Biosynthesis and structure-activity relationship studies of okaramines that target insect glutamate-gated chloride channels

Transcription

1 Supporting Information Biosynthesis and structure-activity relationship studies of okaramines that target insect glutamate-gated chloride channels Naoki Kato 1,, Shogo Furutani 1,2, Junnosuke Otaka 3, Akira Noguchi 2, Kiyomi Kinugasa 1, Kenji Kai 4, Hideo Hayashi 4, Makoto Ihara 2, Shunji Takahashi 1, Kazuhiko Matsuda 2,, and Hiroyuki Osada 3 1 Natural Product Biosynthesis Research Unit, RIKEN Center for Sustainable Resource Science, Wako, Saitama , Japan 2 Department of Applied Biological Chemistry, Faculty of Agriculture, Kindai University, Nara, Nara , Japan 3 Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science, Wako, Saitama , Japan 4 Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka , Japan To whom correspondence should be addressed. naoki-kato@riken.jp or kmatsuda@nara.kindai.ac.jp 1

2 TABLE OF CONTENTS Pages Supporting Methods Plasmid construction 3 Chemical analysis 3 Purification and structure determination of compounds 4 Supporting Tables Table S1. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data of 2 (in acetone-d 6 ) 5 Table S2. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data of 4 (in acetone-d 6 ) 6 Table S3. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data of 5 (in acetone-d 6 ) 7 Table S4. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data of 6 (in acetone-d 6 ) 8 Table S5. Features of the oka gene products of P. simplicissimum AK-40 9 Table S6. Oligonucleotides used for plasmid construction 10 Table S7. Oligonucleotides used for genotyping PCR 11 Supporting Figures Figure S1. Construction of the oka gene deletion mutants in P. simplicissimum AK Figure S2. Nonenzymatic conversion from okaramine A (2) to okaramine H (5) 13 2

3 Supporting methods Plasmid construction To construct the gene deletion plasmids, 2-kb DNA fragments upstream of the start codon and downstream of the stop codon of the target genes were amplified by PCR using chromosomal DNA of P. simplicissimum AK-40 as the template. The primer pairs, oka-uf/oka-ur and oka-df/oka-dr, were used for amplification of the upstream and downstream regions, respectively. The bleomycin-resistant cassette (ble) was used as a selection marker. These DNA fragments were combined in the original orientation in pbi121 in the following order: the upstream regions, ble, followed by the downstream regions (Figure S1A). An In-Fusion cloning system (Clontech) was used for plasmid construction. All DNA fragments amplified by PCR were verified by sequencing. The oligonucleotides that were used for plasmid construction and genotyping are summarized in Tables S6 and S7. Chemical analysis All solvents and reagents were of analytical grade and purchased from commercial sources. UV spectra, optical rotations, and CD spectra were measured using Beckman DU 530 Life Science UV/Vis spectrophotometer (Beckman Coulter), a HORIBA SEPA-300 high sensitive polarimeter (Horiba), and a JASCO J-720 spectropolarimeter (JASCO), respectively. IR spectra were recorded using the ATR method on a HORIBA FT-720 microscopic Fourier transform infrared spectrophotometer (Horiba). NMR spectra (500 MHz for 1 H, 125 MHz for 13 C) were obtained on a JEOL ECA-500 spectrometer in acetone-d 6 or CD 3 OD solution. The acetone-d 6 signal ( 2.05 for 1 H and 29.8 for 13 C) or CD 3 OD signal ( 3.31 for 1 H and 49.0 for 13 C) was used for each reference. High-resolution mass spectra were obtained on a JEOL JMS-T100L High-Resolution Time of Flight LC/MS using electrospray ionization (ESI +, ESI ). Reversed-phase HPLC was performed on a Waters 600 HPLC pump/2996 UV/VIS detector (Waters) with a computerized data station equipped with Waters Empower 2 software using a Senshu PAK PEGASIL ODS SP100 reversed-phase column (10 mm i.d. 250 mm; Senshu Scientific Corporation). Thin layer chromatography was performed on silica gel 60 F254 (Merck). Open column chromatography was carried out using silica gel 60 (40 63 m; Kanto Chemical). For metabolite profiling of the gene deletion mutants, LC/ESI-MS analysis was carried out with a Waters ACQUITY UPLC H-Class system fitted a mass spectrometer (API3200; AB SCIEX). The LC conditions were as follows: column, Waters ACQUITY UPLC BEH C18 (2.1 mm i.d. 50 mm, 1.7 m); flow rate, 0.5 ml min 1 ; solvent A, 0.05% (v/v) aqueous formic acid; solvent B, acetonitrile. After injection of the samples into a column equilibrated with 40% solvent B, the column was developed with a linear gradient from 40% to 60% solvent B over 4 min, 3

4 followed by isocratic elution of 100% solvent B for 2 min. Purification and structure determination of compounds Okaramines A (2) and H (5): The ethyl acetate extract from the okara culture of the okae strain (500 mg) was dissolved in 5 ml MeOH. A part of MeOH solution (1 ml) was supplied into the Sep-Pak C18 cartridge (Waters), followed by purification by semi-preparative ODS-HPLC eluted with 70% acetonitrile (3.0 ml min 1 ) to give 2 (10 mg, min) and 5 (1.5 mg, min). Compound 2: Yellow powder; [ ] 25 D : +470 (c 0.05, MeOH); ESIMS m/z 521 [M+H] +, 519 [M H] ; UV (MeOH) max (log ) 381 (4.30), 285 (4.36), 2.53 (4.45), 227 (4.62) nm. Compound 5: Yellow powder; [ ] 25 D : +509 (c 0.05, MeOH); ESIMS m/z 521 [M+H] +, 519 [M H] ; UV (MeOH) max (log ) 381 (4.22), 284 (4.34), 230 (4.63), 209 (4.68) nm. Cyclo(N 8 -(α,α-dimethylallyl)-l-trp-6a'-(α,α-dimethylallyl)-l-trp) (4): The ethyl acetate extract from the okab strain (136 mg) was dissolved in 5 ml MeOH. After the filtration of MeOH solution using the Sep-Pak C18 cartridge, semi-preparative ODS-HPLC was performed by eluting with 70% acetonitrile (3.0 ml min 1 ) to give 4 (3.0 mg, min). Compound 4: White powder; [ ] D 25 : 83 (c 0.36, CHCl 3 ); ESIMS m/z 511 [M+H] +, 509 [M H] ; UV (MeOH) max (log ) 291 (4.07), 283 (4.13), 223 (4.76) nm; CD (MeOH) 228 ( 2.40) nm. 2-Dehydroxy-3-demethoxy-okaramine B (6): The ethyl acetate extract of the okag strain (650 mg) was partitioned between hexane/90% MeOH, then concentrated in vacuo, affording each extract (hexane extract: 80 mg, MeOH extract: 550 mg). A part of MeOH brown syrup extract (400 mg) was separated by silica gel chromatography (15 mm i.d. 200 mm) eluted with hexane/ethyl acetate (2/1, 200 ml) and CH 3 Cl/MeOH (20/1, 50 ml, 10/1, 50 ml, and 2/1, 50 ml) to give five fractions. The fourth fraction (178 mg) was further purified by semi-preparative ODS-HPLC with a linear gradient from 50% to 100% acetonitrile over 15 min, affording 6 (1.0 mg, min). Compound 6: Yellow powder; [ ] D 25 : +210 (c 0.13, MeOH); ESIMS m/z 521 [M+H] +, 519 [M H] ; UV (MeOH) max (log ) 381 (4.20), 285 (4.28), 232 (4.53), 208 (4.72) nm. 4

5 Table S1. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data of 2 (in acetone-d 6 ) Position C H, (multi, J in Hz) (CH) 4.53, dd (9.5, 5.5) (CH 2 ) 3.11, dd (12.5, 5.0) 2.55, dd (12.5, 9.0) 3a 85.0 (C) 3b (C) (CH) 7.28, dd (7.5, 1.2) (CH) 6.74, td (7.5, 1.0) (CH) 7.05, ddd (8.0, 7.5, 1.2) (CH) 6.90, brs (9.0) 7a (C) 8a 85.7 (CH) 5.52, s (C) (C) (CH) 6.41, dd (17.8, 10.9) (CH 2 ) 5.18, dd (17.8, 1.1) 5.05, dd (10.9, 1.1) (CH 3 ) 1.52, s (CH 3 ) 1.70, s 1' (CH) 7.68, s 2' (C) 4' (CH) 5.88, d (8.0) 5' (CH) 5.61, d (8.0) 6' 36.9 (C) 6a' 7a' (C) (C) 8' (CH) 7.39, brd (7.3) 9' (CH) 7.13, m 10' (CH) 7.16, m 11' (CH) 7.74, dd (7.0, 1.5) 11a' 11b' (C) (C) 12' (C) 13' 28.2 (CH 3 ) 1.69, s 14' 28.2 (CH 3 ) 1.66, s 7'-NH 10.6, brs 5

6 Table S2. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data of 4 (in acetone-d 6 ) Position C H, (multi, J in Hz) (CH) 4.23, m (CH 2 ) 3.25, dd (14.9, 6.5) 3.20, dd (14.9, 4.3) 3a (C) 3b (C) (CH) 7.67, brd (7.4) (CH) 7.09, td (7.4, 1.1) (CH) 7.06, td (7.4, 1.1) (CH) 7.45, d (8.0) 7a (C) 8a 126.3(CH) 7.27, s (C) (C) (CH) 6.11, dd (18.0, 11.0) (CH 2 ) 5.17, d (18.0) 5.13, dd (11.0, 1.0) (CH 3 ) 1.74, s (CH 3 ) 1.72, s 1' 31.6 (CH 2 ) 3.42, dd (14.9, 2.9) 2.26, dd (14.9, 10.3) 2' 57.9 (CH) 4.06, dt (8.5, 1.7) 4' (CH 2 ) 5.01, dd (17.5, 1.2) 4.98, dd (10.0, 1.2) 5' (CH) 6.11, dd (18.0, 11.0) 6' 39.9 (C) 6a' (C) 7a' (C) 8' (CH) 7.20, brd (7.2) 9' (CH) 6.98, ddd (9.0, 6.0, 1.5) 10' (CH) 6.81, ddd (9.0, 6.0, 1.5) 11' (CH) 6.75, d (7.4) 11a' (C) 11b' (C) 12' (C) 13' 28.1 (CH 3 ) 1.51, s 14' 28.2 (CH 3 ) 1.51, s 1-NH 7.12, brs 3'-NH 6.24, brs 7'-NH 9.74, brs 6

7 Table S3. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data of 5 (in acetone-d 6 ) Position C H, (multi, J in Hz) (CH) 4.75, t (10.6, 6.6) (CH 2 ) 2.70, dd (13.0, 6.6) 2.31, dd (13.0, 11.0) 3a 86.7 (C) 3b (C) (CH) 7.19, m (CH) 6.71, t (7.4) (CH) 6.98, brd (7.4) (C) 7a (C) 8a 84.5 (CH) 5.52, d (3.7) (C) (CH 2 ) 3.27, dd (16.0, 7.0) 3.22, dd (16.0, 7.8) (CH) 5.17, m (C) (CH 3 ) 1.72, brs (CH 3 ) 1.71, brs 1' (CH) 7.61, s 2' (C) 4' (CH) 5.80, d (8.5) 5' (CH) 5.95, d (8.5) 6' 36.0 (C) 6a' (C) 7a' (C) 8' (CH) 7.38, brd (6.9) 9' (CH) 7.13, m 10' (CH) 7.16, m 11' (CH) 7.68, d (6.9) 11a' (C) 11b' (C) 12' (C) 13' 26.1 (CH 3 ) 1.76, s 14' 27.4 (CH 3 ) 1.65, s 8-NH 5.54, brd (3.7) 7'-NH 10.70, brs 7

8 Table S4. 1 H (500 MHz) and 13 C (125 MHz) NMR spectroscopic data of 6 (in acetone-d 6 ) Position C H, (multi, J in Hz) (CH) 4.63, t (8.0) (CH 2 ) 2.96, dd (13.5, 7.1) 2.71, dd (13.5, 8.6) 3a 87.9 (C) 3b (C) (CH) 7.45, d (6.9) (CH) 7.00, td (7.4, 1.1) (CH) 7.21, td (10.5, 1.5) (CH) 6.74, d (7.4) 7a (C) 8a 95.9 (C) (C) (C) (CH) 5.31, q (7.5) (CH 3 ) 1.37, d (7.5) (CH 3 ) 1.78, s (CH 3 ) 0.95, s 1' (CH) 7.60, s 2' (C) 4' (CH) 5.69, d (8.0) 5' (CH) 5.92, d (8.0) 6' 36.8 (C) 6a' (C) 7a' (C) 8' (CH) 7.38, brd (6.9) 9' (CH) 7.14, dd (7.0, 1.5) 10' (CH) 7.18, t (8.0, 1.5) 11' (CH) 7.70, d (7.4) 11a' (C) 11b' (C) 12' (C) 13' 27.0 (CH 3 ) 1.74, s 14' 28.3 (CH 3 ) 1.65, s 7'-NH 10.6, brs 8

9 Table S5. Features of the oka gene products of P. simplicissimum AK-40 Gene okaa okab okac okad okae okaf Size (bp/aa) 6075 / / / / / /382 Deduced function Relatives (accession, identity/similarity [%]) a NRPS OxaA from P. oxalicum (AOC84385, 36/56) FMO TqaH from P. aethiopicum (ADY16696, 41/57) DMATS NotF from Aspergillus versicolor (AGC83577, 41/63) P450 FtmE from A. fumigatus (BAH23999, 40/60) AKD FtmF from A. fumigatus (BAH24000, 41/60) MT OmtB from A. flavus (AAS90012, 49/67) 1887 GLAREA_10030 from Glarea lozoyensis (XP_ , okag P450 /538 30/50) a The listed homologous proteins exclude putative proteins derived from genome sequences. 9

10 Table S6. Oligonucleotides used for plasmid construction Oligonucleotide okab_uf( 2000) okab_ur( 1) okab_df(1584) okab_dr(3580) okad_uf( 2002) okad_ur( 1) okad_df(1604) okad_dr(3590) okae_uf( 2018) okae_ur( 5) okae_df(905) okae_dr(2903) okag_uf( 1998) okag_ur( 2) okag_df(1890) okag_dr(3883) ble_f ble_r Sequence (5'-3') GGAAACGACAATCTGTGACATTGTGATATCGGCCAGATTCGAG TTTAATTTGCAAGCTGGTCGATATAAGTGCTAGTTTTGGCAAG CAGTGTCGAAAGATCCATGATCTTTAGCTACTGTCTCATTGTG AACACATTGCGGACGGCTTCAATAATCACTTTTTGCTTATTTC GGAAACGACAATCTGCTTGAAACATACCTACCATTTGGTCTAC TTTAATTTGCAAGCTTGCAACAGGAAGAGAGTACGGCGTGCAG CAGTGTCGAAAGATCGTTTCCGTCCGCAGTATCTATCTTCATG AACACATTGCGGACGGCGGATCACTACAAACAGATTCAACAAC GGAAACGACAATCTGGCATTTCCGAGTCTATGTTCCAAGGCAC TTTAATTTGCAAGCTCTTAAGGGCTATTTCAAGCTGCAAAACG CAGTGTCGAAAGATCTCTGTTTGTAGTGATCCGCGCCTATTTG AACACATTGCGGACGCAAACCACTGGCAGCAGGATGATAATA GGAAACGACAATCTGGATTCTAGTAGGGCTAGTAGTAGAAAAT TTTAATTTGCAAGCTTCTGTAAGTTATTATCGAACGATACTAG CAGTGTCGAAAGATCAGATAGCAGTGAGCAGAGTAGCTTGCTG AACACATTGCGGACGGTTAACGAATACCAAAAGCACAGGAACA GATCTTTCGACACTGAAATACGTC AGCTTGCAAATTAAAGCCTTCGAG 10

11 Table S7. Oligonucleotides used for genotyping PCR Oligonucleotide okab_af okab_br okab_df okab_dr okad_af okad_br okad_df okad_dr okae_af okae_br okae_df okae_dr okag_af okag_br okag_df okag_dr AR BF CF CR Sequence (5'-3') TCTGGAGCAGCCGTTCAG CTGACGGCGCATGCACTG GCTACAAGGACTCCCGAT TAGCGAGTGTGGCGGCAT GAGTTGGGCGTTGCTTGT GCCCTCCTGTCGGCCAAT CGAGTCTCGCTTTGGTTG CGGTCCTTGTGCTCCCAG CGTGAGATACAGGGGCCT ACGGTCCGGAATAACAGC GACTCTCACCGAAAACGT CAACCTCCAACGTTCAAC TACCAGAGCTACTGCGCC GCCCGTGGACCTTCTAAC CAGGCGGAAGCAGATGGT GCGATTGTACTGGGTCTC CGCTCGAAGGCTTTAATTTGC GCAGGCTCGACGTATTTC GACCGGTGACTCTTTCTG GTCCCGGAAGTTCGTGGA 11

12 Figure S1. Construction of the oka gene deletion mutants in P. simplicissimum AK-40. (A) Scheme for the targeted gene inactivation by homologous recombination. The 6.9-kb DNA fragments containing 2 kb upstream and 2 kb downstream regions of the oka genes and the bleomycin-resistance cassette (ble) were used for transformation of the okaramine-producing strain AK-40 (wild-type, W). (B) Genotyping of the oka gene deletion mutants. Six phleomycin-resistant transformants were tested to verify the correct replacement for each deletion construct. PCR products with four pairs of primers were combined and analyzed by agarose gel electrophoresis. The sizes of amplified DNA fragments were in agreement with the predicted fragments. The targeting efficiency of the genes in the fungus was sufficient for examination of the effects of the gene deletion on metabolite production using several mutant strains. 12

13 Figure S2. Nonenzymatic conversion from okaramine A (2) to okaramine H (5). Okaramine A (2, 0.1 mg ml 1 ) was incubated in 50 mm GTA buffer of ph 4 to 7 at 28 C for 24 h and analyzed by UPLC. Okaramine H (5) isolated from the okae mutant was used as a standard. UV detection was carried out at 373 nm. 13