, 43 (d, J = 8.2 Hz, 1H, H6), 9.50 * (s, 1H, C 6 H 5 CH 2 CONH, 12.56 * (br s, 1H, COOH). 13 C NMR (DMSO-d 6 ): d C 42.7 (C 6 H 5 COCH 2 ), 116.6, 117.4 and 123.7 (C2, C5 and C6), vol.7

, 25 g, 30.6 mmol) and water (10 mL) was added methyl 4-chloro-3-nitrobenzoate (5 g, 23.2 mmol) in ethanol (40 mL). The mixture was refluxed for 2 h and diluted with water (40 mL). The solid thus formed was filtered off, washed with n-hexane and dried, yielding 21 as a yellow solid, .56 g, 93%): mp 136-138°C (recryst from MeOH), lit. 138°C. 45 1 H NMR, vol.2

, 126.1 (C1), 126.4, 127.6 and 127.8 (C2, C5 and C4 0 ), vol.128

, 8 mL) refluxed for 30 min with vigorous stirring. The reaction mixture was diluted with EtOAc (40 mL) and filtered over Celite Ò . The filtered solution was washed with 5% aqueous NaHCO 3 (40 mL), followed by water (2· 40 mL), and dried over anhydrous Na 2 SO 4 . The solvent was evaporated in vacuo and the residue obtained was recrystallised from MeOH to give 22 as an off-white solid (4.0 g, 88%): mp 76-77°C lit. 76-78°C. 45 1 H NMR (DMSO-d 6 ): d H 3.79 (s, 3H, OCH 3 ), 4.10 (s, 2H, C 6 H 5 CH 2 S), 5.46 * (s, 2H, A solution of 21 (5 g, 16.5 mmol) in EtOH (10 mL) was treated with iron dust (7.9 g, 137 mmol) and 20% aqueous acetic acid

S. ,

, This solid was filtered, washed with EtOAc and dried, yielding 23 as a white solid (0.8 g, 88%): mp 245-246°C (recryst from EtOH). 1 H NMR (DMSO-d 6 ): d H 3.85 (s, 3H, OCH 3 ), 4.36 (s, 2H, C 6 H 5 CH 2 S), = 8.4 Hz, 1H, H5), 7.73 (d, J = 1.8 Hz, 1H, H2), 7.90 (dd, J = 1.8 and 8.4 Hz, 1H, H6), 9.66 *, vol.7

S. , Cl, 10.1. Found: C, 54

, A solution of 23 (0.3 g, 0.85 mmol) in THF (1 mL) was added to 1 M NaOH (4 mL) and the mixture heated to 70°C for 1 h. The solvent was evaporated, and the residue obtained re-dissolved in water (3 mL) and the pH adjusted to 6-7 with 1 M HCl. A solid slowly came out of solution, which was filtered and dried

, NH 2 ) 2 ), 11.36 * (br s, 1H, NH@C(NH 2 ) 2 ). 13 C NMR (DMSOd 6 ): d C 35.6 (C 6 H 5 CH 2 S), C4 0 ), 128.4 (C2 0 and C6 0 ), 128.7 (C5 and C6), vol.126

+. , Anal. Calcd for C 15, ES À ) m/z 300, vol.100, p.100

S. ,

, Synthesis of 3-amino-4-benzylmercapto-benzoic acid (25). A mixture of 22 (0.42 g, 1.54 mmol), 1 M NaOH (3 mL) and MeOH

M. Hcl, The solution was extracted with EtOAc (30 mL) and the organic phase dried over anhydrous Na 2 SO 4 and evaporated in vacuo, giving 25 as an offwhite solid (0.35 g, 88%): mp 155-157°C (recryst from MeOH). 1 H NMR

. Hz, 31 (d, J = 1.8 Hz, 1H, H2), 12.71 * (br s, 1H, COOH). 13 C NMR (DMSO-d 6 ): d C 36.4 (SCH 2 NC 6 H 5 ) 114.6 and 116.9 (C1 and C6), 122.0 (C4), 1H, H6), 7.17-7.29 (m, 6H, C 6 H 5 CH 2 S and H5), vol.7

, MS (ES À ) m/z 258, p.100

, The filtrate was chromatographed over a silica-gel column, starting with CH 2 Cl 2 with an increasing gradient of CH 2 Cl 2 : EtOAc (up to 30% EtOAc). Fractions containing the product were combined with solid A and solvent removed in vacuo. Successive recrystallisations of the residue from EtOAc and EtOH yielded 26 as a white solid (0.65 g, 33%): mp 193-195°C. 1 H NMR (DMSO-d 6 ): d H 3.83-3.88 (br s, 5H, OCH 3 and NHCOCH 2 NHFmoc), 4.24-4.33 (m, 5H, C 6 H 5 CH 2 S; CH and CH 2 Fmoc, anhydrous DMF (5 mL) cooled in an ice bath were added Fmoc-Gly-OH (1.74 g, 5.9 mmol) and DCC (1.1 g, 5.5 mmol) and the mixture stirred for a few minutes on the ice bath and then at room temperature for 16 h, vol.7

. Hr-es-ms, A solution of 26 (0.125 g, 0.23 mmol) in EtOAc (5 mL) and diethylamine (5 mL) was refluxed for 1 h and the resulting solution evaporated to dryness in vacuo. The resulting residue was suspended in MeOH (30 mL) and filtered. The filtrate was evaporated to dryness and the residue dissolved in THF (2 mL) and 1 M NaOH (4 mL), and refluxed for 1 h. The mixture was filtered and the pH adjusted to $7 with 1 M HCl. The solid formed was collected by filtration and dried, giving 27 as a white solid (0.026 g, 36%): mp colour change to dark brown at 210°C, p.9

, H 5 CH 2 S), vol.40

, NH 3 þ ), 166.8 (COOH). MS (ES À ) m/z 315, vol.128

, Expression and purification of recombinant TcTS The protocol for expression and purification of recombinant TcTS was adapted from Buschiazzo et al. 46 References and notes

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