Biotransformation of labdane and halimane diterpenoids by two filamentous fungi strains

Biotransformation of natural products by filamentous fungi is a powerful and effective approach to achieve derivatives with valuable new chemical and biological properties. Although diterpenoid substrates usually exhibit good susceptibility towards fungi enzymes, there have been no studies concerning the microbiological transformation of halimane-type diterpenoids up to now. In this work, we investigated the capability of Fusarium oxysporum (a fungus isolated from the rhizosphere of Senna spectabilis) and Myrothecium verrucaria (an endophyte) to transform halimane (1) and labdane (2) acids isolated from Hymenaea stigonocarpa (Fabaceae). Feeding experiments resulted in the production of six derivatives, including hydroxy, oxo, formyl and carboxy analogues. Incubation of 1 with F. oxysporum afforded 2-oxo-derivative (3), while bioconversion with M. verrucaria provided 18,19-dihydroxy (4), 18-formyl (5) and 18-carboxy (6) bioproducts. Transformation of substrate 2 mediated by F. oxysporum produced a 7α-hydroxy (7) derivative, while M. verrucaria yielded 7α- (7) and 3β-hydroxy (8) metabolites. Unlike F. oxysporum, which showed a preference to transform ring B, M. verrucaria exhibited the ability to hydroxylate both rings A and B from substrate 2. Additionally, compounds 1–8 were evaluated for inhibitory activity against Hr-AChE and Hu-BChE enzymes through ICER-IT-MS/MS assay.

Biotransformation of natural products by filamentous fungi is a powerful and effective approach to achieve derivatives with valuable new chemical and biological properties. Although diterpenoid substrates usually exhibit good susceptibility towards fungi enzymes, there have been no studies concerning the microbiological transformation of halimane-type diterpenoids up to now. In this work, we investigated the capability of Fusarium oxysporum (a fungus isolated from the rhizosphere of Senna spectabilis) and Myrothecium verrucaria (an endophyte) to transform halimane (1) and labdane (2) acids isolated from Hymenaea stigonocarpa (Fabaceae). Feeding experiments resulted in the production of six derivatives, including hydroxy, oxo, formyl and carboxy analogues. Incubation of 1 with F. oxysporum afforded 2oxo-derivative (3), while bioconversion with M. verrucaria provided 18,19-dihydroxy (4), 18-formyl (5) and 18-carboxy (6) bioproducts. Transformation of substrate 2 mediated by F. oxysporum produced a 7α-hydroxy (7) derivative, while M. verrucaria yielded 7α-(7) and 3β-hydroxy (8) metabolites. Unlike F. oxysporum, which showed a preference to transform ring B, M. verrucaria exhibited the ability to hydroxylate both rings A and B from substrate 2. Additionally, compounds 1-8 were evaluated for inhibitory activity against Hr-AChE and Hu-BChE enzymes through ICER-IT-MS/MS assay.
2017 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. 1

. Introduction
Diterpenoids have attracted outstanding attention because many of these constituents display a wide variety of pronounced biological activities, following the diversity of new structures discovered each year. These metabolites are known to be produced mostly from plants, but they have also been found from microorganisms, such as fungi and bacteria, as well as marine organisms [1][2][3]. Labdanes constitute a large group of diterpenoids, having a chemical framework of C-20 skeleton comprising a decalin core and a C-6 side chain, cyclic or aliphatic. They usually exhibit five stereocentres and can occur in both normal and antipodal series [4]. Halimanes arise from labdanes by migration of C-20 methyl group from C-10 to C-9 position. Among plants producing diterpenoids as regular constituents, Fabaceae deserves to be highlighted. Remarkably, it comprises Hymenaea genus, a small group of about 14 species, which are rich sources of ent-labdane and ent-halimane diterpenoids [5].
Biotransformation mediated by filamentous fungi consists of a powerful method to perform chemical modifications of a variety of starting materials such as bioactive natural products, to obtain derivatives with improved biological properties or even new biological activities [8]. This approach stands out as a promising alternative to conventional chemical methods since fungi contain multi-enzymatic systems with broad specificities and are, therefore, able to catalyse chemo, regio and stereoselective reactions on non-activated molecular sites that are normally unreactive or difficult to reach chemically [9]. Furthermore, microbial transformation is a fast, efficient, cost-effective and ecologically friendly technique because it requires only mild reaction conditions [10].
Although several biotransformation studies of labdane-skeleton have been reported so far [1,8], to the best of our knowledge, there have been no reports on literature concerning biotransformation of halimane-type, even though halimanes are closely related to labdanes. In this work, we report the investigation of the biotransformation-promoting capabilities of compounds 1 and 2 by two filamentous fungi, namely Fusarium oxysporum and Myrothecium verrucaria, together with the inhibitory activity of enzymes Hr-AChE and Hu-BChE of starting compounds and their biotransformation products.

Determination of biotransformed products
Biotransformation of 1 by F. oxysporum afforded one structurally related compound 3 (figure 1), while M. verrucaria provided three closely related derivatives, metabolites 4, 5 and 6 (figure 1). Compound 3 was obtained as a white and amorphous solid. Its molecular formula was determined as C 20 1 H and 13 C NMR spectra were quite similar to those of 1, allowing direct assignments based on comparison (table 1). The main differences between 1 and 3 corresponded to the presence of signals arising from the resonance of a methylene group adjacent to an α,β-unsaturated carbonyl group (δ H 2.37 and 1.92), and deshielding of H-1 signal from 5.37 ppm for 1 to δ H 5.83 for 3. 13 C NMR spectrum confirmed the presence of a signal corresponding to a typical resonance of a keto group (δ C 202.1), which was assigned to C-2. This assignment was further supported by β-effects observed at C-1 (+3.7) and C-3 (+15.6), and confirmed by HMBC correlation (see electronic supplementary material) from H 2 -3 (δ H 2.37; 1.92) to C-2. Therefore, biotransformation product 3 is the 2-oxo-derivative of substrate 1.
Metabolite 4 showed molecular formula as C 20             Therefore, conversion of substrates 1 and 2 into products 3 and 7, respectively, suggests oxidative reactions mediated by cytochrome P450 monooxygenase [9] at C-2 (substrate 1) and C-7 (substrate 2), and subsequent oxidation of 2-hydroxy derivative into its 2-oxo product. Oxidation of substrate 1 at C-2 to provide 3 is probably favoured because these hydrogens are allylic to 1,10-double bond. Previously microbial transformations of diterpenoid substrates performed by Fusarium species encompasses conversion of dehydroabietic acid into its 1β-hydroxy derivative by F. oxysporum [14], modification of sclareol with F. lini leading to 1β-hydroxy and (12S)-12-hydroxysclareol derivatives [16], and oxidation of cupressic acid by F. graminearum to produce four metabolites, including 3β-hydroxy and 7α-hydroxy analogues [17]. Sequential oxidations at position C-2 from diterpenoid substrate to alcohol and then oxo derivatives have been found to occur only with another fungus, Mucor plumbeus [18].
Only one report involving biotransformation process with Myrothecium species, namely M. roridum, was found in the literature, however, dedicated to malachite green (a triphenylmethane dye)        decolorization [19]. Despite the lack of reference, it is well established that fungi can catalyse a series of transformations through their enzymatic machinery and diterpenoids exhibit good susceptibility towards fungal enzymes. Thus, the formation of hydroxy derivatives 4 (C-19), 7 (C-7) and 8 (C-3) by M. verrucaria would also involve the action of P450 monooxygenase, while analogues 5 and 6 would comprise carboxylation of substrates at C-18, to formyl and carboxy products, subsequently.
Oxidations in substrate 1 were observed only in ring A by both microorganisms. However, F. oxysporum had been able to oxidize just position C-7 (7) from substrate 2 showing preference by ring B, while M. verrucaria not only provided the same derivative but also afforded a second 3-hydroxy derivative (8, oxidized in ring A), indicating no specific ring preference. Only the yields of compound 7 could be compared as it was commonly produced by both fungi, showing a higher yield from F. oxysporum, despite M. verrucaria deviates substrate for conversion between two derivatives.

Anticholinesterase assays
Labdane-type diterpenoids [20] and semisynthetic labdane derivatives [21] are reported to exhibit significant anticholinesterase activities. In this context, compounds 1-8 were screened for inhibitor candidates by means of hydrolysis of acetylcholine (ACh) through on-flow screening by Hr-AChE and Hu-BChE-ICER-IT-MS/MS assay (table 3). In this study, substrates 1 and 2, and their biotransformation derivatives demonstrated different inhibitory activities relative to the structural modifications.
The oxidations occurred into substrate 1 gradually increased the inhibition potential of compounds 3, 4 and 5 over enzyme Hr-AChE, at the same time they selectively decreased their activity towards Hu-BChE. Compound 5, bearing an 18-carboxy-substituent was found to be the most active halimane derivative against Hr-AChE, displaying inhibition around 50%. However, substance 6, possessing an 18-formyl group, showed the worse inhibition over the same enzyme.
Hydroxylation of 2 (ring B) was observed to drastically decrease the Hr-AChE inhibition of compound 7, which possesses a 7,8-diol system, while insertion of the 3-hydroxy group (ring A) in derivative 8 showed to selectively improve its Hr-AChE inhibition. Interestingly, these modifications did not affect Hu-BChE activity to an appreciable extent.
The correlations inferred between structure modifications and resulting inhibitory activities of compounds 1 to 8 suggests oxo, hydroxyl, formyl and carboxy substituents play determinant roles in increasing selectivity and potency of these compounds over Hr-AChE since they share similar hydrocarbon backbones, while these groups are responsible for opposite effects towards Hu-BChE. Such findings indicate oxygen atoms, from substituents added to the bioproducts, could be able to interact with Hr-AChE mainly through hydrogen bonds, possibly formed between hydroxyl and carbonyl groups and the residues from the catalytic triad of Hr-AChE active site [21].
As screening results, two hits were found for Hr-AChE, derivatives 5 and 8, with inhibition percentage around 50% in comparison to standard inhibitor galanthamine. However, despite oxidations at position C-18 (5) and at C-3 (8) having increased the inhibitory potential of these derivatives in comparison to the substrates 1 and 2, respectively, compound 5 can be considered to be inactive based on its IC 50 value (greater than 100 µM), whereas 8 was only weakly active (IC 50 = 95.74 µM), showing some enhanced activity but still requiring further structure optimization for better potency.

General experimental procedures
Optical rotations were measured on a Schimdt-Haensh (Berlin, Germany) Polartronic H-100 polarimeter using quartz cells of 1 dm path length, at 25°C. IR spectra were recorded on a FT-IR Vertex 70 Bruker spectrometer, operating in ATR mode. NMR spectra were acquired on a Bruker Avance III HD 600 spectrometer (14.1 T-600. 13 MHz for 1 H and 150.9 for 13 C) equipped with a Triple Inverse TCI Cryo-Probehead  analysed between each AChEI sample. Percentage inhibition displayed by each sample was calculated by comparison between the area of enzymatic activity in the presence of the inhibitor (P i ) and absence (P 0 ), according to the following equation: %inhibition = 100 − P i P 0 × 100

Conclusion
In this work, the microbial transformation of diterpenoid substrates was performed by two filamentous fungi-F. oxysporum and M. verrucaria. As results, six oxidized derivatives were obtained, including four new and two known metabolites. Remarkable catalysed-modifications, including oxidations of unactivated C-H sp 3 bonds, were observed to occur in both rings A and B from substrates into distinct reactivity positions, affording derivatives with further hydroxy and carbonyl functionalitiesnew reactive sites which can enable accession of a greater number of further analogues. The starting compounds and their biotransformation products were also assayed for anticholinesterase inhibition towards AChE and BChE, through ICER-IT-MS/MS screening. However, only compound 8 showed some enhanced potential over AChE. Therefore, based on the structural modifications from the substrates, it is conclusive that both microorganisms proved to be prolific enzymatic sources for biotransformation of poorly reactive diterpenoids, providing structurally diverse derivatives of valuable chemical and biological relevance.
Data accessibility.