Bridged Epipolythiodiketopiperazines from ... - ACS Publications

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Bridged Epipolythiodiketopiperazines from Penicillium raciborskii, an Endophytic Fungus of Rhododendron tomentosum Harmaja Marena Kajula,†,‡ Joshua M. Ward,† Ari Turpeinen,† Mysore V. Tejesvi,§ Juho Hokkanen,‡ Ari Tolonen,‡ Heikki Hak̈ kan̈ en,⊥ Pere Picart,∥ Janne Ihalainen,⊥ Hans-Georg Sahl,∥ Anna Maria Pirttila,̈ § and Sampo Mattila*,† †

Chemistry and §Genetics and Physiology, University of Oulu, PO Box 3000 Linnanmaa, Oulu, Finland, FIN-90014 Admescope Ltd., Typpitie 1, Oulu, Finland, FIN-90620 ⊥ Nanoscience Center, Department of Biological and Environmental Science, University of Jyväskylä, P.O. Box 35, Jyväskylä, Finland, FIN-40014 ∥ Department of Pharmaceutical Microbiology, Institute for Medical Microbiology and Immunology, Meckenheimer Allee 168, 53115 Bonn, Germany ‡

S Supporting Information *

ABSTRACT: Three new epithiodiketopiperazine natural products [outovirin A (1), outovirin B (2), and outovirin C (3)] resembling the antifungal natural product gliovirin have been identified in extracts of Penicillium raciborskii, an endophytic fungus isolated from Rhododendron tomentosum. The compounds are unusual for their class in that they possess sulfide bridges between α- and β-carbons rather than the typical α−α bridging. To our knowledge, outovirin A represents the first reported naturally produced epimonothiodiketopiperazine, and antifungal outovirin C is the first reported trisulfide gliovirin-like compound. This report describes the identification and structural elucidation of the compounds by LC-MS/MS and NMR.

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and anti-insecticidal activity in grasses, and bioactive compounds are often found in endophytes of plants living in the tropical, subtropical, and temperate regions, but endophytes of woody plants and shrubs in the boreal or arctic region are virtually unstudied.15 Labrador tea (Rhododendron tomentosum Harmaja) is a small shrub found widely across the Northern hemisphere. The essential oil and extracts of R. tomentosum have anti-insect,16 antioxidant, and antimicrobial activities,17 and extracts from leaves and flowers have traditionally been used for the treatment of various infections.18 Earlier we screened the endophytes of this plant for antioxidant and antimicrobial activities.19 The aim of the present study was to identify bioactive compounds produced by the endophyte Penicillium raciborskii strain TRT59 of R. tomentosum.

ndophytic fungi are found in every plant species, and they infect all plant host tissues without eliciting symptoms of disease. Endophytic fungi have a range of activities in the host plant tissue, some of which have been characterized. Many endophytes protect the plant host against pathogens by producing various bioactive secondary metabolites.1,2 Sometimes endophytes are reported to produce compounds identical to those isolated from the plant host. Such compounds include paclitaxel, camptothecin, hypericin, podophyllotoxin, and desoxypodophyllotoxin.3−7 However, the majority of the secondary metabolites produced by endophytic fungi are specific to the fungus itself.8 Endophytic fungi are one source for discovering new bioactive compounds. Recently many new biologically active compounds have been isolated from endophytic fungi; these include penicidones,9 xylariol,10 xylarosides,11 and benzoquinone derivatives.12 The diversity of structures identified from natural sources is greater than that found in combinatorial or synthetic compound libraries. Endophytes, already adapted to life within the tissues of another eukaryote, produce sophisticated compounds with novel structures and, often, potent biological activity.13 One strategy for finding new bioactive compounds is to survey endophytes from less studied plants restricted to specific areas14 or with a history of use as a traditional medicinal plant.2 Fungal endophytes are well studied with respect to diversity © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Initial Characterization. Three fractions exhibiting antioxidant activity and sufficient yield based on initial LC/MS screening of TRT59 fungal extracts were selected for structural elucidation. The principal molecules from each of the three fractions differed from each other only by the number of sulfur atoms. Masses and molecular formulas of the [M + H]+ ions Received: October 20, 2014

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DOI: 10.1021/np500822k J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Proposed fragmentation patterns from MS/MS analysis of compounds 1, 2, and 3 and m/z 283. High-resolution masses are depicted with full accuracy. The fragment ion at m/z 283 was observed for all three compounds.

Table 1. Chemical Shifts (in ppm) of Compounds 1, 2, and 3 in CD3OD 1

a

position, type

δC

1, C 2, CH 3, C 4, C 5a, CH2 5b 6, C 7, CH 8, CH 9, CH 10, CH 11, CH 12, CH 13, C 14, C 15, C 16, C 17, CH 18, CH 19, CH3 20, CH3 21, CH3

166.1 65.7 168.8 66.8 28.6 70.8 75.0 130.5 128.0 65.3 86.7 49.3 115.1 148.2 136.2 153.4 102.4 124.6 59.7 54.7 31.3

2 δH (J in Hz)

4.53, d (3.2)

2.61, d (16.2) 2.21, br, d (15.4) 4.42, 5.68, 5.65, 4.48, 4.08, 5.57,

d (2.4) m m dd (7.3, 2.8) d (7.5) d (3.1)

6.49, 7.37, 3.82, 3.85, 3.20,

d (8.8) d (8.8) s s s

δC

3 δH (J in Hz)

166.8 67.6 166.1 69.1 31.5 71.7 74.9 130.4 127.9 65.6 87.3 41.9 116.4 147.9 136.4 153.8 103.3 122.9 60.1 55.3 32.8

4.54, br, sa

2.25, d (16.1) 2.37, br, d (16.1) 4.37, 5.61, 5.64, 4.52, 4.08, 4.67,

br,s m m da d (6.9) br, s

6.55, 7.37, 3.81, 3.86, 3.12,

d (8.9) d (10.4) s s s

δC 164.5 67.1 163.4 76.7 35.9 72.6 74.5 130.1 127.4 65.9 87.8 56.3 117.8 147.2 136.1 153.5 102.7 125.1 59.7 54.9 31.9

δH (J in Hz) 4.68, br, s

2.20, d (15.3) 2.74, dd (15.3, 1.8) 4.37, 5.61, 5.61, 4.51, 4.12, 5.53,

s (2.6) m m dd (7.0, 2.2) dd (7.0, 1.3) d br, s

6.53, 7.09, 3.82, 3.86, 3.26,

d (8.9) d (8.8) s s s

Signal partially obscured.

atoms present:20 m/z 199 (corresponding to C9H10O3S) for 1, m/z 231 (corresponding to C9H10O3S2) for 2, and m/z 263 (corresponding to C 9 H 10 O 3 S 3 ) for 3 (Figure 1 and supplementary Figure S1). The fragment ion at m/z 447 for both 2 and 3 indicated loss of a sulfur bridge together with two hydrogens (S2H2 and S3H2). The corresponding ion was not observed for 1, but a fragment ion appeared at m/z 435, corresponding to losses of H2O and CO (Figure 1).

were m/z 481.1282, C21H25N2O9S (1); m/z 513.1014, C21H25N2O9S2 (2); and m/z 545.0728, C21H25N2O9S3 (3) (Figure 1). The molecular formulas had the best-fitting isotope ratios and less than 2.5 ppm error between calculated and observed masses. MS/MS analysis of the molecular ions yielded, in addition to a common fragment ion appearing at m/z 283 for all three compounds, fragments that varied only in the number of sulfur B



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Further MS/MS fragmentation of the fragment ion at m/z 283 proceeded via multiple pathways, as evidenced by product ions at m/z 219, m/z 167, m/z 139, and m/z 111 (Figure 1). Errors between the calculated and observed masses for all the fragment ions were less than 2.5 mDa. The molecular formulas of the smaller fragments indicated a stable core containing both nitrogen atoms (m/z 167, m/z 139, and m/z 111), suggesting a diketopiperazine (DKP) ring motif. The neutral loss of CO is characteristic for diketopiperazines, while the loss of H2O indicates the presence of aliphatic hydroxy group(s).21 The structural similarity of the three molecules inferred from the MS data was supported by NMR data. The 1D proton spectra shared an overall global pattern, differing only in relative chemical shift differences and coupling magnitudes (Figure S1, Supporting Information). 13C and 1H chemical shifts are presented in Table 1. The COSY and 13C-HMBC correlations were also similar in all three compounds (Figures S2−S12, Supporting Information). Twenty protons were identified in 1D proton spectra collected in deuterated methanol, indicating four exchangeable hydrogens presumed to be hydroxy groups based on the H2O and −OH leaving groups in the MS/MS fragmentation patterns (Figure 1). All 21 atoms of the carbon skeleton were identified via HSQC and HMBC correlations. Structural Elucidation. The structures of the three compounds (Scheme 1) were determined using multidimen-

bond between C2 (δ 67.6) and C12 (δ 41.9) was assigned through COSY, NOE, and HMBC correlations, although the H2 (δ 4.54) and H12 (δ 4.67) peaks appeared as broad singlets rather than typical doublets due to a small 1.5 Hz vicinal coupling (Figure S2). The small coupling indicated a restricted HCCH torsion angle of approximately ±70 or ±100 degrees25 about the C2−C12 bond. This allowed a determination of the relative stereochemistry, as the NOE correlations between H2, H12, H21 (δ 3.12) and H18 (δ 7.37) could only be satisfied by the (R,S) or (S,R) enantiomers at the estimated torsion angle. Assuming the biosynthetic precursor is a standard L-amino acid, as has been shown for gliovirin,22 the (R,S) enantiomer (C2,C12) as depicted in Scheme 1 was considered in modeling efforts. Unusually small allylic couplings from the alkene protons H8 (δ 5.61) and H9 (δ 5.64) to neighboring protons H7 (δ 4.37) and H10 (δ 4.52), respectively, indicate that the proton− proton torsions approach 90 deg, producing slight line broadening rather than complete vicinal splittings in the onedimensional spectra and are characteristic of conduritols and shikimic acid derivatives where the double bond in the ring biases the ring geometry toward a half-chair conformation.26 The H8 to H7 and H9 to H10 couplings were identified via NOE and COSY cross-peaks, but H7 appeared as a singlet at δ 4.37 and H10 (δ 4.52), which was overlapped with H2 (δ 4.54) and evidently coupled to H11 (which appeared as a doublet and manifested COSY and NOE correlations to H2), and showed no sign of more than doublet splitting. The 10 Hz vicinal coupling between the alkene protons H8 and H9 was readily identified, but the allylic couplings to protons H7 and H10 were determined to be only about 1 Hz by simulating the line shape of the alkene peak signals at 5.6 ppm with a four-spin model system. The relative stereochemistry of the four chiral carbons of the conduritol-like ring, C6 (δ 71.1), C7 (δ 74.9), C10 (δ 65.5), and C11 (δ 87.3), was deduced from the NMR data and was found to resemble the structures of gliovirin and related compounds. An NOE was observed between H7 and H11 (δ 4.08), indicating that they are oriented toward the same face of the ring to place them within proximity of each other (2.8 Å according to the molecular model). H10 and the methylene group are oriented toward the opposite face of the ring, in order to satisfy the NOE observed between H10 and methylene proton H5a (δ 2.25, modeled distance 2.6 Å) while separating the methylene protons from H7 (3.7 and 3.8 Å) and H11 (3.9 and 4.3 Å) too far to generate observable NOEs. Were H7 and H10 cis disposed, the NOE correlations between H7 and the methylene protons (modeled distances 2.5 and 2.7 Å) should then be observable and the NOE between H7 and H11 (modeled distance 3.9 Å) unobservable. The asymmetric coupling patterns, distinct chemical inequivalency, and strong 15 Hz geminal coupling observed for the methylene protons H5a and H5b (doublets at δ 2.25 and 2.37, respectively, in Figure S2) suggested a conformationally restricted ring structure. Together with the constrained geometry deduced around the C2−C12 bond these observations prompted the assignment of the diketopiperazine ring. The nitrogen−oxygen bond of the oxazinane ring between the DKP and conduritol-like rings was ultimately settled upon with the aid of computational modeling and structure-based database searching using the assigned carbon skeleton, which revealed the resemblance to gliovirin.22 The torsional angles between the methylene protons and surrounding carbon atoms

Scheme 1. Structures of Compounds 1, 2, and 3, with Numbering Scheme Depicted on Compound 2 Conforming to That of Stipanovich for Gliovirin22

sional NMR and MS/MS methods. The detected COSY, NOESY, and 13C-HMBC correlations for each compound are presented in Table 2. The structures determined from the MS and NMR data were ultimately found to resemble the epidithiodiketopiperazine antibiotics gliovirin,22 FA-2097 (Nmethylgliovirin23), and pretrichodermamide A.24 The numbering scheme of gliovirin was adopted here for compounds 1, 2, and 3. The numbering scheme according to that of pretrichodermamide A24 is also provided in Scheme S1 in the Suppoting Information to facilitate data comparison. The assignment of the trisubsitituted phenyl moiety of each molecule (C13−C18 in Scheme 1) was straightforward. The C



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Table 2. COSY, NOE, and HMBC Correlations for Compounds 1, 2, and 3 in CD3OD 1

2

position, type

COSYa

HMBCb

COSYa

2, CH 5a, CH2 5b 7, CH 8, CH 9, CH 10, CH 11, CH 12, CH 17, CH 18, CH 19, CH3 20, CH3 21, CH3

12 5b 5a, 11 8, 9 10 7 8, 11 5b, 10 2 18 12, 17

1, 3, 21 3, 4, 7 4, 6, 11 5, 6, 8, 9 6, 7, 10, 11 6, 7, 10, 11 8, 9, 11 5, 6, 10 1, 2, 13, 14, 18 13, 14, 15, 16 12, 14, 15, 16 15 16 2, 3

12 5b 5a, 11 8, 9, 10 7, 10 7, 10 7, 8, 9, 11 5b, 10 2 18 17

NOEa 18, 21 10 8, 9, 11 7 10 5a, 8, 9, 11 7, 8, 9, 10 21 18, 20 2, 12, 17 17 2, 12

3 HMBCb

COSYa

HMBCb

1, 3 1, 4, 7 6,11 5, 6, 8, 9 6, 7, 10, 11 6, 7, 10, 11 8, 9, 11 5, 6, 10 2, 3, 13, 14, 18 13, 14, 15, 16 12, 14, 15, 16 15 16, 17 1, 2

12 5b 5a, 11 8, 9, 10 7, 10 7, 10 7, 8, 9, 11 5b, 10 2 18 17

3, 4, 5 6, 6,

4 6, 11 7, 10, 11 7, 10, 11

6, 10 1, 2, 13, 14, 18 13, 15 12, 14, 16 15 16 2, 3

a

COSY and NOE correlations are from the proton(s) stated to the indicated proton(s). bHMBC correlations are from the proton(s) stated to the indicated carbon.

Comparison with Published Compounds. The resemblance of compound 2 to gliovirin,22 FA-2097 (N-methylgliovirin23), and pretrichodermamide A24 was revealed by structure-based database searches. Gliovirin and FA-2097 were studied by NMR. Crystallographic structures have been reported for gliovirin22 (CSDS id PEKYIO) and pretrichodermamide A (CSDS id VENMAD).24 All three compounds contain two sulfurs bridging the α- and β-carbons across the ring. Gliovirin and FA-2097 both feature an epoxide oxygen bridging carbons 6 and 7 rather than two hydroxy groups as in pretrichodermamide A and compound 2. The NMR chemical shifts and IR spectrum of compound 2 agreed well with those of the published compounds. A carbon chemical shift rmsd of 6.8 ppm was obtained with gliovirin (3.7 ppm if the gliovirin epoxide carbons 6 and 7 are omitted from the comparison). The agreement with FA-2097 was similar, with 6.6 ppm rmsd (3.2 ppm if carbons 6 and 7 are omitted). Agreement of the proton chemical shifts was similar to both compounds with 0.4 ppm rmsd (0.2 ppm if hydrogen 7 is omitted). Closer agreement was obtained between compound 2 and pretrichodermamide A, with 2.1 ppm rmsd for carbon and 0.2 ppm rmsd for proton. The IR frequencies resembled the values published for pretrichodermamide A.24 Epithiodiketopiperazines are cyclic dipeptides that feature inter-residual polysulfide bridges, typically between the two αcarbons. The gliovirins are an unusual class in that they exhibit α- to β-carbon bridging.24 Trithio αβ-bridges have been identified in sporidesmins33 and aspirochlorines.34 Klausmeyer et al. reported UV and MS evidence for a tetrathio aspirochlorine but lacked sufficient quantity to confirm the structure by NMR.34 Compound 3 is the first gliovirin-like compound with three sulfurs to be reported. To our knowledge, no naturally occurring one-sulfur epithiodiketopiperazines such as compound 1 have been identified, but a literature review did uncover a synthetic anhydrogliotoxin analogue possessing a methylene sulfide bridge in place of the disulfide bridge.35 The novelty of the compounds emphasizes the diversity available in the secondary metabolites of fungi. Gliovirin-like compounds have been shown to exhibit selective antifungal and anti-inflammatory activities. Stipanovich and Howell originally reported selective activity for gliovirin against members of the Oomycetes.22 Iwatsuki et al.

calculated from model structures agree with the pattern of observed (∼40 or ∼180 deg) and unobserved (∼70 deg) correlations in the HMBC pattern considering a simple Karplus model for 3JCH (maxima at 0 and 180 deg, minima at ±90 deg). Additionally, a 15N-HMBC was acquired for compound 2 to verify the N-methyl substituent C21, which was assigned based on 13C-HMBC and NOE correlations (Table 2). The 15NHMBC spectrum contained no observable signals (data not shown), but this is not entirely unexpected given the highly constrained geometry of the molecule. NMR chemical shift differences were used to assign the αβbridged epithiodiketopiperazine motifs (Table 1 and Scheme 1). The C12 and H12 benzyl group signals exhibited the largest chemical shift differences between molecules, indicating close physical interaction with the sulfur atoms: carbon at δ 49.3 and proton at δ H5.57 for (1), carbon at δ 41.9 and proton at δ 4.67 for 2, and carbon at δ 56.3 and proton at δ 5.53 for 3. The second largest carbon chemical shift differences were observed in C4: at δ 66.8 for 1, δ 69.1 for 2, and δ 76.7 for 3. The methylene group neighboring C4 also exhibited significant chemical shift differences: the chemical shifts for H5a, H5b, and C5 appeared, respectively, at δ 2.61, 2.21, and 28.6 for 1; δ 2.25, 2.37, and 31.5 for 2; and δ 2.20, 2.74, and 35.9 for 3. A few examples of MS fragmentation data from epipolythiodiketopiperazines were found in the literature, all of them having α,α-sulfur bridges. Pseudo MS/MS experiments of gliotoxin and emestrin showed that S2 leaves before H2O and CO.27 Wu et al. made MS3 experiments with nine molecules containing two- or three-atom sulfur chains. When [M + H]+ was used as the precursor ion, losses of S2H2 and S3H2 were observed28 similar to our study. The final structures were supported by quantum mechanical chemical shift calculations. Table S1 lists the calculated shifts alongside the experimental values, along with root mean squared deviations and corrected mean absolute errors. The overall accuracy was good, with an rmsd of about 3.5 ppm and a corrected mean average error of about 2.5 ppm.29−32 Structural confirmation and full determination of the absolute configuration of the molecules by crystallography were additionally desired, but crystallization was hampered by limited sample availability owing to the time and material expense of the slow microbial cultivation and relatively low expression yields. D



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shifts were corrected by linear scaling27,29 with δcalc = 0.95(σTMS − σcalc) + 0.59 (for 13C) and δcalc = 0.95(σTMS − σcalc) + 0.26 (for 1H).28 Antifungal Assay. Quantitative antifungal activity of the compounds was assessed using a microspectrophotometric assay in a 96-well microtiter plate (Nunc F96 microtiter plates) with the following fungal pathogens: Fusarium oxysporum, Botrytis cinerea, and Verticillium dahliae. Each well contained 1000 fungal spores in 100 μL of half-strength potato dextrose broth and the purified compounds at concentrations ranging from 10 to 400 μg/mL. Negative control reactions contained no metabolites and positive controls contained amphotericin B at 10 μg/mL, which completely inhibited the visible growth of each of the fungi tested after 48 h of incubation in the dark. Plates were incubated in the dark at 23 °C for 48 h, after which the activity was scored and expressed as the percentage of growth inhibition. Growth inhibition percentage was defined as 100 × the ratio of the A595 of the control minus the A595 of the sample divided by the A595 of the control.

reported activity for gliovirin against Trypanosoma brucei brucei,36 and Seephonkai et al. reported that pretrichodermide A was active against Mycobacterium tuberculosis.24 Antifungal activity of compound 3 was assayed by microspectrophotometry using a dose−response growth inhibition assay. Compound 3 inhibited growth of all fungal isolates at a low concentration of 0.38 mM (207 μg/mL) but a more significant growth inhibition was observed at the higher concentration of 0.76 mM (413 μg/mL). Compound 3 was most active against Botrytis cinerea (57% inhibition) and slightly less effective against Verticillium dahliae (45% inhibition). In addition to the antifungal activity, Rether et al. suggested a potential antitumor or anti-inflammatory capacity for gliovirin, reporting that it inhibited the expression of cytokines (TNF-alpha and IL-2) and pro-inflammatory enzymes (COX-2, INOS) in T-cells and monocytes/macrophages.37

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ASSOCIATED CONTENT

S Supporting Information *

EXPERIMENTAL SECTION

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/np500822k. Full ref 38, HRESIMS, 1H NMR, COSY, HSQC, and HMBC spectra, and simulated 1H and 13C chemical shifts of compounds 1−3; NOESY and HMBC HSQC spectra of compound 2; figure comparing the gliovirinbased and pretrichodermamide A-based numbering schemes (PDF)

General Experimental Procedures. NMR data were collected on a 500 MHz Bruker DMX spectrometer with a triple-resonance probehead. Chemical shifts were referenced to solvent. Mass spectra were recorded on a Synapt G2 HDMS mass spectrometer (Waters Corp., Milford, MA, USA). Positive mode electrospray was employed with a capillary voltage of 3000 V and cone voltages between 20 and 35 V. HPLC separation was performed with an Acquity UPLC using a BEH C18 1.7 μm, 2.1 × 50 mm column (Waters Corp.). Cultivation and Isolation. The endophytic fungus Penicillium raciborskii (TRT59) was isolated from Rhododendron tomentosum and identified using internal transcribed spacer region, and the sequence was deposited in GenBank with the accession number GQ266152.19 The endophyte was fermented in 3 L Erlenmeyer flasks containing 1000 mL of malt extract broth medium for 4 to 6 weeks at 23 °C under static conditions in two replicates. Fungal mycelia were separated from the culture broth by vacuum filtration. The fermented media were freeze-dried by a Heto PowerDry LL1500 freeze-dryer (ThermoElectron, Mukarov, Czech Republic) and stored at room temperature until used. Compounds were isolated by a preparativescale HPLC system (Waters) as described previously19 with minor changes. An Atlantis Prep T3 OBD, 19 × 50 mm, 5 μm column was used with a modified gradient of 0% to 100% methanol over 20 min. A total of 1.9 g of the dried media was purified. Three fractions possessing sufficient yield and purity for structural elucidation were collected at 7.3 min (containing compound 1), 9.1 min (containing compound 2), and 10.2 min (containing compound 3). Fractions were lyophilized with an SPD 2010 Speed Vac (Thermo Savant, Holbrook, NY, USA) and freeze-dryer. Outovirin A (1): dark yellow powder; 1H and 13C NMR (500/125 MHz, CD3OD) see Table 1; HRESIMS m/z 481.1282 [M + H]+ (calcd for C21H25N2O9S, 481.1281); HRESIMS/MS m/z 435.1223, 199.042, 283.0930; HRESIMS/MS of m/z 283.0930:219.0755, 167.0450, 139.0509, 111.0553. Outovirin B (2): dark yellow powder; IR (NaCl) νmax 3980, 3467, 3344, 2938, 2852, 1693, 1687, 1618, 1610, 1506, 1463, 1434, 1367, 1280, 1245, 1219, 1096, 1072, 1034, and 958 cm−1; 1H and 13C NMR (500/125 MHz, CD3OD) see Table 1; HRESIMS m/z 513.1014 [M + H]+ (calcd for C21H25N2O9S2, 513.1001); HRESIMS/MS m/z 447.1404, 231.0155, 283.0930. Outovirin C (3): dark yellow powder; 1H and 13C NMR (500/125 MHz, CD3OD) see Table 1; HRESIMS m/z 545.0728 [M + H]+ (calcd for C21H25N2O9S3, 545.0722); HRESIMS/MS m/z 447.1404, 263.9877, 283.0930. Ab Initio Simulations. B3LYP/6-31G*-optimized geometries were used in gauge independent atomic orbital method (GIAO) chemical shift calculations at the B3LYP/6-311++G(2d,2p) level of theory in Gaussian09 revision B.01,38 employing program defaults throughout. Reference 13C and 1H nuclear shieldings were calculated from tetramethylsilane with the same protocol. Calculated chemical

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AUTHOR INFORMATION

Corresponding Author

*E-mail: sampo.mattila@oulu.fi. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was funded by the Marie Curie Industry-Academia Partnership and Pathways (IAPP) of the EU 7th Framework Programme, New Antimicrobials (NAM) Project (PIAP-GA2008-218191). The CSC Finnish IT Center for Science is thanked for computational resources and Cecilia Alaye for the picture of Rhododendron tomentosum.

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