Natural Indoles From the Bacterium Pseudovibrio denitrificans P81 Isolated From a Marine Sponge,Aaptos Species

Abstract

A new natural indole, vibrindole B (1), together with known analogs, vibrindole A (2), trisindoline (3), norharmane (4), and 3-(hydroxyacetyl)indole (5), produced by the bacterium Pseudovibrio denitrificans P81, were isolated from a sponge, Aaptos species. The structures of indoles 1 to 5 were established by spectroscopic methods. The proposed biosynthetic pathway of 1 to 5 is also discussed, starting from tryptophan. Moreover, indoles 1 to 3 were found to exhibit cytotoxicity toward T24 tumor cells with IC₅₀ values of 1.71 ± 0.11, 4.53 ± 0.14, and 2.26 ± 0.26 µM, respectively.

Keywords

sponge vibrindole cytotoxicity

Introduction

Marine bacteria belonging to the genus Pseudovibrio (Rhodobacterales) have proven to be not only an important source of bioactive compounds, but also to play an interesting role in marine ecology.^1-5 Pseudovibrio species are described by Shieh¹ as heterotrophic, marine, facultative anaerobes capable of denitrification and fermentation. Since the first type strain, P. denitrificans DN34, was isolated from seawater samples collected in Nanwan Bay, Taiwan,¹ more and more Pseudovibrio species have been found around the world in both marine organisms such as sponges,² corals,⁶ tunicates,⁷ flatworms,⁸ and the environment, such as seawater.^1,5,9

Pseudovibrio species not only live with their hosts, but also provide benefits to them and have diverse genes for producing multiple bioactive compounds.⁴ Some of these have showed activity against other microorganisms. This may prevent their host from infection by other marine microorganisms. For example, heptylprodigiosin, produced by P. denitrificans strain Z143-1 isolated from a Philippine tunicate, displayed anti-Staphylococcus aureus activity;⁷ and a Pseudovibrio species from a marine sponge produced tropodithietic acid (TDA).¹⁰ In a previous study, TDA displayed inhibitory activity against a range of marine bacteria, such as α-Proteobacteria, γ-Proteobacteria, Actinobacteria, and Flavobacteria.¹¹

The metabolites from Pseudovibrio species have displayed extensive pharmacological activities. For instance, P. denitrificans UST4-50 produced compounds belonging to the di(1H-indol-3-yl)methane family, which showed antifouling activity against larval settlement of Balanus amphitrite;¹² P. denitrificans BBCC725 isolated from seawater produced a new indole derivative, tetra(indol-3-yl)ethenone, that displayed cytotoxicity against L929 mouse fibroblasts and A549 human lung epithelial carcinoma cell lines.⁹ In our continuing research aimed at discovering new natural compounds from marine organisms, in an organic extract of bacterium strain P81, identified as Pseudovibrio denitrificans, which was originally isolated from a marine sponge Aaptos sp. collected from Green Island, Taiwan, five indole derivatives were discovered, including one new natural product, vibrindole B (1), along with four known indoles, vibrindole A (2), trisindoline (3), norharmane (4), and 3-(hydroxyacetyl)indole (5) (Figure 1). The fermentation, isolation, structure determination, and bioactivity of these indole derivatives are reported herein.

Figure 1.

Structures of vibrindole B (1), vibrindole A (2), trisindoline (3), norharmane (4), and 3-(hydroxyacetyl)indole (5).

Results and Discussion

Vibrindole B (1) was obtained as a brown amorphous powder. The molecular formula of 1 was established as C₂₀H₂₀N₂ (unsaturation degrees = 12) from a sodiated molecular ion at m/z 311 in the ( + )-electrospray ionization mass spectrum (ESIMS), and was further supported by ( + )-high resolution electrospray ionization mass spectrum (HRESIMS) ions at m/z 311.15163 (calcd for C₂₀H₂₀N₂ + Na, 311.15187). The IR spectrum suggested the presence of an amine (ν_max 3413 cm^–1) group. The ¹³C nuclear magnetic resonance (NMR) spectrum of 1 (Table 1) showed that this compound has only 12 carbons, which were identified by the assistance of heteronuclear single quantum coherence (HSQC) and ¹H NMR spectra as one methyl, two sp³ methylenes, an sp³ methine, five sp² methines, and three sp² non-protonated carbons. The discrepancy of carbon numbers between the ¹³C NMR and mass spectra indicates that part of the structure of 1 is symmetrical.

Table 1.

¹H and ¹³C nuclear magnetic resonance spectroscopic data for 1, 2, and vibrindole A⁹.

C/H	1		2		Vibrindole A
C/H	δ_H ^a (J in Hz)	δ_C ^b, Mult.	δ_H ^a (J in Hz)	δ_C ^b, Mult.	δ_H ^c (J in Hz)	δ_C ^d, Mult.
1	4.50 t (7.8)	33.7, CH	4.69 q (7.2)	28.2, CH	4.61 q (7.6)	28.0, CH
2	2.20 q (7.8)	38.1, CH₂	1.82 d (7.2)	21.7, CH₃	1.74 d (7.6)	21.7, CH₃
3	1.44 sext (7.8)	21.4, CH₂
4	0.95 t (7.8)	14.2, CH₃
1′ & 1"-NH	7.91 br s		7.92 br s		7.85 br s
2′ & 2"	7.01 d (1.8)	121.3, CH	6.85 dd (2.4, 0.6)	121.1, CH	6.86 d (2.2)	121.1, CH
3′ & 3"		120.6, C		121.8, CH		121.8, C
3′a & 3"a		127.2, C		126.9, CH		127.0, C
4′ & 4"	7.60 d (7.8)	119.7, CH	7.58 dd (7.8, 0.6)	119.7, CH	7.50 d (7.9)	119.7, CH
5′& 5"	7.03 t (7.8)	119.0, CH	7.04 td (7.8, 0.6)	119.0, CH	6.96 td (7.9, 0.6)	119.0, CH
6′ & 6"	7.14 t (7.8)	121.6, CH	7.17 td (7.8, 0.6)	121.7, CH	7.09 t (7.9)	121.7, CH
7′ & 7"	7.34 d (7.8)	111.0, CH	7.36 dd (7.8, 0.6)	111.0, CH	7.28 dd (7.9, 0.6)	111.0, CH
7′a & 7"a		136.6, C		136.6, CH		136.6, C

600 MHz, CDCl₃.

150 MHz, CDCl₃.

500 MHz, CDCl₃.

125 MHz, CDCl₃.

The ¹H NMR spectrum of 1 (Table 1) showed an indole-like signal, which included two ortho-disubstituted benzene ring 4-spin systems [δ_H 7.03 (2H, t, J = 7.8 Hz, H-5′ & 5”); δ_H 7.14 (2H, t, J = 7.8 Hz, H-6′ & 6”); δ_H (7.34, 2H, d, J = 7.8 Hz, H-7′ & 7′′); δ_H 7.60 (2H, d, J = 7.8 Hz, H-4′ & 4′′)], two aromatic protons [δ_H 7.01 (2H, d, J = 1.8 Hz, H-2′ & 2′′)], and two amine protons (δ_H 7.91, 2H, br s, NH-1′ & 1′′). The residual proton signals, a methyl [δ_H 0.95 (3H, t, J = 7.8 Hz, H₃-4)], two pairs of methylene protons [δ_H 1.44 (2H, sext, J = 7.8 Hz, H₂-3); δ_H 2.20 (2H, q, J = 7.8 Hz, H₂-2)], and a methine proton [δ_H 4.50 (1H, t, J = 7.8 Hz, H-1)], revealed as an n-butyl group. The ratio of proton signal integration between indole and methine was 2:1. This gave evidence for 1 being a combination of two indoles and an n-butyl group.

The carbon skeleton of 1 was elucidated initially by ¹H–¹H correlation spectroscopy (COSY) and heteronuclear multiple bond coherence (HMBC) spectra (Figure 2). It was possible to establish the separate spin systems that mapped out the proton sequences from H-1/H₂-2/H₂-3/H₃-4 and H-4′/H-5′/H-6′/H-7′. These data, together with the HMBC correlations between protons and non-protonated carbons such as H-1/C-3′, C-3′a; H₂-2/C-3′; H-2′/C-3′, C-7′a; H-4′/C-7′a; H-5′/C-3′a; H-6′/C-7′a; and H-7′/C-3′a, permitted elucidation of the main carbon skeleton of 1. Moreover, a comparison of the one-dimensional (1D) NMR spectroscopic data of 1 and the known compound 2 (Table 1) further demonstrated that 1 was a symmetrical indole alkaloid with an n-propyl group at C-1 in 1 instead of a methyl group in 2. Indole 1 has been obtained as a synthetic product in the reported literature;^13-17 however, to the best of our knowledge, there have been no reports of 1 being obtained from any natural source.

Figure 2.

Main COSY (–) and selective key HMBC () correlations of 1.

Indoles 2 to 5 were identified as vibrindole A,⁹ trisindoline,¹⁸ norharmane (β-carboline),¹⁹ and 3-(hydroxyacetyl)indole,²⁰ by comparison of their spectroscopic data with those in the literature.

A proposed biosynthetic pathway for 1 to 5 is depicted in Figure 3. Previous findings suggested that the amino acid, tryptophan, serves as the biosynthetic precursor.^21-23 Tryptophan can undergo lysis by tryptophanase to indole, which can undergo carbon addition to generate compound I. Vibrindole B (1) and vibrindole A (2) are formed by the combination of I with another indole. Compound II is generated by the hydroxylation of indole. The carbonylation of C-3 and C-2 of the indole ring produces compounds III and IV, sequentially. Further, trisindoline (3) is produced via the nucleophilic addition at C-3 of IV with two indoles. The biosynthetic routes of β-carboline (4) and 3-(hydroxyacetyl)indole (5) have been reported in the literature. Compound V is generated by the cyclization of tryptamine, which is formed via the decarboxylation of tryptophan. Hereafter, β-carboline (4) can be produced via serial oxidation of V and VI.^24,25 3-(Hydroxyacetyl)indole (5) is probably formed from 3-indolylacetaldoxime based on the study by Biihlendor et al.²⁰

Figure 3.

The proposed biosynthetic pathway for 1 to 5, starting from tryptophan.

The cytotoxicity of indole derivatives 1 to 5 against human urinary bladder cancer T24 cells was investigated (Table 2). Indoles 1 to 3 displayed significant cytotoxicity toward T24 cells with IC₅₀ values of 1.7 ± 0.1, 4.5 ± 0.1, and 2.3 ± 0.3 μM, respectively. Comparison of the cytotoxicity of compounds 1 to 5 suggested that the bisindole moiety in 1 to 3 was critical for the cytotoxicity. Furthermore, when the methyl group at C-1 in 2 was replaced with an n-propyl group, as shown in 1, it enhanced the cytotoxicity. This indicated that the chain length of the alkyl group affected cytotoxicity. This interesting sponge species has been transferred to the culture tank located in our institute to provide a large quantity of the potentially useful microorganisms.

Table 2.

Cytotoxic data of indoles 1 to 5 on T24 cells.

Compounds	Cell viability (%) ^a	IC₅₀ (μM)
1	4.8 ± 0.8***	1.7 ± 0.1
2	4.8 ± 0.7***	4.5 ± 0.1
3	5.4 ± 0.7***	2.3 ± 0.3
4	87.6 ± 5.7**	> 10
5	79.3 ± 0.8***	> 10

Percentage of cell viability at 10 μM concentration in 72 h. Results are presented as mean ± S. E. M. (n = 3). **p < 0.01, ***p < 0.001 compared with the control (dimethylsulfoxide, DMSO).

Experimental

General

IR spectra were measured on a Thermo Scientific Nicolet iS5 FT-IR spectrophotometer (Thermo Fisher Scientific). ¹H and ¹³C NMR spectra were recorded on Jeol ECZ NMR spectrometers (Jeol) at either 600 or 400 MHz for ¹H NMR, and either 150 or 100 MHz for ¹³C NMR, respectively, with residual solvent as the internal standard. Positive-ion ESIMS and HRESIMS were acquired on a Bruker 7 Tesla solariX Fourier transform mass spectroscopy (FTMS) (Bruker). Silica gel column chromatography was carried out with C18 (17%) reverse-phase silica gel (230–400 mesh, SiliCycle Inc. ). Precoated silica gel 60 F₂₅₄ plates (E. Merck; 0.25 mm in thickness) were used for thin-layer chromatographic analyses; compounds were visualized by spraying with a 10% solution of H₂SO₄ and heating on a hotplate. Isolation and purification of isolates by normal-phase high performance liquid chromatography (NP-HPLC) and reverse-phase high performance liquid chromatography (RP-HPLC) were performed using (a) a Hitachi L-7110 instrument equipped with a normal-phase column (Supelco Ascentis Si, 250 × 21.2 mm, 5 µm; Sigma-Aldrich); and (b) a Hitachi L-2130 pump, a Hitachi L-2455 photodiode array detector, and a reverse-phase column (Luna 5 µm, 250 × 21.2 mm; Phenomenex).

Culture Conditions, Marine Bacterial Isolation, and Extraction

Marine bacterium number P81 was isolated from a marine sponge, Aaptos species, collected off the coast of Green Island at a depth of − 5 m. P81 was 100% identical to Pseudovibrio denitrificans DN34 (Genebank accession no. NR_029112) on the basis of the 16S rRNA gene sequence. The marine bacterium was cultured in 2.5 L flasks containing 1.0 L M1 broth (1% starch, 0.4% yeast extract, and 0.2% peptone) with 80% seawater and incubated at 25 °C on an orbital shaker at a rotation speed of 120 rpm. After five days of incubation, extraction of the culture broth (45.0 L) with EtOAc (2 × 45.0 L) yielded 4.62 g of crude extract in total. The extract obtained was then stored at − 20°C until further use.

Isolation

The extract was fractionated on a C-18 silica gel column and eluted using a mixture of MeOH and H₂O (stepwise, 3:7 − MeOH) into 8 fractions (1to 8). Fraction 6 was purified separately by NP-HPLC, using n-hexane − acetone (7:3), to afford 18 fractions, 6A to 6R. 6D and 6G were purified using RP-HPLC, using MeCN − H₂O (11:9), to yield 1 (0.9 mg) and 2 (0.3 mg), respectively. Fraction 5 was purified using RP-HPLC, using MeCN − H₂O (3:2), to afford 23 fractions, 5A to 5W. 5H and 5C were further purified using RP-HPLC, using MeCN − H₂O (3:2 for 5H and 2:3 for 5C), to yield 3 (3.2 mg) and 4 (0.9 mg), respectively. Fraction 1 was purified on a C-18 silica gel column, using MeOH − H₂O (stepwise, 1:9 − 3:7) to yield 6 fractions, 1Ato 1F. 1F was repurified using NP-HPLC, using a mixture of dichloromethane − MeOH (20:1), to afford 9 fractions 1F1 to 1F9. 1F2 was further separated using RP-HPLC, using a mixture of MeCN − H₂O (1:4), to afford 5 (1.5 mg).

Vibrindole B (1). Brown amorphous power; IR (attenuated total reflectance [ATR]) ν_max 3413, 3056, 2955, 2926, 2854, 1456, 1338, 1217, 1095, 744 cm⁻¹; ¹H (CDCl_3, 600 MHz) and ¹³C (CDCl₃, 150 MHz) NMR data, see Table 1; ESIMS: m/z 311 [M + Na]⁺; HRESIMS: m/z 311.15163 (calcd for C₂₀H₂₀N₂ + Na, 311.15187).

Vibrindole A (2). Brown amorphous power; IR (ATR) ν_max 3418, 2921, 2851, 1456, 1216, 1101, 762 cm⁻¹; ¹H (CDCl_3, 600 MHz) and ¹³C (CDCl₃, 150 MHz) NMR data were found to be in complete agreement with a previous report;⁹ ESIMS: m/z 283 [M + Na]⁺.

Trisindoline (3). Amorphous power; IR (ATR) ν_max 3405, 3058, 2955, 2924, 2854, 1708, 1456, 1338, 1219, 1100, 743 cm⁻¹; ¹H (CD₃OD_, 600 MHz) and ¹³C (CD₃OD, 150 MHz) NMR data were found to be in complete agreement with a previous report;¹⁸ ESIMS: m/z 386 [M + Na]⁺.

Norharmane (4): Amorphous power; IR (ATR) ν_max 3135, 3058, 2944, 2924, 2864, 1629, 1503, 1451, 1331, 1246, 1035, 737 cm⁻¹; ¹H (CDCl_3, 600 MHz) and ¹³C (CDCl₃, 150 MHz) NMR data were found to be in complete agreement with a previous report;¹⁹ ESIMS: m/z 169 [M + Na]⁺.

3-(Hydroxyacetyl)indole (5). Amorphous power; IR (ATR) ν_max 3135, 3058, 2944, 2924, 2864, 1629, 1503, 1451, 1331, 1246, 1035, 737 cm⁻¹; ¹H (CD₃OD_, 400 MHz) and ¹³C (CD₃OD, 100 MHz) NMR data were found to be in complete agreement with a previous report;²⁰ ESIMS: m/z 189 [M + Na]⁺.

Cell Culture and Cell Viability Assay

T24 human bladder carcinoma cells were cultured at 37°C in Roswell Park Memorial Institute 1640 Medium (RPMI 1640, Gibco) supplemented with 1% antibiotic/antimycotic solution (100 U/mL penicillin, 100 µg/mL streptomycin, and 2.5 µg/mL amphotericin B, Gibco) and 10% fetal bovine serum (Gibco) in a humidified atmosphere with 5% CO₂. Cell viability was assessed using the water soluble tetrazolium salts-1 (WST-1) assay. T24 cells were cultured in RPMI 1640 medium for 24 h. After incubation, cells were incubated with indicated concentrations of test indoles 1 to 5 for 72 h. Then, the WST-1 reagent was added and incubated at 37°C for 2 h. The absorbance was measured spectrophotometrically at 450 nm (Thermo Labsystems). The cytotoxicity of indoles 1 to 5 was assayed with a modification of the MTT[3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric method. Cytotoxicity assays were carried out according to previously described procedures.^26,27

Supplemental Material

sj-docx-1-npx-10.1177_1934578X211033735 - Supplemental material for Natural Indoles From the Bacterium Pseudovibrio denitrificans P81 Isolated From a Marine Sponge, Aaptos Species

Supplemental material, sj-docx-1-npx-10.1177_1934578X211033735 for Natural Indoles From the Bacterium Pseudovibrio denitrificans P81 Isolated From a Marine Sponge, Aaptos Species by Shu-Yen Fang, Sheng-Yuan Chen, You-Ying Chen, Tsu-Jen Kuo, Zhi-Hong Wen, Yu-Hsin Chen, Tsong-Long Hwang and Ping-Jyun Sung in Natural Product Communications

Footnotes

Acknowledgments

The authors are thankful to Ms. Hsiao-Ching Yu and Chao-Lien Ho, the High Valued Instrument Center, National Sun Yat-sen University, for the mass (MS000600) and NMR (NMR001100) spectra (MOST 110-2731-M-110-001).

Author Contributions

S-YF carried out the investigations; S-YC took part in the investigations; Y-YC participated in the investigation; T-JK participated in the investigations; Z-HW took part in the investigations; Y-HC was part of conceptualization, investigation, and draft preparation; T-LH was involved in conceptualization, investigation, and draft preparation; P-JS took part in conceptualization and draft preparation.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Zuoying Branch of Kaohsiung Armed Forces General Hospital (grant number KAFGH-ZY-A-ZBH107-05, MOST107-2320-B-291-001-MY3, MOST109-2320-B-291-001-MY3).

Funding

This research was funded by grants from the National Museum of Marine Biology and Aquarium; Zuoying Branch of Kaohsiung Armed Forces General Hospital (KAFGH-ZY-A-ZBH107-05); and the Ministry of Science and Technology, Taiwan (Grant Nos.: MOST 107-2320-B-291-001-MY3 and 109-2320-B-291-001-MY3), awarded to Zhi-Hong Wen and Ping-Jyun Sung.

Ethical Approval

Not applicable, because this article does not contain any studies with human or animal subjects.

Informed Consent

Not applicable, because this article does not contain any studies with human or animal subjects.

Trial Registration

Not applicable, because this article does not contain any clinical trials.

ORCID iD

Ping-Jyun Sung

Supplemental Material

Supplemental material for this article is available online.

References

Shieh

Lin

Jean

. Pseudovibrio denitrificans gen. nov., sp. nov., a marine, facultatively anaerobic, fermentative bacterium capable of denitrification. Int J Syst Evol Microbiol. 2004;54(6): 2307-2312.

Nicacio

Ióca

Fróes

, et al. Cultures of the marine bacterium Pseudovibrio denitrificans Ab134 produce bromotyrosine-derived alkaloids previously only isolated from marine sponges. J Nat Prod. 2017;80(2):235-240.

Alex

Antunes

. Genus-wide comparison of Pseudovibrio bacterial genomes reveal diverse adaptations to different marine invertebrate hosts. PLoS One. 2018;13:e0194368.

Naughton

Romano

O’Gara

Dobson

ADW

. Identification of secondary metabolite gene clusters in the Pseudovibrio genus reveals encouraging biosynthetic potential toward the production of novel bioactive compounds. Front Microbiol. 2017;8:1494.

Romano

. Ecology and biotechnological potential of bacteria belonging to the genus Pseudovibrio. Appl Environ Microbiol. 2018;84(8):e02516-17.

Chen

Kuo

Sung

, et al. Isolation of marine bacteria with antimicrobial activities from cultured and field-collected soft corals. World J Microbiol Biotechnol. 2012;28(12):3269-3279.

Sertan-de Guzma

Predicala

Bernardo

, et al. Pseudovibrio denitrificans strain Z143-1, a heptylprodigiosin-producing bacterium isolated from a Philippine tunicate. FEMS Microbiol Lett. 2007;277(2):188-196.

Zhang

Tian

Lai

. Pseudovibrio stylochi sp. nov., isolated from a marine flatworm. Int J Syst Evol Microbiol. 2016;66(5):2025-2029.

Bell

Carmeli

Sar

. Vibrindole A, a metabolite of the marine bacterium, Vibrio parahaemolyticus, isolated from the toxic mucus of the boxfish Ostracion cubicus. J Nat Prod. 1994;57(11):1587-1590.

10.

Harrington

Reen

Mooij

, et al. Characterisation of non-autoinducing tropodithietic acid (TDA) production from marine sponge Pseudovibrio species. Mar Drugs. 2014;12(12):5960-5978.

11.

Brinkhoff

Bach

Heidorn

Liang

Schlingloff

Simon

. Antibiotic production by a Roseobacter clade-affiliated species from the German Wadden Sea and its antagonistic effects on indigenous isolates. Appl Environ Microbiol. 2004;70(4):2560-2565.

12.

Wang

, et al. Low-toxicity diindol-3-ylmethanes as potent antifouling compounds. Mar Biotechnol. 2015;17(5):624-632.

13.

Földeák

Czombos

Matkovics

Pórszász

. Synthesis of substances effecting C. N. S. VIII. Synthesis of new diindolylmethane derivatives effecting C. N. S. Acta Phys Chem. 1965;11(3–4):116-125.

14.

Nagarajan

Perumal

. Potassium hydrogen sulfate-catalyzed reactions of indoles: a mild, expedient synthesis of bis-indolylmethanes. Chem Lett. 2004;33(3):288-289.

15.

Bandgar

Bettigeri

Joshi

. Hexamethylenetetraamine-bromine catalyzed rapid and efficient synthesis of bis(indolyl)methanes. Monatsh Chem. 2004;135(10):1265-1273.

16.

Noguchi-Yachide

Tetsuhashi

Aoyama

Hashimoto

. Enhancement of chemically induced HL-60 cell differentiation by 3,3′-diindolylmethane derivatives. Chem Pharm Bull. 2009;57(5):536-540.

17.

Wang

. TFA-catalyzed C-N bond activation of enamides with indoles: efficient synthesis of 3,3-bisindolylpropanoates and other bisindolylalkanes. Tetrahedron. 2013;69(5):1600-1605.

18.

Kobayashi

Aoki

Gato

Matsunami

Kurosu

Kitagawa

. Marine natural products. XXXIV. Trisindoline, a new antibiotic indole trimer, produced by a bacterium of Vibrio sp. Separated from the marine sponge Hyrtios altum Chem Pharm Bull. 1994;42(12):2449-2451.

19.

Parameswaran

Naik

Hegde

. Secondary metabolites from the sponge Tedania anhelans: isolation and characterization of two novel pyrazole acids and other metabolites. J Nat Prod. 1997;60(8): 802-803.

20.

Böhlendorf

Forche

Bedorf

, et al. Antibiotics from gliding bacteria, LXXIII. Indole and quinoline derivatives as metabolites of tryptophan in Myxobacteria. Liebigs Ann. 1996;(1):49-53.

21.

Alkhalaf

Ryan

. Biosynthetic manipulation of tryptophan in bacteria: pathways and mechanisms. Chem Biol. 2015;22(3):317-328.

22.

Kumla

Suwannarach

Matsui

Lumyong

. Biosynthetic pathway of indole-3-acetic acid in ectomycorrhizal fungi collected from northern Thailand. PLOS ONE. 2020;15(1):e0227478.

23.

Bunsangiama

Sakpuntoona

Srisuka

Ohashib

Fujiyamab

Limtonga

. Biosynthetic pathway of indole-3-acetic acid in basidiomycetous yeast Rhodosporidiobolus fluvialis Mycobiology. 2019;47(3):292-300.

24.

Rommelspacher

Wernicke

Lehmann

. β-Carbolines: occurrence, biosynthesis, and biodegradation. In: Isoquinolines and β-carbolines as neurotoxins and neuroprotectants. Current Topics in Neurotoxicity, Springer. 2012;1: pp. 105-113.

25.

Herbert

Mann

. The biosynthesis of the β-carboline alkaloids, harman, and eleagnine. J Chem Soc., Perkin Trans. 1. 1982:1523-1525.

26.

Alley

Scudiero

Monks

, et al. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 1988;48(3):589-601.

27.

Scudiero

Shoemaker

Paull

KD.

, et al. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 1988;48(17):4827-4833.

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