Metabolism of Antifungal Thiochroman-4-ones by Trichoderma viride

Note Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Metabolism of Antifungal Thiochroman-4-ones by Trichoderma viride and Botrytis cinerea Cristina Pinedo-Rivilla, Isidro G. Collado,* and Josefina Aleu* Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz, Campus Río S. Pedro, 11510 Puerto Real, Cádiz, Spain S Supporting Information *

ABSTRACT: Biotransformation of 6-methylthiochroman-4one (1) and 6-chlorothiochroman-4-one (2) was performed using Trichoderma viride in order to obtain new derivatives with antifungal properties against the phytopathogen Botrytis cinerea. Two thiochromanone derivatives are described for the first time. Antifungal activity of these compounds was tested against two different strains of Botrytis cinerea; 1 and 2 gave 100% inhibition of Bc2100 at 100−250 μg/mL, and 3 gave a maximal inhibition of 96% of BcUCA992 at 200 μg/mL. The detoxification mechanism of 1 and 2 by B. cinerea was also investigated.

T

(3),14 6-methylthiochroman-4-ol 1-oxide (4), (S)-6-methylthiochroman-4-ol 1,1-dioxide (5),15 (S)-(−)-6-chlorothiochroman-4-ol (6),16 (1R, 4R)-6-chlorothiochroman-4-ol 1-oxide ((1R,4R)-7), (S)-(−)-6-chlorothiochroman-4-ol 1,1-dioxide (8),17 and 6-chlorothiochroman-4-one 1,1-dioxide (9).18 Compounds 4 and 7 are reported here for the first time. Metabolite 3 was deduced to have the molecular formula C10H12OS from its HREIMS and 13C NMR data (Table 1). The structure of 3 was established as (R)-6-methylthiochroman-4-ol (3), a known compound14 whose spectroscopic data were not found in the literature. The absolute configuration at C-4 was determined by the Mosher ester analysis protocol.13 Specifically, derivatization of 3 with (R)- and (S)-α-methoxy-αphenylacetic (MPA) acids followed by 1H NMR analysis of the resulting diastereomeric esters (S)-MPA-3 and (R)-MPA-3 revealed a negative Δδ (δR − δS) value for H-11 (−0.18) and H-5 (−0.46) and positive Δδ (δR − δS) values for H-2 and H-3 (+0.36, +0.31 and +0.27, +0.27, respectively). Following MPA rules, these data indicated an R-configuration for C-4 in 3. The highest enantiomeric excess (99% ee) for this metabolite was obtained after 7 days of fermentation. Compound 4 gave the molecular formula C10H12O2S, as indicated by HREIMS and 13C NMR data (Table 1). Comparison of its NMR data with those of 3 indicated the presence of a sulfoxide group, suggested by the downfield signal shift of H-2 (δH 3.37 and 3.06), H-3 (δH 2.80 and 2.15), and H8 (δH 7.61) for the major diastereomer. Furthermore, structure 4 was supported by homo- and heteronuclear-2D-correlation experiments. This compound was obtained as a mixture of

hiochromanone compounds are versatile reagents that have been extensively used to synthesize novel heterocycles1 and biologically active compounds. Heterocyclic fragments containing oxygen or sulfur are important cores found in natural and synthetic products used in the flavor, fragrance, and pharmaceutical industries.2 Thiochromanones have been reported to possess cytotoxic,3−5 antifungal,6−8 and other important biological activities. Trichoderma viride is a filamentous fungus widely used as a biological agent against different plant pathogens through induction of plant defense and mycoparasitism.9 It is known to produce hydrolytic enzymes with antagonistic properties against other fungi including Botrytis cinerea.10 Based on previous results regarding the biocatalytic potential of T. viride to biotransform compounds containing sulfur to produce enantiomerically pure derivatives,11,12 we now report on the biotransformation of 6-methylthiochroman-4-one (1) and 6-chlorothiocroman-4-one (2) by T. viride to obtain new derivatives that are active against the phytopathogen B. cinerea. We also report on the biotransformation of 1 and 2 by two strains of B. cinerea constituting part of the fungal detoxification mechanism. The substrates 6-methylthiochroman-4-one (1) and 6chlorothiochroman-4-one (2) were incubated separately with T. viride. The biotransformation products isolated were quantified for each culture (see Experimental Section) and identified by their 1D and 2D 1H and 13C NMR data. The diastereomeric ratio and enantiomeric excesses were determined by HPLC analysis using a Chiralcel OD column. The Mosher method13 was used to determine the absolute configuration of the compounds obtained. Three biotransformation products for 1 (3, 4, 5) and four for 2 (6, 7, 8, 9) were isolated: (R)-6-methylthiochroman-4-ol © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 6, 2017

A

DOI: 10.1021/acs.jnatprod.7b00298 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Note

Scheme 1. Biotransformation of 6-Methylthiochroman-4-one (1) and 6-Chlorothiochroman-4-one (2)

diastereomers with 96% de in favor of the anti-configuration and 94% ee after 10 days of incubation. The anti-configuration for 4 was assigned through the study of the high-field signal pattern of H-3α,3β and H-2α,2β. Enantioselectivity of metabolite 4 was very high, but in the absence of at least one enantiomer of known configuration, we had no way of determining which enantiomer was which. Compound anti-6methylthiochroman-4-ol 1-oxide (4) is reported here for the first time. Metabolite 5 was isolated as a colorless oil with the molecular formula C10H12O3S determined from its HREIMS and corroborated by 13C NMR data. The NMR data were similar to those of 4, but a marked low-field shift for H-2 (δH 3.69 and 3.27), H-3 (δH 2.73 and 2.53), and H-8 (δH 7.80) indicated the presence of a sulfone group for compound 5 (Table 1). Its absolute configuration was determined by comparison of its specific rotation value with that of (R)-6-methylthiochroman-4ol (3), whose absolute configuration is known. Compound 5 presented a negative value, indicating a configuration opposite that of 3 at C-4. Metabolite (S)-6-methylthiochroman-4-ol 1,1dioxide (5) was previously reported as a product from chemical transformations,15 but no spectroscopic data were found for this compound in the literature. These data are reported here for the first time. The highest enantiomeric excess (72% ee) for compound 5 was obtained after 10 days of incubation. Metabolite 6 was established as (S)-(−)-6-chlorothiochroman-4-ol (6), a known compound, by comparing its spectroscopic data with the data found in the literature for the product with configuration R.16 Compound 6 presented a negative specific rotation value, indicating an S-configuration at C-4. Compound 7 was isolated as a colorless oil. Its molecular formula of C9H9O2SCl was determined from its HREIMS and corroborated by 13C NMR data (Table 1). Comparison of the NMR data of 7 with those of 6 indicated the presence of a sulfoxide group by the downfield signal shift of H-2 (δH 3.35 and 3.00), H-3 (δH 2.64 and 2.10), and H-8 (δH 7.60). The absolute configuration of sulfoxide 7 at C-4 was determined by studying its Mosher esters. Reaction of 7 with (R)-MPA and (S)-MPA gave the corresponding Mosher esters,13 which were studied by 1H NMR analysis. In accordance with this method, a negative Δδ (δR − δS) value for H-5 (−0.44) and positive Δδ values for H-2 (+1.29, +1.4) indicated a 4R-configuration in compound 7. This compound was produced by T. viride as the only diastereoisomer, with 99.9% de. The anti-configuration for 7 was assigned through the study of the highfield shift of H-4 and the signal patterns of H-3 and H-2. This metabolite was thus established as (1R,4R)-6-chlorothiochroman-4-ol 1-oxide ((1R,4R)-7) and is reported here for the first time.

The molecular formula of C9H9O3SCl of compound 8 was determined by HREIMS and indicates that 8 possesses one more oxygen atom than the corresponding sulfoxide 7. Moreover, the signals for H-2 (δH 3.67 and 3.32), H-3 (δH 2.74 and 2.56), and H-8 (δH 7.84) in its 1H NMR spectrum presented a marked upfield shift, indicating the presence of a sulfone group (Table 1). A negative value of its specific 16 rotation, [α]23 D = −14.5, suggests an S-configuration at C-4. That compound was produced by T. viride with 90% ee. No spectroscopic data were found for compound (S)-(−)-6chlorothiochroman-4-ol 1,1-dioxide (8) in the literature.17 These data are reported here for the first time. It is very interesting to note that T. viride was also able to produce the compound 6-chlorothiochroman-4-one 1,1-dioxide (9), the sulfone with a ketone group. Spectroscopic data of this product coincided with those reported in the literature.18 Evaluation of the antifungal properties of the biotransformation substrates and products against two different strains of B. cinerea, Bc2100 and BcUCA992, was carried out using the conidial germination assay (Figures S1 and S2, Supporting Information).19 Substrates 6-methylthiochroman-4-one (1) and 6-chlorothiochroman-4-one (2) were active against both strains of B. cinerea. Strain Bc2100 was more sensitive than BcUCA992 to both products, with germination inhibition of 100% for 1 and 2 at 200, 150, and 100 μg/mL concentration. Biotransformation products have proven to be less toxic to fungal growth than 1 and 2, except for product (R)-6methylthiochroman-4-ol (3) (IC50 of 17.1 and 86.0 against BcUCA992 and Bc2100, respectively). Compounds 4, 5, 7, 8, and 9 exhibited significantly less antifungal activity than 1 and 2. The sulfur atom oxidation seems to be a detoxification strategy of the fungus to decrease toxicity. Compounds 8 and 9 were completely inactive against either strain. Compound 7 exhibited some degree of antifungal activity. This compound allowed conidial germination, but hyphal elongation was controlled, which would prevent the start of a possible infection. These results suggest that the presence of a chlorine atom in the molecule does not improve the antifungal activity of this family of compounds. Thus, in order to study the probable fungal detoxification mechanism of the phytopathogenic fungus B. cinerea, biotransformations of 6-methylthiochroman-4-one (1) and 6chlorothiochroman-4-one (2) were carried out by two strains of B. cinerea, Bc2100 and BcUCA992, to identify differences in their enzymatic potential to degrade xenobiotic substrates. Bioconversion of 6-methylthiochroman-4-one (1) by strain Bc2100 afforded syn-(+)-6-methylthiochroman-4-ol 1-oxide (syn-4) and (S)-(−)-6-methylthiochroman-4-ol 1,1-dioxide (5). It is interesting to note that the fungus is able to produce only one enantiomer for compound 4 with 99% ee and 99% de, exhibiting higher enantio- and diastereoselectivity. Compound syn-6-methylthiochroman-4-ol 1-oxide (syn-4) is reported here B


125.4 136.3 139.6 131.6 135.9 139.1 7.60, d (8.3) 130.8 131.8 140.9 7.64, d (8.2) 123.7 137.6 135.1 21.6 2.41, s 2.40, s

7.62, d (7.9)

2.37, s

129.9 135.3 139.1 21.4 7.60, d (7.9)

2.28, s

126.6 129.5 133.9 20.7 7.02, d (8.1)

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined with a PerkinElmer 341 polarimeter. IR spectra were recorded on a Mattson Genesis spectrophotometer, series FTIR. 1H and 13C NMR measurements were obtained on Varian Unity 400 and Varian Unity 600 NMR spectrometers with SiMe4 as the internal reference. Mass spectra were recorded on a GC-MS Thermoquest spectrometer (model: Voyager) and a VG Autospec-Q spectrometer. HPLC was performed with a Hitachi/Merck L-6270 apparatus equipped with a UV−vis detector (L 6200) and a differential refractometer detector (RI-71). TLC was performed on Merck Kiesegel 60 F254, 0.2 mm thick. Silica gel (Merck) was used for column chromatography. Purification by means of HPLC was accomplished with a silica gel column (Hibar 60, 7 m, 1 cm wide, 25 cm long). Chemicals used were from Fluka, BASF, or Aldrich. All solvents used were freshly distilled. Enantiomeric excesses were determined by means of HPLC analyses on a chiral column (Chiralcel OD, Daicel, Japan). Microorganism Cultures and Antifungal Assays. The fungi cultures employed in this work were obtained from grapes from the Domecq vineyard, Jerez de la Frontera, Cádiz, Spain (B. cinerea UCA992) and from the Colección Española de Cultivos Tipo (CECT), Facultad de Biologia,́ Universidad de Valencia, Spain (B. cinerea 2100 and Trichoderma viride UCA06), where cultures of these strains are deposited. Antifungal activity was determined by means of a conidial germination assay. The test compound was dissolved in ethanol− water mixtures to give a final compound concentration of 25−200 mg L−1. Two microliters of a conidial suspension in water containing approximately 5 × 104 conidia/mL of the fungi strains was added to a solution of the test compound (15 μL) and PDB (potato dextrose broth) medium (2 μL) on ELISA plates. The final ethanol concentration was identical in control and treated cultures. Three replicates were made per compound, and the viability of conidia was estimated by measuring their germination immediately after incubation

131.0 134.4 135.8 21.4

7.80, d (8.5)

■

8 9 10 11

7.40, dd (8.3, 2.1) 7.40, dd (8.2, 1.5) 7.33, bd (8.5) 6.97, dd (8.1, 1.6)

66.5 130.9 134.3 129.4 4 5 6 7

3b

2.03, dddd (13.8, 12.3, 3.5, 3.5) 4.74, dd (4.8, 3.5) 7.13, brs

30.2 3a

7.24, d (7.9)

67.3 129.5 143.5 129.7

22.4

2.96, ddd (13.8, 9.1, 5.3) 2.68, dddd (14.1, 9.1, 5.6, 4.7) 2.30, dddd (14.1, 11.4, 5.6, 5.3) 4.77, t (5.6) 7.45, brs 2b

7.29, d (7.9)

65.6 130.2 143.0 130.9

22.4

41.6

for the first time. However, under the same conditions strain BcUCA992 was able to produce both diastereomers, anti- and syn-6-methylthiochroman-4-ol 1-oxide (4), and the compounds (R)-(+)-6-methylthiochroman-4-ol (3) and (S)-(−)-6-methylthiochroman-4-ol 1,1-dioxide (5). Moreover, the following products were obtained when 6chlorothiochroman-4-one (2) was incubated with the strain Bc2100: syn-6-(+)-chlorothiochroman-4-ol 1-oxide (syn-7), (1R,4R)-6-chlorothiochroman-4-ol 1-oxide [(1R,4R)-7], and (R)-(+)-6-chlorothiochroman-4-ol 1,1-dioxide [(R)-8]. However, biotransformation of 2 by BcUCA992 afforded (S)-6chlorothiochroman-4-ol16 (6), syn-(+)-6-chlorothiochroman-4ol 1-oxide (syn-7), (1R,4R)-6-chlorothiochroman-4-ol 1-oxide [(1R,4R)-7], and traces of 6-chlorothiochroman-4-ol 1,1dioxide (8). The increased ability of strain BcUCA992 to biotransform substrates 1 and 2 is in accordance with antifungal assay results. This is a wild strain isolated from a vineyard in 1992 that appears to have genes for oxidases involved in the detoxification of 1 and 2, which may not be present in the genome of strain Bc2100. These data clearly reveal adaptive resources for the wild strain with respect to strain Bc2100 from CECT’s lab collection. The antifungal activity of the detoxification products against B. cinerea was also studied. With the exception of compound 3 against strain BcUCA992, these compounds were less toxic to fungal growth than 1 and 2, thus confirming that B. cinerea has a mechanism to detoxify these compounds probably by reducing the ketone group and oxidizing the sulfur atom.

7.48, dd (8.5, 2.0) 7.84, d (8.5)

65.7 128.9 139.3 130.0 4.90, brs 7.56, d (2.0)

66.9 129.6 138.2 129.2 65.8 129.4 143.8 130.5

29.5

2.32, dddd (14.6, 9.5, 6.2, 4.9) 4.72, t (6.2) 7.50, d (1.5)

22.9 2.98, ddd (13.8, 9.2, 4.9) 2.61, m

4.87, t (3.7) 7.47, d (2.1)

22.9

65.1 130.2 138.2 129.4

2.56, m

29.7

46.8

3.67, ddd, (13.9, 10.2, 3.3) 3.32, ddd (13.9, 8.1, 3.1) 2.74, m 42.2

3.35, ddd (13.9, 10.8, 3.7) 3.00, ddd (13.9, 6.4, 3.7) 2.64, dddd (14.7, 10.8, 3.7, 3.7) 2.10, m 44.2 3.36, m

3.69, ddd (13.9, 10.8, 3.2) 3.27, ddd (13.9, 7.6, 3.2) 2.73, dddd (14.0, 10.8, 3.2, 3.2) 2.53, dddd (14.0, 7.6, 5.0, 3.2) 4.89, brs 7.32, brs

46.6

Note

3.37, ddd (13.9, 11.6, 3.2) 3.06, ddd (13.9, 6.0, 3.2) 2.80, dddd (14.9, 11.6, 3.2, 3.2) 2.15, dddd (14.9, 6.0, 3.2, 3.2) 4.94, t (3.2) 7.30, brs 44.0 3.38, m 21.5

3.28, ddd (12.3, 12.3, 2.9) 2.83, ddd (12.3, 5.6, 3.5) 2.30, m

C H (J in Hz) C H (J in Hz) C H (J in Hz) C H (J in Hz)

position

2a

H (J in Hz) H (J in Hz) H (J in Hz)

syn-7

1 13

5

1 13

anti-4

1 13

syn-4

1

3

13 1

Table 1. NMR Data for Derived Compounds of 6-Methylthiochroman-4-one (1) and 6-Chlorothiochroman-4-one (2)

13

C

1

anti-7

13

C

1

8

13

C


C



Note

flasks (100 mg L−1 per flask) in an orbital shaker after 2 days of growth. The fermentation was allowed to continue for 5 more days for all the flasks. Chromatography of the fermented extract gave (R)(+)-6-methylthiochroman-4-ol (3) (7 mg) {[α]20 D +10.8 (c 0.1, MeOH), 14% ee}, syn-(+)-6-methylthiochroman-4-ol 1-oxide (syn-4) (1 mg) {[α]23 D +25.6 (c 0.4, MeOH), 27% ee, 50% de}, anti-(+)-6methylthiochroman-4-ol 1-oxide (anti-4) (16 mg) {[α]23 D +90 (c 0.3, MeOH), 67% ee}, and (S)-(−)-6-methylthiochroman-4-ol 1,1-dioxide (5) (1 mg) {[α]23 D −21.6 (c 0.1, MeOH), 95% ee}. Biotransformation of 6-chlorothiochroman-4-one (2). Biotransformation by T. viride. 6-Chlorothiochroman-4-one (2) was dissolved in ethanol and then distributed over 12 Roux bottles (150 mg L−1 per bottle) after 2 days’ growth. The fermentation was allowed to continue on surface culture for 4 more days for all the bottles. Biotransformation was carried out only at 4 days because at that time the major product was sulfoxide 7, the most interesting oxidation product from the sulfide, with a yield of 65.5%. Chromatography of the extract fermented for 4 days gave 6-chlorothiochroman-4-one (2) (37 mg), (S)-(−)-6-chlorothiochroman-4-ol21 (6) (1 mg) {[α]20 D −9 (c 0.05, MeOH), 19% ee}, (1R, 4R)-6-chlorothiochroman-4-ol 1-oxide [(1R,4R)-7] (190 mg) {[α]20 D −136.7 (c 1, MeOH), 79% ee, 99.9% de}, (S)-(−)-6-chlorothiochroman-4-ol 1,1-dioxide (8) (3.1 mg) {[α]20 D −14.2 (c 0.2, MeOH), 90% ee}, and 6-chlorothiochroman-4one 1,1-dioxide (9) (1 mg). 6-Chlorothiochroman-4-ol 1-oxide (7): colorless oil; IR νmax (cm−1) 3346, 1559, 1464, 1288, 1011, 820, 756; 1H and 13C NMR data, see Table 1; EIMS (m/z, %) 218 (M+ + 2, 1), 216 (M+, 3), 201 (36), 199 (100), 173 (30), 171 (83); m/z HREIMS (EI, 70 eV) calcd for C9H9O2SCl: 216.0012 [M]+; found 216.0016; HPLC (Chiralcel OD, Daicel, Japan, hexane−IPA, 93:7, 0.6 mL/min) tR 39.4 min (minor) and 45.7 min (major) (anti-7); tR 38.0 min; 43.9 min (major) (syn-7). 6-Chlorothiochroman-4-ol 1,1-dioxide (8): colorless oil; IR νmax (cm−1) 3350, 1584, 1414, 1011, 819; 1H and 13C NMR data, see Table 1; EIMS (m/z, %) 234 (M+ + 2, 13), 232 (M+, 36), 188 (11), 186 (31), 141 (34), 139 (100); m/z HREIMS (EI, 70 eV) calcd for C9H9O3SCl 231.9961 [M]+; found 231.9953; HPLC (Chiralcel OD, Daicel, Japan, hexane−IPA, 93:7, 0.6 mL/min) tR 50.5 min (S) and 56.9 min (R). Biotransformation by B. cinerea 2100. 6-Chlorothiochroman-4one (2) was dissolved in ethanol and then distributed over 5 flasks (100 mg L−1 per flask) in an orbital shaker after 2 days’ growth. The fermentation was allowed to continue for 5 more days for all the flasks. Chromatography of the fermented extract gave syn-6-(+)-chlorothiochroman-4-ol 1-oxide (syn-7) (2 mg) {[α]20 D +124.2 (c 0.4, MeOH), 100% ee}, (1R,4R)-6-chlorothiochroman-4-ol 1-oxide [(1R,4R)-7] (90 mg) {[α]20 D −28.6 (c 0.3, MeOH), 16% ee, 53% de}, and (R)-(+)-6chlorothiochroman-4-ol 1,1-dioxide (8) (2 mg) {[α]20 D +15.1 (c 0.1, MeOH), 96% ee}. Biotransformation by B. cinerea UCA992. 6-Chlorothiochroman-4-one (2) was dissolved in ethanol and then distributed over 5 flasks (100 mg L−1 per flask) in an orbital shaker after 2 days’ growth. The fermentation was allowed to continue for 5 more days for all the flasks. Chromatography of the fermented extract gave 6-chlorothiochroman-4-ol21 (6) (traces), syn-(+)-6-chlorothiochroman-4-ol 1-oxide (syn-7) (7 mg) {[α]20 D +124.2 (c 0.4, MeOH), 100% ee, 50% de}, (1R,4R)-6-chlorothiochroman-4-ol 1-oxide [(1R,4R)-7]] (23 mg) {[α]20 D −130 (c 1, MeOH), 71% ee}, and 6-chlorothiochroman-4-ol 1,1-dioxide (8) (traces). Mosher’s Esters: General Procedure. N,N-Dimethylaminopyridine (4.2 mg, 3.3 × 10−2 mmol), N,N′-dicyclohexylcarbodiimide (6.0 mg, 2.6 × 10−2 mmol), and (R)-MPA or (S)-MPA (5.0 mg, 3.0 × 10−2 mmol) were added to a stirred solution of the corresponding compound, 3 or anti-7 (1.8 × 10−2 mmol), in dry dichloromethane (0.5 mL) at room temperature. The resulting mixture was stirred for 1−2 h and was then concentrated under reduced pressure. The residue was chromatographed using a silica gel column followed by HPLC purification to afford the desired compounds (R)-MPA-3, (S)-MPA-3 and (R)-MPA-anti-7, (S)-MPA-anti-7 with an average yield of 86%.

at 25 °C for 24 h. The commercial fungicide Signum was used as a standard. General Culture Conditions. Fungi were grown at 25 °C on a PDB medium (T. viride) or on a Czapeck-Dox medium (B. cinerea UCA992 and B. cinerea 2100) (150 mL per bottle and 200 mL per flask). The shaken cultures were incubated on an orbital shaker at 150 rpm. The substrates were dissolved in ethanol and then distributed over Roux bottles or flasks, and the fermentation continued for a further period, after which the mycelium was filtered and then washed with brine and ethyl acetate. The broth was extracted three times with ethyl acetate, and the extract was dried over anhydrous sodium sulfate. The solvent was then evaporated, and the residue was chromatographed first on a silica gel column and then with HPLC with a gradient mixture of petroleum ether−ethyl acetate of increasing polarity. Biotransformation of 6-Methylthiochroman-4-one (1). Biotransformation by T. viride. 6-Methylthiochroman-4-one (1) was dissolved in ethanol and then distributed over 24 Roux bottles (150 mg L−1 per bottle) after 2 days of growth. The fermentation was allowed to continue on the surface culture for 4 more days in 12 of the bottles, 7 days in the other six bottles, and 10 more days in the other six. Chromatography of the extract fermented for 4 days gave 6methylthiochroman-4-one (1) (4 mg), (R)-(+)-6-methylthiochroman4-ol (3) (43.7 mg) {[α]20 D +70 (c 0.1, MeOH), 91% ee}, anti-(−)-6methylthiochroman-4-ol 1-oxide (anti-4) (40 mg) {[α]23 D −81 (c 0.3, MeOH), 61% ee, 60% de}, syn-(−)-6-methylthiochroman-4-ol 1-oxide (syn-4) (3 mg) {[α]23 D −42.3 (c 0.1, MeOH), 35% ee}, and (S)-(−)-6methylthiochroman-4-ol 1,1-dioxide (5) (3 mg) {[α]23 D −11.4 (c 0.3, MeOH), 50% ee}. Chromatography of the extract fermented for 7 days gave 6-methylthiochroman-4-one (1) (1 mg), (R)-(+)-6methylthiochroman-4-ol (3) (0.7 mg) {[α]20 D +79 (c 0.1, MeOH), 99% ee}, anti-(−)-6-methylthiochroman-4-ol 1-oxide (anti-4) (39.2 mg) {[α]23 D −110.4 (c 0.5, MeOH), 83% ee, 92% de}, syn-(−)-6methylthiochroman-4-ol 1-oxide (syn-4) (1 mg) {[α]23 D −41 (c 0.1, MeOH), 34% ee}, and (S)-(−)-6-methylthiochroman-4-ol 1,1-dioxide (5) (8.4 mg) {[α]23 D −15.1 (c 0.3, MeOH), 66% ee}. Chromatography of the extract fermented for 10 days gave anti-(−)-6-methylthiochroman-4-ol 1-oxide (anti-4) (59.2 mg) {[α]23 D −124.6 (c 0.3, MeOH), 94% ee, 96% de}, syn-(−)-6-methylthiochroman-4-ol 1-oxide (syn-4) (1 mg) {[α]23 D −98.4 (c 0.1, MeOH), 81% ee}, and (S)-(−)-6methylthiochroman-4-ol 1,1-dioxide (5) (5.5 mg) {[α]23 D −16.2 (c 0.5, MeOH), 72% ee}. 6-Methylthiochroman-4-ol (3): 18 white solid; IR νmax (cm−1) 3380, 1598, 1403, 1284, 1013, 823; 1H and 13C NMR data, see Table 1; EIMS (m/z, %) 180 (M+, 78), 161 (100), 151 (57), 118 (40), 91 (57); m/z HREIMS (EI, 70 eV) calcd for C10H12OS 180.0609 [M]+; found 180.0614; HPLC (Chiralcel OD, Daicel, Japan, hexane−IPA, 95:5, 0.5 mL/min) tR 25.9 min (S) and 29.6 min (R). 6-Methylthiochroman-4-ol 1-oxide (4): yellow oil; IR νmax (cm−1) 3452, 1634, 1274, 1122, 1033, 736; 1H and 13C data, see Table 1; EIMS (m/z, %) 196 (M+, 2), 179 (100), 151 (88), 137 (39), 91 (32); m/z HREIMS (EI, 70 eV) calcd for C10H12O2S 196.0558 [M]+; found 196.0552; HPLC (Chiralcel OD, Daicel, Japan, hexane−IPA, 93:7, 0.6 mL/min) tR 56.1 and 88.6 min (anti-4); tR 43.3 and 48.2 min (syn-4). 6-Methylthiochroman-4-ol 1,1-dioxide (5): colorless oil; IR νmax (cm−1) 3464, 1633, 1407, 1279, 1122, 784, 736; 1H and 13C NMR data, see Table 1; EIMS (m/z, %) 212 (M+, 35), 166 (25), 119 (100), 91 (46); m/z HREIMS (EI, 70 eV) calcd for C10H12O3S 212.0507 [M]+; found 212.0520; HPLC (Chiralcel OD, Daicel, Japan, hexane− IPA, 93:7, 0.6 mL/min) tR 59.7 min (R) and 73.9 min (S). Biotransformation by B. cinerea 2100. 6-Methylthiochroman-4one (1) was dissolved in ethanol and then distributed over 5 flasks (100 mg L−1 per flask) in an orbital shaker after 2 days of growth. The fermentation was allowed to continue for 5 more days for all the flasks. Chromatography of the fermented extract gave syn-(+)-6-methylthiochroman-4-ol 1-oxide (syn-4) (3 mg) {[α]23 D +115.3 (c 0.1, MeOH), 99% ee, 99% de} and (S)-(−)-6-methylthiochroman-4-ol 1,1dioxide (5) (1 mg) {[α]23 D −5.8 (c 0.1, MeOH), 28% ee}. Biotransformation by B. cinerea UCA992. 6-Methylthiochroman-4-one (1) was dissolved in ethanol and then distributed over 5 D



Note

(R)-MPA ester of 6-methylthiochroman-4-ol, (R)-MPA-3: 1H NMR (400 MHz, CDCl3, selected data) δ 6.96 (d, 1H, J = 8.2 Hz, H-8), 6.91 (bd, 1H, J = 8.2 Hz, H-7), 6.55 (brs, 1H, H-5), 5.93 (dd, 1H, J = 5.1 Hz, 3.5 Hz, H-4), 3.13 (ddd, 1H, J = 12.7 Hz, 11.2 Hz, 2.9 Hz, H-2a), 2.87 (ddd, 1H, J = 12.7 Hz, 6.4 Hz, 3.5 Hz, H-2b), 2.35 (m, 1H, H-3a), 2.19 (dddd, 1H, J = 14.5 Hz, 11.2 Hz, 3.5 Hz, 3.5 Hz, H-3b), 2.07 (s, 3H, H-11). (S)-MPA ester of 6-methylthiochroman-4-ol, (S)-MPA-3: 1H NMR (400 MHz, CDCl3, selected data) δ 7.01 (d, 1H, J = 1.2 Hz, H-5), 6.01 (dd, 1H, J = 3.5 Hz, 3.5 Hz, H-4), 2.77 (ddd, 1H, J = 12.6 Hz, 12.6 Hz, 2.6 Hz, H-2a), 2.56 (ddd, 1H, J = 12.6 Hz, 4.1 Hz, 4.1 Hz, H-2b), 2.25 (s, 3H, H-11), 2.08 (m, 1H, H-3a), 1.92 (m, 1H, H-3b). (R)-MPA ester of anti-6-chlorothiochroman-4-ol 1-oxide, (R)MPA-anti-7: 1H NMR (400 MHz, CDCl3, selected data, assignments aided by a COSY experiment) δ 7.63 (d, 1H, J = 8.2 Hz, H-8), 7.41 (dd, 1H, J = 8.2 Hz, 2.0 Hz, H-7), 6.93 (d, 1H, J = 2.0 Hz, H-5), 6.03 (dd, 1H, J = 4.0 Hz, 4.0 Hz, H-4), 3.21 (ddd, 1H, J = 12.7 Hz, 11.2 Hz, 2.9 Hz, H-2a), 3.07 (ddd, 1H, J = 12.7 Hz, 6.4 Hz, 3.5 Hz, H-2b), 2.21 (m, 1H, H-3a), 1.94 (m, 1H, H-3b). (S)-MPA ester of anti-6-chlorothiochroman-4-ol 1-oxide, (S)MPA-anti-7: 1H NMR (400 MHz, CDCl3, selected data, assignments aided by a COSY experiment) δ 7.69 (d, 1H, J = 8.2 Hz, H-8), 7.50 (dd, 1H, J = 8.2 Hz, 2. Hz, H-7), 7.37 (brs, 1H, H-5), 6.06 (dd, 1H, J = 5.1 Hz, 3.5 Hz, H-4), 1.92 (m, 1H, H-2a), 1.67 (m, 1H, H-2b), 1.33 (m, 1H, H-3a), 1.09 (m, 1H, H-3b).

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(8) Wang, G.; Yang, G.; Ma, Z.; Tian, W.; Fang, B.; Li, L. Int. J. Chem. 2010, 2, 19−25. (9) Talla, S. G.; Raju, A. S. R.; Karri, S.; Kumar, Y. S. Afr. J. Biotechnol. 2015, 14, 668−675. (10) Mallikharjuna Rao, K. L. N.; Siva Raju, K.; Ravisankar, H. Braz. J. Microbiol. 2016, 47 (1), 25−32. (11) Pinedo-Rivilla, C.; Aleu, J.; Collado, I. G. J. Mol. Catal. B: Enzym. 2007, 49, 18−23. (12) Pinedo-Rivilla, C.; Carrara Cafêu, M.; Aleu, J.; Regina Araujo, A.; Collado, I. G. Tetrahedron: Asymmetry 2009, 20, 2666−2672. (13) Freire, F.; Seco, J. M.; Quiñoá, E.; Riguera, R. J. Org. Chem. 2005, 70, 3778−3790. (14) Ito, M.; Shibata, A.; Watanabe, A.; Ikariya, T. Synlett 2009, 2009, 1621−162610.1055/s-0029-1217349. (15) Bradamante, S.; Maiorana, S.; Mangia, A.; Pagani, G. J. Chem. Soc. B 1971, 1, 74−78. (16) Stepanenko, V.; de Jesús, M.; Correa, W.; Bermúdez, L.; Vázquez, C.; Guzmán, I.; Ortiz-Marciales, M. Tetrahedron: Asymmetry 2009, 20, 2659−2665. (17) Andersson, L.; Arzel, E.; Berg, S.; Burrows, J.; Hellberg, S.; Huerta, F.; Pedersen, T.; Rein, T.; Rotticci, D.; Staaf, K. PCT Int. Appl., WO 2007040440 A1 20070412, 2007. (18) Song, J.; Jones, L. M.; Kumar, G. D. K.; Conner, E. S.; Bayeh, L.; Chavarria, G. E.; Charlton-Sevcik, A. K.; Chen, S.-E.; Chaplin, D. J.; Trawick, M. L.; Pinney, K. G. ACS Med. Chem. Lett. 2012, 3, 450−453. (19) Prost, I.; Dhondt, S.; Rothe, G.; Vicente, J.; Rodriguez, M. J.; Kift, N.; Carbonne, F.; Griffiths, G.; Rosahl, S.; Castresana, C.; Hamberg, M.; Fournier, J. Plant Physiol. 2005, 139, 1902−1913.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00298. Additional information (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: josefi[email protected]. ORCID

Cristina Pinedo-Rivilla: 0000-0003-1965-3645 Isidro G. Collado: 0000-0002-8612-0593 Josefina Aleu: 0000-0002-5329-9582 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from the MINECO (AGL20015-65684-C2-1-R).

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REFERENCES

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