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M. DellaGreca et al. / Tetrahedron Letters 44 (2003) 2779–2780
The correlations between protons and carbons were
established on the basis of HMQC and HMBC experi-
ments. The H-1 proton, linked to the carbon at l 104.6,
is correlated to the C-1a and C-2 carbons at l 134.3
and 151.2 across two bonds, and to the C-4a, C-3 and
C-9 carbons at l 127.2, 140.8 and 129.9, respectively,
across three bonds. The H-9 proton at l 8.31, linked to
the carbon at l 129.9, shows heterocorrelations across
two bonds with the C-1a and C-8 carbons at l 134.3
and 129.3, and across three bonds with the C-4a, C-2
and C-7 at l 176.1 carbons. The C-7 carbon is also
correlated to the H-6 proton at l 7.85, linked to the
carbon at l 121.0. This proton has correlations with the
C-4a and C-7 carbons. Finally the H-5 proton at l
7.89, linked to the carbon at l 125.8, gives correlations
with the C-4a, C-6 and C-4 carbons.
Thomasidioic acid (6) kept 7 days in BBM was stable
without formation of 6-hydroxy,5,7-dimethoxy-2-naph-
thoic acid and 2,6-dimethoxy-p-benzoquinone, reported
as secondary oxidation products.10
Thomasidioic acid (6) was subsequently used for bio-
transformation, and it was in turn incubated in the
culture of A. braunii for 5 days. The HPLC analysis
results and the spectroscopic data revealed that 6 was
quantitatively biotransformed into benzotropolone 5.
As far as we know, this is a novel type of biosynthesis
of a benzotropolone.
Acknowledgements
Work supported by M.I.U.R. (L488/92) in the frame of
the project Ambiente Terrestre: Chimica per l’Ambiente
(project P2, Cluster 11-A) of Consortium INCA. NMR
experiments were performed on a 500 MHz spectrome-
ter of Consortium INCA (project P0, L488/92, Cluster
11A).
References
1. Pinto, G.; Pollio, A.; Previtera, L.; Temussi, F. Biothec-
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5. Reverse phase C-18 HPLC [H2O–MeOH–CH3CN,
(6:3:1)].
6. Reverse phase C-18 HPLC [H2O–MeOH–CH3CN
(7:2:1)].
7. EIMS: m/z 446; H NMR: l 7.47 (1H, s, H-4), 6.84 (1H,
In order to understand the pathway that leads to the
benzotropolone 5 with A. braunii, we followed the
biotransformation at shorter times. The algal cultures
after 4, 8, 24, 72 h (3 days) and 168 h (7 days) were
directly injected in the HPLC system, after centrifuga-
tion to eliminate the algal suspension. We observed that
in 4 h appeared a peak (compound A) in the HPLC
chromatogram, this peak grew, reached a maximum
after 1 day and disappeared after 7 days, while the
benzotropolone peak appeared after 3 days and reached
a maximum after 7 days.
1
s, H-5), 6.34 (2H, s, H-2% e H-6%), 4.85 (1H, brs, H-1), 3.75
(1H, brs, H-2), 3.69 (3H, s, 8-OMe), 3.57 (6H, s, 3%-OMe
e 5%-OMe), 3.53 (3H, s, 6-OMe); 13C NMR: l 178.9
(2-COOH), 173.8 (3-COOH), 147.5 (C-3%), 147.5 (C-5%),
147.5 (C-6), 145.2 (C-8), 140.2 (C-7), 135.6 (C-4), 135.4
(C-1%), 132.6 (C-4%), 127.9 (C-3), 124.8 (C-9), 123.6 (C-10),
109.0 (C-5), 105.0 (C-2%), 105.0 (C-5%), 61.0 (8-OMe), 56.2
(3%-OMe), 56.2 (5%-OMe), 56.2 (6-OMe), 49.8 (C-2), 40.3
(C-1).
8. Rubino, M. I.; Arntfield, S. D.; Charlton, J. L. J. Agric.
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Compound A was isolated and identified as the already
known thomasidioic acid (6).7 It has been shown that
thomasidioic acid is an air oxidation product of sinapic
acid in alkaline aqueous solutions. The conversion of
sinapic acid into thomasidioic acid is depending from
the solution pH8 and the medium of the A. braunii
culture (pH 6.5) was compatible with the observed
transformation.9
9. Recently, Niwa et al. (Niwa, T.; Doi, U.; Osawa, T.
Bioorg. Med. Chem. Lett. 2002, 12, 963–965) reported
that sinapic acid gave at pH 7.4 thomasidioic acid and
(E,E)-b,b%-bis-sinapic acid, but we have not found this
latter compound in our experimental conditions.
10. Charlton, J. D.; Lee, K. Tetrahedron Lett. 1997, 38,
7311–7312.