molecules Article Characterization of Eight Novel Spiroleptosphols from Fusarium avenaceum Klaus Ringsborg Westphal 1 , Manuela Ilse Helga Werner 1, Katrine Amalie Hamborg Nielsen 1 , Jens Laurids Sørensen 2 , Valery Andrushchenko 3,* , Jacob Winde 1, Morten Hertz 1, Mikkel Astrup Jensen 1, Mathilde Lauge Mortensen 1, Petr Bouˇr 3 , Teis Esben Sondergaard 1 and Reinhard Wimmer 1,* 1 Department of Chemistry and Bioscience-Section for Biotechnology, Aalborg University, Frederik Bajers Vej 7H, 9220 Aalborg Ø, Denmark; [email protected] (K.R.W.); [email protected] (M.I.H.W.); [email protected] (K.A.H.N.); [email protected] (J.W.); [email protected] (M.H.); [email protected] (M.A.J.); [email protected] (M.L.M.); [email protected] (T.E.S.) 2 Department of Chemistry and Bioscience, Aalborg University, Niels Bohrs Vej 8, 6700 Esbjerg, Denmark; [email protected] 3 Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo námˇestí 542/2, 166 10 Prague 6, Czech Republic; [email protected] * Correspondence: [email protected] (V.A.); [email protected] (R.W.); Tel.: +45-9940-8518 (R.W.) Received: 4 September 2019; Accepted: 24 September 2019; Published: 26 September 2019 Abstract: Chemical analyses of Fusarium avenaceum grown on banana medium resulted in eight novel spiroleptosphols, T1, T2 and U–Z (1–8). The structures were elucidated by a combination of high-resolution mass spectrometric data and 1- and 2-D NMR experiments. The relative stereochemistry was assigned by 1H coupling and NOESY/ROESY experiments. Absolute stereochemistry established for 7 by vibrational circular dichroism was found analogous to that of the putative polyketide spiroleptosphol from Leptosphaeria doliolum. Keywords: spiroleptosphol; secondary metabolites; polyketides; polyketide synthases; PKS; Fusarium avenaceum 1. Introduction Filamentous fungi of the genus Fusarium are notorious plant pathogens, causing serious losses of grain every year world-wide. In addition, the fungi produce several harmful compounds during plant infection, which can cause illnesses in both humans and animals. Some of these natural products belong to the group of polyketides (PKs), which are biosynthesized by enzymatic machinery based on the polyketide synthases (PKSs). More than 65 different genes encoding PKSs have so far been identified across the Fusarium genus, of which F. avenaceum is one of the strains with most PKS genes but only a few products are known [1,2]. PKs are a structurally diverse group of compounds named after their biosynthetic origin, where they are assembled from small malonyl and acetyl subunits. Examples of beneficial and harmful PKs from Fusarium are fusarielins, aurofusarin and gibepyrons, which have been shown to have an estrogenic effect on cancer cells, inhibiting probiotic bacteria and nematocidal activity, respectively [3–7]. The secondary metabolome from fungi seldom includes all PKs that potentially could be produced, hence different approaches are needed for activation of responsible gene clusters. One way to trigger gene activation is utilizing different media compositions termed the OSMAC (one strain many compounds) approach [8,9]. A considerable difference between secondary metabolite profiles in fusaria has been demonstrated previously by altering the yeast extract from different suppliers in the growth media or by growth on unusual media [10,11]. Molecules 2019, 24, 3498; doi:10.3390/molecules24193498 www.mdpi.com/journal/molecules Molecules 2019, 24, 3498 2 of 12 Molecules 2019, 24, x 2 of 12 To document the secondary metabolite potential of Fusarium when grown on different media, we isolatedTo eight document related the PKs secondary not reported metabolite before ( 1potential–8). For this of Fusarium purpose, whenF. avenaceum grownwas on different grown on media, banana agarwe isolated medium. eight Below, related we describePKs not reported in detail thebefore structure (1–8). For elucidation this purpose, of 1– 8F.using avenaceum HRMS was and grown NMR. Theon banana relative agar and absolutemedium. stereochemistry Below, we describe was establishedin detail the by structure NMR and elucidation vibrational of circular1–8 using dichroism HRMS (VCD)and NMR. spectroscopies. The relative These and eight absolute compounds stereochem haveistry similar was structural established features by NMR with and spiroleptosphol, vibrational acircular cytotoxic dichroismγ-methylindene-spiro (VCD) spectroscopies. butanolide These isolated eight fromcompounds the ascomycetous have similar fungus structuralLeptosphaeria features doliolumwith spiroleptosphol,and analogous a thereofcytotoxic (Figure γ-methylindene-spiro1)[ 12–23]. Therefore, butanolide they were isolated named from spiroleptosphol the ascomycetous U–Z, withfungus spiroleptosphol Leptosphaeria Tdoliolum existing and in twoanalogous diastereomers thereof (Figure called T1 1) and[12–23]. T2. Therefore, they were named spiroleptosphol U–Z, with spiroleptosphol T existing in two diastereomers called T1 and T2. FigureFigure 1.1. SpiroleptospholsSpiroleptosphols and analogous compounds kn knownown from from literature. literature. 2. Results and Discussion 2. Results and Discussion In an effort to identify the chemical nature of Fusarium secondary metabolism, several isolates In an effort to identify the chemical nature of Fusarium secondary metabolism, several isolates were grown on a variety of different media. The resulting metabolite extracts were profiled using were grown on a variety of different media. The resulting metabolite extracts were profiled using HPLC-DAD-HRMS. Several unusual metabolites were observed (m/z between 250–350 Da) when HPLC-DAD-HRMS. Several unusual metabolites were observed (m/z between 250–350 Da) when F. F. avenaceum strain 05001 was cultivated on solid banana medium (Figure2a,b). Augmented cultivation avenaceum strain 05001 was cultivated on solid banana medium (Figure 2a,b). Augmented of F. avenaceum and metabolite extraction followed by compound isolation by semi-preparative RP cultivation of F. avenaceum and metabolite extraction followed by compound isolation by chromatography resulted in PKs 1–8 (Figure2c). The structures of 1–8 were all elucidated by 1D- and semi-preparative RP chromatography resulted in PKs 1–8 (Figure 2c). The structures of 1–8 were all 7 2Delucidated NMR spectroscopy by 1D- and and2D NMR the absolute spectroscopy configuration and the wasabsolute assigned configuration by VCD for was. assigned by VCD 2.1.for Spiroleptosphol7. U, T1 and T2 (1, 2 and 3) 2.1. CompoundsSpiroleptosphol1– U,3 eluted T1 and as T2 a (1, single 2 and broad 3) peak when analyzed by HPLC-HRMS. This would imply an equilibrium with relatively fast interchange between compounds. The HR-ESI(+)MS returned a Compounds 1–3 eluted as a single broad peak when analyzed by HPLC-HRMS. This would [M + H]+ at 279.1216 Da and a [M H O + H]+ at 261.1112 Da confirmed by a sodium adduct [M + Na]+ imply an equilibrium with relatively− 2 fast interchange between compounds. The HR-ESI(+)MS at 301.1036 Da. This, together with the isotope distribution, was consistent with a chemical formula of returned a [M + H]+ at 279.1216 Da and a [M − H2O + H]+ at 261.1112 Da confirmed by a sodium C H O . 1D and 2D NMR experiments revealed three compounds with similar characteristics of 1H adduct15 18 [M5 + Na]+ at 301.1036 Da. This, together with the isotope distribution, was consistent with a and 13C chemical shifts, 1H–1H couplings, COSY and 1H–13C HMBC correlation (Table1). For all three chemical formula of C15H18O5. 1D and 2D NMR experiments revealed three compounds with similar compounds,characteristics TOCSY of 1H /andCOSY 13C correlations chemical shifts, were 1H– observed1H couplings, for a series COSY of and ten 1 carbonH–13C HMBC atoms initiatingcorrelation at 1 2 5 6 a(Table methyl 1). group For all (H three) and compounds, proceeding throughTOCSY/COSY two trans- correlationsalkenes (Hwere− ),observed a methine for (H a ),series a cis -alkeneof ten 7 8 9 10 13 (Hcarbon− ) and atoms ending initiating with twoat a methinesmethyl group (H − (H)1 both) and withproceedingC chemical through shifts two resonatingtrans-alkenes between (H2−5), 75a 6 10 andmethine 84 ppm (H6), indicating a cis-alkene bonding (H7−8) and to oxygen. ending with HMBC two correlations methines (H from9−10) both H andwith H 13C tochemical a quaternary shifts 11 6 11 carbonresonating (C )between formed 75 a six-carbon and 84 ppm ring indicating from C bondingto C (Figure to oxyge3).n. Additionally, HMBC correlations a resonance from between H6 and 15 9 11 176H10and to a 178quaternary ppm (C carbon) displayed (C11) formed HMBC a correlationssix-carbon ring to C fromand C6 C to Cand11 (Figure can only 3). beAdditionally, explained bya 9 11 15 9 12 formingresonance a γ between-lactone 176 ring and consisting 178 ppm of (C C15)–C displayed,C and HMBC O . correlations Finally, a methine to C9 and (C C11) observedand can only as a 11 1 13 12 singletbe explained showed by HMBC forming correlations a γ-lactone to Cring. consistingH and C of chemical C9–C11, shiftsC15 and of CO9. suggestedFinally, a methine bonding (C to12 an) 13 oxygen.observed For as Compounda singlet showed1, a resonance HMBC correlations for C (δC to212.6) C11. 1H was and attributed 13C chemical to a shifts ketone of whichC12 suggested was not observedbonding forto an2 and oxygen.3. For Compound 1, a resonance for C13 (δC 212.6) was attributed to a ketone which was not observed for 2 and 3. Molecules 2019, 24, 3498 3 of 12 Molecules 2019, 24, x 3 of 12 FigureFigure 2. 2.HPLC-HRMS HPLC-HRMS ofof aa pre-fractionatedpre-fractionated metabolite extract extract from from F.F. avenaceum avenaceum growngrown on on banana banana mediamedia showing showing1 1––88.(. (aa)) BaseBase peakpeak chromatogramchromatogram (black) an andd colored extracted extracted ion ion chromatograms chromatograms ofof1 –1–88. 11––33 andand 55 [M[M + H]H]++ EICEIC = =279.1227279.1227 Da. Da. 4 [M4 [M+ Na]+ Na]+ EIC+ =EIC 315.1204= 315.1204 Da.