Cyanamide

Phenol (99 + %), NaOH (97 + %), conc. HCl (37%), poly(N,N-diallyl -N,N-dimethylammonium) chloride (PDDA-Cl, M_n = 400–500 kg mol^–1, 20% w/w in H₂O), Ludox HS40 silica colloid (40% w/w in H₂O), 1,3-dioxlane (DOL, 99.8%), cyanamide (99%), 1,2-dimethoxymethane (DME, 99.5%), lithium nitrate (LiNO₃, 99.99%), and lithium bis(trifluoromethanesulfonimide) (LiTFSI, 99.95%) were obtained from Sigma-Aldrich. Formaldehyde solution (37% w/w in H₂O), lithium metal strip (0.75 mm thick, 99.9%), and sulfur (99.5%) were obtained from Alfa Aesar. Hydrofluoric acid (48%) was obtained from Acros Organics. Ethanol (88.5–92.5% v/v) was obtained from Macron Fine chemicals. CNF (>98%) was purchased from Sigma-Aldrich.

Animals used in the study were 6–8 weeks old male and female albino MITO-Luc mice [27 (link)] gathered in groups of 4 to 8 animals each. Gavage administration of a solution of ethanol (dose range 3–6 g/kg body weight) in 0.9% saline in a total volume of 150 μl-solution per 10 g of body weight was performed. According to previous reports, this dose range corresponds to a binge-like drinking pattern in humans [32 (link)]. Randomly selected animals received intra-peritoneal (i.p.) administration of the ALDH activator ALDA-1 (5 mg/kg) [33 (link)] or the ALDH inhibitor cyanamide (25 mg/kg) [34 (link)] (Sigma Aldrich, St. Louis, MO, USA) 30 min before ethanol treatment. Similarly, a group of animals received i.p. a solution containing polyphenols (50 mg/kg) (Phenolea Active Complex, kindly provided by Phenofarm, Rieti, Italy) [35 (link)] 2 h before alcohol administration. Methanol was administered (1.5 g/kg body weight) in 0.9% saline solution in a total volume of 150 μL per 10 g of body weight [36 (link)]. Experimental procedures for assessment of cell proliferation in the MITO-Luc mouse model conformed to Animal Care guidelines (D.lgs 26/2014, 4 March 2014).

The synthesis of Fe-N-C was conducted according to the literature [7 (link),18 (link),33 (link),34 (link)]. For the synthesis of Fe-N-C materials, 100 mg of carbon support was impregnated with a mixture of 16.25 mg iron(II) acetate (≥99.99%) and 421 mg of cyanamide (99%), both purchased from Sigma Aldrich (Darmstadt, Germany) in ethanol (Carl Roth) and mixed in a sonication bath until complete evaporation of the solvent and dried in vacuum oven at 30 °C overnight. Pyrolysis was carried out for 1 h at 900 °C at a heating rate of 5 °C/min and 100 L/h of nitrogen flow in a ceramic tube furnace followed by acid leaching with 2 mol/L of H₂SO₄ purchased from Carl Roth for 16 h at 90 °C. After washing the catalyst powder until neutral pH and drying, a second pyrolysis step similar to the first one was performed.

The procedure of coating beads was adopted from Yates et al.^{29 (link)} Carboxylate-modified silica particles (25 mg or 500 μl of 3 μm; Kisker Biotech, Steinfurt, Germany) were washed three times in 1 ml of PBS by vortexing and centrifugation at 2000 g for 1 min. Beads were incubated at room temperature in 25 mg ml⁻¹ cyanamide (Sigma-Aldrich), which works as a cross-linker, in PBS with agitation for 15 min. Beads were washed twice in 1 ml of coupling buffer (0.1 m borate buffer, pH 8.0) and then incubated in 0.5 ml coupling buffer with 1.0 mg defatted bovine serum albumin (Sigma-Aldrich) and 0.1 mg human IgG (Molecular Probes, Eugene, OR, USA) or 0.25 mg ligands (TDM (Enzo Life Sciences) or β-glucan/whole glucan particles (Invivogen, San Diego, CA, USA)) then dispersed for 12 h with agitation. The coated beads were washed three times in 1 ml of quench buffer (250 mM glycine, Sigma-Aldrich) to quench unreacted cyanamide. The beads were re-suspended in 1 ml coupling buffer with 10 μl of 5 mg ml⁻¹ Alexa Fluor 594 succidinimyl ester (Molecular Probes) in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and agitated for 1 h. The beads were washed three times in 1 ml quench buffer, re-suspended in 1 ml PBS with 0.02% sodium azide and stored at 4 °C.

All experiments were conducted in 18 MΩ water processed with a Milli-Q purification system. ¹³C-labelled formaldehyde, dinitrophenylhydrazine (DNPH), glycolaldehyde, paraformaldehyde, d-xylose, d-erythrose, cyanamide, glucose monohydrate, dansyl chloride (DsCl), sodium cyanide, and calcium acetate monohydrate (Ca(OAc)₂·H₂O) were purchased from Sigma-Aldrich. ¹³C-labelled glycolaldehyde was purchased from ChemCruz. 2-Aminooxazole (2-NH₂Ox) was purchased from Combi-Blocks. HPLC-grade acetonitrile was purchased from VWR International Pty Ltd. LCMS-grade acetonitrile and water were purchased from Chem-Supply Pty Ltd. All reagents were used without further purification. 37% formaldehyde stock solutions and 1 M pH 6.7 sodium phosphate buffer were prepared according to established protocols,^{86,87 (link)} and the formose stock was subsequently diluted to 1 M with Milli-Q water. DNPH was dissolved in acetonitrile (ACN) at 3 mg mL⁻¹. Stock solutions of organic compounds were stored at 5 °C and were made fresh after one week. Arabino-, ribo-, and xylo-aminooxazoline were synthesised according to literature procedures.^{50 (link)}

Materials for synthesis and reagents for sonochemical reaction catalytic tests (set up scheme shown in Scheme 1) with 4-hydroxy-3,4-dimethoxybenzylalcohol were purchased from ThermoFisher (Waltham, MA, USA). LUDOX HS-40 colloidal silica (40 wt% suspension in water) and cyanamide (99%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Sucrose and sodium hydroxide were purchased from POCH (Gliwice, Poland). D-glucosamine hydrochloride (≥98%) and low-molecular-weight chitosan (100–300 cps) were purchased from Pol-Aura. Acetic acid (99.5%) was purchased from ChemPur (Karlsruhe, Germany). Milli-Q water was purified using a Millipore Milli-Q lab water system (Burlington, MA, USA). The carbonaceous materials were characterized by nitrogen physisorption, thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FTIR). The equipment used and details of the analysis are shown in the Supporting Information.