Robust routes for the synthesis of N-acylated-l-homoserine lactone (AHL) quorum sensing molecules with high levels of enantiomeric purity

Article abstract of DOI:10.1016/j.tetlet.2011.04.059

The ready availability of native quorum sensing molecules and related structural analogues is of significant biological interest in the development of methods to manipulate bacterial quorum sensing systems in a useful fashion. In this Letter we report robust routes for the synthesis of a range of N-acylated-l-homoserine lactone (AHL) quorum sensing molecules. Crucially, we have analysed the enantiopurity of the final AHLs and in all cases, excellent levels were observed.

Full text of DOI:10.1016/j.tetlet.2011.04.059

Tetrahedron Letters 52 (2011) 3291–3294
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Tetrahedron Letters
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Robust routes for the synthesis of N-acylated-L-homoserine lactone (AHL)
quorum sensing molecules with high levels of enantiomeric purity
James T. Hodgkinson a, Warren R. J. D. Galloway a, Mariangela Casoli a, Harriet Keane a, Xianbin Su a,
George P. C. Salmond b, Martin Welch b, David R. Spring a,
⇑
a Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
b Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
The ready availability of native quorum sensing molecules and related structural analogues is of signif-
icant biological interest in the development of methods to manipulate bacterial quorum sensing systems
in a useful fashion. In this Letter we report robust routes for the synthesis of a range of N-acylated-L-
homoserine lactone (AHL) quorum sensing molecules. Crucially, we have analysed the enantiopurity of
the final AHLs and in all cases, excellent levels were observed.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 25 February 2011
Revised 30 March 2011
Accepted 15 April 2011
Available online 23 April 2011
Keywords:
Quorum sensing
N-acylated-L-homoserine lactones (AHLs)
Synthesis
Enantiomeric purity
Pseudomonas aeruginosa
Quorum sensing is a mechanism of intercellular communication
employed by numerous species of bacteria. This signalling process,
mediated by small diffusible molecules termed autoinducers, al-
lows the cells comprising a bacterial colony to coordinate their
genome expression in a cell-density dependent manner.1 Quorum
sensing has been shown to play a critical role in both pathogenic
and symbiotic bacteria-host interactions.2 For example the bacte-
rium Pseudomonas aeruginosa, one of the most prevalent pathogens
in a range of life-threatening nosocomial infections and the pri-
mary cause of mortality in cystic fibrosis sufferers, uses quorum
sensing to regulate a variety of processes associated with viru-
lence.3 These include the formation of biofilms and the expression
of virulence factors.4
Though quorum sensing systems are used by many bacterial
pathogens to regulate virulence they are not essential for sur-
vival.3a Thus, disruption of quorum sensing (so-called ‘quorum-
quenching’) should attenuate pathogenicity without imposing the
level of selective pressure associated with antibacterial treat-
ments.4c,5,6 Consequently, the targeting of bacterial quorum sens-
ing systems represents an attractive alternative therapeutic
approach for the treatment of human bacterial infections.4c,7 In-
deed, there is proof-of-concept from animal studies that the viru-
lence of the Gram-negative bacterium P. aeruginosa can be
partially attenuated in vivo by the inhibition of quorum sensing.4c,8
The synthesis of small molecules which are capable of modulat-
ing bacterial quorum sensing systems has attracted significant
attention in recent years.9 Many of these molecules are based
around the structures of known, native bacterial auto-inducers.
N-Acylated-L-homoserine lactones (AHLs) are among the most
common signal molecules produced and used by Gram-negative
bacteria for intercellular communication.5 For example, P. aerugin-
osa uses (at least) three types of quorum sensing signals. Two of
these are AHL based (Fig. 1), employing N-(3-oxododecanoyl)-L-
homoserine lactone (OdDHL) and N-butanoyl-L-homoserine lac-
tone (BHL).9
Given the integral role of AHLs in quorum sensing systems it is
unsurprising that there is a large body of work pertaining to their
synthesis and that of other analogous structures.9,10 However, de-
tailed experimental protocols and full analytical data, particularly
enantiomeric purity, has not always been provided. Indeed, access-
ing such compounds with reliably high levels of enantiomeric
O
H
BHL
N
O
O
O
O
OdDHL
H
N
O
O
⇑
Corresponding author. Tel.: +44 1223 336 498; fax: +44 1223 336 362.
E-mail address: spring@ch.cam.ac.uk (D.R. Spring).
Figure 1. Molecules employed by P. aeruginosa in quorum sensing.
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.04.059

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J. T. Hodgkinson et al. / Tetrahedron Letters 52 (2011) 3291–3294
Xu et al. reported a related one-pot procedure for the formation
H
O
O
O
1. pyridine,
of b-ketoamides via the reaction of amines with Meldrum’s acid
adducts 2, with their data suggesting that the reaction requires a
slightly acidic medium for optimum results.12 Thus we initially
decided to attempt one-pot AHL synthesis under acidic conditions.
CH2Cl2, 0 oC
O
O
n
2.
O
O
O
2
O
O
3
Cl
0 oC to rt
n
Proof-of-principle studies were directed towards N-3-oxoacyl-L-
homserine lactone (OOHL) (Scheme 2). Pyridine and hexanoyl
chloride were added to a solution of Meldrum’s acid (3) in dichlo-
romethane. Upon completion of the reaction the solvent was re-
moved in vacuo and the residue was re-suspended in
acetonitrile.13
O
3.
NH3Br
4
O
O
H
N
n
(S)-(À)-a-amino-c-butyrolactone hydrobromide (4) was added
O
followed by trifluoroacetic acid and the resulting solution was stir-
red at 45 °C until the reaction was observed to have proceeded to
completion. The solvent was removed in vacuo and the crude prod-
uct was purified by column chromatography to yield OOHL in a
good overall yield and with an excellent enantiomeric purity
(enantiomeric excess ꢀ95% as determined by chiral HPLC). Our ini-
tial excitement at this result was quickly tempered by the realiza-
tion that the reaction was extremely capricious. Indeed, the results
of our initial experiment could not be obtained on a consistent ba-
sis. A significant problem was the formation of the undesired AHL
side-product 5 at some point during the course of the reaction se-
quence. Unfortunately, 5 was found to co-elute with the desired
product on silica and consequently it proved extremely difficult
to isolate analytically pure product by column chromatography.
It was eventually determined that this side-product arose in the
second stage of the reaction sequence, presumably though amida-
tion of the exocyclic carbonyl group of intermediate adduct 6
(Scheme 2, path b).14 We speculated that the acidic reaction condi-
tions may have influenced the regioselectivity of the nucleophilic
attack on adduct 6 through some unknown mechanism.15 To inves-
tigate this further we repeated the process outlined in Scheme 2
using triethylamine rather than TFA in the third step.16 Under
these basic reaction conditions, no formation of the side-product
5 was observed. Analytically pure OOHL could be isolated by col-
umn chromatography and the reaction proved to be fairly repro-
ducible with a range of acid chlorides to furnish several AHL
derivatives. However, isolated yields of products were relatively
poor (<30%) as were enantiomeric purities (<70% enantiomeric
excess).
O
O
1
Scheme 1. Proposed one-pot synthesis of b-ketoamide AHLs.
purity remains a largely unmet challenge. Herein we report upon
our research towards the development of general and efficient
methods for the synthesis of a variety of AHLs. These studies have
led to robust synthetic routes towards a variety of native AHLs
from readily available starting materials, allowing access to these
biologically important molecules in good yields, and crucially, with
reliably excellent levels of enantiomeric purity. Full experimental
details and analytical data for all the AHLs synthesized are given
(Supplementary data) and we hope that this collection of informa-
tion will be of significant assistance to the practising chemist in
this field.
Our research was directed towards accessing the two major
structural classes of AHLs; those with unfunctionalized acyl chains
(e.g., BHL) and those containing an N-acyl b-diketone moiety
(henceforth known as b-ketoamide AHLs, e.g., OdDHL). Initial stud-
ies focused upon the development of a one-pot method for the syn-
thesis of b-ketoamide AHLs (1). Towards this end, we were inspired
by a report from Chhabra et al. which detailed the synthesis of a
variety of OdDHL analogues using adducts of the form 2, produced
from the DCC-mediated coupling of carboxylic acid derivatives
with Meldrum’s acid (3), as key intermediates (Scheme 1).11 In
their report, these adducts were isolated from the reaction mixture
by a standard work-up procedure and treated in a separate vessel
with the hydrochloride salt of (S)-(À)-
a-amino-c-butyrolactone
Given the problems associated with the one-pot method de-
scribed above, we were interested in exploring alternative routes
towards b-ketoamide AHLs, as well as developing an efficient syn-
thesis of AHLs with unfunctionalized acyl chains. The ability to ac-
cess both of these structural classes of AHLs with reliably excellent
levels of enantiomeric purity was a key goal.
(4) in the presence of a base to afford the corresponding AHL ana-
logue. We envisaged a modification of this method whereby ad-
duct 2, formed by the reaction of Meldrum’s acid with an acid
chloride, could be isolated in situ by solvent removal upon comple-
tion of the reaction, rather than through a work-up process. Addi-
tion of 4 to the same reaction vessel would lead directly to the
corresponding AHL derivative 1 (Scheme 1). Overall, this sequence
would thus comprise a novel one-pot synthesis of AHLs carrying an
N-acyl b-diketone moiety.
AHLs with unfunctionalized acyl chains (7) could be readily
obtained by adaptation of previously reported conditions involving
the reaction of commercially available (S)-(À)-a-amino-c-butyro-
O
H
4
N
O
H
1. pyridine,
Path b
O
O
Undersired
O
O
CH2Cl2, 0oC
O
O
3.
5
O
4
2.
Cl
O
NH3Br
4
O
O
O
O
O
6
4
3
TFA
Path a
0 oC to rt 2 h
then remove
CH2Cl2, add
CH3CN
O
H
N
4
Desired
O
O
O
OOHL
Scheme 2. One-pot synthesis of OOHL.

J. T. Hodgkinson et al. / Tetrahedron Letters 52 (2011) 3291–3294
3293
priate acid chloride generated adducts 2. Subsequent treatment
of this crude material with methanol yielded b-ketoesters 8–11.
Acetal protection generated compounds 12–15 and ester hydroly-
sis furnished acid derivatives 16–19. EDC-mediated coupling with
O
O
H
N
n
O
NH3Br
Na2CO3
O
O
Cl
n
O
CH2Cl2/H2O
(S)-(À)-a-amino-c-butyrolactone hydrobromide (4) proceeded to
7
4
generate the protected amide products 20–23 in good yields. Final-
ly, acid-catalyzed acetal deprotection furnished the final products.
All steps outlined in Scheme 4 generally proceeded in good-to-
excellent yields, and were found to be amenable to scale-up, hav-
ing been performed successfully on multi-gram scale. In all cases
the spectroscopic data of the final compounds were consistent
with those previously reported in the literature (where available),
or those obtained from authentic samples. In addition, the enantio-
meric excesses of the AHL products were excellent indicating that
little racemization occurred during the reaction sequence. Thus
this route provides a reliable, scalable method for synthesizing sev-
eral b-ketoamide AHLs with excellent levels of enantiomeric
purity.
Scheme 3. Synthesis of AHLs using Schotten–Baumann conditions.
Table 1
AHLs synthesized using the Schotten–Baumann coupling procedure
Compound
n
Yield (%)
eea (%)
N-butanoyl-
L
-homoserine lactone (BHL)
-homoserine lactone (HHL)
-homoserine lactone (OHL)
-homoserine lactone (DHL)
2
4
6
8
86
97
96
98
96
>99
>99
>99
>99
>99
N-hexanoyl-
N-octanoyl-
N-decanoyl-
N-dodecanoyl-
L
L
L
L
-homoserine lactone (dDHL)
10
a
ee = enantiomeric excess as determined by chiral HPLC by comparison with
In this Letter we have reported the development of robust and
reliable synthetic routes to a variety of native quorum sensing mol-
ecules. Significantly, we have used chiral HPLC to analyse the enan-
tiopurity of the final AHLs, and in all cases, excellent levels were
observed. These results are timely and address a significant short-
coming of previously reported routes towards such compounds.
Thus the protocols that we have developed serve as arguably the
most reliable means to access a range of AHLs with excellent levels
of enantiomeric purity. Full experimental details and analytical
data for all final compounds reported in this manuscript are given,
and we hope that this collection of information will be of assis-
tance to the practising chemist in this field. Further analogue syn-
theses and structure–activity studies are ongoing and the results of
this work will be reported in due course.
independently synthesized racemic samples.
lactone hydrobromide (4) with a variety of acid chlorides under
Schotten–Baumann conditions (Scheme 3).11,17 Using this method-
ology, a range of AHLs (Table 1) were prepared in good-to-excel-
lent yields. The enantiomeric excesses of the AHL products were
determined by chiral HPLC and found to be consistently excellent
indicating the absence of any racemization during the coupling
process. Full experimental details and comprehensive analytical
data are provided for all final compounds synthesized by this route
(Supplementary data).
A
robust linear route towards the b-ketoamide AHLs,
N-(3-oxooctanoyl- -homoserine lactone (OOHL, n = 4), N-(3-oxo-
decanoyl)- -homoserine lactone (ODHL, n = 6), N-(3-oxododeca-
noyl)- -homoserine lactone (OdDHL, n = 8) and
N-(3-oxotetradecanoyl)- -homoserine lactone (OtDHL, n = 10)
L
L
Acknowledgements
L
L
We are grateful to funding from the EPSRC, BBSRC, MRC, Fran-
ces and Augustus Newman Foundation and Wellcome Trust.
was developed by adaptation of previously reported procedures
(Scheme 4).17a,b,18 Reaction of Meldrum’s acid (3) with the appro-
p-TsOH,
HO(CH2)2OH,
CH(OMe)3, rt
H
O
O
O
O
1. pyridine
O
O
O
CH2Cl2, 0 oC
O
O
MeOH
reflux
O
O
n
O
n
O
n
2.
O
O
O
O
n = 4, 8, 67%
n = 6, 9, 62%
n = 8, 10, 65%
n = 10, 11, 65%
n = 4, 12, 97%
n = 6, 13, 90%
n = 8, 14, 91%
n = 10, 15, 94%
Cl
0
n
3
2
o
to rt
C
(yields over 2 steps)
NaOH, rt
O
NH3Br
O
O
O
O
H
N
H
N
O
O
n
n
TFA, rt
HO
n
O
O
O
O
O
O
O
EDC, DMAP,
CH2Cl2, rt
n = 4, OOHL, 83%, e.e. >99%
n = 6, ODHL, 48%, e.e. >99%
n = 8, OdDHL, 79%, e.e. >99%
n = 10, OtDHL, 87%, e.e. >99%
n = 4, 20, 75%
n = 6, 21, 69%
n = 8, 22, 77%
n = 10, 23, 79%
n = 4, 16, 98%
n = 6, 17, 96%
n = 8, 18, 94%
n = 10, 19, 83%
Scheme 4. Linear route towards b-ketoamide AHLs. rt = room temperature. ee = enantiomeric excess as determined by chiral HPLC by comparison with independently
synthesized racemic samples.

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J. T. Hodgkinson et al. / Tetrahedron Letters 52 (2011) 3291–3294
Chem. Lett. 2008, 18, 5978; (b) Thomas, G. L.; Bohner, C. M.; Williams, H. E.;
Supplementary data
Walsh, C. M.; Ladlow, M.; Welch, M.; Byrant, C. E.; Spring, D. R. Mol. Biosyst.
2006, 2, 132; (c) Persson, T.; Hansen, T. H.; Rasmussen, T. B.; Skinderso, M. E.;
Givskov, M.; Nielsen, J. Org. Biomol. Chem. 2005, 3, 253.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.04.059.
11. Chhabra, S. R.; Harty, C.; Hooi, D. S. W.; Daykin, M.; Williams, P.; Telford, G.;
Pritchard, D. I.; Bycroft, B. W. J. Med. Chem. 2003, 46, 97.
12. Xu, F.; Armstrong, J. D.; Zhou, G. X.; Simmons, B.; Hughes, D.; Ge, Z. H.;
Grabowski, E. J. J. J. Am. Chem. Soc. 2004, 126, 13002. Note that this report did
not deal with the synthesis of b-ketoamide AHLs..
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