Absolute configuration and antimicrobial activity of acylhomoserine lactones

Article abstract of DOI:10.1021/np800127b

(S)-N-Heptanoylhomoserine lactone is an uncommon acyl odd-chain natural product employed by many Gram-negative bacteria as a signaling substance in chemical communication mechanisms known as quorum sensing. The absolute configuration determination of the metabolite produced by the phytopathogen Pantoea ananatis Serrano is reported herein. As with all other substances of this class, the lactone moiety possesses S configuration, corroborating the hypothesis that it shares the same biosynthetic pathway as the (S)-N-hexanoylhomoserine lactone and also that some LuxI homologues can accept both hexanoyl- and heptanoyl-ACP as precursors. Evaluation of the antimicrobial activity of enantiomeric acylhomoserine lactones against three Gram-positive bacteria (Bacillus cereus, B. subtilis, and Staphylococcus aureus) revealed important features between absolute configuration and antimicrobial activity. The N-heptanoylhomoserine lactone was considerably less active than the 3-oxo derivatives. Surprisingly, non-natural (R)-N-(3-oxo-octanoyl)homoserine lactone was as active as the S enantiomer against B. cereus, while the synthetic racemic product was less active than either enantiomer.

Full text of DOI:10.1021/np800127b

1032
J. Nat. Prod. 2008, 71, 1032–1036
Absolute Configuration and Antimicrobial Activity of Acylhomoserine Lactones
Armando M. Pomini and Anita J. Marsaioli*
Chemistry Institute, State UniVersity of Campinas, CP 6154, CEP 13083-970, Campinas, Sa˜o Paulo, Brazil
ReceiVed February 26, 2008
(S)-N-Heptanoylhomoserine lactone is an uncommon acyl odd-chain natural product employed by many Gram-negative
bacteria as a signaling substance in chemical communication mechanisms known as quorum sensing. The absolute
configuration determination of the metabolite produced by the phytopathogen Pantoea ananatis Serrano is reported
herein. As with all other substances of this class, the lactone moiety possesses S configuration, corroborating the hypothesis
that it shares the same biosynthetic pathway as the (S)-N-hexanoylhomoserine lactone and also that some LuxI homologues
can accept both hexanoyl- and heptanoyl-ACP as precursors. Evaluation of the antimicrobial activity of enantiomeric
acylhomoserine lactones against three Gram-positive bacteria (Bacillus cereus, B. subtilis, and Staphylococcus aureus)
revealed important features between absolute configuration and antimicrobial activity. The N-heptanoylhomoserine lactone
was considerably less active than the 3-oxo derivatives. Surprisingly, non-natural (R)-N-(3-oxo-octanoyl)homoserine
lactone was as active as the S enantiomer against B. cereus, while the synthetic racemic product was less active than
either enantiomer.
Chemical communication is a phenomenon explored by many
different organisms and nowadays is one of the most prominent
research areas at the chemistry-microbiology interface. For almost
20 years, science has recognized the ability of different bacteria to
coordinate phenotype expression using signaling substances, a
crucial process for successful environment colonization in plants,
human beings, and other animal hosts. This multicellular-like
behavior is known as quorum sensing.1
Different compound classes are employed by bacteria as signaling
substances. Among Gram-positive bacteria, γ-butyrolactones play
a major role in antibiotic production and sporulation of the
Streptomyces species.2,3 Low molar mass peptides are employed,
for example, by Staphylococcus aureus to control virulence factor
expression.4 The Gram-negative bacterium Pseudomonas aerugi-
nosa employs quinolones in a complex cascade mechanism to
regulate elastase-encoding genes expression.5 However, the most
studied compounds in Gram-negative bacteria signaling mechanisms
are the acylhomoserine lactones (acyl-HSLs).1
from the aminoacid portion of the S-adenosyl methionine
precursor.9,10,12–15 From a chemical point of view, if both odd
and even acyl-HSLs share the same absolute configuration, the
hypothesis of acceptance of similar acyl-ACPs by some LuxI
homologues would have a firmer foundation.
In addition to the important role of acyl-HSLs in chemical
communication, alternative biological activities have been found.
These include interferences in eukaryotic organisms, such as
induction of apoptosis in macrophages and neutrophils with
important reflexes in immunological systems.16–18 The antimicrobial
activity of some acyl-HSLs against Gram-positive bacteria (even
though at high concentrations) is another important characteristic,
especially under Biofilm conditions, where the metabolites, cells,
and enzymes are much more concentrated.19 One of the most
studied substances of this class is (S)-N-(3-oxododecanoyl)-HSL
produced by P. aeruginosa, which can be toxic to several Gram-
positive bacteria, but not for the Gram-negative ones.19
The production of bactericidal acyl-HSLs by P. aeruginosa
confers a competitive advantage to this microorganism in lung
colonization, in relation to Staphylococcus aureus, in cystic fibrosis
patients. It is known that S. aureus is stepwise substituted by P.
aeruginosa in advanced lung diseases. Probably, the bactericidal
property of N-(3-oxododecanoyl)-HSL against S. aureus may
contribute to this process, since this metabolite was found in
relatively high concentrations (600 µM/L) in lung secretions of
affected patients in comparison with its production by P. aeruginosa
under planktonic conditions (5 µM/L).20,21
The antimicrobial activity of acyl-HSLs against S. aureus has
been extensively studied. Generally, acyl-HSLs with no chemical
substituents such as hydroxy or carbonyl groups at position 3 did
not show any activity against this microorganism, while 3-oxo-
acyl-HSLs with alkyl side chains ranging from 8 to 14 carbon atoms
were active at micromolar concentrations.20 The antimicrobial
activity of N-3-oxododecanoyl-HSL was attributed to interferences
in S. aureus quorum-sensing regulated phenotypes, including
important changes in cellular membrane.19,20 In spite of these new
findings, the importance of absolute configuration for antimicrobial
activity has not been mentioned previously.
There is a predominance of even-numbered acyl-HSLs assigned
to the major production of even-numbered fatty acids in microbial
cells. However, the number of odd-chain acyl-HSLs-producing
microorganisms is increasing, e.g., Serratia marcescens,6 Sinorhizo-
bium leguminosarum,7 Edwardisiella tarda,8 Erwinia psidii,9 and
the phytopathogen Pantoea ananatis,10 which produces N-hep-
tanoyl-HSL (1) together with large amounts of N-hexanoyl-HSL.
Long-chain acyl-HSLs such as N-tridecanoyl- and N-pentadecanoyl-
HSL have been found in marine bacteria (Alphaproteobacteria).11
It is worth pointing out that odd-chain derivatives always occur
in trace amounts together with large amounts of even-numbered
acyl-HSL-homologues. In S. leguminosarum, it was demonstrated
that a lower expression of the cinI gene results in smaller production
of three short-chain acyl-HSLs, namely, N-hexanoyl-, N-heptanoyl-,
and N-octanoyl-HSL, suggesting that these substances are produced
by the same acyl-HSL synthesizing protein (RhiI).7 This is very
strong evidence that both even- and odd-acyl-HSLs of similar acyl
length can share the same biosynthetic enzyme (LuxI homologue),
most probably due to the minor structural differences between
substrates (hexanoyl- and heptanoyl-ACP, for example). However,
the absolute configuration of an odd-numbered acyl-HSL at the
lactone moiety has not been reported before. The S absolute
configuration of even-numbered acylhomoserine lactones derives
Thus, we report herein the absolute configuration determination
of the acyl-odd-numbered (S)-N-heptanoylhomoserine lactone
produced in trace amounts by P. ananatis and also the importance
of absolute configuration for antimicrobial activity against Gram-
positive bacteria, including synthetic 3-oxoacyl-HSLs, which are
* Corresponding author. Tel: + 55 3521 3067. Fax: + 55 3521 3023.
E-mail: anita@iqm.unicamp.br.
10.1021/np800127b CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/09/2008

Acylhomoserine Lactone Absolute Configuration
Journal of Natural Products, 2008, Vol. 71, No. 6 1033
Scheme 1. (A) Synthesis of the N-Heptanoyl-HSL Racemic Mixture and the Enantiomers; (B) General Synthetic Procedure for
N-3-Oxo-acyl-HSLs (n ) 1, 5, or 7)
Table 1. Absolute Configuration Determination of Natural
(S)-N-heptanoyl-HSL Produced by Pantoea ananatis CCT 6481
by GC-FID (chiral column)
known to be more active than nonsubstituted acyl-HSLs.20 The
results are discussed in the face of the current theories.
Results and Discussion
R retention time S retention time
ee
(major)
N-heptanoyl-HSL
(min)
(min)
In a previous paper we reported the occurrence of three
acylhomoserine lactones from the phytopathogen P. ananatis
Serrano [(S)-N-hexanoyl-, N-heptanoyl-, and N-octanoyl-HSL, in
decreasing abundance].10 This pathogen was initially described in
pineapple rot disease (Ananas comosus L.).22 Nowadays, it also
represents an important drawback in onion (Allium cepa L.)
production in Georgia (USA)23 and has also been found to be a
pathogen in rice (Oryza satiVa L.),24 eucalyptus (Eucalyptus hyb.),25
and sorghumn (Sorghum sudanense S.).26
(() synthetic
60.87
61.05
61.06
1:1 peaks
97% S
>99% R
90% S
(S) synthetic
(R) synthetic
(S) natural
60.88
60.86
60.85
61.03
61.03
co-injection (()-synthetic
22% S
and natural producta
a Co-injection: 1:1 mixture of (()-N-heptanoyl-HSL (1 mg/mL) and
fraction M (0.02 mg/mL). Fraction M (total mass: 4.8 mg) contains
approximately 1.4% of 1.
In order to determine the absolute configuration of 1 produced
by P. ananatis, 8 L of conditioned cultivation media was extracted
with ethyl acetate. Purification by column chromatography and GC-
MS analyses allowed the detection of one fraction with trace
amounts of 1 eluting with DCM/EtOAc (3:1), sufficiently pure for
chiral column analysis. The natural product was characterized by
MS comparison and co-injection with a synthetic standard.10
Racemic and chiral synthetic standards were obtained from hep-
tanoic acid and (()- or (S)-R-amino-γ-butyrolactone using the
synthetic strategy previously reported (Scheme 1) and were fully
characterized by spectroscopic analyses (1H NMR, 13C NMR, IR,
mass spectra, optical rotation, and circular dichroism).9,12,28 Frac-
tions and extracts from P. ananatis were also evaluated with
bioreporter A. tumefaciens NTL4(pZLR4), and positive bioactivities
were observed in fractions containing the acylhomoserine lactone.
The absolute configuration determination was performed using
the chiral column Chirasil cyclodextrin (Chrompack). Analytical
conditions were optimized using the synthetic racemic product. The
S absolute configuration (90% ee) for the natural product was
determined by co-injection and retention time comparison with
racemic and chiral products. Results are shown in Table 1. Like in
even-numbered acyl-HSLs the S absolute configuration was ob-
served in compound 1, reinforcing the hypothesis that some acyl-
HSL synthesizing proteins (LuxI homologues) are not substrate
specific and may accept similar acyl-ACPs with minor differences
in their side chain length, minimizing the probability of the existence
of LuxI homologues specifically devoted to the synthesis of odd-
numbered acyl-HSL.
In spite of the importance of the acyl-HSLs bactericidal properties
under environmental conditions, the relevance of the absolute
configuration for the antimicrobial activity was not previously
reported. Therefore, we investigated the activities of the synthetic
racemic mixture and the S and R enantiomers of N-heptanoyl-, N-(3-
oxo-octanoyl)-, N-(3-oxo-dodecanoyl)-, and N-(3-oxo-tetradecanoyl)-
HSL against B. subtilis CCT 0089, B. cereus CCT 4060, and S.
aureus CCT 1295, looking for the most active substance. For this
purpose the desired substances were synthesized according to the
route shown in Scheme 1. Each product was fully characterized
by spectroscopic analyses.
In order to select the most appropriate substance/microorganism
set, a preliminary antimicrobial assay was carried out applying a
simple microtiter plate-based colorimetric assay dependent on the
reduction of the tetrazolium dye MTT [3-(4,5-dimethylthiazol-2-
yl) 2,5-diphenyl tetrazolium bromide] to its formazan and on
microorganism growth.30
In general, B. subtilis was the sole microorganism susceptible
to N-heptanoyl-HSL, at 500 ppm. This microorganism seemed to

1034 Journal of Natural Products, 2008, Vol. 71, No. 6
Pomini and Marsaioli
was employed as carrier gas (1 mL/min). The injector and detector
temperatures were 220 and 250 °C, respectively. Routinely, samples
were dissolved in highly pure EtOAc (1 mg/mL) and injected in split
mode (1/100). The temperature program for N-(3-oxo-octanoyl)-HSL
was 50-180 °C (1 °C/min) held for 40 min at 180 °C. For N-heptanoyl-
HSL the temperature program was 50-180 °C (2 °C/min) held for 5
min at 180 °C.
GC-MS Analyses. GC-MS analyses (70 eV) were carried out in an
Agilent 6890 chromatograph coupled to a Hewlett-Packard 5973 mass
detector. Routine trace analyses were carried out with a capillary Si
gel column (HP5 or MDN-5S, 30 m × 0.25 mm × 0.25 µm), with the
temperature program 100-290 °C (10 °C/min) held at 290 °C for 10
min, using helium (1 mL/min) as carrier gas. Samples were injected in
splitless mode (1 µL) in ethyl acetate (1 mg/mL), with the injector at
250 °C.
Table 2. Results of Preliminary Antimicrobial Assays of Some
Acyl-HSLs against Gram-Positive Bacteria: Range of Enantiomers
and Racemic Product Activities
concentration range for growth inhibition
(µg/mL)
solutions
B. cereus
B. subtilis
S. aureus
a
a
a
water/DMSO 20%
cloranfenicol
N-heptanoyl-HSL
15.62-3.91
3.91
500
31.25-7.81
a
a
N-(3-oxo-octanoyl)-HSL
N-(3-oxododecanoyl)-HSL
N-(3-oxotetradecanoyl)-HSL 500
125-62.5
500-125
125-62.5
250-125
500-250
500
500
500
a Inactive even at the highest concentration assayed (µg/mL).
Cultivation of P. ananatis and Detection of (S)-N-Heptanoylho-
moserine Lactone. The inoculum was prepared in a test tube containing
liquid NB medium (10 mL) incubated in BOD at 30 °C without shaking.
After 24 h, the inoculum was transferred to a 2 L Erlenmeyer containing
NB medium (1 L) for incubation at 28 °C for 24 h, under shaking
(110 rpm). After the incubation period, the medium was centrifuged
under refrigeration (18 °C, 5000 rpm, 20 min). The aqueous layer was
extracted with EtOAc (3 × 500 mL), and the combined organic layers
were extracted with distilled water (500 mL) and evaporated under
reduced pressure at 40-45 °C. This procedure was repeated eight times,
yielding 557.0 mg of crude extract. This extract was purified by silica
gel column chromatography (18 g of silica; column diameter 2 cm),
with n-hexane, DCM, EtOAc, and MeOH in increasing polarity as
eluents. Fractions were grouped by TLC similarities and analyzed by
GC-MS. Some fractions were also assayed with A. tumefaciens
bioreporter. The metabolite (S)-N-heptanoyl-HSL was identified in trace
amounts (<1%, by GC-MS chromatogram peak area integration) in
the complex fraction M with 4.8 mg of total mass, eluting at DCM/
EtOAc (3:1) polarity.
be the most susceptible against these substances, while S. aureus
was the most resistant (Table 2). The 3-oxo-acyl-HSLs were more
active than nonsubstituted N-heptanoyl-HSL, while N-(3-oxo-
octanoyl)-HSL was the most active. All substances were consider-
ably less active than the positive control (cloramphenicol).
Therefore for better accuracy the quantitative absorbance assay
using spectrophotometric absorbance measurements (A at 650 nm)19
was chosen together with the B. cereus/N-(3-oxo-octanoyl)-HSL
set. The results are shown in Figure 2. No statistically significant
activity difference (medium SD ) (0.012) between the R and S
enantiomers was observed. This result, per se, is interesting, as the
non-natural (R)-acyl-HSL is about as active as the natural S
enantiomer. Another remarkable result is that the racemic product
is, in general, less active than either of the pure enantiomers.
These results could fit into a model of two independent bacterial
receptors for (R)- or (S)-acyl-HSL, each with a specific affinity to
one enantiomer. Therefore, in the racemic product each enantiomer
would be diluted by half, leading to an almost 50% activity
decrease. The hypothesis of the enantiomers being each other’s
antagonist should also be considered; however the results are more
consistent with the first hypothesis.
Bioassays with Agrobacterium tumefaciens NTL4(pZLR4). The
ꢀ-galactosidase bioassay for detection of acylhomoserine lactones was
carried out with extracts and fractions from P. ananatis cultivation
media, as well as with synthetic products. It was performed as
previously described.9
In summary, several findings concerning the absolute configu-
ration of acylhomoserine lactones, including chiral characterization
of a rare metabolite and also the importance of chirality for
antimicrobial activity against Gram-positive bacteria, have been
reported. Other cross-species correlations involving acylhomoserine
lactones and important Brazilian phytopathogenic bacteria are
currently under investigation.
Synthesis of Acylhomoserine Lactones. General Procedure for
N-Heptanoylhomoserine Lactone Enantiomers.28 To a round-bot-
tomed flask (5 mL) with a magnetic stirrer were added ultrapure Milli-Q
water (2.5 mL), Et3N (0.105 mmol), (S)-, (R)-, or (()-R-amino-γ-
butyrolactone hydrobromide (or hydrochloride) (0.105 mmol), and
heptanoic acid (0.157 mmol). Then, 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride (0.157 mmol) was added. The reaction
mixture was stirred at room temperature. After 24 h, it was extracted
with ethyl acetate (3 × 10 mL), and the combined organic layers were
extracted with 5% aqueous NaHCO3 (2 × 6 mL), 1 mol/L KHSO4 (1
× 6 mL), and saturated NaCl solution (1 × 6 mL). The solvent was
evaporated under reduced pressure, yielding 1 as a white solid.
(()-N-Heptanoylhomoserine Lactone. Yield: 64%. GC-MS (EI,
70 eV): m/z 213 (2), 156 (15), 143 (100), 125 (20), 113 (13), 102 (12),
101 (16), 100 (6), 85 (16), 57 (50). IR (KBr): 3315, 2955, 2858, 1776,
Experimental Section
Materials and Methods. NMR analyses were carried out on a Varian
Inova-500 or a Bruker Gemini-300 spectrometer, using CDCl3 as solvent
and TMS as internal reference. The IR spectra were recorded on a
Bomen Michelson MB spectrophotometer, using KBr (Merck) pellets
as sample support. A Perkin-Elmer 341 polarimeter equipped with a
sodium lamp was employed for [R]20 analyses, using MeOH (HPLC
D
1
1646, 1545, 1384, 1173, 1013 cm-1. H NMR (499.88 MHz, CDCl3,
grade) as solvent. Microplates (96-well) were analyzed with a FlashScan
530 Analytik Jena spectrophotometer. Column chromatography puri-
fications were carried out with Si gel (Acros, 0.035-0.070 mm) and
high-quality solvents. All solvents were treated before using, usually
by drying with anhydrous Na2SO4 followed by distillation with Vigreux
columns. For synthetic purposes, DCM was dried by refluxing and
distilled over CaH2. The reagents were from Aldrich or Sigma.
Microorganisms. The microorganisms Pantoea ananatis CCT 6481
()ATCC 33244, type strain), Bacillus subtilis CCT 0089, B. cereus
CCT 4060, and Staphylococcus aureus CCT 1295 were maintained in
slants containing solid nutrient broth medium (OXOID, 20 g/L), under
refrigeration at 5 °C. The bioreporter Agrobacterium tumefaciens
NTL4(pZLR4) was maintained in Luria-Bertani medium (LB: 1%
peptone, 0.5% yeast extract, 0.5% NaCl) supplemented with gentami-
cine (50 µg/mL) and was provided by Dr. Welington L. Arau´jo
(originally from Dr. Stephen K. Farrand, University of Illinois). Miller-
Hinton medium was from OXOID.
TMS): δ 0.88 (t, 3H, J 7.3 Hz, H-7′); 1.29 (m, 6H, H-4′, H-5′, H-6′);
1.64 (quintet, 2H, J 7.6 Hz, H-3′); 2.14 (m, 1H, H-4); 2.25 (t, 2H, J
8.9 Hz, H-2′); 2.84 (m, 1H, H-4); 4.28 (ddd, 1H, J 11.3; 9.5; 5.8 Hz,
H-5a); 4.47 (t, 1H, J 8.9 Hz, H-3); 4.56 (ddd, 1H, J 11.3; 8.6; 5.8 Hz,
H-5b); 6.15 (d, NH, J 3.7 Hz). 13C NMR (125.71 MHz, CDCl3, TMS):
δ 14.0 (C-7′); 22.4 (C-6′); 25.3 (C-3′); 28.8 (C-5′); 30.5 (C-4); 31.4
(C-4′); 36.1 (C-2′); 49.2 (C-3); 66.1 (C-5); 173.7 (C-1′); 175.5 (C-2).
(S)-(-)-N-Heptanoylhomoserine Lactone. Chemical yield and GC-
MS, IR, 1H, and 13C data are identical to those observed for the racemic
compound. [R]20 -18 (c 0.42 MeOH).
D
(R)-(+)-N-Heptanoylhomoserine Lactone. Chemical yield and GC-
MS, IR, 1H. and 13C data are identical to those observed for the racemic
compound. [R]20 +17 (c 0.42 MeOH).
D
General Procedure for N-3-Oxo-acylhomoserine Lactones.29
Meldrum Acid Derivatives. A round-bottomed flask (50 mL) contain-
ing dry DCM (20 mL), the fatty acid (hexanoic, decanoic, or dodecanoic
acid, 2.0 mmol), 4-DMAP (2.1 mmol), DCC (2.2 mmol), and Meldrum
acid (2,2-dimethyl-1,3-dioxane-4,6-dione; 2.0 mmol) was stirred under
a nitrogen atmosphere for 24 h. Then, the reaction medium was filtered
Chiral GC-FID Analyses. GC-FID analyses were conducted in an
Agilent 6890 chromatograph using the chiral column Chrompack
Chirasil CD (25 m × 0.25 mm × 0.25 µm). Highly pure hydrogen

Acylhomoserine Lactone Absolute Configuration
Journal of Natural Products, 2008, Vol. 71, No. 6 1035
Figure 1. (A) (S)-N-Heptanoyl-HSL produced by P. ananatis. (B) Antimicrobial (S)-N-(3-oxo-octanoyl)-HSL.
(t, 3H, J 6.2 Hz, H-12′); 1.26 (m, 12H, H-11′, H-10′, H-9′, H-8′, H-7′,
H-6′); 1.58 (quintet, 2H, J 7.0 Hz, H-5′); 2.25 (m, 1H, H-4); 2.53 (t,
2H, J 7.3 Hz, H-4′); 2.75 (m, 1H, H-4); 3.47 (s, 2H, H-2′); 4.28 (ddd,
1H, J 11.0; 9.5; 6.2 Hz, H-5); 4.47 (td, 1H, J 9.1; 1.2 Hz, H-3); 4.60
(ddd, 1H, J 11.4; 8.8; 6.7 Hz, H-3); 7.69 (d, NH, J 6.2 Hz). 13C NMR
(75.45 MHz, CDCl3, TMS): δ 14.2 (C-12′); 22.7 (C-11′); 31.87 (C-
10′); 29.0 (C-9′); 29.3 (C-8′); 29.38 (C-7′); 29.41 (C-6′); 23.4 (C-5′);
43.9 (C-4′); 48.2 (C-2′); 65.9 (C-5); 29.8 (C-4); 49.1 (C-3); 166.2 (C-
1′); 174.6 (C-2); 206.3 (C-3′).
(S)-(-)-(3-oxo-dodecanoyl)homoserine Lactone. Global yield and
1
IR, H NMR, and 13C NMR data are identical to those observed for
the racemic compound. [R]20 -19 (c 0.50 MeOH).
D
(R)-(+)-(3-Oxo-dodecanoyl)homoserine Lactone. Global yield and
1
IR, H NMR, and 13C NMR data are identical to those observed for
the racemic compound. [R]20 +15 (c 0.50 MeOH).
D
(()-N-(3-Oxo-tetradecanoyl)homoserine Lactone, 4. Global yield:
46.8%. IR (KBr): 3298, 2920, 2850, 1772, 1716, 1641, 1549, 1178,
1015, 721 cm-1. 1H NMR (300.06 MHz, CDCl3, TMS): δ 0.88 (t, 3H,
J 6.7 Hz, H-14′); 1.26 (m, 16H, H-13′, H-12′, H-11′, H-10′, H-9′, H-8′,
H-7′, H-6′); 1.58 (quintet, 2H, J 7.0 Hz, H-5′); 2.25 (m, 1H, H-4);
2.53 (t, 2H, J 7.3 Hz, H-4′); 2.74 (m, 1H, H-4); 3.47 (s, 2H, H-2′);
4.27 (ddd, 1H, J 11.0; 9.2; 6.1 Hz, H-5); 4.48 (td, 1H, J 9.2; 1.2 Hz,
H-3); 4.62 (ddd, 1H, J 11.0; 8.9; 7.0 Hz, H-5); 7.73 (d, NH, J 6.4 Hz).
13C NMR (75.45 MHz, CDCl3, TMS): δ 14.1 (C-14′); 22.7 (C-13′);
23.4 (C-12′); 29.0 (C-11′); 29.3 (C-9′); 29.4 (C-8′); 29.4 (C-7′); 29.6
(C6′, C5′); 29.7 (C-4); 31.87 (C-10′); 43.9 (C-4′); 48.2 (C-2′); 49.0
(C-3); 65.9 (C-5); 166.5 (C-1′); 175.0 (C-2); 206.6 (C-3′).
Figure 2. Antimicrobial activity of N-(3-oxo-octanoyl)-HSL against
B. cereus evaluated by turbidimetry. Legend: ((): 4; (R): b; (S):
]; cloramphenicol: 9. Blank absorbance: 0.724 ( 0.01 (water/
DMSO 20%). The results represent a quadruplicate average.
through cotton wool, and the organic layer was evaporated under
reduced pressure, producing a yellow oil, which was dissolved in ethyl
acetate (20 mL). This was extracted with aqueous 2 mol/HCl (3 × 10
mL) and distilled H2O (1 × 10 mL) and then dried over anhydrous
MgSO4. The organic layer was filtered and evaporated under reduced
pressure. All Meldrum acid derivatives were used as fast as possible
in the next synthetic step.
(S)-(-)-(3-Oxo-tetradecanoyl)homoserine Lactone. Global yield
N-(3-Oxo-acyl)homoserine Lactones. To a round-bottomed flask
(100 mL) equipped with a refluxing condenser and magnetic stirrer
were added acetonitrile (22.5 mL, HPLC grade), the Meldrum acid
derivative (0.75 mmol), Et3N (1.2 mmol), and (S)-, (R)-, or (()-R-
amino-γ-butyrolactone hydrobromide (or hydrochloride) (0.75 mmol).
The reaction mixture was stirred at room temperature for 2 h and then
under reflux for 3 h. Then, the reaction medium was evaporated under
reduced pressure, and the white solid remaining was dissolved in ethyl
acetate and methanol (20/5 mL). The organic layer was washed with
saturated aqueous NaHCO3 solution (3 × 10 mL), 1 mol/L KHSO4 (3
× 10 mL), and saturated NaCl (3 × 10 mL), dried over anhydrous
MgSO4, filtered, and evaporated, yielding the N-(3-oxo-acyl)homoserine
lactone as a pale yellow solid, which was further purified by Si gel
column chromatography (12 g, Acros, 0.035-0.070 mm particle size;
2 cm diameter column) with hexane, DCM, and EtOAc in increasing
polarity as eluents. The acyl-HSLs eluted at DCM/EtOAc (3:1) polarity.
(()-N-(3-Oxo-octanoyl)homoserine Lactone, 2. Global yield: 28%.
GC-MS (EI, 70 eV): m/z 241 (1), 224 (24), 185 (7), 143 (25), 102 (7),
99 (31), 56 (100). IR (KBr) 3258, 2934, 1777, 1716, 1646, 1546, 1173,
1019, 599 cm-1. 1H NMR (300.06 MHz, CDCl3, TMS): δ 0.89 (t, 3H,
J 7.0 Hz, H-8′); 1.30 (m, 4H, H-6′, H-7′); 1.59 (quintet, 2H, J 7.3 Hz,
H-5′); 2.28 (m, 1H, H-4); 2.53 (t, 2H, J 7.3 Hz, H-4′); 2.75 (m, 1H,
H-4); 3.47 (s, 2H, H-2′); 4.28 (ddd, 1H, J 11.0, 9.1; 6.2 Hz, H-5a);
4.47 (td, 1H, J 8.2; 1.4 Hz, H-3); 4.60 (ddd, 1H, J 11.0; 8.8; 6.2 Hz,
H-5b); 7.70 (d, NH, J 5.1 Hz). 13C NMR (75.45 MHz, CDCl3, TMS):
δ 13.8 (C-8′); 22.32 (C-7′); 23.0 (C-6′); 31.1 (C-5′); 29.8 (C-4); 43.8
(C-4′); 48.1 (C-2′); 49.0 (C-3); 65.8 (C-5); 166.4 (C-1′); 174.8 (C-2);
206.5 (C-3′).
1
and GC-MS, IR, H NMR, and 13C NMR data are identical to those
observed for the racemic compound. [R]20 -14 (c 0.50 MeOH).
D
(R)-(+)-(3-Oxo-tetradecanoyl)homoserine Lactone. Global yield
1
and GC-MS, IR, H NMR, and 13C NMR data are identical to those
observed for the racemic compound. [R]20 +11 (c 0.50 MeOH).
D
Preliminary Antimicrobial Assays: Colorimetric. The preliminary
bioassays were performed against the Gram-positive bacteria S. aureus
CCT 1295, B. cereus CCT 4060, and B. subtilis CCT 0089. The inocula
were prepared in slants with solid NB medium (3 tubes each) and grown
in BOD at 30 °C for 24 h. After growing, the cells were removed with
a flamed wire loop and transferred to autoclaved distilled H2O (10 mL)
tubes until the suspension reached 3 × 108 cells/mL according to the
MacFarland scale. Then 5 mL of this suspension was added to 50 mL
of Miller-Hinton medium, and the mixture was pipetted (100 µL for
each well) to 96-well plates (the plates were previously exposed to a
UV lamp for 1 h). The solutions to be assayed (100 µL for each well)
were added to the top column wells, homogenized, and pipetted in the
following lines (50% dilution factor). The test was performed in
duplicate. A solution of DMSO in H2O (20%) was used as negative
control (blank). Cloramphenicol (1000 µg/mL, positive control) and
synthetic acylhomoserine lactones (1000 µg/mL) were prepared in
DMSO (20%) in distilled H2O. After adding the reagents, the microtiter
plates were incubated at 30 °C for 24 h and analyzed by adding 100
µL of MTT solution (0.025% in H2O). The purple color was indicative
of normal microbial development, while growth inhibition was observed
in the yellow wells.
Quantitative Antimicrobial Assays: Absorption. The quantitative
bioassay was performed against B. cereus CCT 4060 with each N-(3-
oxo-octanoyl)-HSL enantiomer and with the racemic mixture. The
inoculum was prepared by adding 500 µL of a B. cereus 3 × 108 cells/
mL suspension to 50 mL of Miller-Hinton medium. The bioassays were
performed in gamma-sterilized, DNA-free plates. A solution of H2O/
DMSO (4:1) (100 µL) was added to each well. Then, the solutions to
be assayed were added to the top wells (100 µL), homogenized, and
pipetted in the following lines (50% dilution factor). Finally, the
inoculum was added (100 µL). As blank, DMSO (20%) in H2O was
employed. The cloramphenicol and acylhomoserine lactones stock
solutions were also prepared in DMSO (20%)/distilled H2O (2000 µg/
(S)-(-)-(3-Oxo-octanoyl)homoserine Lactone. Global yield and
GC-MS, IR, 1H NMR, and 13C NMR data are identical to those observed
for the racemic compound. [R]20 -17 (c 0.53 MeOH), 94% ee [GC-
D
FID(chiral column)].
(R)-(+)-(3-Oxo-octanoyl)homoserine Lactone. Global yield and
GC-MS, IR, 1H NMR, and 13C NMR data are similar to those observed
for the racemic compound. [R]20 +16 (c 0.51 MeOH), 99% ee [GC-
D
FID(chiral column)].
(()-N-(3-Oxo-dodecanoyl)homoserine lactone, 3. Global yield:
39.5%. IR (KBr): 3298, 2925, 2850, 1782, 1721, 1641, 1546, 1382,
1
1178, 1019, 721 cm-1. H NMR (300.06 MHz, CDCl3, TMS): δ 0.87

1036 Journal of Natural Products, 2008, Vol. 71, No. 6
Pomini and Marsaioli
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Acknowledgment. The authors acknowledge FAPESP (05/02934-
4) for financial support and Prof. Carol H. Collins (IQ/UNICAMP) for
text revision.
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NP800127B