Extended Data Fig. 3: Tracking cellular uptake of [¹⁵N]-Lipoamide using NMR.

a, Methodology for quantitation of [¹⁵N]-lipoamide uptake by HeLa cells, using the trans-amide proton to measure [¹⁵N]-lipoamide concentration (see F- H). Medium with 100 μM [¹⁵N]-lipoamide was incubated for 1 h in the absence or presence of HeLa cells. Following removal of medium, the cells were washed with medium (without arsenate) and detached using EDTA-trypsin. Solution or cell pellet/in-cell NMR was used to determine [¹⁵N]-lipoamide concentration. Example spectra for cells stressed with 3 mM arsenate and incubated with R-(+)-lipoamide are shown with the same y axis scale. b, Cellular uptake was determined by subtracting signal from medium incubated with cells (red) from signal from medium without cells (blue). This was carried out for all four combinations of stressed (3 mM arsenate) or unstressed cells with [¹⁵N]-(R)-(+) or (±)-lipoamide. For stressed cells treated with [¹⁵N]-(R)-(+)-lipoamide the high signal intensity from the washed cell sample (green) is consistent with the large uptake from the medium calculated from the with (red) and without cell (blue) signal intensity. c, Quantitation of B showing percentage uptake and calculated intracellular concentration, assuming that lipoamide is uniformly distributed within cells (see Supplemental Methods). Uncertainty in measurement was approximately 30% and there was no significant difference in uptake between conditions. All measurements indicated substantial uptake of lipoamide and cellular concentrations >1 mM. d, Overview of synthesis of [¹⁵N]-lipoamide, highlighting the trans amide proton (14). e, ¹H NMR spectrum of [¹⁵N]-lipoamide in CDCl₃. Peaks can be unambiguously assigned to individual proton environments. f-h, Controls determining reliability of quantitation of [¹⁵N]-lipoamide using the amide protons in ¹⁵N edited ¹H NMR experiments. f, Dependency of the cis (13) and trans (14) amide proton signal on temperature, at a constant pH of 8.3. Both resonances decreased with increasing temperature, indicating local molecular dynamics and/or interactions with H₂O on ms to µs timescale reduce the signal. Trans amide proton resonance approaches a plateau towards 10 °C. g, Dependency of the cis and trans amide proton signal on pH, at a constant temperature of 10 °C. Together, indicating at 10 °C and below pH 8.6 integrated signal intensity of the trans-amide proton of lipoamide in ¹⁵N edited ¹H NMR experiments is a reliable proxy for concentration. h, Signal intensity of the trans-amide proton of lipoamide, when dissolved in growth medium, decreased over time at 37 °C but not at 10 °C. At 10 °C signal intensity is stable for >10 h experiments.

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