The Strain T436 was isolated from the Arctic ice in the Eastern Weddell Sea.
Our attention in this strain was evoked by its high antibacterial and antifungal activ-ity and the huge metabolic potency. Due to its characteristic and its capacactiv-ity to pro-duce a wide number of nitro compounds, [56] we re-fermented it now. The strain T436 grew after two days from plating on M1 and showed in the first 3-5 days circu-lar, beige colonies with shiny smooth surfaces. The taxonomy was identified on the basis of 16 S-RNA as Salegentibacter holothuriorum.
The crude extract showed in the TLC (CH2Cl2/MeOH 9:1) a series of yellow zones in polare as well as in the apolare range, which showed no colour reaction which anisaldehyde. Several new nitro and chloro derivates were isolated now, which were different from those isolated in the first fermentation, [56] and their struc-tures were established on the basis of NMR data, MS data, and other spectroscopic method and by comparison with known and the previously isolated compounds.
In the first fermentation, 24 aromatic nitro compounds and nitroindoles were isolated: [56,73] Pyriculamide [nitro-cyclo (tyrosylprolyl)] (32), 3’-nitro-daidzein (33) and 3’,5’-dinitro-genistein (34), 2,6-dinitro-4- (2’-nitroethenyl)phenol (35), N- (4-hydroxy-3-nitrophenylethyl)acetamide (36a), N-(hydroxyphenylethyl)-acetamide (36b), hydroxyphenyl-propionic acid (37a), 3,5-dinitro-4-hydroxyphenyl-2-chloro-propionic acid methyl ester (37b), 3,5-dinitro-hydroxyphenyl-ethylchloride (38a), 2-(hydroxy-3-nitrophenyl)ethanol (38b), 4-hydroxy-3-nitrophenyl-acetic acid (39a), 3,5-dinitro-4-hydroxyphenyl-acetic acid methyl ester (39b), hydroxy-3-nitrophenyl-acetic acid methyl ester (39c), 4-hydroxy-3-nitrophenyl-propionic acid (40), 2-nitro-N -acetyltryptamin (41a), 6-nitro-N -acetyl tryptamine (41b) und 7-nitro-N -acetyltryptamin (41c); salegentipyr-rol A (42a), and salegentipyrsalegentipyr-rol B (42b).
Salegentibacter holothuriorum T436 43
The second fermentation produced similar compounds: 3-(4-hydroxy-3,5-dinitrophenyl)propionic acid (37b), 4-hydroxy-3-nitrophenyl-propionic acid (40) and some new nitro compounds 3-(4-hydroxy-3,5-dinitrophenyl)propionic acid methyl ester (37c), dinitro-tyrosol (38c), 4-hydroxy-3,5-nitrophenyl-acetic acid (39d), 3'-nitrogenistein (43), 2-hydroxy-3-(4-hydroxy-3-nitrophenyl)propionic acid methyl ester (45).
40
Figure 24: Work-up scheme of Salegentibacter holothuriorum T436
O2N
Salegentibacter holothuriorum T436 45
5.3.1 3´-Nitrogenistein
The yellow UV active solid 43 showed in the 1H NMR spectrum six signals in the aromatic range, which were attributed due to the coupling constants to a 1,2,4-trisubstituted aromatic ring, two protons in meta position at 6.18 and 6.28 and at the end one singlet at 8.15.
Figure 25 : 1H NMR spectrum (600 MHz) of 3-nitrogenistein (43) in MeOH-d4
Compound 43 showed by (-)-ESI MS a quasimolecular ion at m/z 314 [M-H]-. The search in AntiBase with the proton data and the molecular mass gave 3´-nitrogenistein (43). The fragmentation by LC/MS/MS/MS with a collision energy of 35 eV showed characteristic elimination of HNO2 and two hydroxy groups. The fragmentation at higher collision energy (45 eV) gave only one daughter ion with the maximum abundance at m/z 297 due to the loss of an OH group. By decreasing the collision energy (35 eV), the abundance of product ions increased [m/z 297 (100), 280 (90), 267 (70)] (Figure 26).
Figure 26: (-)-ESI LC/MS/MS spectrum of 3'-nitrogenistein (43)
The structure was confirmed by LC-ESI MS/MS by comparison with the pub-lished spectrum from the synthetic product [74].
It is reported that the loss of those 3 fragments (two OH and HNO2) is untypical for nitro compounds; a proposed fragmentation scheme for this special case is shown in Figure 27 [74,75]. from 3'-nitrogenistein (43) (m/z 314 for [M-H]-)
O
Nitrogenistein had been isolated previously from a genetically engineered Strep-tomyces sp. K3. [76] It showed no significant antimicrobial or phytotoxic activities.
Among the compounds isolated from this strain, it was however, the only one with a mild cytotoxicity against L1210 and the Jurkat cells.
Salegentibacter holothuriorum T436 47
5.3.2 3-(4-Hydroxy-3,5-dinitrophenyl)propionic acid methyl ester
The yellow compound 37c was obtained from fractions A2 and B3. The ESI MS spectrum gave a molecular mass of 270 Dalton and the high resolution delivered the molecule formula C10H10N2O7. The UV-spectrum shows the same maximum as compound 37b. The search in AntiBase gave 3-(4-hydroxy-3,5-dinitrophenyl)propionic acid (37b). Comparison with the spectra and mass led me to conclude that compound 37c was the methyl ester of compound 37b which was pre-viously isolated from the same strain. It was impossible to interpret the proton spec-trum, because the substance seemed to decompose with time. Due to the small amount of material (0.7 mg) there was no possibility to purify it further.
O 5.3.3 3,5-Dinitro-tyrosol
The orange compound 38c showed the same UV absorption as compounds 38a and 38b. It was isolated from fraction A3 and B2. Its 1H NMR spectrum was almost the same as that of compound 38a. Both methylene signals, however, were shifted downfield. ESI and HRESIMS gave a molecular mass of 228 Dalton and a molecular formula C8H7N2O6. Compound 38c is the new 3,5-dinitro-4-hydroxyphenyl-ethanol (dinitro-tyrosol). The chloro derivate 38a had been isolated previously from the same strain.
Due to its small amount it was not possible to measure the 2D NMR spectra for all compounds. The structures were elucidated by means of 1H NMR, APCI, EI, ESI and HRESIMS and finally comparison was made with the derivatives isolated previ-ously from the same strain.
5.3.4 4-Hydroxy-3, 5-dinitrophenylacetic acid
Compound 39d was isolated as orange solid from fraction A1 and showed yel-low fluorescence under UV at 366 nm; it gave no colour reaction with anisaldehyde.
The 1H NMR spectrum showed only two singlets, one at 8.21 for two symmetrical aromatic protons and the other one at 3.60 with the intensity of two protons, which belong to a methylene group.
Figure 28: 1H NMR spectrum (600 MHz) of compound 39d in MeOH-d4
From the (-)-ESI MS spectrum a quasimolecular ion at m/z 241 [M-H]- can be identified and the HR-ESI MS gave the molecular formula C8H6N2O7. The search in the database with the substructure produced many hits including the 4-hydroxy-3,5-dinitrophenyl-acetic acid methyl ester (39b), which was isolated from the same strain cultivated in another medium. [56] By comparison of their proton data, it could be observed that they differed only in one signal at 3.70 (attributed to a methylene group) and the absence of the methoxy signal. Comparison also of the UV spectra showed absorption at 432 nm for a dinitro-substituted phenyl ring. The search in An-tiBase with the proton NMR substructure and the molecular mass gave no hits. Inter-pretations of 1D NMR und MS spectra delivered the free acid corresponding to
com-Salegentibacter holothuriorum T436 49
pound 39b. Due to the small amount it was not possible to measure the 2D NMR spectra.
OH O O
H
NO2 O2N
1 3
5
39d
5.3.5 2-Hydroxy-3-(4-hydroxy-3-nitrophenyl)propionic acid methyl ester The orange compound 45 was isolated from fraction B2 and showed yellow fluorescence under UV at 366 nm. Its 1H NMR spectrum revealed in the aromatic region characteristic signals for a 1,2,4-trisubstituted aromatic ring at δ 7.98, 7.52 und 7.13, one methine multiplet at δ 4.38, which is supposed to be near an hetero atom, one methoxy group at δ 3.70 and in addition two doublets of doublets for a methylene group at δ 3.10 and 2.98 were visible.
Figure 29: 1H NMR spectrum (600 MHz) of compound 45 in MeOH-d4
The (-)-APCI mass spectra gave a quasimolecular ion at m/z 240. The search in AntiBase gave one hit, which was already described in the literature. [56] Comparison with the literature confirmed compound 45 to be 2-hydroxy-3-(4-hydroxy-3-nitrophenyl)propionic acid methyl ester.
O