Virescenosides from the Holothurian-Associated Fungus Acremonium striatisporum Kmm 4401

Ten new diterpene glycosides virescenosides Z9-Z18 (1–10) together with three known analogues (11–13) and aglycon of virescenoside A (14) were isolated from the marine-derived fungus Acremonium striatisporum KMM 4401. These compounds were obtained by cultivating fungus on wort agar medium with the addition of potassium bromide. Structures of the isolated metabolites were established based on spectroscopic methods. The effects of some isolated glycosides and aglycons 15–18 on urease activity and regulation of Reactive Oxygen Species (ROS) and Nitric Oxide (NO) production in macrophages stimulated with lipopolysaccharide (LPC) were evaluated.

During our ongoing search for new natural compounds from marine-derived fungi, we have investigated the strain Acremonium striatisporum KMM 4401 associated with the holothurian Eupentacta fraudatrix. Twenty-one new diterpene glycosides, virescenosides have previously been isolated from this strain under cultivation on solid rice medium and wort agar medium [10][11][12]. Virescenosides Z 5 and Z 7 exhibited an unusual 16-chloro-15-hydroxyethyl group as their side chains in aglycones [12]. So, we attempted directed biosynthesis for the production of other halogenated compounds by culturing the fungus Acremonium striatisporum KMM 4401 in media containing potassium bromide. Unfortunately, we were unable to obtain glycoside derivatives with the incorporation of a bromine atom in a molecule structure. Chromatographic separation of the CHCl 3 -EtOH extract of the culture of fungus has now led to the isolation of ten undescribed diterpene glycosides virescenosides Z 9 -Z 18 EtOH extract of the culture of fungus has now led to the isolation of ten undescribed diterpene glycosides virescenosides Z9-Z18 (1-10) ( Figure 1) together with known virescenosides F (11) and G (12), lactone of virescenoside G (13) and aglycon of virescenoside A (14) ( Figure S1).

Results and Discussion
The CHCl3-EtOH (2:1, v/v) extract of the culture of A. striatisporum was separated by lowpressure reversed-phase column chromatography on Teflon powder Polycrome-1 followed by Si gel flash column chromatography and then by RP HPLC to yield individual compounds 1-14 as colorless, amorphous solids.
The molecular formula of virescenoside Z9 (1) was determined as C26H42O11 based on the analysis of HRESIMS (m/z 529.2656 [M-H] -, calcd for C26H41O11, 529.2654) and NMR data. A close inspection of the 1 H and 13 C NMR data (Tables 1 and 2

Results and Discussion
The CHCl 3 -EtOH (2:1, v/v) extract of the culture of A. striatisporum was separated by low-pressure reversed-phase column chromatography on Teflon powder Polycrome-1 followed by Si gel flash column chromatography and then by RP HPLC to yield individual compounds 1-14 as colorless, amorphous solids.
In HRESIMS virescenoside Z10 (2) gave a quasimolecular ion at m/z 493.2446 [M-H] -. These data, coupled with 13 C NMR spectral data (DEPT), established the molecular formula of 2 as C26H38O9. 1 H and 13 C NMR spectra of 2 (Tables 1 and 2; Figures S9-S13) indicated the presence of a ∆ 15 -pimarenetype aglycon possessing primary alcohol on a quaternary carbon (AB system, coupling at 3.73 d, 10.2 Hz and 4.17 d, 10.2 Hz) and one secondary alcohol function at δC 80.0. The remaining functionality, corresponding to the carbon signals at δ 202.9 (C), 168.7 (C) and 130.3 (C), suggested the presence of the tetrasubstituted enone chromophore. The structure of the aglycon part of 2 was found by extensive NMR spectroscopy to be the same as that of virescenoside P [17]. Interpretation of the COSY data gave rise to spin systems for monosaccharide involving one anomeric proton, four oxymethines and protons of a hydroxymethyl group. A comparison of the 13 C NMR spectrum of 1 with the data published for α-D-altropyranoses and β-D-altropyranoses as well as a good coincidence of carbon signals due to the glycosidic moiety with those of virescenosides O, T, W [10] together with magnitudes of 1 H-1 H spin coupling constants in 1 H NMR spectra of 1 elucidated the presence of a β-D-altropyranoside unit of 4 C 1 form in 1. A long-range correlation H-1 (δ H 5.43)/C-19 as well as the NOESY cross-peak between H-1 and H-19a and downfield chemical shift of C-19 (δ C 74.0) revealed a linkage between the altrose and aglycon. Thus, the structure of virescenoside Z 9 (1) was represented as 19-O-β-D-altropyranosyl-7-oxo-isopimara-15-en-2α,3β,6α,8β-tetraol.
In HRESIMS virescenoside Z 10 (2) gave a quasimolecular ion at m/z 493.2446 [M-H] − . These data, coupled with 13 C NMR spectral data (DEPT), established the molecular formula of 2 as C 26 H 38 O 9 . 1 H and 13 C NMR spectra of 2 (Tables 1 and 2; Figures S9-S13) indicated the presence of a ∆ 15 -pimarene-type aglycon possessing primary alcohol on a quaternary carbon (AB system, coupling at 3.73 d, 10.2 Hz and 4.17 d, 10.2 Hz) and one secondary alcohol function at δ C 80.0. The remaining functionality, corresponding to the carbon signals at δ 202.9 (C), 168.7 (C) and 130.3 (C), suggested the presence of the tetrasubstituted enone chromophore. The structure of the aglycon part of 2 was found by extensive NMR spectroscopy to be the same as that of virescenoside P [17].  The HRESIMS of virescenosides Z 11 (3) showed the quasimolecular ion at m/z 509.2408 [M-H] − . These data, coupled with 13 C NMR spectral data (DEPT), established the molecular formula of 3 as C 26 H 38 O 10 . The structure of the aglycon moiety of 3 was found by extensive NMR spectroscopy ( 1 H, 13 C, HSQC, HMBC and NOESY) (Tables 1 and 2; Figures S14-S18) to be the same as those of virescenoside M [18].
The molecular formula of virescenoside Z 15 (7) was determined as C 27 (Tables 1 and 3; Figures S34-S38) observed for the aglycon part of 7 closely resembled those obtained for virescenoside Z 10 (2) with the exception of the C-1-C-4 carbon and proton signals of ring A. The HMBC correlations from H-5 (δ H 1.75) to C-3 (δ C 84.4), H-3 (δ H 3.00) and from H 2 -1 (δ H 1.23, 2.18) to C-2 (δ C 69.2) and downfield chemical shifts of C-2 placed an additional hydroxy group at C-2 of ring A. The relative stereochemistry of protons on C-2 and C-3 was defined based on the 1 H-1 H coupling constant (J=9.8) and assigned as axial. Previously, a similar aglycon has been described for virescenoside M [10].
The HRESIMS of virescenoside Z 16 (8) showed the quasimolecular at m/z 515.2617 [M + Na] + . These data, coupled with 13 C NMR spectral data (DEPT), established the molecular formula of 8 as C 27 H 40 O 8 (Tables 1 and 3). The structure of the aglycon moiety of 8 was found by 2D NMR experiments ( Figures S39-S43) to be the same as that of virescenoside Z 4 [12].
The HRESIMS of virescenoside Z 18 (10) showed the quasimolecular at m/z 517.2773 [M + Na] + . These data, coupled with 13 C NMR spectral data (DEPT), established the molecular formula of 10 as C 27 H 42 O 8 . The 1 H and 13 C NMR data observed for the aglycon part of 10 (Tables 1 and 3; Figures S49-S54) matched those reported for virescenoside Q [17]. Initial examination of the 1-D proton and one bond correlation NMR data suggested the presence of one sugar (anomeric signal at δ H 4.97, δ C 103.5). The 1 H and 13 C NMR spectra of the sugar part of 10 indicated the presence of the methoxycarbonyl group (δ H 3.64, δ C 51.8, 170.7). A comparison of the 13 C NMR spectrum with the data published for αand β-D-mannopyranoses as well as a good coincidence of carbon signals C-1 -C-4 with those of virescenoside Q together with magnitudes of 1 H-1 H spin coupling constants in 1 H NMR spectrum of 10 elucidated the presence of β-D-mannouronopyranoside unit of 4 C 1 form in 10 [17,22,23]. A long-range correlation H-1 (δ H 4.97)/C-19 (δ C 72.1) as well as the NOESY cross-peak between H-1 and H-19a (δ H 4.26) and downfield chemical shifts of C-19 indicated that sugar moiety was linked at C-19. Thus, the structure of virescenoside Z 18 (10) was determined as 19-O-[(methyl-β-D-mannopyrananosyl)-uronat]-isopimara-7,15-dien-3β-ol.
Since methanol is used in the isolation procedure of virescenosides, it is possible that the methyl esters of the sugar units may be obtained during the course of isolation. Therefore, we separated the part of subfraction II by RP-HPLC using acetonitrile instead of methanol and obtain virescenosides Z 12 (4) and Z 13 (5) which were characterized by 1 H and 13 C NMR spectra. Furthermore, we observed compounds 4-8 and 10 in subfraction II by HPLC-MS method (See Supplementary Figure S2).
The structures of known compounds virescenosides F (11) and G (12), lactone of virescenoside G (13) [19] and aglycon of virescenoside A (14) [13] (See Supplementary Figure S1) were determined based on HRESIMS and NMR data and comparison with literature. The aglycons of virescenosides B (15, 16), C (17) and M (18) (See Supplementary Figure S1, Experimental Section) were prepared as a result of acid hydrolysis of the corresponding glycosides for examination of their biological activity.
Next, we investigated the effects of some isolated compounds and aglycones 15-18 on urease activity and regulation of ROS and NO production in macrophages stimulated with lipopolysaccharide (LPS).
The development of urease inhibitors, usually considered as antiulcer agents, carries a significant interest for medicinal chemists. Urease is an enzyme that is clinically used as diagnostic to determine the presence of pathogens in the gastrointestinal and urinary tracts. It has been described that the bacterial urease causes many clinically harmful infections, like stomach cancer, infectious stones and peptic ulcer formation in human and animal health [24]. Urease is also involved in the pathogenesis of hepatic coma, urolithiasis, urinary catheter encrustation and oral cavity infections by hydrolyzing the salivary urea [25].

General Experimental Procedures
Optical rotations were measured on a Perkin-Elmer 343 polarimeter (Perkin Elmer, Waltham, MA, USA). UV spectra were recorded on a Shimadzu UV-1601PC spectrometer (Shimadzu Corporation, Kyoto, Japan) in methanol. NMR spectra were recorded in CD 3 OD, CDCl 3 , DMSO-d 6 and C 5 D 5 N on a Bruker DPX-500 (Bruker BioSpin GmbH, Rheinstetten, Germany) and a Bruker DRX-700 (Bruker BioSpin GmbH, Rheinstetten, Germany) spectrometer, using TMS as an internal standard. The Bruker Impact II Q-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) was used to record the MS and MS/MS spectra within m/z range 50-1500. The capillary voltage was set to 1300 V, and the drying gas was heated to 150 • C at the flow rate 3 L/min. Collision-induced dissociation (CID) product ion mass spectra were obtained using nitrogen as the collision gas. The instrument was operated using the program otofControl (ver. 4.0, Bruker Daltonics, Bremen, Germany) and the data were analyzed using the DataAnalysis Software (ver. 4.3, Bruker Daltonics, Bremen, Germany).

Extraction and Isolation
At the end of the incubation period, the mycelium and medium were homogenized and extracted three times with a mixture of CHCl 3 -EtOH (2:1, v/v, 2.5 L). The combined extracts (4.5 g) were concentrated to dryness and separated by low pressure RP CC (the column 20 × 8 cm) on Polychrome-1 Teflon powder in H 2 O and 50% EtOH. After elution of inorganic salts and highly polar compounds by H 2 O, 50% EtOH was used to obtain the fraction of amphiphilic compounds, including the virescenosides. After evaporation of the solvent, the residual material (2.6 g) was subjected to Si gel flash CC (7 × 13 cm) chromatography with a solvent gradient system of increasing polarity from 10 to 60% EtOH in CHCl 3

Spectral Data
Virescenoside

Statistical Analysis
Average value, standard error, standard deviation and p-values in all experiments were calculated and plotted on the chart using SigmaPlot 3.02 (Jandel Scientific, San Rafael, CA, USA). Statistical difference was evaluated by t-test, and results were considered as statistically significant at p < 0.05.