Molecular Modeling Studies of β-Sitosterol Extract from Miconia burchellii Triana (Melastomataceae) from Brazilian Cerrado

The Brazilian Cerrado biome is considered one of the 25 hotspots worldwide that contain bioactive compounds due to its great biodiversity; however, the reduction of its native area over time due to the expansion of urbanization and agribusiness may have compromised knowledge of its biological variety. In this context, knowledge about Cerrado species can contribute to its biodiversity preservation. This study aims to describe the isolation, molecular architecture and theoretical calculations of the compound (3S,8S,9S,10R,13R,14S,17R)-17-[(2R,5R)-5 ethyl -6-methylheptan-2 yl]-10,13 dimethyl 2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H cyclopenta[a]phenanthren-3 ol, extracted from the Brazilian Cerrado Miconia burchellii plant. The supramolecular arrangement was described by Hirshfeld surface analysis, demonstrating the intermolecular interactions in the crystalline packing. The structure-property relationship shows the electrostatic potential map analysis, which reveals that the oxygen region is susceptible to electrophilic attack, and the frontier molecular orbital confirmed the kinetic stability of this compound. This study represents another step forward in the knowledge of compounds with pharmacological and medicinal properties extracted from the Cerrado.


Introduction
2][3] It is a highly heterogeneous landscape, and parts of it are severely threatened.Among those parts that need particular attention, the Cerrado-Amazon transition zone stands out, considering it has undergone heavy deforestation and presents highly unusual rupestrian fields. 4,5The unique characteristics of the biome, such as long periods of drought, as well as its relief, altitude, and soil characteristics, have led to its varied phytophysiognomy. 6,7The evaluation of the therapeutic potential of plant species from Cerrado's region and some of their constituents has been the subject of studies that result in the discovery of molecules with great potential for future use as medicinal agents. 8,9However, its native vegetation has decreased considerably due to urbanization and agribusiness, thus resulting in a lack of biological and medicinal knowledge of the Cerrado. 10The Miconia genus is an example of under-researched vegetation and represents about 2% of the studied species. 11Among the few studies carried out, 79 compounds belonging to different classes have been identified, with sterols representing 6% of this distribution. 11he isolation and characterization of sterols from Cerrado plants are not restricted to the genus Miconia but also occur in Caryocar, 12 Genipa, 13 Qualea, 14 Sebastiania, 3 Jatropha, 3 Poincianella, 3 Plathymenia, 15 Cecropia, 16 Myracroduon, 16 Siparuna, 16 Strphnodedron, 16 among others.

Crystallographic characterization
Single-crystal XRD data collection was performed on a Bruker PHOTON-II diffractometer (Agilent SuperNova, Notre Dame, USA) applying a combination of ωand φ-scans of 0.5°. 36Data were corrected for absorption and polarization effects and analyzed for space group determination. 37The structure was solved by dual-space methods 38 and refined by full-matrix least-squares analysis of F 2 against all reflections. 39Anisotropic atomic displacement parameters were used to refine all non-hydrogen atoms.Atomic displacement for the hydrogens was placed to the equivalent isotropic displacement parameter (U iso (H) = 1.5U eq (C) for methyl, 1.2U eq (C) for all others) according to the riding model.The crystallographic information file (CIF) was deposited at the Cambridge Crystallographic Data Center (CCDC) under deposit number 2182940.

Hirshfeld surface
The HS was calculated over the crystallographic structure (obtained experimentally through XRD), without optimization of the geometric parameters.The analysis of the HS was used to visualize and interpret the potential intermolecular interactions, which can produce a 2D fingerprint histogram, by plotting the fraction of points on the surface as a function of the (d i and d e ) pair. 40On an HS the normalized contact distance (d norm ) is defined from the distance of atoms external (d e ), and internal (d i ) to the surface, through CrystalExplorer software, 41 using the van der Waals radius, described in equation 1: (1) where r vdw represented the van der Waals radii of the atoms. 42he graphical representation of d norm uses color coding system to identify intermolecular interactions, with the sum of the van der Waals radii.Different levels of color are associated with the intensity of interactions, where blue and red indicates long and short intermolecular contacts, respectively. 42

Theoretical calculation
The geometric parameters obtained experimentally through XRD were optimized in the gas phase by the Gaussian09 software, 43 the conformers (STR-I and STR-II) were individually optimized through density functional theory (DFT) 44 applying M06-2X/6-311++G(d,p) level of theory, [45][46][47] which is suitable for non-covalent interactions. 45,48rom the results generated in the optimization, FMO, and the MEP were calculated for each conformer.The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) can indicate kinetic stability and chemical reactivity of the molecule, and characterize π* antibonding and nonbonding orbitals and their nucleophilic/electrophilic regions. 49,50The MEP map also contributes to identifying the reactive regions of a molecule and regions of nucleophilic/electrophilic attack; to build this surface of electrostatic potential, we used a function that considers the potential created by the nucleus and electrons, as shown in equation 2: (2) V(r) is a potential created at a defined point, the first term of summation is the electrostatic potential created by the nucleus, while the second term of summation is the electrostatic potential created by electrons. 51

Solid-state characterization
The STR was crystallized in the non-centrosymmetric monoclinic space group P2 1 , with two independent molecules (STR-I and STR-II), complexed with one water molecule (Figure 1), in the asymmetric unit.The conformers differ in the aliphatic chain site, indicating a conformational polymorphism in the crystalline state.Each conformer has nine chiral carbons (C1, C13, C17, C18 and C21 in R conformation; C4, C9, C10 and C14 in S conformation).Their crystallographic parameters and refinement data are shown in Table 1.
The STR compound has three rings with six members and one ring with five members.Rings A and C have a chair conformation, while ring B has a half-chair conformation.The overlap of STR-I and STR-II demonstrates the value of the root mean square (RMS) = 0.0363, which measures the geometric difference in the structures.These differences can be evidenced by the dihedral angles of atoms C18A-C19A-C20A-C21A (-170.3(9)°) and C18-C19-C20-C21 (63.9 (9)°), not overlapping the ethyl radicals and the isopropyl radicals (Figure 2).
The supramolecular arrangement is formed by O1W-H1WA … O1A, O1W-H1WB ... O1A, O1-H1 … O1W and O1A-H1A … O1 interactions (Table 2), appearing as a ring coordinated by two water molecules related by a twofold screw axis along the b axis, which can be described as R 4 5 (10), as shown in Figure 3a.Also, Figure 3b shows the crystal packing of STR, which is formed by a three-dimensional network described as a "chain of rings" lying at the center of the unit cell.To compare the structure of STR with other water-complexed sterols, mainly concerning the supramolecular arrangement, similar structures were selected from the CCDC: stigmast-5-en-3-ol hemihydrate (code 1434206-STS I 53 and code 1985852-STS II). 54It is observed that both compounds exhibit the three-dimensional network which is coordinated by the water molecules, forming the "chain of rings" that can be described as R 4 5 (10).On the other hand, sterols without the water molecules, such as stigmasta-4,25-diene-3β,6β-diol, 55   Formula weight / (g mol -1 ) 847.39 Temperature / K 120(2) Crystal system monoclinic

Molecular modeling
The RMS values, predicted by Mercury software, 58 between conformers (STR-I and STR-II) experimental geometries and theoretical calculation were 0.0179 and 0.0096, respectively.The overlappings of the M062X/6-311+G(d,p) level of theory (yellow) and X-ray (black) for conformers (STR-I and STR-II) are shown in Figures 6a and 6b, respectively.The comparative graphs (experimental geometries and theoretical calculation) for the bond lengths and angles obtained for STR-I and STR-II are shown in Figure 7.The mean absolute percentage deviations (MAPD) were defined by equation 3: (3) where χ XRD and χ DFT represents the geometric parameters for the theoretical calculation and experimental geometrical data, respectively.The MAPD values for STR-I bond lengths and angles were 0.720 and 0.509, respectively.The Pearson   The calculated HOMO orbital for STR conformers was located on rings A and B (both in STR-I and STR-II), while the LUMO orbital was also similar for both (STR-I and STR-II) and was spread in the molecule (Figure 8).The LUMO energy was -20.47 kJ mol -1 for STR-I and -19.87 kJ mol -1 for STR-II, and this orbital characterizes π* antibonding, with negative energy indicating electrophilic regions, susceptible to accept electrons in a chemical reaction.The energy difference between these orbitals (E GAP = E LUMO -E HOMO ) is an important indicator of the kinetic stability and chemical reactivity of the molecule, 49 because it is energetically unfavorable to add electrons to a high-lying LUMO and to  extract electrons from a low-lying HOMO, and so to form the activated complex of any potential reaction. 59The E GAP is 738.92 kJ mol -1 for STR-I and 740.08 kJ mol -1 for STR-II, respectively.
The MEP map calculated for the STR compound is related to the local charges.The red colors indicate regions susceptible to electrophilic attack and are on the O1A (STR-I) and O1 (STR-II) atoms, while blue colors are susceptible to nucleophilic attacks, 51,60,61 they are on the H1A (STR-I) and H1 (STR-II) atoms.It should be noted that these regions correspond to the O1W-H1WB … O1A, O1W-H1WA … O1A, O1-H1 … O1W and O1A-H1A ... O1 interactions, which have been described by geometrical parameters and electronic density.Figure 9 shows the MEP map surfaces with isovalues of ±0.0004.

Vibrational assignments
The main IR absorption bands are in Table 3 and the theoretical and experimental FTIR spectra for STR-I and STR-II conformers are in Figure 10.The values in vibrational frequencies obtained at M06-2X/6-311++G(d,p) level of theory were scaled by Yin and Kong 62 as 0.943.Theoretical measurements of ν(O-H) for STR-I and STR-II conformers, obtained in the gas phase, absorb at 3687 cm -1 , while the experimental measurements occur at 3424 cm -1 .This decrease of the experimental vibrational frequency value for ν(O-H) occurs due to the molecular hydrogen interactions.Absorption peaks appear in the experimental ν(C=C) for STR-I and STR-II conformers at 1651 cm -1 , while the theoretical measurements absorb at 1658 cm -1 .The ν(C sp 3 -H) for STR-I and STR-II conformers is in the range of 2936-2861 cm -1 , while the DFT calculations assigned at the region of 2936-2877 cm -1 .

Conclusions
The β-sitosterol compound was crystallized with two independent conformers and one water molecule in the asymmetric unit.The title compound was overlaid, and differences were evidenced from the dihedral angles of carbons C18-C19-C20-C21, not overlapping the ethyl radicals and the isopropyl radicals.The supramolecular arrangement was stabilized by classical hydrogen O-H ... O bonding, forming a 'chain of rings', which is also observed in similar compounds researched at CCDC.The kinetic stability of the compound was confirmed through the high value found (738.92 kJ mol -1 for STR-I and 740.08 kJ mol -1 for STR-II).The MEP analysis reveals that the oxygen region is susceptible to electrophilic attack.The structural study of a sterol extracted from the Brazilian Cerrado offers a new and deeper understanding of the biodiversity of sterols.

Figure 1 .
Figure 1.Oak Ridge Thermal Ellipsoid Plot (ORTEP) diagram of ellipsoids at 30% probability level with the atomic numbering scheme for (a) STR-I, and (b) STR-II.
cholest-5-en-3-ol 56 and 10,13-dimethyl-17-(5-(2-methylcyclopropyl)hexan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol 57 (code 639110, 1944206 and 1417552, respectively), exhibit only a two-dimensional crystalline packing network.Intermolecular interactions were analyzed by the HS denominated d norm , where high values of distances d i and d e indicate donor and acceptor regions of intermolecular contacts, represented by d and r, respectively.Color scales are used to indicate intensities of contacts, where the blue color represents weaker contacts and the red color represents stronger contacts.In Figure 4a, the molecule inside the surface is STR-I; the red dots (1r) and (2r) correspond to d e contacts indicating that they act as acceptors for O1W-H1WB … O1A and O1W-H1WA … O1A, respectively.In Figure 4b, the molecule inside the surface is STR-II; the red dot (3d) corresponds to d i contacts, indicating where the molecule acts as a donor of O1-H1 … O1W, and the red dot (4r) corresponds to d e contacts, indicating where the molecule acts as an acceptor of O1A-H1A … O1.The combination of d e and d i distance functions provides a mapping of all contacts present in the molecule, and their percentage contribution to each type of interaction present, making the fingerprints unique for each compound.Figure 5 represents the fingerprint of the STR interactions, where the H … H weak contacts (region d e = d i = 1.2 Å) represent the majority of all observed contacts making up 93.7% of the HS.The O … H contacts constitute the second-largest percentage in the STR compound making up 5.4% of the total surface, and were detected as a spike

Figure 2 .
Figure 2. Overlap of STR-I and STR-II.Hydrogen atoms were omitted.

Figure 4 .
Figure 4. Hirshfeld surface d norm mapped indicating intermolecular interactions of STR-I (a), and STR-II (b).The dotted black lines represent hydrogen bonds.

Figure 5 .
Figure 5. Fingerprint and quantification of different types of contacts of STR.

Figure 6 .
Figure 6.Overlapping between the experimental X-ray data (black) and the M062X/6-311+G(d,p) level of theory (yellow) structures for (a) STR-I and (b) STR-II.

Figure 7 .
Figure 7.The comparative graphs of the geometric (a) bond length and (b) angle for STR-I, and (c) bond length and (d) angle for STR-II, obtained by experimental X-ray and theoretical calculation data.

Table 3 . 1 Scaled
Vibrational assignments of the theoretical and experimental FTIR for STR-I and STR-II conformers.These results were obtained at M06-2X/6-311++G(d,p) level of theory in the gas phase Vibrational mode STR-I and STR-II conformers Experimental frequency / cm -groups; b gem-dimethyl group.ν: stretching; d: bending.

Figure 10 .
Figure 10.Overlapping of the theoretical (red) and experimental (black) FTIR (KBr) spectra of STR-I and STR-II conformers.

Table 1 .
Crystal data and structure refinement for STR

Table 2 .
Hydrogen bond distances and angles for STR