The crude extract of the marine bacterium sp. Pic009 showed several bands dur-ing the screendur-ing by TLC, which gave violet-blue colouration with anisalde-hyde/sulphuric acid reagent and heating. Most of them exhibited UV absorption.
Moreover, the extract exhibited activity against Bacillus subtilis, Streptomyces viri-dochromogenes (Tü57), Escherichia coli and Mucor miehei (Tü284). The fermenta-tion was carried out using a 25-liter shaker culture at 28 °C for 4 days. After filtra-tion and extracfiltra-tion with ethyl acetate, the crude extract was fracfiltra-tionated by Sephadex LH-20 column chromatography employing elution with methanol to yield four frac-tions. Further purification of these fractions led to o-hydroxyphenyl acetic acid (177), isoxanthohumol (180), uridine (187), in addition to other known compounds (Figure 78).
Pic 009 (25 l shaker)
Mycelium Water phase
mixing with celite and filtered by filterpress
5 x with EtOAc 5 x with EtOAc
i.vac Crude extraxt
(2.91g)
Fraction I Fraction II Fraction III Fraction IV Sephadex LH-20 (CHCl3/MeOH 6:4),
Chrom. on Sephadex LH-20 (MeOH)
indolyl-3-acetic acid indoly-3-carboxylic acid
PRv HPLC (STM2) Sephadex LH-20
(CHCl3/MeOH 6:4), HPLC (STM2)
o-hydroxyphenylacetic acid isoxanthohumol
uridine
thymine acetyl-tyramine
HPLC (STM2)
Figure 78: Work-up procedure of strain Pic009
ourless solid was isolated by HPLC. The 1H NMR spectrum displayed four proton signals of an 1,2-disubstituted aromatic ring. In addition, one 2H singlet of a methyl-ene group was found at δ 3.60.
The EI mass spectrum delivered the molecular weight of 177 as 152 Dalton. The molecular ion exhibited two further fragments at m/z 134 and 106 due to elimination of H2O and expulsion of acetic acid, respectively.
A search in AntiBase has led to o-hydroxy phenyl acetic acid (177). Compound 177 was further confirmed by comparison with the literature[222]. o-Hydroxyphenyl acetic acid (177) was found as a fungal metabolite e.g. from Rhizoctonia legumini-cola and Ophiostoma crassivaginata[101] as well as plant metabolite e.g. from Astilbe sp.[24].
OH O OH
1 2 3' 1'
5'
177 4.10.2 Isoxanthohumol
Compound 180 was isolated as colourless solid by applying HPLC to fraction III. It exhibited an UV absorbing band, which showed, however, no colour by stain-ing with anisaldehyde/sulphuric acid. Compound 180 exhibited an UV fluorescent pale yellow band at 366 nm when exposed to ammonia. The 1H NMR spectrum of 180 displayed two phenolic hydroxyl groups at δ 9.38, and 8.51, two doublets each with intensity of 2H at δ 7.39 and 6.88 as an indication of an 1,4-disubstituted aro-matic ring. In addition, a deepfield 1H singlet at δ 6.26 could be due to an aroaro-matic proton between two electron-donating OH-groups. Furthermore, an oxymethine pro-ton gave a dd at δ 5.35 (J2,3a = 12.5, J2,3b = 3.5 Hz), which could be adjacent to an additional methylene group present as ABX between δ 2.93-2.61. A 1H triplet at δ 5.20 of an olefinic proton linked to a methylene group was observed as dd between δ 3.30-3.25. Furthermore a 3H singlet at δ 3.73 of a methoxy group were observed.
Finally, it showed two singlets of methyl groups at δ 1.61 and 1.57, which could be linked to an olefinic carbon. The partial structures derived thereof are given below (Figure 79).
O X H CH3 X
O
O CH3
CH3
Figure 79: Partial structures of isoxanthohumol (180)
Figure 80: 1H NMR spectrum ([D6]acetone, 500 MHz) of isoxanthohumol (180) Both positive and negative ESI mass spectra modes determined the molecular weight of compound 180 as 354 Dalton. The EI spectrum confirmed the molecular weight and gave some significant fragments (Figure 81), one of which at m/z 339 was due to the loss of a methyl group and one at m/z 299 was due to the expulsion of an isobutenyl group. Three other fragments (m/z 15, 43 and 55) showed the charac-teristic pattern of an C-prenylated compound[223]. High resolution of the molecular weight gave the corresponding molecular formula C21H22O5.
O
Figure 81: EI mass fragmentation pattern of isoxanthohumol (180)
Depending on the above revealed chromatographic, spectroscopic, molecular for-mula and UV properties of compound 180, four possible flavanone (178) moieties were taken into account, namely 7,4'-dihydroxy-5-methoxy-6-(γ,γ-dimethylallyl)-flavanone (179a), 7,4'-dihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-7,4'-dihydroxy-5-methoxy-6-(γ,γ-dimethylallyl)-flavanone (180), 4'-methoxy-6-(γ,γ-dimethylallyl)-flavanone (181) and 7,5-dihydroxy-4'-methoxy-8-(γ,γ-dimethylallyl)-flavanone (182).
The latter compounds 181 and 182 were excluded due to their chelated peri-hydroxy group (5-OH) which normally displays a characteristic singlet at δ ≥ 12 [224]. A further search in DNP and CA resulted in isoxanthohumol (180), which was con-firmed by direct comparison of 1H NMR data with the literature[225,226,*]. The 6-prenyl isomer 179a is not known as natural or synthetic product until now, although its analogue with a free hydroxy group at 5-position, 6-prenylnaringenin[225]
(sophoraflavanoneB, 179b)[224] is a natural product.
O
There are many 5,7-dihydroxyflavanones having 3,3-dimethylallyl (prenyl or (E)-3,7-dimethyl-2,6-octadienyl (geranyl) side chains at C-6 or C-8. In the NMR spectrum, the signals of 6-H and 8-H are very close together in 5,7-dihydroxyflavanone, and for example in naringenin (183), they are overlapping each other. However, they can be distinguished for instance by the Gibbs test[227], anoma-lous AlCl3 induced UV shift[228], cyclized reaction[229]. Most flavonoid compounds can be identified based on their UV characteristics as follows:
• The flavanones containing a free C5-OH in ring A show an UV absorbing deep purple spot with no change when exposed to NH3. However, when C5 -OH is blocked, a blue UV fluorescence is observed.
• The free 4'-OH group in ring B is affected by NH3 giving a yellow fluores-cent spot under UV, e.g. in naringenin (183). However, when it is blocked, no change by UV/NH3 is observed, e.g. in pincocembrin[230] (184).
• When the 5-OH is occupied and 4'-OH is free, the compound shows a blue UV fluorescence which changes to yellow when exposed to NH3, e.g. in sa-kuranin[231] (185). The latter case is the same as for 180.
thohumol (186), the principle of hop resin. Compound 180 was isolated recently from the roots of Sophora flavescens[226]. Xanthohumol (186) and its isomer isoxan-thohumol (180) are reported as anticarcinogenic, antifungal and antioxidant agents[232].
O
OH O
H
OH O
O O
H
OH O
183 184
O
O O glucosyl
MeO
OH OH
O H
OH O OMe
185 186
4.10.3 Uridine
Through further screening of fraction III by HPLC, compound 187 was isolated as a colourless solid, exhibiting an UV absorbing band which turned blue with anisaldehyde/sulphuric acid. The 1H NMR spectrum of compound 187 displayed the two, uracil doublet, signals, however, with a slightly higher shift, in addition to a 1H doublet at δ 5.78 of an anomeric proton. Furthermore, three oxymethine protons were observed, two at δ 4.08, and the third one at δ .83. Moreover, a multiplet of an oxy-methylene group was exhibited at δ 3.56, and two broad multiplets of 3 OH at δ 5.29 (OH) and 5.02 (2 OH) which disappeared by H/D exchange.
The molecular weight of 187 was confirmed as 244 Dalton by ESI and CI mass spectra. Comparison with authentic spectra and the literature[233,∗] afforded uridine (187), which is widely distributed in nature in free state or in nucleic acid and can be produced by hydrolysis thereof [24].
O
OH OH O
H
N N O
O H 4
1 5
1' 2'