β-Cyclodextrin Inclusion Complexes with Catechol-Containing Antioxidants Protocatechuic Aldehyde and Protocatechuic Acid—An Atomistic Perspective on Structural and Thermodynamic Stabilities

Protocatechuic aldehyde (PCAL) and protocatechuic acid (PCAC) are catechol derivatives and have broad therapeutic effects associated with their antiradical activity. Their pharmacological and physicochemical properties have been improved via the cyclodextrin (CD) encapsulation. Because the characteristics of β-CD inclusion complexes with PCAL (1) and PCAC (2) are still equivocal, we get to the bottom of the inclusion complexation by an integrated study of single-crystal X-ray diffraction and DFT full-geometry optimization. X-ray analysis unveiled that PCAL and PCAC are nearly totally shielded in the β-CD wall. Their aromatic rings are vertically aligned in the β-CD cavity such that the functional groups on the opposite side of the ring (3,4-di(OH) and 1-CHO/1-COOH groups) are placed nearby the O6–H and O2–H/O3–H rims, respectively. The preferred inclusion modes in 1 and 2 help to establish crystal contacts of OH⋅⋅⋅O H-bonds with the adjacent β-CD OH groups and water molecules. By contrast, the DFT-optimized structures of both complexes in the gas phase are thermodynamically stable via the four newly formed host–guest OH⋯O H-bonds. The intermolecular OH⋅⋅⋅O H-bonds between PCAL/PCAC 3,4-di(OH) and β-CD O6–H groups, and the shielding of OH groups in the β-CD wall help to stabilize these antioxidants in the β-CD cavity, as observed in our earlier studies. Moreover, PCAL and PCAC in distinct lattice environments are compared for insights into their structural flexibility.

The literature review above together with our recently reported crystal structures of β-CD complexes with catechol-containing antioxidants in olive [35] raise several questions, leading to a twofold hypothesis of this work. (i) Because PCAC and PCAL are highly structurally related and have an optimum size for the β-CD nanocavity, they probably form an equimolar complex with β-CD with a common inclusion of the aromatic moiety and the protrusion of 3,4-di(OH) group from the O6 side. (ii) Both complexes are thermo-dynamically stable due to the optimal host-guest interactions and the molecular stabilities of PCAC and PCAL are improved upon inclusion in the β-CD cavity. To validate the hypothesis statements and gain an atomic-level understanding on inclusion complexation, we investigate the β-CD encapsulation of PCAL (1) and PCAC (2) by a combined study of single-crystal X-ray diffraction and density functional theory (DFT) full-geometry optimization. Plus, PCAL and PCAC seem to be rather rigid. We further explore to what extent the two antioxidants adapt their rotatable CHO and COOH groups to distinct lattice circumstances (in the free form, encapsulated in β-CD, and in complex with protein).

Results and Discussion
Here, we adopt the atom numbering scheme as used in our previous works [36][37][38][39]. β-CD is conventionally numbered as carbohydrates, viz., atoms C63-O63 denote the methylene C6−H 2 connected to hydroxyl O6−H groups of glucose unit 3 (G3) in the complex β-CD−PCAL (1), Figure 1. PCAL and PCAC are numbered according to the corresponding IUPAC names and are additionally labeled with arbitrary letters L and D in the respective complexes 1 and 2, Figure 1.
The literature review above together with our recently reported crystal structures of β-CD complexes with catechol-containing antioxidants in olive [35] raise several questions, leading to a twofold hypothesis of this work. (i) Because PCAC and PCAL are highly structurally related and have an optimum size for the β-CD nanocavity, they probably form an equimolar complex with β-CD with a common inclusion of the aromatic moiety and the protrusion of 3,4-di(OH) group from the O6 side. (ii) Both complexes are thermodynamically stable due to the optimal host-guest interactions and the molecular stabilities of PCAC and PCAL are improved upon inclusion in the β-CD cavity. To validate the hypothesis statements and gain an atomic-level understanding on inclusion complexation, we investigate the β-CD encapsulation of PCAL (1) and PCAC (2) by a combined study of single-crystal X-ray diffraction and density functional theory (DFT) full-geometry optimization. Plus, PCAL and PCAC seem to be rather rigid. We further explore to what extent the two antioxidants adapt their rotatable CHO and COOH groups to distinct lattice circumstances (in the free form, encapsulated in β-CD, and in complex with protein).

β-CD Macrocycles Are Affected to Some Extent by Inclusion of PCAL and PCAC
The molecular structures of CD hydrates are usually round. The uncomplexed α-, β-, and γ-CDs have respective pseudo-6-, 7-and 8-fold rotational axes passing through the molecular centroid and perpendicular to the molecular plane (O4 plane). This is due to the small differences of the composing glucose units, particularly the rotatable O6-H (freely) and O2-H/O3-H (restricted due to the interglucose H-bonds) groups lining the narrower and wider perimeters, respectively. From one to another inclusion complex, CD macrocycles adapt to some extent to optimally fit to various aromatic guest molecules. Therefore, the host-guest structural changes induced each other upon inclusion complexation deserve a detailed discussion. The inclusion geometries of various polyphenols containing 3,4-dihydroxybenzene in the β-CD cavity are compared in Table 1 and discussed in Section 2.2.

β-CD Macrocycles Are Affected to Some Extent by Inclusion of PCAL and PCAC
The molecular structures of CD hydrates are usually round. The uncomplexed α-, β-, and γ-CDs have respective pseudo-6-, 7-and 8-fold rotational axes passing through the molecular centroid and perpendicular to the molecular plane (O4 plane). This is due to the small differences of the composing glucose units, particularly the rotatable O6-H (freely) and O2-H/O3-H (restricted due to the interglucose H-bonds) groups lining the narrower and wider perimeters, respectively. From one to another inclusion complex, CD macrocycles adapt to some extent to optimally fit to various aromatic guest molecules. Therefore, the host-guest structural changes induced each other upon inclusion complexation deserve a detailed discussion. The inclusion geometries of various polyphenols containing 3,4-dihydroxybenzene in the β-CD cavity are compared in Table 1 and discussed in Section 2.2.  [35]. c One CFA and two CGA molecules in the β-CD dimeric cavity [36]. d Interplanar angle between polyphenol aromatic ring (AR) and CD molecular plane (common O4 mean plane). e Free OH including OH of COOH that are available for intermolecular H-bonding. f When the CD O6-side pointing upwards, the positive(negative) values indicate that the AR centroid of guest is above(beneath) the host molecular plane.
The H-bonding functionality of the β-CD OH groups affecting its structure should be further noted. Whereas O6-H groups are freely rotatable to make H-bonding to the surrounding water molecules and β-CD OH groups, O2-H/O3-H groups are more restricted due to their engagement in the interglucose flip-flop O3···O2 H-bonds [44]. The systematic intramolecular O3···O2 H-bonds act like a belt to secure the annular β-CD conformation, thus these OH groups are less available for interaction intermolecularly. The glucose tilt angle and the interglucose O3(n)···O2(n + 1) distances are closely related parameters, as indicated by the similar peak positions of the radar plots (Figure 2a,b). This is notable in the β-CD-EC inclusion complex [43], of which β-CD is more distorted from a round conformation. The large tilt angles of G1 and G4 (33.7(1) • and 30.6(1) • ) result in longer O31···O22 and O33···O24 distances (3.346 (3) Table S2. O6-H groups contribute significantly to the lattice stability through making OH· · · O hydrogen bond networks, particularly for the complex β-CD-PCAC (2) (Section 2.3, Figure 5, and Tables S3 and S4).

Common Inclusion Geometry of Catechol-Containing Antioxidants
The polyphenolic plant extracts containing an ortho-dihydroxybenzene (catechol) are powerful antioxidant [45] due to the greater molecular stability of intramolecular O-H· · · O H-bond [46] (Figure 1 and Table 2). Principally, on the basis of host-guest space-fit, the opposed 3,4-di(OH) and 1-COOH/1-CHO groups on the aromatic ring of PCAL/PCAC could be placed nearby both β-CD perimeters, giving two equally plausible inclusion modes. However, the belt of O2· · · O3 H-bonds make O2-H, O3-H groups less available and O6-H groups are more ready for H-bonding. Moreover, our previous investigations on the β-CD encapsulation of olive HTY, OLE [35] and coffee CFA, CGA [36] showed that all the guest polyphenols prefer pointing their 3,4-di(OH) groups to the β-CD O6-H side for the energetically stable inclusion complexation. The thermodynamic stabilities of β-CD inclusion complexes with PCAL and PCAC relative to other relevant complexes as inferred from DFT full-geometry optimization are given in Section 2.4. All together, we envisage that both structurally related antioxidants, PCAL and PCAC, are similarly entrapped in the β-CD cavity. The details of inclusion structures in 1 and 2 revealed by X-ray analysis are described below.  In the limited space of the β-CD nanocavity, the aromatic guest molecule with parasubstituted groups does not rotate freely but prefers aligning the aromatic ring (AR) about a right angle against the β-CD molecular plane (O4 plane). This is the case for PCAL (1) and PCAC (2), as indicated by the respective interplanar angle of 74.9(1) • and 71.5(1) • , Table 1. Their AR centroids are 0.787 Å above and 0.739 Å beneath the β-CD O4 plane, facilitating 3,4-di(OH) group nearby the O6-side to H-bond with the adjacent water molecules and O3-H group, and CHO, COOH groups around the O2/O3-side to interact with the surrounding O3-H, O6-H and water molecule ( Figure 4 and Table 2). This results in O-H· · · O H-bonding network stabilizing the solid structures of 1 and 2 ( Figure 5 and Table 2, Tables S3 and S4). The atomic-level inclusion structures of 1 and 2 from single-crystal X-ray diffraction go beyond the overall solution structures derived spectroscopically [27,29].
Moreover, a question rises how 1 and 2 are compared to other polyphenolic complexes and their meanings. Similar inclusion geometries of olive HTY, OLE in the β-CD cavity are observed for the equimolar complexes [35]. However, coffee CFA and CGA with additional CH 2 =CH-C(=O)O and quinic acid (QNA) moieties are bulkier than PCAL and PCAC. This requires an extended space of the β-CD dimeric cavity to optimally anchor CFA and CGA [36]. Certainly, the distinct inclusion structures are observed as only the QNA portion of CGA is outside the wall while CFA is totally embedded in the dimeric cavity, Table 1 [36]. The AR moieties of CFA and CGA are oriented mostly vertically in a larger space of the β-CD dimer interface, Table 1 [36]. Note that the intermolecular OH· · · O H-bonds between o-di(OH) of catechol antioxidants and β-CD OH groups and the shielding of OH groups in the β-CD wall help to improve molecular stability and antioxidant activity in the β-CD cavity [35,36,43]; see Section 2.4. cavity are observed for the equimolar complexes [35]. However, coffee CFA and CGA with additional CH2=CH-C(=O)O and quinic acid (QNA) moieties are bulkier than PCAL and PCAC. This requires an extended space of the β-CD dimeric cavity to optimally anchor CFA and CGA [36]. Certainly, the distinct inclusion structures are observed as only the QNA portion of CGA is outside the wall while CFA is totally embedded in the dimeric cavity, Table 1 [36]. The AR moieties of CFA and CGA are oriented mostly vertically in a larger space of the β-CD dimer interface, Table 1 [36]. Note that the intermolecular OH⋯O H-bonds between o-di(OH) of catechol antioxidants and β-CD OH groups and the shielding of OH groups in the β-CD wall help to improve molecular stability and antioxidant activity in the β-CD cavity [35,36,43]; see Section 2.4.     Table 2,  Table S3). In 2, the extensive complex H-bond network of the adjacent entrapped PCAC molecules as a result from the presence of COOH group is notable. The two H-bond chains linking one PCAC to others are built from bridging water molecules and β-CD O6-H groups: (i) O1D-H· · · O1W-H· · · O67-H· · · O3D; and (ii) O4D-H· · · O3W-H· · · O63· · · H-O2W· · · H-O2D ( Figure 5b and Table 2, Table S4).  Table 2. In 1, the intramolecular O34 H···O25 H-bond stabilizing the round β-CD structure (blue line) is supported by the water network. In 2, PCAC molecul embedded in the neighboring β-CD cavities are linked via bridging water molecules and β-CD O6-H groups. The β-C cavities accommodating the PCAL and PCAC molecules are omitted for clarity. The ORTEP diagrams are drawn wi 30% probability level.  [35]). Names in italics indicate molecules/groups of the adjacent asymmetric units; see also Figure 4 and Table 2. In 1, the intramolecular O34-H···O25 H-bond stabilizing the round β-CD structure (blue line) is supported by the water network. In 2, PCAC molecules embedded in the neighboring β-CD cavities are linked via bridging water molecules and β-CD O6-H groups. The β-CD cavities accommodating the PCAL and PCAC molecules are omitted for clarity. The ORTEP diagrams are drawn with 30% probability level. The well-ordered six water molecules of hydration distributed outside the β-CD cavity, in the intermolecular interstices make H-bonds to the surrounding OH groups of both β-CD rims and to each other. They play a key role as H-bonding mediators in stabilizing the crystal lattice ( Table 2, Tables S3 and S4). Note that the PCAL and PCAC 3,4-di(OH) groups (the key moiety contributing to the antioxidant capacity of flavonoids) in 1, 2 and the β-CD-olive HTY are stabilized through H-bonding to the adjacent β-CD OH groups and water molecules (Figure 5c; [35]). What would happen if the crystal contacts were absent, i.e., the sole host-guest interactions were considered? (see Section 2.4).

Thermodynamic Stabilities of β-CD-PCAL and β-CD-PCAC
A structural chemistry study including DFT complete-geometry optimization and single-crystal X-ray diffraction share several important complementary results. Whereas X-ray analysis provides accurately determined space-and-time average crystal and molecular structures of the crystallizable compounds, computational chemistry (particularly the popular DFT method) gives relevant thermodynamic quantities of molecules and complexes. For the somewhat flexible, large-ring CDs, the available X-ray-derived atomic coordinates were usually used as a starting structure for a better convergence of the DFT energy minimization. The DFT full-geometry optimization in vacuum was adequate to provide meaningful thermodynamic quantities of CD supramolecular complexes in reasonable computing time (especially the stabilization energies). The DFT calculation in implicit solvent took much longer computing time but did not improve the host-guest complexation energies as pointed out in our previous works on the β-CD-tea catechin [43] and β-CD-tricyclic antidepressant [37].
Here, the DFT full-geometry optimization in the gas phase converged smoothly. The host β-CD structures in both complexes are changed to some extent after DFT energy minimization but are still superimposable on the original X-ray structures, as indicated by the rms fits of 0.508 Å (β-CD-PCAL) and 0.478 Å (β-CD-PCAC), Figure 6 and Figure S1; the calculations considered only the non-H atoms of β-CD backbone, excluding O6. Moreover, the O2· · · O3 distances of neighboring glucoses in the DFT-derived β-CD structures tend to be lengthened, compared to the original X-ray structure (Tables S3-S5). This is to facilitate the large tilting of glucose units G3 (30.6 • ) and G2 (27.8 • ) to better space-fit the rising guest molecules, PCAL and PCAC, respectively (see the cyan arrows in Figure 6). Clearly, this is a paradigm of the well-known induced-fit mechanism [47] necessary for CD inclusion complexes and other biological molecules [48]. In both DFT-and X-ray-derived structures, the comparatively round β-CDs are maintained by systematic interglucose O3· · · O2 H-bonds (Tables S3-S5).
Whereas the complex energies (E cpx s) of β-CD-PCAL and β-CD-PCAC cannot be directly compared due to the slight chemical composition differences of the guest molecules, their stabilization and interaction energies (∆E stb and ∆E int ) are applicable. For both complexes, the values of ∆E stb and ∆E int , −6.66 to −23.88 and −12.55 to −30.53 kcal mol -1 are comparable to those of β-CD complexes with other polyphenols, olive HTY, OLE [35] and coffee CFA, CGA [36], and fall in a regular energy range for weak non-covalent interactions (Table S6). Note that although both β-CD-PCAL and β-CD-PCAC complexes have the same type and number of host-guest OH· · · O H-bond interactions (4), β-CD-PCAC is 17.22 and 17.98 kcal mol -1 more stable than β-CD-PCAL, based on ∆∆E stb and ∆∆E int (Tables S5 and S6). This is due mainly to β-CD of β-CD-PCAC is less stable (more deformed) than that of β-CD-PCAL by 13.72 and 13.28 kcal mol -1 , based on their energy differences, ∆∆E β-CD _ opt and ∆∆E β-CD _ sp (Table S6). Moreover, the DFT-optimized structures of both complexes in the gas phase have ascending shifts of PCAL and PCAC to the β-CD O6-H-side for energetically stable inclusion geometries, which are maintained by four intermolecular OH· · · O H-bonds on both β-CD rims. These four newly formed hostguest OH· · · O H-bonds are present in the gas phase to compensate the thermodynamic stability lose due to the extinction of crystal contacts (Figure 7, Figure S1 and Table 2,  Tables S5 and S6). However, the contrary is observed in solution. β-CD-PCAL is at least one order of magnitude more stable than β-CD-PCAC, as indicated by the respective binding constants of 6700 ± 777 M -1 (UV-vis) [29] and 264 ± 29 M −1 (UV-vis) [28], 661 M −1 (NMR) [27].  Figure S1 and Tables S5 and S6. The rms fits are calculated for the non-H atoms using the X-ray-derived β-CD skeleton as a reference structure (excluding O6 atoms). Note that after DFT full-geometry optimization, PCAL and PCAC molecules are shifted up to form O1/O2···O6 H-bonds with β-CD O6-H perimeter ( Table 2).
Whereas the complex energies (Ecpxs) of β-CD-PCAL and β-CD-PCAC cannot be directly compared due to the slight chemical composition differences of the guest molecules, their stabilization and interaction energies (ΔEstb and ΔEint) are applicable. For both complexes, the values of ΔEstb and ΔEint, −6.66 to −23.88 and −12.55 to −30.53 kcal mol -1 are comparable to those of β-CD complexes with other polyphenols, olive HTY, OLE [35] and coffee CFA, CGA [36], and fall in a regular energy range for weak non-covalent interactions (Table S6). Note that although both β-CD-PCAL and β-CD-PCAC complexes have the same type and number of host-guest OH⋯O H-bond interactions (4), β-CD-PCAC is 17.22 and 17.98 kcal mol -1 more stable than β-CD-PCAL, based on ΔΔEstb and ΔΔEint (Tables S5 and S6). This is due mainly to β-CD of β-CD-PCAC is less stable (more deformed) than that of β-CD-PCAL by 13.72 and 13.28 kcal mol -1 , based on their energy differences, ΔΔEβ-CD_opt and ΔΔEβ-CD_sp (Table S6). Moreover, the DFT-optimized structures of both complexes in the gas phase have ascending shifts of PCAL and PCAC to the β-CD O6-Hside for energetically stable inclusion geometries, which are maintained by four intermolecular OH⋯O H-bonds on both β-CD rims. These four newly formed host-guest OH⋯O H-bonds are present in the gas phase to compensate the thermodynamic stability lose due to the extinction of crystal contacts (Figure 7, Figure S1 and Table 2, Tables S5 and S6). However, the contrary is observed in solution. β-CD-PCAL is at least one order of magnitude more stable than β-CD-PCAC, as indicated by the respective binding constants of 6700 ± 777 M -1 (UV-vis) [29] and 264 ± 29 M −1 (UV-vis) [28], 661 M −1 (NMR) [27].  Figure S1 and Tables S5 and S6. The rms fits are calculated for the non-H atoms using the X-ray-derived β-CD skeleton as a reference structure (excluding O6 atoms). Note that after DFT full-geometry optimization, PCAL and PCAC molecules are shifted up to form O1/O2···O6 H-bonds with β-CD O6-H perimeter ( Table 2). Among various β-CD-polyphenol complexes (Table S6), the complex with olive OLE is most stable due to the greater number of host-guest OH⋯O H-bonds (6). By contrast, the β-CD-tricyclic antidepressant (TCA) complexes with the aromatic moiety entrapped and primarily kept in position by CH⋯π interactions [37,38]. The corresponding ΔEstb and ΔEint about −4 to −8 kcal mol -1 of the TCA complexes indicate that they are ~2-4 times less stable than the H-bond-stabilized complexes of polyphenols. Among various β-CD-polyphenol complexes (Table S6), the complex with olive OLE is most stable due to the greater number of host-guest OH· · · O H-bonds (6). By contrast, the β-CD-tricyclic antidepressant (TCA) complexes with the aromatic moiety entrapped and primarily kept in position by CH· · · π interactions [37,38]. The corresponding ∆E stb and ∆E int about −4 to −8 kcal mol -1 of the TCA complexes indicate that they are~2-4 times less stable than the H-bond-stabilized complexes of polyphenols.

Bioactive PCAL and PCAC Are Rather Rigid in Distinct Lattice Circumstances
Theoretically, the induced-fit mechanism [47] of the CD inclusion complexation works such that both host and guest molecules adapt their structures to some extent to attain a thermodynamically stable adduct. We point out in Section 2.1 that the complexed β-CDs in 1 and 2 are distorted to an extent from a round conformation of the uncomplexed β-CD·12H 2 O [42], as indicated by the respective rms fits of 0.277 and 0.708 Å (Section 2.1). However, a reverse situation is observed for the guest PCAL and PCAC molecules as clearly depicted in Figure 8a The corresponding rms fit for each structure pair is indicated by nearby distance. Note that the orientation of C7=O3 group with respect to O1 is emphasized by ball model. Figure 8a displays the perfect overlay of PCAL in the β-CD cavity and uncomplexed PCAL [51], as indicated by the small rms fit of 0.040 Å, excluded the rotatable O3L (flipped to other side, see balls). CHO groups are in the same plane of the aromatic ring with the small rms deviations of least squares plane of 0.013 and 0.024 Å for the embedded and free PCAL, respectively. PCAC·H2O in the triclinic and monoclinic modifications [52][53][54] are isostructural and their COOH groups are coplanar with the aromatic ring because these COOH groups are engaged in the intermolecular OH⋯O H-bonds of the planar PCAC dimer. The planarities of four PCAC molecules in the triclinic asymmetric unit are indicated by the small rms deviations of least squares plane of 0.016-0.035 Å. By contrast, PCAC in complex with β-CD and with Lox [55] allow their COOH groups to deviate from the aromatic plane although their 3,4-dihydroxybenze structures are mostly identical; rms fit = 0.062 Å (Figure 8b). The twisting of the COOH group from the PCAC molecular plane facilitates the H-bonding network in the lattice of the β-CD-PCAC complex (Figure 5b) and the better binding of PCAC in the protein active site [55]. The corresponding rms deviations of least squares plane are 0.051 and 0.221 Å.

Materials
β-CD (>95%) as white solid powder was obtained from Cyclolab, Budapest, Hungary (code CY-2001). PCAL (97%) as tan solid powder was supplied by Sigma-Aldrich, St. Louis, MO, USA, (code D108405). PCAC (97%) as brown crystalline powder was pur- To gain further insight into structural changes of the bioactive PCAL and PCAC, we made a survey through two crystal data banks of small molecules at the Cambridge Crystallographic Data Center (CCDC; www.ccdc.cam.ac.uk; [49]) and of biological molecules at the RCSB Protein Data Bank (RCSB PDB; www.rcsb.org; [50]). The CCDC database search revealed that there is one structure of free PCAL anhydrate (code MASBUD; [51] and two polymorphs of PCAC·H 2 O in the triclinic system (code BIJDON03; [52]; code BIJDON05; [53]) and monoclinic system (code BIJDON04; [54]). Surprisingly, there is just one published data set available in the PDB for PCAC in complex with lipoxygenase (Lox) (code 1N8Q; [55]). Loxs are nonheme iron-containing enzymes for catalysis of lipid deoxygenation, producing the unsaturated fatty acid metabolites, which influence inflammatory diseases and cancer progression. PCAC is a degradation product found nearby the iron site in the soy Lox-quercetin complex [55]. Quercetin in alkaline solution is degraded to PCAC, which is significantly decreased when the catechol moiety of quercetin is embedded in various β-CD cavities [56]. Figure 8a displays the perfect overlay of PCAL in the β-CD cavity and uncomplexed PCAL [51], as indicated by the small rms fit of 0.040 Å, excluded the rotatable O3L (flipped to other side, see balls). CHO groups are in the same plane of the aromatic ring with the small rms deviations of least squares plane of 0.013 and 0.024 Å for the embedded and free PCAL, respectively. PCAC·H 2 O in the triclinic and monoclinic modifications [52][53][54] are isostructural and their COOH groups are coplanar with the aromatic ring because these COOH groups are engaged in the intermolecular OH· · · O H-bonds of the planar PCAC dimer. The planarities of four PCAC molecules in the triclinic asymmetric unit are indicated by the small rms deviations of least squares plane of 0.016-0.035 Å. By contrast, PCAC in complex with β-CD and with Lox [55] allow their COOH groups to deviate from the aromatic plane although their 3,4-dihydroxybenze structures are mostly identical; rms fit = 0.062 Å (Figure 8b). The twisting of the COOH group from the PCAC molecular plane facilitates the H-bonding network in the lattice of the β-CD-PCAC complex (Figure 5b) and the better binding of PCAC in the protein active site [55]. The corresponding rms deviations of least squares plane are 0.051 and 0.221 Å.

Crystallization
In each separate 1.5-mL vial, the 1:2 solid mixtures of β-CD-PCAL (1) and β-CD-PCAC (2) prepared from β-CD 50 mg (0.044 mmol), PCAL 12 mg (0.088 mmol) and PCAC 14 mg (0.088 mmol) were dissolved in pure water 1 mL. After heating at 323 K for three hours in an ultrasonic bath, light yellow concentrated solutions of 1 and 2 were obtained. Then the two vials containing solutions were left undisturbed in an air-conditioned room (298 K). Slow solvent evaporation took place for two weeks, single crystals were harvested.

Diffraction Data Collection
Colorless platelet single crystals with dimensions of 0.02 × 0.14 × 0.40 mm (1) and 0.02 × 0.24 × 0.32 mm (2) each was mounted on the tip of a MiTeGen microloop. Xray diffraction experiment was conducted at 296(2) K on a Bruker APEXII Kappa CCD diffractometer operated at 50 kV, 30 mA, producing monochromatic MoK α radiation (λ = 0.71073 Å). With the help of the APEX2 software suite [57], diffraction image of 600 and 680 frames were collected in ω−φ mode to 0.70 Å atomic resolution with exposure times and scan angles of 8 sec, 1.2 • and 10 sec, 1.2 • for respective complexes 1 and 2. Data processing was carried out with standard programs implemented in the APEX2 software suite [57]. The procedures began from data image integration with SAINT [58], followed by data reduction and scaling together with multiscan absorption corrections using SADABS [57] and completed with data merging using XPREP [58], yielding relatively good room-temperature diffraction data with mostly 100% completeness; see the summary below. Details of data statistics are given in Supplementary Materials, Table S1.

Structure Solution and Refinement
The crystal structures of 1 and 2 were solved by intrinsic phasing method with SHELXTL XT [57], providing all non-H atoms of host β-CD, guest PCAL, PCAC, and water molecules. The structures were refined anisotropically by full matrix least squares on F 2 with SHELXTL XLMP [57]. All H-atom positions (excluding those of flexible OH groups and water molecules) were calculated geometrically and treated with a riding model: C−H = 0.95 Å, U iso = 1.2U eq (C)(aromatic); C−H = 1.00 Å, U iso = 1.2U eq (C)(methine); and C−H = 0.99 Å, U iso = 1.2U eq (C)(methylene). The H atoms of β-CD OH groups and six water molecules were initially located from difference Fourier electron density maps. Then The stabilization energy and interaction energy of the complex (∆E stb and ∆E int ) were calculated straightforwardly. For example, ∆E stb was obtained by subtracting the molecular energies of β-CD and PCAL/PCAC from the energy of the β-CD-PCAL/PCAC complex from the full-geometry optimization. Similarly, ∆E int considered the corresponding singlepoint energies in the complexed states. The DFT-derived inclusion structures of the two complexes together with their O−H· · · O hydrogen bonds, ∆E stb and ∆E int are given in Supplementary Materials, Figure S1 and Tables S5 and S6.
Note that no basis set superposition error (BSSE) correction is applied to the DFTderived energies of the β-CD-catechol antioxidant complexes listed in Table S6. This is because the estimated energy differences (∆∆E stb and ∆∆E int ) are sufficient to interpret the relative thermodynamic stabilities in relation to host-guest interactions and antioxidant properties.

Conclusions
Protocatechuic aldehyde (PCAL) and protocatechuic acid (PCAC) are polyphenol extracts from plants. They contain a catechol moiety and thus having wide therapeutic effects including antioxidant, anti-inflammatory, and anticancer activities. Their physicochemical and pharmacological properties have been improved via cyclodextrin (CD) inclusion complexation, of which their characteristics are still controversial. To address at the atomic-level of the inclusion complexation, we investigate the β-CD encapsulation of PCAL (1) and PCAC (2) by a combined study of single-crystal X-ray diffraction and density functional theory (DFT) full-geometry optimization.
X-ray analysis disclosed that PCAL and PCAC are nearly totally shielded in the β-CD wall. Their aromatic rings are vertically aligned in the β-CD cavity such that the functional groups on the opposite side of the ring (3,4-di(OH) and 1-CHO/1-COOH groups) are placed nearby the O6-H and O2-H/O3-H rims, respectively. The preferred inclusion modes in 1 and 2 help to establish crystal contacts of OH···O H-bonds with the adjacent β-CD OH groups and water molecules, which are similar to our previous works on β-CD inclusion complexes with olive HTY and OLE [35]. By contrast, the DFT-optimized structures in the gas phase of both complexes are thermodynamically stable via the four newly formed host-guest OH···O H-bonds. The intermolecular OH···O H-bonds between PCAL/PCAC 3,4-di(OH) and β-CD OH groups and the shielding of OH groups in the β-CD wall help to stabilize these antioxidants in the β-CD cavity, as observed in our earlier studies. Moreover, PCAL and PCAC bearing CHO and COOH groups in distinct lattice environments (uncomplexed form, confined in β-CD cavity, and in complex with protein) are compared, revealing their structural rigidity. An atomistic perspective of the β-CD encapsulation of catechol antioxidants PCAL and PCAC through the lens of X-ray crystallography and DFT calculation goes beyond the spectroscopically derived inclusion structures in solution.