Introduction

During our investigation of secondary metabolites produced by microorganisms in Arctic and Antarctic habitats, we isolated from Arctic pack ice a psychrotolerant, Gram-negative bacterium T436, which was assigned to a distinct group within the genus Salegentibacter on the basis of 16S rRNA gene profile and physiological characteristics.1 Our interest in this strain was attracted by an antimicrobial activity of crude extracts against Bacillus brevis, B. subtilis, Nematospora coryli and Micrococcus luteus, as well as by an intensively yellow color.1 On TLC, numerous yellow zones were visible over the whole polarity range, which were not because of quinones, xanthones, phenazines, polyenes or other common chromophores. On spraying with tin(II) chloride solution, the yellow spots were reduced to colorless or faintly yellow products, which gave intensively yellow or orange Schiff bases on treatment with 4-dimethylaminobenzaldehyde, as an indication of aromatic nitro compounds. This interpretation was confirmed by two IR signals in the range of ν 1510–1570 and 1320–1350 cm−1, and by typical fragments in the EI mass spectra (see Supplementary Figure 1). Nitro compounds are widespread in microorganisms, but are rather rare in nature. Recent examples are nitrobenzyl alcohols from an endophytic mangrove fungus,2 or nitroresorcinols from myxobacteria.3

On TLC, the mononitrophenols isolated in this study absorbed UV light at 254 nm and showed an absorption maximum at λmax∼350 nm in solution (see Supplementary Figure 2). In contrast, the dinitrophenols emitted a yellow UV fluorescence on TLC at 366 nm, and showed a long-wavelength absorption at λmax∼430 nm. A further specialty in the spectroscopic characterization of nitro compounds was with regard to the 13C NMR signals of the quaternary carbon atoms carrying the nitro group, which were only visible at long relaxation delays (1 s).

So far, 25 nitro, dinitro and trinitrophenols were isolated from this strain.4, 5, 6 Compounds 2–6, 8, 10, 11–17, 23 and 24 were already mentioned in our previous report.1 In this study, we describe their chemical properties in detail and report additional metabolites obtained on re-fermentation.

Results and discussion

Mononitrophenols

For the isolation of further nitro derivatives, a 20 l fermentation conducted under the same conditions as described before1 was extracted at weakly acidic pH; under basic conditions, the extraction was very ineffective. Chromatographic separation on silica gel, Sephadex LH-20 and by HPLC (Supplementary Figure 3) delivered 10 mononitrophenols. The de-replication of known compounds was performed by MS and HRMS, and by comparison with AntiBase data;7 new compounds were elucidated by additional 2D NMR measurements. p-Nitrophenol (1),8 isolated previously from carrot truffle (Stephanaspora caroticolor), was easily identified in this manner. It is a widespread environmental contaminant.

A group of nine compounds, 2–10, showed the characteristic 1H NMR signal pattern of 1,2,4-trisubstituted benzene derivatives. The downfield shift of aromatic 1H NMR signals, the pH-dependent yellow color and the IR data pointed to a group of 4-substituted o-nitrophenols with differences in a side chain at C-4. Compounds 1, 7 and 9 have not been described in our previous report on Salegentibacter sp. T436.1

Beside the signals for a 1,2,4-trisubstituted aromatic ring, the 1H NMR spectrum of 6 revealed an oxymethine multiplet at δ 4.38, a methoxy group at δ 3.70, as well as the AB part of an ABX system at δ 3.10 and 2.98. (−)-APCI mass spectra gave an [M-H]– ion at m/z 240. 2D NMR data revealed the structure of 2-hydroxy-3-(4′-hydroxy-3′-nitrophenyl)propionic acid methyl ester (6). The NMR data of 2-chloro-3-(4′-hydroxy-3′-nitrophenyl)propionic acid methyl ester (7) were very similar, but were downfield shifted. Both compounds are reported in this study for the first time. N-Acetyl-3′-nitrotyramine (9, 0.4 mg l−1) was new in Salegentibacter sp. T436, but had been isolated previously from Pyricularia oryzae,9 the causative agent of pyriculariosis, a widespread disease of rice.

Dinitro and trinitro derivatives

Seven simple dinitro compounds 11–17 had already been isolated previously from Salegentibacter T436.1 Two further compounds 18 and 19 have been added now; all are new from natural sources but some had been obtained by synthesis. Their UV maxima (λmax 432 nm) showed a strong bathochromic shift in alkaline solution similar to mononitro-phenols; however, 1H NMR data indicated clearly symmetrically tetrasubstituted benzene derivatives by 2H singlets as the only aromatic signal; the structural differences were again localized in the side chain.

Their structures were derived from 2D NMR measurements and HR mass spectra as 4′-hydroxy-3′,5′-dinitrophenylacetic acid methyl ester (12, 0.02 mg l−1), 4′-hydroxy-3′,5′-dinitrophenylacetic acid (11),10 4′-hydroxy-3′,5′-dinitrophenylpropionic acid (13, 0.05 mg l−1), 4′-hydroxy-3′,5′-dinitrophenylpropionic acid methyl ester (14)11 and dinitro-tyrosol (15).12

The 1H NMR spectrum of compound 16 (0.03 mg l−1) was similar to that of 13, although the methylene triplets were at different shifts. The ESI mass spectrum showed molecular ions at m/z 246/248 (EIMS) in the ratio of ca. 3:1, typical for chlorine. The IR spectrum gave no hints for carboxy or ester groups. From these data and from the empirical formula (by HRMS), the structure of 4′-hydroxy-3′,5′-dinitrophenylethyl chloride (16) was derived, which was finally confirmed by heteronuclear multibond correlation (HMBC) data (Supplementary Figure 4). Compound 16 is a new natural product and is also unknown from synthesis.

Compound 17 was obtained in the low yield of 0.04 mg l−1. Its 1H NMR spectrum showed signals of a methine group at δ 4.56, a methoxy group at δ 3.73 and signals of diastereotopic methylene protons at δ 3.18 and 3.06. In the APCI mass spectrum, an [M–H]− ion at m/z 303 indicated chlorine by its isotope pattern. The IR spectrum showed a strong absorption at 701 cm−1, which is characteristic of a C-Cl bond. These data were in agreement with the structure of 4′-hydroxy-3′,5′-dinitrophenyl-2-chloropropionic acid methyl ester (17), and HRESIMS confirmed the respective formula, C10H9ClN2O7. Compound 17 had not been described before, but the mononitro derivative has been synthesized.13

The 1H NMR spectrum of 18 (0.14 mg l−1) resembled that of 16 in the aromatic region, whereas the aliphatic signals corresponded with those of 9; moreover, the IR spectrum was very similar to that of 9. The ESI mass spectrum showed an [M–H]− ion at m/z 268. It follows that 18 is the new N-acetyl-3′,5′-dinitrotyramine. The structure was further confirmed by HMBC correlations (see Supplementary Figure 5).

The ESI mass spectrum of 19 indicated an odd number of nitrogen atoms by a pseudomolecular ion at m/z 254 [M–H]−; HRMS delivered the formula C8H5N3O7. In the 1H NMR spectrum, two 1H doublets of a trans-configured double bond appeared at a similar shift as in 10, which resulted in 2,6-dinitro-4-(2′-nitroethenyl)phenol (19); the latter is the first natural trinitro derivative.

Simple dinitro-phenols

A further separation by HPLC yielded two isomeric methoxy-dinitrophenols with the formula C7H6N2O6 (APCI HRMS). Both compounds showed two doublets of meta-coupled protons with strong HMBC correlations to the phenolic carbon, but only one proton in both isomers coupled with the carbon connected with the methoxy group). Therefore, in one isomer, the OH group has to be positioned at 1,3-distance with respect to both protons as in 20, and at 1,2-distance in the other phenol as in 21. Placing the methoxy group in para position to the OH group would result in two symmetrical dinitro compounds and can be excluded: one phenol must therefore be 2-methoxy-4,6-dinitrophenol (20) and the other is 3-methoxy-4,5-dinitrophenol (21). A direct comparison with synthetic materials14, 15 identified the slightly more polar isomer as 20. Both assignments were further confirmed by HMBC data. The NMR data of 2-methoxy-3,5-dinitrophenol and 2-methoxy-4,5-dinitrophenol are additionally listed in the experimental part. It is worth mentioning that the aromatic proton signals of 20 varied up to Δδ 0.2 (Δδ 4 for 13C), depending on the concentration and purity of the sample and the solvent.

4,6-Dinitroguaiacol (20)16 and isomeric 3,5-dinitroguaiacol17 are known as metabolites of the red alga Marginisporum aberrans; they showed antimicrobial activity against B. subtilis. The dinitroresorcinol ether 21 had not been described before.

Additional fermentation products

The isoflavones daidzein and genistein are common by-products in the fermentation of bacteria, if soybean flour or malt extract was used for cultivation. Surprisingly, these compounds were found now in the nitrated form as 3′-nitro-daidzein (22, 0.06 mg l−1; see Supplementary Figure 6), 3′-nitrogenistein (23) and 3′,5′-dinitro-genistein (24, see Supplementary Figure 7). All three compounds had been isolated previously from the genetically engineered Streptomyces sp. K3.18, 19 They showed no significant antimicrobial or phytotoxic activities. In addition, the nitro-diketopiperazine pyriculamide (25, 0.3 mg l−1) was found.7

A yellow compound with a mass of m/z 168 was identified as 2,6-dimethoxy-1,4-benzoquinone (26) and was confirmed by literature data. It is known as a plant metabolite with antitumor activity.20 Metabolite 27 was identified as tryptophol methyl ether, which was known only from synthesis.21 Its spectroscopical data were in accordance with literature. Further polar UV absorbing fractions contained thymine, uracil and p-hydroxybenzoic acid.

ESI fragmentation of aromatic nitro compounds

The MS analysis of nitro compounds was performed best in the negative ESI mode. Under EI conditions, phenolic mononitrocarboxylic acids showed a loss of carbon dioxide and of a 30 Da fragment, a phenomenon that had already been observed in the early 1980s in CIMS experiments22, 23, 24, 25 (see Supplementary Figure 1). This fragmentation may be explained by the reduction of the aromatic nitro group into an amino function in the source during measurement. A better explanation, however, is based on an NO elimination, which was already known from EI experiments.26 This assumption is further supported by the fact that in the full-scan spectra, no [M-H-30]– ions were detected, whereas these signals appeared after the fragmentation process. In several cases, a signal at [M-47-H]– was observed, which indicates the loss of an HNO2 molecule.

The dinitro derivatives investigated in this study showed similar fragmentation patterns as those of mononitro compounds. If the nitrophenols contained further leaving groups, such as chlorine atoms, these were eliminated first (in this study as HCl); thereafter, the elimination of NO occurred.

In the positive ESI mode, only those nitro compounds were detectable, which carried a functional group which can be easily protonated, as in 9 or 18. In these cases, usually an elimination of a 46 Da fragment was observed, which was most likely NO2.

Biosynthetic considerations

For the biosynthesis of natural nitro groups, at least three different pathways are known: the oxidation of anilines, the direct nitration of phenols with reactive nitrogen species (RNS) and the oxidation of nitroso precursors.

By means of feeding experiments, it was shown that p-aminophenylalanine is the precursor of the p-nitrophenylserinol part in chloramphenicol,27, 28 as well as of the nitrophenyl unit of aureothin.29 In the last step of pyrrolnitrin biosynthesis, an amino function is converted into a nitro group by a chloroperoxidase.30, 31 For most of the nitro compounds described in this study, potential amino precursors are, however, not known, and therefore this pathway seems less plausible.

An alternative biosynthetic route to nitro compounds is the oxidation of nitroso precursors,32 which themselves could be formed with nitrite, NO+ or NO• radicals under physiological conditions. Several nitroso compounds related to the nitro compounds isolated in this study were described, for example, 4′-hydroxy-3′-nitrosobenzoic acid from Streptomyces murayamaensis.33 Nitroso compounds such as viridomycin E34 and others7 are strong siderophores and are involved in the accumulation of iron, which is a limiting factor for phytoplankton growth in offshore surface waters of the Antarctic35, 36 and northeast Subarctic Pacific Ocean.37 A search by HPLC/MS in the crude extract of Salegentibacter sp. T436 for nitroso compounds corresponding to the nitro derivatives isolated in this study was, however, not successful.

Fermentation under various conditions has shown that nitrate is a precondition for the production of these nitro compounds. As in the brine channel system of the Arctic bottom sea ice increased nitrate concentrations occur,38 it can be assumed that another pathway is used: the direct nitration by RNS such as peroxynitrite and NO2, formed as secondary products of NO metabolism in the presence of oxidants including superoxide, hydrogen peroxide and transition metal centres. 3′-Nitrotyrosine formed in such a process has been discussed as a biomarker of NO-dependent oxidative stress;39, 40 3′-nitrogenistein is formed under the influence of peroxynitrite,41 and in the case of dioxapyrrolomycin, such a nitration by RNS was proven on the basis of isotope labeling with an 15N/18O-enriched nitrate.42, 43

It seems that tyrosine is the parent compound for most of the nitro aromates described here. Starting with tyrosine, first a mono- and dinitration by RNS occur in the benzene ring. The following formation of nitrated phenylpropionic acids (for example, 5), tyramines, N-acetyltyramines (such as 9), phenyllactic acids and tyrosols (8) is easily explained by reduction, oxidative deamination, decarboxylation and acetylation, and a combination thereof. A similar mechanism has been found for the enzymatic degradation of stephanosporin.8

Even the chloro derivatives 16 and 17, as well as the respective hydroxy derivatives 6 and 8/15, may be produced in this way: It has been shown that diazonium ions can be formed under physiological conditions by the reaction of amines with RNS,44 and more than 30 stable diazonium salts have been reported as natural products.7, 45 The formation of chloro derivatives may be explained by intermediates 28/29, which could react with water or with chloride in the sense of a Sandmeyer reaction (Figure 1). Furthermore, the formation of 27 could be explained by the reaction of tryptamine through the respective diazonium ion with methanol. Further investigations with isotope labeling to confirm these assumptions are, however, presently not realistic because of the very low yield.

Figure 1
figure 1

Sidechain transformations in nitrotyrosines by postulated diazonium intermediates 28/29, R=4′-hydroxy-3′-nitrophenyl and 4′-hydroxy-3′,5′-dinitrophenyl, respectively.

Full size image

Experimental section

Materials and methods

Nuclear magnetic resonance spectra were measured on Varian Unity 300 (300.145 MHz) and Varian Inova 600 (599.740 MHz) spectrometers with tetramethylsilane as internal standard (Varian Dentschland GmbH, Darmstadt, Germany). ESI mass spectra were recorded on a Quattro Triple Quadrupole Mass Spectrometer, Finnegan TSQ 7000 (Thermo Scientific, Dreieich, Germany) with nano-ESI-API-ion source. ESI-HRMS was measured on a 7 Tesla-Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer (APEX IV, Bruker Daltonik GmbH, Bremen, Germany). IR spectra were recorded on a Perkin-Elmer 1600 Series FT-IR spectrometer as KBr pellets. UV-VIS spectra were recorded on a Perkin-Elmer Lambda 15 UV/vis spectrometer (Wallthem, MA, USA). Flash chromatography was carried out on silica gel (230–400 mesh). TLC and determination of Rf values were performed on Polygram SIL G/UV254 (Macherey-Nagel & Co., Düren, Germany). Size exclusion chromatography was carried out on Sephadex LH-20 (Lipophilic Sephadex, Amersham Bioscience Ltd; purchased from Sigma-Aldrich Chemie, Steinheim, Germany).

HPLC Two Jasco Intelligent Prep. pumps PU-987 (Jasco, Easton, MD, USA) with high-pressure mixer, Vertex 4 × 250 mm column with 4 × 4 mm pre-column, Merck Lichrosorb RP C18 7 μm (Merck, Darmstadt, Germany); and preparative separations on Eurochrom Europrep RP 60–10 C18 60 Å 7–12 μm (Knauer, Berlin, Germany) aceotrope (83.7% acetonitrile 16.3% water, b.p. 78.5 °C) were used.

Taxonomy, fermentation and isolation

Details on the producing strain and its cultivation in Erlenmeyer flasks and in a 20-1 fermenter have been published previously.1

Isolation and characterization of metabolites

The crude extract from a 20 l batch fermentation was separated on silica gel with a stepwise CH2Cl2/MeOH gradient as described before;1 the fractions obtained were further separated by chromatography on Sephadex LH-20 (CH2Cl2/MeOH 2:1 und MeOH). Subsequent flash chromatography on silica gel (CH2Cl2/MeOH) afforded 0.4 mg p-nitrophenol (1), 1.8 mg 4′-hydroxy-3′-nitrobenzoic acid (2), 3.0 mg 4′-hydroxy-3′-nitrophenylacetic acid (3), 1.7 mg 4′-hydroxy-3′-nitrophenylpropionic acid (5), 1.7 mg 4′-hydroxy-3′-nitrophenylacetic acid methyl ester (4), 2.6 mg 3′-nitrotyrosol (8), 7.1 mg N-acetyl-3′-nitrotyramine (9), 0.4 mg 2-nitro-4-(2′-nitroethenyl)phenol (10), 0.4 mg 4′-hydroxy-3′,5′-dinitrophenylacetic acid methyl ester (12), 1.1 mg 4′-hydroxy-3′,5′-dinitrophenylpropionic acid (13), 0.6 mg 4′-hydroxy-3′,5′-dinitrophenylethyl chloride (16), 0.7 mg 4′-hydroxy-3′,5′-dinitrophenyl-2-chloropropionic acid methyl ester (17), 2.7 mg N-(4′-hydroxy-3′,5′-dinitrophenylethyl)-acetamide (18), 0.4 mg 2,6-dinitro-4-(2′-nitroethenyl)phenol (19), 6.1 mg pyriculamide (25), 1.1 mg 3′-nitro-daidzein (22), 3.9 mg 3′,5′-dinitro-genistein (24), 1.2 mg 2-methoxy-4,6-dinitrophenol (20), 1.8 mg 3-methoxy-4,5-dinitrophenol (21), 2.1 mg 2,6-dimethoxy-1,4-benzoquinone (26), 1.1 mg 3′-indolylethyl-methylether (27), 4.7 mg thymine, 4.2 mg uracil and 2.2 mg p-hydroxybenzoic acid.

The crude extract (1.5 g) obtained from a second 20 l fermentation was separated on silica gel with ethyl acetate/hexane and then with Sephadex LH-20 (MeOH), followed by preparative HPLC (MeCN+0.001% H3PO4). The following compounds were obtained: 4′-hydroxy-3′-nitrophenyl-propionic acid (5, 1.2 mg), 2-hydroxy-3-(4′-hydroxy-3′-nitrophenyl)propionic acid methyl ester (6, 2 mg), 2-chloro-3-(4′-hydroxy-3′-nitrophenyl)propionic acid methyl ester (7, 2.1 mg), 4′-hydroxy-3′,5′-dinitrophenylacetic acid (11, 2.0 mg), 3-(4′-hydroxy-3′,5′-dinitrophenyl)propionic acid methyl ester (14, 0.7 mg), dinitrotyrosol (15, 2.7 mg), and 3′-mononitrogenistein (23, 1.4 mg). The retention time of an individual compound corresponds to the elution profile, shown in Supplementary Figure 3.

4′-Hydroxy-3′-nitrobenzoic acid (2)

Yellow solid, UV absorbing (254 nm), RF 0.25 (CH2Cl2/MeOH 9:1), Rt 11.9 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 237 (4.38), 340 (3.51) nm. IR (KBr) νmax 3307, 2924, 2853, 1685, 1626, 1574, 1541, 1433, 1338, 1312, 1257, 1175, 1112, 923, 854, 827, 762, 704, 639, 532 cm−1. 1H NMR (CD3OD, 600 MHz) δ 8.66 (d, 4J 1.8 Hz, 1H, 2-H), 8.15 (dd, 4J 1.8 Hz, 3J 8.4 Hz, 1H, 6-H), 7.13 (d, 3J 8.4 Hz, 1H, 5′-H). 13C NMR (CD3OD, 150 MHz) δ 169.8 (COOH), 158.0 (Cq-4), 137.9 (CH-6), 135.5 (Cq-3), 127.8 (CH-2), 126.9 (Cq-1), 120.8 (CH-5). EI-MS (70 eV) m/z (%) 183 ([M]•+, 100), 167 (12), 166 (18), 153 (16), 119 (22), 81 (23), 63 (61), 53 (28). (−)-APCI MS m/z 182 [M–H]–.

4′-Hydroxy-3′-nitrophenylacetic acid (3)

Yellow solid, UV absorbing (254 nm), RF 0.39 (CH2Cl2/MeOH 9:1), Rt 11.3 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 274 (3.84), 354 (3.52) nm. IR (KBr) νmax 3432, 2930, 1698, 1632, 1581, 1493, 1434, 1412, 1331, 1258, 1216, 1180, 1132, 1084, 922, 848, 764 cm−1. 1H NMR (CD3OD, 300 MHz) δ 8.01 (d, 4J 2.1 Hz, 1H, 2-H), 7.54 (dd, 3J 8.5 Hz, 4J 2.1 Hz, 1H, 6-H), 7.10 (d, 3J 8.5 Hz, 1H, 5′-H), 3.63 (s, 2H, CH2COO). EI MS (70 eV) m/z (%) 198 (38), 197 ([M]•+, 23), 152 (100), 106 (20), 77 (19), 51 (18).

4′-Hydroxy-3′-nitrophenylacetic acid methyl ester (4)

Yellow solid, UV absorbing (254 nm), RF 0.76 (CH2Cl2/MeOH 9:1), Rt 13.6 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 273 (4.00), 354 (3.68) nm. IR (KBr) νmax 3480, 3280, 1736, 1634, 1581, 1537, 1486, 1430, 1420, 1356, 1330, 1307, 1262, 1172, 1083, 1002, 918, 926 cm−1. – 1H NMR (CD3OD, 300 MHz) δ 7.99 (d, 4J 2.4 Hz, 1H, 2-H), 7.52 (dd, 3J 8.7 Hz, 4J 2.4 Hz, 1H, 6-H), 7.09 (d, 3J 8.7 Hz, 1H, 5′-H), 3.69 (s, 3H, COOMe), 3.67 (s, 2H, CH2). EI MS (70 eV) m/z (%) 211 ([M]•+, 16), 165 (11), 152 (100), 135 (12), 106 (44), 77 (36), 59 (23), 51 (35).

4′-Hydroxy-3′-nitrophenylpropionic acid (5)

Yellow solid, UV absorbing (254 nm), RF 0.82 (CH2Cl2/MeOH 9:1), Rt 11.4 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 274 (3.65), 356 (3.31) nm. IR (KBr) νmax 3404, 2922, 1714, 1630, 1581, 1539, 1483, 1430, 1404, 1327, 1244. 1180, 1081, 907, 850, 762, 661, 600 cm−1. 1H NMR (CDCl3, 300 MHz) δ 10.46 (s, 1H, 4′-OH), 7.93 (d, 4J 2.1 Hz, 1H, 2-H), 7.43 (dd, 3J 8.4 Hz, 4J 2.1 Hz, 1H, 6-H), 7.08 (d, 3J 8.4 Hz, 1H, 5′-H), 2.93 (t, 3J 7.0 Hz, 2H, CH2CH2COO), 2.67 (t, 3J 7.0 Hz, 2H, CH2CH2COO); (MeOH-d4, 600 MHz) δ 7.96 (d, J 2.1 Hz, 1H, 2-H), 7.52 (dd, J 8.4, 2.1 Hz, 1H, 6-H), 7.08 (d, J 8.4 Hz, 1H, 5′-H), 2.90 (t, J 7.0 Hz, 2H, CH2CH2COO), 2.59 (t, J 7.0 Hz, 2H, CH2CH2COO). EI MS (70 eV) m/z (%) 211 ([M]•+, 32), 193 (46), 175 (20), 152 (100), 151 (56), 147 (38), 106 (15).

2-Hydroxy-3-(4′-hydroxy-3′-nitrophenyl)-propionic acid methyl ester (6)

Yellow solid, RF 0.59 (CH2Cl2/MeOH 9:1), Rt 9.14 min (LC-MS). UV/vis (MeOH) λmax nm (lg ɛ) 274 (3.45), 354 (3.07). IR (KBr) νmax 3433, 2925, 2853, 1740, 1630, 1538, 1489, 1431, 1384, 1329, 1253, 1181, 1098, 824, 765, 678 cm−1. 1H NMR (MeOH-d4, 600 MHz) δ 7.96 (d, 4J 2.1 Hz, 1H, 2-H), 7.50 (dd, 3J 8.4 Hz, 4J 2.1 Hz, 1H, 6-H), 7.08 (d, 3J 8.4 Hz, 1H, 5′-H), 4.38 (ABX, 1H, 2-H), 3.70 (s, 3H, 1-OCH3), 3.06, 2.94 (ABX, JAB 13, JAX 8.4, JBX 4.2 Hz, 2H, 3-H2); see Supplementary Figure 8. EI (70 eV) m/z (%) 241 ([M]•+, 8), 223 (24), 192 (3), 152 (100), 135 (11), 106 (20), 77 (21). (−)-APCI MS m/z 240 [M-H]–. (−)-ESI-HRMS m/z 240.05147 ([M-H]–, calcd. 240.05135 for C10H10NO6).

2-Chloro-3-(4′-hydroxy-3′-nitrophenyl)propionic acid methyl ester (7)

Yellow solid, UV absorbing (254 nm), Rt 10.54 min (LC-MS). –UV/vis (MeOH) λmax (lg ɛ) 270 (3.75), 348 (3.42) nm. IR (KBr) νmax 3423, 2926, 1746, 1631, 1540, 1491, 1436, 1328, 1256, 1177, 824, 765 cm−1. 1H NMR (MeOH-d4, 600 MHz) δ 7.96 (d, 4J 2.1 Hz, 1H, 2-H), 7.50 (dd, 3J 8.4 Hz, 4J 2.1 Hz, 1H, 6-H), 7.08 (d, 3J 8.4 Hz, 1H, 5′-H), 4.63 (t, 1H, 3J 7 Hz, 2-H), 3.73 (s, 3H, 1-OCH3), 3.75, 3.10 (2 m, 2H, 3-H2). – EI (70 eV) m/z (%) 259, 261 ([M]•+, 6, 2), 223 (100), 192 (50), 152 (87), 106 (19), 77 (17). (−)-APCI MS m/z 258, 260 [M-H]–. (−)-ESI-HRMS m/z 258.01767 ([M+-H]–, calcd. 258.01747 for C10H9ClNO5).

3′-Nitrotyrosol (8)

Yellow solid, UV absorbing (254 nm), RF 0.59 (CH2Cl2/MeOH 9:1), Rt 13.2 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 275 (3.93), 358 (3.59) nm. IR (KBr) νmax 3300, 2929, 2847, 1632, 1586, 1540, 1488, 1427, 1326, 1253, 1179, 1058, 1026, 904, 836, 764 cm−1. 1H NMR (CDCl3, 300 MHz) δ 10.46 (s, 1H, 4′-OH), 7.96 (d, 4J 2.2 Hz, 1H, 2-H), 7.46 (dd, 3J 8.5 Hz, 4J 2.2 Hz, 1H, 6-H), 7.08 (d, 3J 8.5 Hz, 1H, 5′-H), 3.86 (t, 3J 6.5 Hz, 2H, CH2CH2OH), 2.84 (t, 3J 6.5 Hz, 2H, CH2CH2OH). EI MS (70 eV) m/z (%) 183 ([M]•+, 35), 152 (100), 135 (39), 106 (31), 77 (20), 51 (9).

N-Acetyl-3′-nitrotyramine (9)

Yellow solid, UV absorbing (254 nm), RF 0.51 (CH2Cl2/MeOH 9:1), Rt 11.1 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 275 (3.86), 357 (3.56) nm. IR (KBr) νmax 3400, 3290, 3079, 2934, 1634, 1558, 1532, 1489, 1424, 1321, 1291, 1257, 1171, 850, 766 cm−1. 1H NMR (CDCl3, 300 MHz) δ 10.44 (s, H/D exchangeable, 1H, 4′-OH), 7.89 (d, 4J 2.1 Hz, 1H, 2-H), 7.41 (dd, 3J 8.4 Hz, 4J 2.1 Hz, 1H, 5′-H), 7.08 (d, 3J 8.4 Hz, 1H, 6-H), 5.72 (br s, H/D exchangeable, 1H, NH), 3.46 (q, 3J 6.9 Hz, 2H, CH2CH2NH), 2.79 (t, 3J 6.9 Hz, 2H, CH2CH2NH), 1.93 (s, 3H, Ac). EI MS (70 eV) m/z (%) 206 ([M]•+, 60), 165 (100), 135 (23), 105 (19), 77 (15), 72 (18), 60 (17), 43 (41).

2-Nitro-4-(2′-nitroethenyl)phenol (10)

Yellow solid, UV absorbing (254 nm), RF 0.83 (CH2Cl2/MeOH 9:1), Rt 13.7 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 222 (4.20), 269 (4.04), 325 (4.20), 435 (sh) (3.54) nm. IR (KBr) νmax 3436, 3110, 2927, 1640, 1624, 1537, 1513, 1504, 1489, 1342, 1275, 1174, 972, 836, 766 cm−1. 1H NMR (CD3OD, 600 MHz) δ 8.36 (d, 4J 2.4 Hz, 1H, 2-H), 8.04 (d, 3J 13.5 Hz, 1H, CHNO2), 7.86 (d, 3J 13.5 Hz, 1H, CHCHNO2), 7.85 (dd, 4J 2.4 Hz, 3J 8.7 Hz, 1H, 6-H), 7.09 (d, 3J 8.7 Hz, 1H, 5′-H). 13C NMR (CD3OD, 150 MHz) δ 160.8 (Cq-4), 145.8 (Cq-3), 138.4 (CHCHNO2), 138.0 (CHCHNO2), 136.5 (CH-6), 129.0 (CH-2), 122.5 (CH-5), 121.8 (Cq-1); see Supplementary Figure 9. EI MS (70 eV) m/z (%) 210 ([M]•+, 24), 163 (100), 118 (38), 89 (70), 63 (58).

4′-Hydroxy-3′,5′-dinitrophenylacetic acid (11)

Yellow solid, RF 0.55 (CH2Cl2/MeOH 9:1), Rt 12.8 min (LC-MS). UV/vis (MeOH/HCl) λmax (lg ɛ) 338, (3.97), 350 (3.65) nm. IR (KBr) νmax 3433, 1696, 1641, 1580, 1544, 1431, 1401, 1352, 1305, 1261, 1155, 910, 729, 609 cm−1. 1H NMR (MeOH-d4, 600 MHz) δ 8.22 (s, 2H, 2′,6′-H), 3.72 (s, 2H, 2-CH2). EI-MS (70 eV) m/z (%) 242 ([M]•+, 65), 197 (100), 151 (20), 105 (16), 76 (16). (−)-APCI m/z 241 [M-H]–. (−)-ESI HRMS m/z 241.01027 ([M-H]–, calcd. 241.00997 for C8H5N2O7).

4′-Hydroxy-3′,5′-dinitrophenylacetic acid methyl ester (12)

Yellow solid, UV absorbing (254 nm), RF 0.36 (CH2Cl2/MeOH 9:1), Rt 12.8 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 228 (4.33), 432 (3.85) nm. IR (KBr) νmax 3437, 2925, 1738, 1631, 1553, 1439, 1406, 1384, 1338, 1265, 1238, 1198, 1177, 1116, 900, 784 cm−1. 1H NMR (CD3OD, 600 MHz) δ 7.96 (s, 2H, 2,6-H), 3.70 (s, 3H, COOCH3), 3.60 (s, 2H, CH2COO). 13C NMR (CD3OD, 150 MHz) δ 173.7 (COO), 159.4 (Cq-4), 143.9 (Cq-3,5), 132.4 (CH-1), 114.6 (CH-2,6), 52.6 (CH2COO), 39.6 (COOCH3). EI MS (70 eV) m/z (%) 256 ([M]•+, 14), 226 (13), 197 (12), 59 (16), 44 (100). EI HRMS m/z 256.03330 ([M]•+, calcd. 256.03315 for C9H8N2O7); (+)-ESI-HRMS m/z 279.02252 ([M+Na]+, calcd. 279.02238 for C9H8N2O7Na).

4′-Hydroxy-3′,5′-dinitrophenylpropionic acid (13)

Yellow solid, UV absorbing (254 nm), Rf 0.16 (CH2Cl2/MeOH 9:1), Rt 11.9 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 356 (3.16), 436 (3.39) nm. IR (KBr) νmax 3412, 1712, 1638, 1542, 1428, 1343, 1262 cm−1. 1H NMR (CD3OD, 600 MHz) δ 8.04 (s, 2H, 2,6-H), 2.89 (t, 3J 7.0 Hz, 2H, CH2CH2COO), 2.62 (t, 3J 7.0 Hz, 2H, CH2CH2COO). 13C NMR (CD3OD, 150 MHz) δ 176.2 (COOH), 152.7 (Cq-4), 141.7 (Cq-3,5), 131.4 (CH-2,6), 128.4 (Cq-1), 36.1 (CH2CH2COO), 30.3 (CH2CH2COO). (−)-ESI MS m/z (%) 533 [2M-2H+Na]– (29), 255 [M-H]– (100). EI-HRMS m/z 256.03320 ([M]•+, calcd. 256.03261 for C9H8N2O7).

3-(4′-Hydroxy-3′,5′-dinitrophenyl)-propionic acid methyl ester (14)

Yellow solid, RF 0.62 (CH2Cl2/MeOH 9:1). EI MS (70 eV) m/z (%) 270 ([M]•+, 20), 260 (45), 210 (100), 187 (10), 180 (10). (−)-ESI HRMS m/z 269.04160 ([M-H]–, calcd. 269.04097 for C10H9N2O7).

Dinitrotyrosol (15)

Yellow solid, RF 0.55 (CH2Cl2/MeOH 9:1). Rt 10.34 min (LC-MS). UV/vis (MeOH/HCl) λmax (lg ɛ) 248 sh (3.80), 351 (3.51) nm. IR (KBr) νmax 3395, 2945, 2834, 1639, 1545, 1384, 1029, 618 cm−1. 1H NMR (CD3OD, 600 MHz) δ 8.14 (s, 2H, 2′,6′-H), 3.78 (t, 2H, 3J 6.8 Hz, 2-H2), 2.82 (t, 3J 6.8 Hz, 2H, 2-H2). EI MS (70 eV) m/z (%) 228 ([M]•+, 32), 197 (28), 180 (100), 151 (28), 105 (12). (−)-APCI MS m/z 227 [M-H]–. (−)-ESI HRMS m/z 227.03102 ([M-H]–, calcd. 227.03095 for C8H7N2O6).

4′-Hydroxy-3′,5′-dinitrophenylethylchloride (16)

Yellow solid, UV absorbing (254 nm), RF 0.44 (CH2Cl2/MeOH 9:1), Rt 15.6 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 231 (4.16), 435 (3.68) nm; λmax 350 nm in acidic solution. IR (KBr) νmax 3407, 2926, 1719, 1638, 1543, 1459, 1338, 1246, 1111, 10455, 914, 783 cm−1. 1H NMR (CD3OD, 600 MHz) δ 7.86 (s, 2H, 2,6-H), 3.70 (t, 3J 7.2 Hz, 2H, CH2CH2Cl), 2.93 (t, 3J 7.2 Hz, 2H, CH2CH2Cl). 13C NMR (CD3OD, 150 MHz) δ 159.6 (Cq-4), 144.2 (Cq-3,5), 132.2 (CH-2,6), 119.1 (Cq-1), 46.0 (CH2CH2Cl), 38.2 (CH2CH2Cl). EI MS (70 eV) m/z (%) 246 ([M]•+, 16), 197 (100), 151 (16), 91 (98). EI HRMS m/z 246.00430 ([M]•+, calcd. 246.00381 for C8H7N2O5Cl).

(4′-Hydroxy-3′,5′-dinitrophenyl)-2-chloropropionic acid methyl ester (17)

Orange solid, UV absorbing (254 nm), RF 0.41 (CH2Cl2/MeOH 9:1), Rt 17.0 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 348 (2.89) nm. IR (KBr) νmax 3427, 2925, 2854, 1740, 1621, 1545, 1399, 1258, 1098, 701 cm−1. 1H NMR (CD3OD, 600 MHz) δ 7.82 (s, 2H, 2′,6′-H), 4.58 (t, 1H, 3J 8.9 Hz, 2-H), 3.73 (s, 3H, 1-OCH3), 3.18, 3.06 (ABX, 2J 15.1 Hz, JAX, JBX 8.9 Hz, 3-H2). (−)-APCI MS m/z 303 [M-H]–. (−)-ESI-HRMS m/z 303.00254 ([M-H]–, calcd. 303.00254 for C10H8ClN2O7).

N-Acetyl-3′,5′-dinitrotyramine (18)

Yellow solid, UV absorbing (254 nm), RF 0.19 (CH2Cl2/MeOH 9:1), Rt 10.3 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 225 (3.80), 443 (3.21) nm. IR (KBr) νmax 3421, 2928, 1637, 1543, 1460, 1384, 1362, 1338, 1310, 1253, 1205, 1027, 1002, 910, 785 cm−1. – 1H NMR (DMSO-d6, 300 MHz) δ 7.82 (t br, 1H, NH), 7.64 (s, 2H, 2,6-H), 3.17 (m, 2H, CH2CH2NH), 2.48 (m, 2H, CH2CH2NH), 1.77 (s, 3H, Ac). 13C NMR (DMSO-d6, 150 MHz) δ 169.0 (CH3CO), 158.5 (Cq-4), 142.6 (Cq-3,5), 130.7 (CH-2,6), 114.4 (Cq-1), 40.0 (CH2CH2NH), 33.2 (CH2CH2NH), 22.5 (CH3CO). (−)-ESI-MS m/z (%) 537 [2M-H]– (45), 268 [M-H]– (100). EI HRMS m/z 269.06480 ([M], calcd. 269.06425 for C10H11N3O6); (+)-ESI HRMS m/z 270.07206 ([M+H]+, calcd. 270.07206 for C10H12N3O6).

2,6-Dinitro-4-(2′-nitroethenyl)phenol (19)

Orange solid, UV absorbing (254 nm), RF 0.53 (CH2Cl2/MeOH 9:1). 1H NMR (CD3OD, 600 MHz) δ 8.24 (s, 2H, 3-H, 5′-H), 7.99 (d, 3J 13.5 Hz, 1H, 2′-H), 7.76 (d, 3J 13.5 Hz, 1H, 1′-H). (−)-ESI MS m/z 531 [2M-2H+Na]–, 254 [M-H]–; (−)-ESI HRMS m/z 254.00545 ([M-H]–, calcd. 254.00546 for C8H4N3O7).

2-Methoxy-4,6-dinitrophenol (20)

Orange solid, UV absorbing (254 nm), RF 0.50 (CH2Cl2/MeOH 9:1), Rt 14.1 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 268 (3.78), 377 (3.89), 420 sh (3.75) nm. IR (KBr) νmax 3440, 1608, 1554, 1493, 1385, 1345, 1235, 1094, 1049, 798, 785, 735, 711 cm−1. 1H NMR (CD3OD, 300 MHz) δ 8.51 (d, 4J 2.7 Hz, 1H, 5-H), 7.75 (d, 4J 2.7 Hz, 1H, 3-H), 3.94 (s, 3H, 2-OCH3). 13C NMR (CD3OD, 150 MHz) δ 157.8 (Cq-1), 153.7 (Cq-2), 135.5 (Cq-6), 134.2 (Cq-4), 116.4 (CH-5), 108.3 (CH-3), 57.1 (2-OCH3). EI MS (70 eV) m/z (%) 214 ([M]•+, 100), 197 (70), 166 (22), 121 (28), 53 (29), 50 (33).

Compound 20 by synthesis according to Ehrlich and Bogert14: 1H NMR (CD3OD, 300 MHz) δ 8.42 (d, 4J 2.8 Hz, 1H, 5-H), 7.97 (d, 4J 2.8 Hz, 1H, 3-H), 4.03 (s, 3H, 2-OCH3); (acetone-d6) δ 8.53 (d, 4J 2.8 Hz, 1H, 5-H), 7.73 (d, 4J 2.8 Hz, 1H, 3-H), 4.02 (s, 3H, 2-OCH3); 13C NMR (CD3OD, 75 MHz) δ 151.4 (Cq-1), 150.3 (Cq-2), 139.9 (Cq-6), 136.4 (Cq-4), 114.0 (CH-5), 110.4 (CH-3), 57.7 (2-OCH3); (CDCl3) δ 150.8 (Cq-1), 150.5 (Cq-2), 139.2 (Cq-6), 132.4 (Cq-4), 112.6 (CH-5), 111.0 (CH-3), 57.0 (2-OCH3); (acetone-d6) δ 157.4 (Cq-1), 153.0 (Cq-2), 139.2 (Cq-6), 132.4 (Cq-4), 112.6 (CH-5), 111.0 (CH-3), 57.3 (2-OCH3).

3-Methoxy-4,5-dinitrophenol (21)

Orange solid, UV absorbing (254 nm), RF 0.51 (CH2Cl2/MeOH 9:1), Rt 14.1 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 213 (4.22), 266 (3.92), 342 (3.68) nm. IR (KBr) νmax 3391, 3109, 2946, 2835, 1778, 1607, 1556, 1449, 1344, 1261, 1092, 1027, 946, 918, 804, 711 cm−1. 1H NMR (CD3OD, 600 MHz) δ 8.59 (d, 4J 2.4 Hz, 1H, 6-H), 7.55 (d, 4J 2.4 Hz, 1H, 2-H), 3.87 (s, 3H, 3-OCH3). 13C NMR (CD3OD, 150 MHz) δ 164.6 (Cq-1), 155.6 (Cq-3), 135.2 (Cq-5), 132.2 (Cq-4), 118.3 (CH-6), 106.7 (CH-2), 57.0 (3-OCH3). (−)-APCI MS m/z 213 [M-H]–. (−)-ESI HRMS m/z 213.01535 ([M-H]–, calcd. 213.01529 for C7H5N2O6).

2-Methoxy-3,5-dinitrophenol

Nitration of guaiacol acetate (2 g) according to Hynning et al.15 resulted in a mixture of 3- and 5-nitroguaiacol acetate of similar polarity. After drying, the orange resin was dissolved at 0 °C in 5 ml fuming nitric acid and the product precipitated after 5 min by the addition of water. The dinitro acetate was hydrolyzed by dissolving in 2N NaOH. After 5 min at room temperature, the orange-red solution was acidified with diluted HCl and extracted with ether. Some polar impurities were separated by filtration over silica gel (column 4 × 10 cm, CH2Cl2). 1H NMR (CD3OD, 300 MHz) δ 8.08 (d, 4J 2.7 Hz, 1H, 4-H), 7.88 (d, 4J 2.7 Hz, 1H, 6-H), 4.02 (s, 3H, 2-OCH3). 13C NMR (CD3OD, 75 MHz) δ 153.7 (Cq-1), 147.6 (Cq-2), 145.2 (Cq-3), 143.7 (Cq-5), 115.3 (CH-6), 111.3 (CH-4), 62.4 (2-OCH3); assignment on the basis of 2D spectra.

2-Methoxy-4,5-dinitrophenol

2-Methoxy-4,5-dinitrophenol was obtained by nitration of veratrole and ether cleavage, according to Ehrlich and Bogert14. –1H NMR (CD3OD, 300 MHz) δ 7.80 (s, 2H, 3, 6-H), 6.96 (s, 3H, 2-OCH3). –13C NMR (CD3OD, 125 MHz) δ 152.8 (Cq-1), 149.3 (Cq-2), 141.9 (Cq-5), 132.9 (Cq-4), 107.7 (CH-3,6), 57.6 (2-OCH3).

3′-Nitro-daidzein (22)

Yellow solid, UV absorbing 254 nm). RF 0.59 (CH2Cl2/MeOH 9:1). Rt 16.0 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 243 (3.99), 300 (sh, 3.67), 359 (3.22) nm. IR (KBr) νmax 3429, 3281, 1620, 1588, 1578, 1537, 1426, 1385, 1310, 1266, 1240, 1179, 1101, 864, 766 cm−1. 1H NMR (DMSO-d6, 300 MHz) δ 10.90 (s br, 1H, OH), 8.45 (s, 1H, 2-H), 8.15 (d, 4J 2.1 Hz, 1H, 2′-H), 7.97 (d, 3J 8.8 Hz, 1H, 5′-H), 7.71 (dd, 3J 8.8 Hz, 4J 2.1 Hz, 1H, 6′-H), 7.12 (d, 3J 8.8 Hz, 1H, 5′-H), 6.94 (dd, 3J 8.8 Hz, 4J 2.1 Hz, 1H, 6-H), 6.88 (d, 4J 2.1 Hz, 1H, 8-H). 13C NMR (DMSO-d6, 150 MHz) δ 174.3 (Cq-4), 162.8 (Cq-7), 157.4 (Cq-8a), 153.7 (CH-2), 153.0 (Cq-4′), 136.5 (Cq-3′), 135.3 (CH-6′), 127.2 (CH-5), 125.2 (CH-2′), 122.0 (Cq-1′), 121.4 (Cq-3), 119.6 (CH-5′), 116.4 (Cq-4a), 115.3 (CH-6), 102.1 (CH-8). (+)-ESI MS m/z (%) 621 [2M+Na]+ (78), 300 [M+H]+ (18). (−)-ESI MS m/z (%) 597 [2M-H]– (40), 298 [M-H]– (100). DCI MS (NH3) m/z (%) 456 [2M+NH4]+ (14), 237 [M+NH4]+ (100). (+)-ESI HRMS m/z 300.05020 ([M+H]+, calcd. 300.05027 for C15H10NO6).

3′-Nitrogenistein (23)

Yellow solid, RF 0.55 (CH2Cl2/MeOH 9:1), Rt 12.49 min (LC-MS). UV/vis (MeOH) λmax (lg ɛ) 265 nm. IR (KBr) νmax 3432, 2963, 2926, 1628, 1537, 1382, 1261, 1092, 1030, 803 cm−1. 1H NMR (MeOH-d4, 600 MHz) δ 8.20 (s, 1H, 2-H), 8.15 (d, J 2.1 Hz, 1H, 6′-H), 7.60 (dd, J 8.8, 1.9 Hz, 1H, 2′-H), 7.01 (d, J 8.8 Hz, 1H, 3′-H), 6.28 (d, J 2.1 Hz, 1H, 8-H), 6.18 (d, J 2.1 Hz, 1H, 6-H); see Supplementary Figure 10. (−)-ESI MS m/z (%) 314.3 [M-H]– (100). (−)-ESI MS/MS (45 eV) m/z (%) 314 [M-H]– (45), 297 (100). (−)-ESI MS/MS (35 eV) m/z (%) 297 (100), 280 (90), 267 (80). (+)-APCI MS m/z 316 [M+H]+. (−)-APCI MS m/z 314 [M-H]−.

3′,5′-Dinitro-genistein (24)

Orange solid, UV absorbing (254 nm), RF 0.21 (CH2Cl2/MeOH 9:1), Rt 12.46 min (LC-MS). –UV/vis (MeOH) λmax (lg ɛ) 268 (4.12), 434 (3.29) nm. IR (KBr) νmax 3410, 1632, 1552, 1503, 1346, 1258, 1108, 1028, 1006, 822, 557, 450 cm−1. 1H NMR (DMSO-d6, 300 MHz) δ 12.78 (br, 1H, OH), 8.27 (s, 1H, 2-H), 8.03 (s, 2H, 2′,6′-H), 6.15 (d, 4J 2.1 Hz, 1H, 8-H), 6.03 (d, 4J 2.1 Hz, 1H, 6-H). 13C NMR (DMSO-d6, 150 MHz) δ 178.9 (Cq-4), 161.6 (Cq-5), 159.0 (Cq-4′), 157.9 (Cq-8a), 152.6 (CH-2), 142.9 (Cq-3′,5′), 130.4 (CH-2′,6′), 119.9 (Cq-1′), 105.7 (Cq-3), 102.3 (Cq-4a), 100.2 (CH-6), 94.3 (CH-8). (−)-APCI MS m/z (%) 359 [M-H]– (100). (+)-ESI HRMS m/z 361.03046 ([M+H]+, calcd. 361.03027 for C15H9N2O9); (−)-ESI HRMS m/z 359.01580 ([M-H]–, calcd. 359.01569 for C15H7N2O9).

Pyriculamide (25)

Yellow solid, UV absorbing (254 nm), RF 0.45 (CH2Cl2/MeOH 9:1), RT 9.5 min (LC-MS). UV/vis (MeOH) λmax 273, 354 nm. IR (KBr) νmax 3439, 3191, 3081, 2930, 2862, 1655, 1623, 1524, 1472, 1445, 1356, 1335, 1280 cm−1. 1H NMR (DMSO-d6, 300 MHz) δ 10.7 (s br, 1H, OH), 8.14 (d, 3J 4.0 Hz, 1H, NH), 7.70 (d, 4J 2.3 Hz, 1H, 5′-H), 7.34 (dd, 3J 8.4 Hz, 4J 2.3 Hz, 1H, 9-H), 7.08 (d, 3J 8.4 Hz, 1H, 8-H), 3.94 (m, 1H, 2-H), 3.48 (m, 1H, 2′-H), 3.46 (m, 1H, 3-HA), 3.27 (m, 1H, 3-HB), 3.03, 2.96 (ABX, 2J 13, 3J 7 Hz, 2H, 5′-H2), 2.06 (m, 1H, 3′-HA), 1.83 (m, 1H, 4′-HA), 1.68 (m, 2H, 3′-HA, 4′-HB). 13C NMR (DMSO-d6, 150 MHz) δ 168.2 (Cq-1), 164.5 (Cq-1′), 151.1 (Cq-7), 136.6 (CH-5), 136.2 (Cq-6), 127.4 (Cq-4), 125.7 (CH-9), 119.1 (CH-8), 57.8 (CH-2), 57.3 (CH-2′), 44.7 (CH2-3), 37.7 (CH2-5′), 28.5 (CH2-3′), 21.4 (CH2-4′). (+)-ESI MS m/z (%) 655 [2M-H+2Na]+ (100), 633 [2M+Na]+ (25). (−)-ESI MS m/z (%) 631 [2M-2H+Na]– (100), 304 [M-H]– (55).

2,6-Dimethoxy-1,4-benzoquinone (26)

Yellow solid, 1H NMR (CDCl3, 300 MHz) δ 5.84 (s, 2H, 3′,5′-H), 3.80 (s, 6H, 2 OCH3). 13C NMR (CDCl3, 150 MHz) δ 186.8 (Cq-1), 176.7 (Cq-4), 157.3 (Cq-2,6), 107.4 (CH-3,5), 56.5 (2 OCH3). EI MS (70 eV) m/z (%) 168 ([M]•+, 5), 104 (39), 91 (21), 80 (20), 69 (100), 53 (29).

3′-Indolylethyl-methyl ether (27)

Colorless oil, UV absorbing at 254 nm, RF 0.80 (CH2Cl2/MeOH 9:1), with anisaldehyde/sulfuric acid red, with Ehrlich's reagent violet. Rt 15.7 min (LC-MS). UV/vis (MeOH) λmax 221, 281 nm. IR (KBr) νmax 3415, 2926, 1620, 1457, 1384, 1339, 1228, 1107, 742 cm−1. 1H NMR (CD3OD, 600 MHz) δ 7.51 (d, 3J 7.8 Hz, 1H, 7′-H), 7.31 (d, 3J 7.8 Hz, 1H, 4′-H), 7.06 (t, 3J 7.8 Hz, 1H, 6′-H), 7.05 (s, 1H, 2′-H), 6.98 (t, 3J 7.8 Hz, 1H, 5′-H), 3.66 (t, 3J 7.2 Hz, 2H, 1-H), 3.37 (s, 3H, OCH3), 2.99 (t, 3J 7.2 Hz, 2H, 2-H). EI MS (70 eV) m/z (%) 175 ([M]•+, 20), 130 (100), 103 (7), 77 (10), 45 (12). EI HRMS m/z 175.0997 ([M]•+, calcd. 175.09917 for C11H13NO).