Fig. 2. Investigation of the AmbI1-3 isonitrile synthase promiscuity in vitro, using differentially substituted halo tryptophans. Standard assay conditions: all assays were carried out in a 1 mL scale at pH 7.4 with 2 (2.5 mM), Rub5P (2.5 mM), α-KG (2.0 mM), (NH₄)₂Fe(SO₄)₂ (0.5 mM) and AmbI1, I2, I3 (20 μM each) for 4 h at 30 °C. All assays were stopped by extraction with ethyl acetate, processed and analysed by HPLC and HR-LCMS in an identical manner as detailed in ESI.† Individual assays were repeated at least in duplicate and representative results are shown. HPLC chromatographs with a UV detector at 315 nm that selectively detects indolyl vinyl isonitriles show: (a) 6-F-l-Trp 2d is a substrate for AmbI1-3 and is processed to give a compound with identical retention time to the synthetic standard 1d, and competitively preferred over the native substrate l-Trp 2a. l-Trp and halogen-substituted l-Trps starting materials, being insoluble in the ethyl acetate extract and not absorbing at 315 nm, are not present in the chromatogram; in all cases an excess of substrate was provided. The average conversion from l-Trp to 1a under standard assay conditions is estimated to be 30%, (b) The conversion of 4-F, 5-F, 6-F and 7-F l-Trp (2b–e) to the corresponding isonitriles by AmbI1-3 is equally effective as that of native substrate 2a, (c) the conversion of 5-F, 5-Cl, 5-Br l-Trp (2c, 2f–g) to the corresponding isonitriles by AmbI1-3 decreases with the increasing size of halogen substituent, (d) the conversion of 7-F, 7-Cl, 7-Br and 7-I l-Trp (2e, 2h–j) to the corresponding isonitriles by AmbI1-3 decreases with the increasing size of halogen substituent. The authenticity of each enzymatic product (1a–j) was ascertained by HRMS (Fig. SI 5–13, ESI†), comparative retention time and UV spectral analysis (Fig. SI 14, ESI†), in addition to HPLC co-elution with authentic standards applied to 1a, 1b, 1f.