Unravelling structures of radicals of kynurenic acid formed in the photoinduced reactions with tryptophan and N-acetyl tyrosine.

Kynurenic acid (KNA) in the triplet state reacts with tryptophan (Trp) at neutral pH via proton-coupled electron transfer (PCET), which includes the stepwise transition of both electron and proton from Trp to triplet KNA. In the case of tyrosine (Tyr), the quenching reaction is H-transfer, a simultaneous transfer of electron and proton. In this work, we used the time-resolved chemically induced dynamic nuclear polarization (TR CIDNP) method to unveil the sites of H/H+ transfer within KNA. For this purpose, we obtained the values of 1H hyperfine coupling constants (HFCCs) and g-factors for different tautomeric forms of KNA radicals by the DFT method, then calculated CIDNP intensities using these g-factors and HFCCs according to the Adrian model. The calculated CIDNP intensities for different protons were correlated with their CIDNP intensities in the geminate spectra detected in the photoreactions of KNA with Trp, N-acetyl Trp, and N-acetyl Tyr. Best-fit proportionality relationships between calculated and experimental CIDNP intensities have shown that the KNA anion radical is present in two of the three possible tautomeric forms, which result from the H/H+ movement to the carbonyl oxygen of keto- and oxo-quinolinate forms of KNA, without any visible contribution of the H/H+ transfer to the nitrogen of the enol form. For 4-hydroxyquinoline (4HQN), being the chromophoric core of KNA and exhibiting the same PCET and H-transfer reactions with Trp and Tyr, a single possible tautomeric form of its radical has been revealed as H/H+ transfer to the carbonyl oxygen of the keto-form.

Exchange interaction in short-lived flavine adenine dinucleotide biradical in aqueous solution revisited by CIDNP (chemically induced dynamic nuclear polarization) and nuclear magnetic relaxation dispersion.

Flavin adenine dinucleotide (FAD) is an important cofactor in many light-sensitive enzymes. The role of the adenine moiety of FAD in light-induced electron transfer was obscured, because it involves an adenine radical, which is short-lived with a weak chromophore. However, an intramolecular electron transfer from adenine to flavin was revealed several years ago by Robert Kaptein by using chemically induced dynamic nuclear polarization (CIDNP). The question of whether one or two types of biradicals of FAD in aqueous solution are formed stays unresolved so far. In the present work, we revisited the CIDNP study of FAD using a robust mechanical sample shuttling setup covering a wide magnetic field range with sample illumination by a light-emitting diode. Also, a cost efficient fast field cycling apparatus with high spectral resolution detection up to 16.4 T for nuclear magnetic relaxation dispersion studies was built based on a 700 MHz NMR spectrometer. Site-specific proton relaxation dispersion data for FAD show a strong restriction of the relative motion of its isoalloxazine and adenine rings with coincident correlation times for adenine, flavin, and their ribityl phosphate linker. This finding is consistent with the assumption that the molecular structure of FAD is rigid and compact. The structure with close proximity of the isoalloxazine and purine moieties is favorable for reversible light-induced intramolecular electron transfer from adenine to triplet excited flavin with formation of a transient spin-correlated triplet biradical F⚫--A⚫+. Spin-selective recombination of the biradical leads to the formation of CIDNP with a common emissive maximum at 4.0 mT detected for adenine and flavin protons. Careful correction of the CIDNP data for relaxation losses during sample shuttling shows that only a single maximum of CIDNP is formed in the magnetic field range from 0.1 mT to 9 T; thus, only one type of FAD biradical is detectable. Modeling of the CIDNP field dependence provides good agreement with the experimental data for a normal distance distribution between the two radical centers around 0.89 nm and an effective electron exchange interaction of -2.0 mT.

Multifrequency Nuclear Magnetic Resonance as an Efficient Tool To Investigate Heterospin Complexes in Solutions.

We report a multifrequency nuclear magnetic resonance (NMR) study of heterospin complexes [Eu(SQ)3Ln], where SQ is 3,6-di(tert-butyl)-1,2-semiquinone, L is tetrahydrofuran (THF), pyridine (Py), or 2,2'-dipyridyl (Dipy), and n is the number of diamagnetic ligands. Multifrequency NMR experiments allowed us to determine the effective paramagnetic shifts of the ligands (L = THF or Py) and the chemical equilibrium constant for [Eu(SQ)3(THF)2]. In addition, we have found a strong magnetic field effect on the NMR line broadening, giving rise to very broad NMR lines at high magnetic fields. We attribute this effect to broadening under fast exchange conditions when the NMR spectrum represents a homogeneously broadened line with a width proportional to the square of the NMR frequency difference of the free and bound forms of L. Consequently, the line width strongly increases with the magnetic field. This broadening effect allows one to determine relevant kinetic parameters, i.e., the effective exchange time. The strong broadening effect allows one to exploit the [Eu(SQ)3(THF)2] complex as an efficient shift reagent, which not only shifts unwanted NMR signals but also broadens them, notably, in high-field NMR experiments. We have also found that [Eu(SQ)3Dipy] is a thermodynamically stable complex; hence, one can study [Eu(SQ)3Dipy] solutions without special precautions. We report an X-ray structure of the [Eu(SQ)3Dipy]·C6D6 crystals that have been grown directly in an NMR tube. This shows that multifrequency NMR investigations of heterospin compound solutions not only provide thermodynamic and kinetic data for heterospin species but also can be useful for the rational design of stable heterospin complexes and optimization of synthetic approaches.

Electron transfer vs. proton-coupled electron transfer as the mechanism of reaction between amino acids and triplet-excited benzophenones revealed by time-resolved CIDNP.

Hyperfine coupling constants (HFCCs) of the short-lived radicals of 4-carboxy, 4,4'-dicarboxy, and 3,3',4,4'-tetracarboxy benzophenones (4-CBP, DCBP, and TCBP, respectively) formed in their photoreaction with tyrosine were obtained from analysis of geminate CIDNP spectra. These HFCCs were compared to HFCCs calculated using density functional theory. From this comparison, it was established that the CIDNP pattern of TCBP originates from contributions of three types of TCBP radical structures: the non-protonated anion radical and two anion radical structures with a protonated carboxylic group at position 3 or 4 (or 3' or 4'). This allowed us to conclude that the mechanism of the quenching reaction is proton coupled electron transfer (PCET): electron transfer is followed by proton transfer to one of four possible positions with similar probabilities. The same CIDNP pattern and therefore the same reaction mechanism was established for histidine. For 4-CBP and DCBP, triplet quenching proceeds also via PCET, again with formation of the anion radical with a protonated carboxylic group.

Kinetics of Reversible Protonation of Transient Neutral Guanine Radical in Neutral Aqueous Solution.

Time-resolved chemically induced dynamic nuclear polarization (TR-CIDNP) is applied to follow transformation of the short-lived neutral guanine radical into a secondary guanine radical by its protonation, presumably at position N7. In the initial step the photoreaction of guanosine-5'-monophosphate (GMP) with triplet excited 3,3',4,4'-tetracarboxy benzophenone (TCBP) leads to formation of the neutral radical G(-H). . The evidence of the radical conversion is based on the inversion of CIDNP sign for TCBP and GMP protons on the microsecond timescale as a result of the change in magnetic resonance parameters in the pairs of TCBP and GMP radicals due to structural changes of the GMP radical. Acceleration of the CIDNP sign change upon addition of phosphate (proton donor) confirms that the radical transformation responsible for the observed CIDNP kinetics is protonation of the neutral guanine radical with formation of the newly characterized cation radical, (G.+ )'. From the full analysis of the pH-dependent CIDNP kinetics, the protonation and deprotonation behaviour is quantitatively characterized, giving pKa =8.0±0.2 of the cation radical (G.+ )'.