Compound 7
2-aminothiazole
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Synthetic procedure: See article for the definitive version of this procedure and for full experimental details.
Formation of 2-aminothiazole (7) in a complex aldehyde/ketone mixture: to a solution of glycolaldehyde (2a), formaldehyde (2d), acetaldehyde (2e), propanal (2f), isobutyraldehyde (2h), 2-methylbutyraldehyde (2i), 3-methylbutyraldehyde (2j), methional (2m), acetone (2o), monohydroxyacetone (2p), dihydroxyacetone (2q), 2-butanone (2r), 2-pentanone (2s), 3-methyl-2-butanone (2t), 3-methyl-2-pentanone (2u), 2-hexanone (2v), L-erythrulose (L-2w), D-sorbose, D-fructose, D-glucose, D-galactose, D-mannose, D-ribose, L-arabinose, D-lyxose, D-xylose, and β-mercaptoacetaldehyde (9a) (all 27 componenents 50mM) in sodium phosphate buffer (0.25M, pH 7, 2 mL) was added cyanamide (3; 4.2 mg, 0.1 mmol) and the reaction mixture was stirred overnight at room temperature. 1H NMR spectra were acquired and 3 major 7–derived species (7 and two 7–(hemi)aminals; Supplementary Figure 21) were observed. The reaction mixture was acidified with DCl (4M) to pD 3, and 1H NMR spectra acquired. Signal amplification for 2-aminothiazole (7) and 7–aminals hydrolysis were observed. No signals corresponding to 2-aminooxazole (4) from the reaction of 9a and cyanamide (3) were observed. 2-aminothiazole (7) was the major aromatic signal at pD 3 (Supplementary Figure 22). Quantification by 1H NMR spectroscopy indicates that 7 was formed in >95 % yield. Aminooxazoles or aminooxazolines were not observed. Data for 2-aminothiazole (7): 1H NMR (600 MHz, D2O, pD 3) δ 6.84 (d, J = 4.5 Hz, 1H, (C4)–H), 6.52 (d, J = 4.5 Hz, 1H, (C5)–H).
Formation of 2-aminothiazole (7) from β-mercaptoacetaldehyde (9a) in the presence of H2S (9b): to a suspension of β-mercaptoacetaldehyde (9a; 7.6mg, 0.1 mmol) and sodium hydrosulfide hydrate (assumed min. 50%, 1-5 equiv.) in sodium phosphate buffer (0.5M, pH 7, 1 mL) was added cyanamide (3; 4.2 mg, 0.1 mmol) and the reaction mixture was stirred overnight at room temperature. The reaction mixture was then diluted with DMSO (5 mL), an aliquot (100 µL) was further diluted with DMSO-d6 (0.6 mL) and NMR spectra were acquired. The formation of 2-aminothiazole (7) was quantified using 1,3,5-trimethoxybenzene as an internal standard. Yields are shown in Supplementary Table 1.
Formation of 2-aminothiazole (7) in a mixture of thiols: to a suspension of β-mercaptoacetaldehyde (9a; 7.6 mg, 0.1 mmol), sodium hydrosulfide hydrate (assumed min. 50%, 0.1 mmol, 9.3 mg), sodium methanethiolate (9c; 0.1 mmol, 7.0 mg), ethanethiol (9d; 0.1 mmol, 7.4 µL), 1-propanethiol (9e; 0.1 mmol, 9.3 µL), 1-butanethiol (9f; 0.1 mmol, 9.0 µL) in sodium phosphate buffer (0.5M, pH 7, 1 mL) was added cyanamide (3; 4.2 mg, 0.1 mmol) and stirred overnight at room temperature. The reaction mixture was then diluted with DMSO (5 mL), an aliquot (100 µL) was further diluted with DMSO-d6 (0.6 mL) and NMR spectra were acquired to reveal only one aromatic compound. 2-Aminothiazole (7) was formed in 86% yield, as quantified using 1,3,5-trimethoxybenzene as an internal standard, and confirmed by spiking with a commercial sample of 2-aminothiazole (7). Data for 2-aminothiazole (7): 1H NMR (400 MHz, DMSO-d6) δ 6.88 (d, J = 3.7 Hz, 1H, (C4)–H), 6.53 (d, J = 3.7 Hz, 1H, (C5)–H).
Formation of 2-aminothiazole (7) from β-mercaptoacetaldehyde (9a) and S-methylisothiourea: to a suspension of β-mercaptoacetaldehyde (9a; 7.6 mg, 0.1 mmol) was added S-methylisothiourea hemisulfate (10; 278 mg, 0.2 mmol) in sodium phosphate buffer (0.5M, pH 7, 1 mL) and stirred overnight at room temperature. The reaction mixture was spiked with D2O (50 µL) and analysed by 1H NMR spectroscopy to reveal the presence of 2-aminothiazole (7). The reaction mixture was then diluted with DMSO (5 mL), an aliquot (100 µL) was further diluted with DMSO-d6 (0.6 mL) and NMR spectra were reacquired for quantification. The formation of 2-aminothiazole (7) was quantified using 1,3,5-trimethoxybenzene as an internal standard (72%), and spiked with commercially available 7 to confirm its presence. Data for 7: 1H NMR (600 MHz, D2O) 7 δ 7.01 (d, J = 3.7 Hz, 1H, (C4)–H), 6.67 (d, J = 3.7 Hz, 1H, (C5)–H).
Formation of 2-aminothiazole (7) from disulfide reduction by cyanide, condition A: potassium cyanide (9.75 mg, 0.15 mmol) in D2O (0.1 mL) was added to a stirred solution of 2n (12 mg, 0.1 mmol) in D2O (0.9 mL) and the solution was adjusted to pD 7 with 4M DCl. Ammonium hydroxide (13M, 39 µL, 0.50 mmol) was added, the solution adjusted to pD 9.2 with 4M DCl/NaOD, and warmed to 60 °C and incubated for 16 h The solution was cooled to room temperature and 1H NMR spectra were acquired. The formation of 2-aminothiazole (7, 14%) was observed (Supplementary Figure 23A). 1H NMR (400 MHz, D2O) δ 7.01 (d, J = 3.7 Hz, 1H, (C4)–H), 6.67 (d, J = 3.7 Hz, 1H, (C5)–H).
Formation of 2-aminothiazole (7) from disulfide reduction by cyanide, condition B: potassium cyanide (9.75 mg, 0.15 mmol) in D2O (0.1 mL) was added to a stirred solution of 2n (12 mg, 0.1 mmol) in D2O (0.9 mL) and the solution was adjusted to pD 7 with 4M DCl. Cyanamide (3; 12.6 mg, 0.30 mmol) was added and the solution adjusted to pD 7.0 with 4M DCl/NaOD. The reaction was incubated at 40 °C for 1 h. The solution was cooled to room temperature and 1H NMR spectra were acquired. The formation of 2-aminothiazole (7, 75%) was observed (Supplementary Figure 23B). 1H NMR (400 MHz, D2O) δ 7.01 (d, J = 3.7 Hz, 1H, (C4)–H), 6.67 (d, J = 3.7 Hz, 1H, (C5)–H).
