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

Authors: Renyer Alves Costa; Maria Lucia Belem Pinheiro; Kelson Mota Teixeira de Oliveira; Andersson Barison; Kahlil Schwanka Salomé; Júlio Rodolfo Iank; Noam Gadelha da Silva; Tiara Souza Cabral; Emmanoel Vilaça Costa
Journal: Journal of Chemistry
 (2016)

---

## Abstract

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 the measured experimental data, with bands assigned. Calculated HOMO/LUMO gaps 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.


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## Body

## 1. 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 [9–13] and was first isolated fromRhazya stricta [14]. Strictosidine has also been obtained in cell suspension cultures and under biomimetic conditions [15, 16].Figure 1
Structure of strictosidine.
(a)
(b)Studies discussing the structure and stereochemistry of strictosidine [16–18] have compared its spectral data with those of similar structures, confirming that its C3 atom has the S configuration or 3α [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 3β [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 fromStrychnos amazonica and the molecule was characterized by NMR (1H, 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.


## 2. 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
n data were recorded on LQC fleetion 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.


### 2.2. Plant Material

The leaves ofStrychnos amazonica were collected in the Adolpho Ducke Forest Reserve located 25 km from the city of Manaus (2°56′01.0′′S, 59°57′45.8′′W), Amazonas, Brazil. The species was identified by the DNA barcoding technique, in which the distance matrices, which were calculated for rcbL and rpoC1 and for the concatenated gene sequences (rbcL + rpoC1), showed null values ​​of genetic divergence between the collected specimen and a voucher specimen ofStrychnos amazonica (INPA 216208) deposited in the herbarium of the National Research Institute of Amazonas (INPA). The rcbL and rpoC1 sequences were deposited in GenBank under accession numbers KJ764797 to KJ764819.


### 2.3. Isolation of Strictosidine

30 g of leaf extract in methanol was submitted to a silica-gel column using hexane/ethyl acetate and ethyl acetate/methanol as the mobile phases. Of the 31 fractions obtained, fractions 10 to 16 showed the presence of an alkaloid. These fractions were pooled and submitted to an ESI-TI-MS analysis, which showed an ion peak atm/z 531. CID analysis revealed fragments atm/z 514,m/z 369, andm/z 356, which are compatible with strictosidine [20]. This fraction was submitted to a silica-gel column using 100% ethyl acetate, 9 : 1 ethyl acetate/methanol, and 100% methanol in succession, resulting in an amorphous brown solid (20 mg) that appeared to be pure in CCD/Dragendorff and ESI-IT-MS analyses (with only one ion peak atm/z 531). Then, it was subjected to NMR (H1, HMBC, and HSQC), UV, and IR analysis for structural confirmation (see Table S1 and Figures S1, S2, and S3 in the Supplementary Material data available online at http://dx.doi.org/10.1155/2016/1752429).


### 2.4. Computational Methods

The theoretical quantum chemical calculations were performed using the Gaussian 03 W Program (Revision E.01) on the Debian Linux (5.0 version) platform on an Intel Quadcore™ PC (8 GB RAM) [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 J
3
H
H 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].


## 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
n data were recorded on LQC fleetion 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.


## 2.2. Plant Material

The leaves ofStrychnos amazonica were collected in the Adolpho Ducke Forest Reserve located 25 km from the city of Manaus (2°56′01.0′′S, 59°57′45.8′′W), Amazonas, Brazil. The species was identified by the DNA barcoding technique, in which the distance matrices, which were calculated for rcbL and rpoC1 and for the concatenated gene sequences (rbcL + rpoC1), showed null values ​​of genetic divergence between the collected specimen and a voucher specimen ofStrychnos amazonica (INPA 216208) deposited in the herbarium of the National Research Institute of Amazonas (INPA). The rcbL and rpoC1 sequences were deposited in GenBank under accession numbers KJ764797 to KJ764819.


## 2.3. Isolation of Strictosidine

30 g of leaf extract in methanol was submitted to a silica-gel column using hexane/ethyl acetate and ethyl acetate/methanol as the mobile phases. Of the 31 fractions obtained, fractions 10 to 16 showed the presence of an alkaloid. These fractions were pooled and submitted to an ESI-TI-MS analysis, which showed an ion peak atm/z 531. CID analysis revealed fragments atm/z 514,m/z 369, andm/z 356, which are compatible with strictosidine [20]. This fraction was submitted to a silica-gel column using 100% ethyl acetate, 9 : 1 ethyl acetate/methanol, and 100% methanol in succession, resulting in an amorphous brown solid (20 mg) that appeared to be pure in CCD/Dragendorff and ESI-IT-MS analyses (with only one ion peak atm/z 531). Then, it was subjected to NMR (H1, HMBC, and HSQC), UV, and IR analysis for structural confirmation (see Table S1 and Figures S1, S2, and S3 in the Supplementary Material data available online at http://dx.doi.org/10.1155/2016/1752429).


## 2.4. Computational Methods

The theoretical quantum chemical calculations were performed using the Gaussian 03 W Program (Revision E.01) on the Debian Linux (5.0 version) platform on an Intel Quadcore™ PC (8 GB RAM) [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 J
3
H
H 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].


## 3. Results and Discussion

### 3.1. Geometry Optimization

Because no crystallographic data for strictosidine are available, the geometry optimization data, which were calculated using the B3LYP/6-31G(d) and B3LYP/6-311++G(2d,p) basis set, were compared with the X-ray data for similar structures [26–29]. Due to the similarity of the data obtained from both basis sets (Table 1), the discussion that follows is based on the B3LYP 6-31G(d) values but should also apply to B3LYP 6-311++G(2d,p).Table 1
Calculated geometrical parameters for strictosidine compared with the experimental data.
Parameter
B3LYP 6-31G(d)
B3LYP 6-311++G(2d,p)
Molina et al. [27]
Dupont and Dideberg [26]
Lentz and Rossmann [29]
Bond length
N1-C2
1.386
1.384
1.363
1.400
C2-C3
1.507
1.506
—
1.488
C3-N4
1.479
1.477
—
1.477
N4-C5
1.472
1.473
—
1.482
C5-C6
1.543
1.542
—
1.523
C6-C7
1.501
1.501
—
1.490
C7-C2
1.375
1.375
1.442
1.429
C7-C8
1.439
1.439
1.457
1.425
C8-C9
1.406
1.407
—
1.432
C8-C13
1.427
1.426
—
1.406
C9-C10
1.392
1.391
—
1.432
C10-C11
1.411
1.410
—
1.377
C11-C12
1.393
1.392
—
1.346
C12-C13
1.399
1.398
1.409
C13-N1
1.381
1.381
1.406
1.379
C14-C3
1.548
1.548
—
1.541
C14-C15
1.552
1.562
—
1.62
C15-C16
1.515
1.520
—
1.48
C16-C17
1.350
1.350
—
1.60∗
C18-C19
1.335
1.334
—
1.51∗
C19-C20
1.512
1.513
—
1.60
C20-C21
1.526
1.526
—
1.50
C21-O
1.401
1.403
—
1.43
C22-C16
1.469
1.471
—
1.60
C23-O
1.437
1.441
—
1.49
C1′-O
1.403
1.401
—
1.42
C1′-C2′
1.528
1.526
—
1.52
C2′-C3′
1.528
1.527
—
1.51
C3′-C4′
1.527
1.528
—
1.55
C4′-C5′
1.537
1.537
—
1.52
C5′-C6′
1.523
1.522
1.52
C5′-O
1.438
1.442
—
1.45
Bond angle
N1-C2-C7
109.570
109.562
108.7
110.5
C2-C7-C8
107.063
107.071
105.7
107.0
C7-C8-C13
106.793
106.792
107.5
107.2
C8-C13-N1
107.347
107.343
—
108.0
C13-N1-C2
109.221
109.221
—
107.3
C2-C3-N4
110.742
110.742
—
106.6
C3-N4-C5
114.580
114.582
—
112.6
N4-C5-C6
114.370
114.370
—
110.1
C5-C6-C7
108.976
108.976
—
109.4
C6-C7-C2
121.873
121.881
128.9
122.1
C7-C2-C3
125.545
125.546
107.9
126.2
C13-C8-C9
118.786
118.771
—
119.7
C8-C9-C10
119.165
119.172
—
115.3
C9-C10-C11
121.057
121.052
—
122.7
C10-C11-C12
121.177
121.181
—
122.2
C11-C12-C13
117.626
117.625
—
117.6
C12-C13-C8
122.186
122.186
—
122.4
C2-C3-C14
111.904
111.903
—
110.8
N4-C3-C14
110.456
110.456
—
116.9
C3-C14-C15
113.558
113.562
—
112.7
C15-C16-C20
112.883
112.883
—
—
C15-C16-C17
112.915
112.914
—
—
107
C16-C17-O
125.412
125.420
—
—
110
C17-O-C21
119.937
119.941
—
—
121
C18-C19-C20
129.038
129.038
—
—
101
C16-C22-O
113.414
113.415
—
—
111
CH3-O-C22
115.912
115.912
—
—
116
C21-O-C1′
115.516
115.516
—
—
114
O-C1′-C2′
108.278
108.278
—
—
106
C1′-C2′-C3′
109.237
109.237
—
—
106
C2′-C3′-C4′
111.945
111.945
110
C3′-C4′-C5′
109.024
109.024
—
—
105
C4′-C5′-O
108.796
108.795
—
—
106
C5′-O-C1′
114.025
114.027
—
—
108
∗Corresponding to single bonds due to the crystallization process of loganin in loganin penta-acetate monomethyl ether bromide.Initially, the bond lengths indicated a small distortion in the pentacyclic tryptophan ring, showing a greater distance for the C7-C8 (1.44 Å) and C8-C13 (1.42 Å) bonds compared to the N1-C2, C2-C7, and C13-N1 bonds. Significant distortions were observed in the second six-membered ring of the tryptophan portion, which was distinct for all connections. Similar distortions were observed in the dihydropyran ring: 1.52 Å (C15-C16), 1.35 Å (C16-C17), 1.34 Å (C7-O), 1.53 Å (C20-C21), and 1.56 Å (C20-C15). These distortions resulted from the presence of double bonds and heteroatoms, which make these bonds shorter than the C-C bonds.Concerning the planar angles, the aromatic ring shows no large deformations, which was similar to the experimental data for previously analyzed indole alkaloids [26, 29]. The planar angles were 118.78° (C9-C8-C13), 119.16° (C8-C9-C10), 121.06° (C9-C10-C11), 121.17° (C10-C11-C12), 117.62° (C11-C12-C13), and 122.18° (C8-C12-C13). The pentacyclic ring showed a nonangular uniformity except for the angles between N1-C2-C7 (109.56°) and C13-C2-N1 (109.22°).1H-1H NMR coupling constants are highly sensitive to the dihedral angles of hydrogen atoms and both the configuration and conformation of a structure can be validated if there is agreement between the experimental and theoretical J
3
H
H values. The calculated initial dihedral angles for the hydrogen atoms in positions 5 and 6 of the third ring of the tryptophan portion were 162.78° (with theoretical J
H
5
a
-
H
6
b
T
=
11.0 Hz) and 46.76° (theoretical J
H
5
a
-
H
6
a
T
=
4.2 Hz). These values were consistent with the experimentally measured values for coupling constants, J
H
5
a
-
H
6
b
E
=
11.9 Hz and J
H
6
a
-
H
5
a
E
=
4.8 Hz, revealing a pseudoaxial-axial relationship between hydrogen atoms H5a-H6b and an axial-equatorial relationship between H5a and H6a. The obtained dihedral angles for hydrogen atoms of the C3-C14-C15 bridge also showed coupling constants values consistent with the experimental values: 172.28° for H3-H14proR with theoretical J
3
-
14
R
T
=
10.7 (experimental J
3
-
14
R
E
=
11.4 Hz), −72.41° for H3-H14proS with J
14
S
-
3
T
=
2.8 Hz (J
14
S
-
3
E
=
3.6 Hz), and −87.53° for H14proR-H15 with J
H
14
R
-
H
15
T
=
0.6 Hz (J
H
14
R
-
H
15
E
=
1.8 Hz). The conformation of the dihedral angles involving H15-H20 (−56.90°), H20-H21 (−178.69°), and H19-H20 (65.23°) is also plausible given the respective experimental hydrogen coupling constants J
H
15
–
H
20
T
=
4.4 Hz (J
E
=
4.8 Hz), J
H
20
-
H
21
T
=
8.1 (J
E
=
8.4 Hz), and J
H
19
-
H
20
T
=
3.0 Hz (J
E
=
3.0 Hz). The similarity between the modeled DFT structure (Figure 2) (with15S,20S, and21S configuration) and the experimental spectroscopic NMR data complements the conformational arrangement study of strictosidine. The15S,20S, and21S configuration was reported in previous experimental X-ray studies of strictosidine precursors [19, 28–30]. Finally, due to the consistency between the geometry calculated using the DFT B3LYP/6-31G(d) and B3LYP/6-31++G(2d,p) methods and the coupling constant values measured from the experimental 1H spectrum, the conformation of the C21-O-C1′ bridge (so far uncertain) was depicted with angles of 81.17° and lengths of 2.65 Å between H21 and H1′ (Figure 2). This depiction is consistent with the NOESY data provided by Patthy-Lukáts et al. [19]. Other interactions between segments found in the NOESY data are justified by the calculated distances between the hydrogen atoms as follows: H3-H5b (3.76 Å), H3-H14S (2.55 Å), H5a-H5b (1.76 Å), H6a-H6b (1.76 Å), H9-H10 (2.48 Å), H10-11 (2.477 Å), H11-H12 (2.488 Å), H14R-H14S (1.75 Å), H14R-H19 (2.83 Å), H14R-H15 (2.66 Å), H14S-H21 (2.59 Å), H15-H20 (2.41 Å), H18Z-H18E (1.85 Å), H18Z-H20 (3.57 Å), H18E-H19 (2.40 Å), and H20-H19 (2.52 Å) (for information about Mulliken charges and natural bond analysis, see Tables S2 and S3 in Supplementary Material data).Figure 2
The optimized geometry of strictosidine with the scheme of atom numbering.


### 3.2. 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 Figure3 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, π
→
π
∗ transitions were involved but the presence of conjugations in the pentacyclic ring of the tryptophan portion and in the β-alkoxyacrylate group suggested that n
→
π
∗ transitions also occurred. The theoretical calculations predicted an intense electronic transition of 5.8964 eV, with an oscillator strength f
=
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 f
=
0.2714 at 220.62 nm. These results showed good agreement with the measured experimental data (222 nm) assigned to the π
→
π
∗ and π
→
σ
∗ transitions of the indole portion. The calculations also predicted a weaker electronic 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 n
→
π
∗ and π
→
π
∗ transitions of the indole and β-alkoxyacrylate groups.Figure 3
Experimental and theoretical ultraviolet-visible spectra of strictosidine in methanol.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 Figure4. 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 β-alkoxyacrylate group. Transitions involving the LUMO contribute minimally to the absorption wavelengths (
λ
) of strictosidine, showing that the transitions in the β-alkoxyacrylate group have no influence, in disagreement with the ancient proposal that β-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].Figure 4
Frontier molecular orbitals of strictosidine.Figure 5
Mechanism proposed for the major fragmentation pathways of protonated strictosidine.


### 3.3. 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(
η
) 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 (
μ
) 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 (
χ
) 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]:(1)
η
=
1
2
∂
2
E
∂
N
2
V
r
=
1
2
∂
μ
∂
N
V
r
,
μ
=
∂
E
∂
N
V
r
,
χ
=
-
μ
=
-
∂
E
∂
N
V
r
,where E and V
r are the electronic energy and the external potential of anN-electron, respectively. Based on Koopmans theorem for closed-shell molecules, these global chemical reactivity descriptors can be simplified and defined as follows: (2)
η
=
I
-
A
2
,
μ
=
-
I
+
A
2
,
χ
=
I
+
A
2
,where A is the ionization potential and I is the electron affinity of the molecule. The ionization energy and electron affinity are obtained from the HOMO and LUMO energies as I
=
-
E
H
O
M
O and A
=
-
E
L
U
M
O. 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 (ω), 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: (3)
ω
=
μ
2
2
η
.This scale permits the classification of organic molecules as strong,ω
>
1.5 eV, moderate, 0.8
<
ω
<
1.5 eV, and marginal, ω
<
0.8 eV, electrophiles. On the other hand, there is a good correlation in the inverse of the electrophilicity (1
/
ω); 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 (N) appears as a different descriptor which gives more information about nucleophilicity and is defined as follows [37]:(4)
N
=
E
H
O
M
O
-
E
H
O
M
O
T
C
E
,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 Table 2. Both HOMO and LUMO are bonding orbitals, resulting in a low excitation energy for strictosidine. The excitation energies, which were calculated as 4.451 eV for 6-31G(d) and 4.408 eV for 6-311++G(2d,p), reflect the low hardness value (2.22) showing strictosidine as a soft molecule with high polarizability. Electronegativity and electrophilicity values of strictosidine indicate that this molecule has significative attractive electron power acting as an electrophile and in addition to its polarization becomes very reactive since the electrons are farther from the nucleus; however, the nucleophilicity index value indicates that strictosidine is a strong nucleophile too. Such characteristic is justified because strictosidine is a large molecule that has many reactive groups with different potentials which forms small polarized points over its surface (as discussed in Section 3.6), which makes strictosidine a versatile molecule in view of the variety of alkaloids which it forms through intramolecular reactions with groups acting as nucleophiles and others as electrophiles [10]. On the other hand, the calculated chemical potential values, −3.06 for B3LYP/6-31G(d) and −3.40 for B3LYP/6-311++G(2d,p), reveal certain stability, indicating that it does not decompose spontaneously; that is, strictosidine molecule is reactive but does not tend to degrade into the components that formed it.Table 2
Calculated energy values for strictosidine in methanol using B3LYP/6-31G(d) and B3LYP/6-311++G(2d,p).
Basis set
B3LYP/6-31G(d)
B3LYP/6-311++G(2d,p)
Energy (a.u.)
−1835.80
−1836.39
Dipole moment
5.30 Debye
5.50 Debye
E
H
O
M
O (eV)
−5.28
−5.60
E
L
U
M
O (eV)
−0.83
−1.19
E
H
O
M
O
-
L
U
M
O (eV)
4.45
4.40
E
H
O
M
O
-
1 (eV)
−5.89
−6.20
E
L
U
M
O
+
1 (eV)
−0.18
−0.62
E
(
H
O
M
O
-
1
)
-
(
L
U
M
O
+
1
) (eV)
5.71
6.80
Hardness (η)
2.22
2.20
Chemical potential (μ)
−3.06
−3.40
Electronegativity (χ)
3.06
3.40
Electrophilicity index (ω)
2.11
2.63
Nucleophilicity index (N)
5.82
5.91


### 3.4. Tandem Mass Identification

The tandem mass spectra of protonated strictosidine (Figure S4 in Supplementary Data) ofm/z 531 showed major fragment ions ofm/z 514, 369, and 352. The ion ofm/z 514 [M + H—17Da] originated by loss of the NH3 group from the tryptophane portion in a manner that was similar to the fragmentation of aporphine alkaloids (Pathway A) [38]. The ESI-MS3 of this ion generated fragment ions ofm/z 352 [M + H—162Da] due to the hydrogen rearrangement in the glycoside followed by heterolytic cleavage of the C21-O-C1′ bridge (Pathway C). The fragment ion ofm/z 369 [M + H—162Da] 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 MS3 spectrum of the ion ofm/z 369. Guided by the ESI-MS2 and MS3 data, a fragmentation mechanism is proposed for protonated strictosidine in Figure 5.


### 3.5. NBO Study

An NBO analysis describes the Lewis-like 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 leadingN/2 members (orN members in the open-shell case) give the most accurate possible Lewis-like description of the totalN-electron density. The Lewis-type NBOs determine the localizednatural 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 [39–42].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 [43–47] within the molecule. This analysis uses the second-order perturbation energies E
(
2
) [donor (i) → acceptor (j)] that involve the most important delocalization instances, which are given as follows:(5)
E
2
=
Δ
i
j
=
q
i
F
i
j
2
ε
j
-
ε
i
.Table3 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). The intramolecular hyperconjugative interactions between C2, C7, C8, C9, C10, C11, C12, C13, and N1 revealed strong stabilization of the indolic portion, principally by π
→
π
∗ interactions between C11-C10 → C8-C9 (20.07 kcal/mol), C9-C8 → C11-C10 (20.77 kcal/mol), C9-C8 → C13-C12 (20.01 kcal/mol), and C13-C12 → C8-C9 (19 kcal/mol) and electron donation from LP(1) N1 to π
∗ C2-C7 (36.44 kcal/mol) and π
∗ C12-C13 (40.91 kcal/mol). Other hyperconjugative interactions, especially the donation of electron density from LP(2) O3 to π
∗ C16-C17 (37.05 kcal/mol), LP(1) O2 to σ
∗ C22-O1 (32 kcal/mol), and LP(2) O1 to π
∗ C22-O2, gave strong stabilization to the dihydropyran ring of strictosidine. In addition, the π
∗
→
π
∗ interactions of C8-C9 → C7-C2 and C21-O2 → C16-C17 provided enormous stabilization of 191.70 kcal/mol and 62.11 kcal/mol, respectively. Table 4 provides all significant values for the hyperconjugative interactions given by the second-order perturbation theory.Table 3
Electron population in Lewis and non-Lewis orbitals for strictosidine.
Orbitals
Electrons
Percentage
Core
75.97
99.96% of 76
Valence Lewis
201.50
97.817% of 202
Total Lewis
277.47
98.39% of 282
Valence non-Lewis
4.02
1.43% of 282
Rydberg non-Lewis
0.50
0.17% of 282
Total non-Lewis
4.53
1.61% of 282Table 4
Selected second-order perturbation energies for strictosidine.
Donor orbital (i)
Type
Acceptor orbital (j)
Type
E
(
j
)
-
E
(
i
) a.u.
E
(
2
) (kcal/mol)
C11-C10
π
C9-C8
π
∗
0.28
20.07
C13-C12
π
∗
0.27
18.90
C9-C8
π
C11-C10
π
∗
0.27
20.77
C12-C13
π
∗
0.27
20.01
C7-C2
π
∗
0.29
14.88
C13-C12
π
C11-C10
π
∗
0.28
21.04
C8-C9
π
∗
0.29
16.32
C7-C2
π
C9-C8
π
∗
0.29
19.71
C16-C17
π
C22-O1
π
∗
0.29
24.34
N1
LP(1)
C13-C12
π
∗
0.29
40.91
N1
LP(1)
C2-C7
π
∗
0.31
36.44
O3
LP(2)
C16-C17
π
∗
0.36
37.05
O4
LP(2)
O3-C21
σ
∗
0.56
15.13
O4
LP(2)
C1′-O5
σ
∗
0.60
13.48
O2
LP(1)
C22
RY∗
1.55
14.52
O2
LP(2)
C16-C22
σ
∗
0.72
16.43
O2
LP(2)
C22-O1
π
∗
0.63
32.65
O1
LP(2)
C22-O2
π
∗
0.32
47.44
C8-C9
π
∗
C7-C2
π
∗
0.01
191.70
C22-O2
π
∗
C16-C17
π
∗
0.03
62.11A 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 Figure2, the most stable fragments arose from the heterolytic breakage of the O4-C1′, N4-C3, and N4-C5 bonds, which have the following weak hyperconjugative interactions: N4-C3 → N2-C3 (σ
→
σ
∗, 3.59 kcal/mol), C5-N4 → C3 (σ
→
σ
∗, 1.5 kcal/mol), N4-H → C5 (σ
→
σ
∗, 1.68 kcal/mol), N4-H → C5-H (σ
→
σ
∗, 2.51 kcal/mol), O4-C1′→ C20-C21 (σ
→
σ
∗, 1.46 kcal/mol), and O3-C21 → C21-O4 (σ
→
σ
∗, 0.51 kcal/mol). These results showed that the NBO study complemented the mass fragmentation study.


### 3.6. Molecular Electrostatic Potential Surface

Figure6 illustrates 3D plots of the molecular electrostatic potential (MEP) 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.Figure 6
Molecular electrostatic potential maps (from two perspectives) for strictosidine calculated using the B3LYP/6-31G(d) basis set.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 (−0.0643 a.u.), over OH on position C6′ (−0.0471 a.u.), and over oxygen atom on C4′ position (−0.0201 a.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.


### 3.7. IR Analysis

Figure7 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).Table 5
Experimental and calculated wavenumbers∗ (cm−1) and assignments for strictosidine.
IR solid
B3LYP 6-31G(d)
Assignment (PED > 5%)
Exp.
IR intensity
Monomer
Dimer
Calculated
IR intensity
Calculated
IR intensity
3767
30
3633
62
Stre. OH (96%)
3732
30
3602
6
Stre. OH (95%)
3430
42
3731
49
3556
225
Stre. OH (96%)
3411
44
3724
44
3535
64
Stre. OH (97%)
3390
43
3656
44
3493
47
Stre. N1-H1 (88%) + tors. H1-N1-C2-C13 (11%)
3382
58
3488
0.27
3475
2.49
Stre. N4-H4 (88%) + bend H4-N4-C5 (11%)
3320
51
3260
4
3260
5
Stre. C18-H18 (70%) + stre. C19-H19 (26%)
3211
20
3246
3
3239
12
Stre. C17-H17 (99%)
3205
38
3183
22
Stre. C9-H9 (36%) + stre. C10-H10 (39%) + stre. C12-H12 (10%)
3180
17
3193
39
3190
28
Stre. C9-H9 (21%) + stre. C10-H10 (16%) + stre. C11-H11 (30%) + C12-H12 (23%)
3152
9
3183
3
3173
2
Stre. C9-H9 (12%) + stre. C10-H10 (24%) + stre. C11-H11 (32%) + stre. C12-H12 (13%)
3150
25
3176
19
3139
1
Stre. sim. C23-H23 (22%) + stre. sim. C23-H23 (19%) + stre. ass. C23-H23 (24%) + stre. C23-H23 (19%)
3140
22
3176
2
3152
14
Stre. C9-H9 (12%) + stre. C10-H10 (17%) + stre. C11-H11 (43%)
3175
14
3140
7
Stre. C19-C18 (41%) + stre. sim. C18-H18 (25%) + stre. C19-H19 (18%)
3129
12
3161
2
3125
17
Stre. C9-H19 (66%) + stre. sim. C18-H18 (26%)
3105
10
3144
23
3122
7
Stre. C23-H23 ass. (92%)
3100
11
3103
35
3104
20
Stre. C6′-H6′ (91%)
3102
35
3082
8
Stre. C5-H5 (47%) + stre. C14-H14 (33%)
3093
25
3075
28
Stre. C14-H14 (74%)
3076
10
3072
27
Stre. C15-H15 (85%) + stre. C3-H3 (11%)
3071
44
3069
28
Stre. sim. C23-H23 (72%)
3070
2
3063
44
Stre. C3-H3 (57%) + stre. C15-H15 (12%)
3061
42.72
3061
1
Stre. C5-H5 (86%)
3060
44
3052
54
Stre. C21-H21 (60%)
3052
6
3050
48
Stre. C14-H14 (65%)
3041
19
3042
14
Stre. ass. C6-H6 (77%)
3040
110
3033
13
Stre. C1′-H1′ (77%)
3020
15
3032
13
3032
28
Stre. C4′-H4′ (62%)
3017
6
3011
37
Stre. C20-H20 (94%)
3016
2
3008
5
Stre. C2-H2 (44%) + stre. C5-H5 (46%)
3013
12
3007
3
Stre. C2′-H2′ (31%) + stre. C4′-H4′ (20%) + stre. C5′-H5′ (35%)
2931
26
2999
50
2997
4
Stre. C6-H6 ass. (71%)
2929
23
2989
1
2992
8
Stre. C3′-H3′ (63%) + stre. C6′-H6′ sim. (18%)
2920
12
2985
44
2988
46
Stre. C6′-H6′ (72%)
1690
62
1767
321
1716
308
Stre. C22=O2 (73%)
1628
41
1694
263
1691
272
Stre. C16=C17 (65%) + bend H17-C17-O3 (12%)
1586
7
1619
2
1591
1.5
Stre. (entire indole portion) (33%)
1527
5
1537
3.5
1519
3.6
Stre. C9=C10 (12%) + bend H10-C10 (20%) + bend H1-N1 (11%)
1531
1.2
1497
4
Bend (scissoring) H6′-C6′ (80%)
1527
9
1496
7
Bend (scissoring) C23-H23 (63%)
1400
20
1418
32
1415
11
Stre. C15-C16 (16%) + bend H17-C17-O3 (27%)
1399
25
1413
4
1413
4
Bend H-C entire indole portion
1377
1399
6
1395
9
Bend H21-C21 (14%) + bend H5′-C5′ (13%) + tors. H5′-C5′-O5-C1′ (10%) + tors. H4′-C4′-OH (16%)
1365
12
1386
3
1375
8
Tors. H14-C14-C3-N4 (11%) + tors. H15-C15-C14-C3 (11%) + H5-C5-N4-C3 (10%)
1360
10
1381
4
1363
1
Bend H1′-C1′ (16%) + bend H4′-C4′ (15%) + tors. C5′-H5′ (19%)
1350
11
1378
32
1362
19
Bend H3′-C3′ (11%) + bend H21-C21 (16%) + bend H1′-C1′ (14%)
1344
18
1367
47
1345
19
Bend H18-C18-C19 (32%) + bend H2′-C2′ (12%)
1328
13
1348
59
1340
52
Bend H17-C17-O3 (15%) + bend H15-C15 (10%)
1345
66
1346
29
Bend H17-C17 (12%) + tors. H15-C15-C14-C20 (10%)
1303
30
1342
53
1342
41
Bend H1′-C1′ (22%) + tors. H5′-C5′-O5-C1′ (25%)
1338
65
1327
9
Bend H20-C20 (15%)
1300
33
1326
22
1320
31
Stre. C13-N1-C2 (29%)
1297
25
1312
7
1301
18
Bend H20-C20 (16%) + bend H3-C3 (10%) + tors. H14-C14-C3-N4 (16%)
1250
22
1267
23
1260
17
Bend H-C entire structure
1257
40
1243
40
Bend O-H (5′) (18%) + bend H4′-C4′ (10%)
1211
13
1253
117
1252
16
Stre. C17-O3 (22%) + H15-C15 (13%)
1157
137
1132
41
Stre. O-C2′ (12%) + stre. O-C3′ (22%) + stre. O4-C21 (13%)
1136
191
1136
26
Stre. C2′-O (28%) + stre. C3′-O (19%)
1130
63
1130
42
Stre. O1-C23 (18%) + O3-C1′ (18%)
1128
225
1128
152
Stre. C5′-C6′ (12%) + stre. O1-C23 (13%)
1123
13
1121
67
Stre. C5′-C4′ (20%) + tors. H6-C6-C5-N4 (15%)
1118
43
1119
53
Stre. C5′-C4′ (50%)
1109
112
1190
120
Stre. O-C6′ (17%)
1102
138
1103
18
Stre. O-C6′ (37%)
1073
30
1100
33
1100
66
Stre. C15-C16 (13%)
1069
38
1090
21
1067
294
Stre. O-C4′ (13%)
1060
40
1079
55
1079
99
Stre. O-C6′ (27%)
1070
230
1046
51
Stre. O5-C1′ (10%)
1058
33
1042
88
Tors. H19-C19-C18-C20 (53%)
1046
10
1036
77
Bend H19-C19-C18 (13%)
1041
12
1030
196
Stre. C10-C11 (15%)
1028
30
1035
33
1027
16
Bend (rocking) H18-C18 (11%)
1020
1028
176
1025
53
Stre. C1′-O5 (32%) + stre. C2′-C3′ (10%) + stre. C5′-C6′ (11%)
989
14
978
23
Stre. C5-C6 (30%)
960
12
988
5
972
14
Stre. sim. C22-O1-C23 (27%)
896
29
883
93
Stre. O5-C5′ (13%) + tors. H6′-C6′-C5′-C4′ (18%)
891
7
894
10
893
11
Bend H4-N4 (22%)
867
10
868
11
Bend H4-N4 (12%)
856
0.6
856
0.7
Tors. H-C (entire aromatic ring) (59%)
844
32
843
16
Stre. C3-C14 (13%) + stre. C15-C16 (11%) + tors. H4-N4-C5-C6 (11%)
763
10
764
30
764
4
Out O1-C22-O2-C16 (63%)
720
11
755
38
754
62
Tors. sim. H-C-C (entire aromatic ring) (40%)
700
11
746
14
739
33
Tors. entire indole portion
619
9
617
5
Tors. entire structure
607
12
606
1.5
606
3
Bend C17-O3-C21 (15%)
603
12
601
2
Tors. entire structure
∗The calculated wavenumbers values in the table are unscaled.Figure 7
Experimental IR spectrum (top), theoretical B3LYP 6-31G(d) strictosidine monomer (middle), and theoretical B3LYP 6-31G(d) (bottom) strictosidine dimer IR spectra, in cm−1.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 CH2 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 CH2 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 (3556.00 cm−1) and H-N1 (3493.18 cm−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.Figure 8
The strictosidine dimer, featuring hydrogen bonds (dashed line) between the tryptophanic and glycosidic moieties.


## 3.1. Geometry Optimization

Because no crystallographic data for strictosidine are available, the geometry optimization data, which were calculated using the B3LYP/6-31G(d) and B3LYP/6-311++G(2d,p) basis set, were compared with the X-ray data for similar structures [26–29]. Due to the similarity of the data obtained from both basis sets (Table 1), the discussion that follows is based on the B3LYP 6-31G(d) values but should also apply to B3LYP 6-311++G(2d,p).Table 1
Calculated geometrical parameters for strictosidine compared with the experimental data.
Parameter
B3LYP 6-31G(d)
B3LYP 6-311++G(2d,p)
Molina et al. [27]
Dupont and Dideberg [26]
Lentz and Rossmann [29]
Bond length
N1-C2
1.386
1.384
1.363
1.400
C2-C3
1.507
1.506
—
1.488
C3-N4
1.479
1.477
—
1.477
N4-C5
1.472
1.473
—
1.482
C5-C6
1.543
1.542
—
1.523
C6-C7
1.501
1.501
—
1.490
C7-C2
1.375
1.375
1.442
1.429
C7-C8
1.439
1.439
1.457
1.425
C8-C9
1.406
1.407
—
1.432
C8-C13
1.427
1.426
—
1.406
C9-C10
1.392
1.391
—
1.432
C10-C11
1.411
1.410
—
1.377
C11-C12
1.393
1.392
—
1.346
C12-C13
1.399
1.398
1.409
C13-N1
1.381
1.381
1.406
1.379
C14-C3
1.548
1.548
—
1.541
C14-C15
1.552
1.562
—
1.62
C15-C16
1.515
1.520
—
1.48
C16-C17
1.350
1.350
—
1.60∗
C18-C19
1.335
1.334
—
1.51∗
C19-C20
1.512
1.513
—
1.60
C20-C21
1.526
1.526
—
1.50
C21-O
1.401
1.403
—
1.43
C22-C16
1.469
1.471
—
1.60
C23-O
1.437
1.441
—
1.49
C1′-O
1.403
1.401
—
1.42
C1′-C2′
1.528
1.526
—
1.52
C2′-C3′
1.528
1.527
—
1.51
C3′-C4′
1.527
1.528
—
1.55
C4′-C5′
1.537
1.537
—
1.52
C5′-C6′
1.523
1.522
1.52
C5′-O
1.438
1.442
—
1.45
Bond angle
N1-C2-C7
109.570
109.562
108.7
110.5
C2-C7-C8
107.063
107.071
105.7
107.0
C7-C8-C13
106.793
106.792
107.5
107.2
C8-C13-N1
107.347
107.343
—
108.0
C13-N1-C2
109.221
109.221
—
107.3
C2-C3-N4
110.742
110.742
—
106.6
C3-N4-C5
114.580
114.582
—
112.6
N4-C5-C6
114.370
114.370
—
110.1
C5-C6-C7
108.976
108.976
—
109.4
C6-C7-C2
121.873
121.881
128.9
122.1
C7-C2-C3
125.545
125.546
107.9
126.2
C13-C8-C9
118.786
118.771
—
119.7
C8-C9-C10
119.165
119.172
—
115.3
C9-C10-C11
121.057
121.052
—
122.7
C10-C11-C12
121.177
121.181
—
122.2
C11-C12-C13
117.626
117.625
—
117.6
C12-C13-C8
122.186
122.186
—
122.4
C2-C3-C14
111.904
111.903
—
110.8
N4-C3-C14
110.456
110.456
—
116.9
C3-C14-C15
113.558
113.562
—
112.7
C15-C16-C20
112.883
112.883
—
—
C15-C16-C17
112.915
112.914
—
—
107
C16-C17-O
125.412
125.420
—
—
110
C17-O-C21
119.937
119.941
—
—
121
C18-C19-C20
129.038
129.038
—
—
101
C16-C22-O
113.414
113.415
—
—
111
CH3-O-C22
115.912
115.912
—
—
116
C21-O-C1′
115.516
115.516
—
—
114
O-C1′-C2′
108.278
108.278
—
—
106
C1′-C2′-C3′
109.237
109.237
—
—
106
C2′-C3′-C4′
111.945
111.945
110
C3′-C4′-C5′
109.024
109.024
—
—
105
C4′-C5′-O
108.796
108.795
—
—
106
C5′-O-C1′
114.025
114.027
—
—
108
∗Corresponding to single bonds due to the crystallization process of loganin in loganin penta-acetate monomethyl ether bromide.Initially, the bond lengths indicated a small distortion in the pentacyclic tryptophan ring, showing a greater distance for the C7-C8 (1.44 Å) and C8-C13 (1.42 Å) bonds compared to the N1-C2, C2-C7, and C13-N1 bonds. Significant distortions were observed in the second six-membered ring of the tryptophan portion, which was distinct for all connections. Similar distortions were observed in the dihydropyran ring: 1.52 Å (C15-C16), 1.35 Å (C16-C17), 1.34 Å (C7-O), 1.53 Å (C20-C21), and 1.56 Å (C20-C15). These distortions resulted from the presence of double bonds and heteroatoms, which make these bonds shorter than the C-C bonds.Concerning the planar angles, the aromatic ring shows no large deformations, which was similar to the experimental data for previously analyzed indole alkaloids [26, 29]. The planar angles were 118.78° (C9-C8-C13), 119.16° (C8-C9-C10), 121.06° (C9-C10-C11), 121.17° (C10-C11-C12), 117.62° (C11-C12-C13), and 122.18° (C8-C12-C13). The pentacyclic ring showed a nonangular uniformity except for the angles between N1-C2-C7 (109.56°) and C13-C2-N1 (109.22°).1H-1H NMR coupling constants are highly sensitive to the dihedral angles of hydrogen atoms and both the configuration and conformation of a structure can be validated if there is agreement between the experimental and theoretical J
3
H
H values. The calculated initial dihedral angles for the hydrogen atoms in positions 5 and 6 of the third ring of the tryptophan portion were 162.78° (with theoretical J
H
5
a
-
H
6
b
T
=
11.0 Hz) and 46.76° (theoretical J
H
5
a
-
H
6
a
T
=
4.2 Hz). These values were consistent with the experimentally measured values for coupling constants, J
H
5
a
-
H
6
b
E
=
11.9 Hz and J
H
6
a
-
H
5
a
E
=
4.8 Hz, revealing a pseudoaxial-axial relationship between hydrogen atoms H5a-H6b and an axial-equatorial relationship between H5a and H6a. The obtained dihedral angles for hydrogen atoms of the C3-C14-C15 bridge also showed coupling constants values consistent with the experimental values: 172.28° for H3-H14proR with theoretical J
3
-
14
R
T
=
10.7 (experimental J
3
-
14
R
E
=
11.4 Hz), −72.41° for H3-H14proS with J
14
S
-
3
T
=
2.8 Hz (J
14
S
-
3
E
=
3.6 Hz), and −87.53° for H14proR-H15 with J
H
14
R
-
H
15
T
=
0.6 Hz (J
H
14
R
-
H
15
E
=
1.8 Hz). The conformation of the dihedral angles involving H15-H20 (−56.90°), H20-H21 (−178.69°), and H19-H20 (65.23°) is also plausible given the respective experimental hydrogen coupling constants J
H
15
–
H
20
T
=
4.4 Hz (J
E
=
4.8 Hz), J
H
20
-
H
21
T
=
8.1 (J
E
=
8.4 Hz), and J
H
19
-
H
20
T
=
3.0 Hz (J
E
=
3.0 Hz). The similarity between the modeled DFT structure (Figure 2) (with15S,20S, and21S configuration) and the experimental spectroscopic NMR data complements the conformational arrangement study of strictosidine. The15S,20S, and21S configuration was reported in previous experimental X-ray studies of strictosidine precursors [19, 28–30]. Finally, due to the consistency between the geometry calculated using the DFT B3LYP/6-31G(d) and B3LYP/6-31++G(2d,p) methods and the coupling constant values measured from the experimental 1H spectrum, the conformation of the C21-O-C1′ bridge (so far uncertain) was depicted with angles of 81.17° and lengths of 2.65 Å between H21 and H1′ (Figure 2). This depiction is consistent with the NOESY data provided by Patthy-Lukáts et al. [19]. Other interactions between segments found in the NOESY data are justified by the calculated distances between the hydrogen atoms as follows: H3-H5b (3.76 Å), H3-H14S (2.55 Å), H5a-H5b (1.76 Å), H6a-H6b (1.76 Å), H9-H10 (2.48 Å), H10-11 (2.477 Å), H11-H12 (2.488 Å), H14R-H14S (1.75 Å), H14R-H19 (2.83 Å), H14R-H15 (2.66 Å), H14S-H21 (2.59 Å), H15-H20 (2.41 Å), H18Z-H18E (1.85 Å), H18Z-H20 (3.57 Å), H18E-H19 (2.40 Å), and H20-H19 (2.52 Å) (for information about Mulliken charges and natural bond analysis, see Tables S2 and S3 in Supplementary Material data).Figure 2
The optimized geometry of strictosidine with the scheme of atom numbering.


## 3.2. 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 Figure3 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, π
→
π
∗ transitions were involved but the presence of conjugations in the pentacyclic ring of the tryptophan portion and in the β-alkoxyacrylate group suggested that n
→
π
∗ transitions also occurred. The theoretical calculations predicted an intense electronic transition of 5.8964 eV, with an oscillator strength f
=
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 f
=
0.2714 at 220.62 nm. These results showed good agreement with the measured experimental data (222 nm) assigned to the π
→
π
∗ and π
→
σ
∗ transitions of the indole portion. The calculations also predicted a weaker electronic 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 n
→
π
∗ and π
→
π
∗ transitions of the indole and β-alkoxyacrylate groups.Figure 3
Experimental and theoretical ultraviolet-visible spectra of strictosidine in methanol.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 Figure4. 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 β-alkoxyacrylate group. Transitions involving the LUMO contribute minimally to the absorption wavelengths (
λ
) of strictosidine, showing that the transitions in the β-alkoxyacrylate group have no influence, in disagreement with the ancient proposal that β-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].Figure 4
Frontier molecular orbitals of strictosidine.Figure 5
Mechanism proposed for the major fragmentation pathways of protonated strictosidine.


## 3.3. 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(
η
) 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 (
μ
) 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 (
χ
) 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]:(1)
η
=
1
2
∂
2
E
∂
N
2
V
r
=
1
2
∂
μ
∂
N
V
r
,
μ
=
∂
E
∂
N
V
r
,
χ
=
-
μ
=
-
∂
E
∂
N
V
r
,where E and V
r are the electronic energy and the external potential of anN-electron, respectively. Based on Koopmans theorem for closed-shell molecules, these global chemical reactivity descriptors can be simplified and defined as follows: (2)
η
=
I
-
A
2
,
μ
=
-
I
+
A
2
,
χ
=
I
+
A
2
,where A is the ionization potential and I is the electron affinity of the molecule. The ionization energy and electron affinity are obtained from the HOMO and LUMO energies as I
=
-
E
H
O
M
O and A
=
-
E
L
U
M
O. 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 (ω), 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: (3)
ω
=
μ
2
2
η
.This scale permits the classification of organic molecules as strong,ω
>
1.5 eV, moderate, 0.8
<
ω
<
1.5 eV, and marginal, ω
<
0.8 eV, electrophiles. On the other hand, there is a good correlation in the inverse of the electrophilicity (1
/
ω); 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 (N) appears as a different descriptor which gives more information about nucleophilicity and is defined as follows [37]:(4)
N
=
E
H
O
M
O
-
E
H
O
M
O
T
C
E
,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 Table 2. Both HOMO and LUMO are bonding orbitals, resulting in a low excitation energy for strictosidine. The excitation energies, which were calculated as 4.451 eV for 6-31G(d) and 4.408 eV for 6-311++G(2d,p), reflect the low hardness value (2.22) showing strictosidine as a soft molecule with high polarizability. Electronegativity and electrophilicity values of strictosidine indicate that this molecule has significative attractive electron power acting as an electrophile and in addition to its polarization becomes very reactive since the electrons are farther from the nucleus; however, the nucleophilicity index value indicates that strictosidine is a strong nucleophile too. Such characteristic is justified because strictosidine is a large molecule that has many reactive groups with different potentials which forms small polarized points over its surface (as discussed in Section 3.6), which makes strictosidine a versatile molecule in view of the variety of alkaloids which it forms through intramolecular reactions with groups acting as nucleophiles and others as electrophiles [10]. On the other hand, the calculated chemical potential values, −3.06 for B3LYP/6-31G(d) and −3.40 for B3LYP/6-311++G(2d,p), reveal certain stability, indicating that it does not decompose spontaneously; that is, strictosidine molecule is reactive but does not tend to degrade into the components that formed it.Table 2
Calculated energy values for strictosidine in methanol using B3LYP/6-31G(d) and B3LYP/6-311++G(2d,p).
Basis set
B3LYP/6-31G(d)
B3LYP/6-311++G(2d,p)
Energy (a.u.)
−1835.80
−1836.39
Dipole moment
5.30 Debye
5.50 Debye
E
H
O
M
O (eV)
−5.28
−5.60
E
L
U
M
O (eV)
−0.83
−1.19
E
H
O
M
O
-
L
U
M
O (eV)
4.45
4.40
E
H
O
M
O
-
1 (eV)
−5.89
−6.20
E
L
U
M
O
+
1 (eV)
−0.18
−0.62
E
(
H
O
M
O
-
1
)
-
(
L
U
M
O
+
1
) (eV)
5.71
6.80
Hardness (η)
2.22
2.20
Chemical potential (μ)
−3.06
−3.40
Electronegativity (χ)
3.06
3.40
Electrophilicity index (ω)
2.11
2.63
Nucleophilicity index (N)
5.82
5.91


## 3.4. Tandem Mass Identification

The tandem mass spectra of protonated strictosidine (Figure S4 in Supplementary Data) ofm/z 531 showed major fragment ions ofm/z 514, 369, and 352. The ion ofm/z 514 [M + H—17Da] originated by loss of the NH3 group from the tryptophane portion in a manner that was similar to the fragmentation of aporphine alkaloids (Pathway A) [38]. The ESI-MS3 of this ion generated fragment ions ofm/z 352 [M + H—162Da] due to the hydrogen rearrangement in the glycoside followed by heterolytic cleavage of the C21-O-C1′ bridge (Pathway C). The fragment ion ofm/z 369 [M + H—162Da] 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 MS3 spectrum of the ion ofm/z 369. Guided by the ESI-MS2 and MS3 data, a fragmentation mechanism is proposed for protonated strictosidine in Figure 5.


## 3.5. NBO Study

An NBO analysis describes the Lewis-like 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 leadingN/2 members (orN members in the open-shell case) give the most accurate possible Lewis-like description of the totalN-electron density. The Lewis-type NBOs determine the localizednatural 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 [39–42].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 [43–47] within the molecule. This analysis uses the second-order perturbation energies E
(
2
) [donor (i) → acceptor (j)] that involve the most important delocalization instances, which are given as follows:(5)
E
2
=
Δ
i
j
=
q
i
F
i
j
2
ε
j
-
ε
i
.Table3 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). The intramolecular hyperconjugative interactions between C2, C7, C8, C9, C10, C11, C12, C13, and N1 revealed strong stabilization of the indolic portion, principally by π
→
π
∗ interactions between C11-C10 → C8-C9 (20.07 kcal/mol), C9-C8 → C11-C10 (20.77 kcal/mol), C9-C8 → C13-C12 (20.01 kcal/mol), and C13-C12 → C8-C9 (19 kcal/mol) and electron donation from LP(1) N1 to π
∗ C2-C7 (36.44 kcal/mol) and π
∗ C12-C13 (40.91 kcal/mol). Other hyperconjugative interactions, especially the donation of electron density from LP(2) O3 to π
∗ C16-C17 (37.05 kcal/mol), LP(1) O2 to σ
∗ C22-O1 (32 kcal/mol), and LP(2) O1 to π
∗ C22-O2, gave strong stabilization to the dihydropyran ring of strictosidine. In addition, the π
∗
→
π
∗ interactions of C8-C9 → C7-C2 and C21-O2 → C16-C17 provided enormous stabilization of 191.70 kcal/mol and 62.11 kcal/mol, respectively. Table 4 provides all significant values for the hyperconjugative interactions given by the second-order perturbation theory.Table 3
Electron population in Lewis and non-Lewis orbitals for strictosidine.
Orbitals
Electrons
Percentage
Core
75.97
99.96% of 76
Valence Lewis
201.50
97.817% of 202
Total Lewis
277.47
98.39% of 282
Valence non-Lewis
4.02
1.43% of 282
Rydberg non-Lewis
0.50
0.17% of 282
Total non-Lewis
4.53
1.61% of 282Table 4
Selected second-order perturbation energies for strictosidine.
Donor orbital (i)
Type
Acceptor orbital (j)
Type
E
(
j
)
-
E
(
i
) a.u.
E
(
2
) (kcal/mol)
C11-C10
π
C9-C8
π
∗
0.28
20.07
C13-C12
π
∗
0.27
18.90
C9-C8
π
C11-C10
π
∗
0.27
20.77
C12-C13
π
∗
0.27
20.01
C7-C2
π
∗
0.29
14.88
C13-C12
π
C11-C10
π
∗
0.28
21.04
C8-C9
π
∗
0.29
16.32
C7-C2
π
C9-C8
π
∗
0.29
19.71
C16-C17
π
C22-O1
π
∗
0.29
24.34
N1
LP(1)
C13-C12
π
∗
0.29
40.91
N1
LP(1)
C2-C7
π
∗
0.31
36.44
O3
LP(2)
C16-C17
π
∗
0.36
37.05
O4
LP(2)
O3-C21
σ
∗
0.56
15.13
O4
LP(2)
C1′-O5
σ
∗
0.60
13.48
O2
LP(1)
C22
RY∗
1.55
14.52
O2
LP(2)
C16-C22
σ
∗
0.72
16.43
O2
LP(2)
C22-O1
π
∗
0.63
32.65
O1
LP(2)
C22-O2
π
∗
0.32
47.44
C8-C9
π
∗
C7-C2
π
∗
0.01
191.70
C22-O2
π
∗
C16-C17
π
∗
0.03
62.11A 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 Figure2, the most stable fragments arose from the heterolytic breakage of the O4-C1′, N4-C3, and N4-C5 bonds, which have the following weak hyperconjugative interactions: N4-C3 → N2-C3 (σ
→
σ
∗, 3.59 kcal/mol), C5-N4 → C3 (σ
→
σ
∗, 1.5 kcal/mol), N4-H → C5 (σ
→
σ
∗, 1.68 kcal/mol), N4-H → C5-H (σ
→
σ
∗, 2.51 kcal/mol), O4-C1′→ C20-C21 (σ
→
σ
∗, 1.46 kcal/mol), and O3-C21 → C21-O4 (σ
→
σ
∗, 0.51 kcal/mol). These results showed that the NBO study complemented the mass fragmentation study.


## 3.6. Molecular Electrostatic Potential Surface

Figure6 illustrates 3D plots of the molecular electrostatic potential (MEP) 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.Figure 6
Molecular electrostatic potential maps (from two perspectives) for strictosidine calculated using the B3LYP/6-31G(d) basis set.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 (−0.0643 a.u.), over OH on position C6′ (−0.0471 a.u.), and over oxygen atom on C4′ position (−0.0201 a.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.


## 3.7. IR Analysis

Figure7 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).Table 5
Experimental and calculated wavenumbers∗ (cm−1) and assignments for strictosidine.
IR solid
B3LYP 6-31G(d)
Assignment (PED > 5%)
Exp.
IR intensity
Monomer
Dimer
Calculated
IR intensity
Calculated
IR intensity
3767
30
3633
62
Stre. OH (96%)
3732
30
3602
6
Stre. OH (95%)
3430
42
3731
49
3556
225
Stre. OH (96%)
3411
44
3724
44
3535
64
Stre. OH (97%)
3390
43
3656
44
3493
47
Stre. N1-H1 (88%) + tors. H1-N1-C2-C13 (11%)
3382
58
3488
0.27
3475
2.49
Stre. N4-H4 (88%) + bend H4-N4-C5 (11%)
3320
51
3260
4
3260
5
Stre. C18-H18 (70%) + stre. C19-H19 (26%)
3211
20
3246
3
3239
12
Stre. C17-H17 (99%)
3205
38
3183
22
Stre. C9-H9 (36%) + stre. C10-H10 (39%) + stre. C12-H12 (10%)
3180
17
3193
39
3190
28
Stre. C9-H9 (21%) + stre. C10-H10 (16%) + stre. C11-H11 (30%) + C12-H12 (23%)
3152
9
3183
3
3173
2
Stre. C9-H9 (12%) + stre. C10-H10 (24%) + stre. C11-H11 (32%) + stre. C12-H12 (13%)
3150
25
3176
19
3139
1
Stre. sim. C23-H23 (22%) + stre. sim. C23-H23 (19%) + stre. ass. C23-H23 (24%) + stre. C23-H23 (19%)
3140
22
3176
2
3152
14
Stre. C9-H9 (12%) + stre. C10-H10 (17%) + stre. C11-H11 (43%)
3175
14
3140
7
Stre. C19-C18 (41%) + stre. sim. C18-H18 (25%) + stre. C19-H19 (18%)
3129
12
3161
2
3125
17
Stre. C9-H19 (66%) + stre. sim. C18-H18 (26%)
3105
10
3144
23
3122
7
Stre. C23-H23 ass. (92%)
3100
11
3103
35
3104
20
Stre. C6′-H6′ (91%)
3102
35
3082
8
Stre. C5-H5 (47%) + stre. C14-H14 (33%)
3093
25
3075
28
Stre. C14-H14 (74%)
3076
10
3072
27
Stre. C15-H15 (85%) + stre. C3-H3 (11%)
3071
44
3069
28
Stre. sim. C23-H23 (72%)
3070
2
3063
44
Stre. C3-H3 (57%) + stre. C15-H15 (12%)
3061
42.72
3061
1
Stre. C5-H5 (86%)
3060
44
3052
54
Stre. C21-H21 (60%)
3052
6
3050
48
Stre. C14-H14 (65%)
3041
19
3042
14
Stre. ass. C6-H6 (77%)
3040
110
3033
13
Stre. C1′-H1′ (77%)
3020
15
3032
13
3032
28
Stre. C4′-H4′ (62%)
3017
6
3011
37
Stre. C20-H20 (94%)
3016
2
3008
5
Stre. C2-H2 (44%) + stre. C5-H5 (46%)
3013
12
3007
3
Stre. C2′-H2′ (31%) + stre. C4′-H4′ (20%) + stre. C5′-H5′ (35%)
2931
26
2999
50
2997
4
Stre. C6-H6 ass. (71%)
2929
23
2989
1
2992
8
Stre. C3′-H3′ (63%) + stre. C6′-H6′ sim. (18%)
2920
12
2985
44
2988
46
Stre. C6′-H6′ (72%)
1690
62
1767
321
1716
308
Stre. C22=O2 (73%)
1628
41
1694
263
1691
272
Stre. C16=C17 (65%) + bend H17-C17-O3 (12%)
1586
7
1619
2
1591
1.5
Stre. (entire indole portion) (33%)
1527
5
1537
3.5
1519
3.6
Stre. C9=C10 (12%) + bend H10-C10 (20%) + bend H1-N1 (11%)
1531
1.2
1497
4
Bend (scissoring) H6′-C6′ (80%)
1527
9
1496
7
Bend (scissoring) C23-H23 (63%)
1400
20
1418
32
1415
11
Stre. C15-C16 (16%) + bend H17-C17-O3 (27%)
1399
25
1413
4
1413
4
Bend H-C entire indole portion
1377
1399
6
1395
9
Bend H21-C21 (14%) + bend H5′-C5′ (13%) + tors. H5′-C5′-O5-C1′ (10%) + tors. H4′-C4′-OH (16%)
1365
12
1386
3
1375
8
Tors. H14-C14-C3-N4 (11%) + tors. H15-C15-C14-C3 (11%) + H5-C5-N4-C3 (10%)
1360
10
1381
4
1363
1
Bend H1′-C1′ (16%) + bend H4′-C4′ (15%) + tors. C5′-H5′ (19%)
1350
11
1378
32
1362
19
Bend H3′-C3′ (11%) + bend H21-C21 (16%) + bend H1′-C1′ (14%)
1344
18
1367
47
1345
19
Bend H18-C18-C19 (32%) + bend H2′-C2′ (12%)
1328
13
1348
59
1340
52
Bend H17-C17-O3 (15%) + bend H15-C15 (10%)
1345
66
1346
29
Bend H17-C17 (12%) + tors. H15-C15-C14-C20 (10%)
1303
30
1342
53
1342
41
Bend H1′-C1′ (22%) + tors. H5′-C5′-O5-C1′ (25%)
1338
65
1327
9
Bend H20-C20 (15%)
1300
33
1326
22
1320
31
Stre. C13-N1-C2 (29%)
1297
25
1312
7
1301
18
Bend H20-C20 (16%) + bend H3-C3 (10%) + tors. H14-C14-C3-N4 (16%)
1250
22
1267
23
1260
17
Bend H-C entire structure
1257
40
1243
40
Bend O-H (5′) (18%) + bend H4′-C4′ (10%)
1211
13
1253
117
1252
16
Stre. C17-O3 (22%) + H15-C15 (13%)
1157
137
1132
41
Stre. O-C2′ (12%) + stre. O-C3′ (22%) + stre. O4-C21 (13%)
1136
191
1136
26
Stre. C2′-O (28%) + stre. C3′-O (19%)
1130
63
1130
42
Stre. O1-C23 (18%) + O3-C1′ (18%)
1128
225
1128
152
Stre. C5′-C6′ (12%) + stre. O1-C23 (13%)
1123
13
1121
67
Stre. C5′-C4′ (20%) + tors. H6-C6-C5-N4 (15%)
1118
43
1119
53
Stre. C5′-C4′ (50%)
1109
112
1190
120
Stre. O-C6′ (17%)
1102
138
1103
18
Stre. O-C6′ (37%)
1073
30
1100
33
1100
66
Stre. C15-C16 (13%)
1069
38
1090
21
1067
294
Stre. O-C4′ (13%)
1060
40
1079
55
1079
99
Stre. O-C6′ (27%)
1070
230
1046
51
Stre. O5-C1′ (10%)
1058
33
1042
88
Tors. H19-C19-C18-C20 (53%)
1046
10
1036
77
Bend H19-C19-C18 (13%)
1041
12
1030
196
Stre. C10-C11 (15%)
1028
30
1035
33
1027
16
Bend (rocking) H18-C18 (11%)
1020
1028
176
1025
53
Stre. C1′-O5 (32%) + stre. C2′-C3′ (10%) + stre. C5′-C6′ (11%)
989
14
978
23
Stre. C5-C6 (30%)
960
12
988
5
972
14
Stre. sim. C22-O1-C23 (27%)
896
29
883
93
Stre. O5-C5′ (13%) + tors. H6′-C6′-C5′-C4′ (18%)
891
7
894
10
893
11
Bend H4-N4 (22%)
867
10
868
11
Bend H4-N4 (12%)
856
0.6
856
0.7
Tors. H-C (entire aromatic ring) (59%)
844
32
843
16
Stre. C3-C14 (13%) + stre. C15-C16 (11%) + tors. H4-N4-C5-C6 (11%)
763
10
764
30
764
4
Out O1-C22-O2-C16 (63%)
720
11
755
38
754
62
Tors. sim. H-C-C (entire aromatic ring) (40%)
700
11
746
14
739
33
Tors. entire indole portion
619
9
617
5
Tors. entire structure
607
12
606
1.5
606
3
Bend C17-O3-C21 (15%)
603
12
601
2
Tors. entire structure
∗The calculated wavenumbers values in the table are unscaled.Figure 7
Experimental IR spectrum (top), theoretical B3LYP 6-31G(d) strictosidine monomer (middle), and theoretical B3LYP 6-31G(d) (bottom) strictosidine dimer IR spectra, in cm−1.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 CH2 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 CH2 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 (3556.00 cm−1) and H-N1 (3493.18 cm−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.Figure 8
The strictosidine dimer, featuring hydrogen bonds (dashed line) between the tryptophanic and glycosidic moieties.


## 4. Conclusion

The strictosidine alkaloid, which was isolated fromStrychnos 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
n analysis, and these modes may contribute to the further characterization of strictosidine analogues and derivatives.



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*Source: 1752429-2016-03-08.xml*