Rational design and synthesis of new quorum-sensing inhibitors derived from acylated homoserine lactones and natural products from garlic

Article abstract of DOI:10.1039/b415761c

Parallel solution-phase synthesis of sulfide AHL analogues (10a-s) by one-pot or a sequential approach is reported. The corresponding sulfoxides 13a-e and sulfones 14a-e were prepared to expand the diversity of the 19-member array of sulfides 10a-s. Likewise, dithianes 12a-c were prepared with similarity both to sulfides 10a-s and to bioactive structures from garlic. Design and biological screening of all compounds presented in this work targeted inhibition of quorum-sensing comprising competitive inhibition of transcriptional regulators LuxR and LasR. The design was based on critical interactions within the binding-site and structural motifs in molecular components isolated from garlic, 7 and 8, shown to be quorum-sensing inhibitors but not antibiotics. A potent quorum-sensing inhibitor N-(heptylsulfanylacetyl)-L-homoserine lactone (10c) was identified. Together with data collected for the other analogues, the resulting structure - activity relationship led to a hypothesis in which competitive binding was assumed.

Full text of DOI:10.1039/b415761c

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A R T I C L E
Rational design and synthesis of new quorum-sensing inhibitors
derived from acylated homoserine lactones and natural products from
garlic
Tobias Persson,a,b Thomas H. Hansen,a,c Thomas B. Rasmussen,c Mette E. Skindersø,c
Michael Givskovc and John Nielsen*a
a Department of Natural Sciences, Bioorganic Chemistry Section, Royal Veterinary and
Agricultural University, Frederiksberg C, DK-1871, Denmark. E-mail: jn@kvl.dk;
Fax: +45 3528 2398; Tel: +45 3528 2428
b Department of Chemistry, Technical University of Denmark, Kgs. Lyngby, DK-2800, Denmark
c Centre for Biomedical Microbiology, BioCentrum, Technical University of Denmark, Kgs.
Lyngby, DK-2800, Denmark
Received 12th October 2004, Accepted 29th October 2004
First published as an Advance Article on the web 1st December 2004
Parallel solution-phase synthesis of sulfide AHL analogues (10a–s) by one-pot or a sequential approach is reported.
The corresponding sulfoxides 13a–e and sulfones 14a–e were prepared to expand the diversity of the 19-member array
of sulfides 10a–s. Likewise, dithianes 12a–c were prepared with similarity both to sulfides 10a–s and to bioactive
structures from garlic. Design and biological screening of all compounds presented in this work targeted inhibition of
quorum-sensing comprising competitive inhibition of transcriptional regulators LuxR and LasR. The design was
based on critical interactions within the binding-site and structural motifs in molecular components isolated from
garlic, 7 and 8, shown to be quorum-sensing inhibitors but not antibiotics. A potent quorum-sensing inhibitor
N-(heptylsulfanylacetyl)-L-homoserine lactone (10c) was identified. Together with data collected for the other
analogues, the resulting structure–activity relationship led to a hypothesis in which competitive binding was assumed.
between the biofilms of the wild-type and those formed by QS
Introduction
mutants were observed.3 Biofilms produced by P. aeruginosa
N-Acyl-L-homoserine lactones (AHLs) play an important role in
are much more tolerant to treatment with antibiotics than
the pathogenicity of many gram-negative bacteria. These AHLs
planktonic cells, hence, infections by bacteria in the biofilm
are signal molecules that accumulate during cell growth in a bac-
mode are difficult to eradicate.4 Blocking the QS receptors with
terial population. At a certain threshold level, a concentration-
dependent burst of target gene expression is mediated in a
3 was recently shown to increase biofilm sensitivity to treatment
with tobramycin.5 Much work has been done on finding such
process termed quorum-sensing (QS).1 N-(3-Oxododecanoyl)-
antagonists (V. fischeri ,6,7 Chromobacterium violaceum,8 and
L-homoserine lactone (1, Fig. 1) forms a complex with LasR,
Agrobacterium tumefaciens9) but strikingly few, considering the
a member of the LuxR (Vibrio fischeri) family of transcription
clinical impact of P. aeruginosa, employed LasR as the target.10
factors. LasR controls multiple target genes in Pseudomonas
One possible explanation is that LasR is less susceptible to
aeruginosa, including biofilm formation and a second QS regu-
structural variations of the signal molecule, 1. In fact, 1 has
latory circuit denoted rhl. The corresponding LasR homologue,
been shown to inhibit both RhlR11 and LuxR,6 whereas the
RhlR, responds to the concentration of another signalling
signal molecules of RhlR and LuxR do not inhibit LasR.
molecule, N-butanoyl-L-homoserine lactone (2, Fig. 1).
Likewise, experience from previous studies12,13,14 on antagonist
identification suggests lower discrimination towards structural
modifications of the homoserine lactone (HSL) moiety in
ligands of LuxR, as compared to LasR. However, there are
excellent examples where the las/rhl cascade was blocked by
non-HSL based molecules,15 especially an in vivo experiment
involving 3 showing attenuation of pathogenicity in a mouse
model.5 Brominated ylidenebutenolide (3) is a biomimetic of
1 and a synthetic analogue of naturally occurring fimbrolide,
4, which is one out of many analogous secondary metabolites
produced by red seaweed Delisea pulchra (Fig. 1). QS inhibitory
activity has been documented for several of these.16
Due to lack of structural information of target receptors,
most inhibitors have primarily been developed based on AHL-
Fig. 1 Signal molecules N-(3-oxododecanoyl)-L-homoserine lactone
(1) and N-butanoyl-L-homoserine lactone (2), and QS inhibitors
(5Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone (3) and (1ꢀR, 5Z)-
4-bromo-3-(1ꢀ-hydroxybutyl)-5-(bromomethylene)-2(5H)-furanone (4).
like structures. Even though pharmacophoric elements have
been suggested, these do not deviate much from the structural
elements revealed by 1.17 Only recently, a 3-D pharmacophore
of the target receptor could be created from a crystal structure
of TraR (A. tumefaciens, Fig. 2).18 Even though the sequence
alignment between TraR and LasR shows great dissimilarities,
all amino acid residues forming hydrogen bonds to the ligand;
Tyr53, Trp57, Asp70, Ala38 (glycine in LasR) and Thr129 (serine
in LasR), are conserved between the two proteins. The two
The involvement of QS in biofilm formation was originally
reported for P. aeruginosa. A mutant unable to produce 1
formed flat and undifferentiated biofilms when compared to
the wild-type or a mutant unable to produce 2, which formed
characteristic microcolonies.2 In other studies, no differences
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 – 2 6 2
2 5 3
©

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elements by Maly et al.,24 has been successfully applied before.25
This possibility was also investigated here.
Results and discussion
In this study we wished to combine rational design with bioactive
scaffolds from natural products. We anticipated that such natural
compounds with quorum-sensing inhibitory activity could be
isolated from garlic. In order to apply these structural motifs to
the rationally derived compounds 10a–e, design and synthesis
of the subsequent analogues were performed side-by-side with
the isolation and identification of non-toxic quorum-sensing
inhibitors from garlic.
Identification of substances in garlic responsible for a QS
inhibitory effect
Toluene extracts of garlic were shown to inhibit LuxR- and
LasR-based QS systems.26 Through bio-assay guided fraction-
ation, six compounds (6a–d, 7 and 8, Fig. 3) were identified by
GC-MS analysis and NMR spectroscopy, that inhibited QS in
a LuxR monitor system. This was also confirmed by screening
synthetic 6a–d, 7 and 8, which are known components from
garlic and obtained readily either from commercial sources (6a
and 6b) or by known synthetic procedures (6c, 6d, 7 and 8).27,28
6a–d antagonized LuxR but they were also toxic to the bacteria.
More significantly, compounds 7 and 8 possessed QS activity
exclusively, albeit only in the LuxR monitor system.
Fig. 2 Ribbon diagram of residues 34–139 of TraR (PDB entry [1L3L]
reproduced with Swiss-Pdb viewer v37)[#346] in an active conformation
showing hydrogen bonds (green) inside the binding-site between the
ligand (N-[3-oxooctanoyl]-L-homoserine lactone) and five amino acid
residues.18
latter interactions are mediated by a water molecule inside the
binding pocket formed between a b-sheet (red) and 4 a-helices
(blue). Ala38 and Thr129 are positioned on the b-sheet together
with some hydrophobic residues responsible for van der Waals
contacts on one side of the acyl chain. Likewise, on the opposite
side of the acyl chain are lipophilic residues belonging to the
a-helices. One striking deviation is Phe62 in TraR, which is
not found in LasR. This residue is found near the end of the
acyl chain, presumably deciding which AHLs to reject, since
most of the AHL signal molecules differ only by length and
functionality of the acyl chain and all embrace a lactone moiety.
Several questions can be raised on how much this part of the
cavity in the receptor changes when transforming into an active
conformation: Are the oxygen atoms necessary for inhibition
of the receptor or only for activation? Is the butyrolactone or
butenolide a prerequisite? If not, can this be combined with other
structural elements known to suppress the active conformation?
Antagonists of LasR, lacking the potential to interact with the
receptor through a 3ꢀ-oxo-group (as in 1) have been identified.
These analogues had low inhibitory activity10 but the reason
is not obvious from the model used. Inhibition was measured
as a degree of competition for the binding site, not the actual
quenching of QS. The general perception of QS inhibition needs
to be further unified to eliminate some of these uncertainties.
The body of literature on QS covers many ways to
synthesize AHLs and other analogous structures, both by
solution-phase19,20 and solid-phase21 techniques. Evidently, the
L-homoserine lactone (HSL) moiety has a central role. Herein,
we present an improved protocol for the synthesis of L-
homoserine lactone hydrobromide (5). A number of procedures
are also included, whereby substances of the general formulae
10 were obtained.
It is known from literature that garlic (genus Allium
sativum L.) possess antibiotic activity.22 The activity has mainly
been attributed to 2-propenesulfenic acid, which in garlic gives
allicin, a thiosulfinate with antibiotic properties.23 During this
study, we have investigated in more details if garlic extracts
contained compounds, which solely inhibit QS, void of antibiotic
activity, which, to the best of our knowledge, has not previously
been investigated. Indeed, some individual molecular compo-
nents inhibiting QS were identified after isolation from garlic
extracts. The components differed structurally from 10 inferring
affinity for another binding-site than for the AHLs, perhaps a
nearby site. Grafting of two individual low-affinity binders to
yield one ligand with high affinity, referred to as linked binding-
Fig. 3 Garlic components found to be antibiotics (6a–d) and QS
antagonists (6a–d, 7 and 8).
Notably, allicin, which is known to possess antibacterial
activity,29,30 did not show inhibitory activity against any of the
QS systems tested (LuxR and LasR).
Synthesis and characterization of QS inhibitors
From the vast collection of QS attenuators reported in liter-
ature, those containing an HSL-moiety are in great majority.
Therefore, most protocols published to date involve a carboxylic
acid being coupled to HSL. The modest representation of
compounds like 10 in literature offer no exception.31,32 Therefore,
to be able to vary R1 and R2 in a one-step procedure, a new
strategy was developed. The chosen strategy provides the N-acyl
part first and then diverts into 10a–q. Since analogues 10a–m
were all derivatives of HSL, they could be produced from N-
bromoacetyl homoserine lactone (9) by method A (Scheme 1).
When 10c and 10n–q were synthesized, method B was applied.
This one-pot approach allowed for high throughput synthesis
of any sulfanyl-acetamides (10) from thiols and amines. All
amines used were commercially available either as halide salts
or their free amines. However, due to the large quantities needed
of L-homoserine lactone hydrobromide (5), we developed an
optimized synthetic protocol. S-Alkylation of L-methionine with
bromoacetic acid and then immediate intramolecular nucle-
ophilic substitution (lactonization) in acidic solution,33 gave 5 by
precipitation with hydrobromic acid. In our hands, the formerly
described precipitation process, with hydrochloric acid,34 was
quite inefficient and it is likely that some product was lost at that
stage. The optimized yield was 81% and the next step, addition
of bromoacetyl bromide to L-homoserine lactone hydrobromide
2 5 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 – 2 6 2

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Table 1 Yield of products 10a–q using the sequential method (method
A) or the one-pot method (method B)
R1
R2
Method Product Yield/%a
A
A
B
10a
10b
10c
10d
10e
10f
10g
10h
10i
32
47
46
25
53
62
25
64
64
A
A
A
A
A
A
Scheme 1 Reagents and conditions: (i) bromoacetyl bromide, TEA,
◦
CH2Cl2, −78 C → RT, 70%; (ii) R2-SH, TEA, EtOH, RT; (iii)
bromoacetyl bromide, TEA, CH2Cl2, −20 ◦C → RT then R2-SH, TEA;
(iv) (COCl)2, DMSO, CH2Cl2, −78 ◦C.
(5), gave 9 in 70%.35 As a consequence of these high yields,
products 10a, 10b and 10d–m were furnished in a satisfying
overall yield (20–56%) from L-methionine in three steps (Table 1
gives the yields of 10a–q starting from the amines). Analogues
10e–m were all synthesized by method A modified by omitting
the washing procedure. Less satisfactory yields were obtained for
analogues 10c and 10n–q using method B (3–46%). Method B
also gave longer reaction times, but was more convenient during
synthesis of analogues 10n–q, due to variations in R1 (starting
materials for 10n: cyclopentylamine, 10o: cyclohexylamine,
10p: trans-2-aminocyclopentanol hydrochloride, 10q: trans-2-
aminocyclohexanol hydrochloride). Two other analogues, 10r
and 10s, were derived from 10p and 10q by Swern oxidation
(Scheme 1). The presence of a sulfide resulted in complications
reflected in the yields 34 and 44%, respectively.
A
10j
64
It is a well-known tactic in medicinal chemistry to combine
several discrete bioactive structural motifs into a larger, more
potent pharmacophore.24 Contemplating garlic component 7,
a second class of analogues, 12a and 12b, conformationally
restricted isosteres of 10, were synthesized from 11a and 11b
(Scheme 2). These starting materials were accessible through
hydrolysis of the corresponding esters,36 commercially available
or synthesized according to a published procedure.37 Synthesis
of 12c served as a model system for optimization of the coupling
reaction and only this product was stable enough for purification
by silica gel flash chromatography, whereas both 12a and 12b had
to be isolated by preparative RP-HPLC.
A
A
A
10k
10l
69
69
64
10m
B
B
B
10n
10o
10p
27
37
3
B
10q
6
a All yields are based on the amines or ammonium salts used as
starting material and the products obtained after purification by flash
chromatography.
Scheme 2 Reagents and conditions: (i) isobutyl chloroformate, TEA,
CH2Cl2, 0 ◦C.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 – 2 6 2
2 5 5

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Table 2 QS inhibition of LuxR and LasR controlled gene expression
with 3 as reference
Sulfinyl- and sulfonyl functionalities are isosteres of ketones
and have analogous reactivity to sulfides and ketones (acidic a-
proton). These inherent properties made analogues of 10 with
such functionalities an interesting target. 13a–e and 14a–e were
produced by oxidation of the corresponding sulfides (10a–e)
with m-CPBA (Scheme 3). 13a–e were obtained as mixtures
of diastereomers and their ratio determined by H NMR. The
diastereomers could not be resolved by the standard RP-HPLC
protocol.
Analogue
LuxRa
LasRb
2
Maximum inductione
3
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
—
+
+
+
—
+
—
—
—
—
—
10%
NAd
NAd
NAd
NAd
NAd
NAd
67%
33%
13%
10%
33%
50%
50%
67%
67%
67%
50%
25%
40%
50%
33%
50%
NAd
50%
50%
NAd
NAd
NAd
67%
33%
50%
NAd
67%
NAd
NAd
NAd
NAd
NAd
c
6a
—
—
—
—
—
—
—
1
c
6b
c
6c
c
6d
c
7
8
c
c
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
10s
12a
12b
12c
13a
13b
13c
13d
13e
14a
14b
14c
14d
14e
300
6
50
300
50
100
c
—
—
—
c
c
100
100
150
150
75
100
c
—
150
150
c
—
—
c
Scheme 3 Reagents and conditions: (i) m-CPBA, CH2Cl2, −10 ◦C; (ii)
m-CPBA, CH2Cl2, RT.
NAd
c
—
300
50
All substances applied in the micro-assay were confirmed as
homogeneous by LC-MS (>99% pure at 215 nm). Exceptions
were the products 10p and 10q, which appeared pure with
NMR spectroscopy but contained a strongly UV-absorbing con-
taminant, tentatively assigned by HRMS as the corresponding
sulfoxide impurity. NMR spectroscopy of all sulfonyls, 14a–e,
was also suggestive of pure products but for some of these,
homogeneity could not be confirmed by LC-MS.
NAd
c
—
NAd
NAd
NAd
NAd
NAd
a
indicates positive or negative response in the QSIS1 system,
respectively.26 b Concentration (lM) required to lower the activity of
a lasB-gfp fusion to 50%.38 c The compound was not able to lower
activation to 50%. d NA = not available. e Maximum induction in relation
to an untreated control of a lasB-gfp fusion in the presence of the highest,
non-toxic, concentration of the test compound.
Biological activity and mechanistic considerations
All experiments made with the LasR system were preceded by
tests in a LuxR screening system. The V. fischeri QS circuit is
less specific, accepting a wider spectrum of ligands (vide supra).
Therefore, qualitative tests were made to select candidates
attractive enough for a more quantitative determination of
QS inhibition in P. aeruginosa (Table 2). The initial screening
employed the QS inhibitor selector system QSIS1, constructed
by Rasmussen et al.26 This system indicates if a compound
is able to block LuxR mediated QS in the presence of N-
(3-oxohexanoyl)-L-homoserine lactone, the natural agonist of
LuxR. All the positive samples from the first screen were
tested with the las system from P. aeruginosa. A fusion of
the lasB promoter to gfp (ASV), harboured by P. aeruginosa
PAO1, was used.38 Grown without inhibitor, this monitor strain
developed green fluorescence when the QS systems are activated.
If an inhibitor is present in the growth medium, the recorded
induction of green fluorescence is decreased or, in case of a strong
inhibitor, abolished. The compounds were tested in this system at
various concentrations to determine the strength of the inhibitor,
in other words to which degree the induction could be prevented.
The values given in Table 2 are the concentrations that gave half
the induction of an untreated control. The maximum inhibition
was also determined at the highest non-toxic concentration.
Screening of 10a–e and 13a–e in the LuxR monitor system
was successful in all cases but for 13d. Therefore, 13d was not
screened in the subsequent LasR monitor system. Neither were
the sulfonyl analogues (14a–e), which were also incapable of
antagonizing LuxR. Even though, as outlined above, the LuxR
monitor system is more susceptible to structural variations of
the signal molecule compared to the LasR monitor system,
there is a risk of overlooking hits. However, at this stage
all three functionalities; sulfides, sulfoxides and sulfones, were
represented with the same acyl groups, usually critical for
differentiating the receptor homologues.
The highest activities were observed for 10c and 13c, thus
the optimal chain length was two carbon atoms shorter than
for the natural ligand. This is not necessarily a consequence
of pharmacodynamics since it was established a long time ago
that the efficiency of diffusion of molecules with a long carbon
chain through membranes is to a great extent determined by
its length.39 Inefficient diffusion has been observed also for the
AHLs40 and there is some evidence of an efflux system active
in P. aerugonosa.41 Another bias in this part of the screening
might be that the metabolic stability towards reduction of the
sulfoxide analogues is low42 and what we are really observing is
another less concentrated set of thioether analogues. However,
this possibility is contradicted by the total absence of activity for
13d. In that case, if it is the activity for the sulfoxide scaffold we
observed, there is an even greater difficulty for the longer chain
derivative 13d than for 10d to cross the cell membrane. It seems
unlikely that the affinity would drop that much for 13d and not
the other sulfoxides (13a–c and 13e) compared to 10a–e.
2 5 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 – 2 6 2

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After the initial biological screening of compounds 10a–e,
13a–e and 14a–e it was concluded that the most potent of
the three types of functionalities was a thioether. Valuable
structure–activity relationships were collected by a larger set
of aromatic analogues. This was also an attempt to get around
the detrimental effect that a highly lipophilic acyl chain could
have on the pharmacokinetic properties but at the same time
preserve the same approximate length of the acyl group. At
this point, the low potency of 10e was relevant only in light of
more potent analogues. Indeed, both 10f and 10g, with a para-
methyl and -hydroxyl group, respectively, were more potent,
the former being the strongest of the aromatic QS inhibitors
that were screened. A set of chloro-aryl analogues ortho (10h),
meta (10i) and para (10j), were also screened to reveal any
steric factors. However, 10h–j were only active in the LuxR
monitor system and therefore it was not possible to quantize the
activity. The impact of the thioether functionality as a part of the
scaffold could be evaluated by comparing all the 4-substituted
phenyl derivatives. Since the activity of a 4-chloro derivative
(10j) vanished compared to non-substituted 10e, and both 10f
and 10g were more potent, this was clearly a −reffect according
to Topliss.43 It is not as clear whether the higher potency of 10f
compared to 10g can be assigned on the basis of a +p effect or
if the −r effect have an influence here as well.
Importantly, the ability of a sulfide group to stabilize a-anions
is considered to be a significant difference. Electronic factors
certainly do have influence on the inert binding caused by
the thioethers. Otherwise the aromatic analogues 10e–n, with
different geometry than 10a–d, would not contain the same
approximate potencies.
The same requirements were not fulfilled by the conformation-
ally restricted garlic-derived analogues 12a–c, most strikingly
12a, for which the effect should be stronger having two sulfur
atoms in the b-position. Both 12a and 12b were antagonists
of LuxR but not LasR and this was also the case for the QS
inhibiting natural products found in garlic, 7 and 8. Compound
12c did not inhibit any of the two QS monitor systems it
was screened against. The grafting strategy outlined at the end
of the introduction was not successful since the two separate
components, 7 and 10c, each had the same or higher potency,
respectively. The reason was perhaps the wrong linker between
the two, or that the double bond in 7 was absent in 12a–c.
Compound 3 was included as a reference in Table 2 since it
is the most potent antagonist of LasR to date. However, the
poor chemical stability observed for 3 under the conditions used
makes it a less suitable candidate.
Conclusion
Extending 10e with a methyl group in the 4-position gave a
more potent antagonist, but moving the phenyl group further
away reduced their potency as inhibitors (10k and 10l) and
worsening as in 10m with a naphthyl group. There are several
aromatic residues in the binding-site likely to be responsible
for favourable interactions and it should be stressed that the
acyl chain of 10b, an inactive analogue, extends to the same
approximate distance as the acyl group of 10f.
We have designed, synthesized and screened 32 compounds
in two QS systems designed from the LuxR (V. fischeri ) and
LasR (P. aeruginosa) systems. A significant portion of these
compounds proved to inhibit either one or both of the QS
systems. When including the compounds lacking activity, the
observed structure–activity relationships (SARs) gave rise to a
preliminary hypothesis explaining the notable potency of the
sulfide class of analogues. Thus, strong inhibition is obtained
by competitive binding of 3ꢀ-sulfide analogues unable to interact
with the b-sheet but with the ability to form other important
contacts inside the binding-pocket. This includes van der Waals
interactions between the a-helices and the lipophilic acyl group.
Apparently, these contacts are critical for antagonizing receptors
like LuxR or LasR. Furthermore, several natural products
isolated from garlic were identified as inhibitors of QS without
affecting microbial growth. Interestingly, these inhibitors were
structurally different from the AHLs and the above-mentioned
sulfide AHL analogues and a small series of conformationally
restricted analogues, in which the sulfide AHL motif had been
grafted together with a dithiane moiety (representing 7), were
synthesized. However, these garlic-derived structures were less
potent than their more flexible sulfide analogues.
In 10n–s the lactone moiety had been changed and all
analogues were antagonists of LuxR. The design of 10p–s was
based on the acyl chain in potent 10c but also on structures
observed as inducers of QS by Smith et al.,15 structures which
had the same cyclic moiety as for 10p–s but with the same acyl
chain as 1. Their corresponding analogues of 10p and 10s were
antagonist of LasR, with the same relative potency as that for
10p and 10s. The thioether functionality and shorter acyl chain
also made 10r an antagonist of LasR. Likewise, the cyclopentyl
and cyclohexyl analogues 10n and 10o, lacking the 1-oxo group,
and thus their ability to form hydrogen bonds to residue Trp57
in LasR, were antagonists.
Even though these findings may provide aid in future archi-
tectures, it is tempting to return to the strongest antagonist 10c.
It was several times more potent than the previously reported
10d32 and 10l31, also included in this work. Whereas Reverchon
et al. screened 10l in a LuxR system and found it to be a potent
QS inhibitor,31 it had low potency compared to 10c in our LasR
assay. The key feature of this type of structure is a shorter acyl
chain than in 1 and a 3ꢀ-oxo group substituted with a sulfur
atom. Obviously, the water-mediated hydrogen bond involving
receptor residues Gly38 (Ala38 in TraR) and Ser129 (Thr129
in TraR), which is important for signal binding, is extremely
unlikely if the lactone/amide part of 10c is positioned like 1 in
the binding-pocket. According to our hypothesis, the b-sheet,
to which residues Gly38 (Ala38 in TraR) and Ser129 (Thr129
in TraR) belong, will then be forced from the position where
it is found in an active receptor–ligand complex (the activated
complex is actually a dimer that binds to an operon1 ). If this
is the reason why LasR is blocked in our biological assays, one
may wonder if compounds like 1 lacking the 3ꢀ-oxo functionality,
despite their less favourable physicochemical properties, also
could be potent inhibitors. This type of compounds are known
as inhibitors but they seem to be considerably weaker as such.10
Even though a methylene and a sulfide group are bioisosteric,
the acyl chain of 10c is tilted because the C–C–C and C–S–C
Experimental
Biology
The QSIS1 assay was performed as described elsewhere.26 The
concentration of N-(3-oxohexanoyl)-L-homoserine lactone was
maintained constant at 100 nM during screening, whereas the
concentrations for the analogues were increased in a gradient
never exceeding 10 mM.
The lasB-gfp assay was performed in accordance with Hentzer
et al.38 This monitor system was prepared from a wild-type strain
and therefore the concentration of 1 was maintained at a natural
level.
Chemistry
Commercially available reagents (Aldrich) were used without
further purification unless otherwise noted. Starting materials
11a and 11b were prepared as previously reported.36,37 Solvents
used for the synthesis were of analytical grade, dried over
˚
activated 4 A molecular sieves when necessary (all solvents
used under dry conditions had a water content <25 ppm).
bond angles (109 and 105◦, respectively) differ and so do the
Triethylamine was distilled from P2O5 and stored over 4 A molec-
˚
44
˚
C–C and C–S bond lengths (1.54 and 1.82 A, respectively).
ular sieves. MilliQ water was used for RP-HPLC. Analytical
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 – 2 6 2
2 5 7

View Article Online
TLC was performed using pre-coated silica gel 60 F254 plates
and visualized using either UV light, phosphomolybdic acid or
potassium permanganate stain. Merck 60H silica gel was used
for VLC. Flash chromatography was performed automatically
on a Biotage Quad 3+ Parallel Flash PurificationTM system with
The product was again evaporated to dryness and purified by
flash chromatography (eluent: 1% MeOH in CH2Cl2,). Yield:
155 mg (70%); mp 126–129 ◦C; [a]D22 = 20.5 (c = 0.0074, CHCl3);
1H NMR (300 MHz, CDCl3) d 7.02 (br s, exch., 1 H, NH), 4.62–
4.25 (m, 3 H, CH2-5 and CH-3), 3.91 (ABq, D = 7 Hz, JAB
=
11 Hz, 2 H, CH2-2ꢀ), 2.87–2.77 (m, 1 H, CH2-4), 2.30–2.14 (m, 1
H, CH2-4); 13C NMR (75 MHz, CDCl3) d 174.3 (1 C, C-2), 166.1
(1 C, C-1ꢀ), 65.8 (1 C, C-5), 49.5 (1 C, C-3), 29.7, 28.0 (2 C, C-4
and C-2ꢀ); LC-MS (ESI) tR = 1.18 min (m/z 222 [MH]+); HRMS
(M + H)+ calcd. for C6H9BrNO3 221.9766, found 221.9787.
˚
pre-packed columns (KP-SIL, 32–63 lm, 60 A). Parallel reac-
tions requiring cooling were performed in a Radley Greenhouse
Parallel SynthesizerTM block. Corrected melting points were
measured in open capillary tubes on a Gallenkamp electrother-
mal melting point apparatus. Optical rotation measurements
were obtained on an Optical Activity Ltd. AA-1000 polarimeter
using a 0.5 dm path length micro cell. Infrared spectrum was
collected on a Perkin Elmer System 2000 FT-IR instrument.
Preparative RP-HPLC was performed on a Waters 600 system
equipped with a Waters 996 photodiode array (PDA) detector
and three consecutive columns (40 × 100 mm prep. NOVA Pak
General procedure for the preparation of 10a, 10b and 10d by
method A. At ambient temperature, triethylamine (1.1 equiv.)
was added to a 0.05 M solution of 9 (1.0 equiv.) in abs. EtOH
turning it dark beige. Half an hour of stirring was followed by
addition of thiol (1.1 equiv., R2-SH, Table 1) and then another
3 h of stirring was ended by addition of 10% HCl. Extraction
(3 × CH2Cl2) of the aqueous phase and drying of the combined
organic phases (MgSO4) gave the pure sulfides after removal
of solvent in vacuo and purification by flash chromatography
(eluent: CH2Cl2).
−1
˚
HR C18 6 lm, 60 A) with a flow of 35 ml min . 80% → 50% A 0–
29 min and then 50% → 0% A 30–65 min (A: water B: CH3CN).
1H and 13C NMR spectral data was recorded on a Bruker
Avance 300 using the deuterated solvent as lock. Chemical shifts
are reported in ppm relative to the residual solvent peak (1H
NMR) or the solvent peak (13C NMR) as the internal standard.
GC-MS was carried out on a Trio 2 VG Masslab fitted with
a 5809A Hewlett Packard gas chromatograph. Accurate mass
and purity determinations were performed on a Waters 2795
system equipped with a Waters 996 PDA detector and a Waters
Symmetry C18 Column (2.1 × 50 mm, 3.5 lm) with a flow
of 0.2 ml min−1. 100% → 0% A 0–10 min (A: 0.1% aq formic
acid, B: 95% CH3CN in 0.05% aq formic acid). The connected
Micromass LCT apparatus was equipped with an AP-ESI probe
calibrated with Leu-Enkephalin (556.2771 g mol−1).
N-(Propylsulfanylacetyl)-L-homoserinelactone(10a). 222 mg
9 gave◦101 mg of a white solid (46% yield, >99% purity); mp
70–71 C; 1H NMR (300 MHz, CDCl3) d 7.38 (br d, 3J = 5 Hz,
1 H, NH), 4.64–4.23 (m, 3 H, CH2-5 and CH-3), 3.25 (ABq,
D = 6 Hz, JAB = 17 Hz, 2 H, CH2-2ꢀ), 2.81–2.72 (m, 1 H, CH2-
4), 2.54 (t, 3J = 6 Hz, 2 H, CH2-4ꢀ), 2.27–2.15 (m, 1 H, CH2-4),
1.61 (m, 2 H, CH2-5ꢀ), 0.97 (t, 3J = 7 Hz, 3 H, CH3 ); 13C NMR
ꢀ
(75 MHz, CDCl3) d 174.9 (1 C, C-2), 169.8 (1 C, C-1ꢀ), 65.8 (1 C,
C-5), 49.2 (1 C, C-3), 35.7, 35.0, 29.9, 22.4 (4 C, C-4, C-2ꢀ and
ꢀ
ꢀ
2 × CH2 ), 13.3 (1 C, CH3 ); LC-MS (ESI) tR = 1.35 min (m/z
218 [MH]+); HRMS (M + H)+ calcd. for C9H16NO3S 218.0851,
found 218.0845.
Separation and identification of garlic components. Garlic
(1800 g) chopped in pieces was soaked in toluene (3750 ml) and
stirred overnight. The toluene was decanted and concentrated
to dryness. The remaining oil was partitioned between hexane
and water–MeOH and the clear hexane-phase dried (Na2SO4)
and concentrated in vacuo to afford 3.61 g of a viscous yellow
oil. Flash chromatography (eluent: 0–10% EtOAc in hexane)
yielded several bioactive fractions from which six known garlic
compounds (6a–d, 7 and 8) were identified by comparison of
data (NMR spectroscopy and GC-MS analysis) with authentic
samples. The authentic samples, either commercially available
or accessible through known synthetic procedures,27,28 served to
verify that 6a–d, 7 and 845 were antagonists of LuxR and that
6a–d were antimicrobials.
N-(Pentylsulfanylacetyl)-L-homoserine lactone (10b). 1.60 g
9 gave◦1.18 g of a white solid (67% yield, >99% purity); mp
73–75 C; 1H NMR (300 MHz, CDCl3) d 7.35 (br d, 3J = 5 Hz,
1 H, NH), 4.64–4.25 (m, 3 H, CH2-5 and CH-3), 3.27 (ABq,
D = 6 Hz, JAB = 17 Hz, 2 H, CH2-2ꢀ), 2.85–2.76 (m, 1 H, CH2-
4), 2.57 (t, 3J = 6 Hz, 2 H, CH2-4ꢀ), 2.26–2.11 (m, 1 H, CH2-4),
ꢀ
1.65–1.55 (m, 2 H, CH2-5ꢀ), 1.41–1.25 (m, 4 H, 2 × CH2 ), 0.89 (t,
3J = 7 Hz, 3 H, CH3 ); 13C NMR (75 MHz, CDCl3) d 174.8 (1 C,
ꢀ
C-2), 169.7 (1 C, C-1ꢀ), 65.9 (1 C, C-5), 49.2 (1 C, C-3), 35.9, 33.1,
ꢀ
30.9, 30.1, 28.8, 22.2 (6 C, C-4, C-2ꢀ and 4 × CH2 ), 13.9 (1 C,
CH3 ); LC-MS (ESI) tR = 6.00 min (m/z 246 [MH]+); HRMS
ꢀ
(M + H)+ calcd. for C11H20NO3S 246.1164, found 246.1145.
N-(Nonylsulfanylacetyl)-L-homoserine lactone (10d). 312 mg
9 gave◦149 mg of a white solid (35% yield, >99% purity); mp
94–95 C; 1H NMR (300 MHz, CDCl3) d 7.34 (br d, 3J = 5 Hz,
1 H, NH), 4.64–4.25 (m, 3 H, CH2-5 and CH-3), 3.27 (ABq,
D = 6 Hz, JAB = 17 Hz, 2 H, CH2-2ꢀ), 2.86–2.77 (m, 1 H, CH2-
4), 2.57 (t, 3J = 6 Hz, 2 H, CH2-4ꢀ), 2.26–2.11 (m, 1 H, CH2-4),
L-Homoserine lactone hydrobromide (5). A mixture of bro-
moacetic acid (1.54 g, 11.0 mmol) and L-methionine (1.52 g,
10.0 mmol) in 14.4 ml of an H2O–2-propanol–AcOH mixture
(5 : 5 : 2 v/v) was refluxed for 9 h. The solvent was then removed
at reduced pressure. Further drying overnight by vacuum pump
left a beige semi-solid. It was partly dissolved in 10 ml of a
4 : 1 mixture (v/v) of 2-propanol–HBr (30% in AcOH). The
title compound was collected by filtration and the purification
procedure repeated starting from evaporation of the orange
filtrate to dryness. Compound 5 was collected as a white powder
after drying of both portions in vacuo (m = 1.47 g, 81%), mp
226–228 ◦C (lit.ref. 34 mp 229–231 ◦C); [a]2D2 = −19.5 (c = 0.05,
H2O); lit.ref. 34 [a]2D5 = −24.4 (c = 0.087, H2O). All other physical
and analytical data were in agreement with those previously
reported.34
ꢀ
1.64–1.54 (m, 2 H, CH2-5ꢀ), 1.39–1.26 (m, 12 H, 6 × CH2 ),
3
ꢀ
0.88 (t, J = 7 Hz, 3 H, CH3 ); 13C NMR (75 MHz, CDCl3) d
174.7 (1 C, C-2), 169.7 (1 C, C-1ꢀ), 65.9 (1 C, C-5), 49.3 (1 C,
C-3), 35.9, 33.1, 31.8, 30.2, 29.4, 29.2, 29.2, 29.1, 28.7, 22.7 (10 C,
C-4, C-2ꢀ and 8 × CH2 ), 14.1 (1 C, CH3 ); LC-MS (ESI) tR
=
ꢀ
ꢀ
7.65 min (m/z 302 [MH]+); HRMS (M + H)+ calcd. for
C15H28NO3S 302.1790, found 302.1770.
General procedure for the preparation of 10e–m (modified
method A). Method A was also applied for the aromatic ana-
logues 10e–m but the work-up preceding flash chromatography
was abandoned. Instead the crude was isolated by removal of
solvent after completion of the reaction using a stream of air.
Then it was dissolved in least amount of CH2Cl2 and absorbed
onto silica gel for purification by flash chromatography.
N-Bromoacetyl-L-homoserine lactone (9). Under dry con-
ditions, triethylamine (290 ll, 2.10 mmol) was added to a
suspension of 5 (182 mg, 1.00 mmol) in CH2Cl2 (50 ml) cooled
at −78 ◦C. 2-Bromoacetyl bromide (96 ll, 1.10 mmol) was then
added dropwise and the temperature allowed to rise to RT during
a period of 1 h. Evaporation to dryness gave a white semi-solid. It
was partly dissolved by EtOAc and the salt remaining separated
off by filtration (HNEt3+Br− exclusively according to 1H NMR).
N-(Phenylsulfanylacetyl)-L-homoserine lactone (10e). 56 mg
9 gave 47 mg of a white solid (75% yield, >99% purity) after flash
chromatography (eluent: 1% MeOH in CH2Cl2); mp 87–89 ◦C;
2 5 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 – 2 6 2

View Article Online
1H NMR (300 MHz, CDCl3) d 7.36–7.22 (m, 5H, S–C6H5), 7.21
(br s, exch., 1 H, NH), 4.56–4.21 (m, 3 H, CH2-5 and CH-3),
3.67 (s, 2 H, CH2-2ꢀ), 2.80–2.70 (m, 1 H, CH2-4), 2.09–1.95 (m,
1 H, CH2-4); 13C NMR (75 MHz, CDCl3) d 174.7 (1 C, C-2),
168.8 (1 C, C-1ꢀ), 134.1, 129.3, 128.9, 127.1 (6 C, S–C6H5), 65.9
(1 C, C-5), 49.3 (1 C, C-3), 37.6 (1 C, C-2ꢀ), 29.9 (1 C, C-4);
LC-MS (ESI) tR = 5.51 min (m/z 252 [MH]+); HRMS (M +
H)+ calcd. for C12H14NO3S 252.0694, found 252. 0738.
chromatography (eluent: 1% MeOH in CH2Cl2); mp 77–79 ◦C;
1H NMR (300 MHz, CDCl3) d 7.32–7.24 (m, 5H, Ar-H), 7.18
(br s, exch., 1 H, NH), 4.54–4.21 (m, 3 H, CH2-5 and CH-3),
3.77 (s, 2 H, CH2-2ꢀ), 3.16 (s, 2 H, CH2-2ꢀ), 2.80–2.70 (m, 1 H,
CH2-4), 2.13–1.99 (m, 1 H, CH2-4); 13C NMR (75 MHz, CDCl3)
d 174.9 (1 C, C-2), 169.3 (1 C, C-1ꢀ), 136.9, 129.1, 128.7, 127.4
(6 C, Ar), 65.9 (1 C, C-5), 49.1 (1 C, C-3), 37.0, 34.9 (2 C, C-2ꢀ
and C-4ꢀ), 29.8 (1 C, C-4); LC-MS (ESI) tR = 5.68 min (m/z
266 [MH]+); HRMS (M + H)+ calcd. for C13H16NO3S 266.0851,
found 266.0847.
N-(p-Tolylsulfanylacetyl)-L-homoserine lactone (10f). 44 mg
9 gave 47 mg of a white solid (88% yield, >99% purity) after
flash chromatography (eluent: 1% MeOH in CH2Cl2); mp 103–
N-(Phenethylsulfanylacetyl)-L-homoserine lactone (10l).
44 mg 9 gave 55 mg of a white solid (99% yield, >99% purity)
after flash ◦chromatography (eluent: 1% MeOH in CH2Cl2);
◦
1
106 C; H NMR (300 MHz, CDCl3) d 7.28 (br s, exch., 1 H,
3
3
NH), 7.26 (d, J = 8 Hz, 2H, Ar-H), 7.21 (d, J = 8 Hz, 2H,
1
Ar-H), 4.57–4.19 (m, 3 H, CH2-5 and CH-3), 3.60 (s, 2 H, CH2-
mp 67–70 C; H NMR (300 MHz, CDCl3) d 7.32–7.19 (m,
2ꢀ), 2.75–2.65 (m, 1 H, CH2-4), 2.13–1.98 (m, 1 H, CH2-4); 13
C
6H, Ar-H and NH), 4.61–4.22 (m, 3 H, CH2-5 and CH-3),
3.26 (s, 2 H, CH2-2ꢀ), 2.94–2.81 (m, 4 H, CH2-4ꢀ and CH2-5ꢀ),
2.76–2.68 (m, 1 H, CH2-4), 2.22–2.07 (m, 1 H, CH2-4); 13C
NMR (75 MHz, CDCl3) d 174.8 (1 C, C-2), 169.5 (1 C, C-1ꢀ),
139.7, 128.5, 128.5, 126.5 (6 C, Ar), 65.8 (1 C, C-5), 49.1 (1 C,
C-3), 35.8, 35.4, 34.3 (3 C, C-2ꢀ, C-4ꢀ and C-5ꢀ), 29.7 (1 C, C-4);
LC-MS (ESI) tR = 5.92 min (m/z 280 [MH]+); HRMS (M +
H)+ calcd. for C14H18NO3S 280.1007, found 280.0978.
NMR (75 MHz, CDCl3) d 174.8 (1 C, C-2), 169.0 (1 C, C-1ꢀ),
137.4, 130.4, 130.1, 129.7 (6 C, Ar), 65.9 (1 C, C-5), 49.3 (1 C,
C-3), 38.3 (1 C, C-2ꢀ), 29.8 (1 C, C-4), 21.0 (1 C, ArCH3); LC-MS
(ESI) tR = 5.83 min (m/z 266 [MH]+); HRMS (M + H)+ calcd.
for C13H16NO3S 266.0851, found 266.0831.
N -(p-Hydroxyphenylsulfanylacetyl)-L-homoserine lactone
(10g). 44 mg 9 gave 19 mg of a colourless oil (36% yield,
>99% purity) after flash chromatography (eluent: 1% MeOH in
N-(Naphthyl-2-sulfanylacetyl)-L-homoserine lactone (10m).
44 mg 9 gave 55 mg of a white solid (91% yield, >99% purity)
after flash chromatography (eluent: 1% MeOH in CH2Cl2); mp
133–134 ◦C; 1H NMR (300 MHz, CDCl3) d 7.79–7.39 (m, 7H,
Ar-H), 7.35 (br d, 3J = 6 Hz, 1 H, NH), 4.55–4.33 (m, 3 H, CH2-
5 and CH-3), 3.74 (s, 2 H, CH2-2ꢀ), 2.67–2.58 (m, 1 H, CH2-4),
2.08–1.93 (m, 1 H, CH2-4); 13C NMR (75 MHz, CDCl3) d 174.7
(1 C, C-2), 168.7 (1 C, C-1ꢀ), 133.7, 132.0, 131.5, 128.9, 127.6,
127.3, 127.3, 126.8, 126.6, 126.2, (10 C, Ar), 65.8 (1 C, C-5),
49.3 (1 C, C-3), 37.5 (1 C, C-2ꢀ), 29.6 (1 C, C-4); LC-MS (ESI)
tR = 6.17 min (m/z 302 [MH]+); HRMS (M + H)+ calcd. for
C16H16NO3S 302.0851, found 302.0832.
1
3
CH2Cl2). H NMR (300 MHz, CD3OD) d 7.33 (d, J = 9 Hz,
2H, Ar-H), 6.72 (d, 3J = 9 Hz, 2H, Ar-H), 4.58–4.20 (m, 3 H,
CH2-5 and CH-3), 3.43 (s, 2 H, CH2-2ꢀ), 2.49–2.39 (m, 1 H,
CH2-4), 2.21–2.07 (m, 1 H, CH2-4); 13C NMR (75 MHz,
CD3OD) d 177.1 (1 C, C-2), 172.2 (1 C, C-1ꢀ), 159.1, 135.8,
135.5, 124.4 (6 C, Ar), 67.2 (1 C, C-5), 50.1 (1 C, C-3), 41.0 (1 C,
C-2ꢀ), 29.5 (1 C, C-4); LC-MS (ESI) tR = 4.92 min (m/z 268
[MH]+); HRMS (M + H)+ calcd. for C12H14NO4S 268.0644,
found 268.0587.
N-(o-Chlorophenylsulfanylacetyl)-L-homoserine lactone (10h).
44 mg 9 gave 53 mg of a white solid (92% yield, >99% purity)
after flash chromatography (eluent: 1% MeOH in CH2Cl2); mp
140–141 ◦C; 1H NMR (300 MHz, CDCl3) d 7.40–7.14 (m, 5H,
Ar-H and NH), 4.55–4.19 (m, 3 H, CH2-5 and CH-3), 3.71 (s,
2 H, CH2-2ꢀ), 2.74–2.65 (m, 1 H, CH2-4), 2.14–1.99 (m, 1 H,
CH2-4); 13C NMR (75 MHz, CDCl3) d 174.5 (1 C, C-2), 168.3
(1 C, C-1ꢀ), 133.6, 133.4, 129.9, 128.8, 127.8, 127.7 (6 C, Ar),
65.8 (1 C, C-5), 49.3 (1 C, C-3), 36.4 (1 C, C-2ꢀ), 29.7 (1 C, C-4);
LC-MS (ESI) tR = 5.85 min (m/z 286 [MH]+); HRMS (M + H)+
calcd. for C12H13ClNO3S 286.0305, found 286.0289.
N-(Heptylsulfanylacetyl)-L-homoserine lactone (10c) by
method B. Under dry conditions triethylamine (3.07 ml, 22.0
mmol) was added to a suspension of 5 (1.86 g, 10.0 mmol)
in CH2Cl2 (50 ml) at RT. While cooling at, −20 ◦C 2-
bromoacetylbromide (11.0 mmol, 0.958 ml) was added dropwise
and the mixture was then allowed to warm to ambient tempera-
ture during a period of 1.5 h whereupon a white solid appeared.
After another 2.5 h of stirring, additional triethylamine (1.54 ml,
11.0 mmol) was added together with 1-heptanethiol (1.69 ml,
11.0 mmol), in that order. The reaction was ended after 17 h
by washing the mixture with 10% HCl (25 ml) and the organic
phase was dried (MgSO4), filtrated and evaporated to dryness.
10c was isolated as a white solid (1.30 g, 46% yield, >99% purity)
after VLC (eluent: CH2Cl2) in); mp 86–89 ◦C; [a]D22 = −9.6 (c =
0.084, CHCl3); mmax(KBr)/cm−1 3307 (CONH), 2924 (CH), 1774
(ring CO), 1649 (amide CO), 1549, 1176; 1H NMR (300 MHz,
CDCl3) d 7.37 (br d, 3J = 5 Hz, 1 H, NH), 4.64–4.24 (m, 3 H,
CH2-5 and CH-3), 3.26 (ABq, D = 6 Hz, JAB = 17 Hz, 2 H,
N-(m-Chlorophenylsulfanylacetyl)-L-homoserine lactone (10i).
44 mg 9 gave 53 mg of a white solid (92% yield, >99% purity)
after flash chromatography (eluent: 1% MeOH in CH2Cl2); mp
◦
1
98–99 C; H NMR (300 MHz, CDCl3) d 7.32–7.19 (m, 5H,
Ar-H and NH), 4.60–4.20 (m, 3 H, CH2-5 and CH-3), 3.66 (s,
2 H, CH2-2ꢀ), 2.75–2.66 (m, 1 H, CH2-4), 2.15–2.01 (m, 1 H,
CH2-4); 13C NMR (75 MHz, CDCl3) d 174.8 (1 C, C-2), 168.3
(1 C, C-1ꢀ), 136.3, 134.9, 130.3, 128.5, 127.1, 126.7 (6 C, Ar),
65.9 (1 C, C-5), 49.3 (1 C, C-3), 37.2 (1 C, C-2ꢀ), 29.7 (1 C, C-4);
LC-MS (ESI) tR = 5.96 min (m/z 286 [MH]+); HRMS (M + H)+
calcd. for C12H13ClNO3S 286.0305, found 286.0296.
3
CH2-2ꢀ), 2.84–2.75 (m, 1 H, CH2-4), 2.56 (t, J = 6 Hz, 2 H,
CH2-4ꢀ), 2.26–2.14 (m, 1 H, CH2-4), 1.63–1.54 (m, 2 H, CH2-
ꢀ
ꢀ
5ꢀ), 1.38–1.25 (m, 8 H, 4 × CH2 ), 0.87 (t, 3J = 7 Hz, 3 H, CH3 );
13C NMR (75 MHz, CDCl3) d 174.8 (1 C, C-2), 169.8 (1 C, C-
1ꢀ), 65.9 (1 C, C-5), 49.2 (1 C, C-3), 35.8, 33.1, 31.6, 30.1, 29.1,
N-(p-Chlorophenylsulfanylacetyl)-L-homoserine lactone (10j).
44 mg 9 gave 53 mg of a white solid (92% yield, >99% purity)
after flash chromatography (eluent: 1% MeOH in CH2Cl2); mp
130–131 ◦C; 1H NMR (300 MHz, CDCl3) d 7.31–7.25 (m, 4H,
Ar-H), 7.22 (br s, exch., 1 H, NH), 4.58–4.20 (m, 3 H, CH2-5
and CH-3), 3.63 (s, 2 H, CH2-2ꢀ), 2.75–2.67 (m, 1 H, CH2-4),
2.14–1.99 (m, 1 H, CH2-4); 13C NMR (75 MHz, CDCl3) d 174.8
(1 C, C-2), 168.5 (1 C, C-1ꢀ), 133.2, 132.6, 130.5, 129.4 (6 C, Ar),
65.9 (1 C, C-5), 49.3 (1 C, C-3), 37.8 (1 C, C-2ꢀ), 29.8 (1 C, C-4);
LC-MS (ESI) tR = 5.96 min (m/z 286 [MH]+); HRMS (M + H)+
calcd. for C12H13ClNO3S 286.0305, found 286.0290.
ꢀ
ꢀ
28.8, 28.7, 22.5 (8 C, C-4, C-2ꢀ and 6 × CH2 ), 14.0 (1 C, CH3 );
LC-MS (ESI) tR = 6.79 min (m/z 274 [MH]+); HRMS (M + H)+
calcd. for C13H24NO3S 274.1477, found 274.1481.
General procedure for the preparation of 10n–q by modified
method B. While cooling a mixture of triethylamine (614 ll,
4.4 mmol. In the synthesis of 10p and 10q only 2.2 mmol triethy-
lamine was added initially since these starting materials were
not salts) and amine or ammonium salt (2.0 mmol) in CH2Cl2
(2 ml) under dry conditions at −20 ◦C, 2-bromoacetylbromide
(2.2 mmol, 196 ll) was added dropwise. The mixture was then
allowed to warm to ambient temperature during a period of
N-(Benzylsulfanylacetyl)-L-homoserine lactone (10k). 44 mg
9 gave 52 mg of a white solid (98% yield, >99% purity) after flash
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 – 2 6 2
2 5 9

View Article Online
1 h. Additional triethylamine (307 ll, 2.2 mmol) was added
together with 1-heptanethiol (355 ll, 2.2 mmol) in that order.
After another 24 h of stirring the crude was isolated by removal
of solvent using a stream of air. It was then absorbed onto silica
gel by aid of the least amount of chromatography eluent possible
(the triethylammonium halide salt was left behind by filtration)
for purification by flash chromatography.
10p or 10q (0.037 mmol) keeping the temperature constant at
−78 ◦C. After 15 min triethylamine (0.16 mmol, 22 ll) was added
and as the mixture warmed to RT it turned opaque. The 0.1 M
HCl (10 ml), added to the reaction mixture after another 10 min
of stirring, was extracted (3 × 10 ml EtOAc) and the combined
organic phases washed (10 ml sat. NaHCO3 and 10 ml brine in
that order) and dried (MgSO4). Concentration to dryness under
reduced pressure gave a crude resolved by flash chromatography
(1% MeOH in CH2Cl2).
N-Cyclopentyl-2-heptylsulfanyl-acetamide (10n). oil. 27%
yield (>99% purity) after flash chromatography (eluent: 10%
1
EtOAc in hexane). H NMR (300 MHz, CDCl3) d 6.95 (br s,
( )-2-Heptylsulfanyl-N-(2-oxo-cyclopentyl)-acetamide (10r).
Yellow oil. 34% yield. 1H NMR (300 MHz, CDCl3) d 7.20 (br s,
exch., 1 H, NH), 4.22–4.13 (m, 1 H, CHNH), 3.25 (ABq, D =
6 Hz, JAB = 17 Hz, 2 H, CH2-2ꢀ), 2.66–1.26 (m, 18 H, CH2-3,
exch., 1 H, NH), 4.28–4.16 (m, 1 H, CH-1), 3.22 (s, 2 H, CH2-
2ꢀ), 2.50 (t, 3J = 8 Hz, 2 H, CH2-4ꢀ), 1.70–1.27 (m, 18 H, CH2-2,
CH2-3, CH2-4, CH2-5 and 5 × CH2 ), 0.88 (t, 3J = 6 Hz, 3 H,
ꢀ
CH3 ); 13C NMR (75 MHz, CDCl3) d 168.3 (1 C, C-1ꢀ), 51.3
ꢀ
ꢀ
3
ꢀ
CH2-4, CH2-5 and 6 × CH2 ), 0.88 (t, J = 7 Hz, 3 H, CH3 );
13C NMR (75 MHz, CDCl3) d 214.5 (1 C, C-1), 169.4 (1 C, C-
1ꢀ), 58.0 (1 C, C-2), 36.0, 34.9, 33.1, 31.7, 29.8, 29.1, 28.8, 28.7,
(1 C, C-1), 36.1, 33.1, 32.8, 31.5, 29.1, 28.6, 28.5, 23.5, 22.4
ꢀ
ꢀ
(11 C, C-2, C-3, C-4, C-5 and 7 × CH2 ), 13.9 (1 C, CH3 ); LC-
MS (ESI) tR = 8.04 min (m/z 258 [MH]+); HRMS (M + H)+
calcd. for C14H28NOS 258.1892, found 258.1911, (M + CH3CN +
H)+ calcd. for C16H31N2OS 299.2157, found 299.2154.
ꢀ
ꢀ
22.6, 18.1 (10 C, C-3, C-4, C-5 and 7 × CH2 ), 13.9 (1 C, CH3 );
LC-MS (ESI) tR = 7.10 min (m/z 272 [MH]+); HRMS (M + H)+
calcd. for C14H26NO2S 272.1684, found 272.1686.
N-Cyclohexyl-2-heptylsulfanyl-acetamide 10o. White solid.
37% yield (>99% purity) after flash chromatography (eluent:
10% EtOAc in hexane); mp 55–56 C; H NMR (300 MHz,
CDCl3) d 6.95 (br d, 3J = 8 Hz, 1 H, NH), 3.82–3.68 (m, 1 H,
CH-1), 3.16 (s, 2 H, CH2-2ꢀ), 2.47 (t, 3J = 8 Hz, 2 H, CH2-4ꢀ),
1.89–1.10 (m, 20 H, CH2-2, CH2-3, CH2-4, CH2-5, CH2-6 and
( )-2-Heptylsulfanyl-N-(2-oxo-cyclohexyl)-acetamide (10s).
Yellow oil. 44% yield. 1H NMR (300 MHz, CDCl3) d 7.68 (br d,
3J = 7 Hz, 1 H, NH), 4.53–4.44 (m, 1 H, CHNH), 3.23 (s, 2 H,
CH2-2ꢀ), 2.66–1.25 (m, 20 H, CH2-3, CH2-4, CH2-5, CH2-6 and
◦
1
6 × CH2 ), 0.87 (t, 3J = 7 Hz, 3 H, CH3 ); 13C NMR (75 MHz,
CDCl3) d 207.2 (1 C, C-1), 168.7 (1 C, C-1ꢀ), 58.1 (1 C, C-2),
41.2, 36.2, 35.3, 33.1, 31.7, 29.1, 28.8, 28.7, 27.9, 24.1, 22.6, (11
ꢀ
ꢀ
5 × CH2 ), 0.88 (t, 3J = 7 Hz, 3 H, CH3 ); 13C NMR (75 MHz,
CDCl3) d 167.7 (1 C, C-1ꢀ), 48.2 (1 C, C-1), 36.3, 33.1, 32.9,
32.9, 31.6, 29.2, 28.7, 28.6, 25.4, 24.6, 24.6, 22.5 (12 C, C-2,
ꢀ
ꢀ
ꢀ
ꢀ
C, C-3, C-4, C-5, C-6 and 7 × CH2 ), 14.1 (1 C, CH3 ); LC-MS
(ESI) tR = 7.59 min (m/z 286 [MH]+); HRMS (M + H)+ calcd.
for C15H28NO2S 286.1841, found 286.1888.
ꢀ
ꢀ
C-3, C-4, C-5 C-6 and 7 × CH2 ), 13.9 (1 C, CH3 ); LC-MS
(ESI) tR = 8.51 min (m/z 272 [MH]+); HRMS (M + H)+ calcd.
for C15H30NOS 272.2048, found 272.2063, (M + CH3CN + H)+
calcd. for C17H33N2OS 313.2314, found 313.2301.
General procedure for the synthesis of 12a–c. While cooling
a slurry of acid (11a or 10b, 1.0 equiv.) and amine or ammonium
salt (1.0 equiv.) in CH2Cl2 (5 ml) under dry conditions at
(
)-2-Heptylsulfanyl-N-(trans-2-hydroxy-cyclopentyl)-acet-
0
◦C, isobutyl chloroformate (1.0 equiv.) and triethylamine
amide (10p). Yellow oil. 3% yield after flash chromatography
(2.2 equiv.). In the synthesis of 12c only 1.1 equiv. triethy-
lamine was added since this starting material was no salt) was
added dropwise and simultaneously over a period of 1 h. The
suspension was stirred for another 2 h while the temperature
was allowed to rise to RT and then 5 ml of 4 M HCl was
added. The resulting mixture was dissolved in 100 ml CH2Cl2
and subsequently washed with 30 ml brine, dried (Na2SO4) and
concentrated in vacuo to give a crude purified by preparative
RP-HPLC or flash chromatography.
1
(eluent: 2% MeOH in CH2Cl2). H NMR (300 MHz, CDCl3)
d 7.02 (br s, exch., 1 H, NH), 4.02–3.80 (m, 2 H, CHOH and
CHNH), 3.22 (s, 2 H, CH2-2ꢀ), 2.53 (t, 3J = 8 Hz, 2 H, CH2-4ꢀ),
ꢀ
2.22–1.26 (m, 16 H, CH2-3, CH2-4, CH2-5 and 5 × CH2 ), 0.88
(t, 3J = 6 Hz, 3 H, CH3 ); 13C NMR (75 MHz, CDCl3) d 171.1
ꢀ
(1 C, C-1ꢀ), 79.5 (1 C, C-1), 60.8 (1 C, C-2), 36.0, 33.3, 32.6,
31.7, 30.3, 29.2, 28.8, 28.7, 22.6, 21.3, (10 C, C-3, C-4, C-5
ꢀ
ꢀ
and 7 × CH2 ), 13.9 (1 C, CH3 ); LC-MS (ESI) tR = 6.85 min
(m/z 274 [MH]+); HRMS (M + H)+ calcd. for C14H28NO2S
274.1841, found 274.1856. The sulfoxide impurity of 10p was
also identified by LC-MS (ESI) tR = 5.70 min (m/z 290 [MH]+);
HRMS (M + H)+ calcd. for C14H28NO3S 290.1790, found
290.1914.
N-[1,3]Dithiane-2-carboxyl-L-homoserine lactone (12a).
530 mg 11a gave 59 mg of a white fluffy solid (7% yield, >98%
purity) after preparative RP-HPLC; mp 177–182 ◦C; 1H NMR
3
(300 MHz, DMSO-d6) d 8.54 (br d, J = 3 Hz, 1 H, NH),
4.60–4.51 (m, 1 H, CH-3), 4.52 (s, 1 H, CH-2ꢀ), 4.49–4.17 (m,
2 H, CH2-5), 3.25–3.16 (m, 2 H, CH2-4ꢀ), 2.77–2.67 (m, 2 H,
CH2-4ꢀ), 2.48–2.38 (m, 1 H, CH2-4), 2.22–2.08 (m, 1 H, CH2-4),
2.00–1.89 (m, 2 H, CH2-5ꢀ); 13C NMR (75 MHz, DMSO-d6) d
174.7 (1 C, C-2), 168.7 (1 C, C-1ꢀ), 65.2 (1 C, C-5), 48.0 (1 C,
C-3), 43.0 (1 C, C-2ꢀ), 28.0, 26.8, 26.7, 24.9 (4 C, C-4, C-5ꢀ and
2 × C-4ꢀ); LC-MS (ESI) tR = 4.84 min (m/z [MH]+); HRMS
(M + H)+ calcd. for C9H14N O3S2 248.0415, found 248.0416.
(
)-2-Heptylsulfanyl-N-(trans-2-hydroxy-cyclohexyl)-acet-
amide (10q). Yellow oil. 6% yield after flash chromatography
(eluent: 2% MeOH in CH2Cl2). 1H NMR (300 MHz, CDCl3) d
7.02 (br d, 3J = 7 Hz, 1 H, NH), 3.69–3.28 (m, 2 H, CHOH and
CHNH), 3.24 (ABq, D = 8 Hz, JAB = 17 Hz, 2 H, CH2-2ꢀ), 2.53
(t, 3J = 8 Hz, 2 H, CH2-4ꢀ), 2.08–1.17 (m, 18 H, CH2-3, CH2-4,
ꢀ
3
ꢀ
CH2-5, CH2-6 and 5 × CH2 ), 0.86 (t, J = 6 Hz, 3 H, CH3 );
13C NMR (75 MHz, CDCl3) d 170.6 (1 C, C-1ꢀ), 75.1 (1 C, C-1),
55.9 (1 C, C-2), 36.2, 34.4, 33.1, 31.6, 31.3, 29.1, 28.8, 28.7, 24.5,
N -[1,3]Dithiane-2-acetyl-L-homoserine lactone (12b).
230 mg 11b gave 21 mg of a white solid (6% yield, >99% purity)
after preparative RP-HPLC (an additional 186 mg was isolated
in fractions containing minor impurities); mp 185–187 ◦C;
1H NMR (300 MHz, CDCl3) d 6.31 (br s, exch., 1 H, NH),
4.58–4.24 (m, 4 H, CH2-5, CH-3 and CH-3ꢀ), 2.99–1.81 (m,
10 H, CH2-4, CH2-2ꢀ, 2 × CH2-5ꢀ and CH2-6ꢀ); 13C NMR
(75 MHz, CDCl3) d 175.1 (1 C, C-2), 169.3 (1 C, C-1ꢀ), 66.2 (1
C, C-5), 49.6 (1 C, C-3), 42.6, 42.0 (2 C, C-2ꢀ and C-3ꢀ), 30.4,
30.2, 30.2 (3 C, C-4 and 2 × C-5ꢀ), 25.2 (1 C, C-6ꢀ); LC-MS
(ESI) tR = 4.96 min (m/z [MH]+); HRMS (M + H)+ calcd. for
C10H16NO3S2 262.0572, found 262.0592.
ꢀ
24.0, 22.5, (11 C, C-3, C-4, C-5, C-6 and 7 × CH2 ), 14.0 (1 C,
CH3 ); LC-MS (ESI) tR = 7.07 min (m/z 288 [MH]+); HRMS
ꢀ
(M + H)+ calcd. for C15H30NO2S 288.1997, found 288.1989.
The sulfoxide impurity of 10q was also identified by LC-MS
(ESI) tR = 5.91 min (m/z 304 [MH]+); HRMS (M + H)+ calcd.
for C14H28NO3S 304.1946, found 304.1859.
Oxidation of 10p and 10q. Swern reagent was prepared under
dry conditions by adding DMSO (0.080 mmol, 5.7 ll) to a
CH2Cl2 solution (0.2 ml) of oxalyl chloride (0.040 mmol, 3.6 ll)
cooled at −78 ◦C. The mixture was transferred immediately
and dropwise to another CH2Cl2 solution (0.2 ml) containing
2 6 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 – 2 6 2

View Article Online
ꢀ
ꢀ
N-[1,3]Dithiane-2-carboxylphenethyl-amide (12c). 260 mg
11a gave 82 mg of a white solid (18% yield, >95% purity)
after flash chromatography (eluent: hexane then 10% EtOAc
in hexane); mp 131–133 ◦C; 1H NMR (300 MHz, DMSO-d6) d
7.27–7.14 (m, 5 H, Ar-H), 5.23 (br s, 1 H, NH), 4.25 (s, 1 H,
CH-2ꢀ), 3.51 (q, J = 22, 2 H, CH2-1), 2.95–2.87 (m, 2 H, CH2-
4ꢀ), 2.79 (t, J = 23, 2 H, CH2-2), 2.65–2.57 (m, 2 H, CH2-4ꢀ),
1.96–1.89 (m, 2 H, CH2-5ꢀ); 13C NMR (75 MHz, DMSO-d6) d
167.6 (1 C, C-1ꢀ), 138.5, 128.8, 128.6, 126.6 (6 C, Ar), 46.7, 41.3
(2 C, C-1 and C-2ꢀ), 35.4 (1 C, C-2), 28.1, (2 C, 2 × C-4ꢀ), 25.1
(1 C, C-5ꢀ); LC-MS (ESI) tR = 6.39 min (m/z [MH]+); HRMS
(M + H)+ calcd. for C13H18NOS2 268.0830, found 268.0831.
(12 C, 2 × C-4, and 6 × CH2 ), 13.9, 13.9 (2 C, 2 × CH3 ); LC-MS
(ESI) tR = 8.06 min (m/z 290 [MH]+); HRMS (M + H)+ calcd.
for C13H24NO4S 290.1426; found 290.1439.
N-(Nonylsulfinylacetyl)-L-homoserine lactone (13d). 207 mg
10d gave 65 mg of a white solid. 36% yield (>99% purity); H
NMR (300 MHz, CDCl3, 57 : 43 diastereomeric mixture) d 7.85
1
3
3
(br d, J = 9 Hz, 0.57 H, NH), 7.82 (br d, J = 9 Hz, 0.43 H,
NH), 4.83–4.22 (m, 3 H, CH2-5 and CH-3), 3.77 (d, 3J = 14 Hz,
0.57 H, CH2-2ꢀ), 3.74 (d, 3J = 14 Hz, 0.43 H, CH2-2ꢀ), 3.38 (d,
3J = 14 Hz, 0.43 H, CH2-2ꢀ), 3.32 (d, 3J = 14 Hz, 0.57 H, CH2-
2ꢀ), 3.14–2.23 (m, 4 H, CH2-4 and CH2-4ꢀ), 1.81–1.68 (m, 2 H,
CH2-5ꢀ), 1.49–1.26 (m, 12 H, 6 × CH2 ), 0.87 (t, 3J = 7 Hz, 6 H,
ꢀ
2 × CH3 ); 13C NMR (75 MHz, CDCl3) d 174.4, 174.4 (2 C, 2 ×
ꢀ
General procedure for the preparation of sulfoxides 13a–e.
Over a period of 15 min, a 0.5 M solution of m-CPBA (1.0 equiv.,
based on the lowest purity reported on the m-CPBA label) in
CH2Cl2 (m-CPBA is potentially explosive when highly purified
and therefore the solution was just dried with MgSO4 and filtered
into the reaction mixture) was added to a clear 0.1 M solution
of sulfide (1.0 equiv.) in CH2Cl2 cooled at −10 ◦C. It turned
yellow–orange and then after a few min colourless again. After
3 h of stirring the reaction mixture was warmed to RT, washed
with 5% NaHCO3 (20 ml), dried (MgSO4) and evaporated to
dryness. The product was isolated as a diastereomeric mixture
by flash chromatography (eluent: 1% MeOH in CH2Cl2).
C-2), 165.0, 164.5 (2 C, 2 × C-1ꢀ), 65.8, 65.6 (2 C, 2 × C-5), 52.8,
52.6, 51.3, 51.0 (4 C, 2 × C-2ꢀ and 2 × C-4ꢀ), 49.5, 48.7 (2 C,
2 × C-3), 31.8, 31.8, 29.2, 29.2, 29.2, 29.1, 29.1, 29.1, 29.1, 29.1,
ꢀ
28.7 28.6, 23.0, 22.9, 22.6, 22.6 (16 C, 2 × C-4, and 14 × CH2 ),
ꢀ
14.1, 14.1 (2 C, 2 × CH3 ); LC-MS (ESI) tR = 6.40 min (m/z
318 [MH]+); HRMS (M + H)+ calcd. for C15H28NO4S 318.1739;
found 318.1716.
N-(Phenylsulfinylacetyl)-L-homoserine lactone (13e). 25 mg
10e gave 15 mg of a white solid. 56% yield (>99% purity); H
1
NMR (300 MHz, CDCl3, 57:43 diastereomeric mixture) d 7.70–
7.53 (m, 5H, Ar-H), 7.45 (br s, 1 H, NH), 4.59–4.17 (m, 3 H,
3
N-(Propylsulfinylacetyl)-L-homoserine lactone (13a). 98 mg
CH2-5 and CH-3), 3.83 (d, J = 14 Hz, 0.57 H, CH2-2ꢀ), 3.82
1
3
3
10a gave 63 mg of a white solid. 60% yield (>99% purity); H
(d, J = 14 Hz, 0.43 H, CH2-2ꢀ), 3.55 (d, J = 14 Hz, 0.43 H,
3
NMR (300 MHz, CDCl3, 53:47 diastereomeric mixture) d 7.98
(br d, 3J = 7 Hz, 0.53 H, NH), 7.92 (br d, 3J = 7 Hz, 0.47 H, NH),
4.77–4.20 (m, 3 H, CH2-5 and CH-3), 3.75 (d, 3J = 14 Hz, 0.53
H, CH2-2ꢀ), 3.72 (d, 3J = 14 Hz, 0.47 H, CH2-2ꢀ), 3.45 (d, 3J =
CH2-2ꢀ), 3.49 (d, J = 14 Hz, 0.57 H, CH2-2ꢀ), 2.71–2.54 (m,
1 H, CH2-4), 2.23–2.07 (m, 1 H, CH2-4); 13C NMR (75 MHz,
CDCl3) d 174.3, 174.2 (2 C, 2 × C-2), 164.2, 163.9 (2 C, 2 ×
C-1ꢀ), 141.2, 140.9, 131.8, 131.7, 129.5, 129.4, 124.1, 124.1 (12
C, Ar), 65.8, 65.7 (2 C, 2 × C-5), 58.1, 58.1 (2 C, 2 × C-2ꢀ), 49.1,
49.0 (2 C, 2 × C-3), 29.6, 29.6 (2 C, 2 × C-4); LC-MS (ESI)
tR = 6.04 min (m/z 268 [MH]+); HRMS (M + H)+ calcd. for
C12H14NO4S 268.0643; found 268.0621.
3
14 Hz, 0.47 H, CH2-2ꢀ), 3.38 (d, J = 14 Hz, 0.53 H, CH2-2ꢀ),
3.07–2.23 (m, 4 H, CH2-4 and CH2-4ꢀ), 1.85–1.71 (m, 2 H, CH2-
5ꢀ), 1.07 (t, 3J = 7 Hz, 3 H, CH3 ); 13C NMR (75 MHz, CDCl3)
ꢀ
d 174.4, 174.4 (2 C, 2 × C-2), 164.9, 164.4 (2 C, 2 × C-1ꢀ), 65.8,
65.7 (2 C, 2 × C-5), 53.9, 53.6, 53.3, 53.0 (4 C, 2 × C-2ꢀ and 2 ×
C-4ꢀ), 49.4, 48.8 (2 C, 2 × C-3), 29.0, 28.5, 16.5, 16.4 (4 C, 2 ×
General procedure for the preparation of 14a–e. At ambient
temperature m-CPBA (2.0 equiv., based on the lowest purity
reported on the m-CPBA label) was added to a 0.1 M solution
of sulfide (1.0 equiv.) in CH2Cl2. The mixture was stirred for
2 h and then washed with 5% NaHCO3. The organic phase was
dried (MgSO4) and evaporated to dryness. Purification by flash
chromatography (eluent: 1% MeOH in CH2Cl2) afforded the
product.
ꢀ
ꢀ
C-4 and 2 × CH2 ), 13.2, 13.2 (2 C, 2 × CH3 ); LC-MS (ESI)
tR = 5.29 min (m/z 234 [MH]+); HRMS (M + H)+ calcd. for
C9H16NO4S 234.0800; found 234.0795.
N-(Pentylsulfinylacetyl)-L-homoserine lactone (13b). 491 mg
1
10b gave 35 mg of a white solid. 7% yield (>99% purity); H
NMR (300 MHz, CDCl3, 56:44 diastereomeric mixture) d 7.85
3
3
(br d, J = 9 Hz, 0.56 H, NH), 7.82 (br d, J = 9 Hz, 0.44
N-(Propylsulfonylacetyl)-L-homoserine lactone (14a). 51 mg
10a gave 21 mg of a white solid. 36% yield (73% purity); mp
90–94 ◦C; 1H NMR (300 MHz, CDCl3) d 7.38 (br d, 3J = 5 Hz,
1 H, NH), 4.65–4.24 (m, 3 H, CH2-5 and CH-3), 3.96 (s, 2 H,
CH2-2ꢀ), 3.24–3.18 (m, 2 H, CH2-4ꢀ), 2.73–2.64 (m, 1 H, CH2-4),
2.44–2.29 (m, 1 H, CH2-4), 1.97–1.84 (m, 2 H, CH2-5ꢀ), 1.09 (t,
3
H, NH), 4.83–4.22 (m, 3 H, CH2-5 and CH-3), 3.77 (d, J =
14 Hz, 0.56 H, CH2-2ꢀ), 3.74 (d, J = 14 Hz, 0.44 H, CH2-2ꢀ),
3
3.38 (d, 3J = 14 Hz, 0.44 H, CH2-2ꢀ), 3.32 (d, 3J = 14 Hz, 0.56
H, CH2-2ꢀ), 3.14–2.23 (m, 4 H, CH2-4 and CH2-4ꢀ), 1.82–1.69
(m, 2 H, CH2-5ꢀ), 1.51–1.30 (m, 4 H, 2 × CH2 ), 0.91 (t, J =
ꢀ
3
7 Hz, 3 H, CH3 ); 13C NMR (75 MHz, CDCl3) d 174.4, 174.4 (2
ꢀ
3J = 7 Hz, 3 H, CH3 ); 13C NMR (75 MHz, CDCl3) d 174.5 (1 C,
ꢀ
C, 2 × C-2), 165.0, 164.5 (2 C, 2 × C-1ꢀ), 65.8, 65.7 (2 C, 2 ×
C-5), 52.8, 52.7, 51.2, 51.0 (4 C, 2 × C-2ꢀ and 2 × C-4ꢀ), 49.5,
48.7 (2 C, 2 × C-3), 30.7, 30.7, 29.2, 28.6, 22.6, 22.6, 22.2, 22.2
C-2), 162.2 (1 C, C-1ꢀ), 66.0 (1 C, C-5), 58.5, 54.9 (2 C, C-2ꢀ and
C-4ꢀ), 49.6 (1 C, C-3), 28.7 (1 C, C-4), 15.7, 13.0 (2 C, C-5ꢀ and
ꢀ
CH3 ); LC-MS (ESI) tR = 4.51 min (m/z 250 [MH]+); HRMS
ꢀ
ꢀ
(8 C, 2 × C-4 and 6 × CH2 ), 13.7, 13.7 (2 C, 2 × CH3 ); LC-MS
(ESI) tR = 5.03 min (m/z 262 [MH]+); HRMS (M + H)+ calcd.
for C11H20NO4S 262.1113; found 262.1126.
(M + H)+ calcd. for C9H16NO5S 250.0749, found 250.0786.
N-(Pentylsulfonylacetyl)-L-homoserine lactone (14b). 123 mg
10b gav◦e 72 mg of a white solid. 29% yield (>99% purity); mp
97–100 C; 1H NMR (300 MHz, CDCl3) d 7.08 (br d, 3J = 5 Hz,
1 H, NH), 4.62–4.25 (m, 3 H, CH2-5 and CH-3), 3.92 (s, 2 H,
CH2-2ꢀ), 3.24–3.18 (m, 2 H, CH2-4ꢀ), 2.78–2.69 (m, 1 H, CH2-4),
2.41–2.27 (m, 1 H, CH2-4), 1.93–1.82 (m, 2 H, CH2-5ꢀ), 1.50–
N-(Heptylsulfinylacetyl)-L-homoserine lactone (13c). 123 mg
1
10c gave 78 mg of a white solid. 60% yield (>99% purity); H
NMR (300 MHz, CDCl3, 54 : 46 diastereomeric mixture) d 7.96
(br d, 3J = 6 Hz, 0.54 H, NH), 7.90 (br d, 3J = 7 Hz, 0.46 H, NH),
4.76–4.20 (m, 3 H, CH2-5 and CH-3), 3.75 (d, 3J = 14 Hz, 0.54
H, CH2-2ꢀ), 3.71 (d, 3J = 14 Hz, 0.46 H, CH2-2ꢀ), 3.44 (d, 3J =
ꢀ
ꢀ
1.33 (m, 4 H, 2 × CH2 ), 0.92 (t, 3J = 7 Hz, 3 H, CH3 ); 13C NMR
(75 MHz, CDCl3) d 174.1 (1 C, C-2), 162.0 (1 C, C-1ꢀ), 65.9 (1 C,
C-5), 58.5, 53.3 (2 C, C-2ꢀ and C-4ꢀ), 49.6 (1 C, C-3), 30.3, 28.9,
3
14 Hz, 0.46 H, CH2-2ꢀ), 3.37 (d, J = 14 Hz, 0.54 H, CH2-2ꢀ),
3.07–2.23 (m, 4 H, CH2-4 and CH2-4ꢀ), 1.78–1.67 (m, 2 H, CH2-
ꢀ
ꢀ
22.0, 21.6 (4 C, C-4 and CH2 ), 13.7 (1 C, CH3 ); LC-MS (ESI)
tR = 5.44 min (m/z 278 [MH]+); HRMS (M + H)+ calcd. for
C11H20NO5S 278.1062, found 278.1085.
ꢀ
ꢀ
5ꢀ), 1.49–1.26 (m, 8 H, 4 × CH2 ), 0.85 (t, 3J = 6 Hz, 3 H, CH3 );
13C NMR (75 MHz, CDCl3) d 174.6, 174.6 (2 C, 2 × C-2), 164.9,
164.4 (2 C, 2 × C-1ꢀ), 65.8, 65.7 (2 C, 2 × C-5), 53.8, 53.5, 51.5,
51.2 (4 C, 2 × C-2ꢀ and 2 × C-4ꢀ), 49.4, 48.8 (2 C, 2 × C-3),
31.4, 31.4, 29.0, 28.7, 28.7, 28.6, 28.6, 28.6, 22.8, 22.7, 22.4, 22.4
N-(Heptylsulfonylacetyl)-L-homoserine lactone (14c). 700 mg
10c gave 60 mg of a white solid. 6% yield (80% purity);
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 – 2 6 2
2 6 1

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mp 103–106 ◦C; 1H NMR (300 MHz, CDCl3) d 7.13 (br d, 3J =
5 Hz, 1 H, NH), 4.64–4.25 (m, 3 H, CH2-5 and CH-3), 3.92 (s,
2 H, CH2-2ꢀ), 3.24–3.18 (m, 2 H, CH2-4ꢀ), 2.68–2.77 (m, 1 H,
CH2-4), 2.42–2.27 (m, 1 H, CH2-4), 1.92–1.81 (m, 2 H, CH2-5ꢀ),
8 K. McClean, M. Winson, A. T. L. Fish, S. Chhabra, M. Camara, M.
Daykin, J. Lamb, S. Swift, B. Bycroft, G. Stewart and P. Williams,
Microbiology, 1997, 143, 3703.
9 J. Zhu, J. W. Beaber, M. I. More´, C. Fuqua, A. Eberhard and S. C.
Winans, J. Bacteriol., 1998, 180, 5398.
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Iglewski, J. Bacteriol., 1996, 178, 5995.
11 E. Pesci, J. Pearson, P. Seed and B. Iglewski, J. Bacteriol., 1997, 179,
3127.
ꢀ
ꢀ
1.50–1.25 (m, 8 H, 4 × CH2 ), 0.88 (t, 3J = 7 Hz, 3 H, CH3 ); 13
C
NMR (75 MHz, CDCl3) d 174.1 (1 C, C-2), 162.0 (1 C, C-1ꢀ), 65.9
(1 C, C-5), 58.5, 53.3 (2 C, C-2ꢀ and C-4ꢀ), 49.6 (1 C, C-3), 31.4,
ꢀ
28.9, 28.6, 28.2, 22.5, 21.9 (6 C, C-4 and 5 × CH2 ), 14.0 (1 C,
CH3 ); LC-MS (ESI) tR = 6.23 min (m/z 306 [MH]+); HRMS
ꢀ
12 J. A. Olsen, R. Severinsen, T. B. Rasmussen, M. Hentzer, M. Givskov
and J. Nielsen, Bioorg. Med. Chem. Lett., 2002, 12, 325.
13 T. Hjelmgaard, T. Persson, T. B. Rasmussen, M. Givskov and J.
Nielsen, Bioorg. Med. Chem., 2003, 11, 3261.
(M + H)+ calcd. for C13H24NO5S 306.1375, found 306.1357.
N-(Nonylsulfonylacetyl)-L-homoserine lactone (14d). 98 mg
10d gave◦81 mg of a white solid. 74% yield (69% purity); mp
14 Unpublished results.
15 K. M. Smith, Y. Bu and H. Suga, Chem. Biol., 2003, 10, 1.
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Joachimiak, Nature, 2002, 417, 971.
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Telford, D. I. Pritchard and B. W. Bycroft, J. Med. Chem., 2003, 46,
97.
20 A. Eberhard and J. B. Schineller, in Chemical Synthesis of Bacterial
Autoinducers and Analogs, Academic Press, New York, 2000, 305, p.
301.
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Tetrahedron Lett., 1998, 39, 297.
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Connolly, C.-H. Chu, R. J. George, D. A. Gordon, H. Jamil, K. G.
Jolibois, L. K. Kunselman, S.-J. Lan, T. J. Maccagnan, B. Ricci, M.
Yan, D. Young, Y. Chen, O. M. Fryszman, J. V. H. Logan, C. L.
Musial, M. A. Poss, J. A. Robl, L. M. Simpkins, W. A. Slusarchyk,
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P. Kristoffersen, M. Ko¨te, J. Nielsen, L. Eberl and M. Givskov,
J. Bacteriol., in press.
1
3
113–115 C; H NMR (300 MHz, CDCl3) d 7.16 (br d, J =
5 Hz, 1 H, NH), 4.63–4.25 (m, 3 H, CH2-5 and CH-3), 3.93 (s,
2 H, CH2-2ꢀ), 3.24–3.19 (m, 2 H, CH2-4ꢀ), 2.77–2.67 (m, 1 H,
CH2-4), 2.42–2.28 (m, 1 H, CH2-4), 1.91–1.81 (m, 2 H, CH2-5ꢀ),
1.50–1.26 (m, 12 H, 6 × CH2 ), 0.88 (t, 3J = 7 Hz, 3 H, CH3 );
13C NMR (75 MHz, CDCl3) d 174.1 (1 C, C-2), 162.0 (1 C, C-
1ꢀ), 65.9 (1 C, C-5), 58.5, 53.4 (2 C, C-2ꢀ and C-4ꢀ), 49.6 (1 C,
C-3), 31.8, 29.2, 29.1, 29.0, 29.0, 28.3, 22.6, 21.9 (8 C, C-4 and
ꢀ
ꢀ
ꢀ
ꢀ
7 × CH2 ), 14.1 (1 C, CH3 ); LC-MS (ESI) tR = 6.93 min (m/z
334 [MH]+); HRMS (M + H)+ calcd. for C15H28NO5S 334.1688,
found 334.1679.
N-(Phenylsulfonylacetyl)-L-homoserine lactone (14e). 20 mg
10e gave ◦13 mg of a white solid. 57% yield (>99% purity); mp
163–164 C; 1H NMR (300 MHz, CDCl3) d 7.99 (d, 3J = 8 Hz,
3
2H, o-SO2–C6H5), 7.71 (d, J = 8 Hz, 1H, p-SO2–C6H5), 7.60
3
3
(d, J = 8 Hz, 2H, m-SO2–C6H5), 7.30 (br d, J = 5 Hz, 1 H,
NH), 4.63–4.23 (m, 3 H, CH2-5 and CH-3), 4.09 (s, 2 H, CH2-
2ꢀ), 2.77–2.67 (m, 1 H, CH2-4), 2.33–2.19 (m, 1 H, CH2-4); 13
C
NMR (75 MHz, CDCl3) d 174.1 (1 C, C-2), 161.1 (1 C, C-
1ꢀ), 137.8, 134.6, 129.5, 128.3 (6 C, S–C5H6), 65.8 (1 C, C-5),
49.4 (1 C, C-3), 49.4 (1 C, C-2ꢀ), 29.3 (1 C, C-4); LC-MS (ESI)
tR = 4.95 min (m/z 222 [MH]+); HRMS (M + H)+ calcd. for
C12H14NO5S 284.0593, found 284.0603.
Acknowledgements
27 G. Derbesy and D. N. Harpp, Tetrahedron Lett., 1994, 35, 5381.
28 G. M. Li, S. Niu, M. Segi, K. Tanaka, T. Nakajima, R. A.
Zingaro, J. H. Reibenspies and M. B. Hall, J. Org. Chem., 2000, 65,
6601.
Financial support was in part obtained from the Royal Vet-
erinary and Agricultural University, the Technical University
of Denmark, Hede Nielsen Foundation, the Danish Technical
Research Council (Grant no. 56-00-0134) and the Villum Kann
Rasmussen Foundation (Grant 1002–12). Dr. J. Øgaard Madsen
is thanked for assistance with GC-MS and Søren Ryborg for
technical assistance. QSI Pharma A/S is acknowledged for their
generous donation of trans-2-aminocyclopentanol hydrochlo-
ride.
29 M. Focke, A. Feld and H. K. Lichtenthaler, FEBS Lett., 1990, 261,
106.
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34 S. R. Angle and R. M. Henry, J. Org. Chem., 1998, 63, 7490.
35 J. Brennan and P. J. Murphy, Tetrahedron Lett., 1988, 29,
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2 6 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 5 3 – 2 6 2