J Mol Model (2014) 20:2084 DOI 10.1007/s00894-014-2084-0
ORIGINAL PAPER
Structure and electronic properties of azadirachtin Elton A. S. de Castro & Daniel A. B. de Oliveira & Sergio A. S. Farias & Ricardo Gargano & João B. L. Martins
Received: 13 May 2013 / Accepted: 8 November 2013 / Published online: 9 February 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract We performed a combined DFT and Monte Carlo 13 C NMR chemical-shift study of azadirachtin A, a triterpenoid that acts as a natural insect antifeedant. A conformational search using a Monte Carlo technique based on the RM1 semiempirical method was carried out in order to establish its preferred structure. The B3LYP/6-311++G(d,p), wB97XD/6-311++G(d,p), M06/6-311++G(d,p), M06-2X/6311++G(d,p), and CAM-B3LYP/6-311++G(d,p) levels of theory were used to predict NMR chemical shifts. A Monte Carlo population-weighted average spectrum was produced based on the predicted Boltzmann contributions. In general, good agreement between experimental and theoretical data was obtained using both methods, and the 13C NMR chemical shifts were predicted highly accurately. The geometry was optimized at the semiempirical level and used to calculate the NMR chemical shifts at the DFT level, and these shifts showed only minor deviations from those obtained following structural optimization at the DFT level, and incurred a much lower computational cost. The theoretical ultraviolet spectrum showed a maximum absorption peak that was mainly contributed by the tiglate group.
E. A. S. de Castro (*) Universidade Estadual de Goiás, Av. Universitária s/n, 73807-250 Formosa, GO, Brazil e-mail: [email protected] D. A. B. de Oliveira : J. B. L. Martins Laboratório de Química Computacional, Instituto de Química, CP 4478, Brasília, DF 70904-970, Brazil S. A. S. Farias Instituto de Ciências da Educação, Universidade Federal do Oeste do Pará, CP 68035-110, Santarém, PA, Brazil R. Gargano Instituto de Física, UnB, CP 04455, Brasília, DF 70919-970, Brazil
Keywords Ultraviolet spectroscopy . Chemical shifts . Density functional theory . Empirical dispersion . Azadirachtin . Monte Carlo
Introduction Azadirachtins (C35H44O16) are a group of triterpenoids obtained from Azadirachta indica A. Juss. (“neem”) trees. There are a total of eight azadirachtins: A, B, C, D, E, F, G, and I [1, 2]. Azadirachtin A, commonly referred to simply as “azadirachtin,” has various useful applications; for example, it acts as an insect growth and reproduction inhibitor as well as a natural insect antifeedant. In addition, it can be applied in medicinal treatments such as fungicidal, anti-inflammatory, and antiulcer drugs [1]. It is obtained as a microcrystalline powder by successive extractions of neem seeds. Different cell populations have been observed to show different sensitivities to neem oil obtained by methanolic extraction; for instance, there is a tumor line that exhibits significantly higher sensitivity to this extracted oil [3]. Azadirachtin has 16 stereocenters, and its structure has been elucidated by NMR spectroscopy [4]. The complete synthesis of azadirachtin was performed by Ley and coworkers after 22 years using 71 steps, with a total yield of 0.00015 % [5, 6]. In order to isolate azadirachtin, HPLC was employed in association with UV detection [7–10]. The wavelength associated with this molecule ranges from 214 to 219 nm depending on the solvent used [1, 7–12]. Quantitative multidimensional conformational analysis of azadirachtin has been performed in order to get a better understanding of the biological activity of this molecule [13]. That work analyzed four torsion angles (chosen from among a total of almost 30 angles) in azadirachtin using the
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AM1 semiempirical method. The three most stable conformers were found by performing a conformational search in which one dihedral angle between the condensed ring groups and three other dihedral angles involving the hydroxyl groups were varied [13]. The main purpose of the study described in the present paper was to check the correlation of theoretical spectroscopic results for azadirachtin with the corresponding experimental data. To achieve this task, we determined the most stable structures among the calculated conformers of azadirachtin using a Monte Carlo technique based on the RM1 semiempirical method [14]. More rigorous density functional theory (DFT) calculations were also used to optimize its geometry, and the consequent theoretical nuclear magnetic resonance (NMR) chemical shifts and ultraviolet (UV) spectroscopic results were compared with experimental data.
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hydrogen bond interactions [21]. The accuracy of geometries predicted using wB97XD has been reported [22]. CAM-B3LYP gives good results for ultraviolet studies [23], so it was also used in the present work. It is important to note that azadirachtin has a large number of torsion angles. Therefore, it was crucial to perform conformational analysis in order to generate a new starting point for geometry optimization. A Monte Carlo method was used to search for possible conformations of this molecule. One hundred structures were retained within an energy interval of 40 kJ mol−1 and with Boltzmann contributions of >0.001. The Monte Carlo method was carried out using the RM1 semiempirical Hamiltonian, as performed with the Spartan program [24]. DFT calculations were carried out using the Gaussian 09 program [25]. The most stable RM1 conformers were studied using the DFT
Table 1 Twenty-seven most stable geometries optimized using Monte Carlo with the RM1 method
Methods DFT was applied to azadirachtin using different functionals, including B3LYP [15], wB97XD [16], M06 [17], M06-2X [17], and the long-range correction CAM-B3LYP (the Coulomb-attenuating method applied to B3LYP) [18]. Although B3LYP does not include dispersion, it has a well-documented database for a large range of properties [19]. We also used the highly parametrized empirical global hybrid meta-GGA M06 and M06-2X functionals with 27 % and 54 % HF exchange, respectively. These functionals describe noncovalent interactions better than other density functionals [20]. The wB97XD functional [16] including empirical dispersion correction was also used, since this method gives the best results overall for
Fig. 1 Structure of azadirachtin A as deduced from X-ray data [26]. Oxygen in red and carbon in gray. Hydrogen atoms are not shown. A and B groups are also shown
Conformer
B3LYP//RM1a Relative energy (kJ mol−1)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
27.20 28.54 10.54 0.47 26.41 9.99 20.04 21.12 20.32 5.30 1.23 0.00 14.45 17.23 11.45 18.63 11.25 22.63
0.00 0.38 0.65 1.57 2.61 2.81 2.96 3.61 4.17 4.52 4.68 5.20 6.23 6.90 7.51 7.60 7.68 8.83
0.181 0.156 0.140 0.096 0.063 0.058 0.055 0.042 0.034 0.029 0.028 0.022 0.015 0.011 0.009 0.008 0.008 0.005
19 20 21 22 23 24 25 26 27
13.90 10.69 9.66 39.35 6.96 27.53 10.48 22.37 31.39
9.60 9.68 9.79 10.65 11.18 11.24 11.39 11.60 11.75
0.004 0.004 0.003 0.002 0.002 0.002 0.002 0.002 0.002
a
RM1 Relative energy (kJ mol−1)
Calculated with the 6-31+G(d,p) basis set
Boltzmann distribution
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B3LYP hybrid exchange correlation functional [15] within the 6–31+G(d,p) basis set. They were obtained using Schlegel’s algorithm [25], where an ultrafine grid was used for two-electron integrals and their derivatives. All optimizations were performed using extremely tight optimization convergence criteria (opt=verytight). The molecular structure obtained from X-ray measurements [26] was also used as a starting point for geometry optimization at the DFT/6–31+G(d,p) level in an attempt to find the best conformer structure. For all optimized geometries, theoretical NMR spectra were obtained using tetramethylsilane (TMS) as a reference to compare with experimental data. For this step, the gaugeincluding atomic orbitals (GIAO) method was used along with the same functional as employed to optimize the geometry, and the more extended 6-311++G(d,p) basis set in order to increase the accuracy of the calculated data. A comparison with the experimental UV spectrum was carried out via the time-dependent (TD) DFT method, using all of the functionals tested in this work and the 6-311++G(d,p) basis set. TD-DFT was calculated with the polarizable
continuum model using the integral equation formalism variant (IEFPCM) method [27]. All geometries were kept frozen for NMR and UV calculations. This methodology was recently used and provided reasonable results [28, 29]. Highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) frontier orbitals were analyzed to gain insight into the molecular orbitals involved in the UV excitation.
Results and discussion Conformational analysis The first structure for azadirachtin based on NMR was published in 1975 [30]; later, in 1985, two new structures were proposed [31], and in 1987 the structure was reinvestigated [4]. In this structure (Fig. 1), the two most important groups, a modified decalin moiety (A) and a tricyclic part (B), rotate about the single bond C8–C14.
Table 2 Main geometrical parameters of DFT-optimized conformers and comparison to the corresponding experimental data O…O distance (Å)
21–15 21–13 15–13 13–19 13-6 19-6 7–6 11–12 Torsion angle (°) O6–C6–C7–O7 C7–C8–C14–C13 C7–C8–C14–C15 C13–C14–C15–C16 O13–C14–C15–O15 O13–C13–C17–C16 C13–C17–C20–C22 C4–C5–C6–O6 C10–C9–C11–O19 O7–C7–C8–C30 O20–C20–C22–C23 C18–C13–C14–C15 O7–C7–C8–C9 O11–C11–C12–O12 RMSD a
12 RM1a 2.206 5.247 3.580 5.495 5.158 5.697 2.891 3.437
B3LYP/ 6-31+G(d,p)
wB97XD/ 6-31+G(d,p)
2.341 5.279 3.634 5.832 5.152 5.859 2.812 2.572
2.322 5.211 3.616 5.736 5.176 5.823 2.780 2.560
M06/6-31+G(d,p)
2.325 5.201 3.610 5.758 5.145 5.825 2.776 2.565
M06-2X/ 6-31+G(d,p)
CAM-B3LYP/ 6-31+G(d,p)
2.324 5.173 3.614 5.708 5.167 5.840 2.775 2.577
2.327 5.223 3.611 5.773 5.127 5.831 2.789 2.561
Expt. [26]
2.279 5.391 3.627 5.900 5.088 5.769 2.969 2.654
56.95 −117.87 85.31 30.37 −147.85 38.55
50.50 −120.25 82.67 30.48 −147.97 37.13
49.47 −123.71 79.66 29.21 −149.87 35.40
49.40 −122.29 81.20 29.85 −148.83 35.87
49.13 −123.15 80.52 29.30 −149.57 35.51
49.92 −120.07 83.29 30.17 −148.25 36.77
59.20 −110.52 89.30 29.77 −147.80 39.39
−180.56 41.67 25.11 −173.37 −120.07 151.64 63.26 135.60 5.509
−188.32 43.69 13.16 −173.65 −123.51 156.23 62.74 −17.80 0.495
−191.01 43.87 14.30 −174.00 −125.90 156.72 62.08 2.90 0.481
168.93 42.87 13.06 −171.82 −125.93 156.50 64.16 −19.68 0.507
167.72 44.71 13.81 −174.21 −127.92 156.33 61.10 −22.18 0.530
170.32 43.96 13.53 −174.13 −125.04 156.17 61.99 −18.99 0.397
−177.48 39.74 14.96 −173.11 −108.37 157.95 62.60 −24.71 –
Monte Carlo conformer 12 optimized at the RM1 level
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A systematic search of the conformer distribution was performed using the Monte Carlo technique in order to identify the set of energetically accessible conformers. This search generated a total of 1,000 initial structures that were optimized using the RM1 semiempirical method, where the 100 conformers with the lowest energies were retained. Table 1 describes the relative energies of the 27 most stable
structures that make Boltzmann contributions of >0.001. Structure 1 is the most stable at the RM1 level and provides the greatest Boltzmann contribution, while structure 12 is the most stable at the B3LYP/6-311++G(d,p)//RM1 level. Therefore, RM1 structures 1 and 12 were reoptimized at the B3LYP/6-31+G(d,p) level, and structure 12 yielded the most stable geometry.
Table 3 Calculated and experimental 13C chemical shifts (ppm) Carbon
B3LYP// RM1 weighted
B3LYP //RM1a
B3LYP
wB97XD
M06
M06-2X
CAM-B3LYP
Expt. [4]
1 2 3 4 5 6 7 8 9 10 11
75.22 33.53 74.05 58.42 43.22 82.90 82.12 48.15 50.85 57.57 106.72
75.14 32.03 73.97 59.35 43.28 84.04 81.83 48.64 50.56 58.29 106.02
77.29 33.71 75.03 63.11 43.68 81.40 82.40 55.27 53.45 62.46 111.66
75.67 33.59 70.16 58.65 40.60 79.10 77.94 50.28 50.29 57.59 106.13
74.38 29.66 68.42 53.56 35.77 76.60 76.92 44.65 45.75 53.10 104.06
78.89 34.99 72.91 61.01 42.06 81.12 80.19 51.01 52.73 59.57 108.74
76.79 33.20 71.24 59.57 41.00 80.44 79.42 51.00 51.31 57.76 107.99
70.88 29.37 66.69 52.52 37.06 73.79 74.37 45.41 44.69 50.19 104.10
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
180.48 67.97 70.35 77.87 36.62 52.54 23.83 78.71 87.86 112.17 115.73 155.51 177.70 21.92 53.13 53.07 80.30 186.48
180.79 71.63 72.50 79.50 33.69 53.41 21.40 79.52 87.80 113.29 115.59 154.00 177.88 21.92 53.15 53.12 79.80 186.41
184.51 75.90 81.01 87.59 27.81 56.95 22.48 76.71 91.13 117.51 114.34 155.19 178.50 22.99 56.20 57.79 79.68 185.93
183.21 70.77 74.26 82.97 25.70 55.50 23.54 74.43 86.87 112.49 114.93 154.18 177.80 23.76 54.83 55.96 76.44 183.76
183.27 66.29 70.16 84.49 24.10 50.57 21.48 74.27 83.94 113.85 115.54 156.85 177.93 21.76 55.97 56.72 76.05 183.75
194.52 71.46 76.15 86.54 26.04 57.57 25.02 77.45 89.15 115.94 128.72 166.85 190.33 24.85 56.29 57.19 78.43 196.38
186.61 72.46 77.14 84.61 26.47 56.68 23.34 75.72 88.40 114.75 116.66 156.94 180.99 23.44 55.57 56.87 77.53 187.58
171.70 68.53 69.69 76.43 25.06 48.67 18.49 69.07 83.55 108.70 107.30 147.00 169.50 20.88 53.52 52.72 76.10 173.20
30 31 32 33 34 35 RMSD
25.81 176.83 136.47 16.25 144.88 17.29 6.50
24.15 176.99 137.54 15.51 143.37 18.47 6.46
22.24 176.29 138.59 15.43 150.03 18.61 8.21
20.97 176.02 135.14 14.82 153.18 17.47 6.12
20.25 175.72 134.76 12.07 153.81 16.47 5.62
21.45 188.75 151.74 14.65 171.36 17.99 12.62
21.23 178.43 138.90 14.29 151.67 17.39 7.46
21.33 166.22 128.60 14.29 137.50 11.94 –
The chemical shift of tetramethylsilane (TMS), calculated at the same level, was used as the reference. The 6-311++G(d,p) basis set was used. B3LYP// RM1 was obtained from a Boltzmann-weighted average a
Monte Carlo conformer 12
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Page 5 of 7, 2084 Table 5 TD/DFT results obtained using the 6-311++G(d,p) basis set and the IEFPCM method (in methanol solvent)
Fig. 2
13
C NMR chemical shifts: experimental versus calculated data
The most relevant geometrical parameters are presented in Table 2. The root mean square deviation (RMSD) was calculated taking into account only the carbon atoms. When compared with the experimental X-ray data, the RM1 geometry 12 gave the largest RMSD (Table 2). The DFT-optimized geometries presented nearly the same RMSD values (Table 2), whereas CAM-B3LYP gave the closest geometry to that observed experimentally. The dihedral angle C7–C8–C14–C15 was reported to be 73.2° when calculated at the AM1 level [13], while the experimentally derived value is 89.30° [26], meaning that the molecule adopts a rather folded configuration. The values of the C7–C8–C14–C15 dihedral angle (79.66–85.31°) we obtained at various levels of DFT (Table 2) are in good agreement with the experimental data. These conformers probably play an important role in determining the electronic and spectroscopic properties of azadirachtin. Therefore, we focused on the most stable conformers in order to determine the NMR chemical shifts, and then compared them with those observed experimentally. NMR and UV The theoretically derived NMR chemical shifts are compared with the corresponding experimental data [4] in Table 3. All chemical shifts were calculated using the 6-311++G(d,p) basis set. Figure 2 plots the calculated against the experimental NMR chemical shifts of the DFT-optimized geometries, while
λmax (nm)
Oscillator strength
B3LYP wB97XD M06 M06-2X
227.75 215.49 227.44 212.32
0.2526 0.3208 0.2714 0.3323
CAM-B3LYP Expt. [11]
214.29 217
0.3192 –
Table 4 provides the calculated parameters from linear regressions of the calculated against the experimental NMR chemical shifts. In general, the NMR chemical shifts calculated at various levels of DFT present almost the same amount of error, although they have different conformations (Table 2). The structure optimized at the wB97XD level has the lowest RMSD between the calculated and experimental chemical shifts (Table 3). Otherwise, linear regressions of the calculated versus experimental results show that the largest correlation coefficient (R) is obtained when CAM-B3LYP is used at the same basis set level (Table 4). sp3 carbon and terminal methyl groups show the largest deviations from experimental values since they are more flexible than the other carbon groups. The most important chemical moieties of azadirachtin, C13–C14 of the epoxide ring and C32–C34 of the tiglate ester, show only small deviations in chemical shift from those seen experimentally [4]. Also, NMR chemical shifts calculated at the DFT level using the geometry optimized at the semiempirical level show only small deviations from those calculated at the same DFT level using the geometries optimized with DFT, and were obtained at much lower computational cost. The theoretical spectroscopic results can be used to identify the chromophore groups present in the azadirachtin molecule. TD-DFT calculations were performed using the optimized geometry obtained at the same level, as well as the nonequilibrium PCM solvation model and the 6-311++G(d,p) basis set. Experimental UV-visible spectral data show that the maximum absorption of the azadaricathin molecule occurs at 215–219 nm, depending on the solvent used [1, 7–12, 32].
Table 4 Results of linear regression of experimental versus calculated chemical shift data AY=A+BX
B3LYP//RM1 weighted
B3LYP//RM1a
B3LYP
wB97XD
M06
M06-2X
CAM-B3LYP
A B R Standard deviation
2.8479 1.0351 0.9981 3.0874
2.6936 1.0379 0.9985 2.7870
4.1967 1.0438 0.9987 2.5894
1.5405 1.0472 0.9988 2.5319
−1.8859 1.0711 0.9985 2.8502
−1.0559 1.1409 0.9965 4.7008
1.3564 1.0663 0.9991 2.2322
a
Monte Carlo conformer 12
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Fig. 3 HOMO (left) and LUMO (right) molecular orbitals calculated at the CAM-B3LYP/6-311++G(d,p) level. Isovalue of 0.02 a.u.
Our theoretical results (Table 5) showed that wB97XD/6311++G(d,p) and CAM-B3LYP/6-311++G(d,p) yield the smallest errors in the maximum absorption of azadaricathin in methanol as compared to the experimental value (also obtained in methanol, at 217 nm) [11, 12]. Otherwise, CAM-B3LYP gives the closest optimized geometry to that observed experimentally. All of the functionals indicated the same contributions from the HOMO and LUMO orbitals (Fig. 3), with the HOMO mainly being spread over the tricyclic part (B, see Fig. 1) while the LUMO is mainly located on the tiglate moiety. However, the TD method shows that the UV transition with the largest oscillator strength occurs from HOMO-1 (molecular orbital 190) to LUMO (molecular
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orbital 192), whereas HOMO (molecular orbital 191) is not involved in this transition. We noted some differences among the results obtained using the various functionals regarding HOMO-1 and HOMO-2 (molecular orbital 189), as depicted in Fig. 4. B3LYP and wB97XD show the inverse picture of HOMO-1 and HOMO-2 in relation to the picture provided by the other functionals. All of the functionals except for B3LYP and wB97XD show that the contribution of HOMO-1 is spread over the tiglate moiety (Fig. 4) and HOMO-2 is localized over the tricyclic part (B, see Fig. 1), while B3LYP and wB97XD show the HOMO-1 over the tricyclic part and HOMO-2 over the tiglate moiety. wB97XD, however, shows a small contribution of the tiglate moiety to the HOMO-1 orbital. Therefore, with the exception of B3LYP and wB97XD, all of the other functionals show that HOMO-1 and HOMO are spread over the C8–C14 bonds and furan group. For the M06, M06-2X, and CAM-B3LYP functionals, the excitation with the greatest oscilator strength occurs from the tiglate ester to the same group, which is the strongest chromophore in the azadirachtin structure, in accordance with the experimental results at 217 nm [11].
Conclusions Theoretical spectroscopic calculations were performed in order to analyze the azadirachtin molecule in relation to the experimental data. We have determined the most stable structures among the calculated conformers of azadirachtin using a Monte Carlo technique with the RM1 method. DFT calculations were used to optimize the geometry and compare the theoretically derived NMR chemical shifts and UV spectroscopic results with the corresponding experimental data. The theoretical chemical shifts were found to be in good agreement with the experimental results. The geometry optimized at the semiempirical level and the NMR chemical shifts calculated using DFT showed minor deviations from the shifts obtained using DFT and the DFT-optimized geometry and incurred a much lower computational cost. The best theoretical results for the UV spectrum of azadirachtin show a maximum peak at 215.49 nm (using wB97XD/6-311++G(d,p)) or 214.29 nm (using CAMB3LYP/6-311++G(d,p)) in methanol, while the maximum in the experimental data occurs at 217 nm. Moreover, theoretical UV spectroscopic data allowed us to identify that the tiglate group is associated with this absorption, in accordance with the experimental data.
Fig. 4a–e HOMO-2 (left) and HOMO-1 (right) molecular orbitals associated with maximum absorption for the azadirachtin, as calculated at the a B3LYP, b wB97XD, c M06, d M06-2X, and e CAM-B3LYP levels using the 6-311++G(d,p) basis set. Isovalue of 0.02 a.u.
Acknowledgments The authors are indebted to the financial support of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Instituto Nacional de Ciência e Tecnologia dos Materiais em Nanotecnologia (INCTMN).
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ORIGINAL PAPER
Structure and electronic properties of azadirachtin Elton A. S. de Castro & Daniel A. B. de Oliveira & Sergio A. S. Farias & Ricardo Gargano & João B. L. Martins
Received: 13 May 2013 / Accepted: 8 November 2013 / Published online: 9 February 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract We performed a combined DFT and Monte Carlo 13 C NMR chemical-shift study of azadirachtin A, a triterpenoid that acts as a natural insect antifeedant. A conformational search using a Monte Carlo technique based on the RM1 semiempirical method was carried out in order to establish its preferred structure. The B3LYP/6-311++G(d,p), wB97XD/6-311++G(d,p), M06/6-311++G(d,p), M06-2X/6311++G(d,p), and CAM-B3LYP/6-311++G(d,p) levels of theory were used to predict NMR chemical shifts. A Monte Carlo population-weighted average spectrum was produced based on the predicted Boltzmann contributions. In general, good agreement between experimental and theoretical data was obtained using both methods, and the 13C NMR chemical shifts were predicted highly accurately. The geometry was optimized at the semiempirical level and used to calculate the NMR chemical shifts at the DFT level, and these shifts showed only minor deviations from those obtained following structural optimization at the DFT level, and incurred a much lower computational cost. The theoretical ultraviolet spectrum showed a maximum absorption peak that was mainly contributed by the tiglate group.
E. A. S. de Castro (*) Universidade Estadual de Goiás, Av. Universitária s/n, 73807-250 Formosa, GO, Brazil e-mail: [email protected] D. A. B. de Oliveira : J. B. L. Martins Laboratório de Química Computacional, Instituto de Química, CP 4478, Brasília, DF 70904-970, Brazil S. A. S. Farias Instituto de Ciências da Educação, Universidade Federal do Oeste do Pará, CP 68035-110, Santarém, PA, Brazil R. Gargano Instituto de Física, UnB, CP 04455, Brasília, DF 70919-970, Brazil
Keywords Ultraviolet spectroscopy . Chemical shifts . Density functional theory . Empirical dispersion . Azadirachtin . Monte Carlo
Introduction Azadirachtins (C35H44O16) are a group of triterpenoids obtained from Azadirachta indica A. Juss. (“neem”) trees. There are a total of eight azadirachtins: A, B, C, D, E, F, G, and I [1, 2]. Azadirachtin A, commonly referred to simply as “azadirachtin,” has various useful applications; for example, it acts as an insect growth and reproduction inhibitor as well as a natural insect antifeedant. In addition, it can be applied in medicinal treatments such as fungicidal, anti-inflammatory, and antiulcer drugs [1]. It is obtained as a microcrystalline powder by successive extractions of neem seeds. Different cell populations have been observed to show different sensitivities to neem oil obtained by methanolic extraction; for instance, there is a tumor line that exhibits significantly higher sensitivity to this extracted oil [3]. Azadirachtin has 16 stereocenters, and its structure has been elucidated by NMR spectroscopy [4]. The complete synthesis of azadirachtin was performed by Ley and coworkers after 22 years using 71 steps, with a total yield of 0.00015 % [5, 6]. In order to isolate azadirachtin, HPLC was employed in association with UV detection [7–10]. The wavelength associated with this molecule ranges from 214 to 219 nm depending on the solvent used [1, 7–12]. Quantitative multidimensional conformational analysis of azadirachtin has been performed in order to get a better understanding of the biological activity of this molecule [13]. That work analyzed four torsion angles (chosen from among a total of almost 30 angles) in azadirachtin using the
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AM1 semiempirical method. The three most stable conformers were found by performing a conformational search in which one dihedral angle between the condensed ring groups and three other dihedral angles involving the hydroxyl groups were varied [13]. The main purpose of the study described in the present paper was to check the correlation of theoretical spectroscopic results for azadirachtin with the corresponding experimental data. To achieve this task, we determined the most stable structures among the calculated conformers of azadirachtin using a Monte Carlo technique based on the RM1 semiempirical method [14]. More rigorous density functional theory (DFT) calculations were also used to optimize its geometry, and the consequent theoretical nuclear magnetic resonance (NMR) chemical shifts and ultraviolet (UV) spectroscopic results were compared with experimental data.
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hydrogen bond interactions [21]. The accuracy of geometries predicted using wB97XD has been reported [22]. CAM-B3LYP gives good results for ultraviolet studies [23], so it was also used in the present work. It is important to note that azadirachtin has a large number of torsion angles. Therefore, it was crucial to perform conformational analysis in order to generate a new starting point for geometry optimization. A Monte Carlo method was used to search for possible conformations of this molecule. One hundred structures were retained within an energy interval of 40 kJ mol−1 and with Boltzmann contributions of >0.001. The Monte Carlo method was carried out using the RM1 semiempirical Hamiltonian, as performed with the Spartan program [24]. DFT calculations were carried out using the Gaussian 09 program [25]. The most stable RM1 conformers were studied using the DFT
Table 1 Twenty-seven most stable geometries optimized using Monte Carlo with the RM1 method
Methods DFT was applied to azadirachtin using different functionals, including B3LYP [15], wB97XD [16], M06 [17], M06-2X [17], and the long-range correction CAM-B3LYP (the Coulomb-attenuating method applied to B3LYP) [18]. Although B3LYP does not include dispersion, it has a well-documented database for a large range of properties [19]. We also used the highly parametrized empirical global hybrid meta-GGA M06 and M06-2X functionals with 27 % and 54 % HF exchange, respectively. These functionals describe noncovalent interactions better than other density functionals [20]. The wB97XD functional [16] including empirical dispersion correction was also used, since this method gives the best results overall for
Fig. 1 Structure of azadirachtin A as deduced from X-ray data [26]. Oxygen in red and carbon in gray. Hydrogen atoms are not shown. A and B groups are also shown
Conformer
B3LYP//RM1a Relative energy (kJ mol−1)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
27.20 28.54 10.54 0.47 26.41 9.99 20.04 21.12 20.32 5.30 1.23 0.00 14.45 17.23 11.45 18.63 11.25 22.63
0.00 0.38 0.65 1.57 2.61 2.81 2.96 3.61 4.17 4.52 4.68 5.20 6.23 6.90 7.51 7.60 7.68 8.83
0.181 0.156 0.140 0.096 0.063 0.058 0.055 0.042 0.034 0.029 0.028 0.022 0.015 0.011 0.009 0.008 0.008 0.005
19 20 21 22 23 24 25 26 27
13.90 10.69 9.66 39.35 6.96 27.53 10.48 22.37 31.39
9.60 9.68 9.79 10.65 11.18 11.24 11.39 11.60 11.75
0.004 0.004 0.003 0.002 0.002 0.002 0.002 0.002 0.002
a
RM1 Relative energy (kJ mol−1)
Calculated with the 6-31+G(d,p) basis set
Boltzmann distribution
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B3LYP hybrid exchange correlation functional [15] within the 6–31+G(d,p) basis set. They were obtained using Schlegel’s algorithm [25], where an ultrafine grid was used for two-electron integrals and their derivatives. All optimizations were performed using extremely tight optimization convergence criteria (opt=verytight). The molecular structure obtained from X-ray measurements [26] was also used as a starting point for geometry optimization at the DFT/6–31+G(d,p) level in an attempt to find the best conformer structure. For all optimized geometries, theoretical NMR spectra were obtained using tetramethylsilane (TMS) as a reference to compare with experimental data. For this step, the gaugeincluding atomic orbitals (GIAO) method was used along with the same functional as employed to optimize the geometry, and the more extended 6-311++G(d,p) basis set in order to increase the accuracy of the calculated data. A comparison with the experimental UV spectrum was carried out via the time-dependent (TD) DFT method, using all of the functionals tested in this work and the 6-311++G(d,p) basis set. TD-DFT was calculated with the polarizable
continuum model using the integral equation formalism variant (IEFPCM) method [27]. All geometries were kept frozen for NMR and UV calculations. This methodology was recently used and provided reasonable results [28, 29]. Highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) frontier orbitals were analyzed to gain insight into the molecular orbitals involved in the UV excitation.
Results and discussion Conformational analysis The first structure for azadirachtin based on NMR was published in 1975 [30]; later, in 1985, two new structures were proposed [31], and in 1987 the structure was reinvestigated [4]. In this structure (Fig. 1), the two most important groups, a modified decalin moiety (A) and a tricyclic part (B), rotate about the single bond C8–C14.
Table 2 Main geometrical parameters of DFT-optimized conformers and comparison to the corresponding experimental data O…O distance (Å)
21–15 21–13 15–13 13–19 13-6 19-6 7–6 11–12 Torsion angle (°) O6–C6–C7–O7 C7–C8–C14–C13 C7–C8–C14–C15 C13–C14–C15–C16 O13–C14–C15–O15 O13–C13–C17–C16 C13–C17–C20–C22 C4–C5–C6–O6 C10–C9–C11–O19 O7–C7–C8–C30 O20–C20–C22–C23 C18–C13–C14–C15 O7–C7–C8–C9 O11–C11–C12–O12 RMSD a
12 RM1a 2.206 5.247 3.580 5.495 5.158 5.697 2.891 3.437
B3LYP/ 6-31+G(d,p)
wB97XD/ 6-31+G(d,p)
2.341 5.279 3.634 5.832 5.152 5.859 2.812 2.572
2.322 5.211 3.616 5.736 5.176 5.823 2.780 2.560
M06/6-31+G(d,p)
2.325 5.201 3.610 5.758 5.145 5.825 2.776 2.565
M06-2X/ 6-31+G(d,p)
CAM-B3LYP/ 6-31+G(d,p)
2.324 5.173 3.614 5.708 5.167 5.840 2.775 2.577
2.327 5.223 3.611 5.773 5.127 5.831 2.789 2.561
Expt. [26]
2.279 5.391 3.627 5.900 5.088 5.769 2.969 2.654
56.95 −117.87 85.31 30.37 −147.85 38.55
50.50 −120.25 82.67 30.48 −147.97 37.13
49.47 −123.71 79.66 29.21 −149.87 35.40
49.40 −122.29 81.20 29.85 −148.83 35.87
49.13 −123.15 80.52 29.30 −149.57 35.51
49.92 −120.07 83.29 30.17 −148.25 36.77
59.20 −110.52 89.30 29.77 −147.80 39.39
−180.56 41.67 25.11 −173.37 −120.07 151.64 63.26 135.60 5.509
−188.32 43.69 13.16 −173.65 −123.51 156.23 62.74 −17.80 0.495
−191.01 43.87 14.30 −174.00 −125.90 156.72 62.08 2.90 0.481
168.93 42.87 13.06 −171.82 −125.93 156.50 64.16 −19.68 0.507
167.72 44.71 13.81 −174.21 −127.92 156.33 61.10 −22.18 0.530
170.32 43.96 13.53 −174.13 −125.04 156.17 61.99 −18.99 0.397
−177.48 39.74 14.96 −173.11 −108.37 157.95 62.60 −24.71 –
Monte Carlo conformer 12 optimized at the RM1 level
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A systematic search of the conformer distribution was performed using the Monte Carlo technique in order to identify the set of energetically accessible conformers. This search generated a total of 1,000 initial structures that were optimized using the RM1 semiempirical method, where the 100 conformers with the lowest energies were retained. Table 1 describes the relative energies of the 27 most stable
structures that make Boltzmann contributions of >0.001. Structure 1 is the most stable at the RM1 level and provides the greatest Boltzmann contribution, while structure 12 is the most stable at the B3LYP/6-311++G(d,p)//RM1 level. Therefore, RM1 structures 1 and 12 were reoptimized at the B3LYP/6-31+G(d,p) level, and structure 12 yielded the most stable geometry.
Table 3 Calculated and experimental 13C chemical shifts (ppm) Carbon
B3LYP// RM1 weighted
B3LYP //RM1a
B3LYP
wB97XD
M06
M06-2X
CAM-B3LYP
Expt. [4]
1 2 3 4 5 6 7 8 9 10 11
75.22 33.53 74.05 58.42 43.22 82.90 82.12 48.15 50.85 57.57 106.72
75.14 32.03 73.97 59.35 43.28 84.04 81.83 48.64 50.56 58.29 106.02
77.29 33.71 75.03 63.11 43.68 81.40 82.40 55.27 53.45 62.46 111.66
75.67 33.59 70.16 58.65 40.60 79.10 77.94 50.28 50.29 57.59 106.13
74.38 29.66 68.42 53.56 35.77 76.60 76.92 44.65 45.75 53.10 104.06
78.89 34.99 72.91 61.01 42.06 81.12 80.19 51.01 52.73 59.57 108.74
76.79 33.20 71.24 59.57 41.00 80.44 79.42 51.00 51.31 57.76 107.99
70.88 29.37 66.69 52.52 37.06 73.79 74.37 45.41 44.69 50.19 104.10
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
180.48 67.97 70.35 77.87 36.62 52.54 23.83 78.71 87.86 112.17 115.73 155.51 177.70 21.92 53.13 53.07 80.30 186.48
180.79 71.63 72.50 79.50 33.69 53.41 21.40 79.52 87.80 113.29 115.59 154.00 177.88 21.92 53.15 53.12 79.80 186.41
184.51 75.90 81.01 87.59 27.81 56.95 22.48 76.71 91.13 117.51 114.34 155.19 178.50 22.99 56.20 57.79 79.68 185.93
183.21 70.77 74.26 82.97 25.70 55.50 23.54 74.43 86.87 112.49 114.93 154.18 177.80 23.76 54.83 55.96 76.44 183.76
183.27 66.29 70.16 84.49 24.10 50.57 21.48 74.27 83.94 113.85 115.54 156.85 177.93 21.76 55.97 56.72 76.05 183.75
194.52 71.46 76.15 86.54 26.04 57.57 25.02 77.45 89.15 115.94 128.72 166.85 190.33 24.85 56.29 57.19 78.43 196.38
186.61 72.46 77.14 84.61 26.47 56.68 23.34 75.72 88.40 114.75 116.66 156.94 180.99 23.44 55.57 56.87 77.53 187.58
171.70 68.53 69.69 76.43 25.06 48.67 18.49 69.07 83.55 108.70 107.30 147.00 169.50 20.88 53.52 52.72 76.10 173.20
30 31 32 33 34 35 RMSD
25.81 176.83 136.47 16.25 144.88 17.29 6.50
24.15 176.99 137.54 15.51 143.37 18.47 6.46
22.24 176.29 138.59 15.43 150.03 18.61 8.21
20.97 176.02 135.14 14.82 153.18 17.47 6.12
20.25 175.72 134.76 12.07 153.81 16.47 5.62
21.45 188.75 151.74 14.65 171.36 17.99 12.62
21.23 178.43 138.90 14.29 151.67 17.39 7.46
21.33 166.22 128.60 14.29 137.50 11.94 –
The chemical shift of tetramethylsilane (TMS), calculated at the same level, was used as the reference. The 6-311++G(d,p) basis set was used. B3LYP// RM1 was obtained from a Boltzmann-weighted average a
Monte Carlo conformer 12
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Page 5 of 7, 2084 Table 5 TD/DFT results obtained using the 6-311++G(d,p) basis set and the IEFPCM method (in methanol solvent)
Fig. 2
13
C NMR chemical shifts: experimental versus calculated data
The most relevant geometrical parameters are presented in Table 2. The root mean square deviation (RMSD) was calculated taking into account only the carbon atoms. When compared with the experimental X-ray data, the RM1 geometry 12 gave the largest RMSD (Table 2). The DFT-optimized geometries presented nearly the same RMSD values (Table 2), whereas CAM-B3LYP gave the closest geometry to that observed experimentally. The dihedral angle C7–C8–C14–C15 was reported to be 73.2° when calculated at the AM1 level [13], while the experimentally derived value is 89.30° [26], meaning that the molecule adopts a rather folded configuration. The values of the C7–C8–C14–C15 dihedral angle (79.66–85.31°) we obtained at various levels of DFT (Table 2) are in good agreement with the experimental data. These conformers probably play an important role in determining the electronic and spectroscopic properties of azadirachtin. Therefore, we focused on the most stable conformers in order to determine the NMR chemical shifts, and then compared them with those observed experimentally. NMR and UV The theoretically derived NMR chemical shifts are compared with the corresponding experimental data [4] in Table 3. All chemical shifts were calculated using the 6-311++G(d,p) basis set. Figure 2 plots the calculated against the experimental NMR chemical shifts of the DFT-optimized geometries, while
λmax (nm)
Oscillator strength
B3LYP wB97XD M06 M06-2X
227.75 215.49 227.44 212.32
0.2526 0.3208 0.2714 0.3323
CAM-B3LYP Expt. [11]
214.29 217
0.3192 –
Table 4 provides the calculated parameters from linear regressions of the calculated against the experimental NMR chemical shifts. In general, the NMR chemical shifts calculated at various levels of DFT present almost the same amount of error, although they have different conformations (Table 2). The structure optimized at the wB97XD level has the lowest RMSD between the calculated and experimental chemical shifts (Table 3). Otherwise, linear regressions of the calculated versus experimental results show that the largest correlation coefficient (R) is obtained when CAM-B3LYP is used at the same basis set level (Table 4). sp3 carbon and terminal methyl groups show the largest deviations from experimental values since they are more flexible than the other carbon groups. The most important chemical moieties of azadirachtin, C13–C14 of the epoxide ring and C32–C34 of the tiglate ester, show only small deviations in chemical shift from those seen experimentally [4]. Also, NMR chemical shifts calculated at the DFT level using the geometry optimized at the semiempirical level show only small deviations from those calculated at the same DFT level using the geometries optimized with DFT, and were obtained at much lower computational cost. The theoretical spectroscopic results can be used to identify the chromophore groups present in the azadirachtin molecule. TD-DFT calculations were performed using the optimized geometry obtained at the same level, as well as the nonequilibrium PCM solvation model and the 6-311++G(d,p) basis set. Experimental UV-visible spectral data show that the maximum absorption of the azadaricathin molecule occurs at 215–219 nm, depending on the solvent used [1, 7–12, 32].
Table 4 Results of linear regression of experimental versus calculated chemical shift data AY=A+BX
B3LYP//RM1 weighted
B3LYP//RM1a
B3LYP
wB97XD
M06
M06-2X
CAM-B3LYP
A B R Standard deviation
2.8479 1.0351 0.9981 3.0874
2.6936 1.0379 0.9985 2.7870
4.1967 1.0438 0.9987 2.5894
1.5405 1.0472 0.9988 2.5319
−1.8859 1.0711 0.9985 2.8502
−1.0559 1.1409 0.9965 4.7008
1.3564 1.0663 0.9991 2.2322
a
Monte Carlo conformer 12
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Fig. 3 HOMO (left) and LUMO (right) molecular orbitals calculated at the CAM-B3LYP/6-311++G(d,p) level. Isovalue of 0.02 a.u.
Our theoretical results (Table 5) showed that wB97XD/6311++G(d,p) and CAM-B3LYP/6-311++G(d,p) yield the smallest errors in the maximum absorption of azadaricathin in methanol as compared to the experimental value (also obtained in methanol, at 217 nm) [11, 12]. Otherwise, CAM-B3LYP gives the closest optimized geometry to that observed experimentally. All of the functionals indicated the same contributions from the HOMO and LUMO orbitals (Fig. 3), with the HOMO mainly being spread over the tricyclic part (B, see Fig. 1) while the LUMO is mainly located on the tiglate moiety. However, the TD method shows that the UV transition with the largest oscillator strength occurs from HOMO-1 (molecular orbital 190) to LUMO (molecular
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orbital 192), whereas HOMO (molecular orbital 191) is not involved in this transition. We noted some differences among the results obtained using the various functionals regarding HOMO-1 and HOMO-2 (molecular orbital 189), as depicted in Fig. 4. B3LYP and wB97XD show the inverse picture of HOMO-1 and HOMO-2 in relation to the picture provided by the other functionals. All of the functionals except for B3LYP and wB97XD show that the contribution of HOMO-1 is spread over the tiglate moiety (Fig. 4) and HOMO-2 is localized over the tricyclic part (B, see Fig. 1), while B3LYP and wB97XD show the HOMO-1 over the tricyclic part and HOMO-2 over the tiglate moiety. wB97XD, however, shows a small contribution of the tiglate moiety to the HOMO-1 orbital. Therefore, with the exception of B3LYP and wB97XD, all of the other functionals show that HOMO-1 and HOMO are spread over the C8–C14 bonds and furan group. For the M06, M06-2X, and CAM-B3LYP functionals, the excitation with the greatest oscilator strength occurs from the tiglate ester to the same group, which is the strongest chromophore in the azadirachtin structure, in accordance with the experimental results at 217 nm [11].
Conclusions Theoretical spectroscopic calculations were performed in order to analyze the azadirachtin molecule in relation to the experimental data. We have determined the most stable structures among the calculated conformers of azadirachtin using a Monte Carlo technique with the RM1 method. DFT calculations were used to optimize the geometry and compare the theoretically derived NMR chemical shifts and UV spectroscopic results with the corresponding experimental data. The theoretical chemical shifts were found to be in good agreement with the experimental results. The geometry optimized at the semiempirical level and the NMR chemical shifts calculated using DFT showed minor deviations from the shifts obtained using DFT and the DFT-optimized geometry and incurred a much lower computational cost. The best theoretical results for the UV spectrum of azadirachtin show a maximum peak at 215.49 nm (using wB97XD/6-311++G(d,p)) or 214.29 nm (using CAMB3LYP/6-311++G(d,p)) in methanol, while the maximum in the experimental data occurs at 217 nm. Moreover, theoretical UV spectroscopic data allowed us to identify that the tiglate group is associated with this absorption, in accordance with the experimental data.
Fig. 4a–e HOMO-2 (left) and HOMO-1 (right) molecular orbitals associated with maximum absorption for the azadirachtin, as calculated at the a B3LYP, b wB97XD, c M06, d M06-2X, and e CAM-B3LYP levels using the 6-311++G(d,p) basis set. Isovalue of 0.02 a.u.
Acknowledgments The authors are indebted to the financial support of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Instituto Nacional de Ciência e Tecnologia dos Materiais em Nanotecnologia (INCTMN).
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