|The manuscript describes the exploration of chemical space of two aromatic amino acids in CW-photo-CIDNP. Whereas TR-photo-CIDNP would be mechanistically more informative, the approach using CW-photo-CIDNP has clear sensitivity advantages. With their approach the authors could demonstrate the existence of several compounds showing strong photo-CIDNP effects. |
In cyclic reactions, as studied here, cancellation of polarization of recombination and escape products (chemically the same in a cyclic reaction) will occur. The observed CIDNP depends critically on the various kinetic rates and nuclear spin relaxation times. Thus not only for understanding the mechanism, but also for optimal CW-CIDNP intensity and identifying new compounds exploration of experimental conditions can play a significant role. The authors could comment on this.
Recommendable overviews for this study would be Hore and Broadhurst (1993), Photo-CIDNP of Biopolymers, Prog. NMR Spectrosc. 25, 345-402 and Kuhn (2013), Photo-CIDNP NMR Spectroscopy of Amino Acids and Proteins. In: Kuhn (eds) Hyperpolarization Methods in NMR Spectroscopy. Topics in Current Chemistry Vol 338, Springer Berlin Heidelberg, pp. 229-300, https://doi.org/10.1007/128_2013_427
A related study showing examples CW-photo-CIDNP of tyrosine and tryptophan derivatives would be Stob and Kaptein (1989), Photo-CIDNP of the amino-acids, Photochem. Photobiol. 49(5):565-577, https://doi.org/10.1111/j.1751-1097.1989.tb08425.x. Also the review by Hore and Broadhurst summarizes several dyes and compounds for photo-CIDNP.
Scheme II and scheme I in the paper by Stob and Kaptein may provide a simple framework to interpret the results in this manuscript as well. That paper also shows a pH and concentration dependency of the CW CIDNP intensity. Fig. 15 in that paper may be a warning, since small [Trp] concentrations can have a steep CIDNP intensity effect, idem Fig. 14 showing strong pH dependency at pH 6. Also the review by Hore and Broadhurst shows such pH dependency for Tyr and Trp (Fig.6, and explained on p.363). The referred paper by Okuno and Cavagnero (2016, J Phys Chem B) also shows possibilities for simulating CW-photo-CIDNP and parameters for that.
The pH dependency of CW-photo-CIDNP intensities could also deviate substantially from the pKa’s of compounds. Thus characterizing CW-photo-CIDNP at a few concentrations and pH values may be recommendable for this type of screening. Whether concentration and pH could indeed play a role, the authors could partially check this already using the equations given by Stob and Kaptein.
Though not really observable when perfect 90° detection pulses were used (better detected with 45° pulses), the spectral difference between Tyr and TyrA may also point to a difference in a multiplet effect, for which there is another Kaptein’ CIDNP rule, and which would be independend of a diference in g-values in the radical pair and may therefore still be present in case of weak net effects.
In Table 1, I would use / add the same short names and order as in Fig. 1 (then short names are also defined, and not partially as now in Fig. 2). Please check SNE’s in Fig .1 and Table 1: e.g. AT12, SNE for TyrA is -60 (Table 1) and -63 (Fig. 1), whereas with the other molecules numbers appear to be the same. Conditions for the experiment are also the same.
The authors may also consider at several instances pKa changes also source for the CIDNP intensity differences:
- Could pKa differences also be explanation for CIDNP intensity changes between HOPI / dH-Trp, IPA / IAA, etc?
- l. 168: interestingly, such pKa changes had been discussed by Stob and Kaptein (1989) for Trp and N-acetyl-Trp. Could such pKa play a role here as well?
- l.184/5: are these the estimated pKa’s for fluorescein that of its radical? Idem for the other (notably ’tryptophan’) pKa’s on p.8.
- l. 205: please note that the precise pKa value and thus changes therein could also play a significant role on the CW-CIDNP effects studied here as well. That would much more subtile than overall charge, and could also be explanation for failure of just using charge alone.
- l. 210: the rates of sidechain dynamics may be much slower than the various rates in CIDNP (reactions, ET, protonation). Dynamics would then rather be: presenting more or less active or conformations, or conformations with different pKa’s.
- l. 222/ Fig.4: the observed correlation of CIDNP SNE and hydrophobicity is interesting. But possibly pKa effects present also a good explanation for observed effetcts. The authors could discuss this as alternative to hydrophobicity.
- ‘Absolute’ may still be hard, but would relative pKa predictions using DFT calculations of classes of ‘tyrosine’ and ‘tryptophan’ compounds be useful for interpreting the CIDNP intensities, as an additional approach to charge/log(P) or TR-photo-CIDNP?