Guignardic acid, a novel type of secondary metabolite produced by the endophytic fungus Guignardia sp.: Isolation, structure elucidation, and asymmetric synthesis

Article abstract of DOI:10.1002/1522-2675(20011219)84:12<3766::AID-HLCA3766>3.0.CO;2-Z

A UV-guided fractionation of the AcOEt extract of the fermentation broth of Guignardia sp., an endophytic fungus from the leaves of the tropical tree Spondias mombin, resulted in the identification of the new metabolite (-)-(2S,5Z)-2-(1-methylethyl)-4-oxo

Full text of DOI:10.1002/1522-2675(20011219)84:12<3766::AID-HLCA3766>3.0.CO;2-Z

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Helvetica Chimica Acta Vol. 84 (2001)
Guignardic Acid, a Novel Type of Secondary Metabolite Produced by the
Endophytic Fungus Guignardia sp.: Isolation, Structure Elucidation, and
Asymmetric Synthesis
by Katia F. Rodrigues-Heerklotza), Konstantin Drandarovb), Jˆrg Heerklotzb), Manfred Hesse*b), and
Christa Werner*b)
a) Departamento de Micologia, Instituto Oswaldo Cruz, FIOCRUZ, CP 926, BR-21045-900, Rio de Janeiro, RJ,
Brazil
b) Organisch-chemisches Institut der Universit‰t Z¸rich, Winterthurerstrasse 190, CH-8057 Z¸rich
A UV-guided fractionation of the AcOEt extract of the fermentation broth of Guignardia sp., an
endophytic fungus from the leaves of the tropical tree Spondias mombin, resulted in the identification of the new
metabolite (À)-(2S,5Z)-2-(1-methylethyl)-4-oxo-5-(phenylmethylene)-1,3-dioxolane-2-carboxylic acid (1), iso-
‡
lated as NH4 salt 1a. The metabolite 1 was designated (À)-(S)-guignardic acid. This first member of a new class
of natural compounds contains a dioxolanone moiety formed by fusion of 2-oxo-3-phenylpropanoic acid
(phenylpyruvic acid) and 3-methyl-2-oxobutanoic acid (dimethylpyruvic acid), products of the oxidative
deamination of phenylalanine and valine, respectively. The structure of 1a was deduced from spectral data (UV,
1
IR, MS, H- and 13C-NMR) and confirmed by asymmetric synthesis.
Introduction. The interest in endophytic fungi as a source of novel bioactive
compounds is increasing mainly because of the difficulty to find new interesting lead
compounds from extensively investigated organisms. In Brazil, Spondias mombin L.
(Anacardiaceae) has been used in traditional medicine because of its antimicrobial
properties [1].
Species of Guignardia (Ascomycetes) and/or its anamorph genus Phyllosticta have
been frequently isolated as endophytes [2 4]. Phyllosticta, a genus of asexual fungi, is a
source of various secondary metabolites. The most abundant, the phytotoxin
phyllosinol, containing an a,b-unsaturated ketone moiety, shows antibacterial activity
[5][6]. Recently the sesquiterpene lactone heptelidic acid and two analogues, which are
toxic against spruce budworm larvae, have been reported [7].
During an initial screening of secondary metabolites produced by strains of
different fungal species isolated as endophytes from S. mombin that could contain
useful antimicrobial properties [8], we selected a Guignardia strain based on some
degree of antibacterial activities demonstrated by its crude extract. In the present
communication, we describe the isolation, structure elucidation, and asymmetric
synthesis of the new metabolite (À)-(S)-guignardic acid (1) as ammonium salts 1a or 1b.
‡
Results and Discussion. (À)-(S)-Guignardic acid (1), isolated as its NH4 salt 1a,
was obtained as amorphous solid and identified as (À)-(2S,5Z)-2-(1-methylethyl)-4-
oxo-5-(phenylmethylidene)-1,3-dioxolane-2-carboxylic acid, as deduced from UV, IR,
MS, 1H-, 13C-NMRspectral data.
We failed to obtain useful data of 1 by the usual positive mode in EI, CI, or ESI mass spectrometric
methods, but the ESI-MS data of ammonium guignardate 1a in the negative mode furnished a [M À H]À peak at

Helvetica Chimica Acta Vol. 84 (2001)
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m/z 261 (molecular mass 262 for C14H14O5). The tandem mass spectrum (MS/MS) from the parent ion at m/z
261 showed two daughter ions: one at m/z 217, which obviously arises by loss of CO2, and one at m/z 189 (the
base fragment ion), which can be written as [M À H À CO2 À CO]À.
The UV spectrum of the salt 1a, showing a strong absorption band at 301 nm, is similarly shaped as those of
(E)-cinnamic acid derivatives and suggests a similarly arranged extended chromophore system. The presence of
the strong bands at 1646 and 1783 cmÀ1 in the IRspectrum of 1a, corresponding to conjugated CˆC and CˆO
bonds, supports such a correlation. Moreover, the strong shorter wavelength shift (at 1783 cmÀ1) of the second
band indicates that the corresponding CˆO group is part of a five-membered lactone ring. In addition, the IR
spectrum of 1a contains a broad band at 3404 cmÀ1 (OH) and a strong band at 1666 cmÀ1 (CˆO), which
evidently belong to a COOH group.
The 13C-NMRspectrum of 1a confirms the presence of two CˆO groups at d(C) 166.5 and 164.6, one
olefinic methin CH at d(C) 104.0, and two quaternary C-atoms of which one is an olefinic (d(C) 139.1) and the
other a quaternary acetal C-atom (d(C) 111.0). The lack of spin-spin coupling of the signal at d(H) 6.22 (s) is
evidence that the corresponding olefinic CH group is located between two quaternary C-atoms and is part of a
conjugated, exocyclic CˆC bond. The signals for one methine C-atom (d(C) 31.6 and d(H) 2.62 (sept.), together
with the signals for two Me groups (d(C) 16.4 and 15.1 ppm and the d at d(H) 0.92 and 0.95, resp.) indicate the
presence of an isopropyl moiety in 1a. The d at d(H) 7.66 and two t at d(H) 7.38 and 7.27 in the 1H-NMR
spectrum of 1a are typical of a mono-substituted benzene ring. The structure elucidation of (À)-guignardic acid
(1) was further supported by 2D NMRexperiments (DEPT, HSQC, and HMBC). In addition, the presence of
the strong UV absorption band at 301 nm, typical for the (E)-cinnamic acid chromophore is evidence for the
(Z)-configuration of the exocyclic double bond of 1 [9][10].
To confirm the structural conclusions described above and to establish its absolute
configuration, (À)-(S)-guignardic acid (1) was asymmetrically synthesized, as shown in
Scheme 1. Starting from benzyl 3-methyl-2-oxobutanoate (3) (obtained from the
commercially available sodium 3-methyl-2-oxobutanoate (2) and phenylmethanol in
the presence of SOCl2) and commercially available (2R)-2-hydroxy-3-phenylpropanoic
acid (4) in the presence of BF3 ¥ Et2O [11], the 6 :1 mixture of the diastereoisomeric
dioxolanones 5 and 6 was prepared in 48% yield similarly to a published procedure. The
mixture was separated by CC. The (2S,5R)-configuration of 5 was established by a
NOESY experiment (NOE between Me2CH and PhCH2ÀC(5)). Similarly, the
(2R,5R)-configuration was deduced for the diastereoisomer 6 (NOE between Me2CH
and HÀC(5)). According to a procedure used for similar compounds [12], diaste-
reoisomer 5 was refluxed for 10 min in the presence of N-bromosuccinimide (NBS) and
a catalytic amount of 2,2'-azobis[isobutyronitrile] (AIBN) in CCl4 under irradiation
with white light to give the brominated derivative 7 in 45% yield. The NOESY
experiment of 7 confirmed its (2R,4S)-configuration (NOE between both Me of
Me2CH and arom. H-atoms of PhCH2ÀC(5)). Thus, the bromination of 5 at C(5)
proceeded in a strictly stereoselective manner with retention of configuration. The
elimination of HBr from compound 7 carried out in acetone in the presence of Et3N,
similarly to a published procedure [12], yielded ester 8 quantitatively. By selective
catalytic hydrogenolysis in the presence of Lindlar catalyst, compound 8 was
transformed to (À)-(S)-guignardic acid (1) almost quantitatively. Because of its
instability under acidic conditions, the synthetically prepared 1 was isolated as its
triethylammonium salt 1b. The synthetic triethylammonium (À)-(S)-guignardate (1b)
was identical to the natural ammonium salt 1a by TLC, HPLC (tR), UV, and chiroptical
properties (Fig.), including its absolute configuration1).
1
1
)
The differences between the H-NMRchemical shifts of the natural (isolated as ammonium salt 1a) and
synthetic (À)-(S)-guignardic acid (prepared as triethylammonium salt 1b) are a function of the different
cationic counterparts.

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Helvetica Chimica Acta Vol. 84 (2001)
Scheme 1
To the best of our knowledge, (À)-(S)-guignardic acid (1) is the first identified
member of a new class of natural compounds. Obviously, the biogenetic precursors of 1
are 3-methyl-2-oxobutanoic acid (dimethylpyruvic acid; 9) and 2-oxo-3-phenylpropa-
noic acid (phenylpyruvic acid; 10), which are products of the oxidative deamination of
the amino acids valine and phenylalanine, respectively. The proposed biogenetic
pathway of these compounds is shown in Scheme 2. The existence of other
combinations of naturally occurring keto acids derived from deaminated a-amino

Helvetica Chimica Acta Vol. 84 (2001)
3769
Figure. 1. CD Spectrum of (À)-(2S)-guignardic acid ammonium salt (1a)
acids is likely. It is known that phenylpyruvic acid exists mainly in its enol-tautomer
form 10b. Obviously the tautomerism of phenylpyruvic acid makes it a favored reaction
partner in the formation of such natural dioxolanones.
The authors are grateful to Mr. Martin Binder of our NMRDepartment for the measurements and
assistance during the interpretation of the NMRspectra of the natural guignardic acid and to Drs. Laurent Bigler
and Aycil Yurdakul for the MS measurements. This study was supported in part by a Grant-in-Aid for Scientific
Research from the Brazilian National Council for Scientific and Technological Research (CNPq), the Swiss
National Science Foundation, and the Dr. Helmut Legerlotz-Stiftung.

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Helvetica Chimica Acta Vol. 84 (2001)
Scheme 2
Experimental Part
General. TLC: Merck precoated plates, silica gel 60 F254; detection by UV (254 or 366 nm). CC: silica gel 60
F 254 (70 230 mesh) from Merck. UV Spectra: Perkin-Elmer 555 spectrophotometer. IRSpectra: Perkin-
Elmer 297 infrared spectrometer; in cmÀ1. NMR: Bruker AMX-600 (600 MHz), AC-300, or AMX-300;
(D6)DMSO or CDCl3; chemical shifts d in ppm, Me4Si as internal standard, J in Hz, ESI-MS and ESI-MS/MS:
Finnigan TSQ-700 mass spectrometer.
Fungal Strain. Guignardia sp. was isolated as endophyte from surface-sterilized leaves of the Spondias
mombin L. tree as previously described [13]. Our fungal culture produced both sexual and asexual stages
(Phyllosticta). This fungus is still under investigation. Apparently, it is a widespread species, which has been
isolated repeatedly from other unrelated host plant families. It had been subjected to molecular studies, showing
identical internal transcribed spacer (ITS) sequences to Phyllosticta telopeae, which is available in the
GeneBank (accession number AF 312015). The isolate is preserved, lyophilized, and deposited in the fungal
culture collection of the Mycology Department (IOC) at FIOCRUZ (Brazil).
Extraction and Isolation. The AcOEt extract from the broth of a 14-day fermentation in malt extract (20 g/l
malt extract, 1 g/l peptone, and 20 g/l glucose) was evaporated and the residue purified by prep. TLC (silica gel,
CHCl3/MeOH/25% aq. NH3 soln. 78 :19 :3). Eight UV-absorbing bands were scraped from the plates and eluted
with MeOH/CHCl3 4 :1. From Fr. 2 (Rf 0.2), (À)-(S)-guignardic acid was isolated by prep. HPLC (Nucleosil-C8
column (7 mm, 10 Â 250 mm; Macherey-Nagel, Oensingen, Switzerland), flow rate 5 ml minÀ1, linear gradient
from 100% H2O to 100% MeOH in 20 min, then 5 min at 100% MeOH).
(À)-Ammonium (2S,5Z)-2-(1-Methylethyl)-4-oxo-5-(phenylmethylene)-1,3-dioxolane-2-carboxylate
ˆ
(À)-(2S)-Guignardic Acid Ammonium Salt; 1a). Yellowish amorphous solid. [a]D ˆ À56.5 (c ˆ 0.20, MeOH).
IR(CHCl 3): 3404 (br., OH), 1783s (CˆO, lactone), 1666s (CˆO, acid), 1646s (CˆC, conj.), 1360m (CÀO).
1H-NMR((D )DMSO): 7.66 (d, J ˆ 7.4, 2 arom. H); 7.38 (t, J ˆ 7.7, 2 arom. H); 7.27 (t, J ˆ 7.4, 1 arom. H); 6.62
6

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(s, 1 H); 2.62 (sept., J ˆ 6.8, 1 H); 0.95 (d, J ˆ 6.9, 1 Me); 0.92 (d, J ˆ 6.9, 1 Me). 13C-NMR((D 6)DMSO): 166.5
(CˆO); 164.6; 139.1; 111.0; 133.3; 129.1; 128.8; 128.1; 104.0; 31.6; 15.1; 16.4. ESI-MS: 261 ([M À H]À,
[C14H14O5 À H]À). ESI-MS/MS (ÀDAU 261, 8 eV): 261 (36, [M À H]À), 217 (2, [M À H À CO2]À), 189 (100,
[M À H À CO2 À CO]À).
Phenylmethyl 3-Methyl-2-oxobutanoate (3). To a suspension of sodium 3-methyl-2-oxobutanoate (2; 3.4 g)
in phenylmethanol (12 ml), SOCl2 (2.9 ml) was added dropwise at 08. The mixture was stirred at 08 for 30 min.
After addition of H2O (20 ml), the mixture was stirred for 10 min and then extracted with AcOEt. The org. layer
was washed with aq. Na2CO3 soln. and H2O and evaporated. The residual oil was purified by CC (silica gel,
hexane, then hexane/THF 10 :0.5): 3 (3.7 g, 82%). Colorless oil. TLC (hexane/THF 19 :1): Rf 0.4. 1H-NMR
(CDCl3): 7.5 7.25 (m, 5 arom. H); 5.28 (s, PhCH2O); 3.24 (m, J ˆ 6.9, Me2CH); 1.14 (d, J ˆ 6.9, Me2CH).
13C-NMR(CDCl 3): 197.74 (CˆO); 161.60 (ester, CˆO); 134.53 (arom. quat. C); 128.59, 128.43, 128.28, 127.66
‡
‡
(arom. C); 67.58 (COOCH2Ph); 37.07 (Me2CH); 16.98 (Me). EI-MS: 205 (3, [M À H] ), 91 (100, C7H7 ).
Phenylmethyl (2S,5R)-2-(1-Methylethyl)-4-oxo-5-(phenylmethyl)-1,3-dioxolane-2-carboxylate (5). A mix-
ture of commercially available (2R)-2-hydroxy-3-phenylpropanoic acid (4; 2 g, 0.012 mol), 3 (1.27 g, 0.006 mol),
and 48% BF3 ¥ Et2O (6 ml) was stirred for 2 h at r.t., then diluted with CH2Cl2, and carefully transferred by small
portions to a separatory funnel containing aq. Na2CO3 soln. After every portion, the funnel was carefully shaken
until the liberation of CO2 was finished. The aq. phase was extracted once more with CH2Cl2, the combined org.
extract washed with H2O and evaporated, and the residue dissolved in toluene (5 ml) and Et3N (1.5 ml) and
purified by CC (silica gel, hexane, then hexane/THF 10 :0.5): 5 (1 g, 48%). Colorless oil. TLC (hexane/THF
19 :1): Rf 0.3 (6: Rf 0.27). [a]D ˆ ‡12 (c ˆ 2.38, CHCl3). 1H-NMR(CDCl 3): 7.45 7.15 (m, 10 arom. H); 5.21
(s, PhCH2O); 4.67, 4.66 (2d, J ˆ 6.8, 4.2, HÀC(5)); 3.21, 3.2 (2d, J ˆ 14.7, 4.2, 1 H, PhCH2CH); 3.07, 3.04 (2d, J ˆ
14.7, 6.8, 1 H, PhCH2CH); 2.37 (m, J ˆ 6.9, Me2CH); 0.81 (d, J ˆ 6.9, Me2CH). 13C-NMR(CDCl 3): 171.5, 166.7
(2 CˆO); 135.21, 134.56 (2 arom. quat. C); 129.56, 128.59, 128.41, 128.10, 127.04 (arom. C); 107.98 (C(2)); 75.75
(C(5)); 67.75 (COOCH2Ph); 36.79 (PhCH2CH); 31.63 (Me2CH); 15.17, 14.62 (Me2CH). EI-MS: 313 (40, [M À
‡
‡
‡
‡
CH2ˆCHCH2] ), 219 (28, [M À PhCH2OCO] ), 191 (20, [M À PhCH2OCO À CˆO] ), 91 (100, C7H7 ).
Phenylmethyl (2R,4S)-4-Bromo-2-(1-methylethyl)-5-oxo-4-(phenylmethyl)-1,3-dioxolane-2-carboxylate
(7). To a soln. of 5 (0.86 g, 0.0024 mol) in CCl4 (5 ml), NBS (0.44 g, 0.0027 mol) and AIBN (12 mg) were
added. The pale yellow suspension was refluxed for 10 min under irradiation with white light (normal-pressure
200-W lamp, placed at ca. 30 cm from the reaction flask) to give a colorless suspension2). The mixture was
diluted with CCl4, the insoluble succinimide removed by filtration, and the filtrate washed with sat. aq. NaHCO3
soln. and evaporated. The residual oil was dissolved in a few ml of hexane containing a few drops of THF and
separated by CC (hexane/THF 10 :0.5): 7 (470 mg, 45%)3). Colorless oil. TLC (hexane/THF 19 :1): Rf 0.4.
[a]D ˆ À63 (c ˆ 0.88, CHCl3). 1H-NMR(CDCl 3): 7.45 7.15 (m, 10 arom. H); 5.27 (s, PhCH2O); 3.81
(s, PhCH2CBr); 1.99 (m, J ˆ 6.9, Me2CH); 0.7 (d, J ˆ 6.9, 3 H, Me2CH); 0.36 (d, J ˆ 6.9, 3 H, Me2CH).
13C-NMR(CDCl 3): 165.66, 165.49 (2 CˆO); 134.15, 133.10 (2 arom. quat. C); 128.79, 128.67, 128.56, 128.50,
127.98 (arom. C); 107.96 (C(2)); 88.45 (CBr); 68.36 (COOCH2Ph); 46.32 (PhCH2CBr); 33.51 (Me2CH); 15.37,
‡
‡
13.66 (Me2CH). EI-MS: 352 (4, [M À HBr] ), 297 (20, [M À PhCH2OCO À H] ), 217 (20, [M À PhCH2OCO À
‡
‡
HBr] ), 91 (100, C7H7 ).
Phenylmethyl (2S,5Z)-2-(1-Methylethyl)-4-oxo-5-(phenylmethylene)-1,3-dioxolane-2-carboxylate (8). To a
soln. of 7 (100 mg) in acetone (4 ml), Et3N (1 ml) was added at r.t. The mixture was stirred for 2 h ( ! crystals of
Et3N ¥ HBr). The solvent was evaporated, the residue dissolved in toluene, the insoluble Et3N ¥ HBr filtered off,
and the filtrate separated by CC (hexane, then hexane/THF 50 :1): 8 (80 mg, quant.). Colorless, glass-like
residue. TLC (hexane/THF 19 :1): Rf 0.4. [a]D ˆ À142 (c ˆ 0.59, CHCl3). 1H-NMR(CDCl 3): 7.8 7.15
(m, 10 arom. H); 6.49 (s, PhCHˆC); 5.26 (s, PhCH2O); 2.68 (m, J ˆ 6.9, Me2CH); 1.04 (d, J ˆ 6.9, Me2CH).
13C-NMR(CDCl 3): 165.17, 152.75 (2 CˆO); 135.66, 134.43, 132.15 (2 arom. ‡ 1 olef. quat. C); 129.79, 128.96,
128.65, 128.53, 127.90 (arom. C); 109.59 (olef. C); 108.4 (C(2)); 68.02 (COOCH2Ph); 32.72 (Me2CH); 15.12,
.
‡
‡
14.36 (Me2CH). CI-MS: 370 (100, [M ‡ NH3 ‡ H] ), 352 (10, M ).
Triethylammonium (2S,5Z)-2-(1-Methylethyl)-4-oxo-5-(phenylmethylene)-1,3-dioxolane-2-carboxylate
(1b). To a suspension of 10% Pd/C (35 mg) in toluene (2.5 ml), a 0.25% soln. of quinoline (0.67 ml) in
toluene and a soln. of 8 in toluene (100 mg in 2 ml) were added. The suspension was stirred 2 h at r.t. under H2.
2
)
Sometimes, the bromination reaction worked poorly; the introduction of a few drops of diluted Br2 soln. in
CCl4 to the starting suspension helped in these cases.
Because of additional bromination of 5 at the benzyl ester CH2 group and following hydrolysis, some
benzaldehyde was formed, which was eluted just before.
3
)

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Helvetica Chimica Acta Vol. 84 (2001)
Then, Et3N (0.5 ml) was added, the catalyst filtered off, and the filtrate evaporated (bath temp. < 458): 1b
(96 mg, 93%). Viscous, colorless oil. TLC (CHCl3/MeOH/25% aq. NH3 soln. 8 :2 :0.3): Rf 0.2. [a]D ˆ À92 (c ˆ
1.8, MeOH). 1H-NMR((D 6)DMSO): 7.69 (d, J ˆ 7.6, 2 arom. H); 7.6 7.2 (m, 3 arom. H); 6.3 (s, 1 olef. H); 3.15
(q, J ˆ 7.3, (MeCH2)3N); 2.72 (m, J ˆ 6.9, Me2CH); 1.23 (t, J ˆ 7.3, (MeCH2)3N); 1.05 (t, J ˆ 6.9, Me2CH).
13C-NMR((D 6)DMSO): 166.43, 163.83 (2 CˆO); 138.02, 132.83 (2 arom. quat. C); 129.55, 128.95, 128.53,
128.06 (arom. C); 110.09 (C(2)); 104.84 (olef. CH); 45.21 ((MeCH2)3N); 31.35 (Me2CH); 15.76, 14.73 (Me2CH);
‡
‡
‡
8.26 ((Me(CH2)3N). ESI-MS: 102 (100, Et3NH ), 364 (20, [M ‡ Et3N ‡ H] ), 386 (40, [M ‡ Et3N ‡ Na] ).
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Received June 20, 2001