Metathesis-based synthesis of jasmonate and homojasmonate lactones, candidates for extracellular quorum sensing molecules in Candida albicans

Article abstract of DOI:10.1039/a807417h

Ring-closing metathesis of jasmonate esters is shown to provide a rapid entry to jasmonate lactones. The lactones were investigated as possible quorum sensing molecules for the fungi Candida albicans. Assays demonstrated no effect on fungal morphology at concentrations up to 96 μM.

Full text of DOI:10.1039/a807417h

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Metathesis-based synthesis of jasmonate and homojasmonate
lactones, candidates for extracellular quorum sensing molecules in
Candida albicans
Su C. Cho,a Patrick H. Dussault,*a Amber D. Lisec,b Ellen C. Jensenb† and Kenneth W.
Nickerson*b
a Department of Chemistry and b School of Biological Sciences, University of Nebraska-Lincoln,
Lincoln, NE 68588, USA
Received (in Cambridge) 23rd September 1998, Accepted 20th November 1998
Ring-closing metathesis of jasmonate esters is shown to provide a rapid entry to jasmonate lactones. The lactones
were investigated as possible quorum sensing molecules for the fungi Candida albicans. Assays demonstrated no effect
on fungal morphology at concentrations up to 96 µM.
Introduction
We recently required a rapid synthetic approach to jasmonate
lactones for screening as quorum-sensing molecules (QSMs) in
fungi. An essential aspect of fungal pathogenicity is the cap-
acity to undergo morphological interconversion. We have been
studying an aspect of yeast-mycelial dimorphism termed the
inoculum size effect in which inoculation above a given thres-
hold level (usually 106 spores mLϪ1) gives single celled, budding
yeast while inoculation below that threshold level gives fila-
mentous mycelial growth.1 This effect is similar to quorum sens-
ing in Gram negative bacteria, a phenomenon based on the
excretion of acyl-homoserine lactones.2 For both Ceratocystis
ulmi, the causative agent of Dutch elm disease, and Candida
albicans, a human pathogen,1,3 we have shown that the inocu-
lum sensing mechanism relies on extracellular QSMs.3,4 For C.
albicans, QSM activity could be extracted into ethyl acetate,
resuspended in hexane or 90% methanol, and eluted from a C8
reverse phase column with 90% methanol. Analysis of the
active fraction by GC-MS showed two major peaks, one con-
taining a molecular ion with a mass of 222 and a fragmentation
pattern reminiscent of jasmonates. The possibility that the
fungal QSMs were jasmonates was attractive because: (1) all
fractions with QSM activity had a jasmonate-like aroma; (2)
Scheme 1 Ring-closing metathesis approach to lactones.
jasmonates are a new group of plant growth regulators which
may act as positive transcription factors;5,6 and (3) jasmonates
structure threatened to require preparation of a large number
of unknown function have been discovered in the culture
filtrates of other fungi.7 For example, 7 of 46 fungal strains
of jasmonate lactones. Although 12-hydroxyjasmonate and the
corresponding lactone (jasmine ketolactone) are known com-
studied by Miersch and co-workers were found to contain
pounds,11 the reported syntheses were either lengthy or were
jasmonates, including jasmonic acid, isojasmonic acid and
unsuitable for the synthesis of isomers or homologs.12–14 We
therefore planned an approach based upon ring-closing
metathesis (RCM) of jasmonate esters.15,16 As our initial targets
for this approach, we investigated the preparation of the known
jasmine ketolactone 1b as well as two isomeric ketolactones, 2b
and 3b. Lactone 2b is a regioisomer of 1b while 3b is a homolog
with the correct molecular mass for the candidate structure. As
shown below, the RCM strategy would in principle allow syn-
thesis of all three targets from readily obtained jasmonate
esters.17
several hydroxylated jasmonic acids.7–10
The mass spectroscopic data, combined with the nonpolar
nature of the unknown, led us to suspect a homologated jas-
monate lactone as the putative QSM. In the course of preparing
candidate structures, we discovered
a remarkably rapid
approach to jasmonate lactones based upon the use of ring-
closing metathesis (RCM). We now report the synthesis of jas-
monate lactones 1b and 3b and their bioassay for the ability to
influence quorum sensing in C. albicans (Scheme 1).
Results and discussion
Synthesis
Given the limited structural information available at the incep-
tion of this project, synthetic elucidation of a candidate
The starting materials for RCM, esters 1a, 2a, and 3a, were
readily prepared through esterification of racemic jasmonic
acid with the appropriate unsaturated alcohol in the presence of
dicyclohexylcarbodiimide (DCC). The individual esters were
subjected to RCM in the presence of Grubb’s ruthenium
† Permanent address: Biological Sciences, St. John’s University,
Collegeville, MN, USA.
J. Chem. Soc., Perkin Trans. 1, 1999, 193–196
193

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and CaH2, respectively. 1H and 13C NMR spectra were recorded
on G.E. Omega-300, or G.E.-500 MHz spectrometers in CDCl3;
individual peaks are reported as (multiplicity, number of
hydrogens, coupling constant in Hz). Infrared spectra were
recorded on an Analect RFX-65 FT-IR spectrophotometer as
neat films unless otherwise stated. Selected absorbances are
reported in wavenumbers (cmϪ1). Elemental analyses were
obtained from M-H-W Laboratories, Phoenix, AZ, USA,
Desert Analytics, Tucson, AZ, USA, or Quantitative Technolo-
gies, Inc., NJ, USA. Mass spectra were obtained at the
Nebraska Center for Mass Spectrometry. Much of the chrom-
atography was performed with recycled ethyl acetate–hexane
reformulated using a reported procedure.18
Scheme 2 Reagents: a. but-3-en-1-ol, DCC; b. but-3-en-2-ol, DCC; c.
pent-4-en-1-ol, DCC; d. (Cy3P)2Cl2RuCHPh.
carbene under high dilution conditions (Scheme 2). Metathesis
of 1a afforded 1b in moderate yield as a mixture of E and
Z isomers which could be separated by preparative HPLC
and identified in comparison with literature reports.17 The
individual geometric isomers were each present as a single
diastereomer. Metathesis of 2a failed to provide isolable
amounts of the corresponding lactone, an outcome presumably
reflecting increased strain in the nine-membered ring lactone.
Metathesis of 3a proceeded in modest yield to furnish lactone
3b as a single isomer.
Isolation of the putative QSM and mass spectroscopic data
Candida albicans A-72 was grown in Fernbach flasks containing
1 L of glucose–phosphate–proline medium.1 Flasks were inocu-
lated at a cell density of 107 per mL and shaken for 24 h at 30 ЊC
and 110 rpm. The cells were removed by centrifugation and the
supernatants were sterilized by passage through 0.2 µm filters.
Active supernatant (total 15.5 L) was extracted with equal vol-
umes of ethyl acetate and the organic layer was concentrated by
rotary evaporation. The dried residue was resuspended in 1.5 L
of 90% MeOH and subdivided into 100 mL aliquots, which
were individually extracted with hexane (2 × 70 mL). The
pooled hexane fractions were concentrated by rotary evapor-
ation and resuspended in 15.5 mL of 90% methanol. A 10.5 mL
portion was loaded onto a C8 cartridge and eluted with 90%
MeOH as 0.5 mL fractions, which were bioassayed for their
ability to prevent germ tube formation. Active fractions were
resuspended in 100% methanol and analyzed by GC-MS, (30
meter DB-5 column) using CI (methane) ionization. The peak
Direct determination of the alkene geometry for 3b was not
1
feasible due to non-first order splitting in the H NMR spec-
trum. However, the presence of an E-alkene could be demon-
strated after cleavage of the lactone to the hydroxy methyl ester
3c [eqn. (1)].
group eluting at 9.87 to 9.90 minutes contained an apparent
ϩ
molecular ion (MϩH) at m/z 223, with other significant ions at
207, 205, 191, 181, 151, 137, 123, 113, and 109.
Comparison of synthetic materials with unknown QSM
As described in the Experimental section, the newly synthesized
jasmonate lactones were compared by silica thin layer chroma-
tography with methyl jasmonate and the unknown QSM. The
Rf values were 0.54 for methyl jasmonate, 0.50 for the unknown
QSM from C. albicans, 0.39 for lactone 3b, and 0.36 for lactone
1b. Thus, the putative QSM for C. albicans is more polar (lower
Rf) than methyl jasmonate but less polar than either of the
jasmonate lactones. The inability to detect any of the molecules
with 254 nm UV light on fluorescent TLC plates indicated the
absence of conjugated functional groups. Finally, the mass
spectroscopic fragmentation patterns of the synthetic lactones
differed significantly from that of the unknown QSM.
But-3-en-l-yl ester of jasmonic acid, but-3-en-1-yl 1,2-trans-3-
oxo-2-[(Z)-pent-2-enyl]cyclopentane-1-acetate (1a)
DCC (22 mmol, 4.54 g) was added slowly into a solution of
jasmonic acid (18.3 mmol, 3.86 g) in CH2Cl2 (91.5 mL) under
N2 in a dry 250 mL flask in a water bath. After stirring for 20
minutes, in the course of which a white precipitate formed, but-
3-en-1-ol (22 mmol, 1.9 mL) was added slowly with a syringe.
DMAP (0.9 mmol, 0.111 g) was added to the solution which
was allowed to stir for 3 days, whereupon TLC indicated com-
pletion of the reaction. Diethyl ether (50 mL) was added to the
mixture to precipitate urea. The concentrated filtrate was puri-
fied by flash chromatography (10% ethyl acetate–hexane) to
give 4.01 g (83%) of the ester as a yellow oil: Rf = 0.43 (10%
Assays for influence on quorum sensing
1
ethyl acetate–hexane); H NMR (500 MHz, CDCl3) 5.74 (ddt,
Lactones (1a and 3a) were bioassayed for the ability to prevent
germ tube formation (GTF) in C. albicans. However, no effect
was observed at concentrations of up to 96 µM. In both assays,
GTF was ≥90% relative to assays conducted without the add-
ition of lactone. For comparison, autoinducers of Gram nega-
tive bacteria such as 3-oxohexanoyl-L-homoserine lactone are
active at nM concentrations, while those plants which respond
to methyl jasmonate often do so at concentrations at or below
40 mM.2,5,6
In conclusion, we have developed a new and rapid approach
to the synthesis of jasmonate lactones. Further investigations
into the identity of the fungal QSMs are in progress and will be
reported in due course.
1H, J = 17.2, 10.4, 6.8), 5.41 (m, 1H), 5.22 (m, 1H), 5.08 (dd,
1H, J = 17.0, 1.6), 5.04 (dd, 1H, J = 10.4, 0.9), 4.12 (t, 2H,
J = 6.6), 2.66 (m, 1H), 2.38–2.17 (7H), 2.10–1.99 (4H), 1.85
(apparent dt, 1H, J = 8.9, 5.6), 1.46 (m, 1H), 0.92 (t, 3H,
J = 7.6); 13C NMR (125 MHz, CDCl3) 218.7, 171.9, 134.0,
133.9, 124.9, 117.2, 63.45, 53.9, 38.9, 38.0, 37.6, 33.0, 27.1, 25.4,
20.5, 14.0; IR (Neat) 2962, 2933, 1746, 1653, 1644, 1465, 1233,
1166, 990, 633 cmϪ1; [HRMS(EI) Calcd. for C16H24O3 (M)ϩ:
264.1725. Found: 264.1724].
But-3-en-2-yl ester of jasmonic acid, but-3-en-2-yl 1,2-trans-3-
oxo-2-[(Z)-pent-2-enyl]cyclopentane-1-acetate (2a)
Compound 2a was prepared in 76% yield from but-3-en-2-ol by
a similar procedure: Rf = 0.44 (10% ethyl acetate–hexane); H
NMR (500 MHz, CDCl3) 5.80 (ddd, 1H, J = 16.1, 10.5, 6.0),
5.41 (m, 1H), 5.33 (m, 1H), 5.22 (m, 1H), 5.21 (dd, 1H, J = 16.9,
1.2), 5.11 (d, 1H, J = 10.9), 2.67 (m, 1H), 2.34–2.17 (5H), 2.10–
1.98 (4H), 1.85 (apparent dt, 1H, J = 10.1, 5.6), 1.47 (m, 1H),
1
Experimental
General
All reagents and solvents were used as supplied commercially,
except THF and CH2Cl2, which were distilled from Na–Ph2CO
194
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1.28 (d, 3H, J = 6.5), 0.91 (t, 3H, J = 7.6); 13C NMR (125 MHz,
CDCl3) 219.59, 171.92, 138.18, 134.66, 125.60, 116.67, 71.81,
54.61, 39.92, 38.70, 38.35, 27.74, 26.06, 21.21, 20.55, 14.72; IR
(Neat) 2967, 2936, 1744, 1463, 1425, 1377, 1233, 1168, 992, 670
cmϪ1; [HRMS(EI) Calcd. for C16H24O3 (M)ϩ: 264.1725. Found:
264.1736].
C13H18O3 (M)ϩ: 222.1256. Found: 222.1257]. Other peaks at m/z
204, 179, 163, 135, 121.
Saponification of homojasmonate lactone {trans-3-oxo-2-
[(Z)-6-hydroxyhex-2-enyl]cyclopentane-1-acetic acid, methyl
ester} (3c). Into a solution of sodium methoxide (0.012 g) in
MeOH (5 mL) under N2 was added homojasmonate lactone 3b
(0.009 g, 0.04 mmole) as a solution in MeOH (0.5 mL). The
reaction was allowed to stir for five days, whereupon TLC
showed the reaction to be complete. The mixture was then
extracted with 10% HCl (2 mL), water (10 mL), and 25% ethyl
acetate–hexane (3 × 10 mL), dried with anhydrous NaSO4, and
the solvent was evaporated under vacuum to yield (0.010 g, ~
100% crude yield) of trans-3-oxo-2-[(Z)-6-hydroxyhex-2-
enyl]cyclopentane-1-acetic acid, methyl ester (3c) which was
Pent-4-enyl ester of jasmonic acid, pent-4-en-1-yl 1,2-trans-3-
oxo-2-[(Z)-pent-2-enyl]cyclopentane-1-acetate (3a)
Compound 3a was prepared in 77% yield by a similar procedure
1
from pent-4-en-1-ol: Rf = 0.37 (10% ethyl acetate–hexane); H
NMR (500 MHz, CDCl3) 5.79 (ddt, 1H, J = 17.3, 10.5, 6.8),
5.44 (m, 1H), 5.25 (m, 1H), 5.02 (dd, 1H, J = 16.9, 1.6), 4.98
(dd, 1H, J = 10.1, 1.2), 4.09 (t, 2H, J = 6.8), 2.68 (m, 1H), 2.39–
2.19 (5H), 2.13–2.01 (6H), 1.87 (apparent dt, 1H, J = 8.9, 5.6),
1.73 (quintet, 2H, J = 6.9), 1.49 (m, 1H), 0.94 (t, 3H, J = 7.6);
13C NMR (125 MHz, CDCl3) 218.4, 171.8, 137.1, 133.8, 124.8,
115.1, 63.7, 53.8, 38.8, 37.9, 37.5, 29.8, 27.6, 27.0, 25.3, 20.4,
13.9; IR (Neat) 2962, 2935, 1745, 1653, 1642, 1464, 1232,
1167, 994, 604 cmϪ1; [HRMS(EI) Calcd. for C17H26O3 (M)ϩ:
278.1882. Found: 278.1882].
1
used without further purification: H NMR (500 MHz) 5.50
(dt, 1H, J = 15, 7), 5.35 (dt, 1H, J = 15, 7), 3.70 (s, 3H), 3.63 (t,
2H, J = 6.4), 2.62–2.68 (m, 1H), 2.39–2.20 (m, 6H), 2.12–2.06
(m, 3H), 1.87 (dt, 1H, J = 9.7, 5), 1.62 (dt, 2H, J = 14.5, 7),
1.54–1.47 (m, 1H); 13C NMR (125 MHz, CDCl3) 218.7, 172.6,
132.8, 126.8, 62.3, 54.0, 51.6, 38.7, 37.7, 37.6, 32.2, 30.9, 28.8,
27.2.
Ring-closing metathesis
Chromatographic comparison with unknown QSM
Jasmine ketolactone [(E and Z)-1,2-trans-8,13-dioxo-7-oxa-
bicyclo[8.3.0]tridec-3-ene] (1b). Into a dry 1000 mL three-neck
round bottom flask under N2 containing CH2Cl2 (200 mL)
was added a solution of bis(tricyclohexylphosphine)benzyl-
idene ruthenium() dichloride (0.34 mmol, 0.280 g) in CH2Cl2
(200 mL) from a pressure equalizing dropping funnel. A solu-
tion of ester 1a (3.4 mmol, 0.900 g) in CH2Cl2 (200 mL) was
added by a syringe pump over ~12 h. The resulting mixture was
refluxed for 2 days, whereupon more ruthenium catalyst (0.1
mmol, 0.08 mg) was slowly added in a solution of CH2Cl2 (50
mL). The reaction was refluxed for ~24 h and then concentrated
in vacuo. The residue was filtered through a short column of
silica with 10–25% ethyl acetate–hexane and the concentrated
filtrate was subjected to flash chromatography (5–25% ethyl
acetate–hexane) to furnish 0.395 g (59%) of jasmine ketolac-
tone as a 2:1 E–Z mixture which could be separated by semi-
preparative HPLC (5% propan-2-ol–hexane, Rainin Dynamax
TLC was performed on 250 µm Analtech GHLF silica plates.
Samples were dissolved in 100% methanol prior to application
(6–10 µL). TLC plates were developed in hexane–ethyl acetate
(3:1) and the bands were visualized by dipping in a 1% aqueous
potassium permanganate solution.
C. albicans bioassay
Washed C. albicans A72 cells stored at 4 ЊC in 50 mM potas-
sium phosphate (pH 6.5) at a cell density of 2 × 109 mLϪ1 were
tested for germ tube forming ability by inoculating them at a
final concentration of 107 mLϪ1 into a prewarmed (37 ЊC)
medium consisting of: 2.5 mM N-acetyl glucosamine, 3 mM
MgSO4ؒ7H2O, 10 mM L-proline, 111 mM glucose, and 50 mM
potassium phosphate (pH 6.5). The cell suspensions (5 mL)
were incubated for 4 hours at 37 ЊC in 25 mL Erlenmeyer flasks
on a New Brunswick Scientific G-2 shaker at 175 rpm. After 4
hours the control cultures with no added QSMs gave ca. 90%
GTF whereas those with active QSMs (such as Fraction 4) gave
≥90% budding yeasts (0–10% GTF).
1
8 µm Si): Rf = 0.21 (25% ethyl acetate–hexane); H NMR (300
MHz, CDCl3) 5.38 (m, 0.66H), 5.12 (m, 1.3H), 4.74 (td, 0.66H,
J = 10.7, 4.7), 4.58 (dt, 0.33H, J = 10.7, 4.6), 3.81 (m, 0.66H),
3.73 (td, 0.33H, J = 10.2, 3.5), 2.78 (d, 0.66H, J = 10.7), 2.70
(dd, 0.33H, J = 14.1, 2.8), 2.61 (m, 0.33H), 2.52 (d, 0.66H,
J = 13.6), 2.47–1.98 (7.4H), 1.89–1.64 (1.7H), 1.53–1.17 (0.9H);
13C NMR (75 MHz, CDCl3) 218.09, 217.67, 173.79, 171.92,
133.72, 130.99, 127.85, 125.89, 62.68, 62.06, 57.22, 55.80, 41.80,
41.18, 40.98, 38.91, 37.20, 37.09, 34.83, 33.91, 32.74, 28.92,
27.71, 25.54; IR (Neat) (E) 2980, 2958, 2908, 1744, 1685, 1288,
1160, 1136, 982, 742; (Z) 3056, 2978, 2857, 1738, 1657, 1286,
1186, 1138, 741, 721, 698, 661 cmϪ1; [HRMS(EI) Calcd. for
C12H16O3 (M)ϩ: 208.1099. Found: 208.1104]. Other peaks at m/z
191, 178, 163, 149, 138, 134, 122.
Acknowledgements
This work was supported by grants to K. W. N. from the
University of Nebraska Center for Biotechnology and the
Consortium for Plant Biotechnology Research. We gratefully
acknowledge a University of Nebraska Graduate Research
Fellowship for Su Cho as well as spectroscopic assistance from
Professor Richard Shoemaker.
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cedure as employed for 1b except that the reaction was refluxed
for three days after the initial addition and then for four more
days following recharge with additional catalyst. Purification as
before furnished 0.054 g (28%) of a yellow oil as a single alkene
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