The eight structures of caffeic acid: a jet-cooled laser ablated rotational study

Abstract

This work reports a complete conformational analysis of caffeic acid, an exceptionally versatile pharmacophore, using laser ablation chirped-pulse Fourier transform microwave spectroscopy. The whole conformational space consisting of eight distinct species has been fully deciphered based on the trend of the rotational constants supported by theoretical computations. We show how rotational spectroscopy can be confidently used to distinguish between conformers even when the structural differences are minimal, such as those involved in the conformational panorama of caffeic acid. Additionally, the structural information here provided, such as the planarity observed in all the conformers, could help to elucidate the mechanisms underlying the biological and pharmacological activity of hydroxycinnamic acids.

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Introduction

In recent decades, natural products have been portrayed as essential scaffolds in drug design and discovery.¹ Despite a myriad of small organic and biomolecules employed in the search for potential therapeutical agents, phenolic acids stand above the rest.^2–5 This family of compounds is widespread in nature, with over 50 000 diverse species identified so far,^6,7 and can be classified into different groups according to their molecular structure.⁸ Among them, of paramount importance are hydroxycinnamic acids, which are present in nearly all plants.^9–11 The principal representative hydroxycinnamic acid is caffeic acid (trans-3-(3,4-dihydroxy phenyl)prop-2-enoic acid), commonly found in food as several simple derivatives such as esters and amides.^12,13 The pharmacological profile of this molecule and its derivatives is quite broad, highlighting their potential use as anti-cancerigenous^14–17 and anti-inflammatory agents,^18–20 as well as manifesting a remarkable antioxidant and scavenging activity.^21–24 It is well known that the aforementioned biomedical properties mainly rely on the corresponding structure–activity relationship.²⁵ Hence, deciphering the shape of these species at the molecular scale is crucial to better understand the mechanisms underlying their biological activity.

What can we expect from the conformational panorama of caffeic acid? Based on purely chemical intuition, we can anticipate a conformational landscape consisting of eight distinct structures due to four different torsional degrees (see Fig. 1). In this context, several experimental investigations have been devoted to studying this archetypal hydroxycinnamic acid using IR, NMR, UV, terahertz, and X-ray diffraction techniques.^26–28 Surprisingly, the prior experimental scenario delineated a somewhat simple conformational panorama of only one configuration.²⁶ Note that these studies are limited to condensed phases, where intermolecular interactions with the surrounding media might alter or even restrict conformational behavior. Thus, a thorough investigation under the isolation conditions of the gas phase is mandatory to provide compelling information on the intrinsic conformational behavior of caffeic acid.

Fig. 1 The entire conformational space of caffeic acid.

High-resolution microwave spectroscopy is a spectroscopic technique capable of facing this challenging problem. Rotational spectroscopy is extremely sensitive to the molecular geometry so that conformers of caffeic acid can be discerned as totally different species, and the assignments rely on the consistency of many rotational lines measured for each conformer. One unique aspect of rotational studies is that even very subtle changes in the molecular structure are translated into a distinctive array of spectroscopic constants.^29–31 Nevertheless, the major constraint of transferring caffeic acid to the gas phase is its solid nature (mp 213 °C) and its inherent vaporization problems. Nowadays, it has become possible to explore the architecture of solid biomolecules in the gas phase by techniques that couple laser ablation (LA), for transferring them intact into the gas phase, with high-resolution Fourier transform microwave (FTMW) techniques in a supersonic expansion. The low-temperature environment of a supersonic expansion provides the ideal medium for preparing individual conformers of caffeic acid in virtual isolation conditions, ready to be interrogated by a short burst of microwave radiation. The Fourier transformation of the temporal profile of the emerging radiation yields rotational spectra of unprecedented resolution and sensitivity. These combined techniques have been established as an exceptionally robust tool to investigate the conformational landscape of many solid biomolecules^31–35 and, particularly, systems showing intricate conformational panoramas.^35–40 This experimental approach has been recently extended to the related trans-cinnamic and trans-p-coumaric phenolic acids.⁴¹

In this work, we have examined the gas phase structure of caffeic acid using our broadband LA-CP-FTMW technique.^33,42 As we will see below, comparing the experimental spectroscopic constants with those predicted theoretically enables us to conclusively identify the conformers of caffeic acid. These findings are of interest to unveil the role of caffeic acid as a versatile pharmacophore and lead the search for more effective caffeic acid derivatives.

Experimental methods

Caffeic acid was obtained commercially and used without further purification. The sample was pulverized and mixed with a small amount of commercial binder, which was needed to provide enough consistency to the rod. Then it was pressed into a cylindrical die to obtain a rod, which was vaporized by the third harmonic (355 nm) of a Nd:YAG picosecond laser. We use a motor controller, which allows a DC motor to rotate and translate the rod up and down along the injection system to achieve homogeneous exploitation of the solid rod. The ablation products, seeded in a flow of neon at a stagnation pressure of 12 bar, were supersonically expanded and characterized by chirped pulse Fourier transform microwave spectroscopy. Details of the instrumental setup working from 2–8 GHz have been reported elsewhere.^43,44 In this experiment, chirped pulses of 4 μs directly generated by the 24 GS s⁻¹ arbitrary waveform generator (Tektronix AWG7122B), were amplified to about 300 W peak power using a traveling wave tube amplifier (IFI, GT186-300). The amplified pulse was broadcasted through microwave horn antennas inside the vacuum chamber, perpendicularly intercepting the pulsed-jet expansion. At a repetition rate of 2 Hz, a total of 62 000 free induction decays (4 FID emissions per gas pulse), each with a 10 μs length, were averaged and digitized using a 50 GS s⁻¹ digital oscilloscope. The frequency-domain spectrum in the 2.75–6.50 GHz frequency range (see Fig. S1a†) was obtained by taking a fast Fourier transform (FFT) following the application of a Kaiser–Bessel window to improve the baseline resolution. We usually attain line widths (full-width-half-maximum, FWHM) of approximately 100 kHz due to the perpendicular direction of the sample injection with respect to the microwave field, which is further translated in a relatively short transit time of the polarized molecular jet. Frequency measurements were extended up to 14 GHz (see Fig. S1b†) using a parabolic reflector system described in detail elsewhere.^42,45 We estimate the uncertainty of the line measurements in both configurations to be better than 20 kHz.

Results and discussion

Theoretical modelling

Prior to the experimental work, we extended the previous DFT calculations on the conformational space of caffeic acid⁴⁶ and performed additional computations to predict molecular conformational properties relevant to the investigation of the rotational spectrum. The method of choice was the hybrid density functional B3LYP, including the Grimme dispersion^47,48 with Becke–Johnson damping.^49,50 As anticipated, the complete conformational panorama consists of eight distinct conformations (see Fig. 1) of remarkable stability, all lying below 400 cm⁻¹. They are therefore expected to be observed in the supersonic jet. Table 1 provides rotational constants and electric dipole moment components to guide the spectral searches and assignments.

Table 1 Calculated spectroscopic parameters for the caffeic acid conformers

	Syn-				Anti-
Parameter	Cis-a	Cis-b	Trans-a	Trans-b	Cis-b	Cis-a	Trans-b	Trans-a
a A, B, and C represent the rotational constants (in MHz); μa, μb and μc are the electric dipole moment components (in D).b Relative energies (in cm^−1) with respect to the global minimum, taking into account the zero point energy (ZPE).c Gibbs energies (in cm^−1) were calculated at 298 K. Theoretical computations were carried out at the B3LYP-GD3/6-311++G(d, p) level of theory.
A^a	2269.4	2269.4	2279.1	2279.6	2479.6	2479.1	2463.5	2463.4
B	321.5	322.1	323.1	323.7	309.6	309.9	312.2	312.5
C	281.6	282.1	283.0	283.4	275.2	275.4	277.1	277.3
μa	1.7	4.2	2.1	4.6	2.0	3.3	2.8	4.2
μb	3.0	1.2	0.2	4.0	0.9	3.8	3.5	1.2
μc	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
ΔEZPE^b	0	139	145	334	65	213	276	364
ΔG^c	0	161	133	336	44	135	223	364

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The candidate structures can be sorted into two syn and anti families according to the different orientations of the pendant ring with respect to the meta-OH group (see Fig. 1). As seen in Table 1, this event gives rise to a specific and substantial change in their moments of inertia, which is further transferred into the rotational constants. For each family, four different conformers arise from a variation in the disposition of the terminal carboxyl group (cis or trans) or a change in the orientation of the catechol hydroxyl group (clockwise, a, and counterclockwise, b). As shown in Table 1, the values of rotational constants are extremely close within each family because of their similar mass distribution. We can thus anticipate that the analysis of the rotational spectrum will be a difficult task.

Broadband LA-CP-FTMW rotational spectrum

We recorded the broadband spectrum of caffeic acid in the 2.75–6.50 GHz range using our laser ablation chirped-pulse Fourier transform microwave (LA-CP-FTMW) spectrometer.^33,42 As usually in our LA experiments,⁵¹ we first removed instrumental artifacts and photo-fragmentation lines from the spectra, including those familiar to the previous study on trans-cinnamic and trans-p-coumaric acids.⁴¹ The resulting spectrum, shown in Fig. 2A, appears highly dense due to the anticipated noticeable number of rotameric species of caffeic acid. At first glance, guided by our theoretical computations, we identified strong progressions dominating the spectrum. They were ascribed to b-type R-branch transitions from a first rotamer I (✶). We extended the assignment to b-type Q-branches and weaker a-type R-branch lines. The corresponding rigid rotor analysis give rise to the experimental spectroscopic constants collected in the first column of Table 2. At first look, the experimental values of the rotational constants are consistent with those predicted for all the syn-family conformers. Rotamer I exhibits an intense b-type and a weak a-type rotational spectrum, according to the predicted values of the dipole moment components for syn-cis-a conformer in Table 1. Thus, it is reasonable to ascribe it to the global minimum syn-cis-a conformer as starting point.

Fig. 2 (A) Broadband CP-FTMW rotational spectrum of caffeic acid from 2.75 to 6.50 GHz showing characteristic R-branch progressions for rotamers I–IV; (B) remaining spectrum resulting from removing the lines ascribable to rotamers I–IV. Transitions belonging to rotamers V–VIII are depicted. Intensity is given in arbitrary units.

Table 2 Experimental spectroscopic parameters for the caffeic acid rotamers I–IV

	Rotamer I	Rotamer II	Rotamer III	Rotamer IV
a A, B, and C represent the rotational constants (in MHz); μa, μb, and μc are the electric dipole moment components.b Δ = Ic − Ia − Ib in μÅ^2. Conversion factor: 505 379.07 μÅ^2 MHz.c Rms deviation of the fit (kHz).d Number of fitted transitions.e Standard error in parentheses in units of the last digit.
A^a	2262.22348 (78)^e	2263.95 (20)	2271.20 (49)	2272.9750 (10)
B	321.88280 (31)	322.45623 (55)	323.51160 (12)	324.00522 (45)
C	281.96625 (23)	282.4286 (54)	283.36153 (98)	283.77359 (41)
Δ^b	−1.1	−1.1	−1.2	−1.2
μa	Observed	Observed	Observed	Observed
μb	Observed	—	—	Observed
μc	—	—	—	—
σ^c	14.4	11.6	18.2	14.9
N^d	55	16	14	27

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Continuing the analysis, we found satellite lines around the transitions of rotamer I that may be attributable to other syn-conformers. We succeeded in assigning three a-type R-branch progressions to the other three rotamers belonging to the syn family; II (●), III (✦), and IV ([hexagon filled, flat-side down]). The rotational constants derived from the corresponding rigid rotor analyses are also listed in Table 2, and the measured rotational transitions are collected in Tables S1–S4 of the ESI.†

At this point, we achieved a complete conformational identification based on a thorough investigation of the trend of the rotational constant's values comparing predicted and experimental changes (see Fig. 3). Hence, when going from rotamer I to rotamer II, the observed changes in the rotational constants are ΔA = 1.7 MHz, ΔB = 0.6 MHz, and ΔC = 0.5 MHz, which perfectly match with the theoretically predicted ones of ΔA = 0.0 MHz, ΔB = 0.6 MHz, and ΔC = 0.5 MHz in going from syn-cis-a to syn-cis-b (see Fig. 3). It points to the identification of rotamer I as syn-cis-a and rotamer II as syn-cis-b, respectively. Fig. 3 shows that the predicted changes in rotational constants in going from the syn-cis-b to syn-trans-a conformer are in excellent agreement with those derived from the experimental values of Table 2 when going from rotamer II to rotamer III, thus leading again to the conclusion that rotamer II is syn-cis-b. The observed changes between rotamers III and IV are only consistent with those calculated for the syn-trans-a and syn-trans-b conformers. Furthermore the b-type R-branch transitions observed for rotamer IV have a similar intensity compared to the a-type transitions, confirming that it belongs to the syn-trans-b conformer, which has a similar value for the predicted μ_a and μ_b dipole moments. Overall, the experimental compliance of the theoretical trend supports our identification and the consistency of the whole conformational assignment.

Fig. 3 Experimental and calculated changes in the values of the rotational constants for rotamers I–IV (in MHz).

Once the four syn conformers were analyzed, we removed all their signals from the broadband spectrum, generating the spectrum of Fig. 2B, on which the identification of the trans family conformers was addressed. We employed the same strategy to the anti-conformers and identified a dominant a-type R-branch progression attributable to rotamer V (■), which was tentatively assigned to the anti-cis-b conformer based on the dipole moment selection rules and line intensities. Then, we searched for lines that fall in the proximity of those belonging to rotamer V and found new a-type progressions ascribable to the three VI (✚), VII ([pentagon filled]), and VIII () rotamers. For rotamers VI and VII, b-type R-branch transitions were also observed. The experimental rotational constants derived from rigid rotor analysis are listed in Table 3, and the measured transitions are reported in Tables S5–S8 of the ESI.† The values of the rotational constants are consistent with those predicted for the anti-conformers in Table 1.

Table 3 Experimental spectroscopic parameters for the caffeic acid rotamers V–VIII

	Rotamer V	Rotamer VI	Rotamer VII	Rotamer VIII
a A, B, and C represent the rotational constants (in MHz); μa, μb and μc are the electric dipole moment components.b Δ = Ic − Ia − Ib in μÅ^2. Conversion factor: 505 379.07 μÅ^2 MHz.c Rms deviation of the fit (kHz).d Number of fitted transitions.e Standard error in parentheses in units of the last digit.
A^a	2473.82 (67)^e	2474.36056 (90)	2456.980 (13)	2458.81 (93)
B	309.7585 (85)	310.02508 (40)	312.2982 (48)	312.6236 (11)
C	275.4663 (83)	275.70948 (25)	277.28203 (49)	277.5706 (12)
Δ^b	−1.2	−1.4	−1.3	−1.4
μa	Observed	Observed	Observed	Observed
μb	—	Observed	Observed	—
μc	—	—	—	—
σ^c	13.9	14.3	15.4	16.1
N^d	14	40	22	7

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Once again, we need to inspect the trend of the rotational constant's values to decipher the subtle structural changes between each anti-conformer and attain a definite conformational identification. For the anti-family, these minimal differences (i.e., the reorganization of the OH groups) produce once more very mild yet specific shifts in the rotational constants, which serve as conclusive proof to assign each structure while “travelling” between conformers (see Fig. 4). Therefore, once we have identified rotamer V as the anti-cis-b conformer, we can tie rotamers VI, VII, and VIII to conformers anti-cis-a, anti-trans-b, and anti-trans-a, respectively. The small experimental changes observed in the values of the rotational constants are again in harmony with the calculated shifts at the B3LYP-GD3/6-311++G(d, p) level of theory in Fig. 4, corroborating the conformational identification. Additionally, the dipole moment selection rules and intensities allow us to confirm further the identification of rotamer VI and VII as conformers anti-cis-a and anti-trans-b.

Fig. 4 Experimental and calculated changes in the values of the rotational constants for rotamers V–VIII (in MHz).

Although we tried to estimate the relative population of the detected conformers based on the intensity measurement of the rotational transitions and the predicted dipole moments, any quantitative information is meaningless as the number of fitted transitions for some of the less stable species is small. Nevertheless, we found that the experimental relative population, based on the spectral intensity produced by each detected conformer, follows the tendency given by the theoretical relative energies in Table 1. This is another point supporting that B3LYP-GD3/6-311++G(d, p) is a good choice for caffeic acid.

Regarding the structure–activity relationship of caffeic acid, it is known that some of the most important features of potent antioxidants consist on the presence of planar phenylopropanoic moieties stabilized by resonance (mainly in radical and radical cation forms) as well as catechol hydroxyl groups (OH groups attached to a benzene ring). Moreover, they can be reinforced by the presence of additional electron donating functional groups such as hydroxyl and methoxyl groups.⁵² The eight structures of caffeic acid exhibit small negative values of the inertial defect, varying between −1.4 and −1.1 μŲ, which implies that all are planar with C_s symmetries. The small negative values are due to the contribution of the out-of-plane C(ph)–C–C–COOH torsion to the inertial deffect.^53–56 Note that c-type transitions were not observed, in accordance with the μ_c = 0 dipole moment component expected for planar structures. This structural feature, present in all the caffeic structures, highlight a deluge of possibilities for the caffeic acid scaffold to act as a pharmacophore.

Conclusions

The present study provides the first experimental information on naked caffeic acid. We have transferred the solid sample into the gas phase by laser ablation and probed it in the isolation conditions of a supersonic expansion by Fourier transform microwave spectroscopy. The spectroscopic data directly compared with those computated theoretically in vacuo constitutes an unmatched tool to achieve the unambiguous identification of the eight planar conformers of caffeic acid that constitute its conformational landscape. High-resolution rotational spectroscopy has allowed us to unveil subtle structural changes between each conformer. We are pushing the limits of rotational spectroscopy, highlighting its potential as an outstanding characterization technique for the investigation of very similar structures exhibiting analogous rotational constants, as in the case of caffeic acid.

The structural information provided in this work should be of interest to comprehend the physical–chemical properties shown by this archetypal hydroxycinnamic acid and shall ultimately shed light on the mechanisms underlying its biological and pharmacological activity. Additionally, our investigation may help as a preliminary step in designing new caffeic acid-based pharmacophores.

We have proven once more the superb potential of rotational spectroscopy on the structural study of relevant biomolecules. Within this framework, we plan to extend our research to unveil the shape of more natural products that have remained, until now, elusive for high-resolution rotational studies.

Conflicts of interest

The authors declare no competing financial interests.

Acknowledgements

The financial funding from Ministerio de Ciencia e Innovación (PID2019-111396GB-I00), and Junta de Castilla y León (VA244P20) is acknowledged. G. J. acknowledges funding from the Spanish “Ministerio de Ciencia, Innovación y Universidades” under the predoctoral FPI Grant (BES-2017-082173).

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Footnote

† Electronic supplementary information (ESI) available: In Section 1 we provide the original LA-CP-FTMW broadband spectrum of caffeic acid (Fig. S1). We collect in Section 2 a complete list of the measured frequencies and residuals (in MHz) for all the observed rotamers of caffeic acid (see Tables S1–S8). Finally, in Section 3 we display the Cartesian coordinates of the caffeic acid conformers optimized at B3LYP-GD3BJ/6-311++G(d, p), which are shown in Tables S9–S16. See DOI: https://doi.org/10.1039/d2ra07124j

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