Chemical investigation of terrestrial and marine streptomycete isolates led to the identification of two new natural pigments, namely, 6-hydroxyisatin (3) and 6-hydroxy-5-methoxyisatin (4). Additionally, the strains delivered numerous known compounds, among them Nβ-acetyltryptamine, N-acetyltyramine, phenylacetamide, N-(2-phenethyl)acetamide, 1-acetyl-β-carboline, tyrosol, 2′-deoxyadenosine, 2′-deoxythymidine, anthranilic acid, 2′-deoxyuridine, indolyl-3-acetic acid, indolyl-3-carboxylic acid, and p-hydroxybenzoic acid. The isatin structures were deduced by NMR and mass studies and further confirmed by synthesis and by X-ray diffraction of the isomeric 5-hydroxy-6-methoxyisatin (5).
Isatin (1) was obtained for the first time by oxidative degradation of indigo with nitric or chromic acid , and was later also isolated from Isatis tinctoria and various other natural sources including microorganisms . Although one of its derivatives, 5-bromo-isatin, is rather widespread as a trace component in mollusks [3, 4], other isatins including 1 are not often found in nature . 4-Hydroxyisatin and the methyl ether, isalexin, were isolated from the tubers of Brassica napus . Melosatins A–D, a series of phenylpentyl-isatins, are produced by Melochia tomentosa . Only five isatins were reported from microorganisms . The embryos of the shrimp Palaemon macrodactylus are colonized by an Alteromonas sp., which synthesizes isatin (1) and thus protects the larvae against fungal infections by Lagenidium callinectes . Isatin (1) was also found in a Pseudoalteromonas sp. T268 isolated from the Antarctic krill Euphausia superb  and a few streptomycetes. It is known as fungal pigment of a Schizophyllum commune mutant  and is an inhibitor of xanthine oxidase  and monoamine oxidase . Further two isatins, 5- and 6-prenylisatin, were isolated from Chaetomium globosum  and Streptomyces albus . From various streptomycetes, we also isolated recently 7-prenylisatin [15, 16] and now – for the first time from nature – 6-hydroxy-isatin (3) and 6-hydroxy-5-methoxyisatin (4). The related 5-chloro-6-methoxy-1-methylisatin was found in Micromonospora carbonacea . The structures of 3 and 4 were assigned by spectroscopic methods, by synthesis, and confirmed indirectly by crystal structure analysis of the synthetic isomer 5.
Indigo is often formed as a trace component during the synthesis, biosynthesis, and biodegradation of indole derivatives. Blue indigotin or red indirubin, or the respective hydroxyindigos, however, were not found in the bacterial extracts investigated here.
2 Results and discussion
New natural products are identified in plants, microorganisms, or other sources not solely on the basis of their biological activity, but often in combination with the so-called chemical screening: extracts were investigated for this purpose in a thin-layer chromatography (TLC)-guided search for fluorescent or fluorescence-quenching zones, for color reactions with special reagents, or even simpler, with the naked eye or with a diode array detector for colored constituents. Even without the presence of biological activities, this sensitive, yet simple, cheap and fast approach has been surprisingly successful worldwide.
In our search for new microbial metabolites by chemical screening, we observed often yellow, orange, or red constituents in trace amounts, which were insufficient for common elucidation procedures, so that they were usually ignored. In a focused search for the nature of these traces, we recently identified a group of new isoquinolinequinones, the orange-red mansouramycins . In the actual case, two further orange trace components in the extracts of the streptomycete strains B1848 and AdM13 were investigated: they did not develop blue or violet colors with sodium hydroxide, so that peri-hydroxyquinones were excluded. With concentrated sulfuric acid, carotenoids turn blue or green, polyene macrolides turn brown or black, and phenoxazinones (e.g. cinnabarin or the actinomycins) stain intensively orange-red, which we also did not observe here. This awakened our interest.
Upscaling and separation of extracts from a marine-derived streptomycete strain B1848  delivered one of these orange trace components in sufficient amounts for further investigation . The obtained solid of moderate polarity displayed a light red coloration with sodium hydroxide, and the UV spectrum in methanol exhibited a broad absorption maximum at λmax=435 nm. The molecular weight was established as m/z=163 by electrospray ionization (ESI)- and electron ionization (EI)-mass spectrometry (MS), and EI-high-resolution mass spectra (HRMS) delivered the molecular formula C8H5NO3. The 1H NMR spectrum in dimethyl sulfoxide ([D6]DMSO) showed only three signals in the aromatic region with the typical pattern of a 1,2,4-trisubstituted benzene, and additionally two exchangeable 1H singlets at δ~11 ppm. The 13C NMR spectrum displayed eight sp2 signals (including two carbonyls) and was thereby similar to that of 1. One methine signal in the aromatic region was exchanged, however, against a quaternary low-field carbon signal, pointing to 5-hydroxy- (2) or 6-hydroxy-isatin (3). Heteronuclear multiple-bond correlations (HMBC) confirmed the isatin skeleton. The weak spectrum did not allow, however, differentiating between 2 and 3, as H-4 and H-7 both showed strong HMBC couplings to a phenolic carbon at δ=167.0 ppm, assigned as C-5 or C-6. The isatins 2 and 3 were therefore synthesized starting from 4- or 3-methoxyaniline, respectively, using Sandmeyer’s procedure . On direct comparison, the natural pigment was identified unequivocally as 6-hydroxy-isatin (3), and the low-field signal was assigned as C-6.
Other fractions of the bacterial extract contained as main components Nβ-acetyltryptamine, N-acetyltyramine, phenylacetamide, N-(2-phenethyl)acetamide, 1-acetyl-β-carboline, tyrosol, 2′-deoxyadenosine, 2′-deoxythymidine, anthranilic acid, 2′-deoxyuridine, indolyl-3-acetic acid, indolyl-3-carboxylic acid, and p-hydroxybenzoic acid [8, 20].
From the extracts of the terrestrial Streptomyces sp. strain AdM13, a similar orange minor component was obtained in a series of chromatographic purification steps as reddish-brown powder. The streptomycete produced additionally the known antibiotics, actinomycin D, and reductiomycin, which were responsible for the high bioactivity of the extract [22, 23].
The molecular formula of the minor component was established as C9H7NO4 by ESI-HRMS of the [M–H]− ion at m/z=192 (ESI-MS). The 1H NMR spectrum displayed a broad NH singlet at δ=10.38 ppm and showed aromatic 1H singlets of two p-positioned protons in the neighborhood of electron-donating groups (δ=6.93 and 6.25 ppm). A 3H singlet at δ=3.71 ppm and the corresponding signal in the 13C NMR spectrum (δ=56.2 ppm) indicated a methyl ether, connected with a carbon at δ=144.2 ppm; a further low-field carbon at δC=160.9 ppm was assigned to a phenol.
In accordance with the molecular formula and the NMR data, this compound was 6-hydroxy-5-methoxyisatin (4) or 5-hydroxy-6-methoxyisatin (5). The methoxy group was in direct neighborhood of the proton at δ=6.93 ppm, as the strong nuclear Overhauser effect between these protons indicated. Because of the small amount, not all expected 13C NMR signals were visible, and the remaining shifts had to be extracted from the 2D data; also some crucial HMBC signals were missing. That the respective aromatic proton was at H-4 and not H-7 was confirmed by the HMBC spectra of a synthetic sample (Fig. 1) and by simulation of the NMR shifts , thereby confirming structure 4.
To further analyze the spectroscopic behavior and the biological properties of hydroxyisatins, we synthesized 4 and further isatins 2–9 using Sandmeyer’s procedure  (Fig. 2 and Supplementary Information). Starting from, for example, 3-hydroxy-4-methoxyaniline (10), we obtained by a reaction with chloralhydrate and hydroxylamine the isonitrosoacetanilide 14, which afforded in concentrated sulfuric acid the isatin 4. The synthetic product had identical properties as the natural 6-hydroxy-5-methoxyisatin (4). The isomeric 5-hydroxy-6-methoxyisatin (5) was synthesized by partial demethylation of 5,6-dimethoxyisatin (8), and also 5-hydroxyisatin (2) was obtained in a similar way.
A sample of 5 crystallized from moist DMSO as hair-like needles, suitable for X-ray diffraction (see the Experimental section, Fig. 3, and Supplementary Information), which further confirmed indirectly also the structure of the isomer 4.
In the 13C NMR spectra in DMSO, the unusual strong downfield shift of the hydroxy carbon C-6 of 3 (δ=167.0 ppm) and 4 (δ=160.9 ppm) is explained by a tautomerism between the carbonyl C3=O and the phenol or phenolate at C-6, resulting in a partial carbonyl character of C6–OH or C6–O−, respectively. Indeed, density functional theory (DFT) calculations confirmed that the Mulliken bond order of C6–OH and C3=O (δ=0.98, 1.88 ppm) in 3 in neutral solution changed to δ=1.59/1.75 ppm for the phenolate; for 4, the respective bond orders changed from δ=1.01/1.88 to 1.57/1.76 ppm (Spartan’14 ). Correspondingly, the proton shifts of 4 were strongly influenced by the measurement conditions. Depending on the solvent (methanol or DMSO), the concentration and especially the pH value, H-4 in 4 was found between δ=6.42 and 7.09 ppm (average of n=8 at δ=6.84 ppm), and H-7 was observed between δ=5.43 and 6.38 ppm (average 6.03 ppm), with a shift difference between both signals of Δδ=0.68 and 0.98 ppm. After the addition of ethyl-diisopropylamine, even the methoxy signal was shifted from δ=3.7–3.8 to 3.58 ppm, as expected for enol ethers (see Tables 1 and 2, and Fig. S22, Supplementary Information). In [D6]DMSO in the presence of trifluoroacetic acid no shifts>0.02 ppm were observed.
|Solvent/Frequency in MHz||DMSO 300||MeOD 300||DMSO 300||MeOD 300||DMSO 300||DMSO 300||MeOD 600||DMSO 300||MeOD 300||DMSO 300||DMSO 300||MeOD 300||MeOD 300|
|H-4||6.83 dd||6.93 dd||7.39 d||7.42 d||6.55 s||6.93 s||6.71 sb||6.85 s||6.92 s||7.07 d||7.47 d||7.09 s||8.54 s|
|2.7, 0.5||2.7, 0.4||8.3||8.5||7.07 sc||2.7||8.5|
|H-5||–||–||6.40 dd||6.43 dd||–||–||–||–||–||–||6.58 dd||–||–|
|8.3, 2.4||8.3, 2.0||8.5, 2.1|
|H-6||7.00 ddd||7.02 dd||–||–||–||–||–||7.19 dd||–||–||–|
|8.4, 2.7, 0.7||8.3, 2.0||8.4, 2.7|
|H-7||6.75 dd||6.77 dd||6.29 d||6.29 d||5.68 s||6.25 s||5.84 sb||6.45 s||6.53 s||6.85 d||6.39 d||6.55 s||5.93 s|
|8.4, 0.5||8.4, 0.4||2.4||2.0||6.32 sc||8.4||2.1|
|5-OMe||–||–||–||–||3.58 s||3.71 s||3.82 s, 3.70 s||–||–||3.75 s||–||3.78 s||–|
|6-OMe||–||–||–||–||–||–||–||3.92 s||3.97 s||–||3.87 s||3.94 s||–|
|OH/NH||10.10 s||–||10.79 s,||–||9.77 s||10.38 s||–||–||–||–||10.92 s||–||–|
Shifts as δ values and coupling constants in Hz.
aSynthetic, after addition of ethyl-diisopropylamine.
b,cSamples independently isolated from different strains.
|Solvent/Frequency in MHz||DMSO 125||MeOD 125||DMSO 125||MeOD 125||DMSO 125||MeOD 150||MeOD 125||DMSO 125||DMSO 125||DMSO 125|
Shifts as δ values.
aSignals may be exchanged.
bCorresponds to data set b) of 4 in Table 1. The carbon shifts of the second sample (c in Table 1) differed by 0 (C-2) up to 14 ppm (C-6) from the values listed here.
In contrast, characteristic for the isomeric 5-hydroxy-6-methoxyisatin (5) is the smaller and solvent-independent shift difference between both 1H signals of Δδ=0.39–0.40 ppm. The bond orders of C5–OH and C3=O in 2 (δ=0.95/1.90, basic 1.53/1.85 ppm) and 5 (δ=0.98/1.89, basic 1.47/1.83 ppm) are also different in neutral and acidic solution, but less than those for the 6-hydroxy isomers.
These differences in the keto/phenol tautomerism were reflected by the UV/Vis spectra in acidic and alkaline solution in an unexpected way. The deep bromine-colored solution of 6-hydroxy-5-methoxyisatin (4) in methanol absorbed at 484 nm, and with base, only a small bathochromic shift to 487 nm was observed. In a similar way, 6-hydroxyisatin (3) showed the long-wavelength maximum in neutral solution at λmax=435 nm, after the addition of sodium hydroxide at λmax=441 nm. Both 6-hydroxyisatins 3 and 4 are obviously rather acidic, so that equilibria between neutral form and phenolate or respective salts were observed already at ambient conditions. This acidity is also indicated by the blood-red color, which is obtained when a solution of 4 in DMSO or methanol was diluted with calcium-containing tap water.
While the basic solution of 4 is orange-red, the yellow solutions of the 5-hydroxyisatins 2 and 5 turned violet (λmax=411→531 and 478→550 nm) after the addition of sodium hydroxide, although no keto/phenolat tautomerism is expectable (Fig. 4). An explanation for this unexpected behavior was given by DFT calculations  (see Table 3), which predicted for the absorption maximum at longest wavelength for 2 and 5 a strong bathochromic shift from acidic to basic solution of Δλmax~300 or 260 nm, respectively (exp. found Δλ=120/72 nm). Only a slight bathochromic shift of ~35/20 nm was predicted for the basic solution of 3 and 4, which agreed well with the experiments (6/3 nm). A qualitative explanation is already given by the energy differences of lowest unoccupied molecular orbital and highest occupied molecular orbital (LUMO-HOMO), which are for the violet anions of 2 and 5 substantially smaller than for those of 3 and 4.
|Isatins||λmax (exp)||∆λmax (exp)||λmax (calcd)||∆λmax (calcd)||Etr||f|
|2 (nt; 5-OH)||411||120||396||299||395.80||0.0319|
|2 (bas; 5-O−)||531||695||695.90||0.0202|
|3 (nt; 6-OH)||435||6||340||35||340.81||0.0347|
|3 (bas; 6-O−)||441||375||375.87||0.2204|
|4 (nt; 6-OH-5-OMe)||484||3||376||16||377.03||0.0411|
|4 (bas; 6-O−-5-OMe)||487||392||392.47||0.1828|
|5 (nt; 5-OH-6-OMe)||478||72||386||258||388.31||0.0444|
|5 (bas; 5-O−-6-OMe)||550||644||643.91||0.0217|
3 Experimental section
3.1 Materials and methods
UV/Vis spectra were recorded on a Perkin-Elmer Lampda 15 UV/VIS spectrometer. NMR spectra were measured on Varian Unity 300 and Varian Inova 600 spectrometers. HPLC-MS: Finnigan LCQ ion trap mass spectrometer coupled with a Flux Instruments quaternary pump Rheos 4000 (Basel, Switzerland) and a HP 1100 HPLC (Nucleosil column EC 125/2, 100-5, C 18) with autosampler (Jasco 851-AS, Jasco Inc., Easton, MD, USA) and a Diode Array Detector (Finnigan Surveyor LC System). HRMS were recorded by ESIMS on an Apex IV 7 Tesla Fourier-Transform Ion Cyclotron Resonance Mass Spectrometer (Bruker Daltonics, Billerica, MA, USA). EI-MS (70 eV) were recorded on a Finnigan MAT 95 spectrometer (Thermo Electron Corp., Bremen, Germany) with perfluorokerosene as reference substance for high-resolution electron impact mass spectroscopy. Flash chromatography was carried out on silica gel (230–400 mesh). Rf values were measured on Polygram SIL G/UV254 (Macherey-Nagel & Co.). Size exclusion chromatography was done on Sephadex LH-20 (Lipophilic Sephadex, Amersham Biosciences Ltd.; purchased from Sigma-Aldrich Chemie, Steinheim, Germany). Amberlite XAD 16 resin was obtained from Rohm and Haas (Frankfurt, Germany).
3.1.1 Taxonomy of Streptomyces sp. isolate B1848
The marine-derived strain S. sp. B1848 has been described previously .
3.1.2 Fermentation of marine-derived Streptomyces sp. isolate B1848
The Streptomyces sp. isolate B1848 was fermented for 3 days at 29°C in a 25-L jar fermenter, containing 21 L of M2+ medium [malt extract (10 g), yeast extract (4 g), and glucose (4 g) were dissolved in artificial seawater (0.5 L) and tap water (0.5 L). Before sterilization, the pH was adjusted to 7.8 by addition of 2 n NaOH].
The obtained faint yellow culture broth was mixed with diatomaceous earth (ca. 1 kg) and filtered under pressure. Both, filtrate and mycelial cake, were extracted four times with ethyl acetate. The mycelial cake was further extracted twice with acetone, followed by concentration in vacuo; the resulting aqueous residue was re-extracted with ethyl acetate (3×). The combined organic phases were concentrated in vacuo to dryness to afford a reddish-brown crude extract (10.53 g). The latter was suspended in methanol and defatted with cyclohexane. The remaining methanol extract was evaporated to dryness (7.0 g) and applied to flash silica gel column chromatography (3×60 cm), using a CHCl3-MeOH gradient, to afford under TLC monitoring five fractions. The orange fraction IV obtained with CHCl3-10% MeOH followed by Sephadex LH-20 (CHCl3-MeOH 6:4) afforded 6-hydroxyisatin (3, 1.5 mg) as orange powder. Other fractions contained Nβ-acetyltryptamine (4.1 mg), N-(2-phenethyl)acetamide (3.8 mg), 1-acetyl-β-carboline (10.4 mg),  anthranilic acid (6.3 mg), 2′-deoxyadenonsine (8.2 mg), tyrosol (3.4 mg), indolyl-3-acetic acid (7.3 mg), phenyl acetamide (2.7 mg), 2′-deoxythymidine (6.2 mg), 2′-deoxyuridine (5.5 mg), indolyl-3-carboxylic acid (8.3 mg), N-acetyltyramine (3.8 mg), and p-hydroxybenzoic acid (2.4 mg).
3.1.3 6-Hydroxyisatin (3)
Intensively orange powder, in MeOH orange-red, no color change with sulfuric acid or NaOH on TLC; Rf=0.71 (CHCl3-10% MeOH), m.p.>300°C. – UV/Vis (MeOH): λmax=215 (4.08), 262 (3.96), 328 (3.74), 435 nm (3.26); (MeOH/HCl): λmax=210 (4.09), 261 (4.13), 324 (3.80), 401 nm (3.06); (MeOH/NaOH): λmax (log ε)=210, 265, 375 sh, 441 nm. – 1H NMR (300 MHz, CD3OD; separated by “/”: shifts before and after final purification): δ=7.42/7.29 (d, 3J=8.3 Hz, 1H, H-4), 6.38/6.08 (dd, 3J=8.3 Hz, 4J=2.4 Hz, 1H, H-5), 6.26/5.90 (d, 4J=2.4 Hz, 1H, H-7). – 13C NMR (150 MHz, CD3OD): see Table 2; in the natural product, the signals of C-3a and 7a were missing due to the small amount of substance, and obtained from the HMBC spectrum (see also Tables 1 and 2, and Fig. S11 ff, Supplementary Information). –MS ((+)-ESI): m/z=186 [M+Na]+. –MS ((−)-ESI): m/z=162 [M–H]−. – MS (EI, 70 eV): m/z (%)=163 (100), 147 (40), 135 (77), 119 (19), 108 (14). – HRMS (EI): m/z=163.0269 (calcd. 163.0264 for C8H5NO3, [M]+).
3.1.4 Taxonomy of Streptomyces sp. strain AdM13
The terrestrial strain ADM13 was isolated from a soil sample near Göttingen (Germany). The almost complete 16S rRNA gene sequence showed high similarities to the 16S rRNA of the type strain of Streptomyces michiganensis NBRC 12797 (GenBank Accession Nr. AB18415) as well as to Streptomyces xanthochromogenes NBRC 12828 (GenBank Accession Nr. AB184176). The partial 16S rRNA sequence of Streptomyces sp. AdM13 can be accessed via GenBank (Accession Nr. H00696). The strain is deposited in the culture collection at the Institute of Organic and Biomolecular Chemistry, Göttingen, Germany.
3.1.5 Fermentation of Streptomyces sp. AdM13
A 24-L shaker culture of the terrestrial Streptomyces sp. AdM13 on M2 medium (see above, however, with tap water instead of seawater) was cultivated for 7 days at 28°C, and the brown-yellow culture broth was worked up in a similar way as for Streptomyces sp. B1848. Fraction III afforded three components, which after purification on the Sephadex LH-20 (MeOH) column resulted in reductiomycin (100 mg), red-orange 5-methoxy-6-hydroxy isatin (4, 39 mg), and orange actinomycin D (300 mg).
3.1.6 6-Hydroxy-5-methoxyisatin (4)
Dark red-orange powder, in MeOH orange-red with NaOH, on TLC no color change with sulfuric acid. Rf=0.45 (CH2Cl2-5% MeOH), Rf=0.79 (CH2Cl2-10% MeOH); m.p. 252°C. – UV/Vis (CH3OH): λmax (log ε)=276 (2.76), 363 (2.72), 484 nm (2.33); (MeOH-HCl): λmax (log ε)=268 (3.01), 325 (2.63), 465 nm (2.26); (MeOH-NaOH): λmax (log ε)=276 (2.76), 363 (2.75), 487 nm (2.35). – 1H, 13C NMR (see Tables S1 and S2, and Fig. S16 ff, Supplementary Information). –MS ((−)-ESI): m/z (%)=407 (56, [2M–2H+Na]−), 192 (100, [M–H]−); – HRMS ((−)-ESI): m/z=192.03023 (calcd. 192.03022 for C9H6NO4, [M–H]−).
3.1.7 Crystal structure determination of 5-hydroxy- 6-methoxy-isatin (5)
A yellow crystal (0.47×0.45×0.02 mm3) was obtained from a solution of 5 in a droplet of DMSO on exposure to moist air. The crystal was mounted at 100 K on a Bruker Smart 6000 CCD diffractometer equipped with a rotating anode generator and Incoatec Helios optics using CuKα radiation (λ 1.54178 Å). A total number of 10529 reflections were measured, of which 1947 were independent. The asymmetric unit consisted of one molecule 5 and one molecule of DMSO. The measured crystal belonged to the monoclinic space group P21/c with Z=4. The structure was determined by Direct Methods. All atoms except hydrogens were refined anisotropically by full-matrix least-squares methods on F2 using SHELXL . All hydrogen atoms were visible in the difference map and refined using a riding model. The final R factor was 4.84%. Table S1 (Supplementary Information) summarizes important crystal structure data.
CCDC 1414741 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
3.2 DFT calculations
For the calculation of UV/Vis spectra, first a conformer distribution of the respective molecule was determined with Spartan’14  using the Merck Molecular Force Field; only one to two conformers were obtained each. For the least-energy conformers, a geometry optimization was performed with Gaussian , using DFT calculations with the wB97XD functional and the 6-31G* basis set. On basis of these geometries, the UV/Vis data were calculated with CAM-B3LYP/6-311++G** td=(nstates=100) SCF=VeryTight IOP(3/33=1) Pop=Full. From the resulting LOG files, the excitation energies and the respective oscillator strengths were extracted with the program AOMix  and fitted with a Gaussian function (band width at peak half-height=3000 cm−1). For the results, see Table 3.
4 Supplementary information
Synthesis of isatins, crystal structure data, 1D and 2D NMR data of isolated and synthesized compounds, and UV/Vis spectra as well as other data associated with this article are given as Supplementary Information available online (http://dx.doi.org/10.1515/znb-2016-0143).
We thank R. Machinek for the NMR spectra, Dr. H. Frauendorf for the mass spectra, and M. Becker, A. Kohl, and F. Lissy for technical support.
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