Agmatine sulfate

Agmatine sulfate (500 mg, 2.2 mmol, Sigma-Aldrich, > 97%) was dissolved in 2.5 ml deionized water. Sodium hydroxide (175 mg, 4.4 mmol, Sigma-Aldrich, 98.6%) was dissolved separately in 2.5 ml deionized water and allowed to cool back to room temperature (ca. 20°C). Half of the aqueous Sodium hydroxide was added to the Agmatine sulfate solution followed by powered cinnamoyl chloride (366 mg, 2.2 mmol, Sigma-Aldrich, 98%) and stirred with a magnetic ‘flea’. The pH was maintained at around 9 to 10 by further dropwise addition of aqueous Sodium hydroxide and when no further change in pH occurred, indicating complete reaction, the mixture was left stirring for an hour. The product was isolated by reverse phase vacuum column chromatography as described above and eluted in the 20% aqueous methanol fraction. This yielded 44 mg of E-cinnamoylagmatine (8% yield). Unchanged agmatine was recovered from a 2% aqueous methanol fraction, dried down and re-cycled through the synthesis. An aliquot (20 mg = 0.077 mmol) of the E-isomer was dissolved in 2 ml aqueous methanol and exposed to UV light (254 nm) for 72 h. The resulting mixture contained Z and E-isomers in the proportion 1:2 which were separated using HPLC as described above. Both the E and Z synthetic isomers were found to be identical with the compounds isolated from the dried plant by HR-ESI-MS and NMR.

Biogenic amines standards methylamine hydrochloride 99% (MA), ethylamine hydrochloride 98%(EA), dimethylamine hydrochloride 99% (DMA), diethylamine hydrochloride 99% (DEA), propylamine hydrochloride 99% (PA), butylamine 99% (BA), 2-phenylethylamine hydrochloride 98% (PHA), tyramine hydrochloride 98% (TYR), tryptamine hydrochloride 99% (TRP), histamine dihydrochloride 99% (HIS), hexylamine 99%(HEA), isopentylamine 99% (isoPA), putrescine dihydrochloride 98% (PUT), cadaverine dihydrochloride 98% (CAD),spermidine trihydrochloride 99.5%(SPD), spermine tetrahydrochloride 99.5% (SPE), agmatine sulfate 98% (AGM) were obtained from Sigma Aldrich (St. Louis, MO, USA). The internal standard (IS) was 1.7-diamoheptane 98% (DAH) purchased from Sigma Aldrich (St. Louis, MO, USA). Formic acid (FA) was obtained from Merck (Darmstadt, Germany). Boric acid and sodium hydroxide were purchased from POCH (Gliwice, Poland). Acetonitrile (ACN) with LC-MS grade, ammonium formate and tosyl chloride (≥99%) were obtained from Sigma Aldrich (St. Louis, MO, USA). Nylon Captiva Econofilters (25 mm diameter, 0.2 µm pore size) were purchased from Agilent Technologies (Santa Clara, CA, USA). Ultrapure water was produced by the HLP5 system from Hydrolab (Wiślina, Poland).

All chemicals, unless otherwise stated, were of the highest quality and were used as supplied. All the standard biogenic amines (putrescine dihydrochloride, agmatine sulfate, histamine dihydrochloride, 2-phenylethylamine, tyramine hydrochloride, spermine tetrahydrochloride, tryptamine hydrochloride, spermidine trihydrochloride and cadaverine dihydrochloride) were purchased from Sigma-Aldrich (St. Louis, MO, USA). High-performance liquid chromatography (HPLC) grade ammonium acetate and acetonitrile were purchased from Merck (Damstadt, Germany).