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Biotransformation of an africanane sesquiterpene by the fungus Mucor plumbeus
Phytochemistry xxx (2016) 1e7
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Biotransformation of an africanane sesquiterpene by the fungus Mucor plumbeus Braulio M. Fraga a, *, Carmen E. Díaz a, **, Leonardo J. Amador a, Matías Reina a, lez-Coloma c pez-Rodriguez b, Azucena Gonza Matías Lo nchez, 3, 38206, La Laguna, Tenerife, Canary Islands, Spain Instituto de Productos Naturales and Agrobiología, CSIC, Avda. Astrof. F. Sa nica “Antonio Gonza lez”, Universidad de La Laguna, Tenerife, Spain Instituto Universitario de Bioorga c Instituto de Ciencias Agrarias, CSIC, Serrano 113, 28016, Madrid, Spain a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 November 2016 Received in revised form 13 December 2016 Accepted 16 December 2016 Available online xxx
Biotransformation of 8b-hydroxy-african-4(5)-en-3-one angelate by the fungus Mucor plumbeus afforded as main products 6a,8b-dihydroxy-african-4(5)-en-3-one 8b-angelate and 1a,8b-dihydroxy-african-4(5)en-3-one 8b-angelate, which had been obtained, together with the substrate, from transformed root cultures of Bethencourtia hermosae. This fact shows that the enzyme system involved in these hydroxylations in both organisms, the fungus and the plant, acts with the same regio- and stereospecificity. In addition another twelve derivatives were isolated in the incubation of the substrate, which were identified as the (20 R,30 R)- and (20 S,30 S)-epoxy derivatives of the substrate and of the 6a- and 1a-hydroxy alcohols, the 8b-(20 R,30 R)- and 8b-(20 S,30 S)-epoxyangelate of 8b,15-dihydroxy-african-4(5)-en-3-one, the hydrolysis product of the substrate, and three isomers of 8b-hydroxy-african-4(5)-en-3-one 2x,3xdihydroxy-2-methylbutanoate. The insect antifeedant effects of the pure compounds were tested against chewing and sucking insect species along with their selective cytotoxicity against insect (Sf9) and mammalian (CHO) cell lines. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Mucor plumbeus Biotransformations Sesquiterpenes Africanane Insect antifeedants Cytotoxic effects
1. Introduction During the past years we have been interested in the study of the biotransformation of diterpenes with different skeleta by the fungus Mucor plumbeus (Hoffmann and Fraga, 1993; Fraga et al., 1998, 2001, 2003a, 2003b, 2004, 2010). The aim of these studies has been to develop models to explain the hydroxylation of these compounds by this microorganism, which possesses a broad specificity in the substrate (Arantes and Hanson, 2007; OliveiraSilva et al., 2013). On the other hand, we have recently investigated the biological activity of africanane sesquiterpenes isolated from transformed root cultures and aerial parts of Bethencourtia hermosae (Pit) Kunkel (Asteraceae) (Fraga et al., 2014). To complement these works, in order to study their structure-activity relationship as potential pesticides, we prepared new structural derivatives with this carbon skeleton by biotransformation of 8b-
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (C.E. Díaz).
(B.M.
Fraga),
hydroxy-african-4(5)-en-3-one angelate (1) (Bohlmann and Zdero, 1978; Fraga et al., 2014) with M. plumbeus. In this way, we obtained the following metabolites: 6a,8b-dihydroxy-african-4(5)-en-3-one 8b-angelate (2), 1a,8b-dihydroxy-african-4(5)-en-3-one 8b-angelate (3), 8b-hydroxy-african-4(5)-en-3-one (4), 6a,8b-dihydroxyafrican-4(5)-en-3-one 8b-(20 R,30 R)-epoxyangelate (5), 6a,8b-dihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)-epoxyangelate (6), 8bhydroxy-african-4(5)-en-3-one (20 S,30 S)-epoxyangelate (7), 8b-hydroxy-african-4(5)-en-3-one (20 R,30 R)-epoxyangelate (8), 1a,8bdihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)-epoxyangelate (9), 1a,8b-dihydroxy-african-4(5)-en-3-one 8b-(20 R,30 R)-epoxyangelate (10), 8b,15-dihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)epoxyangelate (11), 8b,15-dihydroxy-african-4(5)-en-3-one 8b(20 R,30 R)-epoxyangelate (12) and three diol isomers (13e15). The insect antifeedant effect of the pure compounds was tested against chewing (Spodoptera littoralis, Leptinotarsa decemlineata) and sucking (Myzus persicae, Rhopalosiphum padi) insect species along with their selective cytotoxicity against insect (Sf9) and mammalian (CHO) cell lines.
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http://dx.doi.org/10.1016/j.phytochem.2016.12.015 0031-9422/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Fraga, B.M., et al., Biotransformation of an africanane sesquiterpene by the fungus Mucor plumbeus, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.12.015
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B.M. Fraga et al. / Phytochemistry xxx (2016) 1e7
2. Results and discussion The incubation of 8b-hydroxy-african-4(5)-en-3-one angelate (1) with the fungus M. plumbeus led to the isolation of the metabolites 2e15 (Scheme 1). Two of them were identified as 6a,8bdihydroxy-african-4(5)-en-3-one 8b-angelate (2) and 1a,8b-dihydroxy-african-4(5)-en-3-one 8b-angelate (3), which together with the substrate 1 had been obtained from transformed root cultures
of B. hermosae (Fraga et al., 2014). Thus, with this biotransformation we have developed a procedure that allows us to obtain compounds 2 and 3 from the major product 1. Moreover, it should be noted that the isolation of these compounds show that the enzyme system involved in these hydroxylations, in the plant and the fungus, acts with the same regio- and stereospecificity. The alcohol 4 was also isolated in the biotransformation of the substrate 1. It had been obtained from the aerial parts of
Scheme 1. Metabolites 2e15 obtained by biotransformation of 1 by Mucor plumbeus.
Please cite this article in press as: Fraga, B.M., et al., Biotransformation of an africanane sesquiterpene by the fungus Mucor plumbeus, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.12.015
B.M. Fraga et al. / Phytochemistry xxx (2016) 1e7
B. hermosae (Fraga et al., 2014), and by basic hydrolysis of the ester 1 (Bohlmann and Zdero, 1978) . Other metabolites obtained from this incubation were the aand b-epoxide, 5 and 6, of the sesquiterpene 2. Compound 5 showed the molecular ion at m/z 348.1922 in accordance with the molecular formula C20H28O5. In comparison with that of the substrate 1, its NMR spectra showed a new signal of a geminal proton to a hydroxy group at dH 4.41 (s) and the corresponding carbon at dC 74.4 (Table 1). These chemical shifts and forms of resonance signals were identical with those observed for 2, which indicated that this alcohol group was situated at C-6(a). This assertion was confirmed by the correlations of H-6 with C-1, C-4, C-5, C-7, C-12 and C-13 observed in the HMBC experiment. In addition, in the 1H NMR spectrum the disappearance of the vinylic proton of the ester group, and the presence of a new geminal proton to an epoxy group at d 3.06 (q), were observed. The a-stereochemistry of this epoxide was solved by X-ray analysis (Fig. 1). These data permitted assigning to this metabolite the structure of 6a,8b-dihydroxy-african-4,5-en3-one 8b-(20 R0 ,30 R)-epoxyangelate (5). Moreover, this X-ray study also confirmed the structure and stereochemistry of the substrate 1, and of other derivatives, isolated from B. hermosae (Fraga et al., 2014). Compound 6 was an isomer of 5 showing both very similar NMR spectra. A slight difference was observed in the resonances of the H-40 and H-50 methyls at d 1.33 and 1.59 in 6, and 1.35 and 1.58 in 5, respectively. Thus, both compounds should be isomers in the epoxy group and consequently the structure of 6 was determined as 8b(20 S0 ,30 S)-epoxyangelate of 6a,8b-dihydroxy-african-4,5-en-3-one. The simple epoxidation of the substrate 1 by the fungus afforded the isomer metabolites 7 and 8, the latter in minor yield. Both have the molecular formula C20H28O4 and showed similar spectroscopic data, showing in the 1H NMR spectra the substitution of the vinylic hydrogen of the substrate by a geminal proton to the oxirane ring. The epoxy stereochemistry in 7 was determined on the basis of the following considerations: The difference in the molecular rotation between 2 and 1, due to the introduction of the 6a-OH in the molecule of 2, should be similar to that observed between 6 and 7 for the introduction of the same hydroxyl function. In effect, the [a]D difference between 6 (þ18.5 ) and 7 (þ34.4 ) was negative D [a]D ¼ 15.9 , a similar value to that observed between 2 (þ33.0 ) and 1 (þ46.9 ), D [a]D ¼ 13.9 (Fraga et al., 2014). This fact indicated that the stereochemistry of the epoxy group (20 S,30 S) is the
Table 1 13 C NMR data of compounds 1e9. C
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 20 30 40 50
47.4 38.7 208.9 136.9 170.9 44.4 39.2 79.8 27.1 18.6 22.3 22.1 27.9 18.6 7.8 167.3 128.0 137.6 15.7 20.6
42.3 39.4 209.5 137.4 168.9 74.6 42.6 74.7 27.9 18.6 23.1 20.1 23.8 18.7 7.7 167.1 128.1 137.5 15.7 20.6
78.0 50.5 204.5 137.4 169.4 38.8 39.4 78.4 28.1 23.8 16.8 20.9 28.1 21.9 7.8 167.3 128.2 137.5 15.8 20.7
47.5 38.6 209.0 136.7 171.6 44.6 39.7 79.7 29.6 18.3 21.8 20.8 27.9 18.7 7.8 e e e e e
42.2 39.4 209.0 137.6 169.3 74.4 42.3 76.5 27.6 18.8 22.9 19.8 23.5 18.5 7.8 168.5 59.7 59.8 13.8 19.3
42.2 39.4 209.3 137.6 168.8 74.5 42.4 76.9 27.7 18.6 22.9 19.3 23.8 18.7 7.8 168.5 60.2 59.5 13.7 19.8
47.4 38.7 208.7 137.3 170.3 44.3 39.1 81.6 27.0 18.6 22.0 21.9 27.8 18.6 7.9 169.1 59.8 60.1 13.7 19.1
47.4 38.8 208.9 137.2 170.5 44.4 39.0 81.7 27.0 18.8 22.2 21.9 27.8 18.6 7.9 169.5 59.8 59.9 13.8 19.3
78.2 50.5 204.6 137.7 168.9 38.7 39.2 80.0 27.8 23.8 16.6 20.7 28.0 21.8 7.8 168.9 60.1 59.5 13.7 19.2
3
Fig. 1. X-ray molecular structure of compound 5.
same in 6 and 7. Consequently, the isomer epoxide 8 has the opposite (20 R,30 R) configuration. Another pair of epoxy stereoisomers, obtained in this biotransformation, was the b- and a-epoxide, 9 and 10, of the 1a-hydroxy derivative 3. The structure of the major compound 9 was determined as 1a,8b-dihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)epoxyangelate on the following basis: Its mass spectrum showed the molecular ion at m/z 348.1952 (C20H28O5). In addition to the signals of the epoxyangelate group, the 1H NMR spectrum showed similar resonances to those observed for 3, four methyls, one located over a double bond resonating at dH 1.76, three cyclopropane hydrogens, two H-6 at dH 2.43 (s), the H-2 (a) and H-2 (b) as a pair of doublets centred at dH 2.32 and 3.02, respectively, and H-8 which is geminal to the ester group at dH 5.09 (d, J ¼ 8.8 Hz). The astereochemistry of the hydroxyl group at C-1 was assigned considering the downfield shift in the NMR spectrum of H-11 (a) and H-6 (a) at dH 1.27 and d 2.43, compared with the corresponding values in 3 at dH 0.78 and 2.20, respectively. The stereochemistry of the epoxy group in 9 was determined in a similar way to that described above for 7. Thus, the difference in the molecular rotations of 3 (þ17.1 ) and 1 (þ46.9 ) due to the introduction of the 1aOH group was negative, 29.8 (Fraga et al., 2014), and the difference observed between 9 (þ14.6 ) and 7 (þ34.4 ) was also negative, 19.8 . Therefore, the epoxides in 7 and 9 posses the same (20 S,30 S) configuration, and the isomer 10 the opposite (20 R,30 R). Another process observed in the incubation of 1 was the allylic oxidation of the C-15 to form the hydroxy derivative 11, which was contaminated with its isomer 12. Compound 11, C20H28O5, showed in its NMR spectra, in comparison with those of the substrate, the disappearance of the C-15 methyl, and the presence of a new hydroxymethylene group, resonating as a pair of doublets at dH 4.36 and 4.40 (J ¼ 12.0 Hz) and a signal at dC 55.2 (Table 2), and also showing a geminal proton to an epoxy group at dH 3.07. In the HMBC experiment correlations of H-15 with C-3, C-4 and C-5 were observed. The stereochemistry of the epoxy groups was tentatively assigned considering that the (20 S,30 S)-isomers were the major epoxides obtained in this incubation with M. plumbeus. These data permitted assigning to the major compound the structure of 8b,15dihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)-epoxyangelate 11, and then the corresponding (20 R,30 R)-structure to the minor isomer 12. Allylic oxidations in this type of sesquiterpenes have also been described in the biotransformation of african-4(15)-ene by the fungi Aspergillus niger and Rhizopus oryzae (Venkateswarlu et al., 1999). Three metabolites (13e15) with the molecular formula C20H30O5 were also isolated in the biotransformation of 1. These diols could be artefacts formed by electrophilic opening of the oxirane ring in the metabolites 7 or 8, with formation of a C-20
Please cite this article in press as: Fraga, B.M., et al., Biotransformation of an africanane sesquiterpene by the fungus Mucor plumbeus, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.12.015
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B.M. Fraga et al. / Phytochemistry xxx (2016) 1e7
Table 2 13 C NMR data of compounds 10e15. C
10
11
12
13
14
15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 20 30 40 50
78.0 50.4 204.2 n.o. 169.0 38.5 39.0 80.0 27.7 23.7 16.5 20.6 27.7 21.7 7.7 168.9 59.7 59.7 13.5 19.2
47.7 39.0 209.2 139.4 173.2 43.9 39.0 81.4 26.9 18.5 22.1 22. 1 27.8 18.5 55.2 169.1 59.9 59.7 13.7 19.1
47.8 39.0 209.2 139.4 173.2 43.9 39.0 81.4 26.8 18.4 22.3 22.0 27.6 18.4 55.2 169.4 60.1 59.6 13.7 19.2
47.4 38.6 208.7 137.3 170.4 44.3 39.1 82.9 26.5 18.5 21.9 22.1 27.7 18.6 7.9 175.8 77.5 71.4 16.7 22.0
47.8 39.1 209.0 137.3 170.5 44.6 39.5 83.4 27.0 19.0 22.6 22.0 28.1 18.9 8.3 176.1 77.5 72.2 17.2 22.4
47.4 38.7 208.6 137.4 170.0 44.2 39.2 82.8 26.6 18.7 21.9 22.1 27.7 18.5 7.9 175.0 76.8 72.1 17.7 22.5
n.o. not observed.
carbocation and subsequent neutralization, by both faces of the molecule, with a hydroxyl group. They showed in their NMR spectra signals of the sesquiterpene part of the substrate 1 and the substitution of the angelate double bond by a diol grouping. We have already indicated that epoxides with the(20 S,30 S) configuration are preferably formed in this incubation. Thus, to assign the structures to 13e15, we have tentatively considered that the oxirane opening of the major (20 S,30 S)-epoxide (7) should to form the major diols 13 and 15, whilst the minor (20 R,30 R)-epoxide (8) should to lead to 14 and the non isolated 16. Thus, we can tentatively solve their structures by determining the threo and erythro isomerism of each pair of these compounds 13e16. The 1H NMR resonance values of the 2,3-dihydroxy-2-methylbutanoate part of the esters 13e15 can be observed in Table 3. It has been indicated that the erythro and threo isomers of 2,3-dihydroxy2-methyl-butanoate can be distinguished considering the chemical shift of the methyl groups (Jenett-Siems et al., 1993; Liu et al., 2006). Thus, in the trimethylsilylethyl ester of 2,3-dihydroxy-2methyl-butanoic acid, a difference of 0.09 ppm has been found between the chemical shifts of the methyl groups for the threo isomer, and of 0.20 ppm for the erythro isomer. In our case, the observed values of Dd were 0.09, 0.12 and 0.32 for 13e15, respectively, indicating that 13 and 14 are threo isomers (20 b-OH, 30 b-OH and 20 a-OH, 30 a-OH, respectively), and 15 erythro (20 a-OH, 30 b-OH). Consequently, the fourth isomer 16, that was not isolated, must be also an erythro isomer (20 b-OH, 30 a-OH). Table 4 shows the insecticidal and cytotoxic effects of compounds 1e15. The activity of compounds 1 and 2 against these targets has been previously reported (Fraga et al., 2014). Overall, the biotransformed products 2e10 showed stronger antifeedant effects than the substrate 1 (see Fraga et al., 2014), compounds 11e15 showed larval postingestive toxicity (absent in 1), while compounds 4e6 were not active against the insects mentioned. Specifically, compounds 7 and 9 were strong insect antifeedants,
Table 3 1 H NMR data of the 2,3-dihydroxy-2-methylbutanoate ester in 13e15. H 0
3 40 50
13
14
15
4.00 (1H, m) 1.26 (3H, d, J ¼ 6.4 Hz) 1.35 (3H, s)
3.97 (1H, m) 1.25 (3H, d, J ¼ 6.4 Hz) 1.37 (3H, s)
3.84 (1H, m) 1.18 (3H, d, J ¼ 6.4 Hz) 1.50 (3H, s)
followed by 10, 2 (S. littoralis) and 3 (all insects tested). When orally injected into S. littoralis larvae, africananes 13e15 reduced feeding without further toxic effects, while compound 11 strongly reduced larval biomass without affecting ingestion, suggesting postingestive toxicity (pANCOVA2 < 0.05, Table 4). All the tested compounds showed selective cytotoxicity against insect Sf9 cells, 6 and 11 being the most effective (Table 4). These compounds were not cytotoxic to mammalian CHO cells (data not shown). The fact that compounds 11e15 exhibited postingestive effects suggests that the detoxification system of the insect cannot metabolize these already biotransformed products. The insect cytotoxicity did not follow a clear structure-activity pattern. The loss of the angeloxy group at C-8 resulted in the loss of the antifeedant effect (4). Similarly, previous reports showed that the antifeedant activity of the africananes against S. littoralis was associated with the presence of the angeloxy substituent at C-8 (Fraga et al., 2014). Hydroxylation of 1 at C-1 to form 3 resulted in a moderate increase in activity against all insects tested. Epoxidation of the angeloxy group increased the antifeedant effect strongly (7), or moderately with additional hydroxylation (C-1 hydroxy in 9 and 10). A hydroxy group at C-6 also increased the effect, depending on the stereochemistry of the epoxy group (6 versus 5). Hydroxylation of 7 at C-15 resulted in insecticidal effects (postingestive toxicant) (11), while opening of the epoxide gave moderate postingestive effects in the diols 13e15. 3. Conclusions Three main conclusions can be deduced from the results of this work: 1. The microbiological transformation of 8b-hidroxy-african-4(5)en-3-one angelate (1) by the fungus M. plumbeus afforded several main products, e.g. 6a,8b-dihydroxy-african-4(5)-en-3one 8b-angelate (2) and 1a,8b-dihydroxy-african-4(5)-en-3one 8b-angelate (3), which had been obtained, together with the substrate 1, from transformed roots of Bethencourtia hermosae (Fraga et al., 2014). This fact shows that the enzyme system involved in these hydroxylations in both organisms, the fungus and the plant, acts with the same regio- and stereospecificity. 2. Biotransformation of 1 is a highly satisfactory procedure to increase the yield of the sesquiterpenes 2 and 3 obtained from the transformed roots of this plant. 3. We have reported that the aerial parts of B. hermosae contains africanane sesquiterpenes as part of its chemical defence against insect herbivores. Furthermore, the in vitro production of B. hermosae transformed roots gave a high yield of the africanane derivative 1 (Fraga et al., 2014). Now, in this work, the biotransformation of this main compound 1 with M. plumbeus gave 20 ,30 -epoxy derivatives, which we have shown to be stronger insect antifeedants and insecticidal compounds.
4. Experimental 4.1. General experimental procedures Mps were determined with a Reichert Thermovar apparatus and are uncorrected. Molecular rotations were measured at room temperature on a Perkin Elmer 343 polarimeter. IR spectra were taken in a Perkin-Elmer 1600 FT spectrometer. 1H and 13C NMR spectra were recorded in CDCl3 solution at 500.1 and 125.8 MHz, respectively, with a Bruker AMX-500 spectrometer with pulsed field gradient using this solvent as internal standard. Chemical
Please cite this article in press as: Fraga, B.M., et al., Biotransformation of an africanane sesquiterpene by the fungus Mucor plumbeus, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.12.015
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Table 4 Antifeedant and postingestive effects on biomass gain (DB) and consumption (DI) of compounds on S. littoralis L6 larvae. Cytotoxic effects on insect S. frugiperda Sf9 and mammalian CHO cells. Compound
R. padi
M. persicae
Sf9
%FIa EC50(mg/cm2)b
DBc
DId
%FIa EC50 (mg/cm2)b
94.2 ± 8.4
92.7 ± 5.1
48.1 ± 5.2
56.4 ± 6.9
10.3 (6.2e17.1)
91.0 ± 11.7
95.5 ± 7.3
40.5 ± 5.8
44.6 ± 8.7
47.3 (35e63.8)
89.6 ± 9.8
95.2 ± 4.9 90.7 ± 4.8 100.4 ± 24.3 103.2 ± 23.7 101.5 ± 23.9
34.7 (21.8e55.2) nt 4.2 (0.8e20.1) 10.8 (5.0e23,2)
nt
nt
70.4 ± 3.3* 12.9 (9.9e16.9) 36.6 ± 7.8 nt nt 70.6 ± 5.3* 8.8 (6.9e11.1) nt
17.8 (8.7e36.0)
108.9 ± 6.6 95.1 ± 23.1 88.1 ± 20.2 131.7 ± 31.0
75.2 ± 3.9* 10.4 (7.6.-14.2) 54 ± 7.4 nt nt 91.6 ± 3.1* 4.4 (2.7e7.2) nt
10 11
70.8 ± 5.5 15.4 (7.7e30.4) 89.3 ± 5.8* 10.3 (5.5e19.1) 79.4 ± 8.8* 9.5 (3.0e29.4) 36.6 ± 10.8 36.6 ± 15.7 64.9 ± 11.6 93.8 ± 1.9* 3.4 (2.3e5.0) 90.1 ± 7.2 nc 82.7 ± 10.6 23.5 ± 9.7
nt 90.8 ± 23.4
nt 50.3 ± 6.7
nt 52.5 ± 9.3
nt 6.0 (3.2e11.3)
13 14 15
57.4 ± 5.6 33.9 ± 9.1 4.4 ± 4.4
nt 49.0 ± 12.6* (pANCOVA2 ¼ 0.028) 105.5 ± 28.2 112.7 ± 32.5 114.6 ± 29.5
60.4 ± 16.1* 62.2 ± 17.9* 63.2 ± 16.3*
40.5 ± 6.8 40.8 ± 7.2 27.2 ± 7.0
43.8 ± 7.6 37.2 ± 7.1 49.4 ± 7.9
36.1 (15.7e82.8) 25.8 (10.4e64.1) 24.1 (10.6e54.7)
1 2 3 4 5 6 7 9
*
S. littoralis
ED50 (mg/ml)e
12.0 (5.1e28.0)
p < 0.05 Wilcoxon paired rank test. nc. not calculated; nt, not tested. a Feeding inhibition percentage (50 mg/cm2). b EC50: concentration needed to produce 50% feeding inhibition. c Change in insect body weight (mg dry weight). d Mg food consumed (mg dry weight). e ED50: concentration (mg/ml) needed to give 50% cell viability and 95% confidence limits.
shifts are given in ppm (d). Mass spectra were taken in a Micromass Autospec instrument at 70 eV (probe). Dry column chromatographies were prepared on silica gel Merck 0.02e0.063 mm. Semipreparative HPLC was performed in a Beckman System Gold 125P with a Beckman Ultrasphere column (Si 1 25 cm, 5 mm) or an Inertsil Prep-sil (Gasukuro Kogyo) 25 2 i.d. column.
(3H, s, H-13), 1.12 (3H, s, H-12), 1.35 (3H, d, J ¼ 5.4 Hz, H-40 ), 1.58 (3H, s, H-50 ), 1.77 (3H, d, J ¼ 1.1 Hz, H-15), 2.36 (1H, dd, J ¼ 17.9 and 6.8 Hz, H-2a), 2.58 (1H, d, J ¼ 17.8 Hz, H-2b), 2.88 (1H, d, J ¼ 6.8 Hz, H-1a), 3.06 (1H, q, J ¼ 5.4 Hz, H-30 ), 4.41 (1H, s, H-6b), 5.20 (1H, d, J ¼ 9.4 Hz, H-8a); EIMS m/z (rel. int.): 348 [M]þ (3), 232 (11), 216 (14), 217 (3), 161 (4), 109 (100), 69 (13), 55 (15); HRMS: [M]þ at m/z 348.1922; C20H28O5 requires 348.1937.
4.2. Microorganism
M. plumbeus was grown in shake culture at 25 C for two days in 20 conical flasks (500 mL), each containing sterile medium (100 mL) (Fraga et al., 2004). The substrate 1 (220 mg) in EtOH (3.0 mL) was distributed equally between the flasks and the incubation allowed to continue for a further 6 days. The mycelium was filtered and the broth extracted with EtOAc in a soxhlet. The solvent was evaporated to give a residue, which was chromatographed on a silica gel column using a petroleum ether-EtOAc gradient. Fractions collected were purified further by HPLC when necessary. The biotransformation of 1 gave, in polarity order, starting material (1) (75 mg), 7 (17 mg), 8 (1.0 mg), 3 (1.6 mg), 2 (22 mg), 9 (1.7 mg), 10 (0.8 mg), 5 (1.2 mg), 6 (1.8 mg), 4 (1.0 mg), 13 (4.1 mg), 14 (2.9 mg), 15 (7.6 mg) and (11) (7.0 mg), contaminated with its (20 R,30 R)-isomer (12).
4.3.1.1. Crystal structure analysis of 5. Intensity data were collected at 293 K on an Enraf Nonius diffractometer, using Mo Ka (l ¼ 0.71070 Å) radiation and graphite monochromator. Cell refinement and data reduction and were performed with COLLECT (Nonius, 1998) and DENZO (Otwinowsky and Minor, 1997). The structures were resolved by direct methods using SIR97 (Altomare et al., 1999). Refinements were performed with SHELXL-97 (Sheldrick, 1997) using full-matrix least squares, with anisotropic displacement parameters for all the non-hydrogen atoms. The Hatoms were placed in calculated positions, except those bonded to oxygen atoms, which were located in successive difference-Fourier synthesis, and refined maintaining a common temperature factor of U ¼ 0.08. Molecular graphics were computed with the program PLATON (Spek, 2005). Crystal data of 5: C20H28O5, Mw ¼ 348.42; monoclinic space group, P21, Z ¼ 2, a ¼ 10.753(4), b ¼ 8.072 (3), c ¼ 11.670(6) Å, b ¼ 110.21(9)º, V ¼ 950.89(7) Å3, rcalc ¼ 1.22 gm. cm3, S ¼ 1.11, F(000) ¼ 376, m, (Mo Ka) ¼ 0.086 mm1, R ¼ 0.0496 and Rw ¼ 0.1318 for 2168 observed from 2319 independent reflexions (qmax ¼ 27.5 I > 2s (I) criterion and 311 parameters); maximum and minimum residues were 0.23 and 0.18, respectively.
4.3.1. 6a,8b-dihydroxy-african-4(5)-en-3-one 8b-(20 R,30 R)epoxyangelate (5) Colourless crystal, m.p. 174e175 C (petrol-EtOAc); [a]D: þ50.0 (c ¼ 0.16, CHCl3); 1H NMR (500 MHz): d 0.70 (1H, ddd, J ¼ 9.3, 8.3 and 5.5 Hz, H-9b), 0.78 (3H, s, H-14), 0.79e0.89 (2H, m, H-11), 0.99
4.3.2. 6a,8b-dihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)epoxyangelate (6) Colourless crystal, m.p. 174e176 C (petrol-EtOAc); [a]D: þ18.5 (c ¼ 0.13, CHCl3); 1H NMR (500 MHz): d 0.64 (1H, ddd, J ¼ 9.3, 8.0 and 5.5 Hz, H-9b), 0.77 (3H, s, H-14), 0.78e0.87 (2H, m, H-11), 0.99
The fungal strain, Mucor plumbeus CMI 116688, was a gift from Prof. J.R. Hanson, Department of Chemistry, University of Sussex, UK. 4.3. Incubation of 8b-hydroxy-african-4(5)-en-3-one angelate (1)
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(3H, s, H-13), 1.16 (3H, s, H-12), 1.33 (3H, d, J ¼ 5.3 Hz, H-40 ), 1.59 (3H, s, H-50 ), 1.77 (3H, d, J ¼ 1.1 Hz, H-15), 2.36 (1H, dd, J ¼ 17.8 and 6.8 Hz, H-2a), 2.58 (1H, d, J ¼ 17.8 Hz, H-2b), 2.88 (1H, d, J ¼ 6.5 Hz, H-1a), 3.05 (1H, q, J ¼ 5.4 Hz, H-30 ), 4.41 (1H, s, H-6b), 5.19 (1H, d, J ¼ 9.4 Hz, H-8a); EIMS m/z (rel. int.): 348 [M]þ (2), 232 (10), 217 (4), 161 (5), 109 (100), 69 (15), 55 (20); HRMS: [M]þ at m/z: 348.1944; C20H28O5 requires 348.1937. 0
0
4.3.3. 8b-Hydroxy-african-4(5)-en-3-one (2 S,3 S)-epoxyangelate (7) Colourless crystal, m.p. 133e134 C, (petrol-EtOAc); [a]D: þ34.4 (c ¼ 0.80, CHCl3); IR nmax (CHCl3): 2966, 1740, 1696, 1638, 1452, 1382, 1267, 1161 cm1; 1H NMR (500 MHz): d 0.74e0.76 (3H, m, H-9 and 2H-11), 0.79 (3H, s, H-14), 1.03 (3H, s, H-13), 1.06 (3H, s, H-12), 1.32 (3H, d, J ¼ 5.5 Hz, H-40 ), 1.59 (3H, s, H-50 ), 1.72 (3H, br s, H-15), 2.21 (1H, d, J ¼ 14.7 Hz, H-6a), 2.36 (1H, dd, J ¼ 18.0 and 6.8 Hz, H2a), 2.47 (1H, br d, J ¼ 6.8 Hz, H-1a), 2.50 (1H, d, J ¼ 18.0 Hz, H-2b), 2.69 (1H, d, J ¼ 14.7 Hz, H-6b), 3.06 (1H, q, J ¼ 5.4 Hz, H-30 ), 4.84 (1H, d, J ¼ 8.8 Hz, H-8a); EIMS m/z (rel. int.): 332 [M]þ (26), 233 (8), 216 (100), 201 (39), 190 (62), 175 (68), 161 (65); HRMS: [M]þ at m/z 332.1951; C20H28O4 requires 332.1987. 4.3.4. 8b-Hydroxy-african-4(5)-en-3-one (20 R,30 R)-epoxyangelate (8) 1 H NMR (400 MHz): d 0.74e0.78 (3H, m, H-9 and 2H-11), 0.79 (3H, s, H-14), 0.99 (3H, s, H-13), 1.06 (3H, s, H-12), 1.34 (3H, d, J ¼ 5.6 Hz, H-40 ), 1.58 (3H, s, H-50 ), 1.72 (3H, br s, H-15), 2.19 (1H, d, J ¼ 14.7 Hz, H-6a), 2.31 (1H, dd, J ¼ 18.0 and 6.8 Hz, H-2a), 2.47 (1H, br d, J ¼ 6.8 Hz, H-1a), 2.50 (1H, d, J ¼ 18.0 Hz, H-2b), 2.69 (1H, d, J ¼ 14.7 Hz, H-6b), 3.06 (1H, q, J ¼ 5.4 Hz, H-30 ), 4.84 (1H, d, J ¼ 8.8 Hz, H-8a); HRMS: [M]þ at m/z 332.1988; C20H28O4 requires 332.1987. 4.3.5. 1a,8b-dihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)epoxyangelate (9) Colourless crystal, m.p. 170e171 C (petrol-EtOAc); [a]D þ 14.6 (c ¼ 0.13, CHCl3); 1H NMR (500 MHz): d 0.64 (1H, dd, J ¼ 8.5 and 5.0 Hz, H-11b), 0.69 (1H, dt, J ¼ 8.8 and 5.2 Hz, H-9b), 0.88 (3H, s, H14), 0.89 (1H, m, H-11a), 1.00 (3H, s, H-13), 1.07 (3H, s, H-12), 1.27 (1H, br t, J ¼ 4.9 Hz, H-11a), 1.32 (3H, d, J ¼ 5.4 Hz, H-40 ), 1.59 (3H, s, H-50 ), 1.76 (3H, br s, H-15), 2.32 (1H, d, J ¼ 16.7 Hz, H-2a), 2.43 (2H, s, 2H-6), 3.02 (1H, d, J ¼ 16.7 Hz, H-2b), 3.05 (1H, q, J ¼ 5.4 Hz, H-30 ), 5.09 (1H, d, J ¼ 8.8 Hz, H-8a); EIMS m/z (rel. int.): 348 [M]þ (6), 232 (95), 215 (8), 190 (49), 175 (49), 161 (56), 109 (100); HRMS: [M]þ at m/z: 348.1952; C20H28O5 requires 348.1937. 4.3.6. 1a,8b-dihydroxy-african-4(5)-en-3-one 8b-(20 R,30 R)epoxyangelate (10) Colourless gum, [a]D þ 30.0 (c ¼ 0.08, CHCl3); 1H NMR (500 MHz): d 0.69 (1H, dd, J ¼ 9 and 4.5 Hz, H-11b), 0.75 (1H, dt, J ¼ 8.4 and 3.5 Hz, H-9b), 0.89 (3H, s, H-14), 1.00 (3H, s, H-13), 1.03 (3H, s, H-12), 1.27 (1H, br t, J ¼ 5.0 Hz, H-11a), 1.34 (3H, d, J ¼ 5.4 Hz, H-40 ), 1.55 (3H, s, H-50 ), 1.76 (3H, br s, H-15), 2.32 (1H, d, J ¼ 17.0 Hz, H-2a), 2.43 (2H, s, 2H-6), 3.02 (1H, d, J ¼ 17.0 Hz, H-2b), 3.05 (1H, q, J ¼ 5.4 Hz, H-30 ), 5.09 (1H, d, J ¼ 9 Hz, H-8a); EIMS m/z (rel. int.): 348 [M]þ (12), 232 (91), 215 (26), 190 (46), 189 (47), 175 (36), 161 (32), 138 (21), 109 (63); HRMS: [M]þ at m/z: 348.1921; C20H28O5 requires 348.1937. 4.3.7. 8b,15-dihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)epoxyangelate (11) 1 H NMR (500 MHz): d 0.76e0.81 (3H, m, H-9 and 2H-11), 0.84 (3H, s, H-14), 1.05 (3H, s, H-13), 1.09 (3H, s, H-12), 1.34 (3H, d, J ¼ 5.5 Hz, H-40 ), 1.60 (3H, s, H-50 ), 2.29 (1H, d, J ¼ 14.6 Hz, H-6a), 2.45 (1H, dd, J ¼ 18.0 and 6.7 Hz, H-2a), 2.51e2.57 (2H, m, H-1a and
H-2b), 2.77 (1H, d, J ¼ 14.7 Hz, H-6b), 3.07 (1H, q, J ¼ 5.4 Hz, H-30 ), 4.36 and 4.40 (each 1H, d, J ¼ 12.0 Hz, H-15), 4.85 (1H, d, J ¼ 8.9 Hz, H-8a); EIMS (11 þ 12) m/z (rel. int.): 348 [M]þ (7), 232 (57), 217 (20), 214 (100), 203 (23), 199 (66), 187 (20), 175 (33), 161 (30), 150 (64), 135 (19), 105 (50), 91 (65), 55 (62); HRMS: [M]þ at m/z: 348.1892; C20H28O5 requires 348.1936. 4.3.8. 8b,15-dihydroxy-african-4(5)-en-3-one 8b-(20 R,30 R)epoxyangelate (12) 1 H NMR (500 MHz): d 0.76e0.81 (3H, m, H-9 and 2H-11), 0.85 (3H, s, H-14), 1.00 (3H, s, H-13), 1.08 (3H, s, H-12), 1.34 (3H, d, J ¼ 5.5 Hz, H-40 ), 1.59 (3H, s, H-50 ), 2.25 (1H, d, J ¼ 14.6 Hz, H-6a), 2.42 (1H, dd, J ¼ 18.0 and 6.7 Hz, H-2a), 2.51e2.56 (2H, m, H-1a and H-2b), 2.77 (1H, d, J ¼ 14.7 Hz, H-6b), 3.08 (1H, q, J ¼ 5.4 Hz, H-30 ), 4.34 and 4.38 (each 1H, d, J ¼ 13.0 Hz, H-15), 4.85 (1H, d, J ¼ 8.9 Hz, H-8a). 4.3.9. Diol 13 (8b-hydroxy-african-4(5)-en-3-one 2b,3b -dihydroxy-2-methylbutanoate) Colourless crystal, m.p. 147e149 C (petrol-EtOAc); [a]D þ 30.0 (c ¼ 0.35, CHCl3); 1H NMR (500 MHz): d 0.74e0.78 (3H, m, H-9 and 2H-11), 0.81 (3H, s, H-14), 1.05 (3H, s, H-13), 1.07 (3H, s, H-12), 1.26 (3H, d, J ¼ 6.4 Hz, H-40 ), 1.35 (3H, s, H-50 ), 1.73 (3H, s, H-15), 1.95 (1H, d, J ¼ 9.3 Hz, eOH), 2.24 (1H, d, J ¼ 14.7 Hz, H-6a), 2.38 (1H, dd, J ¼ 17.3 and 6.1 Hz, H-2a), 2.46 (1H, d, J ¼ 6.8 Hz, H-1a), 2.49 (1H, d, J ¼ 17.6 Hz, H-2b), 2.69 (1H, d, J ¼ 14.7 Hz, H-6b), 3.40 (1H, s, eOH), 4.00 (1H, m, H-30 ), 4.82 (1H, d, J ¼ 9.0 Hz, H-8a); EIMS m/z (rel. int.): 350 [M]þ (4), 234 (20), 217 (100), 201 (24), 190 (25), 175 (54), 161 (95), 105 (33), 91 (44), 81 (49), 55 (43); HRMS: [M]þ at m/z 350.2076; C20H30O5 requires 350.2093. 4.3.10. Diol 14 (8b-hydroxy-african-4(5)-en-3-one 2a,3a -dihydroxy-2-methylbutanoate) Colourless crystal, m.p. 126e128 C (petrol-EtOAc); [a]D þ 23.3 (c ¼ 0.24, CHCl3); 1H NMR (500 MHz): d 0.75e0.79 (2H, m, H-9 and H-11), 0.80 (3H, s, H-14), 0.85 (1H, m, H-11), 1.02 (3H, s, H-13), 1.06 (3H, s, H-12), 1.25 (3H, d, J ¼ 6.4 Hz, H-40 ), 1.37 (3H, s, H-50 ), 1.73 (3H, s, H-15), 1.95 (1H, br s, eOH), 2.22 (1H, d, J ¼ 14.7 Hz, H-6a), 2.37 (1H, dd, J ¼ 17.5 and 6.5 Hz, H-2a), 2.46 (1H, d, J ¼ 6.8 Hz, H-1a), 2.50 (1H, d, J ¼ 17.9 Hz, H-2b), 2.70 (1H, d, J ¼ 14.7 Hz, H-6b), 3.38 (1H, s, eOH), 3.97 (1H, m, H-30 ), 4.82 (1H, d, J ¼ 9.2 Hz, H-8a); EIMS m/z (rel. int.): 350 [M]þ (4), 234 (16), 217 (100), 201 (22), 190 (27), 175 50), 161 (92), 105 (33), 91 (41), 81 (44), 55 (38); HRMS: [M]þ at m/z 350.2096; C20H30O5, requires 350.2093. 4.3.11. Diol 15 (8b-hydroxy-african-4(5)-en-3-one 2a,3b -dihydroxy-2-methylbutanoate) Colourless crystal, m.p. 132e133 C (petrol-EtOAc); [a]D þ 13.0 (c ¼ 0.30, CHCl3); 1H NMR (500 MHz): d 0.72e0.78 (3H, m, H-9 and 2H-11), 0.81 (3H, s, H-14), 1.02 (3H, s, H-13), 1.06 (3H, s, H-12), 1.18 (3H, d, J ¼ 6.4 Hz, H-40 ), 1.50 (3H, s, H-50 ), 1.73 (3H, s, H-15), 2.13 (1H, d, J ¼ 8.8 Hz, eOH), 2.21 (1H, d, J ¼ 14.7 Hz, H-6a), 2.38 (1H, dd, J ¼ 17.5 and 6.4 Hz, H-2a), 2.46 (1H, d, J ¼ 6.8 Hz, H-1a), 2.50 (1H, d, J ¼ 17.7 Hz, H-2b), 2.70 (1H, d, J ¼ 14.7 Hz, H-6b), 3.40 (1H, s, eOH), 3.84 (1H, m, H-30 ), 4.84 (1H, d, J ¼ 9.1 Hz, H-8a); EIMS m/z (rel. int.): 350 [M]þ (7), 322 (10), 234 (15), 217 (100), 201 (15), 190 (16), 175 (32), 161 (64), 147 (20), 105 (20), 91 (20), 81 (29), 55 (22); HRMS: [M]þ at m/z:350.2091; C20H30O5 requires 350.2093. 4.4. Insect bioassays S. littoralis, L. decemlineata, M. persicae and R. padi colonies were reared on artificial diet (Poitout and Bues, 1974), potato (Solanum tuberosum), bell pepper (Capsicum annuum) and barley (Hordeum vulgare) plants, respectively, and maintained at 22 ± 1 C, >70%
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relative humidity with a photoperiod of 16:8 h (L:D) in a growth chamber. The feeding bioassays were conducted with newly emerged S. littoralis L6 larvae, L. decemlineata adults, or M. persicae/ ~ o-Tapia et al., 2008). R. padi apterous adults as described (Burguen 4.4.1. Oral cannulation This experiment was performed with preweighed, newly emerged S. littoralis L6 larvae under the same environmental conditions as described. Each experiment consisted of 20e25 larvae orally dosed with 40 mg of the test compound in 4 mL DMSO (treatment) or solvent alone (control). A covariance analysis (ANCOVA 1) of food consumption (DI) and biomass gains (DB) with initial larval weight as covariate was performed to test for significant effects of the test compounds on these variables. An additional ANOVA analysis and covariate adjustment on DB with DI as covariate (ANCOVA2) were performed for those compounds significantly reducing DB in order to gain insight into their postingestive mode of action (antifeedant and/or toxic) (Raubenheimer and Simpson, 1992; Horton and Redak, 1993). 4.5. Cytotoxicity tests Sf9 cells derived from S. frugiperda pupal ovarian tissue and Mammalian Chinese hamster ovary cells (CHO) (European Collection of Cell Cultures, ECCC) were grown as previously described lez-Coloma et al., 2002). Cell viability was analyzed by an (Gonza adaptation of the MTT colorimetric assay method (Mosmann, 1983). The active compounds were tested in a dose-response experiment to calculate their relative potency (ED50 values, the effective dose to give 50% cell viability) which was determined from linear regression analysis (% cell viability on log dose). Acknowledgements This work has been supported by grants CTQ2012-38219-C0301 and CTQ2015-64049-C3-1-R, MINECO, Spain. L.J.A. is grateful to the Spanish Research Council (CSIC) and the European Social Foundation for an I3P fellowship. References Altomare, A., Burla, M.C., Camalli, M., Cascarano, G., Giacovazzo, C., Guagliardi, A.R., Moliterni, A.G.G., Polidori, G., Spagna, R., 1999. SIR97: a new tool for crystal structure determination and refinement. J. Appl. Crystallogr. 32, 115e119. Arantes, S.F., Hanson, J.R., 2007. The biotransformation of sesquiterpenoids by Mucor plumbeus. Curr. Org. Chem. 11, 657e663. Bohlmann, F., Zdero, C., 1978. Über einen neuen sesquiterpentyp aus Senecio
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oxyriifolius. Phytochemistry 17, 1669e1671. ~ o-Tapia, E., Castillo, L., Gonza lez-Coloma, A., Joseph-Nathan, P., 2008. Burguen Antifeedant and phytotoxic activity of the sesquiterpene p-benzoquinone perezone and some of its derivatives. J. Chem. Ecol. 34, 766e771. ndez, M.G., 1998. Microbiological Fraga, B.M., Gonz alez, P., Guillermo, R., Herna transformation of manoyl oxide derivatives by Mucor plumbeus. J. Nat. Prod. 61, 1237e1241. pez, M., Sua rez, S., 2001. BiotransforFraga, B.M., Hern andez, M.G., Gonz alez, P., Lo mation of the diterpene ribenone by Mucor plumbeus. Tetrahedron 57, 761e770. rez, S., 2003a. Biotransformation of the diterpenes epiFraga, B.M., Alvarez, L., Sua candicandiol and candicandiol by Mucor plumbeus. J. Nat. Prod. 66, 327e331. ndez, M.G., Arteaga, J.M., Su Fraga, B.M., Herna arez, S., 2003b. The microbiological transformation of the diterpenes dehydroabietanol and teideadiol by Mucor plumbeus. Phytochemistry 63, 663e668. ndez, M.G., Chamy, M.C., Garbarino, J.A., 2004. Fraga, B.M., Guillermo, R., Herna Biotransformation of two stemodane diterpenes by Mucor plumbeus. Tetrahedron 60, 7921e7932. Fraga, B.M., de Alfonso, I., Gonzalez-Vallejo, V., Guillermo, R., 2010. Microbial transformation of two 15a-hydroxy-ent-kaur-16-ene derivatives by Mucor plumbeus. Tetrahedron 66, 227e234. lez-Coloma, A., Fraga, B.M., Díaz, C.E., Amador, L.J., Reina, M., Santana, O., Gonza 2014. Bioactive compounds from transformed root cultures and aerial parts of Bethencourtia hermosae. Phytochemistry 108, 220e228. lez-Coloma, A., Guadan ~ o, A., de Ine s, C., Martínez-Díaz, R., Cortes, R., 2002. Gonza Selective action of acetogenin mitochondrial complex I inhibitors. Z. Naturforsch. 57C, 1028e1034. Hoffmann, J.J., Fraga, B.M., 1993. Microbial transformation of diterpenes: hidroxylation of 17-acetoxy-kolavenol acetate by Mucor plumbeus. Phytochemistry 33, 827e830. Horton, R.D., Redak, R.A., 1993. Further comments on analysis of covariance in insect dietary studies. Entomol. Exp. Appl. 69, 263e275. Jenett-Siems, K., Kaloga, M., Eich, E., 1993. Ipangulines, the first pyrrolizidine alkaloids from the Convolvulaceae. Phytochemistry 34, 437e440. Nonius, 1998. Kappa CCD server software. Nonius, R. V., Delft, The Netherlands. Liu, H., Gunnertoft-Jensen, K., My-Tran, L., Chen, M., Zhai, L., Olsen, C.E., Søhoel, H., Denmeade, S.R., Isaacs, J.T., Brøgger-Christensen, S., 2006. Cytotoxic phenylpropanoids and an additional thapsigargin analogue isolated from Thapsia garganica. Phytochemistry 67, 2651e2658. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: applications to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55e63. lez-Collado, I., 2013. Oliveira-Silva, E., Jacometti, N.A., Furtado, C., Aleu, J., Gonza Terpenoid biotransformations by Mucor species. Phytochem. Rev. 12, 857e876. Otwinowsky, Z., Minor, W., 1997. Processing of X-ray diffraction data collected in oscillation mode. In: macromolecular crystallography, Part, A., Carter, C.E., Sweet, R.M. (Eds.), Methods in Enzymology, vol. 276. Academic Press, New York, pp. 307e326. ces de le pidopte res Noctuidae et de 2 Poitout, S., Bues, R., 1974. Elevage de 28 espe ces d’Arctiidae sur milieu artificiel simplifie . Particularite s selon les espe ces. Ann. Zool. Ecol. Anim. 6, 431e441. espe Raubenheimer, D., Simpson, S.L., 1992. Analysis of covariance: an alternative to nutritional indices. Entomol. Exp. Appl. 62, 221e231. Sheldrick, G.M., 1997. SHELXL-97: program for the Refinement of Crystal Structures. €ttingen, Germany. University of Go Spek, A.L., 2005. PLATON, a Multipurpose Crystallographic Tool. University of Utrecht, The Netherlands. Venkateswarlu, Y., Ramesh, P., Srinivasa-Reddy, P., Jamil, K., 1999. Microbial transformation of D9(15)-africanene. Phytochemistry 52, 1275e1277.
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Biotransformation of an africanane sesquiterpene by the fungus Mucor plumbeus Braulio M. Fraga a, *, Carmen E. Díaz a, **, Leonardo J. Amador a, Matías Reina a, lez-Coloma c pez-Rodriguez b, Azucena Gonza Matías Lo nchez, 3, 38206, La Laguna, Tenerife, Canary Islands, Spain Instituto de Productos Naturales and Agrobiología, CSIC, Avda. Astrof. F. Sa nica “Antonio Gonza lez”, Universidad de La Laguna, Tenerife, Spain Instituto Universitario de Bioorga c Instituto de Ciencias Agrarias, CSIC, Serrano 113, 28016, Madrid, Spain a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 November 2016 Received in revised form 13 December 2016 Accepted 16 December 2016 Available online xxx
Biotransformation of 8b-hydroxy-african-4(5)-en-3-one angelate by the fungus Mucor plumbeus afforded as main products 6a,8b-dihydroxy-african-4(5)-en-3-one 8b-angelate and 1a,8b-dihydroxy-african-4(5)en-3-one 8b-angelate, which had been obtained, together with the substrate, from transformed root cultures of Bethencourtia hermosae. This fact shows that the enzyme system involved in these hydroxylations in both organisms, the fungus and the plant, acts with the same regio- and stereospecificity. In addition another twelve derivatives were isolated in the incubation of the substrate, which were identified as the (20 R,30 R)- and (20 S,30 S)-epoxy derivatives of the substrate and of the 6a- and 1a-hydroxy alcohols, the 8b-(20 R,30 R)- and 8b-(20 S,30 S)-epoxyangelate of 8b,15-dihydroxy-african-4(5)-en-3-one, the hydrolysis product of the substrate, and three isomers of 8b-hydroxy-african-4(5)-en-3-one 2x,3xdihydroxy-2-methylbutanoate. The insect antifeedant effects of the pure compounds were tested against chewing and sucking insect species along with their selective cytotoxicity against insect (Sf9) and mammalian (CHO) cell lines. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Mucor plumbeus Biotransformations Sesquiterpenes Africanane Insect antifeedants Cytotoxic effects
1. Introduction During the past years we have been interested in the study of the biotransformation of diterpenes with different skeleta by the fungus Mucor plumbeus (Hoffmann and Fraga, 1993; Fraga et al., 1998, 2001, 2003a, 2003b, 2004, 2010). The aim of these studies has been to develop models to explain the hydroxylation of these compounds by this microorganism, which possesses a broad specificity in the substrate (Arantes and Hanson, 2007; OliveiraSilva et al., 2013). On the other hand, we have recently investigated the biological activity of africanane sesquiterpenes isolated from transformed root cultures and aerial parts of Bethencourtia hermosae (Pit) Kunkel (Asteraceae) (Fraga et al., 2014). To complement these works, in order to study their structure-activity relationship as potential pesticides, we prepared new structural derivatives with this carbon skeleton by biotransformation of 8b-
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (C.E. Díaz).
(B.M.
Fraga),
hydroxy-african-4(5)-en-3-one angelate (1) (Bohlmann and Zdero, 1978; Fraga et al., 2014) with M. plumbeus. In this way, we obtained the following metabolites: 6a,8b-dihydroxy-african-4(5)-en-3-one 8b-angelate (2), 1a,8b-dihydroxy-african-4(5)-en-3-one 8b-angelate (3), 8b-hydroxy-african-4(5)-en-3-one (4), 6a,8b-dihydroxyafrican-4(5)-en-3-one 8b-(20 R,30 R)-epoxyangelate (5), 6a,8b-dihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)-epoxyangelate (6), 8bhydroxy-african-4(5)-en-3-one (20 S,30 S)-epoxyangelate (7), 8b-hydroxy-african-4(5)-en-3-one (20 R,30 R)-epoxyangelate (8), 1a,8bdihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)-epoxyangelate (9), 1a,8b-dihydroxy-african-4(5)-en-3-one 8b-(20 R,30 R)-epoxyangelate (10), 8b,15-dihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)epoxyangelate (11), 8b,15-dihydroxy-african-4(5)-en-3-one 8b(20 R,30 R)-epoxyangelate (12) and three diol isomers (13e15). The insect antifeedant effect of the pure compounds was tested against chewing (Spodoptera littoralis, Leptinotarsa decemlineata) and sucking (Myzus persicae, Rhopalosiphum padi) insect species along with their selective cytotoxicity against insect (Sf9) and mammalian (CHO) cell lines.
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Please cite this article in press as: Fraga, B.M., et al., Biotransformation of an africanane sesquiterpene by the fungus Mucor plumbeus, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.12.015
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2. Results and discussion The incubation of 8b-hydroxy-african-4(5)-en-3-one angelate (1) with the fungus M. plumbeus led to the isolation of the metabolites 2e15 (Scheme 1). Two of them were identified as 6a,8bdihydroxy-african-4(5)-en-3-one 8b-angelate (2) and 1a,8b-dihydroxy-african-4(5)-en-3-one 8b-angelate (3), which together with the substrate 1 had been obtained from transformed root cultures
of B. hermosae (Fraga et al., 2014). Thus, with this biotransformation we have developed a procedure that allows us to obtain compounds 2 and 3 from the major product 1. Moreover, it should be noted that the isolation of these compounds show that the enzyme system involved in these hydroxylations, in the plant and the fungus, acts with the same regio- and stereospecificity. The alcohol 4 was also isolated in the biotransformation of the substrate 1. It had been obtained from the aerial parts of
Scheme 1. Metabolites 2e15 obtained by biotransformation of 1 by Mucor plumbeus.
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B.M. Fraga et al. / Phytochemistry xxx (2016) 1e7
B. hermosae (Fraga et al., 2014), and by basic hydrolysis of the ester 1 (Bohlmann and Zdero, 1978) . Other metabolites obtained from this incubation were the aand b-epoxide, 5 and 6, of the sesquiterpene 2. Compound 5 showed the molecular ion at m/z 348.1922 in accordance with the molecular formula C20H28O5. In comparison with that of the substrate 1, its NMR spectra showed a new signal of a geminal proton to a hydroxy group at dH 4.41 (s) and the corresponding carbon at dC 74.4 (Table 1). These chemical shifts and forms of resonance signals were identical with those observed for 2, which indicated that this alcohol group was situated at C-6(a). This assertion was confirmed by the correlations of H-6 with C-1, C-4, C-5, C-7, C-12 and C-13 observed in the HMBC experiment. In addition, in the 1H NMR spectrum the disappearance of the vinylic proton of the ester group, and the presence of a new geminal proton to an epoxy group at d 3.06 (q), were observed. The a-stereochemistry of this epoxide was solved by X-ray analysis (Fig. 1). These data permitted assigning to this metabolite the structure of 6a,8b-dihydroxy-african-4,5-en3-one 8b-(20 R0 ,30 R)-epoxyangelate (5). Moreover, this X-ray study also confirmed the structure and stereochemistry of the substrate 1, and of other derivatives, isolated from B. hermosae (Fraga et al., 2014). Compound 6 was an isomer of 5 showing both very similar NMR spectra. A slight difference was observed in the resonances of the H-40 and H-50 methyls at d 1.33 and 1.59 in 6, and 1.35 and 1.58 in 5, respectively. Thus, both compounds should be isomers in the epoxy group and consequently the structure of 6 was determined as 8b(20 S0 ,30 S)-epoxyangelate of 6a,8b-dihydroxy-african-4,5-en-3-one. The simple epoxidation of the substrate 1 by the fungus afforded the isomer metabolites 7 and 8, the latter in minor yield. Both have the molecular formula C20H28O4 and showed similar spectroscopic data, showing in the 1H NMR spectra the substitution of the vinylic hydrogen of the substrate by a geminal proton to the oxirane ring. The epoxy stereochemistry in 7 was determined on the basis of the following considerations: The difference in the molecular rotation between 2 and 1, due to the introduction of the 6a-OH in the molecule of 2, should be similar to that observed between 6 and 7 for the introduction of the same hydroxyl function. In effect, the [a]D difference between 6 (þ18.5 ) and 7 (þ34.4 ) was negative D [a]D ¼ 15.9 , a similar value to that observed between 2 (þ33.0 ) and 1 (þ46.9 ), D [a]D ¼ 13.9 (Fraga et al., 2014). This fact indicated that the stereochemistry of the epoxy group (20 S,30 S) is the
Table 1 13 C NMR data of compounds 1e9. C
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 20 30 40 50
47.4 38.7 208.9 136.9 170.9 44.4 39.2 79.8 27.1 18.6 22.3 22.1 27.9 18.6 7.8 167.3 128.0 137.6 15.7 20.6
42.3 39.4 209.5 137.4 168.9 74.6 42.6 74.7 27.9 18.6 23.1 20.1 23.8 18.7 7.7 167.1 128.1 137.5 15.7 20.6
78.0 50.5 204.5 137.4 169.4 38.8 39.4 78.4 28.1 23.8 16.8 20.9 28.1 21.9 7.8 167.3 128.2 137.5 15.8 20.7
47.5 38.6 209.0 136.7 171.6 44.6 39.7 79.7 29.6 18.3 21.8 20.8 27.9 18.7 7.8 e e e e e
42.2 39.4 209.0 137.6 169.3 74.4 42.3 76.5 27.6 18.8 22.9 19.8 23.5 18.5 7.8 168.5 59.7 59.8 13.8 19.3
42.2 39.4 209.3 137.6 168.8 74.5 42.4 76.9 27.7 18.6 22.9 19.3 23.8 18.7 7.8 168.5 60.2 59.5 13.7 19.8
47.4 38.7 208.7 137.3 170.3 44.3 39.1 81.6 27.0 18.6 22.0 21.9 27.8 18.6 7.9 169.1 59.8 60.1 13.7 19.1
47.4 38.8 208.9 137.2 170.5 44.4 39.0 81.7 27.0 18.8 22.2 21.9 27.8 18.6 7.9 169.5 59.8 59.9 13.8 19.3
78.2 50.5 204.6 137.7 168.9 38.7 39.2 80.0 27.8 23.8 16.6 20.7 28.0 21.8 7.8 168.9 60.1 59.5 13.7 19.2
3
Fig. 1. X-ray molecular structure of compound 5.
same in 6 and 7. Consequently, the isomer epoxide 8 has the opposite (20 R,30 R) configuration. Another pair of epoxy stereoisomers, obtained in this biotransformation, was the b- and a-epoxide, 9 and 10, of the 1a-hydroxy derivative 3. The structure of the major compound 9 was determined as 1a,8b-dihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)epoxyangelate on the following basis: Its mass spectrum showed the molecular ion at m/z 348.1952 (C20H28O5). In addition to the signals of the epoxyangelate group, the 1H NMR spectrum showed similar resonances to those observed for 3, four methyls, one located over a double bond resonating at dH 1.76, three cyclopropane hydrogens, two H-6 at dH 2.43 (s), the H-2 (a) and H-2 (b) as a pair of doublets centred at dH 2.32 and 3.02, respectively, and H-8 which is geminal to the ester group at dH 5.09 (d, J ¼ 8.8 Hz). The astereochemistry of the hydroxyl group at C-1 was assigned considering the downfield shift in the NMR spectrum of H-11 (a) and H-6 (a) at dH 1.27 and d 2.43, compared with the corresponding values in 3 at dH 0.78 and 2.20, respectively. The stereochemistry of the epoxy group in 9 was determined in a similar way to that described above for 7. Thus, the difference in the molecular rotations of 3 (þ17.1 ) and 1 (þ46.9 ) due to the introduction of the 1aOH group was negative, 29.8 (Fraga et al., 2014), and the difference observed between 9 (þ14.6 ) and 7 (þ34.4 ) was also negative, 19.8 . Therefore, the epoxides in 7 and 9 posses the same (20 S,30 S) configuration, and the isomer 10 the opposite (20 R,30 R). Another process observed in the incubation of 1 was the allylic oxidation of the C-15 to form the hydroxy derivative 11, which was contaminated with its isomer 12. Compound 11, C20H28O5, showed in its NMR spectra, in comparison with those of the substrate, the disappearance of the C-15 methyl, and the presence of a new hydroxymethylene group, resonating as a pair of doublets at dH 4.36 and 4.40 (J ¼ 12.0 Hz) and a signal at dC 55.2 (Table 2), and also showing a geminal proton to an epoxy group at dH 3.07. In the HMBC experiment correlations of H-15 with C-3, C-4 and C-5 were observed. The stereochemistry of the epoxy groups was tentatively assigned considering that the (20 S,30 S)-isomers were the major epoxides obtained in this incubation with M. plumbeus. These data permitted assigning to the major compound the structure of 8b,15dihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)-epoxyangelate 11, and then the corresponding (20 R,30 R)-structure to the minor isomer 12. Allylic oxidations in this type of sesquiterpenes have also been described in the biotransformation of african-4(15)-ene by the fungi Aspergillus niger and Rhizopus oryzae (Venkateswarlu et al., 1999). Three metabolites (13e15) with the molecular formula C20H30O5 were also isolated in the biotransformation of 1. These diols could be artefacts formed by electrophilic opening of the oxirane ring in the metabolites 7 or 8, with formation of a C-20
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B.M. Fraga et al. / Phytochemistry xxx (2016) 1e7
Table 2 13 C NMR data of compounds 10e15. C
10
11
12
13
14
15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 20 30 40 50
78.0 50.4 204.2 n.o. 169.0 38.5 39.0 80.0 27.7 23.7 16.5 20.6 27.7 21.7 7.7 168.9 59.7 59.7 13.5 19.2
47.7 39.0 209.2 139.4 173.2 43.9 39.0 81.4 26.9 18.5 22.1 22. 1 27.8 18.5 55.2 169.1 59.9 59.7 13.7 19.1
47.8 39.0 209.2 139.4 173.2 43.9 39.0 81.4 26.8 18.4 22.3 22.0 27.6 18.4 55.2 169.4 60.1 59.6 13.7 19.2
47.4 38.6 208.7 137.3 170.4 44.3 39.1 82.9 26.5 18.5 21.9 22.1 27.7 18.6 7.9 175.8 77.5 71.4 16.7 22.0
47.8 39.1 209.0 137.3 170.5 44.6 39.5 83.4 27.0 19.0 22.6 22.0 28.1 18.9 8.3 176.1 77.5 72.2 17.2 22.4
47.4 38.7 208.6 137.4 170.0 44.2 39.2 82.8 26.6 18.7 21.9 22.1 27.7 18.5 7.9 175.0 76.8 72.1 17.7 22.5
n.o. not observed.
carbocation and subsequent neutralization, by both faces of the molecule, with a hydroxyl group. They showed in their NMR spectra signals of the sesquiterpene part of the substrate 1 and the substitution of the angelate double bond by a diol grouping. We have already indicated that epoxides with the(20 S,30 S) configuration are preferably formed in this incubation. Thus, to assign the structures to 13e15, we have tentatively considered that the oxirane opening of the major (20 S,30 S)-epoxide (7) should to form the major diols 13 and 15, whilst the minor (20 R,30 R)-epoxide (8) should to lead to 14 and the non isolated 16. Thus, we can tentatively solve their structures by determining the threo and erythro isomerism of each pair of these compounds 13e16. The 1H NMR resonance values of the 2,3-dihydroxy-2-methylbutanoate part of the esters 13e15 can be observed in Table 3. It has been indicated that the erythro and threo isomers of 2,3-dihydroxy2-methyl-butanoate can be distinguished considering the chemical shift of the methyl groups (Jenett-Siems et al., 1993; Liu et al., 2006). Thus, in the trimethylsilylethyl ester of 2,3-dihydroxy-2methyl-butanoic acid, a difference of 0.09 ppm has been found between the chemical shifts of the methyl groups for the threo isomer, and of 0.20 ppm for the erythro isomer. In our case, the observed values of Dd were 0.09, 0.12 and 0.32 for 13e15, respectively, indicating that 13 and 14 are threo isomers (20 b-OH, 30 b-OH and 20 a-OH, 30 a-OH, respectively), and 15 erythro (20 a-OH, 30 b-OH). Consequently, the fourth isomer 16, that was not isolated, must be also an erythro isomer (20 b-OH, 30 a-OH). Table 4 shows the insecticidal and cytotoxic effects of compounds 1e15. The activity of compounds 1 and 2 against these targets has been previously reported (Fraga et al., 2014). Overall, the biotransformed products 2e10 showed stronger antifeedant effects than the substrate 1 (see Fraga et al., 2014), compounds 11e15 showed larval postingestive toxicity (absent in 1), while compounds 4e6 were not active against the insects mentioned. Specifically, compounds 7 and 9 were strong insect antifeedants,
Table 3 1 H NMR data of the 2,3-dihydroxy-2-methylbutanoate ester in 13e15. H 0
3 40 50
13
14
15
4.00 (1H, m) 1.26 (3H, d, J ¼ 6.4 Hz) 1.35 (3H, s)
3.97 (1H, m) 1.25 (3H, d, J ¼ 6.4 Hz) 1.37 (3H, s)
3.84 (1H, m) 1.18 (3H, d, J ¼ 6.4 Hz) 1.50 (3H, s)
followed by 10, 2 (S. littoralis) and 3 (all insects tested). When orally injected into S. littoralis larvae, africananes 13e15 reduced feeding without further toxic effects, while compound 11 strongly reduced larval biomass without affecting ingestion, suggesting postingestive toxicity (pANCOVA2 < 0.05, Table 4). All the tested compounds showed selective cytotoxicity against insect Sf9 cells, 6 and 11 being the most effective (Table 4). These compounds were not cytotoxic to mammalian CHO cells (data not shown). The fact that compounds 11e15 exhibited postingestive effects suggests that the detoxification system of the insect cannot metabolize these already biotransformed products. The insect cytotoxicity did not follow a clear structure-activity pattern. The loss of the angeloxy group at C-8 resulted in the loss of the antifeedant effect (4). Similarly, previous reports showed that the antifeedant activity of the africananes against S. littoralis was associated with the presence of the angeloxy substituent at C-8 (Fraga et al., 2014). Hydroxylation of 1 at C-1 to form 3 resulted in a moderate increase in activity against all insects tested. Epoxidation of the angeloxy group increased the antifeedant effect strongly (7), or moderately with additional hydroxylation (C-1 hydroxy in 9 and 10). A hydroxy group at C-6 also increased the effect, depending on the stereochemistry of the epoxy group (6 versus 5). Hydroxylation of 7 at C-15 resulted in insecticidal effects (postingestive toxicant) (11), while opening of the epoxide gave moderate postingestive effects in the diols 13e15. 3. Conclusions Three main conclusions can be deduced from the results of this work: 1. The microbiological transformation of 8b-hidroxy-african-4(5)en-3-one angelate (1) by the fungus M. plumbeus afforded several main products, e.g. 6a,8b-dihydroxy-african-4(5)-en-3one 8b-angelate (2) and 1a,8b-dihydroxy-african-4(5)-en-3one 8b-angelate (3), which had been obtained, together with the substrate 1, from transformed roots of Bethencourtia hermosae (Fraga et al., 2014). This fact shows that the enzyme system involved in these hydroxylations in both organisms, the fungus and the plant, acts with the same regio- and stereospecificity. 2. Biotransformation of 1 is a highly satisfactory procedure to increase the yield of the sesquiterpenes 2 and 3 obtained from the transformed roots of this plant. 3. We have reported that the aerial parts of B. hermosae contains africanane sesquiterpenes as part of its chemical defence against insect herbivores. Furthermore, the in vitro production of B. hermosae transformed roots gave a high yield of the africanane derivative 1 (Fraga et al., 2014). Now, in this work, the biotransformation of this main compound 1 with M. plumbeus gave 20 ,30 -epoxy derivatives, which we have shown to be stronger insect antifeedants and insecticidal compounds.
4. Experimental 4.1. General experimental procedures Mps were determined with a Reichert Thermovar apparatus and are uncorrected. Molecular rotations were measured at room temperature on a Perkin Elmer 343 polarimeter. IR spectra were taken in a Perkin-Elmer 1600 FT spectrometer. 1H and 13C NMR spectra were recorded in CDCl3 solution at 500.1 and 125.8 MHz, respectively, with a Bruker AMX-500 spectrometer with pulsed field gradient using this solvent as internal standard. Chemical
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Table 4 Antifeedant and postingestive effects on biomass gain (DB) and consumption (DI) of compounds on S. littoralis L6 larvae. Cytotoxic effects on insect S. frugiperda Sf9 and mammalian CHO cells. Compound
R. padi
M. persicae
Sf9
%FIa EC50(mg/cm2)b
DBc
DId
%FIa EC50 (mg/cm2)b
94.2 ± 8.4
92.7 ± 5.1
48.1 ± 5.2
56.4 ± 6.9
10.3 (6.2e17.1)
91.0 ± 11.7
95.5 ± 7.3
40.5 ± 5.8
44.6 ± 8.7
47.3 (35e63.8)
89.6 ± 9.8
95.2 ± 4.9 90.7 ± 4.8 100.4 ± 24.3 103.2 ± 23.7 101.5 ± 23.9
34.7 (21.8e55.2) nt 4.2 (0.8e20.1) 10.8 (5.0e23,2)
nt
nt
70.4 ± 3.3* 12.9 (9.9e16.9) 36.6 ± 7.8 nt nt 70.6 ± 5.3* 8.8 (6.9e11.1) nt
17.8 (8.7e36.0)
108.9 ± 6.6 95.1 ± 23.1 88.1 ± 20.2 131.7 ± 31.0
75.2 ± 3.9* 10.4 (7.6.-14.2) 54 ± 7.4 nt nt 91.6 ± 3.1* 4.4 (2.7e7.2) nt
10 11
70.8 ± 5.5 15.4 (7.7e30.4) 89.3 ± 5.8* 10.3 (5.5e19.1) 79.4 ± 8.8* 9.5 (3.0e29.4) 36.6 ± 10.8 36.6 ± 15.7 64.9 ± 11.6 93.8 ± 1.9* 3.4 (2.3e5.0) 90.1 ± 7.2 nc 82.7 ± 10.6 23.5 ± 9.7
nt 90.8 ± 23.4
nt 50.3 ± 6.7
nt 52.5 ± 9.3
nt 6.0 (3.2e11.3)
13 14 15
57.4 ± 5.6 33.9 ± 9.1 4.4 ± 4.4
nt 49.0 ± 12.6* (pANCOVA2 ¼ 0.028) 105.5 ± 28.2 112.7 ± 32.5 114.6 ± 29.5
60.4 ± 16.1* 62.2 ± 17.9* 63.2 ± 16.3*
40.5 ± 6.8 40.8 ± 7.2 27.2 ± 7.0
43.8 ± 7.6 37.2 ± 7.1 49.4 ± 7.9
36.1 (15.7e82.8) 25.8 (10.4e64.1) 24.1 (10.6e54.7)
1 2 3 4 5 6 7 9
*
S. littoralis
ED50 (mg/ml)e
12.0 (5.1e28.0)
p < 0.05 Wilcoxon paired rank test. nc. not calculated; nt, not tested. a Feeding inhibition percentage (50 mg/cm2). b EC50: concentration needed to produce 50% feeding inhibition. c Change in insect body weight (mg dry weight). d Mg food consumed (mg dry weight). e ED50: concentration (mg/ml) needed to give 50% cell viability and 95% confidence limits.
shifts are given in ppm (d). Mass spectra were taken in a Micromass Autospec instrument at 70 eV (probe). Dry column chromatographies were prepared on silica gel Merck 0.02e0.063 mm. Semipreparative HPLC was performed in a Beckman System Gold 125P with a Beckman Ultrasphere column (Si 1 25 cm, 5 mm) or an Inertsil Prep-sil (Gasukuro Kogyo) 25 2 i.d. column.
(3H, s, H-13), 1.12 (3H, s, H-12), 1.35 (3H, d, J ¼ 5.4 Hz, H-40 ), 1.58 (3H, s, H-50 ), 1.77 (3H, d, J ¼ 1.1 Hz, H-15), 2.36 (1H, dd, J ¼ 17.9 and 6.8 Hz, H-2a), 2.58 (1H, d, J ¼ 17.8 Hz, H-2b), 2.88 (1H, d, J ¼ 6.8 Hz, H-1a), 3.06 (1H, q, J ¼ 5.4 Hz, H-30 ), 4.41 (1H, s, H-6b), 5.20 (1H, d, J ¼ 9.4 Hz, H-8a); EIMS m/z (rel. int.): 348 [M]þ (3), 232 (11), 216 (14), 217 (3), 161 (4), 109 (100), 69 (13), 55 (15); HRMS: [M]þ at m/z 348.1922; C20H28O5 requires 348.1937.
4.2. Microorganism
M. plumbeus was grown in shake culture at 25 C for two days in 20 conical flasks (500 mL), each containing sterile medium (100 mL) (Fraga et al., 2004). The substrate 1 (220 mg) in EtOH (3.0 mL) was distributed equally between the flasks and the incubation allowed to continue for a further 6 days. The mycelium was filtered and the broth extracted with EtOAc in a soxhlet. The solvent was evaporated to give a residue, which was chromatographed on a silica gel column using a petroleum ether-EtOAc gradient. Fractions collected were purified further by HPLC when necessary. The biotransformation of 1 gave, in polarity order, starting material (1) (75 mg), 7 (17 mg), 8 (1.0 mg), 3 (1.6 mg), 2 (22 mg), 9 (1.7 mg), 10 (0.8 mg), 5 (1.2 mg), 6 (1.8 mg), 4 (1.0 mg), 13 (4.1 mg), 14 (2.9 mg), 15 (7.6 mg) and (11) (7.0 mg), contaminated with its (20 R,30 R)-isomer (12).
4.3.1.1. Crystal structure analysis of 5. Intensity data were collected at 293 K on an Enraf Nonius diffractometer, using Mo Ka (l ¼ 0.71070 Å) radiation and graphite monochromator. Cell refinement and data reduction and were performed with COLLECT (Nonius, 1998) and DENZO (Otwinowsky and Minor, 1997). The structures were resolved by direct methods using SIR97 (Altomare et al., 1999). Refinements were performed with SHELXL-97 (Sheldrick, 1997) using full-matrix least squares, with anisotropic displacement parameters for all the non-hydrogen atoms. The Hatoms were placed in calculated positions, except those bonded to oxygen atoms, which were located in successive difference-Fourier synthesis, and refined maintaining a common temperature factor of U ¼ 0.08. Molecular graphics were computed with the program PLATON (Spek, 2005). Crystal data of 5: C20H28O5, Mw ¼ 348.42; monoclinic space group, P21, Z ¼ 2, a ¼ 10.753(4), b ¼ 8.072 (3), c ¼ 11.670(6) Å, b ¼ 110.21(9)º, V ¼ 950.89(7) Å3, rcalc ¼ 1.22 gm. cm3, S ¼ 1.11, F(000) ¼ 376, m, (Mo Ka) ¼ 0.086 mm1, R ¼ 0.0496 and Rw ¼ 0.1318 for 2168 observed from 2319 independent reflexions (qmax ¼ 27.5 I > 2s (I) criterion and 311 parameters); maximum and minimum residues were 0.23 and 0.18, respectively.
4.3.1. 6a,8b-dihydroxy-african-4(5)-en-3-one 8b-(20 R,30 R)epoxyangelate (5) Colourless crystal, m.p. 174e175 C (petrol-EtOAc); [a]D: þ50.0 (c ¼ 0.16, CHCl3); 1H NMR (500 MHz): d 0.70 (1H, ddd, J ¼ 9.3, 8.3 and 5.5 Hz, H-9b), 0.78 (3H, s, H-14), 0.79e0.89 (2H, m, H-11), 0.99
4.3.2. 6a,8b-dihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)epoxyangelate (6) Colourless crystal, m.p. 174e176 C (petrol-EtOAc); [a]D: þ18.5 (c ¼ 0.13, CHCl3); 1H NMR (500 MHz): d 0.64 (1H, ddd, J ¼ 9.3, 8.0 and 5.5 Hz, H-9b), 0.77 (3H, s, H-14), 0.78e0.87 (2H, m, H-11), 0.99
The fungal strain, Mucor plumbeus CMI 116688, was a gift from Prof. J.R. Hanson, Department of Chemistry, University of Sussex, UK. 4.3. Incubation of 8b-hydroxy-african-4(5)-en-3-one angelate (1)
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(3H, s, H-13), 1.16 (3H, s, H-12), 1.33 (3H, d, J ¼ 5.3 Hz, H-40 ), 1.59 (3H, s, H-50 ), 1.77 (3H, d, J ¼ 1.1 Hz, H-15), 2.36 (1H, dd, J ¼ 17.8 and 6.8 Hz, H-2a), 2.58 (1H, d, J ¼ 17.8 Hz, H-2b), 2.88 (1H, d, J ¼ 6.5 Hz, H-1a), 3.05 (1H, q, J ¼ 5.4 Hz, H-30 ), 4.41 (1H, s, H-6b), 5.19 (1H, d, J ¼ 9.4 Hz, H-8a); EIMS m/z (rel. int.): 348 [M]þ (2), 232 (10), 217 (4), 161 (5), 109 (100), 69 (15), 55 (20); HRMS: [M]þ at m/z: 348.1944; C20H28O5 requires 348.1937. 0
0
4.3.3. 8b-Hydroxy-african-4(5)-en-3-one (2 S,3 S)-epoxyangelate (7) Colourless crystal, m.p. 133e134 C, (petrol-EtOAc); [a]D: þ34.4 (c ¼ 0.80, CHCl3); IR nmax (CHCl3): 2966, 1740, 1696, 1638, 1452, 1382, 1267, 1161 cm1; 1H NMR (500 MHz): d 0.74e0.76 (3H, m, H-9 and 2H-11), 0.79 (3H, s, H-14), 1.03 (3H, s, H-13), 1.06 (3H, s, H-12), 1.32 (3H, d, J ¼ 5.5 Hz, H-40 ), 1.59 (3H, s, H-50 ), 1.72 (3H, br s, H-15), 2.21 (1H, d, J ¼ 14.7 Hz, H-6a), 2.36 (1H, dd, J ¼ 18.0 and 6.8 Hz, H2a), 2.47 (1H, br d, J ¼ 6.8 Hz, H-1a), 2.50 (1H, d, J ¼ 18.0 Hz, H-2b), 2.69 (1H, d, J ¼ 14.7 Hz, H-6b), 3.06 (1H, q, J ¼ 5.4 Hz, H-30 ), 4.84 (1H, d, J ¼ 8.8 Hz, H-8a); EIMS m/z (rel. int.): 332 [M]þ (26), 233 (8), 216 (100), 201 (39), 190 (62), 175 (68), 161 (65); HRMS: [M]þ at m/z 332.1951; C20H28O4 requires 332.1987. 4.3.4. 8b-Hydroxy-african-4(5)-en-3-one (20 R,30 R)-epoxyangelate (8) 1 H NMR (400 MHz): d 0.74e0.78 (3H, m, H-9 and 2H-11), 0.79 (3H, s, H-14), 0.99 (3H, s, H-13), 1.06 (3H, s, H-12), 1.34 (3H, d, J ¼ 5.6 Hz, H-40 ), 1.58 (3H, s, H-50 ), 1.72 (3H, br s, H-15), 2.19 (1H, d, J ¼ 14.7 Hz, H-6a), 2.31 (1H, dd, J ¼ 18.0 and 6.8 Hz, H-2a), 2.47 (1H, br d, J ¼ 6.8 Hz, H-1a), 2.50 (1H, d, J ¼ 18.0 Hz, H-2b), 2.69 (1H, d, J ¼ 14.7 Hz, H-6b), 3.06 (1H, q, J ¼ 5.4 Hz, H-30 ), 4.84 (1H, d, J ¼ 8.8 Hz, H-8a); HRMS: [M]þ at m/z 332.1988; C20H28O4 requires 332.1987. 4.3.5. 1a,8b-dihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)epoxyangelate (9) Colourless crystal, m.p. 170e171 C (petrol-EtOAc); [a]D þ 14.6 (c ¼ 0.13, CHCl3); 1H NMR (500 MHz): d 0.64 (1H, dd, J ¼ 8.5 and 5.0 Hz, H-11b), 0.69 (1H, dt, J ¼ 8.8 and 5.2 Hz, H-9b), 0.88 (3H, s, H14), 0.89 (1H, m, H-11a), 1.00 (3H, s, H-13), 1.07 (3H, s, H-12), 1.27 (1H, br t, J ¼ 4.9 Hz, H-11a), 1.32 (3H, d, J ¼ 5.4 Hz, H-40 ), 1.59 (3H, s, H-50 ), 1.76 (3H, br s, H-15), 2.32 (1H, d, J ¼ 16.7 Hz, H-2a), 2.43 (2H, s, 2H-6), 3.02 (1H, d, J ¼ 16.7 Hz, H-2b), 3.05 (1H, q, J ¼ 5.4 Hz, H-30 ), 5.09 (1H, d, J ¼ 8.8 Hz, H-8a); EIMS m/z (rel. int.): 348 [M]þ (6), 232 (95), 215 (8), 190 (49), 175 (49), 161 (56), 109 (100); HRMS: [M]þ at m/z: 348.1952; C20H28O5 requires 348.1937. 4.3.6. 1a,8b-dihydroxy-african-4(5)-en-3-one 8b-(20 R,30 R)epoxyangelate (10) Colourless gum, [a]D þ 30.0 (c ¼ 0.08, CHCl3); 1H NMR (500 MHz): d 0.69 (1H, dd, J ¼ 9 and 4.5 Hz, H-11b), 0.75 (1H, dt, J ¼ 8.4 and 3.5 Hz, H-9b), 0.89 (3H, s, H-14), 1.00 (3H, s, H-13), 1.03 (3H, s, H-12), 1.27 (1H, br t, J ¼ 5.0 Hz, H-11a), 1.34 (3H, d, J ¼ 5.4 Hz, H-40 ), 1.55 (3H, s, H-50 ), 1.76 (3H, br s, H-15), 2.32 (1H, d, J ¼ 17.0 Hz, H-2a), 2.43 (2H, s, 2H-6), 3.02 (1H, d, J ¼ 17.0 Hz, H-2b), 3.05 (1H, q, J ¼ 5.4 Hz, H-30 ), 5.09 (1H, d, J ¼ 9 Hz, H-8a); EIMS m/z (rel. int.): 348 [M]þ (12), 232 (91), 215 (26), 190 (46), 189 (47), 175 (36), 161 (32), 138 (21), 109 (63); HRMS: [M]þ at m/z: 348.1921; C20H28O5 requires 348.1937. 4.3.7. 8b,15-dihydroxy-african-4(5)-en-3-one 8b-(20 S,30 S)epoxyangelate (11) 1 H NMR (500 MHz): d 0.76e0.81 (3H, m, H-9 and 2H-11), 0.84 (3H, s, H-14), 1.05 (3H, s, H-13), 1.09 (3H, s, H-12), 1.34 (3H, d, J ¼ 5.5 Hz, H-40 ), 1.60 (3H, s, H-50 ), 2.29 (1H, d, J ¼ 14.6 Hz, H-6a), 2.45 (1H, dd, J ¼ 18.0 and 6.7 Hz, H-2a), 2.51e2.57 (2H, m, H-1a and
H-2b), 2.77 (1H, d, J ¼ 14.7 Hz, H-6b), 3.07 (1H, q, J ¼ 5.4 Hz, H-30 ), 4.36 and 4.40 (each 1H, d, J ¼ 12.0 Hz, H-15), 4.85 (1H, d, J ¼ 8.9 Hz, H-8a); EIMS (11 þ 12) m/z (rel. int.): 348 [M]þ (7), 232 (57), 217 (20), 214 (100), 203 (23), 199 (66), 187 (20), 175 (33), 161 (30), 150 (64), 135 (19), 105 (50), 91 (65), 55 (62); HRMS: [M]þ at m/z: 348.1892; C20H28O5 requires 348.1936. 4.3.8. 8b,15-dihydroxy-african-4(5)-en-3-one 8b-(20 R,30 R)epoxyangelate (12) 1 H NMR (500 MHz): d 0.76e0.81 (3H, m, H-9 and 2H-11), 0.85 (3H, s, H-14), 1.00 (3H, s, H-13), 1.08 (3H, s, H-12), 1.34 (3H, d, J ¼ 5.5 Hz, H-40 ), 1.59 (3H, s, H-50 ), 2.25 (1H, d, J ¼ 14.6 Hz, H-6a), 2.42 (1H, dd, J ¼ 18.0 and 6.7 Hz, H-2a), 2.51e2.56 (2H, m, H-1a and H-2b), 2.77 (1H, d, J ¼ 14.7 Hz, H-6b), 3.08 (1H, q, J ¼ 5.4 Hz, H-30 ), 4.34 and 4.38 (each 1H, d, J ¼ 13.0 Hz, H-15), 4.85 (1H, d, J ¼ 8.9 Hz, H-8a). 4.3.9. Diol 13 (8b-hydroxy-african-4(5)-en-3-one 2b,3b -dihydroxy-2-methylbutanoate) Colourless crystal, m.p. 147e149 C (petrol-EtOAc); [a]D þ 30.0 (c ¼ 0.35, CHCl3); 1H NMR (500 MHz): d 0.74e0.78 (3H, m, H-9 and 2H-11), 0.81 (3H, s, H-14), 1.05 (3H, s, H-13), 1.07 (3H, s, H-12), 1.26 (3H, d, J ¼ 6.4 Hz, H-40 ), 1.35 (3H, s, H-50 ), 1.73 (3H, s, H-15), 1.95 (1H, d, J ¼ 9.3 Hz, eOH), 2.24 (1H, d, J ¼ 14.7 Hz, H-6a), 2.38 (1H, dd, J ¼ 17.3 and 6.1 Hz, H-2a), 2.46 (1H, d, J ¼ 6.8 Hz, H-1a), 2.49 (1H, d, J ¼ 17.6 Hz, H-2b), 2.69 (1H, d, J ¼ 14.7 Hz, H-6b), 3.40 (1H, s, eOH), 4.00 (1H, m, H-30 ), 4.82 (1H, d, J ¼ 9.0 Hz, H-8a); EIMS m/z (rel. int.): 350 [M]þ (4), 234 (20), 217 (100), 201 (24), 190 (25), 175 (54), 161 (95), 105 (33), 91 (44), 81 (49), 55 (43); HRMS: [M]þ at m/z 350.2076; C20H30O5 requires 350.2093. 4.3.10. Diol 14 (8b-hydroxy-african-4(5)-en-3-one 2a,3a -dihydroxy-2-methylbutanoate) Colourless crystal, m.p. 126e128 C (petrol-EtOAc); [a]D þ 23.3 (c ¼ 0.24, CHCl3); 1H NMR (500 MHz): d 0.75e0.79 (2H, m, H-9 and H-11), 0.80 (3H, s, H-14), 0.85 (1H, m, H-11), 1.02 (3H, s, H-13), 1.06 (3H, s, H-12), 1.25 (3H, d, J ¼ 6.4 Hz, H-40 ), 1.37 (3H, s, H-50 ), 1.73 (3H, s, H-15), 1.95 (1H, br s, eOH), 2.22 (1H, d, J ¼ 14.7 Hz, H-6a), 2.37 (1H, dd, J ¼ 17.5 and 6.5 Hz, H-2a), 2.46 (1H, d, J ¼ 6.8 Hz, H-1a), 2.50 (1H, d, J ¼ 17.9 Hz, H-2b), 2.70 (1H, d, J ¼ 14.7 Hz, H-6b), 3.38 (1H, s, eOH), 3.97 (1H, m, H-30 ), 4.82 (1H, d, J ¼ 9.2 Hz, H-8a); EIMS m/z (rel. int.): 350 [M]þ (4), 234 (16), 217 (100), 201 (22), 190 (27), 175 50), 161 (92), 105 (33), 91 (41), 81 (44), 55 (38); HRMS: [M]þ at m/z 350.2096; C20H30O5, requires 350.2093. 4.3.11. Diol 15 (8b-hydroxy-african-4(5)-en-3-one 2a,3b -dihydroxy-2-methylbutanoate) Colourless crystal, m.p. 132e133 C (petrol-EtOAc); [a]D þ 13.0 (c ¼ 0.30, CHCl3); 1H NMR (500 MHz): d 0.72e0.78 (3H, m, H-9 and 2H-11), 0.81 (3H, s, H-14), 1.02 (3H, s, H-13), 1.06 (3H, s, H-12), 1.18 (3H, d, J ¼ 6.4 Hz, H-40 ), 1.50 (3H, s, H-50 ), 1.73 (3H, s, H-15), 2.13 (1H, d, J ¼ 8.8 Hz, eOH), 2.21 (1H, d, J ¼ 14.7 Hz, H-6a), 2.38 (1H, dd, J ¼ 17.5 and 6.4 Hz, H-2a), 2.46 (1H, d, J ¼ 6.8 Hz, H-1a), 2.50 (1H, d, J ¼ 17.7 Hz, H-2b), 2.70 (1H, d, J ¼ 14.7 Hz, H-6b), 3.40 (1H, s, eOH), 3.84 (1H, m, H-30 ), 4.84 (1H, d, J ¼ 9.1 Hz, H-8a); EIMS m/z (rel. int.): 350 [M]þ (7), 322 (10), 234 (15), 217 (100), 201 (15), 190 (16), 175 (32), 161 (64), 147 (20), 105 (20), 91 (20), 81 (29), 55 (22); HRMS: [M]þ at m/z:350.2091; C20H30O5 requires 350.2093. 4.4. Insect bioassays S. littoralis, L. decemlineata, M. persicae and R. padi colonies were reared on artificial diet (Poitout and Bues, 1974), potato (Solanum tuberosum), bell pepper (Capsicum annuum) and barley (Hordeum vulgare) plants, respectively, and maintained at 22 ± 1 C, >70%
Please cite this article in press as: Fraga, B.M., et al., Biotransformation of an africanane sesquiterpene by the fungus Mucor plumbeus, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.12.015
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relative humidity with a photoperiod of 16:8 h (L:D) in a growth chamber. The feeding bioassays were conducted with newly emerged S. littoralis L6 larvae, L. decemlineata adults, or M. persicae/ ~ o-Tapia et al., 2008). R. padi apterous adults as described (Burguen 4.4.1. Oral cannulation This experiment was performed with preweighed, newly emerged S. littoralis L6 larvae under the same environmental conditions as described. Each experiment consisted of 20e25 larvae orally dosed with 40 mg of the test compound in 4 mL DMSO (treatment) or solvent alone (control). A covariance analysis (ANCOVA 1) of food consumption (DI) and biomass gains (DB) with initial larval weight as covariate was performed to test for significant effects of the test compounds on these variables. An additional ANOVA analysis and covariate adjustment on DB with DI as covariate (ANCOVA2) were performed for those compounds significantly reducing DB in order to gain insight into their postingestive mode of action (antifeedant and/or toxic) (Raubenheimer and Simpson, 1992; Horton and Redak, 1993). 4.5. Cytotoxicity tests Sf9 cells derived from S. frugiperda pupal ovarian tissue and Mammalian Chinese hamster ovary cells (CHO) (European Collection of Cell Cultures, ECCC) were grown as previously described lez-Coloma et al., 2002). Cell viability was analyzed by an (Gonza adaptation of the MTT colorimetric assay method (Mosmann, 1983). The active compounds were tested in a dose-response experiment to calculate their relative potency (ED50 values, the effective dose to give 50% cell viability) which was determined from linear regression analysis (% cell viability on log dose). Acknowledgements This work has been supported by grants CTQ2012-38219-C0301 and CTQ2015-64049-C3-1-R, MINECO, Spain. L.J.A. is grateful to the Spanish Research Council (CSIC) and the European Social Foundation for an I3P fellowship. References Altomare, A., Burla, M.C., Camalli, M., Cascarano, G., Giacovazzo, C., Guagliardi, A.R., Moliterni, A.G.G., Polidori, G., Spagna, R., 1999. SIR97: a new tool for crystal structure determination and refinement. J. Appl. Crystallogr. 32, 115e119. Arantes, S.F., Hanson, J.R., 2007. The biotransformation of sesquiterpenoids by Mucor plumbeus. Curr. Org. Chem. 11, 657e663. Bohlmann, F., Zdero, C., 1978. Über einen neuen sesquiterpentyp aus Senecio
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oxyriifolius. Phytochemistry 17, 1669e1671. ~ o-Tapia, E., Castillo, L., Gonza lez-Coloma, A., Joseph-Nathan, P., 2008. Burguen Antifeedant and phytotoxic activity of the sesquiterpene p-benzoquinone perezone and some of its derivatives. J. Chem. Ecol. 34, 766e771. ndez, M.G., 1998. Microbiological Fraga, B.M., Gonz alez, P., Guillermo, R., Herna transformation of manoyl oxide derivatives by Mucor plumbeus. J. Nat. Prod. 61, 1237e1241. pez, M., Sua rez, S., 2001. BiotransforFraga, B.M., Hern andez, M.G., Gonz alez, P., Lo mation of the diterpene ribenone by Mucor plumbeus. Tetrahedron 57, 761e770. rez, S., 2003a. Biotransformation of the diterpenes epiFraga, B.M., Alvarez, L., Sua candicandiol and candicandiol by Mucor plumbeus. J. Nat. Prod. 66, 327e331. ndez, M.G., Arteaga, J.M., Su Fraga, B.M., Herna arez, S., 2003b. The microbiological transformation of the diterpenes dehydroabietanol and teideadiol by Mucor plumbeus. Phytochemistry 63, 663e668. ndez, M.G., Chamy, M.C., Garbarino, J.A., 2004. Fraga, B.M., Guillermo, R., Herna Biotransformation of two stemodane diterpenes by Mucor plumbeus. Tetrahedron 60, 7921e7932. Fraga, B.M., de Alfonso, I., Gonzalez-Vallejo, V., Guillermo, R., 2010. Microbial transformation of two 15a-hydroxy-ent-kaur-16-ene derivatives by Mucor plumbeus. Tetrahedron 66, 227e234. lez-Coloma, A., Fraga, B.M., Díaz, C.E., Amador, L.J., Reina, M., Santana, O., Gonza 2014. Bioactive compounds from transformed root cultures and aerial parts of Bethencourtia hermosae. Phytochemistry 108, 220e228. lez-Coloma, A., Guadan ~ o, A., de Ine s, C., Martínez-Díaz, R., Cortes, R., 2002. Gonza Selective action of acetogenin mitochondrial complex I inhibitors. Z. Naturforsch. 57C, 1028e1034. Hoffmann, J.J., Fraga, B.M., 1993. Microbial transformation of diterpenes: hidroxylation of 17-acetoxy-kolavenol acetate by Mucor plumbeus. Phytochemistry 33, 827e830. Horton, R.D., Redak, R.A., 1993. Further comments on analysis of covariance in insect dietary studies. Entomol. Exp. Appl. 69, 263e275. Jenett-Siems, K., Kaloga, M., Eich, E., 1993. Ipangulines, the first pyrrolizidine alkaloids from the Convolvulaceae. Phytochemistry 34, 437e440. Nonius, 1998. Kappa CCD server software. Nonius, R. V., Delft, The Netherlands. Liu, H., Gunnertoft-Jensen, K., My-Tran, L., Chen, M., Zhai, L., Olsen, C.E., Søhoel, H., Denmeade, S.R., Isaacs, J.T., Brøgger-Christensen, S., 2006. Cytotoxic phenylpropanoids and an additional thapsigargin analogue isolated from Thapsia garganica. Phytochemistry 67, 2651e2658. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: applications to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55e63. lez-Collado, I., 2013. Oliveira-Silva, E., Jacometti, N.A., Furtado, C., Aleu, J., Gonza Terpenoid biotransformations by Mucor species. Phytochem. Rev. 12, 857e876. Otwinowsky, Z., Minor, W., 1997. Processing of X-ray diffraction data collected in oscillation mode. In: macromolecular crystallography, Part, A., Carter, C.E., Sweet, R.M. (Eds.), Methods in Enzymology, vol. 276. Academic Press, New York, pp. 307e326. ces de le pidopte res Noctuidae et de 2 Poitout, S., Bues, R., 1974. Elevage de 28 espe ces d’Arctiidae sur milieu artificiel simplifie . Particularite s selon les espe ces. Ann. Zool. Ecol. Anim. 6, 431e441. espe Raubenheimer, D., Simpson, S.L., 1992. Analysis of covariance: an alternative to nutritional indices. Entomol. Exp. Appl. 62, 221e231. Sheldrick, G.M., 1997. SHELXL-97: program for the Refinement of Crystal Structures. €ttingen, Germany. University of Go Spek, A.L., 2005. PLATON, a Multipurpose Crystallographic Tool. University of Utrecht, The Netherlands. Venkateswarlu, Y., Ramesh, P., Srinivasa-Reddy, P., Jamil, K., 1999. Microbial transformation of D9(15)-africanene. Phytochemistry 52, 1275e1277.
Please cite this article in press as: Fraga, B.M., et al., Biotransformation of an africanane sesquiterpene by the fungus Mucor plumbeus, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.12.015