ZHANG Zhenkun, WANG Jiaxiang, CAO Fei, ZHOU Xiaojian, WU Jingshuai,FU Xiumei, CHEN Min, *, and WANG Changyun, *

Histone Deacetylation Modifier Induced One New Resorcylic Acid Lactone 7′()-zeaenol from the Zoanthid-Derived Fungus

ZHANG Zhenkun1), 2), 3), WANG Jiaxiang2), CAO Fei4), ZHOU Xiaojian2), WU Jingshuai1), 3),FU Xiumei1), 3), CHEN Min2), *, and WANG Changyun1), 3), *

1),,266003,2),,225127,3),,266237,4),,071002,

Chemical epigenetic manipulation was applied to the zoanthid-derived fungus(TA26-46) with a his- tone deacetylation modifier (100μmolL−1nicotinamide), resulting in the isolation of a new 14-membered resorcylic acid lactone named7′()-zeaenol(1), together with six known analogues (2−7) from the treated broth. The planar structure of 1 was determined by comprehensive NMR spectroscopy and HRESIMS data. The absolute configuration of 1 was elucidated by ECD spectrum,13CNMR shift calculations, and on the basis of biogenetic considerations.Compound 5 exhibited cytotoxic activity against the human tumor cell lines A549, HCT-116, HT-29, Hela, MCF-7, and K562 with the IC50values ranging from 2.54 to 7.44μmolL−1.

; 14-membered resorcylic acid lactone; chemical epigenetic manipulation; cytotoxic activity

1 Introduction

Marine-derived fungi have been researched for decades due to their prospective reservoir for structurally multitu- dinous and biologically significant secondary metabolites(Carroll., 2019; Nagabhishek and Madankumar, 2019).Fungal secondary metabolites include alkaloids, polyketides,meroterpenoids, terpenoids, steroids, and peptides, which have a wide range of biological activities, such as antimi- crobial, antiviral, antioxidant, anti-fouling, cytotoxicity, in-hibition of various enzyme,. (Hafez Ghoran., 2022; Zhang., 2022). Enzymes that build secondary meta- bolites are encoded by colocalized biosynthetic gene clus- ters (BGCs). A lot of genome sequencing results have re- cently revealed that many BGCs are ‘silent’ or conveyed at low levels under standard laboratory conditions (Tomm., 2019; Xu., 2021).For exploring this incomple- tely tapped trove, it is a significant method to activate the silent BGCs of marine-derived fungi by modification of cul- ture conditions, such as chemical epigenetics (Zarins-Tutt., 2016; Zhang., 2022). Nowadays, it is demon- strated that chemical epigenetic manipulation is an effectivetechnique to remodel the fungal epigenome to acquire morelatent fungal secondary metabolites (Guo., 2020; Zhao., 2018), which may encounter large manipulation in a pathway-specific regulator and these changes give an access to the production of novel secondary metabolite (Bharati- ya., 2021).

In our previous work, a series of 14-membered resorcy- lic acid lactones (RALs) have been isolated from marine in-vertebrate-derived fungi collected from the South China Sea (Shao., 2011; Liu., 2014; Zhang., 2017; Xu., 2021). For example, seven RALs with potent anti- fouling activities, including cochliomycins A–C, zeaenol, LL-Z1640-1, LL-Z1640-2 and paecilomycin F, were first- ly isolated from a gorgonian-derived fungus(Shao., 2011). Eleven RALs with antifouling and fungicidal activities, cochliomycins D–F together witheight analogues, were obtained from a sea anemone-derivedfungus(TA26–46) (Liu., 2014). RALs are a family of benzannulated macrolides, and constitute a 14-membered macrocyclic ring fused to a β-resorcylic acid moiety (Lai., 2016). Our previous investigation reveal- ed a variety of biological properties of the isolated RALs and their semisynthetic derivatives, including anntifuling, antimalarial, cytotoxic, antiparasitic, antiviral, fungicidal and kinase inhibitory activities (Xu., 2019). In order to explore the products of silent secondary metabolic path- ways, chemical epigenetic manipulation has been applied to the fungal strain(TA26–46), while sodium butyrate and SAHA lead to the isolation of brominated resorcylic acid lactones (Zhang., 2014), 5-azacyti- dine lead to the isolation of diethylene glycol phthalate esters(Chen., 2016) and α-pyrones (Wu., 2019). Many inducers inactivate the enzyme histone deacety-lase and DNA containing gene material to transfer is tran-scribed and translated for expression of a gene. So, theaddition of external inducer reinforced the transcriptionprocess (Bharatiya., 2021). In the present study, an- other type of chemical epigenetic agent, a histone deace- tylation modifier, nicotinamide (100μmolL−1), was added to the fermentation of(TA26–46), which re- sulting in remarkable difference in the secondary meta- bolites compared with the control. From the treated broth, a new RAL, 7′()-zeaenol (1), together with six known analogues (2–7), as shown in Fig.1, were isolated. Herein, we report the isolation, structural characterization, and bio- activity evaluation of these RALs.

Fig.1 Structures of compounds 1−7.

2 Materials and Methods

2.1 General Experimental Procedures

Optical rotations were measured on an MCP300 automa- tic polarimeter (Anton Paar, Austria) at 20℃. ECD spec- tra were performed on a J-810 Circular Dichroism Spec- trometer (JASCO, Japan). IR experiments were conduct- ed on a Cary 610/670 spectrometer (Agilent, America) us- ing KBr pellets. NMR spectra were measured with an AV- ANCE 600 NMR spectrometer (Bruker, Germany) (600MHz for1H and 150MHz for13C), using TMS as internal standard. HRESIMS spectra were acquired from Agilent 1290 Infinity II UHPLC/6530 Q-TOF MS (Waters, Ame- rica). Semi-preparative HPLC was performed on a HITA- CHI system using a semi-preparative C18(Kromasil, 5μm, 10mm×250mm) column coupled with a 2400 UV detec- tor. Silica gel (Qing Dao Hai Yang Chemical Group Co.; 100–200 and 200–300 mesh), octadecylsilyl silica gel (Uni-corn; 45–60μm) and Sephadex LH-20 (Amersham Bio- sciences) were used for column chromatography (CC). Pre- coated silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were employed for thin layer chromatography (TLC).

2.2 Fungal Material and Culture Conditions

The separation and authentication of the zoanthid-de- rived fungus(TA26–46) have been reported previously (GenBank JF819163) (Liu., 2014).The fungal strain was inoculated in a malt extract broth (malt 30gL−1, peptone 5gL−1, artificial sea salt 30gL−1) containing a kind of histone deacetylation modifier (100μmolL−1nicotinamide). The cultivation was incubated for 30 days at room temperature.

2.3 Extraction and Isolation

The cultivation was percolated to separate the broth from the mycelium. The mycelium was extracted three timeswithethyl acetate (EtOAc) (200mL for each flask) and two times with CH2Cl2–MeOH (1:1, v/v) (200mL for each flask).Meanwhile the fermented broth was extracted with a three- fold volume of EtOAc for three times. The mycelium ex- traction and the fermented broth extraction were combin- ed and concentrated in vacuo pump to produce a crude ex-tract (60.7g). Then the crude extract was separated by sili-ca gel column chromatography (CC) using a step gradient elution of petroleum ether (PET)–EtOAc mixtures with in-creasing polarity (100:0–0:100) to produce six subtractions(Fr.1–Fr.6). Fr.4 and Fr.5 were purified respectively by si-lica gel Sephadex LH-20, octadecyl silane CC, and semi- preparative HPLC to yield compounds 1 (5.3mg), 2 (15.4mg), 3 (6.1mg), 4 (6.6mg), 5 (3.6mg), 6 (4.3mg), and 7 (1.65mg).

2.4 Physical-Chemical Property

2.5 Biological Assays

2.5.1 Cytotoxic activity

Cytotoxic activity was measured by using A549 (human pulmonary adenocarcinoma), HCT116 (human colorectal adenocarcinoma), HT-29 (human colorectal adenocarcino- ma), Hela (human cervical carcinoma), MCF-7 (Humanbreast carcinoma) and K562 (Chronic myeloid leukemia) cell lines. The assessment was conducted through the 96- well microtitre plates (Skehan., 1990; Repetto., 2008), with adriamycin as positive control, and DMSO as

negative control. The inhibition ratios of adriamycin were 79.75%, 61.19%, 75.02%, 69.10%, 82.97% and 62.27% res- pectively against the tested cell lines when its concentra- tion is 1μmolL−1.

2.5.2 Antibacterial activity

Antibacterial activity was assayed on 13 bacterial strains, including Gram-negative,,,,,,,,,,,Gram-positive, and Me- thicillin-resistant. The evaluation was conduct- ed through 96-well microtitre plates (Appendino., 2008), with ciprofloxacin and SeaNine 211 as positive con- trols, DMSO as negative control, and Luria-Bertani broth as blank control.

2.5.3 Microalgae growth inhibition activity

The microalgae growth inhibition activity was evaluated on,,,andBoh- lin.The measurements were conducted using24-well cell culture plates (Eisentraeger., 2003), with SeaNine 211 as a positive control and DMSO as negative control.

3 Results and Discussion

3.1 Structure Determination

Compound(1) was isolated as a white, amorphous pow-der.Its molecular formula was confirmed as C19H24O7(eight degrees of unsaturation) based on the analysis with HR- ESI-MS ion (Fig.2) at387.1405 [M+Na]+(calcd for C19H24NaO7, 387.1414). Careful inspection of the 1D and 2D NMR (Table 1) spectra of 1 revealed that 1 is a RAL possessing the same planar structure as that of zeaenol (2), the major product of(TA26–46). The signifi- cant differences between 1 and 2 were the respective con- figuration of double bond at C-7′. In the1H NMR spectrum (Fig.3), the small coupling constant=11.3Hz between H-7′ (H5.84, dd,=11.3, 9.7Hz) and H-8′ (H5.60, ddd,=11.3, 4.2, 2.4Hz) suggested that the double bond at C-7′ is-configuration in 1,which is different from the 7′- configuration in 2. Careful analysis of the13C NMR spec- trum (Fig.4) showed that the primary differences between 1 and 2 were the chemical shifts of C-6′ (C64.3 in 1. 73.1 in 2), hence it seems that the stereogenic center C-6′ in 1 was inverted to the corresponding center in 2.

Fig.2 HRESIMS spectrum of compound 1.

The absolute configuration of 1 was determined by its ECD spectrum,13C NMR shift calculations, and biogene- tic considerations. The absolute configuration of C-10′ was assigned by the analysis of ECD spectra (Fig.5), in which the appearance of a negative Cotton effect around 275nm implied the 10′configuration (Liu., 2014). It is a chal- lenge to determine the absolute configurations of C4′, C5′, and C6′ of 1. The acetonide reaction of 1 failed due to the complex products which were difficult to be separated. For- tunately, two known 4′, 5′, 6′-triol RALs, zeaenol (2) (Su- gawara., 1992) and 7-epi-zeaenol (3) (Ayers., 2011) with 4′, 5′configurations were isolated simultaneously from(TA26–46). On account of biogenetic con- siderations, the configurations at C-4′ and C-5′ of 1 were 4′, 5′. The absolute configuration of C-6′ was difficult to be determined as the molecule conformational flexibi- lity reflect two kinds of possible absolute configurations (Fig.6, 1a with 4′, 5′, 6′, 10′and 1b with 4′, 5′, 6′, 10'). Consequently, the absolute configuration of 1 was determined by using calculation of GIAO NMR shift (Cao., 2019a, 2019b) at the B3LYP/6-311+G(d,p) level. The absolute configuration of 1b was more likely than1a to be the real configuration,(100.00% 1b.0.00% 1a in both unscaled shift data and shielding tensor data) (Fig.7) when the parameter of DP4 plus probability was considered(Grimblat., 2015). Therefore, the absolute configura- tion of 1 was proposed as 4′,5′,6′,10′. The significant up-field chemical shift of C-6′ may be caused by the shield-ing effect of the double bond at C-7′. Thus, compound 1 was elucidated as 7′()-zeaenol.

Table 1NMR Spectroscopic Data for1 (CDCl3)

Note: Measured at 600MHz for1H NMR and 150MHz for13C NMR in CDCl3.

Fig.3 A part of 1H NMR (600MHz, CDCl3) spectrum of compound 1.

Fig.4 13C NMR (150MHz, CDCl3) spectrum of compound 1.

Fig.5 ECD spectrum of compound 1.

Fig.6Two possible absolute configurations (1a and 1b) for 1.

The known 14-membered RALs2–7have been deter- mined to be zeaenol (2)(Sugawara., 1992), 7-epi-zea-enol (3)(Ayers., 2011), LL-Z1640-1 (4), LL-Z1640-2 (5)(Ellestad., 1978), paecilomycin G (6)(Bujarani- palli and Das, 2016), and-aigialomycin C(7)(Ba- jwa and Jennings, 2008) based on their NMR data, and by the comparison with the data previously reported.

It is interesting that when 100μmolL−1nicotinamide was added into the malt medium, multiple new peaks emerged inthe HPLC profile between 35 and 50min (Fig.8). Through further analysis, we found that RALs were focused on 45 to 50min.

Fig.8 HPLC profiles of EtOAc extracts of C. lunatus (TA26–46) cultured in malt extract broth with 100μmolL−1 nicotinamide.

3.2 Bioactivity

More than 130 naturally occurring 14-membered RALs have been described from many fungal genera since thefirst-discovered RAL radicicol was isolated in 1953 (Xu., 2022). A literature survey revealed that many 14- membered RALs showed a variety of bioactivities, includ- ing but not limited to cytotoxic (Ayers., 2011) and an- tifouling (Xu., 2019) functions. Based on these reports, all isolated RALs (1–7) were first detected for their cyto- toxic activity towards A549, HCT116, HT-29, Hela, MCF-7and K562 cell lines. The new compound (1) displayed weakcytotoxic activity to inhibit HT-29 cell line growing. Com- pound 5 exhibited potent cytotoxic activity against A549, HCT116, HT-29, Hela, MCF-7 and K562 cell lines with the IC50values of 6.97, 2.54, 4.95, 4.25, 6.22 and 7.44μ molL−1, respectively. However, the very similar compound 4 showed no such activities. Comparing the cytotoxic acti- vities of 4 and 5, the configuration of C-5′ plays a critical role in the cytotoxic activities. Compounds 1–7 were al- so tested withtheir antibacterial activity against 13 patho- genic bacteria strains, and antifouling activity against 5 mi- croalgaes. Unfortunately, no activity was found for these RALs.

4 Conclusions

In conclusion, by introducingchemical epigenetic mani- pulation on the zoanthid-derived fungus(TA26–46) with a histone deacetylation modifier (100μmolL−1nicotinamide), a new 14-membered RAL 7′()-zeaenol (1) and six analogues (2–7)can be isolated from the malt ex- tract broth. Compound 5shows strong cytotoxic activity and is worthy of further study. The results indicated once again that chemical epigenetic manipulation is an effec- tive tool for exploring the products of fungal silent secon- dary metabolic pathways.

Acknowledgements

This work was supported by the National Natural Sci-ence Foundation of China (Nos. 81673350, 81703411, 417 76156), the National Science and Technology Major Pro- ject for Significant New Drugs Development, China (No. 2018ZX09735-004), the Fundamental Research Funds for the Central Universities of China (No. 201962002), and the Taishan Scholars Program, China.

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(February 16, 2020; revised May 16, 2020; accepted July 7, 2020)

© Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2023

Corresponding authors. E-mail:dieying0719@163.com

E-mail:changyun@ouc.edu.cn

(Edited by Qiu Yantao)