The general procedure for the synthesis of N-biotinocaproic acid from 2,5-dioxopyrrolidin-1-yl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate and 6-aminohexanoic acid is as follows: ε-aminohexanoic acid (966 mg, 8.4 mmol) was dissolved in 30 mL of 1 M NaHCO3 aqueous solution . Subsequently, N-hydroxysuccinimide biotin (3.09 g) dissolved in 35 mL of DMF was slowly added dropwise to this solution and the reaction mixture was stirred at room temperature for 16 hours. Upon completion of the reaction, some of the solvent was removed by concentration under reduced pressure, then 150 mL of aqueous citric acid (100 g/L) was added and stirred at 60 °C for 30 min. The precipitate was collected by filtration and washed with distilled water. The resulting precipitate was dissolved in a solvent mixture of isopropanol and water (8:2, v/v) and stored at 4 °C to finally obtain pure N-biotin hexanoic acid (1.455 g, 50% yield) as pale yellow crystals. The product was detected by IR (KBr) showing characteristic absorption peaks: 3200-3500, 3070, 2932, 2859, 1696, 1644, 1543, 1475, 1391, 1324, 1265, 1118 cm-1. 1H NMR (600 MHz, DMSO-d6) data: δ 7.75 (1H, t, J = 5.4 Hz, C6-NH), 6.45 (1H, s, C13-NH), 6.38 (1H, s, C14-NH), 4.35-4.27 (1H, m, H-14), 4.12 (1H, m, H-13), 3.09 (1H, m, H-12), 3.00 (1H, m, H-6), 2.82 (1H, dd, J = 12.4, 5.1 Hz, H-15a), 2.57 (1H, d, J = 12.4 Hz, H-15b), 2.19 (2H, t, J = 7.4 Hz, H-2), 2.04 (2H, t, J = 7.3 Hz, H-8), 1.20-1.67 (14H, m).13C NMR (150 MHz, DMSO-d6) data: δ 171.84 (C-1, C-7), 162.74 (C-16), 61.06 (C-13), 59.20 (C-14), 55.44 (C-12), 39.68 (C-15), 38.25 (C-6), 35.21 (C-8), 29.05 (C-2), 28.92, 28.21, 28.04, 26.04, 25.73, 25.36.
