Elsinopirins A–D, Decalin Polyketides from the Ascomycete Elsinoё pyri

In course of our screening for new secondary metabolites from ecological niche specialized, phytopathogenic fungi, the plant pathogen Elsinoё pyri, strain 2203C, was found to produce four novel compounds (1–4), which were named elsinopirins A–D, in addition to the known metabolite elsinochrome A (5). After isolation by preparative high-performance liquid chromatography (HPLC), their structures, including relative stereochemistry, were elucidated by 1D and 2D nuclear magnetic resonance (NMR) and mass spectrometry (MS) data. Finally, absolute stereochemistry was assigned by chemical shifts of Mosher’s esters (α-methoxy-α-trifluoromethylphenylacetic acid; MTPA) derivatives of elsinopirin B (2). The compounds were found to be devoid of significant antibacterial, antifungal, and cytotoxic activities.


Introduction
Elsino ë pyri is a fruit and leaf pathogen causing anthracnose disease symptoms and spots on apple and pear in temperate regions worldwide.The species is easily overlooked and its disease symptoms can be confused with the common pathogen Venturia inaequalis, the major apple scab agent in commercial production settings.Disease management strategies applied against V. inaequalis effectively suppress E. pyri, as the latter is commonly encountered in ecological production settings or private gardens [1][2][3].The host specificity of Ep, its ability to compete with other plant cuticle-inhabiting fungi, and its slow in vitro growth triggered our interest in the secondary metabolite production of this fungus.According to our literature search, the species was hitherto untapped with respect to secondary metabolites.However, some pigments belonging to the class of perylene quinones named elsinochromes had been reported previously from a different species of Elsino ë [4,5].We recently found moderate antimicrobial effects in cultures of strain 2202C, which was identified as E. pyri in our ongoing studies on bioactive compounds from plant-associated Ascomycota [6][7][8].To assess its potential for secondary metabolite production, strain 2203C was cultivated on potato-dextrose-agar (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) plate medium; extracts showed the presence of five metabolites that were isolated using preparative high-performance liquid chromatography (HPLC) (Figure 1).Herein we describe their isolation, structure elucidation, and biological characterization.

Structure Elucidation
Elsinopirin A (1) was isolated as a colorless oil; its HRESIMS (high-resolution electrospray ionization mass spectrometry) data indicated a molecular formula of C19H28O3, indicating a molecule with six degrees of unsaturation. 1 H and HSQC (heteronuclear single quantum correlation spectroscopy) spectra revealed the presence of four methyl, two methylene, four olefinic, and seven aliphatic methine groups.The 13 C spectrum furthermore indicated the presence of one ketone and one carboxylic acid.Starting from the methyls 16-H3, 17-H3, 18-H3, 19-H3 and the olefinic protons 11-H-15-H, one large spin system was assembled by 1 H, 1 H correlation spectroscopy (COSY) and total correlated spectroscopy (TOCSY) correlations (Figure 2). 1 H, 13 C heteronuclear multiple bond (HMBC) correlations from 13-H/14-H to C-15 connected the carboxylic acid group to the trans diene (Δ 11,12 15.2 Hz, Δ 13,14 15.3 Hz), and correlations from 1-H, 2-H, 3-H, 17-H3 as well as 5-H, 9-H2, 10-H resulted in the assignment of the planar structure of 1.The relative configuration was addressed by 1 H, 1 H ROESY (rotating-frame nuclear Overhauser effect correlation spectroscopy) correlations and coupling constants.The large coupling constants of 4-H, 5-H,6-H, 7-Hb, 8-H, 9-Hb, 10-H around 10 Hz, which were confirmed by a J-resolved spectrum due to signal overlaps, indicated axial orientations of these atoms.Consequently, equatorial orientations of 18-H3, 19-H3 and the pentenoic side chain were deduced.ROESY correlations (Figure 3) were observed in ring B between 5-H, 7-Hb, 9-Hb on the β face of the molecule, and between 6-H, 8-H, and 10-H on the α face.For ring A, ROESY correlations between 2-H, 4-H, and 10-H indicated pseudoaxial orientations for these protons on the α face, resulting in equatorial orientations of 17-H3 and the pentenoic side chain.Moreover, a strong ROESY correlation was observed between 5-H and 16-H3 on the β face, indicating an axial orientation of 16-H3.Since the absolute stereochemistry of the metabolite family was established with 2 by Mosher's method (see below), the configuration of elsinopirin A (1) was assigned as 2R, 3R, 4S, 5R, 6R, 8S, 10S.

Structure Elucidation
Elsinopirin A (1) was isolated as a colorless oil; its HRESIMS (high-resolution electrospray ionization mass spectrometry) data indicated a molecular formula of C 19 H 28 O 3 , indicating a molecule with six degrees of unsaturation. 1 H and HSQC (heteronuclear single quantum correlation spectroscopy) spectra revealed the presence of four methyl, two methylene, four olefinic, and seven aliphatic methine groups.The 13 C spectrum furthermore indicated the presence of one ketone and one carboxylic acid.Starting from the methyls 16-H 3 , 17-H 3 , 18-H 3 , 19-H 3 and the olefinic protons 11-H-15-H, one large spin system was assembled by 1 H, 1 H correlation spectroscopy (COSY) and total correlated spectroscopy (TOCSY) correlations (Figure 2). 1 H, 13 C heteronuclear multiple bond (HMBC) correlations from 13-H/14-H to C-15 connected the carboxylic acid group to the trans diene (∆ 11,12 15.2 Hz, ∆ 13,14 15.3 Hz), and correlations from 1-H, 2-H, 3-H, 17-H 3 as well as 5-H, 9-H 2 , 10-H resulted in the assignment of the planar structure of 1.The relative configuration was addressed by 1 H, 1 H ROESY (rotating-frame nuclear Overhauser effect correlation spectroscopy) correlations and coupling constants.The large coupling constants of 4-H, 5-H,6-H, 7-Hb, 8-H, 9-Hb, 10-H around 10 Hz, which were confirmed by a J-resolved spectrum due to signal overlaps, indicated axial orientations of these atoms.Consequently, equatorial orientations of 18-H 3 , 19-H 3 and the pentenoic side chain were deduced.ROESY correlations (Figure 3) were observed in ring B between 5-H, 7-Hb, 9-Hb on the β face of the molecule, and between 6-H, 8-H, and 10-H on the α face.For ring A, ROESY correlations between 2-H, 4-H, and 10-H indicated pseudoaxial orientations for these protons on the α face, resulting in equatorial orientations of 17-H 3 and the pentenoic side chain.Moreover, a strong ROESY correlation was observed between 5-H and 16-H 3 on the β face, indicating an axial orientation of 16-H 3 .Since the absolute stereochemistry of the metabolite family was established with 2 by Mosher's method (see below), the configuration of elsinopirin A (1) was assigned as 2R, 3R, 4S, 5R, 6R, 8S, 10S.
The molecular formula C 19 H 28 O 4 of elsinopirin B (2) indicated the presence of an additional oxygen atom compared to 1.The nuclear magnetic resonance (NMR) spectra of 2 were highly similar to those of 1, with the key difference being the replacement of one methylene by an oxymethine group.Since both methyls 18-H 3 and 19-H 3 showed HMBC correlations to this additional oxymethine, it was identified as C-7.Because 7-H shows two large coupling constants around 9 Hz to both 6-H and 8-H, and ROESY correlations to 5-H/18-H 3 /19-H 3 , a β-axial orientation was assigned for 7-H.The secondary hydroxyl function of 2 was utilized to assign the absolute configuration of the elsinopyirin family by Mosher's method.The positive ∆δ SR values of 6-H/19-H 3 and negative ones for 8-H/9-H 2 /18-H 3 were the characteristic for a 7R configuration (Figure 4).In this experiment, COSY and TOCSY determined the chemical shifts of protons in the vicinity of the secondary alcohol function of C-7.An HMBC correlation from 18-H 3 to methylene C-9, which was clearly identified by a phase-sensitive HSQC experiment, was utilized to distinguish 18-H 3 from 19-H 3 .
Hz, which were confirmed by a J-resolved spectrum due to signal overlaps, indicated axial orientations of these atoms.Consequently, equatorial orientations of 18-H3, 19-H3 and the pentenoic side chain were deduced.ROESY correlations (Figure 3) were observed in ring B between 5-H, 7-Hb, 9-Hb on the β face of the molecule, and between 6-H, 8-H, and 10-H on the α face.For ring A, ROESY correlations between 2-H, 4-H, and 10-H indicated pseudoaxial orientations for these protons on the α face, resulting in equatorial orientations of 17-H3 and the pentenoic side chain.Moreover, a strong ROESY correlation was observed between 5-H and 16-H3 on the β face, indicating an axial orientation of 16-H3.Since the absolute stereochemistry of the metabolite family was established with 2 by Mosher's method (see below), the configuration of elsinopirin A (1) was assigned as 2R, 3R, 4S, 5R, 6R, 8S, 10S.The molecular formula C19H28O4 of elsinopirin B (2) indicated the presence of an additional oxygen atom compared to 1.The nuclear magnetic resonance (NMR) spectra of 2 were highly similar to those of 1, with the key difference being the replacement of one methylene by an oxymethine group.Since both methyls 18-H3 and 19-H3 showed HMBC correlations to this additional oxymethine, it was identified as C-7.Because 7-H shows two large coupling constants around 9 Hz to both 6-H and 8-H, and ROESY correlations to 5-H/18-H3/19-H3, a β-axial orientation was assigned for 7-H.The secondary hydroxyl function of 2 was utilized to assign the absolute configuration of the elsinopyirin family by Mosher's method.The positive Δδ SR values of 6-H/19-H3 and negative ones for 8-H/9-H2/18-H3 were the characteristic for a 7R configuration (Figure 4).In this experiment, COSY and TOCSY determined the chemical shifts of protons in the vicinity of the secondary alcohol function of C-7.An HMBC correlation from 18-H3 to methylene C-9, which was clearly identified by a phase-sensitive HSQC experiment, was utilized to distinguish 18-H3 from 19-H3.The molecular formula C19H28O4 of elsinopirin B (2) indicated the presence of an additional oxygen atom compared to 1.The nuclear magnetic resonance (NMR) spectra of 2 were highly similar to those of 1, with the key difference being the replacement of one methylene by an oxymethine group.Since both methyls 18-H3 and 19-H3 showed HMBC correlations to this additional oxymethine, it was identified as C-7.Because 7-H shows two large coupling constants around 9 Hz to both 6-H and 8-H, and ROESY correlations to 5-H/18-H3/19-H3, a β-axial orientation was assigned for 7-H.The secondary hydroxyl function of 2 was utilized to assign the absolute configuration of the elsinopyirin family by Mosher's method.The positive Δδ SR values of 6-H/19-H3 and negative ones for 8-H/9-H2/18-H3 were the characteristic for a 7R configuration (Figure 4).In this experiment, COSY and TOCSY determined the chemical shifts of protons in the vicinity of the secondary alcohol function of C-7.An HMBC correlation from 18-H3 to methylene C-9, which was clearly identified by a phase-sensitive HSQC experiment, was utilized to distinguish 18-H3 from 19-H3.Elsinopirin C (3) was analyzed for the same molecular formula C19H28O4 as elsinopirin B (2).Again, the NMR spectra of 3 were highly similar to those of 1.The key difference was identified in the replacement of a methine by an oxygenated quaternary carbon atom.This was located to be C-8 by an HMBC correlation of 18-H3.Because 8-OH exhibits ROESY correlations to 6-H and 10-H, whereas 18-H3 correlated to 7-Ha and 9-Ha, an 8R configuration was assigned for C-8.Elsinopirin C (3) was analyzed for the same molecular formula C 19 H 28 O 4 as elsinopirin B (2).Again, the NMR spectra of 3 were highly similar to those of 1.The key difference was identified in the replacement of a methine by an oxygenated quaternary carbon atom.This was located to be C-8 by an HMBC correlation of 18-H 3 .Because 8-OH exhibits ROESY correlations to 6-H and 10-H, whereas 18-H 3 correlated to 7-H a and 9-H a , an 8R configuration was assigned for C-8.
Elsinopirin D (4) was analyzed as C 17 H 26 O 3 by HRESIMS.This accounted for the formal loss of a C 2 H 2 fragment compared to the parental metabolite 1.The NMR spectra of 4 were highly similar to those of 1, with the key difference being the presence of only two olefinic methines instead of four.Consequently, 4 was assigned the same metabolite core as 1, but bearing a propenoic acid instead of the pentadienoic side chain.Nearly identical 13 C chemical shifts indicated identical relative and absolute configurations.

Semisynthetic Conversions
Since a methyl ester moiety was proven to be necessary for the activity of a structurally close relative of 1-4 [9], methyl esters of elsinopirins A-D (6-9) were generated by the methylation of 1-4 through diazomethane (Figure 5).Elsinopirin D (4) was analyzed as C17H26O3 by HRESIMS.This accounted for the formal loss of a C2H2 fragment compared to the parental metabolite 1.The NMR spectra of 4 were highly similar to those of 1, with the key difference being the presence of only two olefinic methines instead of four.Consequently, 4 was assigned the same metabolite core as 1, but bearing a propenoic acid instead of the pentadienoic side chain.Nearly identical 13 C chemical shifts indicated identical relative and absolute configurations.

Semisynthetic Conversions
Since a methyl ester moiety was proven to be necessary for the activity of a structurally close relative of 1-4 [9], methyl esters of elsinopirins A-D (6-9) were generated by the methylation of 1-4 through diazomethane (Figure 5).

Discussion
Elsinopirins A-D (1-4) belong to a group of metabolites containing the 'decalin' motif [11].This group comprises compounds from the isoprenoid and polyketide pathways and includes the famous compound lovastatin.Interestingly, the polyketide decalin skeleton is believed to be biosynthesized by an enzymatic intramolecular Diels-Alder reaction.More specifically, 1-4 belong to a subgroup possessing a penta-2,4-dienoic side chain connected to the decalin core.The majority of these were detected in cultures of Penicillium species and show a plethora of interesting bioactivities.Tanzawaic

Discussion
Elsinopirins A-D (1-4) belong to a group of metabolites containing the 'decalin' motif [11].This group comprises compounds from the isoprenoid and polyketide pathways and includes the famous compound lovastatin.Interestingly, the polyketide decalin skeleton is believed to be biosynthesized by an enzymatic intramolecular Diels-Alder reaction.More specifically, 1-4 belong to a subgroup possessing a penta-2,4-dienoic side chain connected to the decalin core.The majority of these were detected in cultures of Penicillium species and show a plethora of interesting bioactivities.Tanzawaic acid B from Penicillium citrinum inhibited superoxide anion production in human neutrophils [12].Tanzawaic acids A, E, and K showed inhibition of the conidial germination in the rice blast fungus Magnaporthe oryzae in concentrations of about 25 µg/mL [13].Hynapenes A-C, isolated from a soil-inhabiting Penicillium species, were effective against monensin-resisant Eimeria tenella [14].
However, no bioactivity could be detected for the elsinopirins A-D (1-4).In the case of the structurally closely related metabolite coprophilin, the metabolites carboxylic acid moiety was methylated and showed anticoccidial activity [9], whereas its free acid derivative was inactive.It was speculated that the differences in biological activity are mainly based on differences in cellular uptake.Consequently, the free carboxylic acids 1-4 were methylated by diazomethane.However, the methyl ester derivatives 6-9 did not show any activity in our broad test panel either.Therefore, we cannot speculate deeply about the biological function of these metabolites.

Fungal Material
Ripe apples showing the colonization of sooty blotch fungi and scab-like symptoms were collected in Vesela Gora, Slovenia, by H. J. Schroers in October 2009.The E. pyri strain 2203C was isolated by Ajda Medjedović.Taxonomic identification was based on morphological characters and confirmed by generating the DNA barcode sequence of the internal transcribed spacer regions 1 and 2 and the 5.8S rDNA of the rRNA gene cluster using reference sequences KX887267 and KX887268 from Gen Bank (www.ncbi.nlm.nih.gov) of strains CBS 163.29 and CBS 179.82 [3].

Fermentation and Downstream Processing
The solid-state cultivation of strain 2203C was carried out on PD agar plates (Potato Dextrose Agar, Carl Roth).The plates (ca.30 mL medium) were inoculated with 3 mL of a liquid pre-culture in YMG medium (1.0% malt extract, 0.4% glucose, 0.4% yeast extract, pH 6.3).The agar of the culture changed to a red color and was harvested after 14 days of growth at 23 • C.
The agar plates were cut into small pieces and poured into a glass bottle, overlaid with ca. 100 mL of ethyl acetate.The bottle was then incubated in an ultrasonic bath for 30 min.Thereafter, the ethyl acetate was filtered and evaporated in vacuo to yield 72.7 mg of an oily crude extract.
The crude extract was dissolved in 1 mL of methanol and filtered through a reversed phase solid phase cartridge (Strata-X 33 mm, Polymeric Reversed Phase; Phenomenex, Aschaffenburg, Germany).The filtrate was subsequently subjected to preparative RP HPLC using a Kromasil precolumn (Kromasil, Amsterdam, The Netherlands, 100 C18; 50 × 20 mm; 7 µm) and a Nucleodur column (Nucleodur 100 C18ec; 250 × 21 mm; 5 µm) with the following conditions: mobile phase with solvent A (deionized water) and solvent B (acetonitrile), linear gradient of solvent B from 40 to 80% in 45 min, an increase to 100% in 5 min, followed by isocratic conditions at 100% solvent B for 6 min, flow rate of 15 mL/min, UV peak detection at 254 and 210 nm.The obtained fractions were combined according to the main peaks, leading to four main fractions which were evaporated and analyzed by HPLC-MS.The fraction with a retention time of 22 min contained 4 (1.6 mg), the one with 28 min contained 1 (1.8 mg), and the one with 34 min contained metabolite 5 (2.0 mg).The first fraction, which was eluted at around 10 min, contained two different substances (2 and 3) and was therefore forwarded to another preparative HPLC.The conditions were the same as before, besides the gradient which was set to a linear gradient of solvent B from 20 to 45% followed by an increase to 100% in 5 min and isocratic conditions for 6 min.The fractions were again combined according to the peaks, evaporated, and analyzed by HPLC-MS to yield 2 (2.4 mg) and 3 (2.7 mg).Five pure compounds were finally obtained, and their physicochemical characteristics are summarized below.NMR and mass spectra are provided in the Supplementary Materials.  1C NMR (175 MHz, CDCl 3 ): see Table 1; 1 H NMR (700 MHz, CDCl 3 ): see Table 2. HRESIMS m/z 305.2110 ([M + H] + , calcd for C 19 H 29 O 3 305.2111).

Semisynthetic Conversions
Diazomethane (0.4 mmol) in ether (1 mL), which was freshly produced in a Sigma-Aldrich (Sigma-Aldrich Corporation, Deisenhofen, Germany) Diazomethane Generator according to the manufacturer's instructions, was added stepwise to 1 (1.0 mg) in aqueous methanol (50%, 1 mL) at 0 • C. The reaction was monitored by analytical HPLC and stopped by removing the solvent in vacuo after all starting material had been converted.This provided the methyl ester of elsinopirin A (6) without further purification.

Serial Dilution and Cytotoxicity Assay
Minimum inhibitory concentrations were determined in a serial dilution assay carried out in a manner similar to that previously described [10].Various test organisms were used to assess the antibacterial and antifungal activities.The in vitro cytotoxicity assay with the mouse cell line L929 was performed as previously described [10].

Conclusions
In summary, we isolated four novel metabolites from E. pyri strain 2203C, and elucidated their structures including absolute stereochemistry.Since no significant bioactivity was detected, their ecological role remains elusive.

Figure 3 .
Figure 3. ROESY (rotating-frame nuclear Overhauser effect correlation spectroscopy) correlations indicative for the relative configuration of elsinopirin A (1); solid arrows are above main plane; dotted arrows are below.

Figure 3 .
Figure 3. ROESY (rotating-frame nuclear Overhauser effect correlation spectroscopy) correlations indicative for the relative configuration of elsinopirin A (1); solid arrows are above main plane; dotted arrows are below.

Figure 3 .
Figure 3. ROESY (rotating-frame nuclear Overhauser effect correlation spectroscopy) correlations indicative for the relative configuration of elsinopirin A (1); solid arrows are above main plane; dotted arrows are below.