Figure 1.

In vivo production of (2-ethyl-pyridin-4-yl)methanol (5) from ETA by whole cells of MTb. A culture of MTb strain H37Rv at OD₆₅₀ of 1.0 was concentrated 10-fold in 7H9 media, and [1-¹⁴C]ETA at 0.01 μg/ml was added. (A) After incubation at 37°C metabolism of [1-¹⁴C]ETA was visualized by TLC and autoradiography. Lanes a–h correspond to sequential filtered culture samples taken at 0.2, 0.25, 0.75, 1.5, 2.5, 5.0, 8.5, and 25 h, respectively. Lane i represents media autooxidation after 25 h of incubation without bacterial cells. The metabolites observed cochromatographed with commercial and characterized synthetic samples of ETA (1), ETA S-oxide (2), ETA nitrile (3), and ETA amide (4). (B) Mycobacteria from the same sequential culture aliquots (500 μl) were collected by filtration onto 0.22-μm filter disks under vacuum, they were washed twice with PBS (500 μl), and the cell-associated radioactivity was measured. (C) The reversed-phase HPLC retention time of the unknown major metabolite (5) was used to guide cold large-scale ETA feeding experiments where we isolated unlabeled metabolite that gave a mass of 137 (137.9 MH⁺). We assigned this as (2-ethyl-pyridin-4-yl)methanol and confirmed the identity of (5) by cochromatography with a synthetic characterized alcohol standard. The upper HPLC continuous radiodetector spectrum corresponds to A lane i, media control and the lower spectrum; lane d, time point 1.5 h, where the UV₂₅₄ trace of (2-ethyl-pyridin-4-yl)methanol is superimposed in gray.