1100 lc msd

Liquid chromatography-mass spectrometry (LC/MS), NMR spectroscopy, and elemental analysis methods were applied to confirm the structure and purity of all synthesised compounds.
LC/MS analysis was performed on an 1100 LC (Agilent Technologies) with ELSD, UV (DAD 200–400 nm), and mass detection (1100 LCMSD, Agilent Technologies, APCI, and ES positive ionization). The most used column was the Onix C18 50 × 4.6 mm; eluent 1–0.1% TFA in water; eluent 2–0.1% TFA in acetonitrile, gradient—eluent 1–2.9 min, eluent 2–0.2 min, eluent 1—rinsing, flow rate—3.75 mL/min.
The structures of key target final compounds were unambiguously confirmed by 1H, 13C{1H},13C apt, COSY, HSQC, and NOESY NMR spectroscopy. For structure confirmation of intermediates, 1H NMR spectroscopy was applied, and in some cases, if it was necessary, 13C{1H} NMR spectra were also registered. NMR spectra were registered on spectrometers Bruker DRX 400 (400.13 MHz for protons, 100.61 MHz for carbons, 376.50 MHz for fluorine), Bruker Avance II+ 600 (600.11 MHz for protons, 150.93 MHz for carbons), Avance IIIHD 500 (500.13 MHz for protons, 125.78Mhz for carbons), and spectrometer Bruker Avance III 400 UltraShield Plus (400 MHz). CDCl3 and DMSO-d6 were used as solvents.
Elemental analysis was performed on Vario MICRO cube CHNS analyser (Elementar Analysensysteme GmbH, Hanau, Germany).

The details of the preparation of benzofulvene derivatives and their spontaneous polymerization are described in (ESI†). NMR spectra were recorded with either a Bruker DRX-400 AVANCE or Bruker DRX-500 AVANCE spectrometer in the indicated solvents (TMS as internal standard): the values of the chemical shifts are expressed in ppm and the coupling constants (J) in Hz. An Agilent 1100 LC/MSD operating with an electrospray source was used in mass spectrometry experiments.

UV spectra were acquired with a Cary 300 spectrometer. IR spectra were obtained using a Nicolet 5700FTIR microscope spectrometer. Analytical HPLC was conducted on an Agilent system with a 1260 Quat-Pump and DAD detector. For semi-preparative HPLC, a reverse-phase C₁₈ column (Spursil 5μm C₁₈ column: 250 × 10.0 mm) was used with MeCN-H₂O as a solvent system. LC-MS was performed on a 1100–6410 Triple Quad from Agilent or an Agilent 1100 LC/MSD with a G1946D single quadrupole mass spectrometer. High-resolution mass spectrometry was carried out on a XEVO G2-XS QTof from Waters. NMR data were collected using a Bruker-600 or an ADVANCE HD 800 MHz and a Bruker Avance Ⅲ HD 700 MHz spectrometer, where chemical shifts (δ) were reported in ppm and referenced to DMSO-d₆ solvent signal (δ_H 2.49 and δ_C 39.5), CDCl₃ solvent signal (δ_H 7.26 and δ_C 77.0) and acetone-d₆ solvent signal (δ_H 2.04 and δ_C 206.0). 3T3-L1 fibroblast cell (pre-adipocyte) line was purchased from the Cell Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences.

Melting points were recorded on a Mel-Temp melting point apparatus and are uncorrected. Unless otherwise stated, all the materials were obtained from the commercial suppliers and are used without further purification. Chromatography was carried on silica gel (100–200 mesh). All the reactions were monitored by thin-layer chromatography and the spots were visualized under UV light. The ¹H and ¹³C NMR spectra were recorded on Bruker FT-NMR spectrometer operating at 400 MHz for ¹H and 100 MHz for ¹³C using TMS as an internal standard. Chemical shifts are expressed in δ (ppm) and coupling constants J in Hertz (Hz). Mass spectra were recorded on an Agilent 1100 LC/MSD.

The details of the synthesis and characterization of compounds 3a–h and their spontaneous polymerization are described in Supporting Information. The synthetic intermediates and the solvents were purchased from Sigma-Aldrich (Saint Louis, MO, USA) unless otherwise stated. NMR spectra were recorded with a Bruker (Karlsruhe, Germany) AC200, a Varian (Palo Alto, CA, USA) Mercury-300, a Bruker DRX-400 AVANCE, or a Bruker DRX-600 AVANCE spectrometer in the indicated solvents (TMS as internal standard). The values of the chemical shifts are expressed in ppm and the coupling constants (J) in Hz. An Agilent (Santa Clara, CA, USA) 1100 LC/MSD operating with an electrospray source was used in mass spectrometry experiments.

This peptide was synthesised manually by coupling resin 12 (0.14 mmol) scale with Fmoc-Ala-OH followed by Z-Val-OH using standard solid phase Fmoc procedures. Prior to each coupling the Fmoc was removed by treating the resin with (3 × 10 mL) of 20% piperidine in DMF for 10 min. HATU (4.5 equiv.) and DIPEA (10 equiv.) were used as the coupling reagents with 5-fold excess of each amino acid. Cleavage was performed by treatment of the peptide-resin with an ice-cold solution of (95% TFA, 2.5% H₂O, 2.5% TIPS) for 30 min. Following filtration to remove the linker retaining resin, ether was added to the filtrate. The peptide was ether soluble so following evaporation of ether the peptide was solubilised in water and acetonitrile prior to lyophilisation. Aliquots of the peptide were solubilised in a (2 : 1 : 1) mixture of H₂O–MeCN–MeOH (2 mL) and purified on a C8 reverse phase HPLC column (Agilent PrepHT Zorbax 300SB-C8, 21.2 × 250 mm, 7 m) using a linear solvent gradient of 15–60% (MeCN + 0.08% TFA) and (H₂O + 0.08% TFA) over 40 min at a flow rate of 8 mL min⁻¹. Fractions containing the product were combined and lyophilised to dryness to give peptide 13 (4.4 mg, 6.7%). The purified peptide was analysed by LCMS on an Agilent 1100 LC-MSD; estimated purity 65%; MS (ES⁺) m/z: 468.0 [M + H]⁺, 489.9 [M + Na]⁺; calcd for C₂₂H₃₀N₃FO₇: 467.2.