Deuterium oxide 99.9 d

Liquid
chromatography–mass spectrometry
(LC–MS)-grade solvents (acetonitrile), deuterium oxide 99.9%_D, and additives (formic acid, formic acid-d₂, ammonium acetate, and ammonium acetate-d₇) were obtained from Sigma-Aldrich Srl (Milano, Italy).
Milli-Q water was produced in-house (18.2 MΩ, Sartorius Arium
mini, Sartorius Italy Srl, Varedo, MB, Italy). A sample of Lagrein
wine (2016 harvest) used to develop the SPE method was supplied by
a local winery (Kellerei Bozen, Bolzano, Italy).

The cysteine residue in the N-terminus of peptide P4 was exploited for the binding with the maleimide derivative of the Doxo (Doxo-EMCH). In particular, 600 μL of Doxo-EMCH (2.4 mM) in water/dimethylformamide (1:1 v/v) was mixed gently with 600 μL of peptide P4 (1.2 mM) in PBS using an electromagnetic agitator for 1.5 h at 27 °C in the dark. Doxo-EMCH was added dropwise at 25 μL/min [25 (link),26 (link)]. Then, the crude compound was lyophilized and purified by dialysis with the membrane MWCO Da11000.
The reaction between P4 peptide and Doxo-EMCH was followed by NMR spectroscopy; NMR spectra were recorded on a Varian Unity Inova 600 MHz spectrometer equipped with a cold probe. Spectra were acquired at 298 K on samples at roughly 200 µM concentration and volumes equal to 500 µL in either DMSO (Dimethyl-Sulfoxide-d6, 99.9% D, Sigma-Aldrich, Milan, Italy) and in D₂O (Deuterium Oxide 99.9% D, Sigma-Aldrich, Milan -. 2D [¹H, ¹H] TOCSY (Total Correlation Spectroscopy) [27 (link)] experiments were recorded with mixing times equal to 70 ms with a number of scans ranging from 16 to 64 scans, 128–256 FIDs in t1, 1024 or 2048 data points in t2. NMR spectra were processed with VNMRJ 1.1D (Varian by Agilent Technologies, Milan, Italy); 2D TOCSY spectra were analyzed with the software NEASY [28 (link)] enclosed in CARA (http://www.nmr.ch/) (accessed on 12 April 2022).