Rois Benassi
email: rois.benassi@unimore.it

Erika Ferrari

Sandra Lazzari

Francesca Pignedoli

Ferdinando Spagnolo

Monica Saladini

Saladini

physical-chemical properties of Curcumin: an experimental and theoretical study

Keywords: Curcumin, DFT, keto-enolic structure, glucosyl-curcuminoids Curcumin, DFT, β-keto-enolic structure, glucosyl-curcuminoids Curcumin, DFT, β-keto-enolic structure, glucosyl-curcuminoids

A theoretical conformational study on glycosyl curcuminoids is performed. In order to correlate their predicted spectroscopic properties with IR, UV-vis and NMR experimental data we extended the theoretical study on electronic properties in different solvents (H 2 O, MeOH, ACN, DMSO). Introduction Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), a yellow spice extracted from Curcuma Longa L. rhizomes, is used in a wide range of applications, from industrial dyes to pharmaceutical treatments. [1][2][3] It is proved that curcumin holds selective metal-chelating properties that are pharmaceutically interesting. [4] In the field of medicinal chemistry one of the most promising properties of curcumin is its metal ligating ability towards Gallium and Iron. [5,6] This feature can be exploited for a variety of pharmaceutical aims like metal overload detoxification, metal delivery system and radio imaging. Despite these potential pharmaceutical applications, curcumin has low water solubility and limited bioavailability that makes it difficult to handle for pharmaceutical use. To improve chemical properties of curcumin several derivatives were synthesized and studied by means of theoretical and experimental data. [7,8] The aromatic ring glucosylation was found to enhance curcumin water-solubility and kinetic stability which is a fundamental feature for drug bioavailability. [9] The compounds were characterized and their ability to act as metal-chelating agents was also evaluated. [9,10] Biological properties of these molecules were also tested and they showed cytotoxicity towards human ovarian carcinoma cell line leading to an improvement of Cisplatin efficacy with higher selectivity towards cancer than non-cancer cells. [11] In order to elucidate chemical-physical properties of these molecules and to correlate their electronic structures with the ability to act as metal chelating agents, in the present study we employ DFT calculations for a conformational analysis of the compounds reported in Figure 1. A full optimization of compounds' geometry is followed by a conformational search in order to study the potential energy surface (PES) as a function of the O-C exacyclic dihedral angle (φ) rotation (Figure1). It was reported that the presence of different solvents perturbs intra-or inter-molecular hydrogen bonds in curcumin, modifying its photophysical behavior. [12] Therefore now we perform also a theoretical study on electronic properties of glucosyl-curcuminoids in different solvents in

Methods

Computational details

The computations of all studied compounds were performed by DFT approach, the structures were fully optimized using hybrid-functional B3LYP applied to 6-31G* basis set (B3LYP/6-31G*) [13][14][15] by means of Gaussian 03 [16] package of programs.

In a previous study we described curcumin structure using both B3LYP/6-311G** and B3LYP/6-31G* levels. [17] By comparing the obtained results, B3LYP/6-31G* showed to be a good compromise between accuracy and precision. All the calculated properties agreed well with those obtained using B3LYP/6-311G**. Therefore we decided to employ B3LYP/6-31G* in order to study the influence of glucosylation on curcumin properties. GaussView 03 [18] was used as plotting tool for data visualization.

Thermodynamics were obtained from vibrational analysis employing general procedures.

The analysis of the calculated vibrational properties always confirmed the conformational minimum (GS) or rotational transition state (TS), characterized as saddle point, for the considered structure.

Atom charges were calculated from the optimized geometries at B3LYP/6-31G* level with the CHELPG approach as implemented in Gaussian03. The molecular electrostatic potential maps (MEPs) were plotted by Gaussview and reported onto 0.02 e/Bohr isosurface of electrondensity, representations of HOMO and LUMO orbital density were referred to an isovalue of 0.0004.

The solvent effects were evaluated by employing the self-consistent reaction field (SCRF) method with polarized continuum model (PCM). [19][20][21] The absorption wavelengths and oscillator strengths were calculated by means of timedependent density functional theory (TD-DFT) as implemented in Gaussian03. The magnetic

Results and discussion

Conformational analysis

Previous studies [7,17,26] demonstrated that the keto-enolic tautomer of curcumin is the most stable form, so we decided to use it to describe the core of all curcumin derivatives reported in

The first remarkable result is that for all compounds the curcuminic core maintains a completely planar conformation with the same geometrical parameters of curcumin, [17] allowing Curcumin-like electronic conjugation of aromatic/π electrons. This was found to be a fundamental feature to allow metal coordination through dissociated enolic moiety. [27] The introduction of a glucose molecule may perturb curcumin skeleton by means of hydrogen bond interactions or conjugation effects, therefore we decided to scan the dihedral angle φ as it defines the sugar values obtained from conformational analysis of all studied compounds.

A plot of ∆G values as a function of φ angle for A type compounds is reported in Figure 2.

From the analysis of rotational free energy, two states of rotational maximum (TS 1 and TS 2 ) and two states of minimum (GS 1 and GS 2 ) are found.

The main differences in φ values are observed for the absolute minimum conformation (GS 1 ), ranging from 71° to 118° (Table 1). The φ value increases following the decrease of aromatic substituent steric hindrance (H<OH<OCH 3 ). GS 2 ground state is less influenced by substituent bulkiness, since the sugar moiety is rotated thus minimizing the interaction with the substituent on the aromatic ring (Figure 3). As a consequence, φ values in GS 2 are similar, ranging from 282° to 293°.

Free Activation Energy of rotation (∆G*) of TS 1 for A2 and A3 are similar (A2 7.67 Kcal mol -1 ; A3 7.45 Kcal mol -1 ), while for A1 ∆G* assumes an higher value (9.43 Kcal mol -1 ). In A1 the geometrical analysis of GS 1 (Figure 3) provides the evidence of an hydrogen bond between OH phenolic group and an hydroxylic oxygen atom of the sugar molecule (O33-H60⋅⋅⋅O49 1.96 Å, O-H⋅⋅⋅O angle 158.6°). The hydrogen bond breakage in A1 strongly contributes to the higher ∆G*. In A2 and A3 only steric effects are responsible for the ∆G* value and no evidence of significant Hbond is observed. Previous study on similar β-diketo-mono-glucosylated derivatives [27] showed the formation of an hydrogen bond involving OCH 3 aromatic substituent and glucosydic moiety (OH⋅⋅⋅O 2.23 Å, OHO angle 155°). Instead in A2 no hydrogen interaction is observed suggesting that the greater extent of π delocalization with respect to the previously studied compound, probably makes OCH 3 group less capable to form hydrogen bond.

In di-glucosylated C compounds all the possible dispositions of the two sugar molecules with respect to the planar skeleton of curcumin were optimized and the results for C2 are reported C compounds show the same trend as mono-glucosylated (A series) regarding PSE. The ∆G* and dihedral angles for A and C homologues are superimposable with a maximum difference of ±2° (Table 1).

For each C compound, in all states of maximum and minimum, the value of the not scanned θ dihedral angle is constant and equal to the φ value obtained in the ground state (GS 1 ) with opposite sign. This evidence suggests that the two sugar molecules rotate independently and do not influence each other during the rotation. We suggest that θ angle can rotate with the same potential energy of φ since C compounds maintain the same symmetric structure observed in curcumin. For all compounds the relatively low ∆G* values (<10.04 kcal mol -1 ) allow a "free" rotation of the glucosydic group around φ and θ dihedral angle at room temperature. Anyway the free rotation of the glucose moieties does not exert any steric hindrance on keto-enolic group in any conformation, maintaining it accessible to a metal ion for the coordination reaction.

We observed also the formation of an hydrogen bond interaction between OH phenolic group and sugar molecule (O33-H82⋅⋅⋅O56 1.96 Å, O-H⋅⋅⋅O angle 158.7°) in C1. In a previous study on C compounds we discovered that the nature of meta substituent on the aromatic ring affects lipophilicity in the order OH>H>OCH 3 [10] Today computational data may account for the unexpected increased lipophilicity of C1 due to the formation of the intra-molecular hydrogen bond interactions with the glucosydic moiety.

Molecular Orbitals

HOMO and LUMO molecular orbitals where calculated for both GS and TS conformers for A and C series. Figure 5 shows HOMO and LUMO molecular orbitals at the GS 1 conformation, HOMO and LUMO for the other conformations GS 2 , TS 1 , TS 2 are perfectly overlapping with GS 1 .

HOMO and LUMO are delocalized through the whole curcuminic core, allocating the electron density both on the dienic and benzenic moieties as found for curcumin. [17,26] The introduction of one or two glucose molecules does not raise any interaction between the newly attached substituent and aromatic groups, thus not affecting the peculiar conjugation of aromatic/π electrons of curcumin core confirming the results of conformational analysis.

MEPs

Figure 6 shows the MEPs of GS 1 state. In all compounds, the negative charge of the curcuminic core is located on the β-keto-enolic oxygen atoms, resembling the same trend of curcumin. [17,26] suggesting the same potential metal coordination ability of curcumin. [6] The presence of OH groups in A1 and C1 introduces a further center of negative charge that might affects physical-chemical properties. Although enolic and phenolic oxygens show almost the same negative charge values the involvement of phenolic oxygen in metal coordination is prevented by the higher pK a value [pK a (phenolic) 9.4; pK a (enolic) 8.2]. [11] The negative charge on OH aromatic substituent evidences its strong electron withdrawing ability, which probably influences the pK a value of the β-keto-enolic group, in fact C1 was found to be the most acidic compound in aqueous solution and the more effective Fe(III) and Ga(III) chelating agent. [11] A and C compounds show the same electronic properties (MEPs, CHELPG) suggesting similar coordination ability, therefore the analysis of solvent effects is limited to C compounds in 2.

We can observe a general increase of the negative charge on ketonic oxygen atom in solvent with respect to vacuum. This effect seems to be directly related to solvent polarity, in fact H 2 O

gives major increase in negative charge. On the contrary the ability to form hydrogen bonds seems not to influence charge distribution. The high charge density on enolic function may account for the replacement of hydrogen atom by metal ion with a lower pK a value with respect to free ligand system, enabling metal coordination and preventing hydroxide precipitation.

MEPs do not take into account higher electrostatic effects as polarization which may be very important in metal complexation. Anyway the higher charge density on keto-enolic group with respect to sugar oxygen atoms suggests that the keto-enolic function is the only potentially involved in metal coordination. In addition chelating effect and enolic proton acidity enforces this assumption. Previous NMR study on C compounds [9] established that sugar moiety was never involved in metal ligation as no 1 H and 13 C chemical shifts were observed upon metal addition.

NMR spectroscopy

1 H and 13 C NMR spectra were recorded for C compounds and were compared with the calculated values. The GIAO (Gauge-Indipendent-Atomic-Orbitals) isotropic magnetic shielding tensors (σ calc )

were calculated for C1, C2 and C3 in vacuum, H 2 O, MeOH, and DMSO (Table 1-3, supplementary material). Bearing in mind the experimental molecular symmetry of glucosyl-curcuminoids, the average value of σ calc ( calc σ ) between the two keto-enolic limit structures was calculated and reported in Table 3 for each solvent system.

13

C and1 H experimental chemical shift assignments were supported by 2D COSY, HSQC and HMBC experiments, data are reported in Table 4 and Table 5.

Plotting the experimental 13 C and 1 H chemical shifts (δ exp ) in MeOD and DMSO versus the calc σ in vacuum or in the same solvent system, a linear regression is observed:

δ exp = a• calc σ + b (a, b and R 2
parameters are given in Table 4 and Table 5.). This relationship is then used to predict the chemical shifts (δ pred ). [17,28] As already observed, [29] the correlation between experimental chemical shifts and calculated isotropic screening constants is better for 13 C than for 1 H, in fact the correlation coefficient R2 ranges from 0.9834 to 0.9939 for 13 C and from 0,9186 to 0.9718 for 1 H.

A comparison with previously reported Curcumin predicted 13 C NMR data [18] highlights how for glucosyl-curcuminoids the presence of the sugar moiety experiences the solvent effect, in fact linear regressions are definitely better when calculated on the basis of calc σ in the same solvent system than in vacuum. -11), in fact this proton is not well predicted by calculation as a consequence of the intra-molecular hydrogen bond between the OH phenolic group and the sugar moiety.

F

3.4.UV-Vis spectroscopy

Each compound shows a strong intense absorption band in the 300-500 nm wavelength region. Absorption maxima are reported in Table 6 together with calculated values in the corresponding solvent. The red shift in DMSO with respect to ACN medium follows the order C1>C2>C3 and it is related to the higher polarity of the phenolic substituent (OH>OCH 3 >H) interacting with different polar solvent. A solvent dependent red shift in maximum was found also in curcumin [12] and was related to solvent polarity and proticity, in fact methanol, a strong hydrogen bond-donating as well as hydrogen bond accepting gave the greatest maximum red shift.

In our case the three compounds in H 2 O and MeOH behaves similarly, probably because the hydrogen bond effect is masked by the presence of sugar moiety. The same trend is observed in predicted values.

Infra Red Spectroscopy

Usually the calculated harmonic vibrational wavenumbers are higher than the experimental ones, because of the anharmonicity, incomplete treatment of electron correlation and use of finite 12 one-particle basis set. [29] A linear relationship with a good correlation coefficient is obtained plotting the experimental wavenumbers versus the calculated ones ( calc ν ) for all C compounds;

Figure 10 reports C1 data. This result suggests that the over-estimation of calculated wave numbers is quite systematic and allows to predict FT-IR spectra. The slope of the linear regression (a= 0.9991) is greater than the value (0.9613) estimated at B3LYP/6-31G* level for a great number of different compounds. [30] A list of the experimental and calculated frequencies together with a tentative assignment is reported as supplementary material (Table 4 supplementary material)

Conclusions

We have demonstrated that the addition of bulky sugar molecule does not affect electronic properties of curcuminic core, allowing metal coordination through keto-enolic moiety.

Glucose molecule interacts only with meta-substituent on aromatic ring influencing lipophilicity.

Solvent effect is a critical aspect in predicting spectroscopic data which well agree with experimental ones.

F

C1 C2 C3 δ exp (m) δ pred (v) δ pred (m) δ exp (d) δ pred (v) δ pred (d) δ exp (m) δ pred (v) δ pred (m) δ exp (d) δ pred (v) δ pred (d) δ exp (m) δ pred (v) δ pred (m) δ exp (d) δ pred (d) δ exp (m

C1 C2 C3

1 H δ exp (m) The hydrogen interaction is represented by dots. dyes to pharmaceutical treatments. [1][2][3] It is proved that curcumin holds selective metal-chelating properties that are pharmaceutically interesting. [4] In the field of medicinal chemistry one of the most promising properties of curcumin is its metal ligating ability towards Gallium and Iron. [5,6] This feature can be exploited for a variety of pharmaceutical aims like metal overload detoxification, metal delivery and radio imaging. Despite these potential pharmaceutical applications, curcumin has a low water solubility and limited bioavailability that makes it difficult to handle for pharmaceutical use. To improve chemical properties of curcumin, several derivatives were synthesized and studied by means of theoretical and experimental data. [7,8] The glucosylation of the aromatic ring was found to enhance curcumin water-solubility and kinetic stability which is a fundamental feature for drug bioavailability. [9] The compounds were characterized and their ability to act as metal-chelating agents was also evaluated. [9,10] Biological properties of these molecules were also tested and they showed cytotoxicity towards human ovarian carcinoma cell line leading to an improvement of Cisplatin efficiency with higher selectivity towards cancer than non-cancer cells. [11] In order to elucidate chemical-physical properties of these molecules and to correlate their electronic structures with the ability to act as metal chelating agents, in the present study we employ DFT calculations for a conformational analysis of the compounds reported in Figure 1. A full optimization of the geometry is followed by a conformational search in order to study the potential energy surface (PES) as a function of the rotation of the O-C exocyclic dihedral angle (φ) (Figure 1). It was reported that the presence of different solvents perturbs intra-or inter-molecular hydrogen bonds in curcumin modifying its photophysical behavior. [12] Therefore now we perform also a theoretical study on the electronic properties of glucosyl-curcuminoids in different solvents in order to correlate their predicted spectroscopic properties with IR, UV-vis and NMR experimental data.

δ pred (v) δ pred (m) δ exp (d) δ pred (v) δ pred (d) δ exp (m) δ pred (v) δ pred (m) δ exp (d) δ pred (v) δ pred (d) δ exp (m) δ pred (v) δ pred (m) δ exp (d) δ pred (d) δ pred (m

Methods

Computational details

The computations of all the studied compounds were performed by the DFT approach. The structures were fully optimized using hybrid-functional B3LYP applied to 6-31G* basis set (B3LYP/6-31G*) [13][14][15] by means of Gaussian 03 [16] package of programs.

In a previous study we described curcumin structure using both B3LYP/6-311G** and B3LYP/6-31G* levels. [17] By comparing the obtained results, B3LYP/6-31G* showed to be a good compromise between accuracy and precision. All the calculated properties agreed well with those obtained using B3LYP/6-311G**. Therefore we decided to employ B3LYP/6-31G* in order to study the influence of glucosylation on curcumin properties. GaussView 03 [18] was used as a plotting tool for data visualization.

Thermodynamics were obtained from vibrational analysis employing general procedures.

The analysis of the calculated vibrational properties always confirmed the conformational minimum (GS) or rotational transition state (TS), characterized as a saddle point, for the considered structure.

Atom charges were calculated from the optimized geometries at B3LYP/6-31G* level with the CHELPG approach as implemented in Gaussian 03. The molecular electrostatic potential maps (MEPs) were plotted by Gaussview and reported onto 0.02 e/Bohr isosurface of electrondensity;

representations of HOMO and LUMO orbital density were referred to an isovalue of 0.0004.

The solvent effects were evaluated by employing the self-consistent reaction field (SCRF) method with polarized continuum model (PCM). [19][20][21] The absorption wavelengths and oscillator strengths were calculated by means of timedependent density functional theory (TD-DFT) as implemented in Gaussian 03. The magnetic isotropic shielding tensors (σ) for 1 H and 13 C NMR were calculated using the standard GIAO (Gauge-Independent Atomic Orbital) [22] at B3LYP/6-31G* approach with the Gaussian 03 program package.

Conformational analysis

Starting with the mono glucosyl-compounds, Series A (Figure 1), all structures were fully optimized with the DFT approach at B3LYP/6-31G* level.

We utilized the geometries of the full reoptimized structures as the starting point for a rigid PES scan of the O-C exocyclic dihedral angle (φ) (Figure 1). The dihedral angle φ was rotated at 15° step size up to a complete 360° turn.

Minimum and maximum points on rigid PES were optimized and characterized as GS and TS states from the vibrational analysis. For all structures the starting point was confirmed to be the most stable structure.

Geometry of compounds C (Figure 1) was built from the more stable structure of the corresponding series A, adding a second glucose molecule. All the possible dispositions of the second sugar molecule with respect to curcumin planar skeleton were examined and optimized.

Calculations on C compounds were then run using the same approach (rigid PES search of φ dihedral angle, optimization of the obtained structures, vibrational analysis, CHELPG charges calculation, plots of HOMO and LUMO molecular orbitals and MEPs).

Spectroscopy

Spectroscopic data were collected only for previously synthesized compounds C. [10] NMR spectra were recorded at 300 K on a Bruker Avance AMX-400 spectrometer with a Broad Band 5 mm probe (inverse detection). Nominal frequencies were 100.13 MHz for 13 C and 400. 13 MHz for 1 H. The typical acquisition parameters for 1 H were as follows: 20 ppm spectral bandwidth (SW), 6.1 µs pulse width (90° pulse hard pulse on 1 H), 0.5-1 s pulse delay, 216-512 For 2D H,H-Homonuclear Correlated Spectroscopy (COSY) [23] typical parameters were used. For 2D H,X-Hetero Correlated Spectroscopy (HMBC [24] and HMQC [25] ) opportune parameters were used (50-90° pulses; 32k data points; 1 s relaxation delay; 8-64k transients; 1 J H-C 125-145 Hz; 3 J H-C 5-15 Hz). Methanol-d 4 (MeOD) and DMSO-d 6 (DMSO) were used as NMR solvents. D 2 O spectra showed line broadening due to hydrogen bond network forbidding their assignment.

UV-Vis measurements were performed using Jasco V-570 spectrophotometer at 25.0 ± 0.1°C in the 200-600 nm spectral range employing a 1 cm quartz cell. 2.5 × 10 -5 M solutions were prepared using different solvents: H 2 O, ACN, DMSO and MeOH.

The infrared spectra of solid compounds were obtained by means of a Bruker FTIR VERTEX 70, with a MCT Mid-Band detector in the 4000-600 cm -1 spectral range using an ATR Golden-Gate (Heated-Diamond top-plate).

Results and discussion

Conformational analysis

Previous studies [7,17,26] demonstrated that the keto-enolic tautomer of curcumin is the most stable form, so that we decided to use it to describe the core of all curcumin derivatives reported in

The first remarkable result is that for all compounds the curcuminic core maintains a completely planar conformation with the same geometrical parameters as curcumin. [17] allowing Curcumin-like electronic conjugation of aromatic/π electrons. This was found to be a fundamental feature to allow metal coordination through dissociated enolic moiety. [27] The introduction of a glucose molecule may perturb curcumin skeleton by means of hydrogen bond interactions or conjugation effects, therefore we decided to scan the dihedral angle φ as it defines the sugar values obtained from conformational analysis of all studied compounds.

A plot of ∆G values as a function of φ angle for A type compounds is reported in Figure 2.

From the analysis of rotational free energy, two states of rotational maximum (TS 1 and TS 2 ) and two states of minimum (GS 1 and GS 2 ) are found.

The main differences in φ values are observed for the absolute minimum conformation (GS 1 ), ranging from 71° to 118° (Table 1). The φ value increases following the decrease of aromatic substituent steric hindrance (H<OH<OCH 3 ). GS 2 ground state is less influenced by substituent bulkiness, since the sugar moiety is rotated thus minimizing the interaction with the substituent on the aromatic ring (Figure 3). As a consequence, φ values in GS 2 are similar, ranging from 282° to 293°.

Free activation energies of rotation (∆G*) of TS 1 for A2 and A3 are similar (A2 7.67 kcal mol -1 ; A3 7.45 kcal mol -1 ), while for A1 ∆G* assumes an higher value (9.43 kcal mol -1 ). In A1 the geometrical analysis of GS 1 (Figure 3) provides evidence for a hydrogen bond between OH phenolic group and a hydroxylic oxygen atom of the sugar moiety (O33-H60⋅⋅⋅O49 1.96 Å, O-H⋅⋅⋅O angle 158.6°). The hydrogen bond break in A1 strongly contributes to the higher ∆G*. In A2 and A3 only steric effects are responsible for the ∆G* value and no evidence of significant H-bond is observed. Previous study on similar β-diketo-mono-glucosylated derivatives [27] showed the formation of a hydrogen bond involving OCH 3 aromatic substituent and glucosydic moiety (OH⋅⋅⋅O 2.23 Å, OHO angle 155°). Instead in A2, no hydrogen interaction is observed suggesting that the greater extent of π delocalization with respect to the previously studied compound, probably makes OCH 3 group less capable to form a hydrogen bond.

In di-glucosylated compounds C, all the possible dispositions of the two sugar moieties with respect to the planar skeleton of curcumin were optimized and the results for C2 are reported in (Table 1).

For each compound C, in all states of maximum and minimum, the value of the not scanned θ dihedral angle is constant and equal to the φ value obtained in the ground state (GS 1 ) with opposite sign. This evidence suggests that the two sugar molecules rotate independently and do not influence each other during the rotation. We suggest that θ angle can rotate with the same potential energy as φ since compounds C maintain the same symmetric structure observed in curcumin. For all compounds the relatively low ∆G* values (<10.04 kcal mol -1 ) allow a "free" rotation of the glucosydic group around φ and θ dihedral angle at room temperature. Anyway the free rotation of the glucose moieties does not exert any steric hindrance on keto-enolic group in any conformation, maintaining it accessible to a metal ion for the coordination reaction.

We observed also the formation of a hydrogen bond interaction between the OH phenolic group and the sugar moiety (O33-H82⋅⋅⋅O56 1.96 Å, O-H⋅⋅⋅O angle 158.7°) in C1. In a previous study on compounds C we discovered that the nature of a meta substituent on the aromatic ring affects its lipophilicity in the order OH>H>OCH 3 [10] Today computational data may account for the unexpected increased lipophilicity of C1 due to the formation of an intra-molecular hydrogen bond with the glucosydic moiety.

Molecular Orbitals

HOMO and LUMO molecular orbitals were calculated for both GS and TS conformers for A and C series. Figure 5 shows HOMO and LUMO molecular orbitals at the GS 1 conformation, HOMO and LUMO for the other conformations GS 2 , TS 1 , TS 2 are perfectly overlapping with GS 1 .

HOMO and LUMO are delocalized through the whole curcuminic core, allocating the electron density both on the dienic and benzenic moieties as found for curcumin. [17,26] The introduction of one or two glucose molecules does not add any interaction between the newly attached substituent and the aromatic groups, thus not affecting the peculiar conjugation of aromatic/π electrons of curcumin core confirming the results of conformational analysis.

MEPs

Figure 6 shows the MEPs of GS 1 states. In all compounds, the negative charge of the curcuminic core is located on the β-keto-enolic oxygen atoms, resembling the same trend of curcumin. [17,26] Table 2 reports charge density values of  β-keto-enolic and phenolic oxygen atoms of A1 and C1 in GS 1 state. By comparing the values obtained in vacuum with those of curcumin we can observe variation of ≈ 0.004 in the negative charge of the β-keto-enolic oxygen atoms, thus suggesting the same potential metal coordination ability of curcumin. [6] The presence of OH groups in A1 and C1 introduces a further center of negative charge that might affect their physicalchemical properties. Although enolic and phenolic oxygens show almost the same negative charge values the involvement of phenolic oxygen in metal coordination is prevented by the higher pK a value [pK a (phenolic) 9.4; pK a (enolic) 8.2]. [11] The negative charge on OH aromatic substituent evidences its strong electron withdrawing ability, which probably influences the pK a value of the βketo-enolic group. In fact C1 was found to be the most acidic compound in aqueous solution and the more effective Fe(III) and Ga(III) chelating agent. [11] Compounds A and C show the same electronic properties (MEPs, CHELPG) suggesting similar coordination ability. Therefore the analysis of solvent effects is limited to C compounds in 2.

We can observe a general increase in the negative charge on ketonic oxygen atom in solvents with respect to vacuum. This effect seems to be directly related to the solvent polarity. In fact H 2 O gives the major increase in negative charge. On the contrary the ability to form hydrogen bonds seems not to influence charge distribution. The high charge density on the enolic function may account for the replacement of hydrogen atom by metal ion with a lower pK a value with respect to free ligand system, enabling metal coordination and preventing hydroxide precipitation.

MEPs do not take into account higher electrostatic effects than polarization which may be very important in metal complexation. Anyway the higher charge density on keto-enolic group with respect to sugar oxygen atoms suggests that the keto-enolic function is the only group potentially involved in metal coordination. In addition to the chelating effect, the enolic proton acidity enforces this assumption. Previous NMR study on compounds C [9] established that sugar moiety was never involved in metal ligation as no 1 H and 13 C chemical shifts were observed upon metal addition.

NMR spectroscopy

1 H and 13 C NMR spectra of compounds C were recorded and compared with the calculated values. The GIAO (Gauge-Indipendent-Atomic-Orbitals) isotropic magnetic shielding tensors (σ calc )

were calculated for C1, C2 and C3 in vacuum, H 2 O, MeOH, and DMSO (Table 1-3, supplementary material). Bearing in mind the experimental molecular symmetry of glucosyl-curcuminoids, the average value of σ calc ( calc σ ) between the two keto-enolic limit structures was calculated and reported in Table 3 for each solvent system.

13

C and 1 H experimental chemical shift assignments were supported by 2D COSY, HSQC

and HMBC experiments. The data are reported in Table 4 and Table 5.

Plotting the experimental 13 C and 1 H chemical shifts (δ exp ) in MeOD and DMSO versus the calc σ in vacuum or in the same solvent system, a linear regression is observed:

δ exp = a• calc σ + b (a, b and R 2
parameters are given in Table 4 and Table 5.). This relationship is then used to predict the chemical shifts (δ pred ). [17,28] As already observed, [29] the correlation between experimental chemical shifts and calculated isotropic screening constants is better for 13 C than for 1 H. In fact the correlation coefficient R 2 ranges from 0.9834 to 0.9939 for 13 C and from 0,9186 to 0.9718 for 1 H.

A comparison with previously reported Curcumin predicted 13 C NMR data [18] highlights how for glucosyl-curcuminoids the presence of the sugar moiety experiences the solvent effect; in fact linear regressions are definitely better when calculated on the basis of calc σ in the same solvent system than in vacuum.

1 H δ pred values (Table 5) provide good correlation with δ exp with exception of H-1, due to the fact that the GIAO σ calc has almost the same value in the two resonance limit structures of ketoenolic moiety. As a consequence electronic delocalization cannot be predicted accurately. -11), in fact this proton is not well predicted by calculation as a consequence of the intra-molecular hydrogen bond between the OH phenolic group and the sugar moiety.

3.4.UV-Vis spectroscopy

Each compound shows a strong intense absorption band in the 300-500 nm wavelength region. Absorption maxima are reported in Table 6 together with calculated values in the corresponding solvent. The red shift in DMSO with respect to ACN medium follows the order C1>C2>C3 and it is related to the higher polarity of the phenolic substituent (OH>OCH 3 >H) interacting with different polar solvents. A solvent dependent red shift in maximum was found also in curcumin [12] and was related to solvent polarity and proticity. In fact methanol, a strong hydrogen bond-donating as well as hydrogen bond accepting solvent gave the greatest maximum red shift. In our case the three compounds in H 2 O and MeOH behaves similarly, probably because the hydrogen bond effect is masked by the presence of sugar moiety. The same trend is observed in the predicted values.

Infra Red Spectroscopy

Usually the calculated harmonic vibrational wavenumbers are higher than the experimental ones, because of the anharmonicity, incomplete treatment of electron correlation and use of finite 12 one-particle basis set. [29] A linear relationship with a good correlation coefficient is obtained plotting the experimental wavenumbers versus the calculated ones ( calc ν ) for all compounds C; Figure 10 reports C1 data. This result suggests that the over-estimation of calculated wave numbers is quite systematic and allows to predict FT-IR spectra. The slope of the linear regression (a= 0.9991) is greater than the value (0.9613) estimated at B3LYP/6-31G* level for a great number of different compounds. [30] A list of the experimental and calculated frequencies together with a tentative assignment is reported as supplementary material (Table 4 supplementary material)

Conclusions

We have demonstrated that the addition of bulky sugar groups does not affect the electronic properties of the curcuminic core, allowing metal coordination through keto-enolic moiety. A glucose moiety interacts only with meta-substituents on the aromatic ring influencing its lipophilicity.

The solvent effect is a critical aspect in predicting spectroscopic data which well agree with the experimental ones.

F

  their predicted spectroscopic properties with IR, UV-vis and NMR experimental data.

in Figure 4 ,

 4 C1 and C3 show the same trend. The sin-sin configuration is the most stable therefore it is used as the starting point for conformational analysis on φ dihedral angle. For all C compounds the value of θ dihedral angle results close to the corresponding φ angle in GS 1 state of A compound with opposite sign.

  their electronic properties with experimental data. MEPs calculated in different solvents look like those in vacuum; the corresponding density charge values are reported in Table

Figure 7

 7 reports 1 H NMR spectrum of C1 in DMSO-d 6 . Experimental data evidence only one set of signals corresponding to the keto-enolic form; the complete symmetry (C 2v ) of the molecule, due to resonance between the two possible keto-enolic limit structures, is supported by the equivalence of double bonds and aromatic rings, and by13 C NMR spectrum giving undistinguishable and equivalent peaks for ketonic and enolic carbons. C2 and C3 behave the same.The fit between experimental chemical shifts of glucosyl-curcuminoids in DMSO and MeOD for both 1 H (Figure8A) and 13 C (Figure9B) illustrates very small variation due to experimental conditions.

Figure 1 :

 1 Figure 1: General structure of the studied curcumin glucosyl derivatives.

Figure 2 :

 2 Figure 2: ∆G for compounds A1, A2 and A3 as function of the rotational dihedral angle φ.

Figure 3 :

 3 Figure 3: Final optimized structures of A compounds at B3LYP/6-31G* level at GS 1 and GS 2 state.

Figure 4 :

 4 Figure 4: Top view of C2 optimized structures at B3LYP/6-31G* level showing the different sugar

Figure 5 :

 5 Figure 5: Representation of HOMO and LUMO orbital density for A and C series at B3LYP/6-

Figure 6 :

 6 Figure 6: Molecular electrostatic maps in vacuum of A and C compounds in their GS 1 state at

Figure 7 :Figure 8 :

 78 Figure 7: 1 H NMR spectrum of C1 in DMSO-d 6 ( * residual DMSO) Figure 8: Plots of DMSO-d 6 vs. MeOD experimental 1 H (A) and 13 C (B) chemical shifts (δ) for

Figure 9 :

 9 Figure 9: Plot of 1 H and 13 C δ exp vs. calc σ for C1. Solid circle is referred to H-1 proton, dotted

Figure 10 :Figure 4 δδ

 104 Figure 10: Correlation between experimental and B3LYP/6-311G* in vacuum calculated wavenumbers for C1. Equation: ; a = 0.9991, R 2 = 0.9983. calc a ν ν ⋅ = exp

Figure 4 ,

 4 Figure 4, C1 and C3 show the same trend. The syn-syn configuration is the most stable therefore it

  their electronic properties with experimental data. MEPs calculated in different solvents look like those in vacuum; the corresponding density charge values are reported in Table

Figure 7

 7 reports 1 H NMR spectrum of C1 in DMSO-d 6 . Experimental data evidence only one set of signals corresponding to the keto-enolic form; the complete symmetry (C 2v ) of the molecule, due to resonance between the two possible keto-enolic limiting structures, is supported by the equivalence of double bonds and aromatic rings, and by13 C NMR spectrum giving undistinguishable and equivalent peaks for ketonic and enolic carbons. C2 and C3 behave similarly.The fit between experimental chemical shifts of glucosyl-curcuminoids in DMSO and MeOD for both 1 H (Figure8A) and 13 C (Figure9B) illustrates the very small variations due to experimental conditions.

Figure 1 :

 1 Figure 1: General structure of the studied curcumin glucosyl derivatives.

Figure 2 :

 2 Figure 2: ∆G for compounds A1, A2 and A3 as a function of the rotational dihedral angle φ.

Figure 3 :

 3 Figure 3: Final optimized structures of A compounds at B3LYP/6-31G* level at GS 1 and GS 2

Figure 4 :

 4 Figure 4: Top view of C2 optimized structures at B3LYP/6-31G* level showing the different sugar

Figure 5 :

 5 Figure 5: Representation of HOMO and LUMO orbital density for A and C series at B3LYP/6-

Figure 6 :

 6 Figure 6: Molecular electrostatic maps in vacuum of compounds A and C in their GS 1 states at

Figure 7 : 1 HFigure 8 :

 718 Figure 7: 1 H NMR spectrum of C1 in DMSO-d 6 ( * residual DMSO) Figure 8: Plots of DMSO-d 6 vs. MeOD experimental 1 H (A) and 13 C (B) chemical shifts (δ) for

Figure 9 :

 9 Figure 9: Plot of 1 H and 13 C δ exp vs. calc σ for C1. Solid circle is referred to H-1 proton; dotted

Figure 10 : 25 Figure 4 δδ

 10254 Figure 10: Correlation between experimental and B3LYP/6-311G* in vacuum calculated wavenumbers for C1. Equation: ; a = 0.9991, R 2 = 0.9983. calc a ν ν ⋅ = exp

Table 1

 1 reports energies, thermodynamic quantities and dihedral angles (φ, θ)

Table 2

 2

reports charge density values of  β-keto-enolic and phenolic oxygen atoms of A1 and C1 in GS 1 state. By comparing the values obtained in vacuum with those of curcumin we can observe variation of ≈ 0.004 in the negative charge of the β-keto-enolic oxygen atoms, thus

Table 1 -

 1 θ and φ dihedral angles, total electronic energies (E), zero point vibrational energies (E +

	o r
	P
	e
	e r
	R
	e
	v i e
	w

2 -121.64 353.48 -2255.965306 -2255.313594 -2255.269723 -2255.396658 5.

Table 2 -

 2 CHELPG charges calculated with B3LYP/6-31G* basis-set

	Molecule	CHELPG Charges
		=O	-OH	O32	O33
	Curcumin -0.595*	-0.579*
	A1	-0.584	-0.572	-0.553	-0.544
	A2	-0.594	-0.576
	A3	-0.597	-0.569
	C1	-0.591
		-0.646 (w)

*Values taken from Ref 15. Values calculated in vacuum if not specified, (w) water, (m) MeOH, (a) ACN, (d) DMSO.

Table 3 -

 3 Calculated averaged GIAO magnetic isotropic shielding tensors ( .23 115.72 115.9 116.54 115.88 116.26 116.88 116.26 13 114.94 115.9 115.4 113.79 114.9 114.23 114.3 115.37 114.81 14 119.61 120.01 119.61 119.83 120.26 119.81 120.2 120.63 120.2 15 114.05 115.18 114.52 114.92 116.03 115.35 113.73 114.89 114.26 16 126.04 126.7 126.47 126.08 126.87 126.56 125.87 126.58 126.31 .19 24.91 25.03 25.16 25.01 25.12 25.34 25.06 25.18 10 25.22 24.89 25.01 24.97 24.72 24.83 24.61 24.32 24.43 11 27.82 26.98 27.53 26.89 26.73 26.86 27.36 26.69 27.15 12 28.49 28.23 28.47 28.43 28.21 28.47 28.39 28.18 28.

			C1			C2			C3
	13 C	v	m	d	v	m	d	v	m	d
	1 88.34 87.94 88.01	88.5 88.19 88.29 88.75 88.64 88.62
	2 17.99 17.36 17.65 18.13 17.46 17.77 18.14 17.54 17.82
	3 72.92	72.8 72.91 73.34 73.18 73.37 74.28 74.47 74.47
	4 54.21 53.84 54.01 54.69	54	54.2 55.27 54.56 54.82
	5 62.48 63.47 63.29 64.33 64.36 64.45 66.69 67.35 67.23
	6 73.19 73.98 73.96 79.16 77.99 78.37 60.88 60.51 60.69
	7 45.32 46.03 45.79 44.92 44.85 44.83 76.35 76.59 76.57
	8 49.42	48.2 48.58 48.22	48.5 48.41 36.48	35.8 36.03
	9 71.79 71.02 70.95 68.91	69.3	69.1 78.25 77.66 77.72
	10 81.35	79.7 80.11 78.51 77.95 78.11 70.32 69.18 69.41
	11 84.48 85.92 85.26 89.05	89.5 89.35 86.84	87.8 87.33
	12 115.66 1161 H
	1 27.27 26.76 26.96 27.22 26.74 26.94 27.28 26.79 26.99
	3 26.05 25.64 25.82 26.02 25.63 25.82 26.08	25.7 25.86
	4 24.85 24.85	24.9 24.87	24.8 24.86 24.85 24.83 24.89
	6 25.57 25.42 25.57 25.89 25.52	25.7	25.1 24.89 25.01
	7	/	/	/	/	/	/	25.16 25.06 25.13
	9 2542
	13	28.6 28.19 28.44	28.7 28.28 28.57	28.6 28.17 28.43
	14	28.2 28.03 28.22 28.21 28.04 28.25	28.2 28.04 28.24
	15 28.94 28.37 28.69 29.09 28.53 28.88 28.87 28.27	28.6
	16	28 27.86	27.9 28.16	28 28.06 28.01 27.85	27.9
	16 28.14 28.12 28.14 28.41 28.39 28.42 28.22	28.2 28.23

calc σ ) for C1, C2 and C3 in vacuum (v), MeOH (m) and DMSO (d).

F o r P e e r R e v i e w

18

Table 4 -

 4 Experimental (δ exp ) and predicted ( pred δ = a• calc

σ + b)

13

C chemical shifts (ppm) for C1, C2 and C3 in vacuum (v), MeOH (m) and DMSO (d).

Table 5 -

 5 ) 1 102.80 101.85 102.50 101.71 103.19 103.62 102.80 102.08 102.56 101.32 103.50 103.76 102.8 102.4 102.8 101.74 103.82 104.01 2 185.40 184.41 184.20 183.50 180.20 180.27 185.60 184.88 184.43 183.68 180.57 180.57 185.6 186.8 186.1 183.57 181.87 181.69 3 124.50 119.95 120.04 122.77 120.07 120.07 124.50 119.92 119.89 122.89 120.11 120.01 124.5 119.7 119.5 122.81 119.81 119.54 4 142.10 141.90 141.98 140.56 140.55 140.66 142.10 141.86 142.12 140.69 140.53 140.89 142.1 142.4 142.6 140.31 140.82 141.10 5 132.60 132.20 131.22 129.54 131.50 130.55 132.10 130.52 130.25 128.91 129.97 129.73 131.6 128.8 128.0 128.78 128.20 127.48 6 116.20 119.63 118.83 121.45 119.78 118.92 112.10 113.07 114.07 111.60 113.73 114.57 131.6 135.7 135.7 130.32 134.62 134.66 7 147.70 152.34 151.53 147.27 150.28 149.61 149.60 153.36 152.99 149.47 151.23 151.10 118.9 117.2 117.0 116.94 117.52 117.24 8 149.40 147.53 148.29 147.53 145.80 146.57 150.10 149.47 148.84 148.85 147.61 147.20 161.6 164.9 164.7 159.5 161.60 161.71 9 118.80 121.27 122.31 116.31 121.31 122.20 117.30 125.13 124.83 115.42 124.96 124.67 118.9 115.0 115.7 116.94 115.42 115.97 10 122.80 110.06 111.67 114.98 110.84 112.22 122.50 113.84 114.37 122.83 114.44 114.85 131.6 124.4 125.4 130.32 124.19 125.09 11 96.30 106.38 105.70 101.58 107.42 106.61 99.40 101.43 101.32 99.97 102.90 102.61 96.5 104.7 104.4 100.34 105.93 105.43 12 68.10 69.79 70.34 76.96 73.28 73.43 68.10 69.84 70.52 73.52 73.50 73.71 68.4 Experimental (δ exp ) and predicted ( pred

	69.5	70.3 73.55 73.41 73.69

δ = a• calc σ + b) 1 H chemical shifts (ppm) for C1, C2 and C3 in vacuum (v). MeOH (m) and DMSO (d).

Table 6 -

 6 Uv-vis band maximum position (nm) in different solvents for C1 compound.

	)

Table 1 :

 1 NMR values for C1 compound calculated in vacuum, H 2 O, MeOH, ACN and DMSO.

	ATOM N.	vacuum	H2O	MeOH	ACN	DMSO
	1	25.37	24.94	24.95	25.17	25.17
	2	10.62	9.72	9.77	10.15	10.15
	4	74.48	74.58	74.63	74.69	74.69
	5	26.18	25.74	25.77	25.95	25.95
	6	55.32	54.75	54.74	54.92	54.93
	7	24.95	24.92	24.92	24.98	24.98
	8	71.37	70.91	70.98	71.14	71.14
	9	25.93	25.49	25.52	25.7	25.7
	10	53.11	52.98	52.95	53.11	53.11
	11	24.75	24.79	24.79	24.84	24.84
	12	62.48	63.4	63.38	63.23	63.23
	13	49.51	48.29	48.32	48.69	48.68
	14	81.55	79.78	79.87	80.3	80.28
	15	73.57	74.35	74.3	74.26	74.27
	16	45.3	46.06	46.03	45.78	45.78
	17	71.73	71.01	70.99	70.94	70.92
	18	25.26	24.9	24.92	25.05	25.05
	19	25.62	25.45	25.46	25.61	25.61
	20	25.19	24.92	24.92	25.04	25.03
	21	62.49	63.59	63.56	63.34	63.35
	22	49.34	48.06	48.09	48.49	48.49
	23	72.82	73.72	73.67	73.64	73.65
	24	81.16	79.45	79.54	79.95	79.94
	25	71.86	71.06	71.05	71	70.99
	26	45.36	46.07	46.04	45.8	45.81
	27	25.53	25.38	25.39	25.54	25.54
	28	25.19	24.84	24.86	24.99	24.99
	29	25.19	24.91	24.92	25.03	25.03
	34	88.34	87.9	87.95	88.02	88.02
	35	27.27	26.73	26.76	26.97	26.97
	36	189.29	193.45	193.26	192.29	192.32
	37	18.36	18.58	18.57	18.54	18.54
	38	84.49	86.03	85.93	85.26	85.27
	39	115.67	116.25	116.24	115.72	115.72
	40	27.82	26.92	26.98	27.54	27.53
	41	114.06	115.26	115.18	114.51	114.52
	42	114.93	115.95	115.9	115.38	115.39
	43	28.5	28.23	28.23	28.47	28.47
	44	119.61	120.01	120.01	119.62	119.62
	45	28.94	28.34	28.38	28.7	28.69
	46	28.6	28.17	28.19	28.45	28.45
	47	28.2	28.04	28.04	28.23	28.23
	48	84.47	86.02	85.92	85.26	85.26
	49	115.67	116.25	116.24	115.72	115.72
	50	27.83	26.92	26.99	27.54	27.54

Table 1 -

 1 continued from previous page

	ATOM N.	vacuum
	51	114.95
	52	28.5
	53	114.05
	54	119.63
	55	28.6
	56	28.95
	57	28.21
	61	30.54
	63	30.08
	65	29.99
	67	29.99
	69	30.08
	71	30.54
	72	126.03
	73	28
	74	28.15
	76	32.03
	77	126.05
	78	28.01
	79	28.15
	81	32.03
	82	25.17
	83	25.16

Table 2 :

 2 NMR values for C2 compound calculated in vacuum, H 2 O, MeOH, ACN and DMSO. Oxygen atom chemical shifts are omitted.

	ATOM N.	vacuum	H2O	MeOH	ACN	DMSO
	1	25.5	24.99	25.01	25.24	25.24
	2	10.76	9.88	9.93	10.32	10.32
	4	74.94	74.98	75.02	75.17	75.17
	5	26.15	25.74	25.76	25.95	25.95
	6	55.83	54.9	54.91	55.13	55.13
	7	24.98	24.86	24.87	24.94	24.94
	8	71.75	71.28	71.34	71.57	71.58
	9	25.9	25.49	25.51	25.69	25.69
	10	53.57	53.11	53.09	53.3	53.28
	11	24.77	24.73	24.73	24.79	24.78
	12	64.32	64.26	64.26	64.37	64.38
	13	48.31	48.6	48.63	48.52	48.52
	14	78.72	78.04	78.12	78.28	78.28
	15	79.53	78.2	78.3	78.68	78.68
	16	44.9	44.85	44.86	44.84	44.83
	17	68.85	69.41	69.26	69.07	69.06
	18	25.01	24.73	24.75	24.87	24.87
	19	25.95	25.53	25.56	25.74	25.74
	20	25.16	25.02	25.02	25.13	25.13
	21	64.34	64.46	64.46	64.51	64.52
	22	48.14	48.37	48.39	48.33	48.32
	23	78.8	77.59	77.68	78.08	78.07
	24	78.32	77.7	77.79	77.94	77.94
	25	68.99	69.48	69.34	69.15	69.14
	26	44.95	44.86	44.86	44.86	44.85
	27	25.85	25.45	25.48	25.67	25.67
	28	24.94	24.68	24.7	24.81	24.81
	29	25.17	25.01	25.01	25.13	25.13
	34	137.41	137.57	137.56	137.34	137.34
	35	28.57	28.35	28.36	28.43	28.43
	36	28.14	28.06	28.06	28.1	28.1
	37	28.65	28.51	28.52	28.59	28.59
	38	137.39	137.55	137.53	137.32	137.32
	39	28.63	28.5	28.5	28.57	28.57
	40	28.14	28.05	28.05	28.09	28.09
	41	28.55	28.33	28.35	28.42	28.42
	42	88.51	88.15	88.2	88.3	88.3
	43	27.23	26.71	26.74	26.95	26.95
	45	18.33	18.52	18.51	18.49	18.49
	46	89.08	89.77	89.52	89.37	89.37
	47	115.9	116.53	116.54	115.88	115.88
	48	26.89	26.47	26.72	26.85	26.86
	49	114.93	116.16	116.04	115.36	115.36
	50	113.79	114.93	114.89	114.23	114.23

Table 2 -

 2 continued from previous page

	ATOM N.	vacuum
	51	28.43
	52	119.82
	53	29.09
	54	28.7
	55	28.22
	56	89.04
	57	115.91
	58	26.91
	59	113.8
	60	28.43
	61	114.92
	62	119.84
	63	28.7
	64	29.09
	65	28.22
	69	30.67
	71	30.18
	73	30.5
	75	30.5
	77	30.18
	79	30.67
	80	126.08
	81	28.17
	82	28.41
	84	32.17
	85	126.09
	86	28.17
	87	28.42
	89	32.17

Table 3 :

 3 NMR values for C3 compound calculated in vacuum, H 2 O, MeOH, ACN and DMSO. Oxygen atom chemical shifts are omitted.

	ATOM N.	Vacuum	H2 O	MeOH	ACN	DMSO
	1	25.43	24.95	24.97	25.18	25.18
	2	10.86	10.08	10.13	10.47	10.47
	4	76.13	76.66	76.7	76.58	76.58
	5	26.24	25.82	25.85	26.02	26.01
	6	56.42	55.38	55.38	55.71	55.71
	7	24.97	24.91	24.91	24.98	24.98
	8	72.44	72.2	72.25	72.36	72.36
	9	25.94	25.52	25.55	25.72	25.72
	10	54.12	53.76	53.74	53.96	53.95
	11	24.74	24.76	24.76	24.8	24.8
	12	67.23	67.9	67.89	67.74	67.74
	13	37.49	36.71	36.72	37.03	37.02
	14	70.18	68.76	68.85	69.07	69.05
	15	61.48	61.18	61.15	61.35	61.35
	16	77.04	77.78	77.69	77.6	77.61
	17	81.5	80.29	80.39	80.56	80.55
	18	24.65	24.32	24.34	24.45	24.45
	19	25.14	24.94	24.94	25.06	25.06
	20	25.39	25.01	25.05	25.16	25.16
	21	66.15	66.86	66.83	66.71	66.72
	22	35.48	34.87	34.88	35.07	35.06
	23	60.28	59.88	59.89	60.04	60.05
	24	70.47	69.46	69.51	69.78	69.77
	25	75.01	74.96	74.94	74.9	74.89
	26	75.66	75.48	75.51	75.53	75.53
	27	25.06	24.85	24.85	24.97	24.97
	28	24.58	24.29	24.31	24.42	24.42
	29	25.3	25.07	25.08	25.21	25.2
	32	88.76	88.6	88.64	88.63	88.63
	33	27.28	26.77	26.8	27	26.99
	35	18.33	18.56	18.55	18.52	18.52
	36	84.51	85.52	85.42	84.97	84.97
	37	115.85	116.52	116.52	115.88	115.87
	38	27.37	26.68	26.76	27.17	27.17
	39	113.59	114.88	114.8	114.13	114.13
	40	114.15	115.28	115.25	114.66	114.67
	41	28.45	28.23	28.23	28.47	28.47
	42	119.84	120.29	120.28	119.85	119.84
	43	28.94	28.33	28.36	28.7	28.7
	44	28.55	28.1	28.12	28.39	28.39
	45	28.24	28.07	28.07	28.27	28.27
	46	89.18	90.29	90.19	89.71	89.71
	47	116.67	117.26	117.26	116.65	116.65
	48	27.37	26.53	26.63	27.14	27.14

Table 3 -

 3 continued from previous page

	ATOM N.	VACUO
	49	114.46
	50	28.34
	51	113.87
	52	120.56
	53	28.65
	54	28.8
	55	28.18
	59	30.52
	61	30.06
	63	30.44
	65	30.26
	67	30.11
	69	30.46
	70	125.83
	71	28.05
	72	28.24
	74	32.08
	75	125.92
	76	27.98
	77	28.2
	79	32.05
	80	24.96
	81	25.38

Table 4 :

 4 Vibrational modes of C1: ν, stretching; δ, γ in-plane and out-plane bending, respectively; τ, torsion; superscript: Ph, aromatic ring vibrations; A, vibrations connected with 'enolic' part of the molecule(left); B, vibrations connected with 'keto' part of the molecule(right).

	νIRexp Ass νIRCalc A CalcIRkm • mol-1		Assignment
		601	23.75	ν CC	γCOC
					γC=CH
	605	γCOH605	142.03		γCOH
					γCC=O
		622	5.09		γ P h
					CCC
		623	23.69	δCCC
				δ P h	B
				CCC
		628	9.09		γ P hB CCC
		644	40.35		τCCOH
		646	173.64		τCCOH
	663	δCCC
		δ P h
		CCC
		γCCC
		γ P h CCC
		678	1.19	δCC=C
				δ P hA CCC
		682	2.06		γ S CCC
		683	12.31		γ S CCC
		693	0.42		γ P h	B
					C=CH
					γC=C-OH
					γ P h	A
					C=CH
	711	γCCCP h
		725	2.68		γ P hA CCC
		732	4.65		γ P hA CCC
					Continued on next page

Table 4

 4

				-continued from previous page
	νIRexp Ass νIRCalc A CalcIRkm • mol-1		Assignment
					γ P h	B
					CCC
	734	γCCCP h
		δ P h CCC
		δCCC
		γCCH
		747	3.92		γ P hB CCC
					γCK=CH
		754	8.52	δ P h	A
				CCC
				δ P h	B
				CCC
				δ K CCO
		774	11.89		γCK=CH
	795	γCCCP h
		804	66.38		γ P hB CCC
					γ P hA CCC
	810	γ P h
		CCH
		812	22.66		γ P h	B
					CCH
					γ P h	A
					CCH
		815	1.49		γ P h	B
					CCH
					γ P hA CCH
		829	22.98		γ P hB CCH
					γ P hA CCH
		833	10.35	δCC=C	γ P h CCH A
					γ P h	B
					CCH
		852	3.77	δ K CC=O
					Continued on next page

Table 4

 4

				-continued from previous page
	νIRexp Ass νIRCalc A CalcIRkm • mol-1			Assignment
					δ K CCC
					δCC=C
					δ P h	A
					CCC
					δ P hB CCC
	862	γCCHP h
		873	4.58			γCCH
						γ P hB CCH
						γ P h	A
						CCH
		878	36.47			γ P h	B
						CCH
	900	γCCC900	0.42			γCCH
		δCCO
		τCCOH
		903	1.22			γCCH
		909	20.99		δ S CC2OH	γ S CCH
		909	2.52		δ S CC2OH	γ S CCH
		935	12.41			γ P h	A	τ K
						CCH	CCOH
		936	3.52			γ P h	B
						CCH
		938	89.84			γ P h CCH A	τ K CCOH
		969	24.33		δ S CC B 2	OH	γ SB CCH
		969	18.38		δ S CC A 2	OH	γ SA CCH
	972	νCO
		νCC
		δCCC
		975	7.42	ν K CO	δCC=C
						Continued on next page

Table 4

 4

			-continued from previous page
	νIRexp Ass νIRCalc A CalcIRkm • mol-1				Assignment
	1591 δCCH
	1620 623.04	νCC	δ P h COH B
			ν K CO	δCCH
			ν P hB CC	δ P hB CCH
	1626	191.81	ν P hB CC	δ P hA CCH
					δ P hA COH
					δCCH
	1629 νCC
	νCO
	δCCH
	δCOH
	1631 115.11	νCC	δ P hB COH
			ν K CO	δCCH
			ν P hB CC	δ P hB CCH
	1652 714.33	ν K CC	δ K CCH
			νCC		δ K
					COH
	1664 7.05	ν P h	A	δ P h	A
			CC	COH
			νC=C	δCCH
					δ P hA CCH
	1666 9.15	νCC	δ P hB COH
			ν K C=O	δ P hB CCH
					δ K COH
	1694 886.87	νC=C	δ K
					COH
			νCC	δ K C=CH
						Continued on next page

Table 4

 4

		-continued from previous page
	νIRexp Ass νIRCalc A CalcIRkm • mol-1		Assignment
		ν K C=O
		ν K C=C
	1710 46.78	νC=C	δ K COH
		νCC	δ K C=CH
		ν K C=O
		ν K C=C
	1997 νCC
	2053 νCC
	2127 νCC
	2170 νCO
	2296
	2331
	2863 νCH
	2935 νCH
	2943 426.05	ν K CH
	2977 νCH
	2984 1.81
	2984 1.81
	2989 2.61
	2990 2.87	ν S CH
	2998 44.62	ν S CH
	2998 63.85	ν S CH
	3026 50.4	ν S
		CH
	3026 61.7	ν S CH
			Continued on next page

Table 4

 4

		-continued from previous page
	νIRexp Ass νIRCalc A CalcIRkm • mol-1	Assignment
	3046 26.37	ν S CH
	3046 27.24	ν S CH
	3073 32.19	ν S CH
	3073 22.72	ν S CH
	3077 35.93	ν S CH
	3077 50.39	ν S CH
	3177 0.1	νCH
	3186 12.25	νCH
	3188 0.11	νCH
	3197 11.89	νCH
	3207 3.83	ν P h CH
	3207 3.48	ν P h CH
	3209 6.39	ν P h CH
	3209 5.41	ν P h CH
	3222 4.73	ν P h CH
	3222 1.11	ν P h
		CH
	3223 18.14	νC34-H35
	3567 947.75	νO32-H83
	3569 589.9	νO33-H82
	3724 54.31	ν S OH
	3724 67.51	ν S OH
	3726 54.7	ν S OH
	3726 36.68	ν S
		OH
	3742 69.03	ν S OH
		Continued on next page

Table 4

 4 Curcuma Longa L. rhizomes, is used in a wide range of applications, from industrial

		-continued from previous page
	νIRexp Ass νIRCalc A CalcIRkm • mol-1	Assignment
	3742 59.96	ν S OH
	3766 24.08	ν S OH
	3766 32.37	ν S OH

Table 1

 1 reports energies, thermodynamic quantities and dihedral angles (φ, θ)

Table 5

 5 data confirm the importance of the solvent environment in predicting correct 1 H δ pred; in fact the gap ΝΜR data of C1 are better predicted in MeOH than in DMSO, C3 behaves in the opposite way while for C2 the two solvents show the same effect. This behavior suggests that the OH and OCH 3 groups in C1 and C2 respectively may interact with the solvent through hydrogen bond and/or dipolar interactions. Figure 9 reports the plotting of 1 H and 13 C δ exp vs. calc σ for C1 together with the linear relationships. C1 behaves differently from C2 and C3 as it concerns anomeric proton (H

	in R 2 value between in vacuum and solvent prediction is higher for 1 H (0.0117-0.0319) than 13 C
	(0.0003-0.0044).

Table 1 -

 1 θ and φ dihedral angles, total electronic energies (E), zero point vibrational energies (E + ZPE), thermodynamic quantities (G, H) and relative differences calculated at B3LYP/6-31G*. a) Not rotated dihedral angle; b) rotated dihedral angle; c) a.u. ; d) kcal mol-1

	o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2 -

 2 CHELPG charges calculated with B3LYP/6-31G* basis-set

	Molecule	CHELPG Charges
		=O	-OH	O32	O33
	Curcumin -0.595*	-0.579*
	A1	-0.584	-0.572	-0.553	-0.544
	A2	-0.594	-0.576
	A3	-0.597	-0.569
	C1	-0.591
		-0.646 (w)

*Values taken from Ref 15. Values calculated in vacuum if not specified, (w) water, (m) MeOH, (a) ACN, (d) DMSO.

Table 3 -

 3 Calculated averaged GIAO magnetic isotropic shielding tensors ( calc σ ) for C1, C2 and C3 in vacuum (v), MeOH (m) and DMSO (d). .19 88.29 88.75 88.64 88.62 2 17.99 17.36 17.65 18.13 17.46 17.77 18.14 17.54 17.82 3 72.92 72.8 72.91 73.34 73.18 73.37 74.28 74.47 74.47 4 54.21 53.84 54.01 54.69 54 54.2 55.27 54.56 54.82 5 62.48 63.47 63.29 64.33 64.36 64.45 66.69 67.35 67.23 6 73.19 73.98 73.96 79.16 77.99 78.37 60.88 60.51 60.69 7 45.32 46.03 45.79 44.92 44.85 44.83 76.35 76.59 76.57 8 49.42 48.2 48.58 48.22 48.5 48.41 36.48 35.8 36.03 9 71.79 71.02 70.95 68.91 69.3 69.1 78.25 77.66 77.72 10 81.35 79.7 80.11 78.51 77.95 78.11 70.32 69.18 69.41 11 84.48 85.92 85.26 89.05 89.5 89.35 86.84 87.8 87.33 12 115.66 116.23 115.72 115.9 116.54 115.88 116.26 116.88 116.26 13 114.94 115.9 115.4 113.79 114.9 114.23 114.3 115.37 114.81 14 119.61 120.01 119.61 119.83 120.26 119.81 120.2 120.63 120.2 15 114.05 115.18 114.52 114.92 116.03 115.35 113.73 114.89 114.26 16 126.04 126.7 126.47 126.08 126.87 126.56 125.87 126.58 126.31 7.61

			C1		C2			C3
	13 C	v	m	d	v	m	d	v	m	d
	1 88.34 87.94 88.01	88.5 88

Table 6 -

 6 Uv-vis band maximum position (nm) in different solvents for compounds C.

		Vacuum		H 2 O	MeOH		ACN	DMSO
		calc	exp	calc	exp	calc	exp	calc	exp	calc
	C1	402	414 423 411 422 405 419 423 421
	C2	413	413 432 411 432 413 428 425 431
	C3	403	408 425 406 425 405 420 416 423

http://mc.manuscriptcentral.com/poc Journal of Physical Organic Chemistry

H δ pred values (Table5) have good correlation with δ exp with exception of H-1, due to the fact that the GIAO σ calc has almost the same value in the two resonance limit structures of ketoenolic moiety, as a consequence electronic delocalization can't be predicted accurately. Table5data confirm the importance of solvent environment in predicting correct 1 H δ pred , in fact the gap in

R 2 value between in vacuum and solvent prediction is higher for 1 H (0.0117-0.0319) than13 C

Journal of Physical Organic Chemistry

Acknowledgments

We are thankful to the "Consorzio Interuniversitario per il Calcolo Automatico dell'Italia Nord Orientale -CINECA" and to the "Laboratorio di Calcolo Scientifico Avanzato Interdipartimentale dell'Università degli Studi di Modena e Reggio Emilia" for computing facilities.

We are grateful to the "Centro Interdipartimentale Grandi Strumenti -C.I.G.S." of the University of Modena and Reggio Emilia and to the "Fondazione Cassa di Risparmio di Modena" for supplying NMR spectrometer.

Acknowledgments

We are thankful to the "Consorzio Interuniversitario per il Calcolo Automatico dell'Italia Nord Orientale -CINECA" and to the "Laboratorio di Calcolo Scientifico Avanzato Interdipartimentale dell'Università degli Studi di Modena e Reggio Emilia" for computing facilities.

We are grateful to the "Centro Interdipartimentale Grandi Strumenti -C.I.G.S." of the University of Modena and Reggio Emilia and to the "Fondazione Cassa di Risparmio di Modena" for supplying NMR spectrometer.

F o r P e e r R e v i e w

18

C1 C2 C3

1 H δ exp (m)


Krzysztof Zborowski
email: zborowsk@chemia.uj.edu

Miquel Sola

Jordi Poater

Leonard M Proniewicz

Krzysztof ; Zborowski

Krzysztof K Zborowski

Miquel Solà

Theoretical studies on aromaticity of selected hydroxypyrones. Part 3 # . Chelatoaromaticity phenomenon in metalcomplexes of hydroxypyrones

Keywords: hydroxypyrones, chelatocomplexes, aromaticity, metalloaromaticity, density functional theory hydroxypyrones, chelatocomplexes, aromaticity, metalloaromaticity, density functional theory

Theoretical studies on aromaticity of selected hydroxypyrones. Part 3. Chelatoaromaticity phenomenon in metalcomplexes of hydroxypyrones.

Introduction

The family of hydroxypyrones together with closely related hydroxypyridinones forms a class of ligands with many potential applications in medicinal chemistry. [1] After deprotonation, these ligands easily form a number of stable complexes with divalent and trivalent metal ions. Because of that, and due to negligible toxicity, they can be used as agents for in situ metal ion complex formation and subsequent removal of metal ions excess from the body. On the other hand, their metalcomplexes are tested as new drugs designed for improving absorption and biodistribution of metal ions. Diseases such as anemia, thallassemia and diabetes are the most important examples. Potential applications of hydroxypyrones and hydroxypyridinones in diabetic therapy [2][START_REF] Thompson | Medicinal Inorganic Chemistry[END_REF] , iron distribution [START_REF] Liu | [END_REF] and aluminium chelation [5][6] were summarized in several critical reviews.

Metal ion complexes are formed by deprotonated hydroxypyrone units, also referred as anions of hydroxypyrones (see Fig. 1). Deprotonated 3-hydroxy-4-pyrone consists of a sixmembered heterocyclic pyran ring with an oxygen atom in para to the ketone group and two ortho exocyclic oxygen atoms (from the ketone and the deprotonated hydroxyl groups). The two exocyclic oxygens and the carbon-carbon bridge between them constitute the so-called OCCO group that is directly responsible for the metal ion complexation. The presence of π electrons in hydroxypyrone structures imposes the possibility of aromatic properties of the heterocyclic rings. In addition, the electron delocalization can also be determined in the OCCO group and aromaticity can be analyzed in the heterocyclic and metallocyclic rings after complexation too. Aromaticity of carbocyclic and heterocyclic compounds is a very popular topic in physical organic chemistry. On the other hand, chelatoaromaticity (aromaticity of chelate complexes) has received rather sporadic interest until recently, despite the fact that such idea was decribed firstly long time ago by Calvin and Wilson. [7] Recently, the concept of chelatoaromaticity has been renewed due to the synthesis of new chelatoaromatic complexes, new developments in the theory of aromaticity, and the recognition of the importance of chelatoaromaticity in prediction of the reaction mechanisms. This concept may also be useful in explaining many properties of chelates that may have appeared unlinked. For example, the electronic, vibrational, and NMR spectra, as well as the electrochemistry, and X-ray structure can be interpreted as a result of chelatoaromaticity induced by the metal ions. [8] For us, the most important point is that aromatic effects should influence the stability of metalcomplexes of hydroxypyrones. Chelatoaromatic stabilization can be important in cases where a nonaromatic or antiaromatic ligand coordinates to the metal ion and after coordination it gains aromatic character. An increase of the aromatic character in the metalcomplex provides an additional stabilization of the complex.

Aromaticity is a term that still is not precisely defined. Some aspects of its definition are still discussed. [9][10] That's why in the last few years we have focused our studies on selected, very well define group of ligands, i.e. hydroxypyrones to support such studies. In this paper we want to show how procedure used by us can be useful in developments of this topics in chemistry of chelate compounds. Thus, in the present work, we investigate the chelatoaromaticity phenomenon in metalcomplexes of some hydroxypyrones (pyromeconic acid, maltol and ethylmaltol) with several metal ions, like Al 3+ , Ga 3+ and In 3+ . Gallium and indium complexes of hydroxypyrones have potential practical importance as they are considered new useful radiopharmaceuticals. [11][12] On the other hand, the aluminium ionmaltol complex is used to try to understand the origin of the Alzheimer disease. [13] It is also important that all these metals belong to the 13 th main group of the periodic table. Their ions are closed shell species. Because of that, they are more convenient for computational studies than many of transition metal ions.

Many aromaticity indices are used in the present paper. It is important because it is known from the principal component analysis [14][15] that aromaticity may have a multidimensional character. As a result of that, it is recommended to use more than one aromaticity index in order to recognize all aspects of this phenomenon. [16][17][18]

Computational details

In order to study the basis set effects on the obtained results we performed calculations using three basis sets (LANL2DZ, [20] 3-21+G** [21][22] and 6-311++G** [23] ) of increasing quality. LANL2DZ basis set is a standard core potential approach widely used in metalcomplexes calculations. It is a mixture of the Los Alamos effective core potential with the Dunning/Huzinaga valence double zeta basis set for the first row of elements and a double zeta basis set for the valence electrons for the heavier elements. On the other hand, the 6-311++G** is a well-qualified basis set enriched with diffusion and polarization functions.

Unfortunately, this basis set is unavailable for indium. Because of that, available for all studied metals 3-21+G** basis set was also used. Geometries of all studied compounds were computed at the B3LYP [24][25][26] level of theory using all three aforementioned basis sets. All geometries calculated in this work correspond to geometrical minima with no imaginary frequencies. Obtained geometries were used for the calculation of magnetic properties (under the GIAO approximation [27] ) at the same level of theory. All these calculations were computed with the Gaussian03 program. [28] Atoms in Molecules [START_REF] Bader | Atoms in Molecules: A Quantum Theory[END_REF] properties were integrated using the AIMPAC package. [START_REF] Biegler-König | [END_REF] HOMA index (Harmonic Oscillator Model of Aromaticity [31] is calculated using bond lenghts of the studied systems by means of the equation:

( ) ∑ = - - = n i i opt R R n HOMA 1 2 1 α (1)
where R opt and R i are optimal bond lengths and bond lengths in the real system, respectively.

The variable α is an empirical factor chosen to provide a HOMA value of 0 for the Kekulé structure of benzene and a value of 1 for the real benzene with "aromatic" bond lengths.

Finally, n is the number of bonds in the system under consideration. In this paper, the HOMA index has been used for evaluation of electron delocalization efficiency in the heterocyclic ring and the OCCO group of all studied compounds.

The NICS (Nucleus Independent Chemical Shift) index of aromaticity is based on the magnetic properties of aromatic compounds. [32] It is defined as the negative value of the absolute magnetic shielding. It can be calculated in the center of the aromatic ring or fragment determined by the non-weighted mean of the heavy atoms coordinates (NICS(0)), or at 1 Å above it (NICS(1)). Negative NICS values denote efficient electron delocalization. In this work, NICS values were determined for heterocyclic and metallocyclic rings of studied ligands and metalcomplexes. More comprehensive NICS approach is the NICS scan method. [33] Magnetic properties necessary for NICS scan presentation were calculated from the ring center to 4.9 Å above it, with a step size of 0.1 Å along the line (normal) perpendicular to the ring plane.

Another very important property of aromatic compounds is their higher stability with respect to a structurally analogue model system which has no cyclic electronic delocalization.

For such purpose, the calculation of the Aromatic Stabilization Energy (ASE) is the core of defining the reasonable non-aromatic reference state. [34] One of the highly recommended methods for the estimation of ASE is the "isomerization method" based on the enthalpy difference between a methyl derivative of the aromatic system and its non-aromatic exocyclic methylene isomer. [35] There are several possible places for isomerization in the case of studied hydroxypyrones. Among them we have chosen the one presented on the Scheme below (example of the neutral molecule of hydroxypyrones). Such localization of the isomerization place let us avoid changes in the type of the metal ion interactions with investigated ligands.

Using the mentioned reaction, ASE iso were calculated for neutral form of studied ligands as well as for their anions, cations and metalcomplexes.

Scheme 1, here

Recently, electronic indices of aromaticity have been introduced as a new tool in aromaticity research. [36] The simplest index from this group is the PDI (para delocalization index). It is defined as the mean of the electronic delocalization indices (DI) calculated in the frame of the AIM theory for para-related atoms in the rings. [37] PDI values can be only calculated for six-membered rings. FLU is another aromaticity index based directly on electron delocalization that can be applied to heterocyclic rings of hydroxypyrones and their metalcomplexes. FLU measures the number of electrons shared between consecutive atoms in the ring. [38] The small drawback of the FLU index in our calculation is that there is no reference FLU parameter for the carbon-oxygen bond (because it is difficult to find the fully aromatic compound with such a bond in its ring). For this reason, the FLU parameters determined for the carbon-carbon bond have been used for the carbon-oxygen bonds too.

Finally, so-called multicenter aromaticity indices, namely: I ring , MCI, and KMCI, have been also calculated. These indices do not estimate delocalization between pairs of atoms, but 8 take into account delocalization in the whole ring. Details are described in original papers. [39- 41] I ring , MCI and KMCI indices are useful as aromaticity indicators of both heterocyclic and metallocyclic rings of studied compounds. In recent papers [19,42] , it has been shown that the MCI and I ring indices are among the most reliable measures of aromaticity.

Results and Discussion

This section is divided into two parts. First the aromaticity of various forms (neutral, cationic, anionic) of three hydroxypyrones together with their metal complexes is presented.

Then, the effect of the basis set in the results obtained is briefly discussed.

A. Aromaticity of hydroxypyrones

Aromaticity properties of three hydroxypyrones: pyromeconic acid (3-hydroxy-4Hpyran-4-one, abbreviated hereafter as Hpa), maltol (3-hydroxy-2-methyl-4H-pyran-4-one, Hma) and ethylmaltol (3-hydroxy-2-ethyl-4H-pyran-4-one, Hema) have been studied in this project. The neutral, cationic and anionic forms of these compounds, together with their complexes with aluminium, gallium and indium ions, have been taken into account.

Molecular structures of neutral, anionic and cationic forms of the pyromeconic acid molecule together with complexes formed by the pyromeconic acid anion with aluminium ion (two isomers: fac and mer are considered) are depicted in Fig. 1.

HOMA, NICS(0), and NICS(1) data for studied ligands and their charged species, calculated at the B3LYP/6-311++G** level of theory, were published previously. [43,44] Aromaticity of the heterocyclic ring [43] and electronic delocalization in the OCCO group [44] were considered. Performed calculations revealed that aromaticity of hydroxypyrone cations is higher than for neutral molecules, and anions present the lowest one (cations > neutral > anions). It was also previously documented that aromaticity of the deprotonated hydroxypyrone units in metalcomplexes of oxovanadium(IV) with maltol and closely related kojic acid are somewhere between the aromaticity of neutral and cationic forms. [45] Aromaticity data calculated at the B3LYP/6-311++G** level by geometric, magnetic (except NICS scan ) and electronic indices involved in the investigation are presented in Fig. 2. Fig. 2a depicts the HOMA data for the different forms of the pyromeconic acid and its complexes with Al 3+ and Ga 3+ ions. One can observe that, the aromaticity of the heterocyclic ring strongly increases from anion to cation. This correlates with the increase of electronic delocalization in the OCCO group from the anion to the cation as well. The aromaticity of the heterocyclic pyran ring in the metalcomplexes is positioned, more or less, in the middle between the neutral pyromeconic acid and its cation. In the case of the OCCO group, the metal complexed systems present an electron delocalization similar to that of the cation. In both cases aromaticity/electron delocalization of the aluminium complexes is slightly higher than for the galium ones. is poor and these rings should be described as non-aromatic. It can be noticed that, contrary to HOMA results, the aromaticity of gallium metalcomplexes is slightly higher.

Values of the electronic indices of aromaticity are presented in Figs. 2d-f. PDI and FLU data can be calculated in our case for the heterocyclic pyran ring only. PDI results perfectly follow the order of the relative aromaticity described for both HOMA and NICS indices. FLU data also follow the same trend with the only exception of the anionic form, which is found to be slightly more aromatic than the neutral. MCI, KMCI and I ring data for the heterocyclic ring perfectly follow the aromaticity predictions provided by the previous criteria. Predictions of these methods for metallocyclic rings (Fig. 2f) support the conclusion evaluated on the base of previously presented NICS data. Aromaticity of the metallocyclic rings is low. curves for all types of heterocyclic rings suggest its aromaticity. [33] From the differences in the depths of minima on the isotropic chemical shift and its out-of-plane component curves we can conclude that aromaticity increases in the order anion < neutral molecule < cation.

Aromaticity of the heterocyclic ring of the metalcomplex is between aromaticity of neutral molecule and that of cation. Ring in which metal cation is involved in shows shapes of the NICS scan curves similar to the ones observed for the anti-aromatic cyclobutadiene. [33] Figure 3, here Finally, results of the ASE iso calculations are presented in Table 1. These data, in line with the results from the NICS scan curves, suggest that all studied compounds are aromatic, and that the level of aromaticity increases from anion through neutral molecule and metalcomplexes to the cation. Table 1, here Therefore, with respect to the presented above aromaticity analysis at the B3LYP/6-311++G** level of theory, it is observed how all geometry-, magnetic-, energetic-and electronic-based (except FLU) criteria assign an increase of aromaticity of the heterocyclic ring of the pyromeconic acid when going from its anionic to neutral and to cationic forms. In addition, the aromaticity of this ring in the complexed systems is almost the same for both Al and Ga, and is located between that of the previous neutral and cationic forms. Measured by the HOMA method, electron delocalizations of the OCCO group for various forms of hydroxypyrones are similar to the corresponding aromaticity levels of the heterocyclic rings.

In addition, all aromaticity indices predict the strong increase of aromaticity of the anion with complexation.

The results presented above for the heterocyclic rings of neutral species, cations and anions can be easily rationalized in several ways. For example, if the Lewis structures depicted in the Scheme 2 are considered, one can recognize that, the most important resonance structure in all cases is the non-aromatic structure a. However the importance of aromatic structures b and b' decreases in the order cation > neutral > anion. The reason is the fact that in the cation, structures a and b-type differ only by the migration of the positive charge from the oxygen of the ketone group to the oxygen atom of the heterocyclic ring. In the neutral system, the b-type structures have a charge separation that is not present in the However, this system is additionally stabilized by the presence of the very polar carbonyl group, which causes that the actual number of π-electrons within the cyclic conjugated system gets closer to the aromatic reference of 6. [46][47] Deprotonation of the hydroxyl group in the anionic form of hydroxypyrones results in a strong π-donating substituent leading to an increase of the actual number of π-electrons within the heterocyclic ring, making it less aromatic. A similar effect may be obtained by decreasing the polarity of the exocyclic double bond from C=O to C=CH 2 . [48][49] On the other hand, the protonation of the carbonyl oxygen in Finally, complexation with the metal withdraws π-electrons from the pyran ring increasing its aromaticity.

B. Effect of the basis set on the aromaticity results

In order to show the influence of the basis set quality we performed calculations of aromaticity with three different basis sets: LANL2DZ, 3-21+G** and 6-311++G**.

Calculations have been carried out at the optimized geometries obtained with each of the basis set. Calculated values of the aromaticity indices in these three basis sets for all pyromeconic acid forms and complexes are presented in Fig. 4. These data lead to the conclusion that aromaticity measures are affected by the chosen basis set, the extent of that influence depends on the chosen criterion of aromaticity.

Figure 4, here

In the case of the HOMA (see Figs. 4a and4b), we have a simple and direct relationship, that is, the better the basis set the higher the HOMA values, which means higher aromaticity. It is true for both the heterocyclic ring and the electronic delocalization in the OCCO group, although in the case of the heterocyclic ring the difference between 3-21+G** and LANL2DZ is really small. The largest difference is observed between 6-311++G** and LANL2DZ basis sets. Data provided by the middle quality 3-21+G** basis set is closer to LANL2DZ. It must be said that all three basis sets follow the same aromaticity trends, already discussed in the previous section. The variation of both NICS(0) and NICS(1) values with the basis set (Fig. 4c and4d) are not so pronounced as for the HOMA method. In this case there is no direct relationship, and even the curves present crossings, although again all basis sets give the expected trend of an increase of aromaticity for the heterocyclic rings of studied compounds from the anionic to the cationic forms, and the aromaticity of the metal complexes located between those of the neutral and cationic species. Aromaticity of metallocyclic rings is found to be poor for all basis sets.

In the case of PDI and FLU indices (Fig. 4e) different tendencies are observed depending on the basis set. FLU index shows how the better basis set (6-311++G**) gives smaller aromaticity for hydroxypyrone's anion than for neutral molecule, against to all other indices. It is interesting that the FLU with the 3-21+G** reproduces correctly the aromaticity of the pyromeconic acid anion. The aromaticity order obtained in the frame of the PDI method follow the expected trend for the two basis sets.

On the other hand, shapes of the aromaticity curves of the three multicenter indices (I ring , MCI, and KMCI) are qualitatively similar to each other (see Fig. 4f), and all of them follow the expected trend for the heterocyclic ring. The only difference is the gap between values predicted by both basis sets for a particular index, as larger differences are observed for the I ring and MCI indices, whereas the values predicted for the KMCI method are closer, but in all cases the smaller basis set predicts a higher aromaticity. It is likely that multicenter indices provide the best data for the metallocyclic ring (it is possible that NICS predictions for the metallocyclic rings are somewhat perturbed by the presence of the metal at close distance [50] ). It is clearly seen how the aromaticity predicted by these multicenter indices for the metallocyclic ring is always much lower than for the pyran rings. It is also observed how, in the case of metallocyclic rings, the results are quite insensitive to the aromaticity index and the basis set used for the calculation. On the basis of these results and presented above NICS Finally, we want to compare the aromaticities of the Hpa metalcomplexes with those formed by other studied ligands, Hma and Hema, using data obtained based on the 6-311++G** basis set. The corresponding tables enclosing all aromaticity measures calculated on the B3LYP/6-311++G** level of theory can be found in the Supporting Information. As expected from the structures in Scheme 2, both Hma and Hema present the same behavior as Hpa. Similarly, we observe an increase of aromaticity from the anionic form to the cationic one, and that of the metalcomplexes is located between the neutral and the cationic forms.

Even if the aromaticity values for the equivalent forms with the different ligands are quite similar, the substitution of the hydrogen atom by a methyl or an ethyl group at the position 2 of the ring affects neither the aromaticity of the heterocyclic ring nor the electron delocalization in the OCCO group. This shows that in order to have a significant substituent effect on the aromaticity of the heterocyclic ring one needs to place in R position substituents with strong π-donor (such as NR 2 ) or π-acceptor character (such as NO 2 ) that can modify the π-population of the ring. Preliminary calculations suggest that strong π-donor or π-acceptor character substituents can modify the π-population of the ring and significantly change its aromaticity.

Conclusions

In the present work we have shown that electronic indices of aromaticity (FLU, PDI, I ring , MCI, and KMCI) support the aromaticity order of various hydroxypyrones forms, previously established on the base of the results served by some "classical" indices like 16 HOMA and NICS. The same is observed for the NICS scan and ASE iso data. This order states that aromaticity of the anionic form of hydroxypyrones is very low, aromaticity of neutral molecules is moderate, and hydroxypyrone cations are aromatic.

Existence of the chelatoaromaticity phenomenon in complexes of studied hydroxypyrones with aluminium, gallium, and indium ions has been documented. When low aromatic anions of studied ligands coordinate to metal ions, their aromatic properties change very significantly, and they become quite aromatic species. Aromaticity of the heterocyclic ring in studied metalcomplexes is located somewhere between the neutral and the cationic forms of the hydroxypyrone. It seems that the electronic delocalization increase takes also place in the part of the ligand that is directly bonded to the metal ion. On the other hand, the analysis of the metallocyclic rings show that electron delocalization in the whole metallocyclic ring is low. For this reason, the metallocyclic rings must be classified as nonaromatic.

Our study also show that the different hydroxypyrones (pyromeconic acid, maltol and ethylmaltol), present very similar aromaticities in groups of their anionic, neutral or cationic forms, as well as the corresponding metalcomplexes (with either Al 3+ , Ga 3+ or In 3+ ), respectively.

It has to be stressed out that the used for calculations basis set plays crucial role. One cannot use different basis sets for comparison (aromaticity values) of obtained results for the same ligands, although the trends in aromaticity trends remains almost invariant.

Higher aromaticity denotes higher stability. So, an increase of the electron delocalization in hydroxypyrones with complexation should have an important impact on the stability of the studied metalcomplexes. Further research is underway trying to estimate their (chelato)aromatic stabilization energies in order to know if they can be important for their chemistry.

Figure 1

 1 Figure 1, here

  work, numerous aromaticity indices were used: geometric (HOMA), magnetic (NICS(0), NICS(1) and NICS scan ), energetic (ASE iso ) and various electronic indices of aromaticity (PDI, FLU, I ring , MCI and KMCI).

  on energetic basis. Unfortunately, there are a lot of problems with

Figure 2

 2 Figure 2, here

Figure 3

 3 Figure 3 depicts the behaviour of the NICS scan values of the heterocyclic and

  Scheme 2, here

  causes the opposite trend, as becoming a strong π-withdrawal group, thus decreasing the actual number of π-electrons in the ring and making the ring more aromatic.

  metallocyclic rings we can conclude that rings containing metal ions are nonaromatic. The aromaticity of both the heterocyclic and the metallocyclic rings is only slightly changed along the series of Al +3 , Ga +3 to In +3 .

Figure 1 .

 1 Figure 1. Structure of pyromeconic acid in neutral, anionic and cationic form, and the corresponding metal complexes with Al 3+ (two isomers). Structures of maltol and ehylmaltol

Figure 2 .

 2 Figure 2. Aromaticity data for pyromeconic acid and its metalcomplexes at the B3LYP/6-

Figure 3 .

 3 Figure 3. NICS values as a function of distance from the ring center.

Figure 4 .

 4 Figure 4. Comparison of aromaticity data for pyromeconic acid and its metalcomplexes

Figure 1 .

 1 Figure 1. Structure of pyromeconic acid in neutral, anionic and cationic form, and the corresponding metal complexes with Al3+ (two isomers). Structures of maltol and ehylmaltol are also depicted.

Figure 2 .

 2 Figure 2. Aromaticity data for pyromeconic acid and its metalcomplexes at the B3LYP/6-311++G** level.

Table 1 .

 1 Calculated (B3LYP/6-311++G**) ASE iso values of the isomerization reaction from Scheme 1 for different form of the pyromeconic acid, [kJ/mol]. Reaction used for ASE iso calculations. Most important resonance structures of hydroxypyrones (R = H, CH 3 , and CH 2 CH 3 ) in neutral, anionic and cationic forms.

	Compound	ASE iso
	pa -	77.7
	Hpa	81.4
	H 2 pa	118.7
	Alpa mer	107.0
	Alpa fac	106.1
	Gapa mer	104.5
	Gapa fac	103.9

Table S1 .

 S1 B3LYP/6-311++G** aromaticity data for heterocyclic rings of studied compounds.

		HOMA NICS(0) NICS(1) PDI	FLU	I ring	MCI KMCI
	pa -	-0.49	-1.45	-3.75 0.0296 0.1100 0.0038 0.0002 0.0014
	Hpa	0.04	-2.75	-4.69 0.0326 0.1114 0.0073 0.0034 0.0027
	H 2 pa	0.64	-7.28	-7.93 0.0545 0.0752 0.0153 0.0149 0.0057
	Alpa fac	0.32	-4.08	-5.81 0.0383 0.0937 0.0084 0.0045 0.0032
	Alpa mer	0.32	-4.11	-5.88 0.0389 0.0932 0.0085 0.0045 0.0032
	Gapa fac	0.29	-4.02	-5.87 0.0384 0.0945 0.0084 0.0044 0.0032
	Gapa mer	0.30	-4.01	-5.86 0.0383 0.0942 0.0085 0.0045 0.0032
	ma -	-0.47	-1.27	-3.77 0.0290 0.1186 0.0039 0.0003 0.0015
	Hma	0.08	-2.63	-4.58 0.0321 0.1196 0.0071 0.0037 0.0027
	H 2 ma Alma fac Alma mer Gama fac Gama mer ema -	0.76 0.31 0.31 0.28 0.29 -0.47 F -7.10 -4.03 -4.02 -3.88 -3.90 -1.46 o r	-7.79 0.0536 0.0831 0.0146 0.0146 0.0055 -5.76 0.0376 0.1030 0.0083 0.0049 0.0031 -5.71 0.0383 0.1022 0.0083 0.0050 0.0031 -5.66 0.0377 0.1037 0.0083 0.0048 0.0031 -5.66 0.0382 0.1027 0.0083 0.0049 0.0031 -3.82 0.0289 0.1187 0.0039 0.0003 0.0014
	Hema H 2 ema Alema fac Alema mer Gaema fac Gaema mer	0.03 0.61 0.31 0.31 0.28 0.29	-2.76 -6.99 P -4.56 0.0316 0.1229 0.0069 0.0033 0.0026 -7.72 0.0533 0.0839 0.0145 0.0145 0.0055 -4.13 -5.79 0.0380 0.1026 0.0083 0.0050 0.0031 -4.12 e -5.69 0.0384 0.1019 0.0083 0.0050 0.0031 -4.02 -5.70 0.0425 0.1020 0.0076 0.0046 0.0035 -4.00 -5.63 0.0538 0.1038 0.0079 0.0040 0.0035 e r
				R
				e
				v i e
					w

Table S2 .

 S2 B3LYP/6-311++G** aromaticity for OCCO groups/metallocyclic rings of studied compounds .

		HOMA NICS(0) NICS(1) I ring	MCI KMCI
	pa -	-0.51
	Hpa	0.05
	H 2 pa	0.46
	Alpa fac	0.50	-0.55	-1.57 0.0010 0.0012 0.0006
	Alpa mer	0.51	-0.31	-1.63 0.0011 0.0012 0.0006
	Gapa fac	0.45	-1.68	-2.16 0.0028 0.0031 0.0017
	Gapa mer	0.45	-1.56	-2.13 0.0028 0.0031 0.0017
	ma -	-0.40
	Hma	0.06
	H 2 ma	0.44
	Alma fac	0.55	-0.64	-1.41 0.0010 0.0012 0.0006
	Alma mer	0.56	-0.41	-1.66 0.0010 0.0012 0.0006
	Gama fac	0.50	-1.62	-1.94 0.0027 0.0030 0.0017
	Gama mer	0.51	-1.47	-1.88 0.0027 0.0030 0.0017
	ema -	-0.39
	Hema	0.06
	H 2 ema	0.43
	Alema fac	0.55	-0.41	-1.34 0.0010 0.0012 0.0006
	Alema mer	0.56	-0.58	-1.26 0.0010 0.0012 0.0006
	Gaema fac	0.50	-1.66	-186	0.0026 0.0025 0.0020
	Gaema mer	0.51	-1.56	-1.83 0.0033 0.0029 0.0018

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Acknowledgements

The work has been performed under the Project HPC-EUROPA (RII3-CT-2003-506079), with the support of the European Community -Research Infrastructure Action under the FP6 "Structuring the European Research Area" Programme. We thank the Spanish MEC (project CTQ2008-03077/BQU) and the Catalan DIUE (project 2009SGR-637) for their financial support. J.P. thanks the MEC for the Ramón y Cajal contract. Support for the research of M.S. was received through the ICREA Academia 2009 prize for excellence in research funded by the DIUE of the Generalitat de Catalunya. Authors kindly acknowledge Barcelona Supercomputer Center -Centre Nacional de Supercomputació (BSC-CNS) and Warsaw University's Interdisciplinary Centre for Mathematical and Computational Modelling "ICM" (project number G17-8) for computational facilities.

Supplementary material

Numerical values of aromaticity indices for all studied compounds calculated at the B3LYP/6-311++G** level of theory. This material is freely available in Wiley Interscience.