Assessment of Novel C-4 Methylated Tetraketide by HPLC-SPE-TT in Saccharicola sp., an Endophytic Fungus in Eugenia jambolana Lam

A new compound named methyl (6S,7S,2E,4E)-6,7-dihydroxy-4,6-dimethyl octanoate was isolated from Saccharicola sp. using high performance liquid chromatography-solid phase extraction-transfer tube (HPLC-SPE-TT), together with four known compounds, fusaric acid, trans-4-hydroxymellein, thymidine and adenosine. Saccharicola sp. was isolated from the stems of Eugenia jambolana Lam. (Myrtaceae) as endophytic fungus. The structure of this novel substance was established based on its spectroscopic data, including H and C nuclear magnetic resonance (NMR), 2D NMR techniques and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). This paper deals with the first report of its kind on this compound in Saccharicola sp.


Introduction
The scientific community has focused its attention on the secondary metabolites from the fungi kingdom for the discovery of numerous biologically active compounds, including antifungal, new antibiotics, chemotherapeutic agents and agrochemicals.][3] The plant species Eugenia jambolana Lam.(Myrtaceae) has been exploited chemically and biologically as a result of its popular use besides the fact that it presents several pharmacological activities. 4Dametto et al. 5 demonstrated the extracts and fractions from E. jambolana are promising as prototypes of new chemopreventive compounds.The study of endophytic fungi, associated with this species, unfolds a remarkably promising strategic window in the identification of potentially bioactive substances.This plant species exhibits a wide range of endophytes, among them Saccharicola sp. was selected for chemical study owing to the activities it presented against phytopathogenic fungi C. sphaerospermum, apart from its antioxidant and anticholinesterase activities. 6Preliminary studies of Saccharicola sp.presented unprecedented oxygenated cyclohexanoids. 7These compounds have stimulated myriad works of synthesis as result of their reported antifungal, antibacterial and antitumor activities. 8Zhao et al. 9 showed new isoprenylated cyclohexanols, which were isolated from the sponge-associated fungus Truncatella angustata.Besides the truncateol M exhibited potent inhibitory effect against influenza-A viral infection. 9he present work was aimed at conducting chemical assessment of the endophytic fungus Saccharicola sp.isolated from Eugenia jambolana Lam.Herein we discuss the isolation, structure elucidation of new compound (6S,7S,2E,4E)-6,7-dihydroxy-4,6-dimethyl octanoate (1)  and the known compounds mycotoxin fusaric acid, trans-4-hydroxymellein, thymidine and adenosine (2-5).The absolute configuration of compound 1 was determined by comparing experimental and calculated electronic circular dichroism (ECD) spectroscopy data.

General experimental procedures
The compound 1 was isolated using high performance liquid chromatography-solid phase extraction-transfer tube (HPLC-SPE-TT) comprising a solid phase extractor (SPE) Bruker/Spark Prospekt II as an interface between an HPLC in the analytical mode, an Agilent 1260 infinity series HPLC (HP1260 infinity, Agilent, USA) with diode array ultraviolet detector (DAD) and an automatic nuclear magnetic resonance (NMR) sampler and tracer (TT).
An interface (Bruker) operating in the stop-flow mode was used for the treatment of samples for NMR tubes in a totally automated way.The sample was solubilized in MeOH:H 2 O (90:10) and cleaned using RP-18 silica cartridges coupled to Millipore ® membrane (0.2 μm).At the end of this procedure, the sample (10 mg mL -1 ) was stored in a vial.The system employed for HPLC-SPE-TT analysis was a Luna Phenomenex octadecyl silane (RP-18) analytical column (Phenomenex Luna 250 × 4.60 mm, 5 μm, 100 Å).Mass spectra were acquired by quadrupole time-offlight (Q-TOF) Bruker MaXis Impact mass spectrometer using electrospray ionization mass spectrometry (ESI-MS) and MeOH:H 2 O (1:1) as the eluent.Infrared (IR) spectra were recorded on a Jasco FTIR-4600 series spectrometer equipment, while the FTIR spectrum was recorded with 128 scans per spectrum with a resolution of 4 cm -1 .ECD and UV spectra were obtained on a JASCO J-815 spectrometer.Optical rotations were measured using a PerkinElmer polarimeter equipped with a sodium lamp operating at 30 ºC and a sample cell volume of 1 mL using methanol (MeOH) as solvent.

Plant material
The ripe fruits as leaves of Eugenia jambolana were collected on March 27, 2008 in a square in the city of Araraquara, state of São Paulo.The specie was identified by Dra Maria Inês Cordeiro and a voucher specimen No. SP 454124 has been deposited in the Herbarium "Maria Eneida Kauffmann", of the Botanic Garden of São Paulo, Brazil.

Growth of Saccharicola sp., extraction and isolation of the compounds 1-5
The endophytic fungus Saccharicola sp. was inoculated into three Petri dishes containing PDA (potato dextrose agar) and incubated for 10 days at 25 °C.
To obtain the crude extract in Czapek, one third of mycelium from each Petri dish was inoculated into 40 Erlenmeyer flasks (500 mL) containing 300 mL of liquid medium (Czapek).Saccharicola sp. was incubated for 28 days in static mode at 25 °C.After this time, the accumulated biomass was separated from the aqueous medium by filtration, and then the filtrate was subjected to liquid-liquid partition with ethyl acetate (EtOAc, 3 × 2.7 L).
The EtOAc fraction was evaporated to dryness, to give 350.0 mg of crude extract.
To obtain the crude extract in corn, Saccharicola sp. was cultivated in 9 Erlenmeyer flasks (500 mL), each containing 90 g of corn and 75.0 mL of distilled water.The medium was autoclaved three times (in three consecutive days) at 121 °C for 20 min.Following sterilization, the medium was inoculated with the endophyte and incubated while stationary at 25 °C for 21 days.At the end of the incubation period, the cultures were combined, ground and extracted with EtOAc (5 × 200 mL).The solvent was evaporated, yielding a crude EtOAc extract (5.16 g) which was portioned with H 2 O (3 × 5 mL).The EtOAc extract was dissolved in acetonitrile (CH 3 CN) and defatted with hexane by liquid partitioning.The CH 3 CN fraction was evaporated to yield 540.0 mg of crude extract.
For the isolation of compounds 1 and 2 the EtOAc crude extract (250.0 mg) was fractionated in SPE cartridge (solid phase extraction), and a total of 6 fractions were obtained.The fraction 3 was subjected to study via the HPLC-SPE-TT technique using an RP-C 18 column, eluted by H 2 O:MeOH (90:10-0:100) in 40 min in the analytical mode, at flow rate of 0.8 mL min -1 , λ = 254 nm and 30 μL injection volume, to give compound 1 (0.5 mg).The fraction 1 was analyzed by HPLC using an RP-C 18 column in the semi-preparative mode and as eluent a gradient of H 2 O:CH 3 CN (90:10-0:100) in 36 min, flow rate of 3.5 mL min -1 , λ = 254 nm and injection volume of 150 μL, to give compound 2 (0.9 mg).
The CH 3 CN fraction (540.0 mg) from the corn culture medium was subjected to HPLC using RP-C 18 column in the semi-preparative mode and as eluent a gradient of H 2 O:CH 3 CN (90:10-0:100) in 36 min, flow rate of 3.5 mL min -1 , λ = 254 nm and injection volume of 150 μL resulting in the isolation of the compound 3 (0.8 mg).
To obtain compounds 4 and 5, an aqueous fraction was added to a 20.0 × 2.5 cm glass column packed with approximately 20.0 g Amberlite XAD-16N resin previously hydrated with water.Approximately 200.0 mg of the aqueous fraction was diluted in 100% water and added to the column.Once loaded into the aqueous resin fraction, it was fractionated with 600 mL of water followed by 300 mL of 100% methanol.The methanol fraction (80.0 mg) was collected and concentrated by evaporation at low pressure.This CH 3 OH fraction was analyzed by HPLC using RP-C 18 column in the semi-preparative mode, and as eluent an isocratic system of H 2 O:CH 3 OH (90:10) in 36 min in a flow rate of 3.5 mL min -1 and λ = 254 nm, resulting in the isolation of thymidine (4) (1.5 mg) and adenosine (5) (1.2 mg).
Compound 1 possesses two stereogenics centers at C-6 and C-7, therefore four stereoisomers will be assumed namely: 6S,7S; 6R,7R; 6R,7S; 6S,7R.The experimental ECD data of 1 revealed a sequential positive and negative Cotton effects (CE) at 256 and 220 nm, respectively.These effects are due to π→π* transitions of diene group.The in silico calculation of ECD spectra for all possible stereoisomers are represented in Figure 3.Among the calculated ECD spectra the data from 6S,7S showed very close similarity with two sequential positive and negative CEs around 255 and 220 nm, where the others showed distinct spectra.Therefore, the absolute configuration of compound 1 was determined as methyl (6S,7S,2E,4E)-6,7-dihydroxy-4,6-dimethyl octanoate.
Tetraketides with methylation at C-2 and C-6, as well as at C-3 and C-6 are common in nature and are found as building blocks in epi-citreodiol, citreoviral, preaurovertin, aurovertin, citreoviridin, citreoviridinols and asteltoxin, citreodiol and 6,7-dihydroxy-3,6-dimethyl octa-2(E),4(E)-dienoic acid with important biological properties. 18However, tetraketides with methylation at C-4 and C-6 have been rarely reported in the literature.To the best of our knowledge and as far as the literature is concerned, they are only described as building blocks of coarctatin. 19ethyl (6S,7S,2E,4E)-6,7-dihydroxy-4,6-dimethyl octanoate (1) can be obtained by condensing four acetate units with methylation at C-4 and C-6.The base skeleton of this compound can be found in (R)-and (S)-N,R dimethylbenzylamines (DMBA), which are polypeptide macrolides that have apoptotic activity. 20nterestingly, reports on compounds similar to 1 suggest that the precursor of its biosynthesis is malonyl-CoA. 21he extract of half corn was obtained (without inoculation of the microorganisms), and an evaluation by 1 H NMR and HR-ESI-MS was performed aiming to evaluate the presence of substances 4 and 5 in the control.They were not observed, proving that they were products of biosynthesis of Saccharicola sp.

Conclusions
Five compounds including a new one were isolated from Saccharicola sp., an endophytic fungus associated with Eugenia jambolana, a vegetal species used in Brazilian traditional medicine.A few reports describe Saccharicola genus chemistry, therefore, this paper could contribute with chemistry data that can help studies about species from Saccharicola genus.The new natural products isolated methyl (6S,7S,2E,4E)-6,7-dihydroxy-4,6-dimethyl octanoate (1) belongs to a rarely class of tetraketides compounds with methylation at C-4 and C-6.Reports about similar compounds to 1 suggest that the precursor of its biosynthesis is malonyl-CoA.The studies with endophytic fungi of medicinal plants lead us to obtain unknown metabolites as well metabolites with biological activities, which increase the prospects of using endophytes providing benefits to humanity.

Figure 3 .
Figure 3.Comparison of of time-dependent density functional theory (TDDFT) calculated spectra for four possible stereoisomers (a) with experimental data of compound 1 (b).

Table 1 .
13R data for compound 1 in CD 3 OD ( 1 H NMR at 600 MHz and13C NMR at 150 MHz) NO: not observed; d: chemical shift; J: coupling constant.