Synthesis and free radical scavenging activity of a novel metabolite from the fungus Colletotrichum gloeosporioides

Article abstract of DOI:10.1016/j.bmcl.2006.08.071

A novel metabolite (-)-1 was isolated as its peracetylated derivative, (-)-2-(3′,4′-diacetoxyphenyl)-3,4-diacetoxytetrahydrofuran (2), from a strain of the phytopathogenic fungus Colletotrichum gloeosporioides CECT 20122. The synthesis of (-)-1 was carrie

Full text of DOI:10.1016/j.bmcl.2006.08.071

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5836–5839
Synthesis and free radical scavenging activity of a novel metabolite
from the fungus Colletotrichum gloeosporioides
a
b
a
´
´
´
´
´
´
Marienca Femenıa-Rıos, Carlos M. Garcıa-Pajon, Rosario Hernandez-Galan,
a
a,
*
´
´
Antonio J. Macıas-Sanchez and Isidro G. Collado
aDepartamento de Quı´mica Orga´nica, Facultad de Ciencias, Universidad de Ca´diz, s/n, Apdo. 40, 11510 Puerto Real, Ca´diz, Spain
bFacultad de Ciencias, Escuela de Quı´mica de la Universidad Nacional de Colombia sede Medellı´n, A.A.1027 Medellı´n, Colombia
Received 26 June 2006; revised 12 August 2006; accepted 14 August 2006
Available online 6 September 2006
Abstract—A novel metabolite (ꢀ)-1 was isolated as its peracetylated derivative, (ꢀ)-2-(30,40-diacetoxyphenyl)-3,4-diac-
etoxytetrahydrofuran (2), from a strain of the phytopathogenic fungus Colletotrichum gloeosporioides CECT 20122. The synthesis
of (ꢀ)-1 was carried out by ring-closing metathesis of diene 6 and stereoselective dihydroxylation of a dihydrofuran derivative 7 as
key steps. The tetraol (ꢀ)-1 showed free radical scavenging activity comparable to that of BHT, caffeic acid or protocatechuic acid.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Colletotrichum fungal species include some of the most
destructive post-harvest pathogens of a wide range of
plants including cereals, legumes, fruits and vegeta-
bles.1,2 As a part of our research programme on the ra-
tional control of phytopathogenic fungi,3 we have
focussed on the relationship between the infective capa-
bilities of certain phytopathogenic fungi and the role of
their secondary metabolites in the production of active
oxygen species.4–7 These may prove to be a vital re-
source for medically applicable free radical scavengers
in important conditions such as atherosclerosis, stroke,
myocardial infarction, trauma, arthritis and cancer.8–10
rylhydrazyl radical (DPPHÅ) due to its easy and its wide-
spread use.20–22
A liquid culture (65 L) of the fungus C. gloeosporioides
CECT 2012223 was filtered and the resulting broth was
repeatedly extracted with ethyl acetate. After solvent
drying and evaporation, a crude extract (9.3 g) was
obtained, which was fractionated by silica gel column
chromatography. The ethyl acetate fraction (2.6 g) was
acetylated (Ac2O/py) and further analysed (silica gel,
petroleum ether–ethyl acetate gradients) to yield
(ꢀ)-(2R(S),3R(S),4S(R))-2-(30,40-diacetoxyphenyl)-3,4-
diacetoxytetrahydrofuran ((ꢀ)-2) (4 mg), together with
acetates of previously known compounds.24
A wide variety of fungi have produced novel antioxi-
dants.11,12 These include Penicillium roquefortii,13
Aspergillus candidus,14–16 Mortierella sp.,17 Emericella
falconensis18 and fungi of the genus Acremonium.19 In
this paper, we report the synthesis and radical scaveng-
ing activity of the tetrahydroxylated compound (ꢀ)-1,
which was isolated as a peracetylated derivative from
the culture broth of a strain of the fungus Colletotrichum
gloeosporioides (Penz.) Penz. and Sacc. (teleomorph
Glomerella cingulata). Radical scavenging activity has
been determined using the stable 2,2-diphenyl-1-pic-
5
O
2
4
OR
3
1'
OR
4'
RO
3'
OR
1 R = H
2 R = Ac
Compound (ꢀ)-2 was obtained as a yellow oil which
25
D
gave a negative optical specific rotation (½aꢁ ꢀ34 (c
2 mg/mL, CHCl3)). EIMS gave a quasi-molecular ion
peak at m/z 381 [M+1]+, while HREIMS gave a frag-
ment ion peak at m/z 320.0895 [MꢀAcOH], which is
consistent with the formula C16H16O7 (calcd 320.0896).
This, together with the analysis of the 13C NMR and
Keywords: Isolation; Stereoselective synthesis; Antioxidants; Fungi.
*
Corresponding author. Tel.: +34 956 016368; fax: +34 956 016193;
e-mail: isidro.gonzalez@uca.es
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.071

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M. Femenıa-Rıos et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5836–5839
5837
DEPT spectra, which shows the presence of 4 methyl
groups, 1 methylene, 6 methines and 7 quaternary car-
bons, allows the molecular formula C17H20O9 to be as-
signed to compound (ꢀ)-2. The H NMR spectrum of
O
a
H
3 R = H
b
1
RO
4 R = TBS
OR
compound (ꢀ)-2 showed signals attributed to a 1,3,4-tri-
substituted phenyl moiety at d 7.17 (1H, d, J = 8.4 Hz,
H-50), 7.23-727 (2H, H-20, H-60), 4 methyls from acetate
groups at d 2.09 (3H, s, CH3CO on C-4), 2.11 (3H, s,
CH3CO on C-3), 2.28 (6H, s, 2CH3CO on C-30,40),
and a 2,3,4 trisubstituted tetrahydrofuran moiety at d
3.97 (1H, dd, J = 10.3,5.6 Hz, H-5), 4.43 (1H, dd,
J = 10.3, 5.6 Hz, H-50), 4.94 (1H, d, J = 7.0 Hz, H-2),
5.03 (1H, dd, J = 6.8, 5.2 Hz, H-3), 5.41 (1H, ddd,
J = 5.3, 5.2,4.0 Hz, H-4). The connectivity among the
described fragments was determined by HMBC experi-
ments. The data are consistent with the proposed struc-
ture assigned to compound (ꢀ)-2 as (ꢀ)-(2R(S),
3R(S),4S(R))-2-(30,40-diacetoxyphenyl)-3,4-diac-etoxy-
tetrahydrofuran.
O
OH
c
RO
RO
OR
OR
(+/-)-6 R = TBS
(+/-)-5 R = TBS
O
d
e
RO
OR
(+/-)-7 R = TBS
O
O
H
Relative stereochemistry at C-2 for compound 2 was de-
duced from J coupling analysis but it could not be
directly confirmed by NOE analysis. A chemical syn-
thesis of compound ( )-2 and the related tetraol,
(2R(S),3R(S),4S(R))-2-(30,40-dihydroxyphenyl)tetra-
hydrofuran-3,4-diol (( )-1), would support the regio
and stereochemical assignations and would produce
further material for testing.
H
OH
OH
+
OH
OH
RO
RO
OR
OR
(+/-)-8a R = TBS
(+/-)-8b R = TBS
g
f
(+/-)-8b
(+/-)-2
(+/-)-1
The chemical synthesis of the metabolite relied on a
stereoselective hydroxylation of a non-conjugated
dihydrofuran precursor ( )-7, which was prepared via
a ring-closing metathesis (Scheme 1).
Scheme 1. Synthesis of ( )-1 and ( )-2. Reagents and conditions: (a)
TBSCl, imidazole, THF, 0 ꢁC (87%); (b) vinyl bromide, magnesium,
THF, 25 ꢁC (73%); (c) allyl bromide, (TMS)2NLi, THF, 0 ꢁC, 150, then
90 ꢁC, 12 h (98%); (d) 10 mol% Grubbs catalyst ((PCy3)2Cl2-
RuCH@Ph), CH2Cl2, reflux (99%); (e) OsO4, Me3NO, t-BuOH,
H2O, pyridine, reflux, (45%, 1:8 8a vs 8b diastereoisomeric ratio); (f)
iodine, MeOH, reflux (37%); (g) Ac2O, pyridine, 25 ꢁC (34%).
Diallylether ( )-6 was prepared by treatment of the pro-
tected 3,4-dihydroxybenzaldehyde (4)25 with vinyl
bromide and magnesium turnings, in THF,26 to yield
1-[(30,40-bis-(tert-butyldimethyl-silyloxy)phenyl)]prop-2-
en-1-ol (( )-5) (71%). O-Allylation of compound ( )-5
was efficiently carried out by treatment with (TMS)2NLi
and allyl bromide in THF, which allowed to obtain the
allyl ether ( )-6 in 98% yield.
H
H
H
H
O
H
Ring-closing metathesis on the non-conjugated diene
( )-6, mediated by the first generation Grubbs’ cata-
lyst,27 gave a high yield (99%) of the non-conjugated
dihydrofuran ( )-7 after 2 h reaction time. This com-
pound was dihydroxylated with the OsO4/Me3NO28 sys-
tem to give a 1:8 mixture of syn-diols (( )-8a–( )-8b;
45% yield). The observed product ratio reflected the
preference of the reagent for the least hindered face of
the olefin in the precursor compound ( )-7.
H
HO
OH
H
RO
H
RO
H
RO
The relative stereochemistry of minor compound ( )-8a
could be determined by NOE experiments as
2S(R),3R(S),4S(R) (Fig. 1). Compounds ( )-8a and
( )-8b, syn diols obtained by OsO4 mediated dihydroxy-
lation, are diastereoisomers which only differ on their
configuration at C-2. Therefore, once the relative stereo-
chemistry for ( )-8a had been determined as stated
above, the relative stereochemistry for its diastereocom-
plementary diol ( )-8b could be assigned as
2R(S),3R(S),4S(R).
H
H
H
O
H
RO
H
OH
H
OH
H
Figure 1. Selected NOE correlations for compounds ( )-8a (above)
and ( )-8b (below); R = TBS.

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5838
Deprotection of the major diol ( )-8b with I2/MeOH29
gave the tetrahydroxylated compound ( )-1, which after
peracetylation with Ac2O/py gave compound ( )-2. In
order to obtain compound (ꢀ)-1 and its peracetate
(ꢀ)-2, major diol ( )-8b was treated with (ꢀ)-(2R)-2-
methoxy-2-phenylacetic acid, DCC and a catalytical
amount of DMPA in CH2Cl2 at room temperature for
1 hour. This produced two diastereoisomeric diesters,
which after separate treatment with KOH/MeOH yield-
ed, respectively, the enantiomeric diols (ꢀ)-8b and
(+)-8b. Separate treatment of (ꢀ)-8b and (+)-8b with
I2/MeOH gave, respectively, the synthetic tetrols (ꢀ)-1
and (+)-1.30 Treatment of compound (ꢀ)-1 with Ac2O/
Table 1. Comparison of DPPH radical scavenging activity for
compound (ꢀ)-1, BHT, caffeic acid and protocatechuic acid
ARP (AE)b Kinetic behaviourc
a
Compound
EC50
(ꢀ)-1
0.14
0.23
7.14
4.20
7.14
9.1
Slow
Slow
Slow
Slow
BHTd
Protocatechuic acidd 0.14
Caffeic acidd
0.11
a Efficient concentration = EC50 ((mol/L) (ꢀ)-1/(mol/L) DPPHÅ).
bAntiradical power (ARP) = antiradical efficiency (AE) = 1/EC50
.
c Time required to reach steady state in the DPPHÅ scavenging exper-
iment (fast <1 min, intermediate >1 min, <30 min, slow >30 min).
d Ref. 22.
py yielded a synthetic product which had identical phys-
25
ical (½aꢁ ꢀ34 (c 2.2 mg/mL, CHCl3)) and spectral prop-
D
behaviour against the DPPHÅ scavenging, as more than
half an hour is needed to reach a steady state in the reac-
tion (no decrease in percentage of remaining DPPHÅ
with time).
erties to the isolated natural product (ꢀ)-2.
The radical scavenging activity of the synthetic natural
product (ꢀ)-1 was estimated according to the procedure
described by Brand-Williams et al.22 This assay involved
measuring the decrease in absorbance at 515 nm that oc-
curred when the DPPH radical was reduced by the test-
ed antioxidant. A 90 lM methanolic solution of DPPHÅ
(Fluka) was employed and compound (ꢀ)-1 was assayed
at final concentrations in the range between 30 and
5 lM in methanol. For every evaluated concentration,
an aliquot of the DPPHÅ solution (950 lL) was mixed
with an aliquot of the test solution (50 lL) in such a
fashion that the desired concentration was achieved
and the mixture was incubated in darkness at 25 ꢁC.
Simultaneously, a control solution of DPPHÅ was pre-
pared by mixing an aliquot of the DPPHÅ stock solution
(950 lL) with MeOH (50 lL), which was also incubated
in darkness at 25 ꢁC. The absorbance of the mixtures of
compound (ꢀ)-1 and DPPHÅ and their corresponding
controls at 515 nm were determined immediately and
every 10 min for 120 min. Methanol was used to zero
the spectrophotometer. Absorbancies were measured
using a Varian model Cary 50 Bio UV–vis spectropho-
tometer. The measurements were made in triplicate.
The inhibition percentage (IP) values referred to the
DPPH radical at different times (t) were calculated using
the following equation: IP = 100 · (Abs controlt ꢀ Abs
testt)/Abs controlt. Percentage of remaining DPPHÅ (cal-
culated as 100-IP) at steady state for every tested con-
centration of compound (ꢀ)-1 was obtained from a
plot of the percentage of remaining DPPHÅ against time
for the above-mentioned concentrations, according to
the procedure described by Brand-Williams et al.22,31
In conclusion, we have described the isolation of a novel
natural compound (ꢀ)-1 as its tetraacetate (ꢀ)-2, and
established its constitution and relative stereochemistry
via spectroscopic studies and stereoselective synthesis.
The tetrahydrofuran derivative (ꢀ)-1, obtained by
chemical resolution, proved to be an effective inhibitor
of the DPPH radical.
Acknowledgments
This research was supported by grants from MCYT,
AGL2002-04388-C02-01, and CICYT, AGL2003-6480-
´ ´
C02-01 (Spain). M. F.-R. thanks to Fundacion Ramon
Areces (Spain) for a studentship.
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In order to carry out a comparison of DPPHÅ scaveng-
ing activity of compound (ꢀ)-1, the amount of com-
pound necessary to decrease the DPPHÅ concentration
by 50% efficient concentration = EC50 ((mol/L) (ꢀ)-1/
(mol/L) DPPHÅ) was calculated from a plot of the stea-
dy-state values (120 min) of the percentage of remaining
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25
30. Compound (ꢀ)-1. Colourless oil. ½aꢁD ꢀ38 (c 1.85 mg/mL,
MeOH). 1H NMR (400 MHz, CD3OD): d (ppm) 3.80 (dd,
J = 2.4, 8.8 Hz, 1H, H-5b), 3.87 (dd, J = 4.2, 7.6 Hz, 1H,
H-3), 4.22 (ddd, J = 2.4, 4.2, 4.6 Hz, 1H, H-4), 4.25 (dd,
J = 4.6, 8.8 Hz, 1H, H-5a), 4.51 (d, J = 7.6 Hz, 1H, H-2),
6.70 (2H, H-50, H-60), 6.80 (d, J = 2.0 Hz, H-20); 13C
NMR (100 MHz, CD3OD): d (ppm) 72.4 (d, C-4), 74.2 (t,
C-5), 79.7 (d, C-3), 084.5 (d, C-2), 1014.5 (d, C-20), 116.1 (d,
C-60), 119.0 (d, C-5 ), 133.3 (s, C-1 ), 146.1 (s, C-3*), 146.3
(s, C-4*) (*interchangeable signals); selected HMBC cor-
relations: C10 ! H-2, H-3, H-50, H-60; IR (neat) m 3334,
1520; EIMS m/z (rel int.) 212 [M]+ (73), 194 (8), 152 (10),
139 (100); HREIMS m/z calcd for C10H12O5 = 212.0685,
25
found 212.0679. Compound (+)-1. ½aꢁD +38 (c
1.7 mg/mL, MeOH).
31. Concentrations of compound (ꢀ)-1 were expressed as
moles of (ꢀ)-1/mol of DPPHÅ. The DPPHÅ concentration
at a given time in the reaction medium was calculated
20. Blois, M. S. Nature 1958, 181, 1199.
´
21. Sanchez-Moreno, C.; Larrauri, J. A.; Saura-Calixto, F.
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from
a
calibration curve with the equation:
22. Brand-Williams, W.; Cuvelier, M. E.; Berset, C. Lebensm.
Wiss. Technol. 1995, 28, 25.
Abs515 nm = 25.607 · ConcDPPHÅ (mg/mL) + 0.0064 (R =
0.9982).