General remarksAll reagents were commercially available and used without further purification. Solvents used for extraction and purification were distilled before use. Myceliophthora thermophila laccase (Novozym 51003) was a generous gift from Novozymes (Copenhagen, Denmark). Reactions were monitored by thin-layer chromatography (TLC) using silica gel 60 F254. All organic synthesis products were purified by preparative thin-layer chromatography (TLC), (CAMAG ® instrument, in-house prepared 20 × 20 cm silica plates) and characterized by NMR spectroscopy. 1H and 13C NMR spectra were recorded at 300 (75) MHz on a Bruker Avance spectrometer using DMSO-d6 as a solvent. The chemical shifts were referenced to the solvent signals at δH/C 2.49/39.50 ppm (DMSO-d6) relative to TMS. Melting points were determined with a Thermo Scientific 9100 melting point apparatus and were uncorrected. Mass spectra were recorded with an Agilent Technologies (HP) 5973 mass spectrometer.Synthesis of 5-phenyl-1,3,4-oxadiazol-2-thiones (1a–1e)Oxadiazoles 1a–1e were synthesized according to previous literature reports13. Related benzoic acid (40 mmol), sulfuric acid (catalytic amount), and 60 ml of methanol were heated to 80 °C under reflux for 24 h. Excess solvent was then eliminated by vacuum evaporation, followed by the addition of a sodium bicarbonate-saturated aqueous solution to neutralize the excess acid. Ethyl acetate (3 × 30 mL) was used to extract the methyl benzoate product.In the following stage, hydrazine hydrate (85%) (90 mmol, 2.88 mL) and synthesized methyl benzoates (30 mmol) were mixed in ethanol (50 mL) and refluxed overnight. After the reaction was completed, the reaction mixture was concentrated in vacuo, and the product was filtered and collected. The precipitate was washed with distilled water to remove any remaining hydrazine.Benzohydrazide (18.3 mmol), potassium hydroxide (32 mmol), and carbon disulfide (36 mmol) dissolved in a 5:1 ratio of ethanol to water were heated to 90 °C under reflux for 24 h. After reducing the volume in vacuo, the pH was adjusted to 2–3 with 1M HCl. The precipitate was subsequently collected by filtration, and the purity of the product was determined using TLC (n-hexane/ethyl acetate, 6:1) and NMR analysis.Synthesis of 5-phenyl-1,3,4-thiadiazol-2-thiones (1f–1h)Reaction between 4.9 mmol of benzohydrazide, 9.8 mmol of CS2, 6.37 mmol of KOH, and 10 mL of EtOH at room temperature for 3 h, resulted in a precipitate. The precipitate was filtered, washed with ice-cold ethanol, and dried at room temperature. After that, the precipitate was dissolved in 2 mL of concentrated H2SO4 and the reaction mixture was stirred for 2 h in an ice bath. Then, ice water was added to the reaction mixture, and a white precipitate was formed. This precipitate was washed with ice water and dried at room temperature. Finally, the dried precipitate was dissolved in 10% NaOH to obtain a transparent solution, which was filtered and HCl 1M was added to it to adjust the pH to 2–358. By reducing the pH, 5-phenyl-1,3,4-thiadiazol-2-thione was formed.Synthesis of 2,5-disubstituted-1,3,4-oxa(thia)diazole thioethers (3a–3m)A reaction mixture containing 5-(4-methoxyphenyl)-1,3,4-oxadiazole-2-thiol (0.1 mmol), catechol (0.15 mmol) and laccase (1 mL, 1000 U/mL) in citrate buffer (10 mM, pH 4.5, 10 mL) as a solvent and ethyl acetate (5 mL) as a co-solvent was stirred in a 100 mL round bottom flask at room temperature. The progress of the reaction was detected by TLC. After ensuring the completion of the reaction, the reaction mixture was diluted with EtOAc, the aqueous and organic phases were separated, and the aqueous phase was extracted by a separatory funnel using EtOAc (3 × 20 mL). After that, the organic phase was dried with anhydrous sodium sulfate, filtered, and finally, the solvent was eliminated under reduced pressure. The desired product was separated by preparative TLC (n-hexane: ethyl acetate, 2:1) and identified using proton and carbon NMR and also mass spectrometry.Characterization of products (3a–3m)4-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)benzene1,2-diol (3a)Solid (brown), melting point: 190–192 °C, isolated yield (81%), 1H NMR (300 MHz, DMSO-d6) δ 9.70 (s, 2H), 7.90 (m, 2H), 7.59 (m, 3H), 7.09 (s, 1H), 7.02 (d, J = 8.2 Hz, 1H), 6.86 (d, J = 8.3 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) δ 165.9, 163.8, 148.3, 146.8, 132.6, 129.9, 126.8, 126.5, 123.4, 121.6, 117.1, 114.2. MS (EI, 70 eV): m/z = 286.1 [M+], Anal. Calcd for C14H10N2O3S: C 58.73; H 3.52; N 9.78; Found: C 58.22 H 3.81, N 9.21.4-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)thio)benzene-1,2-diol (3b)Solid (yellow), melting point: 178–180 °C, isolated yield (46%), 1H NMR (300 MHz, DMSO-d6) δ 8.40 (d, J = 8.5 Hz, 2H), 8.15 (d, J = 8.6 Hz, 2H), 7.12 (s, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) δ 165.3, 164.4, 149.5, 148.5, 146.7, 128.9, 128.1, 126.8, 125.0, 121.9, 117.1, 113.6. MS (EI, 70 eV): m/z = 331.1 [M+], Anal. Calcd for C14H9N3O5S: C 50.75; H 2.74; N 12.68; Found: C 50.72 H 2.61, N 12.71.4-((5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)benzene-1,2-diol (3c)Solid (brown), melting point: 150–152 °C, isolated yield (94%), 1H NMR (300 MHz, DMSO-d6) δ 10.13–9.08 (s, 2H), 7.79 (d, J = 8.5 Hz, 2H), 7.06 (m, 3H), 6.99 (d, J = 8.5 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 3.80 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 165.8, 163.0, 162.5, 148.2, 146.7, 128.6, 126.4, 121.5, 117.0, 115.7, 115.2, 114.6, 55.9. MS (EI, 70 eV): m/z = 316.1 [M+], Anal. Calcd for C15H12N2O4S: C 56.95; H 3.82; N 8.86; Found: C 56.92 H 3.51, N 8.91.4-((5-(m-tolyl)-1,3,4-oxadiazol-2-yl)thio)benzene-1,2-diol (3d)Solid (pale brown), melting point: 178–180 °C, isolated yield (89%), 1H NMR (300 MHz, DMSO-d6) δ 9.65 (s, 2H), 7.78–7.65 (m, 2H), 7.46 (m, 2H), 7.10 (d, J = 2.3 Hz, 1H), 7.01 (dd, J = 8.2, 2.2 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 2.39 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 166.0, 163.7, 148.3, 146.7, 139.4, 133.3, 129.8, 127.1, 126.4, 124.0, 123.3, 121.6, 117.1, 114.2, 21.2. MS (EI, 70 eV): m/z = 300.1 [M+], Anal. Calcd for C15H12N2O3S: C 59.99; H 4.03; N 9.33; Found: C 60.12 H 4.01, N 9.11.4-methyl-5-((5-(m-tolyl)-1,3,4-oxadiazol-2-yl)thio)benzene-1,2-diol (3e)Solid (brown), melting point: 168–170 °C, isolated yield (93%), 1H NMR (300 MHz, DMSO-d6) δ 7.75–7.64 (m, 2H), 7.46 (m, 2H), 7.06 (s, 1H), 6.80 (s, 1H), 2.39 (s, 3H), 2.28 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 165.9, 163.6, 148.6, 144.6, 139.4, 133.7, 133.2, 129.8, 127.0, 124.0, 123.3, 123.0, 118.6, 112.9, 21.3, 20.1. MS (EI, 70 eV): m/z = 314.1 [M+], Anal. Calcd for C16H14N2O3S: C 61.13; H 4.49; N 8.91; Found: C 61.20 H 4.88, N 8.81.4-methyl-5-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)benzene-1,2-diol (3f)Solid (brown), melting point: 160–162 °C, isolated yield (89%), 1H NMR (300 MHz, DMSO-d6) δ 9.56 (s, 1H), 9.29 (s, 1H), 7.86 (m, 2H), 7.55 (m, 3H), 7.08 (s, 1H), 6.81 (s, 1H), 2.28 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 165.8, 163.7, 148.6, 144.5, 133.8, 132.5, 129.8, 126.7, 123.4, 123.1, 118.5, 113.0, 20.1. MS (EI, 70 eV): m/z = 300.1 [M+], Anal. Calcd for C15H12N2O3S: C 59.99; H 4.03; N 9.33; Found: C 59.92 H 4.01, N 9.11.4-((5-(3-nitrophenyl)-1,3,4-oxadiazol-2-yl)thio)benzene-1,2-diol) (3g)Solid (brown), melting point: 220–222 °C, isolated yield (57%),1H NMR (300 MHz, DMSO-d6) δ 9.65 (s, 1H), 9.50 (s, 1H), 8.51 (s, 1H), 8.40 (d, J = 8.4 Hz, 1H), 8.27 (d, J = 7.8 Hz, 1H), 7.84 (t, J = 8.1 Hz, 1H), 7.07 (s, 1H), 7.05–6.96 (m, 1H), 6.84 (d, J = 8.2 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) δ 164.9, 164.2, 148.5, 148.4, 146.7, 132.8, 131.8, 126.7, 124.8, 121.8, 121.2, 117.1, 113.7. MS (EI, 70 eV): m/z = 331.1 [M+], Anal. Calcd for C14H9N3O5S: C 50.75; H 2.74; N 12.68; Found: C 50.71 H 2.69, N 12.48.4-methyl-5-((5-(3-nitrophenyl)-1,3,4-oxadiazol-2-yl)thio)benzene-1,2-diol (3h)Solid (pale brown), melting point: 208–210 °C, isolated yield (69%), 1H NMR (300 MHz, DMSO-d6) δ 9.54 (s, 1H), 9.25 (s, 1H), 8.49 (s, 1H), 8.38 (d, J = 8.5 Hz, 1H), 8.24 (d, J = 7.9 Hz, 1H), 7.82 (t, J = 8.1 Hz, 1H), 7.04 (s, 1H), 6.77 (s, 1H), 2.26 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 159.6, 159.0, 143.6, 143.4, 139.4, 128.8, 127.6, 126.6, 121.5, 119.7, 118.0, 116.0, 113.3, 107.3, 14.9. MS (EI, 70 eV): m/z = 345.1 [M+], Anal. Calcd for C15H11N3O5S: C 52.17; H 3.21; N 12.17; Found: C 52.23 H 3.16, N 12.25.4-((5-phenyl-1,3,4-thiadiazol-2-yl)thio)benzene-1,2-diol (3i)Solid (pale brown), melting point: 168–170 °C, isolated yield (62%), 1H NMR (300 MHz, DMSO-d6) δ 9.67 (s, 2H), 7.89–7.81 (m, 2H), 7.49 (m, 3H), 7.12 (d, J = 2.4 Hz, 1H), 7.08 (dd, J = 7.8, 2.7 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H). 13C NMR (75 MHz, Chloroform-d) δ 176.3, 173.0, 153.7, 151.9, 136.5, 134.6, 134.4, 132.6, 131.9, 126.7, 123.0, 122.3. MS (EI, 70 eV): m/z = 447 [M+], Anal. Calcd for C14H10N2O2S2: C 55.61; H 3.33; N 9.26; Found: C 55.52 H 3.41, N 9.41.4-methyl-5-((5-phenyl-1,3,4-thiadiazol-2-yl)thio)benzene-1,2-diol (3j)Solid (pale brown), melting point: 198–200 °C, isolated yield (81%), 1H NMR (300 MHz, DMSO-d6) δ 7.93–7.75 (m, 2H), 7.49 (m, 3H), 7.11 (s, 1H), 6.86 (s, 1H), 2.29 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 171.3, 168.1, 149.2, 145.0, 133.8, 131.6, 129.8, 129.7, 127.8, 123.0, 118.9, 117.4, 19.9. MS (EI, 70 eV): m/z = 316.1 [M+], Anal. Calcd for C15H12N2O2S2: C 56.94; H 3.82; N 8.85; Found: C 57.22 H 3.93, N 8.91.4-((5-(p-tolyl)-1,3,4-thiadiazol-2-yl)thio)benzene-1,2-diol (3k)Solid (brown), melting point: 150–152 °C, isolated yield (68%), 1H NMR (300 MHz, DMSO-d6) δ 9.62 (s, 1H), 7.72 (d, J = 7.6 Hz, 2H), 7.28 (d, J = 7.8 Hz, 2H), 7.15–7.02 (m, 2H), 6.90 (d, J = 8.2 Hz, 1H), 2.34 (s, 1H). 13C NMR (75 MHz, DMSO-d6) δ 170.9, 168.3, 148.9, 147.1, 141.7, 130.3, 127.7, 127.1, 127.0, 121.9, 118.3, 117.5, 21.4. MS (EI, 70 eV): m/z = 447 [M+], Anal. Calcd for C15H12N2O2S2: C 56.94; H 3.82; N 8.85; Found: C 57.02 H 3.75, N 8.71.4-((5-(m-tolyl)-1,3,4-thiadiazol-2-yl)thio)benzene-1,2-diol (3l)Solid (pale brown), melting point: 178–180 °C, isolated yield (74%), 1H NMR (300 MHz, DMSO-d6) δ 9.70 (s, 2H), 7.66 (s(br), 2H), 7.35 (m, 2H), 7.16–7.02 (m, 2H), 6.90 (d, J = 8.2 Hz, 1H), 2.34 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 171.5, 168.4, 148.8, 147.0, 139.3, 132.3, 129.6, 129.4, 128.1, 127.1, 124.7, 121.8, 118.3, 117.5, 21.1. MS (EI, 70 eV): m/z = 316.1 [M+], Anal. Calcd for C15H12N2O2S2: C 56.94; H 3.82; N 8.85; Found: C 56.67 H 3.51, N 8.61.3-methyl-4-((5-phenyl-1,3,4-thiadiazol-2-yl)thio)benzene-1,2-diol (3m)Solid (brown), melting point: 148–150 °C, isolated yield (78%), 1H NMR (300 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.82 (s, 1H), 7.80 (d, J = 7.1 Hz, 2H), 7.45 (d, J = 7.9 Hz, 3H), 7.11 (d, J = 8.3 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H), 2.27 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 171.6, 168.0, 148.6, 145.3, 131.6, 129.8, 129.7, 129.2, 127.9, 127.8, 118.8, 114.3, 14.4. MS (EI, 70 eV): m/z = 316.1 [M+], Anal. Calcd for C15H12N2O2S2: C 56.94; H 3.82; N 8.85; Found: C 56.85 H 3.51, N 8.89.
