Centerband‐Only Detection of Exchange NMR with Natural‐Abundance Correction Reveals an Expanded Unit Cell in Phenylalanine Crystals

Abstract

The NMR pulse sequence CODEX (centerband‐only detection of exchange) is a widely used method to report on the number of magnetically inequivalent spins that exchange magnetization via spin diffusion. For crystals, this rules out certain symmetries, and the rate of equilibration is sensitive to distances. Here we show that for 13C CODEX, consideration of natural abundance spins is necessary for crystals of high complexity, demonstrated here with the amino acid phenylalanine. The NMR data rule out the C2 space group that was originally reported for phenylalanine, and are only consistent with a larger unit cell containing eight magnetically inequivalent molecules. Such an expanded cell was recently described based on single crystal data. The large unit cell dictates the use of long spin diffusion times of more than 200 seconds, in order to equilibrate over the entire unit cell volume of 1622 Å3.

SARS-CoV-2 Envelope Protein Forms Clustered Pentamers in Lipid Bilayers

“…Since the same equation with an error of π 2 was used both to calibrate and to determine unknown distances, the previously reported distances are correct, and only the numerical value of F (0) was mis-reported. Correcting for this π 2 factor, the true F (0) value should be 3.7 μs for 19 F and 8.0 μs for 13 C. Other literature values for F (0) of 13 C spins range from 1.2 to 11.4 μs, , in agreement with the revised value.…”

“…It is related to the normalized zero-quantum line shape at zero frequency. As in the existing literature, ,,− here we treat F (0) as a parameter to be calibrated from known crystal structures. 19 F CODEX of 5- 19 F-trptophan (Figure S4a) shows an exponential decay to S/S 0 = 0.50 at 400 ms. Fitting to 0.5 + 0.5e – ct shows excellent agreement with the experimental data ( R 2 = 0.9935) for the time constant c = 183 s –1 (Figure S4b).…”

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“…It is worth mentioning that 13 C-or 15 N-detected exchange experiments may further enhance the utilization of exchange techniques for spin counting in pharmaceutical applications. 61…”

“…Future applications of CODEX aim to investigate the impact of formulation composition, process, and stressed condition on the molecular packing of various ASDs to achieve a mechanistic understanding of dissolution and recrystallization. It is worth mentioning that 13 C- or 15 N-detected exchange experiments may further enhance the utilization of exchange techniques for spin counting in pharmaceutical applications. − …”

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“…This confers high sensitivity in 19 F-detected NMR experiments and allows 19 F to 19 F spin diffusion to be sensitive to a relatively large distance range of 5 to 20 Å. For the CODEX experiment specifically, 19 F detection does not need natural abundance correction either, like in its 13 C-detected [228] counterpart, making precise distance determination easier. 19 F has been used as a popular probe for biological NMR, because 19 F is almost absent in proteins.…”