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Paecilodepsipeptide D, a cyclohexadepsipeptide from cultures of the whitefly pathogenic fungus Conoideocrella luteorostrata BCC 76664
Phytochemistry Letters 34 (2019) 65–67
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Paecilodepsipeptide D, a cyclohexadepsipeptide from cultures of the whitefly pathogenic fungus Conoideocrella luteorostrata BCC 76664
T
Masahiko Isaka , Somporn Palasarn, Pranee Rachtawee, Kitlada Srichomthong, Suchada Mongkolsamrit ⁎
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand
ARTICLE INFO
ABSTRACT
Keywords: Conoideocrella luteorostrata Invertebrate pathogenic fungi Cyclodepsipeptide Antimalarial activity
Paecilodepsipeptide D (1), a new cyclohexadepsipeptide was isolated together with known paecilodepsipeptides A–C (2–4) from the whitefly pathogenic fungus Conoideocrella luteorostrata BCC 76664. The structure of 1 was elucidated by spectroscopic analyses and was further confirmed by the chemical correlation to paecilodepsipeptide A (2) by converting both compounds into the same di-O-prenyl derivative (5). A comparison of the antimalarial activities of 1, 2, and 5 suggested that a polar phenolic (OH) functionality at the D-tyrosine (Tyr-2) is crucial for the antimalarial activity, while a lipophilic O-alkyl side-chain at the other D-tyrosine (Tyr-1) is preferred.
1. Introduction Conoideocrella luteorostrata (Clavicipitaceae) is an invertebrate pathogenic fungus that most commonly attacks scale-insects (Johnson et al., 2009). The anamorphic state of this fungus was formerly known as Paecilomyces cinnamomeus. Previous chemical investigations of this species have revealed the presence of various structural groups of bioactive secondary metabolites such as depsipeptides (paecilodepsipeptides ACe; antimalarial activity) (Isaka et al., 2007a), bioxanthracenes and their monomers (ES-242 derivatives; antimalarial and cytotoxic activities) (Saepua et al., 2015), and prenylated tryptophan derivatives with an oxime functionality (luteorides ACe) (Asai et al., 2011). Recently, we reported the isolation of conoideoxime A, an antibacterial heterodimer of luteorides, from cultures of C. luteorostrata BCC 76664, which is a rare strain isolated from a dead whitefly host (Isaka et al., 2019). Due to our interest in the unique chemical skeleton of conoideoxime A and its antibacterial activity, the producing fungal strain (BCC 76664) has been reinvestigated. Although no new derivative of conoideoxime A was found in the new fermentation batch, the study led to the isolation of a new cyclohexadepsipeptide, named paecilodepsipeptide D (1), together with known paecilodepsipeptides ACe (2–4) (Isaka et al., 2007a; Lang et al., 2006) (Fig. 1). 2. Results and discussion Paecilodepsipeptide D (1) was obtained as a colorless solid. The molecular formula of 1 was determined to be C35H39N5O9 by HRESIMS.
⁎
The IR spectrum displayed an intense and broad absorption band at 1653 cm−1, suggesting the presence of carbonyl groups. The 1H and 13C NMR spectra of 1 in DMSO-d6 were very similar to those of the known cometabolite paecilodepsipeptide A (2). The significant difference was that 1 lacked the prenyl group of the O-prenyl-tyrosine unit in 2. The planar structure, elucidated by analysis of 2D NMR data (Fig. 2), was found to be a hexadepsipeptide composed of a hydroxycarboxylic acid and five amino acid residues, showing six carbonyl carbon signals at δC 172.0–167.6 and five amide NH protons at δH 8.53–7.65. The structure of 3-phenyllactic acid (3-Ph-Lac) residue was elucidated based on the COSY and HMBC correlations. An oxymethine at δC 73.2 (C-2) was attached to δC 36.9 methylene (C-3), which was flanked by a phenyl group. Five amino acid residues were also identified on the basis of COSY and HMBC data to be a glycine (Gly), two alanine (Ala-1 and Ala2), and two tyrosine (Tyr-1 and Tyr-2) residues (Table 1). The sequence of the six residues was established by analysis of HMBC data (Fig. 2). Amide protons of Ala-2, Gly, Tyr-2, Ala-1, and Tyr-1 were correlated to carbonyl carbons of 3-Ph-Lac, Ala-2, Gly, Tyr-2, and Ala-1, respectively. The HMBC correlation from H-2 of 3-Ph-Lac to C-1 of Tyr-1 indicated the presence of an ester linkage of Tyr-3-Ph-Lac to form a cyclohexadepsipeptide. This sequence was further supported by the NOESY correlations: H-2 of 3-Ph-Lac with NH of Ala-2; H3-3 of Ala-2 with NH of Gly; H2-2 of Gly with NH of Tyr-2; H-2 and H2-3 of Tyr-2 with NH of Ala-1; H3-3 of Ala-1 with NH of Tyr-1; and H2-3 of Tyr with H-2 of 3-PhLac. Therefore, the sequence of 1 was proven to be the same as 2. The absolute configurations at C-2 of the 3-Phe-Lac and five amino acid residues in paecilodepsipeptide A (2), isolated from another strain
Corresponding author. E-mail address: [email protected] (M. Isaka).
https://doi.org/10.1016/j.phytol.2019.09.017 Received 5 August 2019; Received in revised form 25 September 2019; Accepted 27 September 2019 1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 34 (2019) 65–67
M. Isaka, et al.
obtained by the prenylation of 1. Consequently, the absolute configurations of 1 were confirmed: D-Tyr (for Tyr-1), D-Ala (for Ala-1), D-Tyr (for Tyr-2), L-Ala (for Ala-2), and (2S)-L-3-Ph-Lac. It was previously shown that paecilodepsipeptide A (2) exhibited antimalarial activity against Plasmodium falciparum K1 (multidrug-resistant strain), while 3 and 4, linear hydrolysis and methanolysis derivatives, were inactive (Isaka et al., 2007a). In the present study, the antimalarial activities of the compounds 1, 2, and 5 were tested for comparison, and displayed IC50 values of > 50, 6.3, and > 50 μg/ml, respectively. Dihydroartemisinin was used as the standard antimalarial drug (IC50 0.63 ng/ml). These results suggested that a polar phenolic (OH) functionality at the D-Tyr (Tyr-2) is crucial for the antimalarial activity, while lipophilic O-alkyl side-chain at the other D-Tyr (Tyr-1) is preferred. Compounds 1, 2, and 5 were noncytotoxic against Vero cells (African green monkey kidney fibroblasts) at 50 μg/ml. 3. Experimental 3.1. General procedures Optical rotations were determined using a JASCO P-1030 digital polarimeter. UV spectra were recorded on an Analytik-jena SPEKOL 1200 spectrophotometer. FTIR spectra were acquired using a Bruker ALPHA spectrometer. NMR spectra were recorded on Bruker DRX400 and AV500D spectrometers. ESITOF mass spectra were measured using a Bruker micrOTOF mass spectrometer. Preparative HPLC was performed on a Waters 600 System equipped with a Waters 2296 photodiode array detector (Waters, Milford, USA). Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) and Silica gel 60H (particle size, 90% < 45 μM; Merck KGaA, Darmstadt, Germany) were used for column chromatography. 3.2. Fungal material The fungus used in this study was isolated from a whitefly (Hemiptera) at Chanthaburi Agricultural Research and Development Center, Chanthaburi Province, Thailand, on December 17, 2014. This fungus was deposited at the Thailand BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology, Pathumthani, Thailand, as BCC 76664. The identification of this fungus was based on the morphology and ITS rDNA sequence data (GenBank accession number: MH188543): phylum Ascomycota, order Hypocreales, family Clavicipitaceae, genus Conoideocrella, species luteorostrata (Isaka et al., 2019).
Fig. 1. Structures of compounds 1–5.
3.3. Fermentation, extraction, and isolation Conoideocrella luteorostrata BCC 76664. was fermented in 20 × 1000 ml Erlenmeyer flasks containing 250 ml of minimum salt medium (yeast extract 1.0 g/l, glucose 20 g/l, NH4NO3 3.0 g/l, KH2PO4 0.5 g/l, NaH2PO4 0.5 g/l, MgSO4·7H2O 0.5 g/l, and CaCl2 0.5 g/l) at 25 °C for 23 days under static conditions. The cultures were filtered to separate mycelia and filtrate (broth). The culture broth was extracted with EtOAc (2 × 5 l) and the combined organic phase was concentrated to obtain a brown gum (extract A, 1.50 g). Wet mycelia were macerated in MeOH (1.0 l, room temperature, 2 days) and then filtered. The filtrate was defatted by partitioning with hexanes (1.0 l). The MeOH layer was concentrated under reduced pressure. The residue was diluted with H2O (50 ml), extracted with EtOAc (2 × 800 ml), and concentrated under reduced pressure to leave a brown solid (extract B, 2.14 g). Extract A was subjected to Sephadex LH-20 column chromatography (CC) (3.5 × 60 cm, MeOH) to obtain 4 pooled fractions, A1 – A4. Fraction A3 (524 mg) was further fractionated by silica gel CC (2.8 × 15 cm, EtOAc/CH2Cl2, step gradient elution from 0:100 to 100:0), and preparative HPLC using a reverse-phase column (Waters NovaPak C18 OBD, 25 × 100 mm, 6 μm; mobile phase MeCN/H2O, 30:70, flow rate 10 ml/min) to yield 1 (8.2 mg, tR 10.5 min). Extract B
Fig. 2. COSY and selected HMBC and NOESY correlations for 1, indicating the sequence of five amino acids and 3-Ph-Lac.
(BCC 9616) of the same fungal species, were previously determined by the combination of chiral column HPLC analysis of the acid hydrolysate and Marfey’s method (Isaka et al., 2007a). The absolute configuration was later confirmed through total synthesis by Yang and coworkers (Yang et al., 2009). The absolute configuration of 1 was determined by chemical correlation to 2. The reaction of 2 with prenyl bromide and K2CO3 (s) in DMF at room temperature for 16 h gave a di-O-prenyl derivative 5 (Isaka et al., 2007b). The same compound (5) was also 66
Phytochemistry Letters 34 (2019) 65–67
M. Isaka, et al.
Table 1 NMR spectroscopic data for paecilodepsipeptide D (1) in DMSO-d6 (500 MHz for 1H, 125 MHz for No.
δC, type
D-Tyr-1 1 2 3
171.3, C 56.0, CH 35.9, CH2
4 5,9 6,8 7 7-OH NH D-Ala 1 2 3 NH
126.1, 130.2, 115.1, 156.4,
C CH CH C
172.0, C 47.7, CH 18.6, CH3
δH, mult. (J in Hz)
4.23, m 2.87, dd (14.1, 6.3); 2.77, dd (14.1, 6.3) 6.90, d (8.0) 6.71, d (8.0) 9.22, s 8.53, br d (2.1) 4.23, m 1.27, d (6.8) 7.65, d (8.0)
No.
δC, type
D-Tyr-2 1 2 3
170.2, C 55.6, CH 35.5, CH2
4 5,9 6,8 7 7-OH NH Gly 1 2
128.1, 129.7, 115.0, 155.7,
NH
–
13
C).
δH, mult. (J in Hz)
C CH CH C
169.1, C 43.0, CH2
4.19, m 2.96, m; 2.73, m 6.97, d (8.0) 6.62, d (8.0) 9.34, s 8.24, d (8.2) 3.58, dd (15.9, 6.3); 3.33, m 8.07, m
No. L-Ala 1 2 3 NH L-3-Ph-Lac 1 2 3 4 5,9 6,8 7
δC, type 171.8, C 48.6, CH 18.2, CH3 167.6, C 73.2, CH 36.9, CH2 135.5, 129.7, 127.9, 126.6,
C CH CH CH
δH, mult. (J in Hz)
4.27, m 1.06, d (6.7) 7.67, d (6.7) – 5.12, m 3.00, dd (14.5, 6.1); 2.51, dd (14.5, 3.1) 6.82, m 7.19, m 7.17, m
was also subjected to similar chromatographic procedures to yield 2 (8.9 mg), 3 (2.9 mg), 4 (2.3 mg), and conoideoxime A (6.2 mg).
assay (Hunt et al., 1999). Ellipticine was used as the positive control in the cytotoxicity assay (IC50 1.58 μg/ml).
3.3.1. Paecilodepsipeptide D (1) MeOH Colorless powder; [α]23 (nm) D +22 (c 0.25, MeOH); UV λmax (log ε): 218 (3.67), 277 (3.24); IR νmax ATR (cm−1): 1743, 1653, 1516, 1234; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSOd6) data, Table 1; HRESIMS (m/z): 696.2632 [M + Na]+ (calc. for C35H39N5O9Na, 696.2640).
Declaration of Competing Interest The authors have no competing interests to declare. Acknowledgements Financial support from the National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA) is gratefully acknowledged.
3.4. Preparation of Compound 5 from paecilodepsipeptide A (2) A mixture of 2 (30 mg, 0.040 mmol; a sample previously isolated from another strain of the same species) (Isaka et al., 2007a), 3,3-dimethylallyl bromide (125 μl, 1.1 mmol), and K2CO3 (30 mg, 0.22 mmol) in DMF (0.5 ml) was stirred at room temperature for 16 h. The mixture was diluted with EtOAc and washed with H2O. The organic layer was concentrated under reduced pressure. The residue was purified by preparative HPLC using a reverse-phase column (MeCN/H2O, 55:45) to yield compound 5 (16 mg; 50%).
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2019.09.017. References Asai, T., Yamamoto, T., Oshima, Y., 2011. Histone deacetylase inhibitor induced the production of three novel prenylated tryptophan analogs in the entomopathogenic fungus Torrubiella luteorostrata. Tetrahedron Lett. 52, 7042–7045. Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D., 1997. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 16, 710–718. Hunt, L., Jordan, M., De Jesus, M., Wurm, F.M., 1999. GFP-expressing mammalian cells for fast, sensitive, noninvasive cell growth assessment in a kinetic mode. Biotechnol. Bioeng. 65, 201–205. Isaka, M., Palasarn, S., Lapanun, S., Sriklung, K., 2007a. Paecilodepsipeptide A, an antimalarial and antitumor cyclodepsipeptide from the insect pathogenic fungus Paecilomyces cinnamomeus BCC 9616. J. Nat. Prod. 70, 675–678. Isaka, M., Berkaew, P., Intereya, K., Komwijit, S., Sathitkunanon, T., 2007b. Antiplasmodial and antiviral cyclohexadepsipeptides from the endophytic fungus Pullularia sp. BCC 8613. Tetrahedron 63, 6855–6860. Isaka, M., Palasarn, S., Choowong, W., Mongkolsamrit, S., 2019. Conoideoxime A, antibacterial bis-oxime prenyl-tryptophan dimer from the whitefly pathogenic fungus Conoideocrella luteorostrata BCC 76664. Tetrahedron Lett. 60, 154–156. Johnson, D., Sun, G.H., Hywel-Jones, N.L., Luangsa-ard, J.J., Bischoff, J.F., Kepler, R.M., Spatofora, J.W., 2009. Systematics and evolution of the genus Torrubiella (Hypocreales, Ascomycota). Mycol. Res. 113, 279–289. Lang, G., Mitova, M.I., Ellis, G., van der Sar, S., Phipps, R.K., Blunt, J.W., Cummings, N.J., Cole, A.L.J., Munro, M.H.G., 2006. Bioactivity profiling HPLC/microtiter-plate analysis: application to a New Zealand marine alga-derived fungus, Gliocladium sp. J. Nat. Prod. 69, 621–624. Saepua, S., Kornsakulkarn, J., Choowong, W., Supothina, S., Thongpanchang, C., 2015. Bioxanthracenes and monomeric analogues from insect pathogenic fungus Conoideocrella luteorostrata Zimm. BCC 31648. Tetrahedron 71, 2400–2408. Yang, M.J., Wu, J., Yang, Z.D., Zhang, Y.M., 2009. First total synthesis of paecilodepsipeptide A. Chin. Chem. Lett. 20, 527–530.
3.4.1. Prenylated derivative 5 Colorless powder; IR νmax ATR (cm−1): 1644, 1510, 1240; 1H NMR (400 MHz, DMSO-d6) and 13C NMR (100 MHz, DMSO-d6) data, Table S1; HRESIMS (m/z): 832.3891 [M + Na]+ (calc. for C45H55N5O9Na, 832.3892). 3.5. Preparation of Compound 5 from paecilodepsipeptide D (1) A mixture of 1 (1.0 mg, 0.0015 mmol), 3,3-dimethylallyl bromide (100 μl, 0.87 mmol), and K2CO3 (30 mg, 0.22 mmol) in DMF (0.3 ml) was stirred at room temperature for 16 h. The mixture was diluted with EtOAc and washed with H2O. The organic layer was concentrated under reduced pressure. The residue was purified by preparative HPLC using a reverse-phase column (MeCN/H2O, 55:45) to yield compound 5 (0.9 mg; 74%). Compound 5 was identified by comparison (1H NMR, HRESIMS, and HPLC analysis) with the compound (5) synthesized from 2. 3.6. Biological assays Antimalarial activity against Plasmodium falciparum (K1, multidrugresistant strain) was performed using the microculture radioisotope technique (Desjardins et al., 1997). Cytotoxicity to Vero cells was evaluated using the green fluorescent protein (GFP)-based microplate
67
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Paecilodepsipeptide D, a cyclohexadepsipeptide from cultures of the whitefly pathogenic fungus Conoideocrella luteorostrata BCC 76664
T
Masahiko Isaka , Somporn Palasarn, Pranee Rachtawee, Kitlada Srichomthong, Suchada Mongkolsamrit ⁎
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand
ARTICLE INFO
ABSTRACT
Keywords: Conoideocrella luteorostrata Invertebrate pathogenic fungi Cyclodepsipeptide Antimalarial activity
Paecilodepsipeptide D (1), a new cyclohexadepsipeptide was isolated together with known paecilodepsipeptides A–C (2–4) from the whitefly pathogenic fungus Conoideocrella luteorostrata BCC 76664. The structure of 1 was elucidated by spectroscopic analyses and was further confirmed by the chemical correlation to paecilodepsipeptide A (2) by converting both compounds into the same di-O-prenyl derivative (5). A comparison of the antimalarial activities of 1, 2, and 5 suggested that a polar phenolic (OH) functionality at the D-tyrosine (Tyr-2) is crucial for the antimalarial activity, while a lipophilic O-alkyl side-chain at the other D-tyrosine (Tyr-1) is preferred.
1. Introduction Conoideocrella luteorostrata (Clavicipitaceae) is an invertebrate pathogenic fungus that most commonly attacks scale-insects (Johnson et al., 2009). The anamorphic state of this fungus was formerly known as Paecilomyces cinnamomeus. Previous chemical investigations of this species have revealed the presence of various structural groups of bioactive secondary metabolites such as depsipeptides (paecilodepsipeptides ACe; antimalarial activity) (Isaka et al., 2007a), bioxanthracenes and their monomers (ES-242 derivatives; antimalarial and cytotoxic activities) (Saepua et al., 2015), and prenylated tryptophan derivatives with an oxime functionality (luteorides ACe) (Asai et al., 2011). Recently, we reported the isolation of conoideoxime A, an antibacterial heterodimer of luteorides, from cultures of C. luteorostrata BCC 76664, which is a rare strain isolated from a dead whitefly host (Isaka et al., 2019). Due to our interest in the unique chemical skeleton of conoideoxime A and its antibacterial activity, the producing fungal strain (BCC 76664) has been reinvestigated. Although no new derivative of conoideoxime A was found in the new fermentation batch, the study led to the isolation of a new cyclohexadepsipeptide, named paecilodepsipeptide D (1), together with known paecilodepsipeptides ACe (2–4) (Isaka et al., 2007a; Lang et al., 2006) (Fig. 1). 2. Results and discussion Paecilodepsipeptide D (1) was obtained as a colorless solid. The molecular formula of 1 was determined to be C35H39N5O9 by HRESIMS.
⁎
The IR spectrum displayed an intense and broad absorption band at 1653 cm−1, suggesting the presence of carbonyl groups. The 1H and 13C NMR spectra of 1 in DMSO-d6 were very similar to those of the known cometabolite paecilodepsipeptide A (2). The significant difference was that 1 lacked the prenyl group of the O-prenyl-tyrosine unit in 2. The planar structure, elucidated by analysis of 2D NMR data (Fig. 2), was found to be a hexadepsipeptide composed of a hydroxycarboxylic acid and five amino acid residues, showing six carbonyl carbon signals at δC 172.0–167.6 and five amide NH protons at δH 8.53–7.65. The structure of 3-phenyllactic acid (3-Ph-Lac) residue was elucidated based on the COSY and HMBC correlations. An oxymethine at δC 73.2 (C-2) was attached to δC 36.9 methylene (C-3), which was flanked by a phenyl group. Five amino acid residues were also identified on the basis of COSY and HMBC data to be a glycine (Gly), two alanine (Ala-1 and Ala2), and two tyrosine (Tyr-1 and Tyr-2) residues (Table 1). The sequence of the six residues was established by analysis of HMBC data (Fig. 2). Amide protons of Ala-2, Gly, Tyr-2, Ala-1, and Tyr-1 were correlated to carbonyl carbons of 3-Ph-Lac, Ala-2, Gly, Tyr-2, and Ala-1, respectively. The HMBC correlation from H-2 of 3-Ph-Lac to C-1 of Tyr-1 indicated the presence of an ester linkage of Tyr-3-Ph-Lac to form a cyclohexadepsipeptide. This sequence was further supported by the NOESY correlations: H-2 of 3-Ph-Lac with NH of Ala-2; H3-3 of Ala-2 with NH of Gly; H2-2 of Gly with NH of Tyr-2; H-2 and H2-3 of Tyr-2 with NH of Ala-1; H3-3 of Ala-1 with NH of Tyr-1; and H2-3 of Tyr with H-2 of 3-PhLac. Therefore, the sequence of 1 was proven to be the same as 2. The absolute configurations at C-2 of the 3-Phe-Lac and five amino acid residues in paecilodepsipeptide A (2), isolated from another strain
Corresponding author. E-mail address: [email protected] (M. Isaka).
https://doi.org/10.1016/j.phytol.2019.09.017 Received 5 August 2019; Received in revised form 25 September 2019; Accepted 27 September 2019 1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 34 (2019) 65–67
M. Isaka, et al.
obtained by the prenylation of 1. Consequently, the absolute configurations of 1 were confirmed: D-Tyr (for Tyr-1), D-Ala (for Ala-1), D-Tyr (for Tyr-2), L-Ala (for Ala-2), and (2S)-L-3-Ph-Lac. It was previously shown that paecilodepsipeptide A (2) exhibited antimalarial activity against Plasmodium falciparum K1 (multidrug-resistant strain), while 3 and 4, linear hydrolysis and methanolysis derivatives, were inactive (Isaka et al., 2007a). In the present study, the antimalarial activities of the compounds 1, 2, and 5 were tested for comparison, and displayed IC50 values of > 50, 6.3, and > 50 μg/ml, respectively. Dihydroartemisinin was used as the standard antimalarial drug (IC50 0.63 ng/ml). These results suggested that a polar phenolic (OH) functionality at the D-Tyr (Tyr-2) is crucial for the antimalarial activity, while lipophilic O-alkyl side-chain at the other D-Tyr (Tyr-1) is preferred. Compounds 1, 2, and 5 were noncytotoxic against Vero cells (African green monkey kidney fibroblasts) at 50 μg/ml. 3. Experimental 3.1. General procedures Optical rotations were determined using a JASCO P-1030 digital polarimeter. UV spectra were recorded on an Analytik-jena SPEKOL 1200 spectrophotometer. FTIR spectra were acquired using a Bruker ALPHA spectrometer. NMR spectra were recorded on Bruker DRX400 and AV500D spectrometers. ESITOF mass spectra were measured using a Bruker micrOTOF mass spectrometer. Preparative HPLC was performed on a Waters 600 System equipped with a Waters 2296 photodiode array detector (Waters, Milford, USA). Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) and Silica gel 60H (particle size, 90% < 45 μM; Merck KGaA, Darmstadt, Germany) were used for column chromatography. 3.2. Fungal material The fungus used in this study was isolated from a whitefly (Hemiptera) at Chanthaburi Agricultural Research and Development Center, Chanthaburi Province, Thailand, on December 17, 2014. This fungus was deposited at the Thailand BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology, Pathumthani, Thailand, as BCC 76664. The identification of this fungus was based on the morphology and ITS rDNA sequence data (GenBank accession number: MH188543): phylum Ascomycota, order Hypocreales, family Clavicipitaceae, genus Conoideocrella, species luteorostrata (Isaka et al., 2019).
Fig. 1. Structures of compounds 1–5.
3.3. Fermentation, extraction, and isolation Conoideocrella luteorostrata BCC 76664. was fermented in 20 × 1000 ml Erlenmeyer flasks containing 250 ml of minimum salt medium (yeast extract 1.0 g/l, glucose 20 g/l, NH4NO3 3.0 g/l, KH2PO4 0.5 g/l, NaH2PO4 0.5 g/l, MgSO4·7H2O 0.5 g/l, and CaCl2 0.5 g/l) at 25 °C for 23 days under static conditions. The cultures were filtered to separate mycelia and filtrate (broth). The culture broth was extracted with EtOAc (2 × 5 l) and the combined organic phase was concentrated to obtain a brown gum (extract A, 1.50 g). Wet mycelia were macerated in MeOH (1.0 l, room temperature, 2 days) and then filtered. The filtrate was defatted by partitioning with hexanes (1.0 l). The MeOH layer was concentrated under reduced pressure. The residue was diluted with H2O (50 ml), extracted with EtOAc (2 × 800 ml), and concentrated under reduced pressure to leave a brown solid (extract B, 2.14 g). Extract A was subjected to Sephadex LH-20 column chromatography (CC) (3.5 × 60 cm, MeOH) to obtain 4 pooled fractions, A1 – A4. Fraction A3 (524 mg) was further fractionated by silica gel CC (2.8 × 15 cm, EtOAc/CH2Cl2, step gradient elution from 0:100 to 100:0), and preparative HPLC using a reverse-phase column (Waters NovaPak C18 OBD, 25 × 100 mm, 6 μm; mobile phase MeCN/H2O, 30:70, flow rate 10 ml/min) to yield 1 (8.2 mg, tR 10.5 min). Extract B
Fig. 2. COSY and selected HMBC and NOESY correlations for 1, indicating the sequence of five amino acids and 3-Ph-Lac.
(BCC 9616) of the same fungal species, were previously determined by the combination of chiral column HPLC analysis of the acid hydrolysate and Marfey’s method (Isaka et al., 2007a). The absolute configuration was later confirmed through total synthesis by Yang and coworkers (Yang et al., 2009). The absolute configuration of 1 was determined by chemical correlation to 2. The reaction of 2 with prenyl bromide and K2CO3 (s) in DMF at room temperature for 16 h gave a di-O-prenyl derivative 5 (Isaka et al., 2007b). The same compound (5) was also 66
Phytochemistry Letters 34 (2019) 65–67
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Table 1 NMR spectroscopic data for paecilodepsipeptide D (1) in DMSO-d6 (500 MHz for 1H, 125 MHz for No.
δC, type
D-Tyr-1 1 2 3
171.3, C 56.0, CH 35.9, CH2
4 5,9 6,8 7 7-OH NH D-Ala 1 2 3 NH
126.1, 130.2, 115.1, 156.4,
C CH CH C
172.0, C 47.7, CH 18.6, CH3
δH, mult. (J in Hz)
4.23, m 2.87, dd (14.1, 6.3); 2.77, dd (14.1, 6.3) 6.90, d (8.0) 6.71, d (8.0) 9.22, s 8.53, br d (2.1) 4.23, m 1.27, d (6.8) 7.65, d (8.0)
No.
δC, type
D-Tyr-2 1 2 3
170.2, C 55.6, CH 35.5, CH2
4 5,9 6,8 7 7-OH NH Gly 1 2
128.1, 129.7, 115.0, 155.7,
NH
–
13
C).
δH, mult. (J in Hz)
C CH CH C
169.1, C 43.0, CH2
4.19, m 2.96, m; 2.73, m 6.97, d (8.0) 6.62, d (8.0) 9.34, s 8.24, d (8.2) 3.58, dd (15.9, 6.3); 3.33, m 8.07, m
No. L-Ala 1 2 3 NH L-3-Ph-Lac 1 2 3 4 5,9 6,8 7
δC, type 171.8, C 48.6, CH 18.2, CH3 167.6, C 73.2, CH 36.9, CH2 135.5, 129.7, 127.9, 126.6,
C CH CH CH
δH, mult. (J in Hz)
4.27, m 1.06, d (6.7) 7.67, d (6.7) – 5.12, m 3.00, dd (14.5, 6.1); 2.51, dd (14.5, 3.1) 6.82, m 7.19, m 7.17, m
was also subjected to similar chromatographic procedures to yield 2 (8.9 mg), 3 (2.9 mg), 4 (2.3 mg), and conoideoxime A (6.2 mg).
assay (Hunt et al., 1999). Ellipticine was used as the positive control in the cytotoxicity assay (IC50 1.58 μg/ml).
3.3.1. Paecilodepsipeptide D (1) MeOH Colorless powder; [α]23 (nm) D +22 (c 0.25, MeOH); UV λmax (log ε): 218 (3.67), 277 (3.24); IR νmax ATR (cm−1): 1743, 1653, 1516, 1234; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSOd6) data, Table 1; HRESIMS (m/z): 696.2632 [M + Na]+ (calc. for C35H39N5O9Na, 696.2640).
Declaration of Competing Interest The authors have no competing interests to declare. Acknowledgements Financial support from the National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA) is gratefully acknowledged.
3.4. Preparation of Compound 5 from paecilodepsipeptide A (2) A mixture of 2 (30 mg, 0.040 mmol; a sample previously isolated from another strain of the same species) (Isaka et al., 2007a), 3,3-dimethylallyl bromide (125 μl, 1.1 mmol), and K2CO3 (30 mg, 0.22 mmol) in DMF (0.5 ml) was stirred at room temperature for 16 h. The mixture was diluted with EtOAc and washed with H2O. The organic layer was concentrated under reduced pressure. The residue was purified by preparative HPLC using a reverse-phase column (MeCN/H2O, 55:45) to yield compound 5 (16 mg; 50%).
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2019.09.017. References Asai, T., Yamamoto, T., Oshima, Y., 2011. Histone deacetylase inhibitor induced the production of three novel prenylated tryptophan analogs in the entomopathogenic fungus Torrubiella luteorostrata. Tetrahedron Lett. 52, 7042–7045. Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D., 1997. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 16, 710–718. Hunt, L., Jordan, M., De Jesus, M., Wurm, F.M., 1999. GFP-expressing mammalian cells for fast, sensitive, noninvasive cell growth assessment in a kinetic mode. Biotechnol. Bioeng. 65, 201–205. Isaka, M., Palasarn, S., Lapanun, S., Sriklung, K., 2007a. Paecilodepsipeptide A, an antimalarial and antitumor cyclodepsipeptide from the insect pathogenic fungus Paecilomyces cinnamomeus BCC 9616. J. Nat. Prod. 70, 675–678. Isaka, M., Berkaew, P., Intereya, K., Komwijit, S., Sathitkunanon, T., 2007b. Antiplasmodial and antiviral cyclohexadepsipeptides from the endophytic fungus Pullularia sp. BCC 8613. Tetrahedron 63, 6855–6860. Isaka, M., Palasarn, S., Choowong, W., Mongkolsamrit, S., 2019. Conoideoxime A, antibacterial bis-oxime prenyl-tryptophan dimer from the whitefly pathogenic fungus Conoideocrella luteorostrata BCC 76664. Tetrahedron Lett. 60, 154–156. Johnson, D., Sun, G.H., Hywel-Jones, N.L., Luangsa-ard, J.J., Bischoff, J.F., Kepler, R.M., Spatofora, J.W., 2009. Systematics and evolution of the genus Torrubiella (Hypocreales, Ascomycota). Mycol. Res. 113, 279–289. Lang, G., Mitova, M.I., Ellis, G., van der Sar, S., Phipps, R.K., Blunt, J.W., Cummings, N.J., Cole, A.L.J., Munro, M.H.G., 2006. Bioactivity profiling HPLC/microtiter-plate analysis: application to a New Zealand marine alga-derived fungus, Gliocladium sp. J. Nat. Prod. 69, 621–624. Saepua, S., Kornsakulkarn, J., Choowong, W., Supothina, S., Thongpanchang, C., 2015. Bioxanthracenes and monomeric analogues from insect pathogenic fungus Conoideocrella luteorostrata Zimm. BCC 31648. Tetrahedron 71, 2400–2408. Yang, M.J., Wu, J., Yang, Z.D., Zhang, Y.M., 2009. First total synthesis of paecilodepsipeptide A. Chin. Chem. Lett. 20, 527–530.
3.4.1. Prenylated derivative 5 Colorless powder; IR νmax ATR (cm−1): 1644, 1510, 1240; 1H NMR (400 MHz, DMSO-d6) and 13C NMR (100 MHz, DMSO-d6) data, Table S1; HRESIMS (m/z): 832.3891 [M + Na]+ (calc. for C45H55N5O9Na, 832.3892). 3.5. Preparation of Compound 5 from paecilodepsipeptide D (1) A mixture of 1 (1.0 mg, 0.0015 mmol), 3,3-dimethylallyl bromide (100 μl, 0.87 mmol), and K2CO3 (30 mg, 0.22 mmol) in DMF (0.3 ml) was stirred at room temperature for 16 h. The mixture was diluted with EtOAc and washed with H2O. The organic layer was concentrated under reduced pressure. The residue was purified by preparative HPLC using a reverse-phase column (MeCN/H2O, 55:45) to yield compound 5 (0.9 mg; 74%). Compound 5 was identified by comparison (1H NMR, HRESIMS, and HPLC analysis) with the compound (5) synthesized from 2. 3.6. Biological assays Antimalarial activity against Plasmodium falciparum (K1, multidrugresistant strain) was performed using the microculture radioisotope technique (Desjardins et al., 1997). Cytotoxicity to Vero cells was evaluated using the green fluorescent protein (GFP)-based microplate
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