Structural , Vibrational , and Electronic Properties of the Glucoalkaloid Strictosidine : A Combined Experimental and Theoretical Study

A detailed structural analysis and spectral behavior of the glucoalkaloid strictosidine, a precursor of all monoterpene indole alkaloids, are discussed. The experimental NMR, FTIR, and UV results were compared to the theoretical DFT spectra calculated by Becke using the three-parameter Lee-Yang-Parr (B3LYP) function with 6-31G(d) and 6-311++G(2d,p) basis sets. The theoretical geometry optimization data were compared with the X-ray data for precursors and similar structures in the associated literature. The similarity between the theoretical and experimental coupling constants values made it possible to affirm the values of dihedral angles and their configuration, reinforcing findings from previous stereochemical studies.Theoretical UV analysis agreed well with themeasured experimental data, with bands assigned. CalculatedHOMO/LUMOgaps show low excitation energy for strictosidine, justifying its stability and reaction kinetics. The molecular electrostatic potential map shows opposite potentials regions that form hydrogen bonds that stabilize the dimeric form, which were confirmed by excellent agreement of the dimeric form theoretical wavenumbers with the experimental IR spectrum. ESI-MS/MS data revealed patterns for the fragmentation of the protonated strictosidine molecule outlined by an NBO study.


Introduction
Indole alkaloids play a very important role in the chemistry of natural products and are especially recognized for their use in clinical medicine as an adjunct to anesthetics.The finding of several clinic uses has driven intense study of this class of substances, and many antiplasmodial [1,2], cytotoxic [3], antibacterial [4], antifungal [5], spasmodic [6], hypotensive [7], and anti-inflammatory [8] properties have been related to indole alkaloids.Strictosidine (Figure 1) is a key glucomonoterpene indole alkaloid precursor of all indole monoterpene alkaloids.This crucial molecule, which originates from a reaction between tryptamine and the monoterpene glycoside secologanin, is found in several plant species [9][10][11][12][13] and was first isolated from Rhazya stricta [14].Strictosidine has also been obtained in cell suspension cultures and under biomimetic conditions [15,16].
Studies discussing the structure and stereochemistry of strictosidine [16][17][18] have compared its spectral data with those of similar structures, confirming that its C3 atom has the S configuration or 3 [S].This configuration is identical to that of known monoterpene indole alkaloids, in disagreement with the proposal that vincoside was the precursor of indole alkaloids with 3 [R] configuration, as previously thought.Patthy-Lukáts et al. [19] studied the stereochemistry of strictosidine based on experimental NMR analysis, determining its spatial configuration.However, there are no X-ray studies because this molecule has not been obtained yet in crystalline form [19] and no theoretical molecular modeling study that discusses the bond lengths and planar and dihedral angles was previously presented.The determination of the relationship between theoretical vibrational frequencies and experimental IR absorbance bands and between theoretical electronic transitions and experimental UV bands of strictosidine has not been investigated yet.Therefore, a detailed theoretical DFT and experimental investigation of the structure and spectral behavior of this molecule, providing a comprehensive description of strictosidine, have been reported.Initially, the alkaloid has been isolated from Strychnos amazonica and the molecule was characterized by NMR ( 1 H, HSQC, and HMBC), MS (ESI-MS/MS), UV, and FTIR.The theoretical data (optimized geometry, UV, IV, MEP, and NBO calculations) were compared with the experimental data to answer questions regarding structure, electronic transitions involved in the UV spectrum, vibrational assignments, and other physical properties of strictosidine.

Experimental Section
2.1.General Procedures.Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 600 AVANCE spectrometer with 12.4-Tesla magnetons at 295 K in deuteriomethanol containing 0.06% TMS as an internal standard.The ESI-MS  data were recorded on LQC fleet ion trap Thermo Scientific spectrometer equipped with an electrospray ionization (ESI) ion source in the positive mode with the following parameters: capillary voltage at 26 V; spray voltage at 5 kV; tube lens offset at 100 V; capillary temperature at 225 ∘ C; auxiliary gas at 5%; and sheet gas at 12%.The UV data were recorded in methanol using a PDA Detector plus Finnigan Surveyor (Thermo Scientific).The FTIR data were recorded in KBr pellet technique (solid phase) using an ABB FTLA200.[21].The DFT approach was used to optimize the geometry using the 6-31G(d) and 6-311G++(2d,p) basis sets and the B3LYP functional.The molecular geometries were fully optimized by the force gradient method using Berny's algorithm, and the potential energy surfaces were characterized using standard analytical harmonic vibrational analysis to confirm that the stationary points corresponded to the minima of the potential energy surfaces (no imaginary frequencies or negative eigenvalues were found).The theoretical 3  HH coupling constants were calculated using the NMR protocols implemented using the DFT B3 LYP 6-31G(d) and 6-311++G(2d,p) basis sets in the Gaussian 03 software.These coupling constants were compared with the measured values that were experimentally obtained, showing RMSD values of 1.45 Hz for DFT B3LYP 6-31G(d) and 1.25 Hz for 6-311++G(2d,p).The UV spectra were calculated using the RTD-B3LYP-FC functional and 6-31G(d) and 6-311G++(2d,p) basis set [22,23].The NBO values were obtained with NBO 3.1, as implemented in the Gaussian 03 package using the 6-31G(d) basis set and the B3LYP functional.The harmonic frequencies were calculated at the B3LYP/6-31G(d) level using the optimized structural parameters.The assignments of the calculated wavenumbers were aided by the animation option of the GaussView program, which gives a visual presentation of the vibrational modes [24].The potential energy distribution (PED) was calculated with the help of the VEDA4 software package [25].

UV Analysis.
The electronic spectrum of the molecule in a methanol solution was compared with the calculated spectrum (in the gas phase) at time dependent density functional using the B3LYP 6-31G(d) and B3LYP 6-311G++(2d,p) basis sets, as Figure 3 shows.The bands located at 222 and 272 nm could be experimentally observed and were in agreement with the presence of chromophores.Because strictosidine is an aromatic compound,  →  * transitions were involved but the presence of conjugations in the pentacyclic ring of the tryptophan portion and in the -alkoxyacrylate group suggested that  →  * transitions also occurred.The theoretical calculations predicted an intense electronic transition of 5.8964 eV, with an oscillator strength  = 0.548 at 210.27 nm for B3LYP 6-31G(d).For B3LYP 6-311++G(2d,p), an electronic transition of 5.62 eV was predicted, with an oscillator strength  = 0.2714 at 220.62 nm.These results showed good agreement with the measured experimental data (222 nm) assigned to the  →  * and  →  * transitions of the indole portion.The calculations also predicted a weaker electronic The highest occupied molecular orbitals (HOMO) and the lowest-lying unoccupied molecular orbitals (LUMO), also called frontier molecular orbitals (FMOs), play an important role in many properties of a compound as well as in its quantum chemistry and UV-Vis spectra.The energy gap between the HOMO and LUMO energies is the basis for the chemical stability and reactivity of a molecule.The quantum bonding features of strictosidine are depicted by a plot of the HOMO, HOMO−1, LUMO, and LUMO−1 in Figure 4.In the UV-Vis spectrum, the two maximum calculated absorption wavelengths corresponded to the contributions of the electronic transitions from HOMO → LUMO+4 (50.7%) and HOMO−1 → LUMO+1 (23.74%) for 210 nm and HOMO → LUMO+1 (89.8%) for 265 nm in B3LYP 6-31G(d).In B3LYP 6-311G++(2d,p), the wavelengths corresponded to HOMO → LUMO+16 (26%), HOMO → LUMO+19 (14%), and HOMO → LUMO+18 (5.7%) for 220.6 nm and HOMO → LUMO+2 (19%) and HOMO → LUMO+3 (70.81%) for 278.3 nm.It is clear from Figure 5 and Figures S5 and S6 (see Supplementary Information) that the major transitions were restricted to the indolic portion because the HOMO, HOMO−1, and LUMO+1 are located in this region and only the LUMO is located in the -alkoxyacrylate group.Transitions involving the LUMO contribute minimally to the absorption wavelengths () of strictosidine, showing that the transitions in the -alkoxyacrylate group have no influence, in disagreement with the ancient proposal that alkoxyacrylate group influences strictosidine UV spectrum [14].This prediction was proven by the similarity of the experimental spectrum of strictosidine to the spectra of several other indole alkaloids that lack alkoxyacrylate groups [31].

Global and Local Reactive Descriptors.
The energy gap between the HOMO and LUMO is very important for determining the electrical properties, kinetic stability, optical polarizability, and chemical reactivity descriptors, such as hardness and softness, of a molecule.
The concept of hardness () and softness is related to a compound's reactivity and is a property that measures the extent of chemical reactivity to which the addition of a charge stabilizes the system.The chemical potential () provides a global reactivity index and is related to charge transfer from a system of higher chemical potential to one of lower chemical potential.Electronegativity () is the power to attract electrons and is directly related to all the previously mentioned properties.All these properties are defined as follows [32,33]: where  and  () are the electronic energy and the external potential of an N-electron, respectively.Based on Koopmans theorem for closed-shell molecules, these global chemical reactivity descriptors can be simplified and defined as follows: where  is the ionization potential and  is the electron affinity of the molecule.The ionization energy and electron affinity are obtained from the HOMO and LUMO energies as  = − HOMO and  = − LUMO .In terms of chemical hardness, a large HOMO-LUMO gap indicates a hard molecule and is related to more stable molecules, whereas a small gap indicates a soft molecule and is related to a more reactive molecule.
Another important descriptor is the electrophilicity index (), a global maximum reactivity index that is similar to chemical hardness and chemical potential.The electrophilicity index measures the global electrophilic nature of a molecule and was proposed by Parr et al. [34,35] as a measure of energy lowering due to charge transfer.The electrophilicity index is defined as follows: This scale permits the classification of organic molecules as strong,  > 1.5 eV, moderate, 0.8 <  < 1.5 eV, and marginal,  < 0.8 eV, electrophiles.On the other hand, there is a good correlation in the inverse of the electrophilicity (1/); thus, molecules located at the bottom of the electrophilicity scale are classified as marginal electrophiles, corresponding with good nucleophiles [36].However, when the molecule bears more than one functional group with opposite electrical charge, its nucleophilic character cannot be straightforwardly associated with the inverse of the electrophilicity.Thus, the nucleophilicity index () appears as a different descriptor which gives more information about nucleophilicity and is defined as follows [37]: where tetracyanoethylene (TCE) is taken as reference.All these properties were calculated using these equations for strictosidine in methanol through B3LYP/6-31G(d) and B3LYP/6-311++G(2d,p) basis sets and their values are shown in      NBO analysis is a helpful tool for understanding the delocalization of the electron density from the occupied Lewis-type (donor) NBOs to properly unoccupied non-Lewis-type (acceptor) NBOs [43][44][45][46][47] within the molecule.This analysis uses the second-order perturbation energies  (2)  [donor () → acceptor ()] that involve the most important delocalization instances, which are given as follows: Table 3 shows more-detailed breakdown of the Lewis and non-Lewis occupancies, confirming the quality of the natural Lewis structure description.The total Lewis occupancy was 98.395% and the non-Lewis occupancy was 1.605%.The NBO analysis revealed strong intramolecular interactions formed by the orbital overlaps between C-C bonding and C-C antibonding and by overlaps between the N and O lone pairs (LP) and C-C antibonding.These interactions led to intramolecular electron-density transfer that caused the stabilization of the molecular system (Table 4).  4 provides all significant values for the hyperconjugative interactions given by the second-order perturbation theory.
A relationship between the ESI-IT-MS study and the stabilization caused by orbital overlap between bonds in the second-order perturbation theory could be established.The strongest stabilization energies for strictosidine involved hyperconjugative interactions in the indolic portion (aromatic and pentacyclic rings) and in the dihydropyran portion, explaining the small number of cleavages and the absence of breakages in the indolic portion and in the dihydropyran ring.In the fragmentation mechanism proposed in Figure 2, the most stable fragments arose from the heterolytic  surface for the strictosidine molecule.An MEP is a plot of the electrostatic potential mapped onto the constant electron density surface and is used primarily for predicting sites and relative reactivities towards electrophilic and nucleophilic attacks.MEPs are used in studies of biological recognition and interactions between the same molecules (e.g., in forming clusters and crystal structures) or other molecules.MEPs also correlate and predict a wide range of macroscopic properties [48,49].The color code of these maps ranges from −0.08 a.u.(deepest red) to 0.08 a.u.(deepest blue), where blue indicates a minimal concentration of electrons and red indicates a high density of electrons.The MEPs for strictosidine indicated regions with positive potentials over H1 (0.0627 a.u.) and over the hydrogen atom of the OH group in position 4  (0.0862 a.u.).Regions with negative potentials were located over the aromatic ring (−0.0429 a.u.), over the carbonyl group O2 (−0.0576 a.u.), between O4 and the OH group of C2  position (−0.0643a.u.), over OH on position C6  (−0.0471 a.u.), and over oxygen atom on C4  position (−0.0201a.u.).The predominance of light green color region indicates great charge dispersion.Strictosidine is a large molecule with polarized points scattered over its surface which promotes various possible forms of intramolecular and intermolecular interaction (between strictosidine molecules); in addition, the nonflat shape hinders the chain interactions between strictosidine molecules which facilitate formation of a packed crystal.

IR Analysis.
Figure 7 shows the experimental and theoretical IV spectra.The differences can be attributed to the fact that the theoretical DFT calculations were made for the molecule in the gas phase, whereas intermolecular interactions occur in solution.The assignment of the experimental bands to the normal modes of vibration was made using the optimized structure with the lowest potential energy, considering the potential energy distribution (PED) by using the B3LYP/6-31G(d) level.A total of 210 normal vibration modes were obtained but were compared with the experimental spectrum only between 400 and 4000 cm −1 (Table 5).
Modes between 3021 and 3640 cm −1 were related to the following vibrations: the strong O-H stretching of the glycoside portion; the H-C stretching of the aromatic ring; the H-C stretching of dihydropyran ring; the N4-H1 and N1-H1 stretching; and the H3-C3, H5-C5, H6-C6, H14-C14, and H15-C15 stretching.The region from 2900 to 3000 cm −1 showed a strong band at 2932 cm −1 that was assigned to the symmetric CH 2 stretching in C6.
The region between 1060 and 1700 cm −1 was related to the following vibrations: the C=O stretching of C22, C=C stretching modes of C16=C17, the H1-N1 bending mode, and the CH 2 scissoring modes (C6  and C23) in 1400-1690 cm −1 ; the H-C bending of the glycoside portion and the dihydropyran ring in 1250-1399 cm −1 ; the O-C stretching of the glycoside portion between 1060 and 1211 cm −1 .
The region between 600 and 1030 cm −1 was related to the C-C stretching of C10 and C11, the C-O and C-C stretching of the glycosidic moiety, the H-N bending of the N4 position (band at 891 cm −1 was noteworthy), the out-of-plane mode of C22 (at 763 cm −1 ), and the torsion modes of the entire structure.
The large differences from 3500 to 4000 cm −1 are related to the H-N1 and O-H stretching, which are indicative that the interactions between strictosidine molecules occur between the tryptophanic and glycosidic regions.These interactions make sense based on the electrostatic potential map (Figure 6), which showed greater polarization of these two regions relative to the entire molecule.The optimized geometry of a strictosidine dimer (Figure 8) showed stabilization due to the existence of intermolecular hydrogen bonds (N1-H1---O-C4  and C4  -OH---O2=C22) and the values assigned to the stretching of O-H (3556.00cm −1 ) and H-N1 (3493.18cm −1 ) groups are closer to the experimental ones (3430 and 3390 cm −1 ).The value assigned to the stretching of carboline group in position C22 (1690 cm −1 ) shows to be closer to the experimental one in the dimer too (1767 cm −1 to the monomer and 1716 cm −1 to the dimer form), implying that the interaction between the carbonyl and OH is plausible and decreasing the stretching frequency oscillator related to C=O bond.These interactions directly influenced the infrared spectrum by decreasing the stretching frequency oscillator related to these bonds, causing reduction in the wavelengths (see Figure 8) and in the RMSD values.For the monomer, the RMSD is 84.40 cm −1 ; for the dimer, the value is 46.41.Applying the empirical scaling factor of 0.9613, the RMSD values feature a visible reduction, 66.33 cm −1 for monomer and 30.66 cm −1 for dimer.

Conclusion
The strictosidine alkaloid, which was isolated from Strychnos amazonica, was comprehensively characterized.The interatomic distances and angles proved to be plausible compared to the X-ray data for similar molecules.The similarity between the theoretical and experimental coupling constants values reveals that the theoretical hydrogen dihedral angles of the C14-C15-C20-C21-C19 positions are plausible, showing that the modeled structure justifies the experimental NMR data.The UV analysis was able to explain the similarity between the UV spectra of strictosidine and related indole alkaloids, showing that the transitions involving the indole moiety are energetically more significant and such characteristic can be used as a "fingerprint" for detecting indole alkaloids.The HOMO-LUMO gap is directly related to the reactivity of a compound reflecting the amount of important properties such as chemical hardness, electrophilicity, nucleophilicity index, and electronegativity.Strictosidine theoretically appears to be a nucleophile and electrophile that in addition to its polarizability behaves as a soft molecule.This indicates low charge states and faster reactions, which makes strictosidine a versatile molecule, justifying its high reactivity and its role as a precursor of indole alkaloids.The comparative IR studies revealed that interactions of strictosidine dimers (between the tryptophanic and glycosidic regions) influenced the infrared spectrum by decreasing the stretching frequency oscillator of the groups which forms hydrogen   bonds and revealed several characteristic vibrations that may be used as a diagnostic tool for other indole alkaloids, simplifying their identification and structural characterization.The NBO calculations showed that the strongest stabilization energies for strictosidine involved hyperconjugative interactions in the indolic portion (aromatic and pentacyclic rings) and in the dihydropyran portion, justifying the few fragmentation modes for its protonated molecule observed in the MS  analysis, and these modes may contribute to the further characterization of strictosidine analogues and derivatives.

Figure 2 :
Figure 2: The optimized geometry of strictosidine with the scheme of atom numbering.

Figure 3 :
Figure 3: Experimental and theoretical ultraviolet-visible spectra of strictosidine in methanol.

3. 4 .
Tandem Mass Identification.The tandem mass spectra of protonated strictosidine (Figure S4 in Supplementary Data) of m/z 531 showed major fragment ions of m/z 514, 369, and 352.The ion of m/z 514 [M + H-17 Da] originated by loss of the NH 3 group from the tryptophane portion in a manner that was similar to the fragmentation of aporphine alkaloids (Pathway A) [38].The ESI-MS 3 of this ion generated fragment ions of m/z 352 [M + H-162 Da] due to the hydrogen rearrangement in the glycoside followed by heterolytic cleavage of the C21-O-C1  bridge (Pathway C).The fragment ion of m/z 369 [M + H-162 Da] possibly arose directly from the heterolytic breakage of the glycoside portion of strictosidine via the cleavage of the C21-O-C1  bridge (Pathway B), which occurred through a similar mechanism to Pathway C. No fragment ions were observed in the MS 3 spectrum of the ion of m/z 369.Guided by the ESI-MS 2 and MS 3 data, a fragmentation mechanism is proposed for protonated strictosidine in Figure 5.

3. 5 .
NBO  Study.An NBO analysis describes the Lewislike molecular bonding pattern of electron pairs (or of individual electrons in the open-shell case) in the optimally compact form of the molecule.More precisely, NBOs are orthonormal sets of localized "maximum occupancy" orbitals whose leading N/2 members (or N members in the open-shell case) give the most accurate possible Lewis-like description of the total N-electron density.The Lewis-type NBOs determine the localized natural Lewis structure (NLS) representation of the wave function, while the remaining "non-Lewis-type" NBOs complete the span of the basis and describe the residual "delocalization effects" (i.e., departures from a single localized Lewis structure).NBOs provide therefore a valence bond-type description of the wave function that is closely linked to classical Lewis structure concepts[39][40][41][42].

Figure 5 :
Figure 5: Mechanism proposed for the major fragmentation pathways of protonated strictosidine.
*Corresponding to single bonds due to the crystallization process of loganin in loganin penta-acetate monomethyl ether bromide.transition at 265.72 nm (4.66 eV) for B3LYP 6-31G(d) and at 278.26 nm (4.45 eV) for B3LYP 6-311++G(2d,p), which were equivalent to the band at 272 nm in the experimental spectrum.These values were assigned to the sum of the  →  * and  →  * transitions of the indole and -alkoxyacrylate groups.

Table 2 .
Both HOMO and LUMO are bonding orbitals, resulting in a low excitation energy for strictosidine.

Table 3 :
Electron population in Lewis and non-Lewis orbitals for strictosidine.