Chloroacetic acid

A single ant was quickly frozen using liquid N₂. The brain of a frozen ant was dissected out in the ice-cold ant saline (128.3 mM NaCl, 4.7 mM KCl, 1.63 mM CaCl₂, 6 mM NaHCO₃, 0.32 mM NaH₂PO_4, 82.8 mM trehalose, pH 7.4). A single brain of a queen was collected into a micro glass homogenizer and homogenized in 50 µl of ice-cold 0.1 M perchloric acid containing 5 ng of 3, 4-dihydroxybenzylamine (DHBA, SIGMA, St Louis, MO, USA) as an internal standard. After centrifugation of the homogenate (0°C, 15000 rpm, 30 min), 40 µl of supernatant was collected. Biogenic amine in the brain was measured using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD). The HPLC-ECD system was composed of a pump (EP-300, EICOM Co., Kyoto, Japan), an auto-sample injector (M-504, EICOM Co., Kyoto, Japan) and a C18 reversed-phase column (250 mm×4.6 mm internal diameter, 5 µm average particle size, CAPCELL PAK C18MG, Shiseido, Tokyo, Japan) heated to 30°C in the column oven. A glass carbon electrode (WE-GC, EICOM Co.) was used for electrochemical detection (ECD-100, EICOM Co.). The detector potential was set at 890 mV versus an Ag/AgCl reference electrode, which was also maintained at 30°C in a column oven. The mobile phase containing 0.18 M chloroacetic acid and 16 µM disodium EDTA was adjusted to pH 3.6 with NaOH. Sodium-1-octanesulfonate at 1.85 mM as an ion-pair reagent and CH₃CN at 8.40% (v/v) as an organic modifier were added into the mobile phase solution. The flow rate was kept at 0.7 ml/min. The chromatographs were acquired using the computer program PowerChrom (eDAQ Pty Ltd, Denistone East, NSW, Australia). The supernatants of samples were injected directly onto the HPLC column. After the acquisition, they were processed to obtain the level of biogenic amines in the same sample by the ratio of the peak area of substances to the internal standard DHBA. We used a standard mixture for quantitative determination that contained amines, precursors and metabolites. Twenty compounds at 100 ng/ml each were DL-3, 4-Dihydroxy mandelic acid (DOMA), L-β-3,4-Dihydroxyphenylalanine (DOPA), L-Tyrosin (Tyr), N-acetyloctopamine (Nac-OA), (−)-noradrenaline (NA), 5-Hydroxy-L-tryptophan (5HTP), (−)-adrenaline (A), DL-Octopamine (OA), 3,4-Dihydroxybenzylamine (DHBA, as an internal standard), 3,4-Dihydroxy phenylacetic acid (DOPAC), N-acetyldopamine (Nac-DA), 3,4-Dihydroxyphenethylamine (DA), 5-Hydroxyindole-3-acetic acid (5HIAA), N-acetyltyramine (Nac-TA), N-Acetyl-5-hydroxytryptamine (Nac-5HT), Tyramine (TA), L-Tryptophan (Trp), 3-Methoxytyramine (3MTA), 5-Hydroxytryptamine (5HT), 6-Hydroxymelatonin (6HM). Nac-OA Nac-DA and Nac-TA were synthesized by Dr. Matsuo (Keio University, Japan). All other substances were purchased from SIGMA.
Differences in the levels of biogenic amines were tested using Student’s t-test (P<0.05).

GO sheets were exfoliated from natural graphite according to the modified Hummers’ method12 29 . GO-COOH was synthesized under strongly basic condition to convert hydroxyl groups to carboxylic acid moieties by activating the GO sample with chloroacetic acid (ClCH₂CO₂H)12 . In a typical process, 1.2 g of chloroacetic acid, 1 g of NaOH and 50 mg of GO were added to 100 mL DI water and then sonicated for 3 h to speed up the elimination of sodium chloride. The resulting GO-COOH solution was neutralized by HNO₃, and purified with acetone and water for several times.
For the synthesis of GO-COOH-CuS nanocomposite, we herein developed a facile and scalable method to anchor CuS nanoparticles (NPs) on large GO-COOH sheets at room temperature. Briefly, various amounts (1 mL, 5 mL, 10 mL and 20 mL) of 1 mg mL⁻¹ GO-COOH solution were mixed with 1 mmol of Cu(NO₃)₂, 1 mmol of C₂H₅NS and 10 mL of DMSO. DMSO acted as a solvent and co-reactant to furnish the sulfur source in the form of H₂S. The mixture was stirred for 24 h at ambient temperature to ensure CuS NPs anchored on GO-COOH sheets. The products were centrifuged and rinsed with acetone, ethanol and DI water several times to remove the residual. The resulting solid samples were freeze-dried for 2 h under −50 °C to obtain the final GO-COOH-CuS catalysts. Here, the samples prepared by addition of various amounts GO-COOH solutions (1 mL, 5 mL, 10 mL and 20 mL) were named as GO-COOH-CuS-1, GO-COOH-CuS-5, GO-COOH-CuS-10 and GO-COOH-CuS-20, respectively. The synthesis process is illustrated in Fig. 13.

Compounds 1–23 were synthesized and characterized as published in Mendes et al. [18 (link)]. Briefly, a series of substituted aromatic isocyanides, benzaldehydes, and aniline derivatives were reacted with chloroacetic acid through Ugi four-component reactions to produce a series of the corresponding Ugi adducts [17 (link)]. Such adducts were subsequently subjected to cyclization through intramolecular N-alkylation to produce the corresponding 2,5-DKPs. As depicted in the scheme of Table 1, 2,5-Diketopiperazines were obtained from substituted aromatic isocyanides, benzaldehydes, aniline derivatives, and chloroacetic acid through intramolecular N-alkylation of Ugi adducts. The chemical structures of all 2,5-DKPs were undoubtedly confirmed by spectrometric, spectroscopic, and crystallography experiments. All data (FT-IR-ATR, 1 D and 2 D NMR, IEMS, HRMS, and X-ray crystallographic data) are presented and discussed in [18 (link)]. For activity tests in bioassays, the compounds were dissolved in DMSO at concentrations of 20 mg/mL, and their identity and purity were confirmed by UHPLC-HRMS.