Zq 4000

Samples were run on an Acquity UPLC I-Class (Waters, Mildford, MA, USA) using a BEH C18 column (1.7 μm particle size, 2.1 mm × 100 mm) and acetonitrile and water containing 0.1% of trifluoroacetic acid as mobile phase. A gradient was used from 10 to 99% of acetonitrile in 10 min and a flow rate of 0.5 mL/min. For HPLC/MS analysis, a Waters ZQ4000 system was used connected to an HPLC 2695/2795 (An Alliance chromatographic system coupled to a SunFire C18 column (3.5 μm particle size, 2.1 mm × 150 mm) and a 996 PDA detector. Acetonitrile and MQ water + formic acid 0.1% were used as the mobile phase and elution was performed with an isocratic hold with acetonitrile (10%) for 4 min followed by a linear gradient of acetonitrile (10–88%) over 30 min (0.25 mL/min). Mass analysis was performed by ESI (electrospray ionization) in the positive mode with a capillary voltage of 3 kV and cone voltage of 20 kV. The Empower 3.0 program was used to compare and analyse the chromatograms obtained from each sample.

A peptide corresponding to the 21-amino acid residues located at the N-terminal of the mature atTic20 (N-ASKDVPSSFRFPPMTKKPQWW-C) was synthesized by standard solid-phase Fmoc chemistry procedures on Wang resin, as previously described [33 (link)]. Briefly, HCTU, HOBt, and DIPEA in DMF were used to activate the C-terminus of amino acids, and a 20% piperidine solution in DMF was utilized for Fmoc deprotection. The peptide was purified by a Waters 600E reversed-phase high-performance liquid chromatography (RP-HPLC) system. Peptide identification and purity were examined using analytical RP-HPLC and electrospray mass spectrometry (Waters ZQ4000). A Luna C5 (Phenomenex) column was used for both analytical and semi-preparative RP-HPLC experiments. Concentration of the peptide was determined using Trp absorption at 280 nm.

The ¹H NMR spectra, J-resolved, ¹H–¹H homonuclear,
and inverse-detected ¹H–¹³C correlation
experiments were recorded at 25 °C on
a NMR instrument Varian Inova (Milan, Italy), operating at the ¹H frequency of 600 MHz, equipped with an indirect triple resonance
probe. CD₃OD was used as an internal lock for polar extracts
and CDCl₃ for chloroform extracts. For ¹H NMR
profiling, the relaxation delay was 2.0 s, observed pulse 5.80 μs,
number of scans 256, acquisition time 16 min, and spectral width 9595.78
Hz (corresponding to δ 16.0). For the aqueous samples, a presaturation
sequence (PRESAT) was used to suppress the residual H₂O
signal at δ 4.83 (power = −6 dB, presaturation delay
2 s). ESI-MS analyses were performed by the direct injection of MeOH
solutions of the compounds using a Waters ZQ 4000 (Milford, MA USA)
mass spectrometer.

All reagents and solvents were purchased by Sigma Aldrich and used without further purification, unless otherwise stated. Melting points were measured on a Buchi SMP-20 apparatus and are uncorrected. Direct infusion ESI-MS spectra were recorded on Waters ZQ 4000 apparatus. NMR spectra were recorded on Varian VRX 200 and 400 MHz instruments. Chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane (TMS), and spin multiplicities are given as s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), or m (multiplet). The elemental compositions of the compounds agreed to within ±0.4 % of the calculated value. When the elemental analysis is not included, crude compounds were used in the next step without further purification. Chromatographic separations were performed on silica gel (Kieselgel 40, 0.040–0.063 mm; Merck) by flash chromatography. Reactions were followed by thin-layer chromatography (TLC) on glass-backed pre-coated silica gel plates (0.25 mm, 60 F254; Merck), then visualized in an iodine chamber or with a UV lamp. The term “dried” refers to the use of anhydrous Na₂SO₄. Compounds were named following IUPAC rules as applied by ChemBioDraw Ultra 11.0.

¹H NMR spectra, J-resolved (J-res), ¹H-¹H homonuclear, and inverse detected ¹H-¹³C correlation experiments were recorded at 25 °C on a Varian Inova 600 MHz NMR instrument (600 MHz operating at the ¹H frequency) equipped with an indirect triple resonance probe. CD₃OD was used for an internal lock. For ¹H NMR profiling, the relaxation delay was 2.0 s, observed pulse 5.80 µs, number of scans 256, acquisition time 16 min, and spectral width 9595.78 Hz (corresponding to δ 16.0). For the aqueous samples, a presaturation sequence (PRESAT) was used to suppress the residual H₂O signal at δ 4.83 (power = −6dB, presaturation delay 2 s).
ESI-MS analyses were performed by direct injection of MeOH solutions of the compounds using a WATERS ZQ 4000 (Milford, MA, USA) mass spectrometer.

The reagents used, unless stated otherwise, were purchased from Sigma-Aldrich (Milan, Italy). CH₂Cl₂ was anhydrified by distillation over P₂O₅. Chromatographic purifications (FC) were carried out on glass columns packed with silica gel (Merck grade 9385, 230–400 mesh particle size, 60 Å pore size) at medium pressure. Thin-layer chromatography (TLC) was performed on silica gel 60 F254-coated aluminum foils (Fluka, Buchs, Switzerland). The spots related to 9-methoxy-9-oxononanoic acid were revealed using a bromocresol green solution (6% in ethanol).
The nuclear magnetic resonance spectra were recorded at 25 °C on Varian spectrometers Mercury 400 or Inova 600 (Varian, Palo Alto, CA, USA) operating at 400 or 600 MHz (for ¹H-NMR) and 100.56 or 150.80 MHz (for ¹³C-NMR), respectively. Signal multiplicities were established by DEPT-135 experiments. Chemical shifts were measured in δ (ppm) with reference to the solvent (δ= 7.26 ppm and 77.00 ppm for CDCl₃, for ¹H- and ¹³C-NMR, respectively). J-values are given in Hz. Electrospray ionization (ESI)-MS and ESI high-resolution (HR)MS spectra were recorded using a Waters ZQ 4000 and Xevo instrument, respectively. Melting points (m.p.) were measured on a Büchi 535 apparatus (Flawil, Zwitzerland) and are uncorrected.

Commercial reagents were used without additional purification. Melting points were tested with an Electrothermal IA9100 (Electrothermal, Staffordshire, UK) micro-melting point apparatus and were uncorrected. NMR spectra were performed with a Varian Unity 400 MHz spectrometer using tetramethylsilane (TMS, Merck, Darmstadt, Germany) as an internal standard and DMSO-d₆ as solvent. Chemical shifts were described as (ppm). Splitting patterns are presented as follows: s = singlet; d = doublet; t = triplet; q = quartet; dd = double doublet; m = multiplet. Analytical thin-layer chromatography (TLC) was carried out by an Art. 5554 Kieselgel 60 GF254 (Merck, Darmstadt, Germany) made by E. Merck. The spots of compounds were examined with a UV light indicator irradiated at 254 and 366 nm. Art. 7734 Kieselgel 60 GF254 (70–400 mesh, Merck, Darmstadt, Germany) produced by E. Merck was used for preparing column chromatography. Waters ZQ-4000 (Waters, Milford, MA, USA) liquid chromatography electrospray ionization mass spectrometry was used to record mass spectra.