5-(2-methylphenyl)-4-pentenoic acid from a terrestrial Streptomycete

Article abstract of DOI:10.1515/znb-2002-0312

From a terrestrial Streptomycete, GW 10/2517, the new 5-(2-methylphenyl)-4-pentenoic acid (1a) was isolated. The structure of 1a was proven by a detailed spectroscopic analysis and by synthesis.

Full text of DOI:10.1515/znb-2002-0312

5-(2-Methylphenyl)-4-pentenoic Acid from a Terrestrial Streptomycete
a
Venugopal J. R. V. Mukku , Rajendra P. Maskeya, Peter Moneckea,
Iris Grün-Wollnyb, and Hartmut Laatscha
a
Department of Organic Chemistry, University of Goettingen, Tammannstrasse 2,
D-37077 Goettingen, Germany
b
bioLeads, Waldhofer Str. 104, D-69123 Heidelberg, Germany
* Reprint requests to Prof. Dr. H. Laatsch. Fax: +49(0)551-399660. E-mail: hlaatsc@gwdg.de
Z. Naturforsch. 57b, 335Ð337 (2002); received November 12, 2001
Streptomycete, 5-(2-Methylphenyl)-4-pentenoic Acid
From a terrestrial Streptomycete, GW 10/2517, the new 5-(2-methylphenyl)-4-pentenoic
acid (1a) was isolated. The structure of 1a was proven by a detailed spectroscopic analysis
and by synthesis.
Introduction
ortho-Methyl phenyl alkenoic acids are periodi-
cally encountered in nature. Examples include the
bronchodilatatoric rubrenoic acids [1] from Al-
teromonas rubra, o-methylcinnamide [2] (U-
77863, which showed antitumor properties) from
Streptomyces griseoluteus, and serpentene [3] from
Streptomyces sp. Tü 3851. In our continuing search
for secondary metabolites of bacteria we have now
isolated a new member (1a) of this class of com-
pounds from a terrestrial Streptomycete GW 10/
2517. From the same Streptomycete, furan-2,5-di-
Fig. 1. Structures of 5-(2-methylphenyl)-4-pentenoic acid
methanol [4] was isolated along with 2-(4-hydroxy- (1a), its methyl ester 1b, and 5-(3-methylphenyl)-4-pen-
phenyl)-ethanol, 2Ј-deoxythymidine, and 2Ј-deox-
tenoic acid methyl ester (2).
yuridine. We report the taxonomy of the
producing strain, isolation, structure determina-
tion, and synthesis of compounds 1 and 2 (Fig. 1).
The 13C NMR spectrum showed 12 carbon sig-
nals, one of them in the carbonyl region (δ 179.2).
A free carboxylic acid was inferred based on the
fact that there were no ester carbonyl signals in
Results and Discussion
the 13C NMR. This was also confirmed by the for-
Compound 1a was obtained as a colourless solid mation of a methyl ester on treatment with diazo-
with the formula C12H14O2 based on the HR mass methane. This implied a 5-arylpentenoic acid
spectrum. The 1H NMR spectrum showed where the double bond is in the 4-position, based
multiplets (4 H) for an ortho- or meta-disubsti- on the chemical shift and splitting of its proton
tuted aromatic ring. In the aliphatic region it signals. This structure was confirmed by a H,H
showed a 3 H singlet (δ 2.32) indicating the pres- COSY spectrum. However, whether the methyl
ence of a methyl group attached to the aromatic group was in ortho or meta position of the aro-
ring, and a broad singlet (δ 2.58, 4 H) due to two matic ring remained uncertain due to strong signal
methylene groups. Additionally two olefinic pro- overlapping. Since HSQC and HMBC spectra and
ton signals were observed at δ 6.65 (d, J = 15.6 even spectral simulations did not allow us to dif-
Hz) and 6.08 (dm, J = 15.7 Hz) indicating an E- ferentiate, both isomers were synthesized using
disubstituted double bond.
standard procedures. Direct comparison con-
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D
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336
V. J. R. V. Mukkuet al. · 5-(2-Methylphenyl)-4-pentenoic Acid from a Terrestrial Streptomycete
menter containing 18 l of culture medium de-
scribed above. The medium was adjusted to
pH 7.0, incubation was carried out at 28 ∞C for 72
h with automatic addition of 2 N HCl or 2 N
NaOH to maintain the pH between 6.0Ð7.2, and
Niax PPG 2025 (Union Carbide, Belgium N. V.
Zwindrecht) to control foaming. Sterile air was
supplied (5 l/min) and agitation was at 120 rpm.
Active principles were extracted with ethyl acetate
from the filtered broth and the cell residue and
the extracts combined.
firmed the isolated compound to be 5-(2-methyl-
phenyl)-4-pentenoic acid (1a). Both acids, as well
as their methyl esters, were inactive against bacte-
ria and fungi in the usual agar diffusion tests.
The known compounds 3,5-furandimethanol, 4-
hydroxyphenyl ethanol, 2Ј-deoxythymidine, and
2Ј-deoxyuridine were isolated from the more polar
fractions of the Streptomycete GW 10/2517 and
identified by comparison with AntiBase [5] data.
After defatting with cyclohexane, the ethyl ace-
tate extract of the fermentation broth of strain
GW 10/2517 was chromatographed on silica gel
using a chloroform/methanol gradient (0 Ð 10%
methanol). The fractions were monitored by TLC
(UVabsorption and anisaldehyde/sulfuric acid
spray). Nine fractions were collected which were
re-chromatographed on Sephadex LH-20 (metha-
nol) and by HPLC (MeCN / water).
Purification of the second fraction led to the iso-
lation of compound 1a. Fraction 7 yielded furan-
2,5-dimethanol, fraction 8 4-hydroxyphenyl etha-
nol, and fraction 9 2Ј-deoxythymidine, and 2Ј-
deoxyuridine.
Experimental Section
NMR spectra were measured on Varian Unity
300 (300.145 MHz) and Varian Inova 500 (499.876
MHz) spectrometers in CDCl3 with TMS as in-
ternal standard. CIMS was recorded on a Finnigan
MAT 95 A instrument using NH3 as the collision
gas. Preparative HPLC was performed using a
RP18 column with a diode array detector. Flash
chromatography was carried out on silica gel
(230Ð400 mesh).
Strain GW10/2517
The culture was obtained from the strain collec-
tion of bioLeads in Heidelberg, Germany. This or-
ganism was Gram-positive, non-acid fast, grew
aerobically, and differentiated into substrate and
aerial mycelium. The aerial mycelium was mono-
podially branched with flexuous spore chains. Nei-
ther aerial hyphae nor substrate mycelium showed
fragmentation. Other morphological features such
as sporangia, or motile spores, were not observed.
The aerial spore mass colour was light pink on
yeast extract-malt agar and white on oatmeal and
soil extract agar. The substrate mycelium was
brown on most media. A light red diffusible pig-
ment was formed on yeast extract-malt extract
agar and on soil extract agar. Melanin pigments
were not produced on tyrosine agar. The diami-
nopimelic acid isomer and the sugar composition
indicated that the strain had cell walls of type I
(L,L-diaminopimelic acid, no characteristic sugars)
and belongs to the genus Streptomyces.
5-(2-Methylphenyl)-4-pentenoic acid (1a)
Colourless wax-like solid, 5 mg from fraction 2,
Rf = 0.75 (CHCl3-MeOH 9:1); UV/vis (MeOH):
λmax = 210, 249 nm; IR (KBr): ν = 3049, 2923, 1715
1
(C=O), 1431, 1299, 1208, 970 cmÐ1. Ð H NMR
(300 MHz, CDCl3): δ = 7.39 (t, J = 4.5 Hz, 1 H),
7.13 (m, 3 H), 6.65 (d, 1 H, J = 15.8 Hz, 5Ј-H), 6.08
(dm, J = 15.7 Hz, 1 H, 4Ј-H), 2.58 (m, 4 H, 2Ј and
3Ј-CH2), 2.32 (s, 3 H, 7Ј-CH3). Ð 13C NMR (50.3
MHz, CDCl3): δ = 179.2 (C-1), 136.4 (C-2Ј) , 135.1
(C-1Ј), 130.2 (C-3Ј), 129.3 (C-5), 129.2 (C-4), 127.1
(C-4Ј), 126.0 (C-5Ј), 125.5 (C-6Ј), 33.9 (C-3), 28.2
(C-2), 19.8 (C-7Ј). Ð MS (EI, 70 eV): m/z (%) =
190 (73) [M+], 131 (100) [M Ð CH2COOH]+. Ð
HREIMS: m/z = 190.0993 (calcd. for C12H14O2,
190.09938).
2,5-Furandimethanol
Colourless solid (3 mg) from fraction 7, Rf =
0.34 (CHCl3-MeOH 9:1); spectroscopical data
were identical with those of a commercial sample.
Fermentation
Three litres of culture medium composed of
malt extract (30 g), yeast extract (12 g), and glu-
cose (12 g) in tap water (3 l) were adjusted to
pH 7.8 and distributed into 15 1 l-Erlenmeyer
flasks. After sterilization, the flasks were inocu-
lated with slant cultures of Streptomyces sp. GW
10/2517 and incubated for 72 hours at 28 ∞C. This
Syntheses
3-Carboxypropyl-triphenylphosphonium bromide
4-Bromobutyric acid (4.59 g, 27.5 mmol) and tri-
culture was used to inoculate a 20-litre jar fer- phenyl phosphine (7.20 g, 27.5 mmol) were heated
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V. J. R. V. Mukkuet al. · 5-(2-Methylphenyl)-4-pentenoic Acid from a Terrestrial Streptomycete
337
under N2 for 3 h at 180 ∞C. After cooling to r.t., gel (CH2Cl2); yield 530 mg (52% for both steps). Ð
the solid was suspended in dry CH2Cl2 (50 ml), dry 1H NMR (300 MHz, CDCl3): δ = 7.38 (t, J = 4.5
ether (100 ml) was added, and the precipitate was Hz, 1 H), 7.15 (m, 3 H), 6.63 (d, J = 15.8 Hz, 1 H),
filtered off under nitrogen and dried; yield 9.48 g 6.06 (dd, J = 15.8, J’ = 6.4 Hz, 1 H), 3.69 (s, 3 H,
(80%). Ð 1H NMR (300 MHz , CD3OD): δ = 7.80 OMe), 2.53 (m, 4 H, 2 CH2), 2.32 (s, 3 H, Ar-Me).
(m, 15 H), 3.43 (m, 2 H, P+-CH2), 2.59 (t, J = 6.6
Hz, 2 H, α-CH2-COOH), 1.90 (m, 2 H, ꢀ-CH2).
The pure acid 1a was obtained by saponification
of 1b with sodium hydroxide. The NMR data were
identical with those of the natural product.
5-(2-Methylphenyl)-4-pentenoic acid methyl ester
(1b)
5-(3-Methylphenyl)-4-pentenoic acid methyl ester
(2)
In the same way as for 1b, the ester 2 was ob-
tained starting with m-tolylaldehyde. Yield 540 mg
(53%). Ð H NMR (300 MHz, CDCl3): δ = 7.15
(m, 3 H), 7.02 (d, J = 7.2 Hz, 1 H), 6.40 (d, J =
16.4 Hz, 1 H), 6.18 (dt, J = 16.4, J’ = 6.4 Hz, 1 H),
3.69 (s, 3 H, OMe), 2.50 (m, 4 H, 2 CH2), 2.33 (s,
3 H, Ar-Me).
To a solution of hexamethyldisilazane (3.45 ml,
16.5 mmol) in dry THF (20 ml), n-BuLi solution in
pentane (9.75 ml, 15 mmol) was slowly added un-
der nitrogen at 0 ∞C. A suspension of the above
phosphonium salt (2.15 g, 5 mmol) in dry THF
(10 ml) was added and the mixture stirred for 1 h
at 20 ∞C. When the salt was dissolved and the solu-
tion turned red, a solution of o-tolylaldehyde
(600 mg, 5 mmol) in THF (3 ml) was added drop-
wise. After stirring for 90 min at r.t., water (50 ml)
1
Acknowledgements
We would like to thank D. Fechner and F. Lissy
and diethyl ether (20 ml) was added. The ether for technical assistance. VJRVM is thankful to the
was discarded after washing with water, the aque- Alexander von Humboldt Foundation, RPM
ous layers were combined, acidified and extracted thanks the DAAD for financial support. This work
several times with ether to obtain 1a.
was granted by the Bundesministerium für Bil-
The crude acid 1a was methylated with excess dung und Forschung (BMBF, grant 0310735) and
diazomethane and the ester 1b purified on silica by the Fonds der Chemischen Industrie.
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[3] M. Ritzau, H. Drautz, H. Zähner, A. Zeeck, Liebigs
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[5] H. Laatsch, AntiBase 2000, A Database for Rapid
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http://www.gwdg.de/~ucoc/laatsch/.
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