Long-chain acyl-homoserine lactones from Methylobacterium mesophilicum: Synthesis and absolute configuration

Article abstract of DOI:10.1021/np900043j

The acyl-homoserine lactones (acyl-HSLs) produced by Methylobacterium mesophilicum isolated from orange trees infected with the citrus variegated chlorosis (CVC) disease have been studied, revealing the occurrence of six long-chain acyl-HSLs, i.e., the saturated homologues (S)-N-dodecanoyl (1) and (S)-N-tetradecanoyl-HSL (5), the uncommon odd-chain N-tridecanoyl-HSL (3), the new natural product (S)-N-(2E)-dodecenoyl-HSL (2), and the rare unsaturated homologues (S)-N-(7Z)-tetradecenoyl (4) and (S)-N-(2E,7Z)-tetradecadienyl-HSL (6). The absolute configurations of all HSLs were determined as 3S. Compounds 2 and 6 were synthesized for the first time. Antimicrobial assays with synthetic acyl-HSLs against Gram-positive bacterial endophytes co-isolated with M. mesophilicum from CVC-infected trees revealed low or no antibacterial activity.

Full text of DOI:10.1021/np900043j

J. Nat. Prod. 2009, 72, 2125–2129
2125
Long-Chain Acyl-Homoserine Lactones from Methylobacterium mesophilicum: Synthesis and
Absolute Configuration
Armando M. Pomini,† Pedro L. R. Cruz,† Cla´udia Gai,‡ Welington L. Arau´jo,‡ and Anita J. Marsaioli*,†
Chemistry Institute, UniVersity of Campinas, CP 6154, 13083-970, Campinas, Sa˜o Paulo, Brazil, and Genetics Department, ESALQ, UniVersity
of Sa˜o Paulo, CP 83, 13400-970, Piracicaba, SP, Brazil
ReceiVed April 30, 2009
The acyl-homoserine lactones (acyl-HSLs) produced by Methylobacterium mesophilicum isolated from orange trees
infected with the citrus variegated chlorosis (CVC) disease have been studied, revealing the occurrence of six long-
chain acyl-HSLs, i.e., the saturated homologues (S)-N-dodecanoyl (1) and (S)-N-tetradecanoyl-HSL (5), the uncommon
odd-chain N-tridecanoyl-HSL (3), the new natural product (S)-N-(2E)-dodecenoyl-HSL (2), and the rare unsaturated
homologues (S)-N-(7Z)-tetradecenoyl (4) and (S)-N-(2E,7Z)-tetradecadienyl-HSL (6). The absolute configurations of
all HSLs were determined as 3S. Compounds 2 and 6 were synthesized for the first time. Antimicrobial assays with
synthetic acyl-HSLs against Gram-positive bacterial endophytes co-isolated with M. mesophilicum from CVC-infected
trees revealed low or no antibacterial activity.
Brazil has the largest orange groves in the world and holds an
important slice of the international concentrated orange juice market.
Sa˜o Paulo State (Southeast Brazil) represents almost 90% of the
national production.1 However, the orange groves suffer from
several plagues and diseases. Citrus variegated chlorosis (CVC) is
one of the most important problems affecting sweet orange (Citrus
sinensis Osbek) cultivation and is caused by the fastidious bacterium
Xylella fastidiosa, which is normally found in the xylem of infected
trees.2,3
spp. from different sources such as potable water, soil, and rice
endophytes with the aid of bioreporters.9 The acyl-HSLs are
important signaling compounds in Gram-negative bacteria, em-
ployed in chemical communication mechanisms.10 This phenom-
enon is known as quorum-sensing and plays an important role in
several phenotypic factor expressions such as enzymes and mul-
ticellular behavior in many bacteria.10 From 18 studied species,
12 promoted the induction of phenotypic factors on bioreporter
assays, suggesting the production of acyl-HSLs.9
The strain M. extorquens AM1 is the sole species of this genus
thoroughly studied for acyl-HSL production.6 Interestingly, this
strain produces unsaturated long-chain acyl-HSLs under methan-
otrophic growth conditions, while short-chain ones are produced
under non-methanotrophic growth. Later work demonstrated that
these signaling compounds are involved in exopolysaccharide
production control.11
It was also reported that acyl-HSLs can exhibit antimicrobial
activities against Gram-positive bacteria.12-14 Continuing our
efforts in this field, we hypothesized that acyl-HSLs produced by
endophytic Methylobacterium could be an additional factor con-
tributing to its large prevalence over other bacteria in CVC-diseased
orange trees. Therefore, the aim of this work was to investigate if
the Gram-negative orange-isolated strain M. mesophilicum SR 1.6/6
produces acyl-HSLs and also to evaluate the antimicrobial activity
of the ensuing compounds against Gram-positive orange endo-
phytes, such as Bacillus and Curtobacterium.
Plants affected by CVC display remarkable reduction in fruit
size and early maturation.4 The groves may be contaminated from
infected shoots and also by xylem sapsucker insects belonging to
the families Cercopidae and Cicadellidae.5,6
Up to now there are no effective control technologies for CVC,
even though X. fastidiosa was the first phytopathogen whose
genome was successfully sequenced.7 However, there is significant
evidence that other endophytic orange bacteria may be associated
with CVC, and some remarkable differences were found in
endophytic flora between diseased, resistant, and healthy citrus
individuals.2,3
The main endophytes found in orange trees from the Brazilian
states of Sa˜o Paulo and Minas Gerais are bacteria belonging to the
genera Bacillus, Curtobacterium, Enterobacter, Nocardia, Pantoea,
Streptomyces, Xanthomonas, and several Methylobacterium species
(M. extorquens, M. mesophilicum, M. fujisawaense, M. radiotol-
erans, and M. zatmanii).1 A seasonal study demonstrated the large
prevalence of Methylobacterium species in symptomatic plants
infected by X. fastidiosa in practically all seasons.1 The presence
of these bacteria also has a positive correlation with the severity
of the CVC disease.
Bacteria belonging to the Methylobacterium genus may fix
atmospheric nitrogen and can survive using MeOH and other
compounds with one carbon (C1) as the sole carbon source. They
are found in different environments, e.g., soil, rhizosphere, and H2O;
however, they are frequently found in symbiotic relationships with
plants, contributing to them by producing many phytohormones
such as indolacetic acid, kitocynins, and vitamin B12 and receiving
from the plants MeOH as a carbon source.7,8
Results and Discussion
The bacterium M. mesophilicum SR 1.6/6 can grow in a specific
cultivation medium (CHOI3) with MeOH as the sole carbon
source.8 It is an interesting characteristic offering advantages during
secondary metabolite isolation and purification.
Initially, M. mesophilicum (5 L) was extracted with EtOAc (16.6
mg) and subjected to silica gel filtration to remove cellular and
macromolecular impurities, enriching the sample in secondary
metabolites.17 This semipurified extract (8.0 mg) was subjected to
a ꢀ-galactosidase assay with reporter Agrobacterium tumefaciens
NTL4(pZLR4), which can detect several exogenous acyl-ho-
moserine lactones.16 In positive assays, a blue coloration is observed
coming from X-Gal reagent degradation by ꢀ-galactosidase en-
zymes, which are expressed when the exogenous acyl-HSLs bind
to the transcriptional regulator protein TraR, which controls the
transcription of an operon containing the lacZ gene cloned from
Escherichia coli.16 The semipurified extract from M. mesophilicum
provided positive biological activity.17-19
Recently, Poonguzhali and co-workers characterized the produc-
tion of acyl-homoserine lactones (acyl-HSLs) by Methylobacterium
* Corresponding author. Tel: + 55 3521 3067. Fax: + 55 3521 3023.
E-mail: anita@iqm.unicamp.br.
† University of Campinas.
‡ University of Sa˜o Paulo.
10.1021/np900043j  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/17/2009

2126 Journal of Natural Products, 2009, Vol. 72, No. 12
Pomini et al.
Absolute Configuration Determination. The absolute con-
figuration at the lactone moiety of the natural acyl-HSLs produced
by M. mesophilicum was first assessed via GC-FID equipped with
a Chrompack Chirasil chiral column and synthetic standards.17-19
However analysis of the six natural acyl-HSLs mixture showed peak
overlapping of the unsaturated homologues. To overcome this
problem, a microscale catalytic hydrogenation reaction using Pd/C
transformed the mixture of HSL into N-tetradecanoyl-HSL and
N-dodecanoyl-HSL. The reaction efficiency was attested by GC-
MS, clearly showing a complete conversion of all unsaturated
homologues into the saturated ones (see Supporting Information),
allowing retention time comparisons and co-injection with racemic
and chiral HSL standards.
Figure 1. Acyl-HSLs produced by the orange endophyte M.
All natural acyl-HSLs displayed S absolute configuration without
any traces of natural R enantiomers (Table 1). The absolute
configuration of these compounds had not been characterized before.
Consequently this simple and elegant procedure allowed the
absolute configuration determination of five compounds in just
one experiment. Up to now, only a few papers have described the
absolute configuration of acyl-HSLs and consistently reported the
occurrence of only S enantiomers at the homoserine lactone
moiety.19
Synthesis of Acyl-Homoserine Lactones. The final unequivo-
cal structural characterization relied on the synthesis of compounds 1,
2, 3, 5, and 6 for GC-MS mass spectra comparisons and co-injection
with natural products. Compounds 1, 3, and 5 were synthesized by
coupling R-amino-γ-butyrolactone salts with dodecanoic, tridecanoic,
or tetradecanoic acids, as previously described.17-19,30
Compound 2 was synthesized in four steps starting from a
Hell-Volhard-Zelinsky bromination of dodecanoic acid followed
by nucleophilic substitution with iodide and thermal elimination,
furnishing the target (2E)-dodecenoic acid.31,32 The last step was
analogous to that employed with the saturated homologues, i.e.,
carbodiimide-mediated coupling with the homoserine lactone
moiety. The MS of synthetic 2 displayed a less abundant fragment
at m/z 143 (21%) than other HSLs similar to that of the natural
product.
mesophilicum.
GC-MS analysis of M. mesophilicum semipurified extract
revealed the presence of at least six different acyl-HSLs. A cleaner
fraction, more suited for spectroscopic investigations, was prepared
from the EtOAc extract from 8 L of M. mesophilicum cultivation
medium and extensive silica gel column chromatography. F22,
eluted with CH2Cl2/EtOAc (7:3), contained 1.1 mg of the mixture
of six acyl-HSLs. They were identified as (S)-N-dodecanoyl- (1),
(S)-N-(2E)-dodecenoyl- (2), N-tridecanoyl- (3), (S)-N-(7Z)-tetrade-
cenoyl- (4), (S)-N-tetradecanoyl- (5), and (S)-N-(2E,7Z)-tetradeca-
dienyl-homoserine lactone (6) (Figure 1).
Unsaturation Positions and Geometries. The minute amounts
of the natural products prevented isolation and an extensive NMR
structural study. To overcome this problem, the dimethyl disulfide
(DMDS) microderivatization protocol was applied with the semi-
purified EtOAc extract from the CHOI3 medium followed by GC-
MS analysis of the DMDS derivatives, aiming to determine the
unsaturation positions.21,22
The DMDS derivative of compound 4 provided fragments at m/z
145 and 258, resulting from characteristic derivative cleavage, and
also at m/z 157 originating from the fragment at m/z 218 undergoing
homoserine lactone loss. These results were in agreement with the
literature, permitting establishment of the double-bond position at
C-7 of the acyl side chain.23
Compound 6 was synthesized in five steps in 67% overall yield
(Scheme 1) using Meldrum reaction with commercially available
(5Z)-dodecenoic acid and 2,2-dimethyl-1,3-dioxane-4,6-dione.34 The
corresponding (5Z)-dodecenoyl derivative of Meldrum acid was
subjected to nucleophilic attack and a thermal decarboxylation
reaction with (S)-R-amino-γ-butyrolactone hydrobromide, furnish-
ing the 3-oxo intermediate 7. The ketone group was reduced using
NaBH4 to the corresponding secondary alcohol, which was mesy-
lated and treated with DBU to afford compound 6 (76% yield).
This is the first synthesis of compound 6.
The DMDS derivative of compound 6 provided a mass spectrum
with the molecular ion at m/z 401, suggesting that only one of the
two double bonds was derivatized, producing a characteristic
fragment at m/z 256, two mass units smaller than that observed for
the DMDS derivative of 4. Therefore, one of the double bonds was
present at C-7, while the other was located between the carbonyl
group and the unsaturation at C-7. It is known that carbon-carbon
double bonds conjugated to carbonyl groups are electron-deficient
and do not react with DMDS under the usual conditions.24 This,
together with a less abundant m/z 143 fragment in the MS of the
parent compound, is evidence that the second unsaturation of
compound 6 was conjugated to the carbonyl group of the acyl side
chain.
Perfect peak overlaps were observed under GC-MS co-injection
with all synthetic compounds and natural products, as well as
identical mass spectra.
All synthetic and natural products were also assayed with reporter
A. tumefaciens NTL4(pZLR4), and a blue coloration was observed,
indicating that these acyl-HSLs were detected by the biosensor.
Consequently these acyl-HSLs were responsible for the positive
test observed with EtOAc extracts from M. mesophilicum.
Influence of Cultivation Media on Acyl-HSL Production.
The influence of the cultivation media on the production of acyl-
HSLs by M. mesophilicum was also investigated. Two non-
methanotrophic conditions were assayed: (1) CHOI3 cultivation
medium with glucose instead of MeOH as carbon source and (2)
complex trypticase soy broth cultivation medium (TSB). The
estimated yields of acyl-HSLs were 137.5 µg/L in CHOI3 medium,
50 µg/L in CHOI3/glucose, and 287.5 µg/L in the TSB medium,
which was the highest acyl-HSL production. Natural products from
non-methanotrophic growth were identified by retention index
comparison with previously studied acyl-HSLs from CHOI3
medium.34 The microorganisms produced only long-chain acyl-
Interestingly, compound 2 did not react with DMDS, as observed
for the energetically less favorable carbonyl-conjugated unsaturation
in 6. This and the smaller abundance of the m/z 143 fragment
reinforced the presence of a conjugated amide moiety in the
molecule.
1
The double-bond geometries were determined by the H NMR
spectrum of the mixture of all natural acyl-HSLs (1.1 mg). Clearly
the double bond conjugated to the carbonyl group of compound 6
possesses trans geometry (5.83 ppm, H-2′, doublet; 6.92 ppm, H-3′,
doublet of doublets; J 15.3 Hz). Both compounds 6 and 4 had
double bonds at C-7 and shared overlapping signals in the 1H NMR
spectrum. A relatively pure 4 (2.3 mg total mass; 70% compound
4) obtained from M. mesophilicum grown in TSB cultivation
medium showed a cis configuration at C-7, determined by compar-
ing the 13C NMR chemical shifts of the allylic carbons (26.9 and
27.2 ppm assigned to C-6′ and C-9′) with those of cis (27.0 and
27.3 ppm) and trans (32.2 and 32.6 ppm) 7-tetradecenoic acids.26

Acyl-Homoserine Lactones from Methylobacterium
Journal of Natural Products, 2009, Vol. 72, No. 12 2127
Table 1. Absolute Configuration of Acyl-HSLs (dodecanoyl and tetradecanoyl homologues) Produced by M. mesophilicum via GC-FID
(Chrompack Chirasil column)
sample
retention time (min) and % (R)
N-Tetradecanoyl-HSL (5)
103.8 (50%)
retention time (min) and % (S)
ee (major)
(()-N-tetradecanoyl-HSL synt.
(S)-N-tetradecanoyl-HSL synt.
F22 natural product
105.4 (50%)
105.4 (97%)
105.4 (>99%)
105.3 (71%)
1:1 peaks
94% (S)
>99% (S)
13% (S)
co-injection (()-N-tetradecanoyl-HSL
103.6 (29%)
and hydrogenated
natural producta
N-Dodecanoyl-HSL (1)
(()-N-dodecanoyl-HSL synt.
(S)-N-dodecanoyl-HSL synt.
F22 natural product
46.9 (50%)
47.7 (50%)
47.7 (97%)
47.7 (>99%)
47.5 (68%)
1:1 peaks
94% (S)
>99% (S)
4% (S)
co-injection (()-N-dodecanoyl-HSL
46.8 (32%)
and hydrogenated
natural productb
a Co-injection: 1:1 mixture of (()-N-tetradecanoyl-HSL at 1 mg/mL and fraction F22 at 1 mg/mL. b Co-injection: 1:1 mixture of (()-N-dode-
canoyl-HSL at 0.1 mg/mL and fraction F22 at 1 mg/mL.
Scheme 1. Synthesis of Compound 6
Table 2. MIC (ppm) of Synthetic Acyl-HSLs against Orange
Endophytic Bacteria
HSLs in the three different media. However, some changes were
observed, such as compound 2 was produced in smaller amounts
in the TSB medium, and compound 3 was not detected in both
non-methanotrophic conditions. Compound 6 was produced in
smaller proportions under non-methanotrophic growth. Compound
4 was almost 70% pure in TSB cultivation media, allowing a more
extensive NMR structural study.
(S)-1
(S)-2
(S)-5
(S)-6
a
Bacillus sp. CL 15
Bacillus sp. CL 16
C. flaccumfaciens ER 1/5
Nocardiopsis sp.
-
-
-
-
-
1000
1000
500
-
-
1000
-
-
1000
1000
500
a - Inactive until the highest concentration assayed (1000 ppm).
Antimicrobial Assays against Gram-Positive Orange
Endophytes. The large prevalence of Methylobacterium over other
endophytic bacteria in orange tissues stimulated the investigation
on whether the acyl-HSLs produced could constitute an evolutionary
and colonization weapon, by Gram-positive bacteria suppression
caused by acyl-HSL antimicrobial activity.12-14 To test this
hypothesis, the microorganisms Bacillus sp. CL15, Bacillus sp.
CL16, Curtobacterium flaccumfaciens ER 1/5, and Nocardiopsis
sp., all isolated from CVC-affected orange tissues, together with
M. mesophilicum, were assayed against the compounds (S)-1, (S)-
2, (S)-5, and (S)-6. The assays were conducted according to a
previously adapted colorimetric protocol.14 Results are shown in
Table 2.
In general, all compounds showed negligible antibacterial activity
if one takes into consideration that M. mesophilicum produces only
0.1375 ppm of acyl-HSLs in CHOI3 medium (1.1 mg for 8 L of
cultivation media), while the most active compound was active at
about 500 ppm.
Therefore, it is improbable that compounds produced by M.
mesophilicum have an effective role in suppressing Gram-positive
orange endophytes. The absence of antimicrobial activity can be
explained in several ways. First, the Gram-positive bacteria may
be resistant to the acyl-HSLs produced by M. mesophilicum, or
the produced concentration may be too small for effective activity.
However, it is also known that 3-oxo-acyl-HSLs are much more
active than those without the 3-keto group.12-14 In this case, the
acyl-HSLs produced by M. mesophilicum do not have this
oxygenation level.
This is the first study of metabolites produced by M. mesophi-
licum, a strain isolated from citrus variegated chlorosis disease. Six
rare acyl-HSLs were detected and synthesized; compound 2 being
a new natural product. In addition, the first syntheses for compounds
6 and 2 and the first absolute configuration characterization of five
acyl-HSLs in just one chromatographic analysis, based upon an
elegant hydrogenation protocol, are reported. The antimicrobial
assays showed that the metabolites produced by M. mesophilicum
do not seem to be associated with antimicrobial competition against
orange endophytic Gram-positive bacteria. Currently, our group is
making efforts to understand the effects of the synthetic acyl-HSLs
on the Xylella fastidiosa gene expression profile.

2128 Journal of Natural Products, 2009, Vol. 72, No. 12
Pomini et al.
Experimental Section
(S)-N-(2E,7Z)-Tetradecadienyl-HSL (6) (GC-MS, EI, 70 eV): m/z
307 (M+•, 2), 236 (14), 206 (20), 169 (30), 143 (55), 125 (17), 121
(38), 102 (20), 81 (100), 68 (48), 55 (59), 43 (28), 41 (45).
Biological Assays with Reporter Agrobacterium tumefaciens
NTL4(pZLR4). The bioreporter assays were performed essentially as
previously reported17-19 with semipurified EtOAc extracts from
different cultivation media, fractions, and synthetic products. Ethanol
was employed as blank and synthetic (S)-N-(3-oxo-octanoyl)-HSL as
positive control. The stock solutions were prepared in EtOH at 1-4
mg/mL.
Natural Acyl-HSL Dimethyl Disulfide (DMDS) Derivatization.
To a round-bottom flask (25 mL) were added 0.5 mL of CH2Cl2 and
0.5 mg of semipurified EtOAc fraction from M. mesophilicum cultivated
in CHOI3 medium. To this mixture were added 0.5 mL of DMDS and
40 µL of an I2 solution (60 mg/mL in Et2O). The mixture was stirred
at 40 °C for 22 h. Then, CH2Cl2 (3 mL) and 5% sodium thiosulfate (3
mL) were added. The mixture was vigorously stirred for 5 min, and
the organic layer was dried over magnesium sulfate, filtered, and
evaporated under a N2 flow, yielding a crude oil, which was subse-
quently analyzed by GC-MS.
Syntheses and Spectroscopic Characterization of (S)-N-Dode-
canoyl-, (()-N-Tridecanoyl-, (S)-N-Tetradecanoyl-, (S)-N-Dode-
cenoyl-, and (S)-N-(2E,7Z)-Tetradecadienyl-HSL. Complete synthetic
procedures and spectroscopic data are provided in the Supporting
Information.
Absolute Configuration Determination of Natural Acyl-HSLs
Produced by M. mesophilicum. Catalytic Hydrogenation Micro-
derivatization Reaction. To a round-bottom flask (5 mL) were added
0.3 mg of fraction F22 from the M. mesophilicum CHOI3 cultivation
(containing the mixture of acyl-HSLs), EtOAc (400 µL), and a catalytic
amount of Pd/C (10%, Merck). The flask was sealed with a rubber
septum, connected to a hydrogen reservoir (1 atm), and magnetically
stirred for 6 h. The reaction was filtered through a silica gel column (2
g, 1 cm diameter) and eluted with EtOAc (150 mL). Approximately
0.3 mg of reduction product was obtained, which was submitted to
GC-MS and GC-FID (chiral column) analyses.
General Experimental Procedures. Optical rotations were measured
with a Perkin-Elmer 341 polarimeter or Jasco J-720 spectropolarimeter
using MeOH (HPLC grade) as solvent. The IR spectra were obtained
with Bomem Michelson MB equipment using KBr pellets as sample
supports for solids and NaCl crystals for oils (films). NMR spectra
were obtained with Varian Inova-500, Bruker Gemini-300, Bruker-
250, or Bruker-300 equipment, using CDCl3 as solvent and TMS as
internal reference (δ 0.0). The chemical shifts are provided in ppm
and the coupling constants in hertz (Hz). GC-MS analyses were carried
out on an Agilent 6890 gas chromatograph equipped with an HP 5973
mass selective detector operating in electron impact mode at 70 eV.
Separations were carried out on HP-5 or MDN-5S columns (30 m ×
0.25 mm × 0.25 µm). Highly pure He was employed as carrier gas (1
mL/min flow rate). The injector and interface temperatures were 250
and 280 °C, respectively. Chromatographic separations were carried
out with Acros 0.035-0.070 mm silica gel columns. TLC analyses
were performed on Merck 60 F254 aluminum sheets and revealed under
exposure to UV light or chemically with acidic anisaldehyde solution.
All solvents were of high analytical grade, distilled before use. Reagents
for acyl-HSL synthesis and X-GAL were ordered from Sigma-Aldrich
(S.Paulo, Brazil).
Cultivation Media. Nutrient broth (NB, 20 g/L in distilled H2O)
and Mu¨ller-Hinton media were from Oxoid. Luria-Bertani medium was
prepared with 1% peptone (Oxoid), 0.5% NaCl, and 0.5% yeast extract
(Oxoid). For solid media, 2% agar was added (Sigma). Trypticase soy
broth (TSB, 20 g/L in distilled H2O) medium was from Oxoid. CHOI3
medium was prepared as previously reported.15 The adapted CHOI3/
glucose medium was prepared with glucose (10 g/L, separately
autoclaved) instead of MeOH as carbon source.
Bacterial Strains. The bacterium M. mesophilicum SR 1.6/6 was
isolated as an endophyte in CVC-diseased citrus plants and conserved
under refrigeration (5 °C) in slants with selective solid CHOI3 medium
for routine use or under ultrafreezer refrigeration (-80 °C) for storage.
The bioreporter Agrobacterium tumefaciens NTL4(pZLR4) was pre-
pared by Cha and co-workers16 and was maintained in solid LB medium
supplemented with gentamicine (50 µg/mL). Bacillus sp. CL15, Bacillus
sp. CL16, Curtobacterium flaccumfaciens ER 1/5, and Nocardiopsis
sp. were isolated as endophytes from infected CVC orange trees and
were maintained under refrigeration in slants containing nutrient broth
medium (NB, Oxoid).
General Procedures for Extract Preparation. The inoculums of
M. mesophilicum were prepared in test tubes containing 10 mL of liquid
CHOI3, CHOI3/glucose, or TSB medium, incubated at 30 °C without
shaking. After four days, the inoculums were transferred to Erlenmeyer
flasks (2 L) containing 1 L of cultivation media, incubated at 29 °C
for five days under shaking (110 rpm). The media were filtered with
conventional paper filters (the cells were densely aggregated), and the
aqueous phases (1 L) were extracted with EtOAc (3 × 500 mL). The
organic layers were washed with distilled H2O (1 × 500 mL) and
evaporated under reduced pressure at 40-45 °C. For the biological
assays with reporter A. tumefaciens NTL4(pZLR4) the EtOAc extracts
from 1 L of cultivation media (CHOI3, CHOI3/glucose, or TSB) were
filtered with silica gel columns (3 g, 1 cm diameter column) with
n-hexane (50 mL), EtOAc (100 mL), and MeOH (50 mL). However,
for more accurate purifications aiming at spectral studies, the combined
extracts from 8 L of cultivation media were purified by silica gel
chromatography using n-hexane, CH2Cl2, and EtOAc in mixtures of
increasing polarity. Fractions were analyzed by GC-MS.
GC-FID (chiral column). Chiral discrimination analyses were
carried out with an Agilent 6850 gas chromatograph equipped with a
Chrompack Chirasil CB chiral column (25 m × 0.25 mm × 0.25 µm).
Highly pure H2 was employed as carrier gas (1 mL/min flow rate).
The injector temperatures were 240 °C for N-tetradecanoyl-HSL and
220 °C for N-dodecanoyl-HSL. Analytes (hydrogenated natural product
and synthetic standards) were dissolved in EtOAc (1 mg/mL) and
injected in a 1/100 split mode. The oven was maintained isotermically
at 180 °C for 150 min. The detector temperatures were 240 °C for
N-tetradecanoyl-HSL and 280 °C for N-dodecanoyl-HSL. Results are
summarized in Table 1.
Antimicrobial Assays. The antimicrobial assays were performed
with synthetic acyl-HSLs and the Gram-positive bacteria Curtobacte-
rium flaccumfaciens ER 1/5, Bacillus sp. CL16, Nocardiopsis sp., and
Bacillus sp. CL15.
Inoculums of Curtobacterium flaccumfaciens ER 1/5 and Bacillus
sp. CL16 were prepared in slants with solid nutrient broth medium,
grown at 30 °C for 24 h in BOD. Inoculum of Bacillus sp. CL15 was
prepared in the same way, but using 48 h of incubation. After growth,
the cells were collected with flamed wire loops and suspended in sterile
distilled H2O (10 mL), until a 3 × 108 cells/mL solution was obtained,
according to the McFarland scale. For Nocardiopsis sp., the inoculum
was prepared in an Erlenmeyer containing liquid Mu¨ller-Hinton medium
(50 mL) incubated under shaking for 72 h at 30 °C (100 rpm). The
cells were decanted, washed with sterile distilled H2O (2×), suspended
in distilled H2O (10 mL), vortexed, and diluted until the desired
concentration. The antimicrobial assays were performed as previously
adapted, using sterile distilled water and DMSO (20%) as blank and
chloramphenicol as positive control.14 The compounds (S)-1, (S)-2, (S)-
5, and (S)-6 were assayed, using stock solutions at 2 mg/mL. The
highest concentration assayed was 1000 ppm.
(S)-N-Dodecanoyl-HSL (1) (GC-MS, EI, 70 eV): m/z 283 (M+•,
4), 170 (2), 156 (19), 143 (100), 125 (17), 102 (20), 83 (15), 57 (46),
43 (24).
(S)-N-(2E)-Dodecenoyl-HSL (2) (GC-MS, EI, 70 eV): m/z 281
(M+•, 11), 180 (13), 156 (11), 143 (25), 125 (46), 102 (31), 101 (30),
83 (27), 67 (23), 57 (34), 41 (30).
N-Tridecanoyl-HSL (3) (GC-MS, EI, 70 eV, SIM mode): m/z 297
(M+•, 7), 201 (23), 156 (20), 143 (100), 125 (17), 102 (21), 83 (22),
57 (54), 43 (35).
For Bacillus sp. CL16 and Curtobacterium flaccumfaciens ER 1/5
addition of coloring agent was performed after 24 h of microplate
incubation at 30 °C, while Nocardiopsis sp. and Bacillus sp. CL15
were incubated for 48 h.14
(S)-N-(7Z)-Tetradecenoyl-HSL (4) (GC-MS, EI, 70 eV): m/z 309
(M+•, 19), 228 (12), 208 (23), 156 (40), 143 (100), 125 (28), 102 (77),
55 (76), 43 (39), 41 (52).
Acknowledgment. The group is indebted to FAPESP for financial
support (AMP 05/02934-4) and to C. H. Collins (IQ, Unicamp) for
manuscript language revision.
(S)-N-Tetradecanoyl-HSL (5) (GC-MS, EI, 70 eV): m/z 311 (M+•,
5), 156 (20), 143 (100), 125 (14), 102 (20), 83 (14), 57 (38), 43 (23).

Acyl-Homoserine Lactones from Methylobacterium
Journal of Natural Products, 2009, Vol. 72, No. 12 2129
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