Protonated Forms of Naringenin and Naringenin Chalcone: Proteiform Bioactive Species Elucidated by IRMPD Spectroscopy, IMS, CID-MS, and Computational Approaches

Naringenin (Nar) and its structural isomer, naringenin chalcone (ChNar), are two natural phytophenols with beneficial health effects belonging to the flavonoids family. A direct discrimination and structural characterization of the protonated forms of Nar and ChNar, delivered into the gas phase by electrospray ionization (ESI), was performed by mass spectrometry-based methods. In this study, we exploit a combination of electrospray ionization coupled to (high-resolution) mass spectrometry (HR-MS), collision-induced dissociation (CID) measurements, IR multiple-photon dissociation (IRMPD) action spectroscopy, density functional theory (DFT) calculations, and ion mobility-mass spectrometry (IMS). While IMS and variable collision-energy CID experiments hardly differentiate the two isomers, IRMPD spectroscopy appears to be an efficient method to distinguish naringenin from its related chalcone. In particular, the spectral range between 1400 and 1700 cm–1 is highly specific in discriminating between the two protonated isomers. Selected vibrational signatures in the IRMPD spectra have allowed us to identify the nature of the metabolite present in methanolic extracts of commercial tomatoes and grapefruits. Furthermore, comparisons between experimental IRMPD and calculated IR spectra have clarified the geometries adopted by the two protonated isomers, allowing a conformational analysis of the probed species.


Nar_1
(0.0) S14/53 Figure S14. Optimized geometries and relative free energies (kJ mol −1 , in parenthesis) of conformers in s-cis configuration, belonging to the ChNar_1 family, calculated at the B3LYP/6-311++G(d,p) level of theory. The relative free energies are calculated with respect to the global minimum ChNar_1. Distances are given in Å.  Figure S20. IRMPD spectrum (orange profile) compared to the calculated IR spectra (black profiles) of the four lowest lying conformers of protonated naringenin chalcone, belonging to the ChNar_1 family in 1) s-cis configuration (left, orange panel), or 2) in s-trans configuration (right, yellow panel) whose geometries are shown in Figure 6 and S17-18. Free energies relative to ChNar_1, are reported in parenthesis (kJ mol −1 ). Calculated IR intensities (Y axis) are plotted in the scale: 0-3500 and 0-2500 Km mol −1 for the s-cis and s-trans conformers, respectively.

Tomato and grapefruit sample preparation
Fresh red grapefruits (Star Ruby, Citrus Paradisi) were washed and the albedos (white part of peel) were separated from the flavedos (orange part of peel). Fresh albedos were cut in small pieces for a hot methanol extraction. In a flask, 33 mL of methanol was added to 5.00 g of fresh albedos. The mixture was heated at 60°C for 3 hours and constantly stirred with a magnetic mixer. Then, 1 μL of extraction solution was diluted with 1 mL methanol.
Tomato skin was obtained from "datterino" peel, a variety of cherry tomatoes (Solanum lycopersicum L.). Tomato extraction was prepared using 1.57 g of tomato skin treated with 9 mL methanol and heated at 60°C for 3 hours while constantly stirred with a magnetic mixer. Finally, 1 μL of extraction solution was diluted with 1 mL methanol.
After the extraction, the diluted solutions were directly submitted to ESI in an ion trap mass spectrometer to verify the presence of the ion at m/z 273 in the natural extracts.

IRMPD experiments
Positively charged ions generated by ESI were mass-isolated in the cell of an Amazon Speed ETD mass spectrometer prior to irradiation with 1 macropulse from FELIX operated at a repetition rate of 10 Hz. Each pulse has an energy of 20 to 160 mJ and bandwidth of 0.5% of the center frequency. The IRMPD spectrum is obtained by plotting the photofragmentation yield R (R= −ln[1 − ∑I(fragment ions)/∑I(all ions)]) as a function of the wavenumber of the IR radiation. A linear correction is applied for variations in the laser power, and the wavelength is calibrated using a grating spectrometer.

Computational Details
The        [a]Vibrational frequencies calculated at the B3LYP/6-311++G(d,p) level of theory are scaled by a factor of 0.975. The computed intensities (km mol −1 ) are given in parenthesis. Bands with intensity lower than 20 km mol −1 are not reported.
[c] this calculated IR feature belongs of Nar_1a conformer    [a] Vibrational frequencies calculated at the B3LYP/6-311++G(d,p) level of theory are scaled by a factor of 0.975. The computed intensities (km mol −1 ) are given in parenthesis. Bands with intensity lower than 20 km mol −1 are not reported.

XYZ coordinates of optimized geometries of the most stable conformer of each family of rotamers/isomers.
Cartesian coordinates of all the other structures mentioned in this paper are available from the authors upon request.