Two benzaldehyde derivatives and their artefacts from a gorgonian-derived Eurotium sp. fungus

Abstract Two new benzaldehyde derivatives, named 3′-OH-tetrahydroauroglaucin (1) and(3′S*,4′R*)-6-(3′,5-epoxy-4′-hydroxy-1′-heptenyl)-2-hydroxy-3-(3′′-methyl-2′′-butenyl)benzaldehyde (2), were isolated from a gorgonian-derived Eurotium sp. fungus. Their structures were determined by extensive spectroscopic analysis including NMR and MS spectra. Dissolved 1 in CDCl3 for several days could be detected its 2H-chromene skeleton derivatives (1a/1b), a pair of enantiomers with opposite configurations at C-3′. Compound 2 was also found to chemically convert to a pair of epimers non-enzymatically. The plausible mechanism to form the 2H-chromene artefacts with racemisation at C-3′ undergoing nucleophilic substitution (SN1) was proposed.


Introduction
Benzaldehydes, such as flavoglaucin and its derivatives, are a class of polyketides frequently isolated from fungi especially the genus of Eurotium. These types of benzaldehyde derivatives contain a C 7 alkyl side chain with several double bonds or oxygenated substituents at their basic skeletons and were reported to show important bioactivities including antioxidative, cytotoxic and antibacterial activities (Miyake et al. 2009(Miyake et al. , 2010Gao et al. 2011aGao et al. , 2011bWang et al. 2015). As part of our studies on secondary metabolites from marine organisms of the South China Sea (Chen et al. 2014a(Chen et al. , 2014b, we have investigated the chemical constituents obtained from the gorgonian-derived fungus Eurotium sp. recently. Two new benzaldehyde derivatives (1, 2) (Figure 1) were isolated from the fungal cultures, and interestingly, both of them could non-enzymatically transform into pairs of enantiomers or epimers, respectively, with opposite configurations at C-3′. Herein, we report the isolation, structure elucidation, and chemical conversions of these benzaldehyde derivatives.

Results and discussion
Compound 1 was isolated as a yellow oil. Its molecular formula of C 19 H 26 O 4 (seven degrees of unsaturation) was determined based on its negative HReSIMS. The 1 H NMR spectrum revealed the presence of one hydrogen-bonded phenolic proton at δ H 11.76 (2-OH), one formyl proton at δ H 10.08, one aromatic proton at δ H 7.01 (s), three olefinic protons at δ H 6.76 (d, J = 16.2 Hz), 5.99 (dd, J = 16.2, 5.4 Hz), 5.28 (brt, J = 7.2 Hz), three methyl groups at δ H 1.75 (s), 1.69 (s) and 0.93 (t, J = 6.6 Hz) and one oxygenated methine group at δ H 4.39 (dd, J = 6.0, 5.4 Hz), as well as four methylene groups. The 13 C NMR and DePT spectroscopic data exhibited 19 carbon signals including one formyl carbon at δ C 195.1, one oxygenated methine at δ C 71.7, four olefinic carbons and one aromatic pentasubstituted benzene. The above spectroscopic evidence suggested 1 was a benzaldehyde derivative, very similar to tetrahydroauroglaucin (Gao et al. 2011a). The HMBC correlation ( Figure S1) from formyl proton H-7 to C-1 showed the formyl was anchored at C-1 of the benzene ring. The contiguous sequence of COSY correlations from H-1′ to H-7′ indicated the connection from C-1′ to C-7′; therefore, a C 7 aliphatic chain was assigned which contained a double bond lied at C-1′ with E configuration (J 1′, 2′ = 16.2 Hz) and a hydroxy group substituted at C-3′. The HMBC correlations from H-1′ to C-6 and C-5 suggested that this C 7 aliphatic chain was located at C-6 of the benzene ring. Additionally, the methine proton (H-2′′) showed connectivity to the methylene doublet (H-1′′) and two tertiary methyl groups (H-4′′) and (H-5′′) according to the COSY and HMBC spectra, which implied a 3-methyl-2-butenyl group. The HMBC correlations from H-1′′ to C-2, C-3 and C-4 suggested that this 3-methyl-2-butenyl group was located at C-3 of the benzene ring. Two hydroxy groups at C-2 and C-5 of the benzene ring were deduced from their respective HMBC correlations. Therefore, the planar structure of 1 was established. Compound 1 was named as 3′-OH-tetrahydroauroglaucin.
Interestingly, compound 1 was not so stable when dissolved in CDCl 3 via intramolecular dehydration slowly changing into a 2H-chromene skeleton (1a/1b), which was confirmed by NMR and HReSIMS data. Its negative HReSIMS data showed a molecular formula of C 19 H 24 O 3 , meaning one 'H 2 O' group less than that of 1. In the 1 H NMR spectrum, the relative small coupling constant value of two olefinic protons (J 1′, 2′ = 9.6 Hz) suggested the Z configuration of the double bond at C-1′. Additionally, the proton signal of C-3′ methine was found to shift to downfield significantly (H-3′, δ H 4.73 in 1a/1b vs δ H 4.39 in 1). All of the above signals indicated the presence of a 2H-chromene structure for 1a/1b whose pyran oxygen was connected to the C-3′ methine carbon and to C-5 of the benzene ring. A possible mechanism from 1 to the 2H-chromene structure through a nucleophilic substitution (S N 1) was proposed (Scheme 1, i). The phenolic hydroxyl group (5-OH) acted as a nucleophile and attacked C-3′ to produce a pair of racemic 2H-chromene structures (Scheme 1, i, 1a/1b), reflecting the same NMR spectra. The racemic feature of 1a/1b was confirmed by the facts that 1a/1b showed not any detectable optical rotation and CD maximum.
Similar chemical change was also found while compound 2 was dissolved in CDCl 3 for few days. Two series of proton signals corresponding to two epimeric 2H-chromene structures with a ratio of 1:1 approximately could be assigned from the 1 H NMR spectrum, of which one was identical to that of 2. A plausible S N 1 mechanism from 2 to the pair of epimers 2/2a, with racemisation at C-3′, was proposed (Scheme 1, ii). Scheme 1. Proposed mechanism to form 2H-chromene artefacts with racemisation at c-3′.
Benzaldehyde derivatives with C-7 open chains, such as flavoglaucin, tetrahydroauroglaucin and related structures, were often reported from several fungal species including the genus of Eurotium (Miyake et al. 2009(Miyake et al. , 2010Gao et al. 2011aGao et al. , 2011b. Many chromene or 2H-chromene-containing derivatives derived from benzaldehydes were also reported (Wang et al. 2006;li et al. 2008). These compounds, including compound 2 in this study, may be formed non-enzymatically. In other words, they are possibly artefacts formed during the extraction/isolation process. The biological activity was not tested for these isolated compounds for their low yields and instability.

General experimental procedures
Optical rotations were measured on a JASCO P-1020 digital polarimeter. CD spectra were recorded on JASCO J-810 circular dichroism spectrometer. NMR spectra were acquired using a JeOl JeM-eCP NMR spectrometer (600 MHz for 1 H and 150 MHz for 13 C), using TMS as internal standard. eSIMS spectra were obtained from a Micromass Q-TOF spectrometer. Semipreparative HPlC was performed on a Waters 1525 system using a semi-preparative C 18 (Kromasil, 5 μm, 10 mm × 250 mm) column coupled with a Waters 2996 photodiode array detector. Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200-300 mesh) and octadecylsilyl silica gel (unicorn; 45-60 μm) were used for column chromatography (CC). Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for thin layer chromatography.

Fungal material and culture conditions
The fungus Eurotium sp. was isolated from the inner part of a fresh gorgonian, Subergorgia suberosa, which was collected from the Xisha Islands coral reef in the South China Sea in December 2009. The strain was deposited at the Key laboratory of Marine Drugs, the Ministry of education of China, School of Medicine and Pharmacy, Ocean university of China, Qingdao, People's Republic of China, with the Genebank (NCBI) accession number, HM991283. The fungal strain was cultivated in a rice medium (12 g of natural sea salt, 100 g of rice, 0.6 g of peptone, 100 ml of H 2 O) in 1 l erlenmeyer flasks (30 flasks) for 35 days at room temperature.

Conclusions
In summary, two new benzaldehyde derivatives (1, 2) were isolated from a gorgonian-derived Eurotium sp. fungus. Their structures were elucidated by using comprehensive spectroscopic methods. An S N 1 mechanism from 1 and 2 to 2H-chromene artefacts with racemisation at C-3′ was proposed.