Biotransformation of rutabaga phytoalexins by the fungus Alternaria brassicicola: Unveiling the first hybrid metabolite derived from a phytoalexin and a fungal polyketide

Accepted Manuscript Biotransformation of rutabaga phytoalexins by the fungus Alternaria brassicicola: Unveiling the first hybrid metabolite derived from a phytoalexin and a fungal polyketide M. Soledade C. Pedras, Abbas Abdoli PII: DOI: Reference:

S0968-0896(16)31026-4 http://dx.doi.org/10.1016/j.bmc.2016.11.017 BMC 13384

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

19 October 2016 8 November 2016 11 November 2016

Please cite this article as: Pedras, M.S.C., Abdoli, A., Biotransformation of rutabaga phytoalexins by the fungus Alternaria brassicicola: Unveiling the first hybrid metabolite derived from a phytoalexin and a fungal polyketide, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.017

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Biotransformation of rutabaga phytoalexins by the fungus Alternaria brassicicola: unveiling the first hybrid metabolite derived from a phytoalexin and a fungal polyketide

M. Soledade C. Pedras* and Abbas Abdoli Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK, S7N 5C9, Canada

Corresponding author:

M. Soledade C. Pedras Email: [email protected]; Telephone: 1-306- 966-4772; Fax: 1-306-966-4730

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Abstract The biotransformations of the rutabaga phytoalexins rutalexin, brassicanate A, isalexin and rapalexin A by the plant pathogenic fungus Alternaria brassicicola are reported. While the biotransformations of rutalexin, brassicanate A, and isalexin are fast, rapalexin A is resistant to fungal transformation. Unexpectedly, biotransformation of rutalexin yields a hybrid metabolite named rutapyrone, derived from rutalexin metabolism and phomapyrone G, a fungal metabolite produced by A. brassicicola. These fungal transformations are detoxification reactions likely carried out by different enzymes. The discovery of rapalexin A resistance to detoxification suggests that this phytoalexin in combination with additional phytoalexins could protect crucifers against this pathogen. Phytoalexins resistant to degradation by A. brassicicola are expected to provide the producing plants with higher disease resistance levels.

Keywords:

Alternaria

brassicicola;

antifungal;

Brassicaceae;

biotransformation;

brassicanate A; crucifer; detoxification; phytoalexin; rapalexin A; rutalexin; rutapyrone.

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Introduction In a broad sense biotransformation is a chemical modification of a compound carried out

by living cells or enzymes. Biotransformations of organic compounds using microbial cells such as bacteria or fungi have been often used to obtain either products of great value,1,2,3 or to get rid of highly detrimental materials such as organic pollutants, pesticides and explosives.4 In an ecological context, microbial biotransformations are often a survival strategy of the microbe to cope with environmental stress. For example, to colonize plant tissues, fungal pathogens produce enzymes that can transform antimicrobial plant defenses, namely phytoalexins,5,6 to non-toxic products.7,8 Conceptually these fungal transformations, highly detrimental to plants, could be prevented if inhibitors of the enzymes mediating these transformations were available. Such inhibitors, known as paldoxins,9 could be applied to protect crops from fungal attack.8,10 To design paldoxins, an understanding of the metabolic detoxification pathways of phytoalexins carried out by fungal plant pathogens is crucial. Because the intermediates and products of these pathways are in general unique to each fungal species, it is necessary to evaluate separately the interaction of each phytoalexin with each phytopathogenic fungal species. Toward this end, we have been investigating the detoxification of cruciferous phytoalexins by fungal pathogens of economically important crops (crucifer crops, e.g. canola, Brassica napus, B. rapa, B. juncea) and chemical strategies to inhibit these processes.8,9,10,11 The fungal plant pathogen Alternaria brassicicola (Schwein.) Wiltshire, together with A. brassicae (Berk.) Sacc. causes black spot disease and substantial yield losses of various crucifer crops.12,13 It has been established that A. brassicicola is able to transform and detoxify several cruciferous phytoalexins including brassinin (1)14 cyclobrassinin (2),15 and camalexin (4),16 but its resistance to the phytoalexins of rutabaga (Brassica napus L. ssp. rapifera) remains unknown.

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Brassinin (1) cyclobrassinin (2) and rutalexin (3) accumulate in leaves of B. juncea infected with A. brassicicola,14 whereas camalexin (4) is produced in wild crucifer species.8 Rutalexin (3), brassicanate A (5) and isalexin (6) are phytoalexins first isolated from rutabaga,17 together with rapalexin A (7),18 brassinin (1), cyclobrassinin (2) and others. In continuation of that work, we have analyzed the biotransformation of phytoalexins 3, and 5–7 in cultures of A. brassicicola and report here results of these studies. This investigation demonstrates that A. brassicicola can efficiently metabolize rutalexin (3), brassicanate A (5), and isalexin (6), but not rapalexin A (7). A hybrid metabolite resulting from the interaction "rutalexin – A. brassicicola" was discovered that could alleviate oxidative stress of fungal cells exposed to this phytoalexin. Importantly, resistance of rapalexin A (7) to A. brassicicola suggests that this phytoalexin in combination with additional defense metabolites like camalexin (4) could effectively protect crucifers against this pathogen. Phytoalexins resistant to degradation by A. brassicicola are expected to provide the producing plants with higher disease resistance levels.

Figure 1. Phytoalexins produced by crucifers: brassinin (1), cyclobrassinin (2), rutalexin (3), camalexin (4), brassicanate A (5), isalexin (6) and rapalexin A (7).

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Results and discussion

2.1 Chemical synthesis of phytoalexins Rapalexin A (7) was synthesized and purified as previously reported, 19 whereas the syntheses of rutalexin (3), brassicanate A (5), and isalexin (6) were modified, as summarized below. Rutalexin (3) was previously prepared in modest yield (24% overall yield) by oxidation of N-t-Boc-2-chloroindole-3-carboxaldehyde (8) to the corresponding acid, followed by conversion into amide 9 upon treatment with thionyl chloride and methanamine.17 The resulting amide 9 was allowed to react with NaHS to yield the key intermediate sulfanylamide 10. However, during the current work it was discovered that oxidation of sulfanylamide 10 to disulfide 13 occurred spontaneously during acidic work-up and chromatographic fractionation. The dimerization of 10 to 13 could explain the lower yield of rutalexin (3) obtained previously.17 Similar dimerization of thiols/thiones have been previously reported under various conditions.20,21 The structure of sulfanylamide 10 was confirmed by methylation of the thiol group followed by deprotection using TFA (20%) in DCM to afford 12 in 91% overall yield; the disulfide 13 was deprotected under similar conditions to yield 14 in 90% yield (Scheme 1). In the current rutalexin synthesis, to prevent formation of disulfide 13, methyl chloroformate was added to the crude reaction mixture containing sulfanylamide 10, to yield compound 11 in almost quantitative yield (95%) after 30 min. Cyclization of compound 11 in the presence of Et3N followed by deprotection using TFA proceeded smoothly; after concentration of the crude reaction mixture to dryness, the crude residue was rinsed with Et2O to yield rutalexin (3) (93% yield, vi - vii; ca. 81% overall yield). Disulfide 14 was previously synthesized in three steps from 1phenylsulfonyl-1H-indol-3-carbonyl chloride to evaluate its ability to inhibit tyrosine kinase

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activity.20

Scheme 1. Synthesis of rutalexin (3). Reagents and conditions: i) NaClO2, KH2PO4, 2-methyl-2butene, tert-butanol, H2O, rt, 4 h; ii) SOCl2, THF/DMF, rt, 3 h; iii) CH3NH2, THF, 0 °C, 1 h, 95% (i–iii); iv) NaHS, DMF/H2O, 0 °C, 1 h; v) methyl chloroformate, 30 min, 95%; vi) Et3N, THF, 50 °C, 3 h; vii) 20% TFA-DCM, rt, 4 h, 93% (vi-vii); viii) CH3I, THF, 30 min, rt, 97%; ix) 20% TFA-DCM, rt, 4 h, 90%; x) 20% TFA-DCM, rt, 4 h, 90%.

Brassicanate A (5) was previously synthesized in 51% overall yield from oxidation of aldehyde 8 to the corresponding acid, followed by methylation with diazomethane.17 In this work, brassicanate A (5) was synthesized in quantitative yield from indole-2-thione (15) by methylation with MeI followed by treatment with phosgene (COCl2) and MeOH (Scheme 2). Isalexin (6) was previously prepared by directed O-lithiation of N-Boc-m-anisidine with n-BuLi in 71% overall yield.17 To avoid n-BuLi, 4-methoxyindole (18) was oxidized with PCC and AlCl3 to yield isalexin (6) in 53% yield (Scheme 3). Sriram et al. have reported the oxidation of indole (16, 100 mmol scale) to isatin (19) using PCC–SiO2 and AlCl3 in DCE in 88% yield;22 our attempts to optimize the reaction conditions of a smaller scale reaction (1 mmol) yielded isatin (19) and isalexin (6) in much lower yields (Scheme 3). Similar oxidation of 4-methoxyindole (18) using

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PCC, but replacing SiO2 with PANI yielded isalexin in 26% yield.23

Scheme 2. Synthesis of brassicanate A (5). Reagents and conditions: i) CH3I, Na2CO3, acetone, rt, 10 h; ii) COCl2, THF, rt, 2 h; iii) MeOH, rt, 30 min, quantitative.

Scheme 3. Synthesis of isalexin (6). Reagents and conditions: i) CH3I, K2CO3, acetone, 35 °C, 18 h, 90%; ii) PCC, AlCl3,, DCE, 80 °C, 2 h, 53%; iii) PCC-PANI, DCE, 80 °C, 7 h, 26%; iv) PCC, AlCl3,, DCE, 80 °C, 2 h, 42%; iii) PCC-PANI, DCE, 80 °C, 7 h, 25%.

2.2 Biotransformations To establish the biotransformation pathways of rutalexin (3), brassicanate A (5), isalexin (6) and rapalexin A (7) in cultures of A. brassicicola, the concentration was chosen based on the solubility and inhibitory activity of compounds (0.10 mM). Mycelial cultures of A. brassicicola (48-h-old) were incubated with each phytoalexin and samples were withdrawn from cultures immediately after addition of each compound (zero hours) and then up to 96 h. Culture samples were either frozen or immediately subjected to neutral, acidic and basic extractions, and the concentrated extracts were analyzed by HPLC-DAD-ESI-MS, as reported in the Experimental Section 4.3.1. Samples of medium incubated with each phytoalexin separately (control solutions)

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were extracted and analyzed similarly to determine chemical stability and recovery. Similarly, samples of cultures of A. brassicicola were extracted and analyzed to evaluate metabolite production in the absence of phytoalexins (control cultures). 2.2.1 Rutalexin (3) The HPLC chromatograms of extracts of cultures of A. brassicicola incubated in medium (or water) containing rutalexin (3) showed a fast decrease in the area of the peak corresponding to 3 (tR = 8.8 ± 0.2 min) and the appearance of new peaks at tR = 9.6 ± 0.2 min and 10.2 ± 0.2 min after 6 h (not detected in control cultures). The ESI-MS and the UV spectra of the peak at 9.6 min was not available in our virtual libraries. After complete metabolism of 3, the area of the peak at 9.6 min remained constant, whereas the area of peak at 10.2 min increased up to 12 h and decreased there on (not detected after 48 h). No other significant peaks could be detected in the chromatograms. The ESI-MS of the compound at 10.2 min displayed an ion at m/z 409 [M-1]that suggested it to be disulfide 14, obtained during rutalexin synthesis (summarized in Scheme 1 and described in Section 4.2.5). The progress curves for transformation of rutalexin (3) by A. brassicicola either in medium or in water indicated that it was metabolized quite fast (t1/2 = <6 h, Fig. 2); rutalexin (3) incubated in medium or in water was stable for the duration of the experiments.

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0.12

0.10

Conc. (mM)

0.08

0.06

0.04

0.02

0.00 0

12

24 36 Incubation time (h)

48

Figure 2. Progress curves of transformation of rutalexin (3) (), formation of metabolites 21() and 14 () in cultures of Alternaria brassicicola, and recovery of rutalexin (3) from medium () (HPLC, method A).

Furthermore, a peak at tR = 6.7 min was observed in the chromatograms of both control cultures and cultures incubated with rutalexin (3). This peak was detected after 6 h in cultures incubated with rutalexin (3), whereas in control cultures (no rutalexin (3)) it was detected only after 24 h of incubation; production in both cultures was comparable after 48 h. Analysis of the peak areas corresponding to this metabolite (established to be phomapyrone G (20), ESI-MS and UV spectra of an authentic sample)14,24 suggested that it was produced in larger amounts in rutalexin (3) amended cultures than in control cultures (Fig. 3).

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1800

Peak area

1500 1200 900 600 300 0 0

12

24

36

48

60

72

84

Incubation time (h)

Figure 3. Progress curves of formation of metabolite at tR = 6.7 min, phomapyrone G (20), in cultures of Alternaria brassicicola incubated with rutalexin (3) () and in control cultures (no rutalexin) () (HPLC, method A).

Larger scale cultures of A. brassicicola incubated with rutalexin (3) up to 72 h allowed the isolation of the metabolite with tR = 9.6 min and its spectroscopic characterization, as described in Section 4.3.2. The HREI-MS spectrum (456.1719 Da, C24H28N2O5S) suggested that this compound had higher MW than rutalexin (3) (232.0306 Da, C11H8N2O2S), i.e. an additional moiety with the molecular formula C13H20O3. The 1H NMR spectrum obtained in CD3CN displayed five indolyl protons (δH: 9.90, br s, 1H; 7.95, d, 1H; 7.30 d, 1H; 7.14 dd, 1H; 7.07 dd, 1H), three protons at δH 7.33 (br, 1H), 6.37 (s, 1H), 6.10 (s, 1H), six methyl groups at δH 3.84 (s, 3H), 2.90 (d, 3H), 2.10 (s, 3H), 1.80 (s, 3H), 1.51 (s, 3H), 1.39 (d, 3H) and one methine proton at

δH 3.56 (q, 1H). The 13C NMR spectrum displayed signals for 24 carbons, namely eight indolyl carbons with chemical shifts similar to those of rutalexin (3), six methyl carbons, four carbons above 160 ppm and six other carbons. The NMR spectroscopic data indicated the presence of an indolyl moiety containing an N-methyl amide (δH 2.90, d, J = 5 Hz, 3H), corresponding to the moiety derived from rutalexin (3). Detailed analysis of HSQC and HMBC spectroscopic data showed correlations corresponding to the additional C13H20O3 moiety. For example, in the HMBC

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spectrum, the singlet proton at δH 6.37 correlated to carbons at δC 160.5, 76.8 and 26.8 (C6', C9' and C12', respectively), the singlet proton at δH 6.10 correlated to carbons at δC 102.5, 160.5 and 129.7 (C3', C6' and C7', respectively) and the methyl protons at δH 1.39 (d, J = 7.2 Hz) correlated with carbon at δC 76.8 (C9) (Table 1). These signals suggested a structure resulting from condensation of a rutalexin-derived moiety with one of the pyrone metabolites produced by A. brassicicola.14,24 Based on this consideration and all spectroscopic data, the hybrid structure 21 derived from rutalexin-phomapyrone G was deduced (Fig. 4). This structure is consistent with NMR and UV spectroscopic data and the predicted chemical reactivity of phomapyrone G (20) (C14H18O4), a 2pyrone polyketide first isolated from Leptosphaeria biglobosa.25 In addition, NOE data showed correlations between H5' and H13', and H13' and H8', which indicated the configuration of the double bond as cis, consistent with the structure of phomapyrone G (20). The chemical structure of the hybrid metabolite suggests that it results from addition of sulfanylamide 23 to the least sterically hindered carbon of the epoxide ring of phomapyrone G (20). To the best of our knowledge, compound 21 is the first hybrid natural product derived from a phytoalexin and a fungal metabolite, for this reason it is named rutapyrone (21).

Figure 4. Chemical structure of rutapyrone (21) and selected HMBC and NOE correlations.

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Table 1 1

H (600 MHz) and 13C NMR (150 MHz) spectroscopic data of rutapyrone (21) in CD3CN Atom #

δC

1 2 3 3a 4 5

130.9 115.6 127.8 111.9 124.1

6

121.7

7 7a 8 9 10

121.8 137.3 166.4 26.4

δH, multiplicity (J, Hz) 9.90, br s 7.30, d (8.4) 7.07, dd (7.8, 7.2) 7.14, dd (7.2, 8.4) 7.95, d (7.8) 7.33, br s 2.90, d (4.8)

Atom #

δC

δH, multiplicity (J, Hz)

1′ 2′ 3′ 4′ 5′ 6′

165.2 102.5 167.1 94.0 160.5

6.10, s -

7′

129.7

-

8′ 9′ 10′ 11′ 12′ 13′ 14′ OH (O)CH3

136.8 76.8 57.6 17.6 26.8 13.5 9.0 57.3

6.37, s 3.56, q (7.2) 1.39, d (7.2) 1.51, s 2.10, s 1.80, s 4.20, br s 3.84, s

In addition, isolation of a very small amount of the metabolite with tR = 6.7 min from the larger scale cultures confirmed it to be phomapyrone G (20). As expected from analysis of the time-course experiments, disulfide 14 was not isolated from these large scale cultures because it was not present in cultures after 48 h of incubation. It is likely that disulfide 14 results from spontaneous oxidation of sulfanylamide 23, similar to results obtained during the chemical synthesis of rutalexin (3). Furthermore, disulfide 14 was not stable either in culture medium or water, oxidizing spontaneously to various undetermined products, including sulfonic acid 22. Sulfonic acid 22 was obtained from disulfide 14 upon oxidation with m-CPBA (Scheme 4). Due

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to its low chemical stability the fate of disulfide 14 in cultures could not be determined.

Scheme 4. Synthesis of sulfonic acid 22. Reagents and conditions: i) m-CPBA, MeOH, 2.5 h, rt, 59%.

The pathway for transformation of rutalexin (3) by A. brassicicola is proposed in Scheme 5. That is, in fungal cultures rutalexin (3) is enzymatically hydrolyzed and decarboxylated to sulfanylamide 23, which in turn adds to the least hindered epoxide carbon C10' of phomapyrone G (20) to produce rutapyrone (21). This addition is most likely an enzyme mediated transformation. As well, sulfanylamide 23 can oxidize spontaneously to disulfide 14 (the major product), which is similarly unstable in aqueous or organic solutions and oxidizes to several other products including sulfonic acid 22.

Scheme 5. Proposed pathway of biotransformation of rutalexin (3) in cultures of Alternaria brassicicola with formation of hybrid metabolite rutapyrone (21).

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Albeit produced in small amount, formation of a hybrid metabolite like rutapyrone (21) derived from plant (rutalexin (3)) and fungus (polyketide phomapyrone G (20)) metabolism is an intriguing process posing a few questions. Specifically, A. brassicicola produces several 2pyrones such as 20, but until now no ecological functions, including phytotoxicity, have been established.24,25 Based on this work we suggest that in fungal cultures rutalexin (3) induces the biosynthesis of phomapyrone G (20) by oxidation of phomapyrone A (24), to reduce the oxidative stress caused by the presence of rutalexin (3) (Scheme 6). Formation of phomapyrone G (20) can quench and hence decrease the levels of reactive oxygen species (ROS), including hydrogen peroxide, generated in mycelial cells incubated with rutalexin (3). The quick depletion of hydrogen peroxide and/or other ROS can mitigate their deleterious effects on the pathogen. In fact some phytoalexins are known to cause oxidative stress on fungal plant pathogens.26 For example, we have shown that camalexin (4) induced production of superoxide dismutase (SOD) in A. brassicicola.10 SOD is an enzyme produced by various organisms, including fungi, to protect cells from ROS produced in cellular responses to oxidative stress. In short, oxidation of phomapyrone A (24) (and stereo isomers) to phomapyrone G (20) can contribute to decrease oxidative stress levels in A. brassicicola by lowering the concentration of ROS species. These considerations suggest that enzymatic coupling of sulfanylamide 23 and phomapyrone G (20) is likely to occur in the cell site where 20 is synthesized and rutalexin (3) is hydrolyzed, i.e. the coupling enzyme is located in the same subcellular compartment where rutalexin is metabolized.

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Scheme 6. Proposed oxidation of phomapyrone A and potential isomers (24) to phomapyrone G (20) in cultures of Alternaria brassicicola.

2.2.2 Brassicanate A (5) The metabolism of brassicanate A (5) was investigated as reported above for rutalexin (3). The HPLC chromatograms of the neutral extracts of cultures of A. brassicicola incubated with brassicanate A (5) showed a fast decrease of the area of the peak corresponding to 5 (t = 15.8 ± R

0.2 min) and the appearance of a new peak at tR =12.0 ± 0.2 min (Fig. 5). Brassicanate A (5) was not detected in cultures after 4 h of incubation; the new peak at t = 12.0 min was not detected in R

control cultures or medium incubated with brassicanate A (5). The UV spectrum of this component was similar to that of 5, whereas the ESI-MS at m/z 205.8 [M-H]- suggested the presence of a carboxylic acid. The structure of this metabolite was confirmed as 2methylsulfanediylindole-3-carboxylic acid (26), after isolation from larger scale cultures of A. brassicicola incubated with brassicanate A (5) for 4 h. The structure was further confirmed by direct comparison with an authentic synthetic sample, prepared as summarized below.

0.12

Conc. (mM)

0.10 0.08 0.06 0.04 0.02 0.00 0

1

2

3 4 Incubation time (h)

5

6

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Figure 5. Progress curves of transformation of brassicanate A (5) () and formation of metabolite 26 () in cultures of Alternaria brassicicola, and recovery of brassicanate A from medium () (HPLC method B).

2-Methylsulfanediylindole-3-carboxylic acid (26) was prepared by treatment of 25 with phosgene followed by aqueous work up (Scheme 7). Next, to establish the metabolic fate of 2methylsulfanediylindole-3-carboxylic acid (26), it was incubated in medium with A. brassicicola and in medium only. Unexpectedly, HPLC analysis of extracts showed a slower decrease in the concentration of 2-methylsulfanediylindole-3-carboxylic acid (26) (t = 12.0 ± 0.2 min) in fungal R

cultures than in medium. These results suggested that the faster decrease of the concentration of carboxylic acid 26 in medium was likely due to spontaneous transformation. It was hypothesized that the faster transformation of acid 26 in medium was favored by the more acidic pH (ca. 6.5) of medium than the pH of cultures of A. brassicicola (increases from 6.5–8.5 in 48 h). To verify this hypothesis, solutions of medium only or water only at different pHs were incubated with acid 26 and each solution analyzed by HPLC every 2 h. Progress curves of the recovery of acid 26 over a 12 h period (Fig. 6) confirmed a much faster decrease of the concentration of acid 26 in solutions at pH 6.0 than at pH 8.5. In addition, the concomitant appearance of a new peak at t = R

23.2 ± 0.2 min and determination of its structure demonstrated that carboxylic acid 26 was quickly transformed to 2-methylsulfanediylindole (25) (Scheme 7).

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Scheme 7. Synthesis and spontaneous decomposition of 2-methylsulfanediylindole-3-carboxylic acid (26). Reagents and conditions: (i) COCl2, THF, 2 h, rt; (ii) H2O, 30 min, rt, 60% (i–ii); (iii) spontaneous decarboxylation in H2O or medium. A 7000 6000

Peak area

5000 4000 3000 2000 1000 0 0

2

4

6

8

10

12

14

Incubation time (h)

B 10000 9000 8000

Peak area

7000 6000 5000 4000 3000 2000 1000 0 0

2

4

6 8 Incubation time (h)

10

12

14

Figure 6. Progress curves of decarboxylation of 2-methylsulfanediylindole-3-carboxylic acid (26) (A) and formation of 2-methylsulfanediylindole (25) (B) in medium (blue) and H2O (blackdashed) at different pHs: 8.5 (), 7.1 () and 6 () (HPLC method C).

Although not detected in cultures of A. brassicicola incubated with brassicanate A (5), brassicanate A sulfoxide (27) was thought to be a likely metabolite caused by ROS. To prove this

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hypothesis, brassicanate A sulfoxide (27) was prepared by oxidation of brassicanate A (5) (Scheme 8) and its biotransformation in culture and in medium was investigated. The HPLCDAD-ESI-MS of neutral extracts revealed that brassicanate A sulfoxide (27) remained in cultures for the duration of the incubation (48 h), that is, A. brassicicola did not transform brassicanate A sulfoxide (27). In addition, brassicanate A sulfoxide (27) was stable in medium for 48 h (Fig. 7).

Scheme 8. Synthesis of brassicanate A sulfoxide (27). Reagents and conditions: i) m-CPBA, MeOH, 0 °C, 30 min, 79%.

0.14

Conc. (mM)

0.12 0.10 0.08 0.06 0.04 0.02 0.00 0

6

12

18

24 30 36 Incubation time (h)

42

48

54

Figure 7. Progress curves of recovery of brassicanate A sulfoxide (27) from cultures of Alternaria brassicicola () and medium () (HPLC method B).

Based on the results described above, the transformation of brassicanate A (5) by A. brassicicola is proposed as shown in Scheme 9. In fungal cultures brassicanate A (5) is enzymatically

hydrolyzed

to

2-methylsulfanediylindole-3-carboxylic

spontaneously decarboxylates in aqueous solutions of pH<7.

acid

(26),

which

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Scheme 9. Proposed pathway of biotransformation of brassicanate A (5) in cultures of Alternaria brassicicola with spontaneous decarboxylation (I, proposed intermediate).

2.2.3 Isalexin (6) The HPLC chromatograms of the neutral extracts of cultures of A. brassicicola incubated with isalexin (6) showed a decrease in the area of the peak corresponding to 6 (t = 9.3 ± 0.2 min) R

and the appearance of new peak at tR = 8.6 ± 0.2 min. Isalexin concentration decreased rapidly (0.12 to ca. 0.03 mM) in 6 h, but remained detectable up to 72 h of incubation, whereas in control medium its concentration remained constant (Fig. 8). The recovery of isalexin (6) from neutral extracts was ca. 75%, small amounts remained in both acidic and basic extracts; hence data points of the progress curve in Fig. 8 represent the amount of 6 detected in the neutral, acidic and basic extracts (obtained by combining the extracts). The peak at 8.6 min was not detected in control cultures or medium incubated with isalexin (6). The ESI-MS (positive mode) spectrum of the peak at 8.6 min displayed an ion at m/z 202 [M+Na]+, which suggested this metabolite to be 1,3-dihydro-3-hydroxy-4-methoxyindol-2-one (28). This structure was confirmed by direct comparison with an authentic synthetic sample prepared by NaBH4 reduction of isalexin (6). Further incubation of cultures with 1,3-dihydro-3-hydroxy-4-methoxyindol-2-one (28) and HPLC analysis indicated that this compound spontaneously oxidized to isalexin (6) and other undetermined polar compounds.

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0.14 0.12

Conc. (mM)

0.10 0.08 0.06 0.04 0.02 0.00 0

12

24

36

48

60

72

Incubation time (h)

Figure 8. Progress curves of transformation of isalexin (6) () and formation of metabolite 28 () in cultures of Alternaria brassicicola, and recovery of isalexin from control medium () (HPLC method B).

Scheme 10. Proposed biotransformation of isalexin (6) in cultures of Alternaria brassicicola.

2.2.4 Rapalexin A (7) The HPLC chromatograms of the neutral extracts of cultures of A. brassicicola incubated with rapalexin A (7) showed a consistent area for the peak corresponding to 7 (tR = 12.1 ± 0.2 min) for the duration of the experiments (96 h). That is, under standard experimental conditions, rapalexin A (7) was not metabolized by A. brassicicola.

0.14 0.12

Conc. (mM)

0.10 0.08 0.06 0.04 0.02 0.00 0

12

24

36

48

60

Incubation time (h)

72

84

96

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Figure 9. Progress curves of recovery of rapalexin A (7) () in cultures of Alternaria brassicicola and recovery from medium () (HPLC method A).

2.3 Antifungal activity of phytoalexins and biotransformation products against Alternaria brassicicola The antifungal activities of synthetic rutalexin (3), brassicanate A (5), isalexin (6), rapalexin A (7) and compounds 12, 14, 21, 22, 25, 26 and 28 against A. brassicicola were evaluated as described in Section 4.4. Due to the low solubility of rutalexin (3) and some metabolites, all phytoalexins and metabolites were tested in liquid medium. Results of antifungal bioassays against A. brassicicola are summarized in Table 2. Among the four phytoalexins, rapalexin A (7) displayed the highest antifungal activity, causing complete growth inhibition of mycelia at 0.50 mM and 89% at 0.20 mM, followed by brassicanate A (5) displaying 100% inhibition at 0.50 mM and 47% at 0.20 mM (Table 2). Due to its low solubility, rutalexin (3) could only be tested at concentrations • 0.10 mM, mM whereas its metabolites were tested at higher concentration. Metabolite 14 at 0.10 mM caused slightly higher growth inhibition (25% vs. 20%) than rutalexin (3), whereas 21 caused substantially lower growth inhibition (6% vs. 20%, at 0.10 mM). The latter result suggests that transformation of rutalexin (3) to rutapyrone (21) is a detoxification reaction. Compound 12 was tested to compare its inhibitory activity with that of metabolite 21, which was shown to be significantly less inhibitory than 12 at 0.10 mM. In addition, sulfonic acid 22 was virtually devoid of antifungal activity. Interestingly, the antifungal activity of metabolite 26 obtained from biotransformation of brassicanate A (5) by A. brassicicola was similar to that of the parent compound (5). Because compound 25 resulted from spontaneous decarboxylation of metabolite 26, the growth inhibitory activity of 25 was evaluated to compare with that of 5 and 26. Compound 25 is significantly less inhibitory than 5 or 26 at 0.50 mM, thus the apparent biotransformation of 26 to 25 is a

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detoxification reaction. Hence the overall transformation of brassicanate A (5) by A. brassicicola is a detoxification. The biotransformation of isalexin (6) to 28 yielded a metabolite displaying significantly lower inhibitory activity, hence this biotransformation is a detoxification reaction as well.

Table 2 Antifungal activitya of rutalexin (3), brassicanate A (5), isalexin (6), rapalexin A (7) and compounds 12, 14, 21, 22, 25, 26 and 28 against Alternaria brassicicola (in liquid medium). Compound (#)

Inhibition ± SD (%) 0.50 mM

0.20 mM

0.10 mM

Rutalexin (3)

n.d.

n.d.

20 ± 4d,e,f

Brassicanate A (5)

100 ± 0b

47 ± 8c

26 ± 4c,d

Isalexin (6)

40 ± 4d

25 ± 3d,e

18 ± 2e,f

Rapalexin A (7)

100 ± 0b

89 ± 2b

66 ± 3b

N-Methyl 2-methylsulfanediyl-1H-indole-3carboxamide (12) 2,2'-Disulfanediylbis(Nb-methyl-1H-indole-3carboxamide) (14) Rutapyrone (21) 3-(N-Methylcarbamoyl)-1H-indole-2-sulfonic acid (22) 2-Methylsulfanediyl-1H-indole (25)

29 ± 3e

20 ± 3e

15 ± 4f

n. d.

32 ± 0d

25 ± 3c

34 ± 3e

21 ± 3e

6 ± 3g

5 ± 2f

0g

0h

75 ± 3c

47 ± 3c

23 ± 3c,d

2-Methylsulfanediylindole-3-carboxylic acid (26) 1,3-Dihydro-3-hydroxy-4-methoxy-indole-2-one (28)

100 ± 0b

32 ± 4d

22 ± 3c,d,e

31 ± 2e

11 ± 4f

0h

a

The percentage of inhibition was calculated using the formula: % inhibition = 100 – [(growth on amended/growth in control) × 100]; values are averages of six independent experiments conducted in triplicate ± standard deviations; n.d. =not determined due to low solubility. For statistical analysis, one-way ANOVA tests were performed followed by Tukey’s test with adjusted αset at 0.05; b–h n = 6; different letters in the same column ( ) indicate significant differences (P < 0.05).

3.

Conclusion In conclusion, here we have developed a more efficient and simple method for the

preparation of the cruciferous phytoalexin rutalexin (3) in 81% yield starting from a simple

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starting material (8), shorter than any of the currently published methods.17, 27 Similarly, brassicanate A (5) was synthesized quantitatively from indoline-2-thione (15), a substantial improvement over a previous synthesis (51%).17 This work is the first report of the formation of a hybrid metabolite between a phytoalexin detoxification metabolite (23) and a fungal metabolite (20), i.e. rutapyrone (21). Nonetheless, further work that would include the isolation of the enzyme catalyzing the coupling of hypothetical metabolite 23 and phomapyrone G (20) needs to be carried out to understand the significance and implications of this discovery. Previous results of the transformation of the phytoalexins brassinin (1), cyclobrassinin (2), and camalexin (4) indicated that A. brassicicola could detoxify 1 (ca. 12 h at 0.10 mM)14 and 2 (ca. 8 h at 0.10 mM)28 quickly, whereas 4 was detoxified relatively slower (>120 h at 0.05 mM).16 In this work, the biotransformation of brassicanate A (5) was the fastest in ca. 4 h (at 0.10 mM), followed by rutalexin (3) (ca. 24 h). By contrast, the transformation of isalexin (6) was much slower (ca. 72 h), yielding several products, including product 28 that re-oxidized spontaneously to the parent compound. These transformations are detoxification reactions likely carried out by different enzymes. Importantly, rapalexin A (7) was not metabolized by A. brassicicola, suggesting that higher concentrations of rapalexin A could protect the producing plant species from infection by this pathogen. In conclusion, it is suggested that since rapalexin A (7) is resistant to A. brassicicola and camalexin (4) is transformed very slowly, engineering the biosynthetic pathway of 4 and increasing production of 7 in cultivated Brassica species could have a great positive impact in obtaining resistance to A. brassicicola.

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Experimental

4.1 General Chemicals were purchased from Alpha Aesar, Ward Hill, MA or Sigma-Aldrich Canada Ltd., Oakville, ON; solvents were HPLC grade and used as such. Flash column chromatography (FCC) was carried out using silica gel grade 60, mesh size 230 – 400 Å or WP C18 prepscale bulk packing 275 Å (J.T. Baker, NJ, USA). Organic extracts were dried over Na2SO4 and concentrated using a rotary evaporator. NMR spectra were recorded on Bruker 500 or 600 MHz Avance spectrometers, for 1H, 500.3 MHz or 600.2 and for 13C, 125.8 or 150.9 MHz; chemical shifts (δ) are reported in parts per million (ppm) relative to TMS; spectra were calibrated using the solvent peaks; spin coupling constants (J) are reported to the nearest 0.5 Hz. FTIR data were recorded on a Bio-Rad FTS-40 spectrometer and spectra were measured by the diffuse reflectance method on samples dispersed in KBr. HREI-MS were obtained on a VG 70 SE mass spectrometer employing a solids probe or on a Jeol AccuToF GCv 4G mass spectrometer [field desorption (FD)] by direct insertion. HPLC analysis was carried out with Agilent high performance liquid chromatographs equipped with quaternary pump, automatic injector, and photodiode array detector (DAD, wavelength range 190 - 600 nm), degasser, and a column Eclipse XDB-C18 (5 μm particle size silica, 4.6 i.d. ×150 mm), having an in-line filter, using methods: A, mobile phase 50% H2O 50% CH3OH to 100% CH3OH for 25.0 min, linear gradient, at a flow rate of 0.75 mL/min; B, mobile phase 90% H2O – 10% MeOH to 100% MeOH for 45.0 min, linear gradient, at a flow rate 0.75 mL/ min; C, mobile phase 100% H2O – TFA (0.10%) to 100% MeOH – TFA (0.10%) for 40.0 min, linear gradient, at a flow rate 1.0 mL/min.

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4.2 Synthesis 4.2.1 Rutalexin (4) A solution of NaClO2 (1 g, 6.80 mmol) and KH2PO4 (1.2 g, 8.8 mmol) in water (6 mL) was added to a mixture of 1-t-Boc-2-chloroindole-3-carboxaldehyde (8, 255 mg, 0.900 mmol), tert-butanol (6.4 mL) and 2-methylbut-2-ene (6.4 mL). The reaction mixture was stirred for 4 h at rt. The organic phase was separated and the aqueous layer was acidified pH 3 (HCl, 0.50 M) and extracted with EtOAc. The organic layer and extracts were combined, dried and concentrated to dryness to afford 1-t-Boc-2-chloroindole-3-carboxylic acid (270 mg, 0.900 mmol, 100%). Thionyl chloride (250 μL, 3.75 mmol) was added to a solution of 1-t-Boc-2-chloroindole-3carboxylic acid (90 mg, 0.30 mmol) in dry THF (3 mL) at 0 °C followed by catalytic amount of DMF (5 μL). The reaction mixture was stirred for 2 h at rt. The excess of thionyl chloride was evaporated, the reaction mixture was cooled to 0 °C and a solution of CH3NH2 in THF (2 M, 4 mL, 8 mmol) was added slowly (10 min). The reaction mixture was stirred for an additional 1 h at 0 °C, was diluted with water and extracted with EtOAc. The combined extracts were dried and concentrated to dryness and the residue was subjected to FCC (silica gel, EtOAc-hexane, 1:1) to afford amide 9 (88 mg, 0.29 mmol, 95%) as colorless oil. A solution of sodium hydrogen sulfide (360 mg, 3.20 mmol) in water (100 μL) was added to a mixture of amide 9 (50 mg, 0.25 mmol) in DMF (1.5 mL) at 0 °C and the reaction mixture was stirred at 0 °C for 1 h. Methyl chloroformate (1.2 mL, 14 mmol) was added dropwise to the reaction mixture, the ice bath was removed and the reaction mixture was stirred for an additional 30 min at rt. The reaction mixture was diluted with water, extracted with EtOAc, and the combined extracts were dried and concentrated to dryness to afford 11 (56 mg, 0.15 mmol, 95%) as colorless oil (spectra in Supplementary Data). Triethylamine (50 μL) was added to a solution of amide 11 (30 mg, 0.08 mmol, 1 eq) in THF (1

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mL) and the reaction mixture was stirred at 50 °C for 3 h. After amide 11 was consumed, the reaction mixture was cooled to rt and a mixture of 20% TFA in DCM (2 mL) was added. The reaction mixture was kept stirring for an additional 4 h at rt, was concentrated to dryness and the residue was rinsed with diethyl ether (2 mL × 2) to afford rutalexin (3, 17 mg, 0.07 mmol, 90%) as white powder. The spectroscopic data were identical to those previously reported.17 4.2.2 Brassicanate A (5) Methyl iodide (280 mg, 123 μL, 1.97 mmol, 2.7 eq) was added to a solution of indole-2thione (15, 100 mg, 0.74 mmol, 1 eq) and sodium carbonate (120 mg, 1.13 mmol, 1.5 eq) in acetone (2 mL) and the mixture was stirred for 10 h at rt.29 The reaction mixture was diluted with brine (10 mL), extracted with EtOAc and the combined extracts were dried and concentrated to dryness to afford 2-methylsulfanediylindole (25, 109 mg, 0.67 mmol) as yellow oil in quantitative yield. Phosgene (130 μL, 0.6 mmol, 4 eq) was added to a solution of 2methylsulfanediylindole (25, 25 mg, 0.06 mmol, 1 eq) in THF (0.5 mL), the reaction mixture was stirred for 2 h at rt, MeOH (1 mL) was added and the reaction mixture was stirred for an additional 30 min at rt. The reaction mixture was diluted with water, extracted with EtOAc, the combined extracts were dried and concentrated to afford brassicanate A (5) as brownish solid in quantitative yield (13.5 mg, 0.06 mmol). The spectroscopic data were identical to those previously reported.17 4.2.3 Isalexin (6) A solution of 4-methoxyindole (18, 25 mg, 0.17 mmol, 1 eq) in 1,2-dichloroethane (DCE) (2 mL) was added to a solution of pyridinium chlorochromate (PCC) (273 mg, 1.36 mmol, 8 eq) in DCE (2 mL) followed by aluminum chloride (7 mg, 0.05 mmol, 0.3 eq). The reaction mixture

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was heated at 80 °C for 2 h and was concentrated to dryness. The residue was subjected to FCC (EtOAc-hexane, 1:1) to afford isalexin (6, 16 mg, 0.09 mmol, 53%) as a yellow powder. The spectroscopic data were identical to those previously reported.17 4.2.4 N-Methyl-2-(methylsulfanediyl)-1H-indole-3-carboxamide (12) A solution of NaHS (108 mg, 1.19 mmol) in water (100 μL) was added to a mixture of amide 9 (30 mg, 0.15 mmol) in DMF (1 mL) at 0 °C and the reaction mixture was stirred at 0 °C for 1 h. Methyl iodide (187 μL, 3.00 mmol) was added dropwise to the reaction mixture, the ice bath was removed and the reaction mixture was stirred for an additional 30 min at rt. The reaction mixture was diluted with water, extracted with EtOAc, and the combined extracts were dried and concentrated to dryness to afford t-Boc protected 12 as a light yellow solid, which was treated with a solution of 20% TFA in DCM (2 mL). The reaction mixture was stirred for 4 h at rt, concentrated to dryness and the residue was subjected to FCC (silica gel, EtOAc-hexane, 1:1) to afford carboxamide 12 (15 mg, 0.070 mmol, 94%) as yellow oil. H NMR (500 MHz, CDCl3): δ8.80 (br s, 1H), 8.30 (d, J= 7.0 Hz, 1H), 7.33 (d, J= 7.0 Hz,

1

1H), 7.25-7.21 (m, 2H), 3.08 (d, J= 3.5 Hz, 3H), 2.55 (s, 3H). 13C NMR (500 MHz, CDCl3): δ 166.3, 136.3, 132.2, 127.6, 123.5, 121.8, 121.6, 112.3, 111.0, 26.5, 19.3. HPLC tR = 2.8 ± 0.2 min. UV (HPLC) λmax (nm): 220, 300. FTIR (KBr) νmax cm-1: 2926, 1725, 1618, 1543, 1413, 1313, 1148, 750. HRMS-EI m/z: measured 220.0668 ([M]+, calcd. 220.0670 for C11H12N2OS) (100%). 4.2.5 2,2'-Disulfanediylbis(N-methyl-1H-indole-3-carboxamide) (14) A solution of NaHS (108 mg, 1.92 mmol) in water (100 μL) was added to a solution of amide 9 (30 mg, 0.15 mmol) in DMF (1 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h, diluted with brine (10 mL) acidified (pH 3, HCl, 0.50 M), extracted with EtOAc, and the

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combined extracts were dried and concentrated to dryness. The residue was dissolved in 20% TFA in DCM (1.50 mL), was stirred for 4 h at rt and was concentrated to dryness. The final residue was rinsed with chloroform to yield 14 in quantitative yield (20 mg, 0.02 mmol) as an off-white powder, mp 232 – 234 °C. HPLC tR = 10.2 ± 0.2 min. UV (HPLC) λmax (nm): 220, 310. FTIR (KBr) νmax cm-1: 1614, 1535, 1438, 1409, 1209, 1026, 742. HRMS-FD m/z: measured 410.0885 ([M]+, calcd. 410.0871 for C20H18N4O2S2) (100%). The 1H and 13C NMR spectroscopic data were identical to those previously reported.20 4.2.6 3-(N-Methylcarbamoyl)-1H-indole-2-sulfonic acid (22) A solution of m-CPBA (90 mg, 0.50 mmol, 16.60 eq) in DCM (1 mL) was added to a solution of disulfide 14 (14 mg, 0.03 mmol, 1 eq) in MeOH (0.50 mL). The reaction mixture was stirred for 2 h at rt followed by addition of dimethyl sulfide (120 μL, 0.12 mmol). The reaction mixture was stirred for an additional 30 min at rt and concentrated to dryness. The residue was fractionated using a reverse-phase column (MeOH-H2O) to afford sulfonic acid 22 (10 mg, 0.04 mmol, 59%) as white powder, mp > 300 °C. H NMR (500 MHz, DMSO-d6): δ 11.56 (br s, 1H), 9.07 (d, J = 3.5 Hz, 1H), 8.22 (d, J =

1

8.0 Hz, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.12 (dd, J = 7.0, 7.5 Hz, 1H), 7.05 (dd, J = 7.5, 7.5 Hz, 1H), 3.16 (s, 1H), 2.78 (d, J = 4 Hz, 3H). 13C NMR (125.8 MHz, DMSO-d6): δ 164.6, 141.8, 132.9, 127.7, 122.4, 122.4, 120.5, 112.1, 105.8, 25.7. HPLC tR = 1.0 ± 0.2 min (HPLC method C). UV (HPLC) λmax (nm): 220, 280. FTIR (KBr) ν••• cm-1: 1600, 1463, 1368, 1118, 1072, 802. HRMS-FD m/z: measured 254.0351 ([M]+, calcd. 254.0361 for C10H10N2O4S) (100%).

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4.2.7 2-Methylsulfanediylindole-3-carboxylic acid (26) Phosgene (195 μL, 2.7 mmol, 9 eq) was added to a solution of 2-methylsulfanediylindole (25, 50 mg, 0.3 mmol, 1 eq) in THF (2 mL) at 0 °C. The ice bath was removed and the reaction mixture was stirred for 2 h at rt. Water (5 mL) was added to the reaction mixture and the mixture was stirred for an additional 30 min at rt. The reaction mixture was diluted with water, extracted with EtOAc and the combined extracts concentrated to dryness. The residue was subjected to FCC on silica gel (DCM-MeOH, 98:2) to afford acid 26 (33 mg, 0.16 mmol, 52 %) as white solid, mp 167 - 170 °C. H NMR (500 MHz, CD3CN): δ 9.85 (br s, 1H), 7.92-7.90 (m, 1H), 7.45-7.43 (m, 1H),

1

7.19-7.14 (m, 2H), 2.60 (s, 3H). 13C NMR (500 MHz, CD3CN): δ 167.0, 146.4, 137.6, 128.6, 122.8, 122.7, 120.9, 111.7, 103.7, 14.7. HPLC tR = 12 ± 0.2 min (HPLC method B). UV (HPLC) λmax (nm): 240, 270, 300. FTIR (KBr) νmax cm-1: 1639, 1498, 1485, 1448, 1301, 1201, 1060, 748. HRMS-ESI m/z: measured 206.0276 ([M-H]-, calcd. 206.0281 for C10H8N1O2S1). 4.2.8 Brassicanate A sulfoxide (27) A solution of m-CPBA (20 mg, 0.1 mmol, 1.2 eq) in DCM (0.50 mL) was added to a solution of brassicanate A (5, 20 mg, 0.09 mmol, 1 eq) in MeOH (1 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min, dimethyl sulfide (6.8 mg, 0.80 mL, 0.11 mmol, 1.2 eq) was added and the mixture was stirred for an additional 30 min at rt. The reaction mixture was concentrated to dryness and the residue was subjected to FCC on silica gel (EtOAc-hexane, 1:1) to afford sulfoxide 27 (16.6 mg, 0.07 mmol, 79%) as white solid, mp 207 °C (decomposed). H NMR (500 MHz, CDCl3): δ11.19 (br s, 1H), 8.09 (d, J= 7.5 Hz, 1H), 7.61 (d, J= 8.0

1

Hz, 1H), 7.37-7.27 (m, 2H), 3.98 (s, 3H), 3.11 (s, 3H). 13C NMR (500 MHz, CDCl3): δ 165.2,

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145.5, 136.4, 127.0, 124.5, 123.0, 121.7, 112.9, 105.8, 51.9, 42.3. HPLC tR = 13.6 ± 0.2 min (HPLC method B). UV (HPLC) λmax (nm): 210, 230, 300. FTIR (KBr) νmax cm-1: 3196, 1680, 1510, 1448, 1270, 1223, 1171, 1037, 786, 747. HRMS-FD m/z: measured 237.0459 ([M]+ , calcd. 237.0460 for C11H11NO3S). 4.2.9 1,3-Dihydro-3-hydroxy-4-methoxy-indol-2-one (28) Sodium borohydride (5.0 mg, 0.15 mmol, 1 eq) was added portion wise to a solution of isalexin (6, 25.0 mg, 0.15 mmol, 1 eq) in EtOH (95%, 2 mL) at 0 °C and the reaction mixture was stirred at 0 °C for 20 min. The resulting suspension was purred into cold water (5 mL), acidified (pH 5, HCl, 0.50 M) and extracted with EtOAc. The combined extracts were concentrated and the residue was subjected to FCC on silica gel (EtOAc-hexane, 1:1) to afford 1,3-dihydro-3hydroxy-4-methoxy-indol-2-one (28, 20 mg, 0.010 mmol, 80%) as light yellow solid, mp 195 196 °C. H NMR (500 MHz, CD3OD): δ 7.22 (dd, J= 8.0, 8.0 Hz, 1H), 6.66 (d, J= 8.5 Hz, 1H),

1

6.49 (d, J= 8.0 Hz, 1H), 4.91 (s, 1H), 3.86 (s, 3H). 13C NMR (500 MHz, CD3OD): δ180.4, 158.8, 144.9, 132.5, 107.2, 104.5, 70.0, 56.0, 31.0. HPLC tR= 8.5 ± 0.2 min. UV (HPLC) λmax (nm): 219, 294. FTIR (KBr) νmax cm-1: 3197, 1681, 1511, 1448, 1270, 1222, 1171, 1037, 786, 748. HRMSFD m/z: measured 202.0481 ([M + Na]+ , calcd. 202.0475 for C9H9NO3Na).

4.3 Fungal cultures and biotransformations A. brassicicola, isolate UAMH 7474 was obtained from the University of Alberta Microfungus Collection & Herbarium, Edmonton, AB, and subcultured on PDA plates under constant light for 10 days at 23 ± 1 °C. Liquid cultures were initiated by inoculating A. brassicicola spores (106 per 100 mL) in a chemically defined medium24 (MM) at 23 ± 1 °C, under

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constant light, on a shaker at 110 rpm. 4.3.1 Biotransformations Solutions of rutalexin (3), brassicanate A (5), isalexin (6) and rapalexin A (7) in DMSO or CH3CN were added to liquid shake cultures (50 mL MM in 150 mL Erlenmeyer flasks, final concentration 0.10 mM). Cultures were incubated on a shaker, samples (5 mL) were withdrawn at appropriate times, frozen or immediately extracted with EtOAc, the extracts were concentrated, dissolved in CH3CN or MeOH and analyzed by HPLC-DAD-ESI-MS. Experiments were performed in triplicate, control flasks, containing the mycelia in minimal medium or compounds in minimal medium were incubated under similar conditions. For experiments carried out in H2O, mycelia of cultures grown in MM were filtered off after 72 h of incubation in medium and transferred into sterile H2O. Cultures were incubated on a shaker at rt and treated as reported above.

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Table 3 HPLC retention times (tR, HPLC methods A, B or C, Section 4.1) of phytoalexins and metabolites. Compound (#) Rutalexin (3) Brassicanate A (5) Isalexin (6) Rapalexin A (7) N-Methyl-2-(methylsulfanediyl)-1H-indole-3-carboxamide (12) 2,2'-Disulfanediylbis(N-methyl-1H-indole-3-carboxamide) (14) Phomapyrone G (20) Rutapyrone (21) 3-(N-Methylcarbamoyl)-1H-indole-2-sulfonic acid (22) 2-Methylsulfanediylindole (25) 2-Methylsulfanediylindole-3-carboxylic acid (26) Brassicanate A sulfoxide (27) 1,3-Dihydro-3-hydroxy-4-methoxy-indol-2-one (28)

tR ± 0.2 min (method) 8.8 (A) 15.8 (B) 9.3 (B) 12.1 (A) 2.8 (A) 10.2 (A) 6.7 (A) 9.6 (A) 1.0 (A) 23.2 (C) 12.0 (B) 13.6 (B) 8.6 (B)

4.3.2 Large scale culture to isolate rutapyrone (21) To obtain sufficient amounts for spectroscopic characterization of the minor product (21) of metabolism of rutalexin (3), larger scale cultures were carried out as follows. Three-days-old mycelia of A. brassicicola (10 flasks, 1 L of MM) were filtered off separately and the mycelial cake of each flask aseptically transferred to an Erlenmeyer flask (10) containing sterile water. Rutalexin (3, 23 mg, 1 L, 10 flasks) was administered to cultures and incubated as above. After 72 hours the mycelia were filtered off, the filtrate was concentrated to a small volume by freezedrying (ca. 30 mL) and extracted with EtOAc. The combined organic extracts were dried, concentrated to dryness and the residue was subjected to reverse-phase chromatography and eluted with CH3CN-H2O (20:80, 15 mL; 25:75, 15 mL; 30:70, 15 mL). Fractions were analyzed by HPLC and the fractions containing rutapyrone (21) were combined and concentrated to dryness to afford rutapyrone (21, 2.8 mg, 0.006 mmol); a very small amount of crude

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phomapyrone G (20) was also obtained (<1 mg). Rutapyrone (21) 1

H NMR (600 MHz) and 13C NMR see Table 1. HPLC tR = 9.6 ± 0.2 min (HPLC method

A). [α]D +9.3 (c 0.36, MeOH). UV (HPLC) λmax (nm): 220, 300, 350; FTIR (KBr) νmax cm-1: 1680, 1551, 1463, 11383, 1226, 1171, 1013, 751; HRMS-ESI m/z: measured 457.1777 ([M+H]+, calcd. 457.1792 for C24H29N2O5S) (100%); mp 250 – 252 °C.

4.4 Antifungal bioassays The antifungal activity of phytoalexins and metabolites against A. brassicicola was evaluated using a mycelial radial growth assay carried out in liquid MM. First, to use as mycelial inoculum, cultures of A. brassicicola were grown on potato dextrose agar (PDA) plates (10 cm diameter) at 23 ± 1 °C under constant light for 7 days. Next, DMSO solutions of each compound (0.50, 0.20, 0.10 and 0.05 mM) in liquid MM (1% DMSO final concentration) were added to each well of six-well plates (1.0 mL per well) and a mycelial plug (4 mm diameter) cut from edges of 7-d-old solid cultures was placed upside down in the center of each well; control cultures containing 1% DMSO in MM were prepared similarly. Plates were incubated under constant light and the diameter of mycelial mat (mm) in each well was measured (72 h). Each assay was conducted in triplicate and repeated three times. The percentage of growth inhibition was calculated as reported in Table 2.

Acknowledgements Financial support for the authors’ work was obtained from the Natural Sciences and Engineering Research Council of Canada (Discovery Grant to M.S.C.P.), the Canada Research Chairs program, Canada Foundation for Innovation, the Saskatchewan Government, and the

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University of Saskatchewan (graduate assistantship to A.A.). We acknowledge the technical assistance of K. Brown (NMR) and K. Thoms (MS) from the Department of Chemistry.

Appendix A. Supplementary Data Supplementary data associated with this article (characterization data of compound 11 and NMR spectra for new compounds 11, 21, 22, 26, 27 and 28) can be found in the online version at doi: //////.

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29. Pedras and Khan, 1996.

Graphical Abstract

O

O

NH

N O S N H

S A. brassicicola

CO2CH3 N H

OCH3

SCH3 A. brassicicola

N H

OCH3 NCS

O HO

O

A. brassicicola

N H

COOH

N H

SCH3

No transformation