% B for 1 min, followed by a wash (95% B) and re-equilibration (5% A) phase. ESI source settings were 350 °C, 10 L/min, 40 psig nebulizer nitrogen gas, 200 V fragmentor and 4,500 V capillary. Figures were created by extraction of the total ion count and deconvolution of charge envelopes using Agilent Qualitative Analysis software with BioConfirm. NMR for Nisin analogs: 1 H/ 13 C NMR spectra were recorded at ambient temperature on Bruker DMX 400 (400 MHz for 1 H NMR and 100 MHz for 13 C NMR) and DRX 600 CryoProbe (600 MHz for 1 H NMR and 150 MHz for 13 C NMR) instruments in the specified deuterated solvents. Chemicals shifts are given in ppm with respect to residual undeuterated solvent signal as internal standard. Coupling constants are reported as J-values in Hz. The following abbreviations were used to explain S2 multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet, m = multiplet, br = broad. Chemical synthesis 1 H and 13 C-NMR spectrum of 13 C-labelled (1): Chloroacetamide-containing ncAA and its 13 C-labelled version were prepared as follows: Boc-4-Amino-L-Phenylalnine (purchased by Ark chemicals) (2g, 7.1 mmol, 1 eq) and NaHCO 3 (3g, 35 mmol, 5 eq) were suspended in anhydrous THF (50 mL) and brought to 0 °C using an icewater bath. Subsequently, chloroacetyl chloride or chloroacetyl chloride-2-13 C for isotope labeling experiment (3.5 mL, 43.74 mmol, 6.25 eq) in anhydrous THF (17.5 mL) was added dropwise, and the reaction stirred overnight shielded from light using aluminum wrap. LC-MS analysis showed full conversion to the desired product, at this point, 4% NaHCO 3 (aq) (10 mL) was added, and the mixture extracted with EtOAc (30 mL 3X). Organic layers were pooled, dried over Na 2 SO 4 , filtered and dried in vacuo. The residue was then taken up in CH 2 Cl 2 (15 mL), brought to 0 °C and then treated with anhydrous TFA (3 mL). Upon complete Boc deprotection, as observed by LC-MS, the reaction mixture was dried in vacuo, and the residue taken up in 1:1 H 2 O-CH 3 CN and freeze-dried to obtain a white powder in 70-85% yield. Products were used without any further purification. 13 C-labelled chloroacetamide 1, was prepared as described previously but using chloroacetyl chloride-2-13 C, purchased from Sigma-Aldrich (CAS# 604097).