1. Introduction
Salicylic acid (2-hydroxybenzoic acid, SA) is a widely used compound with applications in food science and pharmacology. It is a plant hormone that plays an important role in plant defense against stress through different mechanisms, having a significant impact on photosynthesis, transpiration, the uptake and transport of ions, or plant growth [
1]. SA was first identified in willow (genus
Salix) bark, which has been used since ancient times to alleviate pain and reduce fevers due to its analgesic, antipyretic, and anti-inflammatory properties [
2]. Furthermore, SA is the main precursor of aspirin [
3]. Nowadays, SA is widely employed in dermatology as a keratolytic and bacteriostatic agent [
4] or for the treatment of acne, psoriasis, and other cutaneous diseases [
5]. All these biological activities related to the physical and chemical properties of SA are defined by the molecular structure of this biomolecule.
The molecular structure of SA is governed by the
ortho disposition of its two functional carboxyl and hydroxyl groups. The possible intramolecular interactions in
ortho isomers confer them properties different from those corresponding to
meta or
para derivatives [
6,
7]. Moreover, in benzene derivatives, the presence of functional groups that are asymmetric, with respect to the substitution axis of the phenyl ring, is associated with an alteration of the ring structure and the electronic aromatic behavior. This is due to the stabilization of one of the canonical forms of the aromatic ring over the other, a phenomenon known as the angular-group-induced bond alternation (AGIBA) effect [
8,
9]. The knowledge of the molecular structure of SA and of the interaction between its two groups in
ortho disposition have been the subject of different investigations [
10,
11,
12,
13,
14,
15]. Two conformers of SA, I and II (see
Figure 1), were reported to exist from a study of the jet-cooled IR-UV double-resonance spectrum [
10]. By contrast, only the global minimum I form was identified from the free jet millimeter-wave absorption spectrum [
11]. Both I and II conformers present planar structures with an intramolecular hydrogen bond (HB) O-H⋯O from the hydroxyl group to the carbonyl (I) or the hydroxyl (II) moieties of the carboxylic acid group. The molecular structure of SA-I has been determined by electron diffraction [
12], suggesting that the intramolecular HB interaction is further stabilized by resonance-assisted hydrogen bonding (RAHB) [
16]. In other studies, SA has also been employed as a model of the investigation of keto-enol tautomerism observed by excited-state intramolecular proton transfer (ESIPT) [
13,
14,
15].
Investigation of microsolvated molecular systems is a relevant subject in chemistry and biochemistry [
17,
18,
19]. Microwave spectroscopy techniques are exceptional tools to determine their structures, adequate for this purpose due to their inherent high resolution [
20]. There are a high number of microwave studies of microsolvated organic molecules [
19,
21,
22,
23,
24,
25], providing models for a better understanding of the water interactions with biomolecules [
26], of hydrogen bond (HB) cooperativity [
27], and of the way in which solvation induces structural changes in the solute molecule [
22,
23,
27,
28]. These studies have also led to an understanding of the role of association processes and the interplay between the self-association of water and solvation. With few exceptions [
22], in complexes with several H
2O molecules, water prefers to link other water molecules, forming chains or cycles. When the solute has only one HB acceptor site, the structures reflect a balance between maximizing the number of water-solute interactions and the minimum-energy structures of the pure water clusters [
21]. In solutes with double HB donor/acceptor character, water molecules close sequential cycles, as observed in amides [
28], acids [
29,
30,
31,
32], or esters [
33]. In the last years, studies of molecules forming clusters with a high number of water molecules has increased. The microsolvation of benzene derivatives is also of interest [
34], since they are archetypal molecules in chemistry, and their water complexes serve as model chromophores to investigate solute-solvent interactions. Understanding these interactions in aromatic compounds is fundamentally important for studying more complex systems encountered in many natural chemical reactions [
34]. In the specific case of SA, the study of its microsolvates is particularly relevant and of interest in atmospheric chemistry [
35].
In a recent study [
36], we observed the presence of SA as a thermal recombination product of
o-anisic acid, together with methyl salicylate [
37], methyl 2-methoxybenzoate [
38], and their complexes with water [
38,
39]. Water could interact with SA in multiple ways due to the presence of several donor or acceptor groups in this molecule. As inferred from gas-phase microsolvation studies of related acids [
29,
31,
40,
41], the most favorable interaction sites correspond to the carboxyl group, with which water molecules may easily close cycles. Another aspect to consider is how microsolvation affects intramolecular interactions. A special situation is observed in the monohydrate of
o-anisic acid [
36], where each of the two observed conformers of the monomer forms its corresponding water cluster. One complex maintains an intramolecular interaction with a
trans-COOH arrangement, and the other presents a
cis carboxylic acid disposition that establishes the typical sequential ring of the carboxyl group with water.
In this work, we have taken advantage of the potential of the chirped-pulse Fourier transform microwave spectroscopy (CP-FTMW) technique, aided by supersonic jets, to study SA microsolvates with multiple water molecules. We present the gas-phase molecular structure of SA, determined through extensive isotopic species measurements. We have experimentally analyzed the structural properties of SA, such as the AGIBA effect [
8,
9] and the intramolecular HB interaction. We have observed and analyzed the spectra of several SA hydrates with up to four water molecules to gain information on how water molecules accumulate around an acid group, as well as information on the structures adopted by these clusters. Another aspect on which we have focused our work is the identification of pieces of evidence of cooperative effects that further stabilize HB intramolecular interactions in the complexes [
42] as the number of associated interacting molecules increases.
4. Conclusions
In this work, we have recorded the rotational spectra of SA, its monosubstituted 13C isotopologues, several 2H species, and the SA-wn (n = 1–4) hydrates using CP-FTMW spectroscopy. The analysis of the experimental data has been complemented with computational chemistry calculations, including NBO, NCI, and QTAIM analyses, to gain a better understanding of the structural behavior of SA and its hydrates and to characterize the different inter- and intramolecular HB interactions established in these species.
The experimentally determined SA structure has allowed us to characterize the O-H⋯O intramolecular HB between the carboxylic acid and the hydroxyl functional group in ortho position. This HB forms a sequential six-membered cycle further stabilized by π cooperativity (RAHB), which reinforces the planarity and rigidity of the molecule. The AGIBA effect governed by the alcohol group is reflected in the benzene ring bond lengths. However, in proximity to the carboxylic acid or hydroxyl groups, the structure of the ring appears to be dominated by the RAHB effects.
The analysis of the spectra of the hydrated complexes revealed interesting insights into how water aggregates around an acid group. As we have already mentioned, the possible aggregates around the phenolic OH group all have high energies (see
Figures S2–S5). In the complex phenol-w, the O-H group acts preferably as a proton donor. The corresponding structure is predicted to be much more stable than the form in which water behaves as the proton donor [
83]. However, in the most stable forms of SA, the proton donor capacity of the phenolic OH group is employed in the intramolecular interaction, so the possible SA-water complexes with water interacting with the phenol O-H group are expected to have higher energies, as it is confirmed by the calculations.
In the mono-, di-, and trihydrated clusters, water, or its dimer or trimer, forms a chain, closing a sequential cycle with the two HB donor OH and acceptor C=O ends of the carboxyl group. In the tetrahydrated species, water molecules form a tetramer cycle, bonding to the SA carboxyl group with contacts similar to those established in the SA-w
2 cluster. While the mono- and dihydrated clusters maintain the planarity of the heavy atom skeleton of the complex, the spectra of SA-w
3 and SA-w
4 evidence of their non-planarity, as shown by the planar moment values
Pcc, further corroborated by the theoretical computations. The trihydrated cluster has two of the water molecules slightly out of the plane. In the SA-w
4 complex, two of the water molecules are in the plane of SA bonded to the carboxylic acid group, keeping the form of SA-w
2 species, while the other two lie above the plane of SA in an arrangement where the plane of the water molecules presents an angle of about 110° with respect to the SA-w
2 plane. The cyclic structures of the hydrates of SA are comparable with those reported by Howard and coworkers on organic acid mono-, di-, and trihydrates [
29,
30,
31,
32]. However, in contrast with the non-planar heavy atom skeleton observed here for the trihydrate of SA, the trihydrates of di- and trifluoroacetic acids seems to have nearly planar heavy atom skeletons.
In the work on formamide-(H
2-O)
3 complexes [
28], the structural relationship between the formamide-(H
2O)
n clusters and the pure water clusters (H
2O)
n+2 was pointed out. The structures determined in this work for the SA-w
n complexes could be related in the same way to the structures determined or predicted for the (H
2O)
n+2 clusters [
20,
64,
65,
66,
84]. The pieces of evidence found of the enhanced hydrogen bonding (HB), due to the cooperativity associated with the increased number of water molecules in the clusters, are remarkable. The evolution of the HB features reflected in the changes of the O··H and O⋯O distances, the BSSE corrected dissociation energies per HB, the HB strengths estimated from the QTAIM analysis, and the stabilizing delocalization energies predicted by the NBO calculations evidence the existence of σ cooperativity. The differences between the intermolecular HBs of I-w-a species and those of the monohydrated species of benzoic acid [
40] indicate the strong influence of the intramolecular interaction altering the proton acceptor properties of the C=O functional group. In conclusion, the interaction with the water molecules in the clusters increases the strength of the intramolecular HB, which is the dominant interaction in complex