Thiourea-catalyzed aminolysis of N-acyl homoserine lactones

Article abstract of DOI:10.1039/c3cc00268c

Thiourea catalysts accelerate aminolysis of N-acyl homoserine lactones (AHLs), molecules integral to bacterial quorum sensing. The catalysts afford rate enhancement of up to 10 times the control in CD₃CN. Mild catalysis in other polar aprotic s

Full text of DOI:10.1039/c3cc00268c

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Thiourea-catalyzed aminolysis of N-acyl homoserine
lactones†
Cite this: Chem. Commun., 2013,
49, 2055
a
b
a
´
Michael A. Bertucci, Stephen J. Lee and Michel R. Gagne*
Received 30th November 2012,
Accepted 22nd January 2013
DOI: 10.1039/c3cc00268c
www.rsc.org/chemcomm
Thiourea catalysts accelerate aminolysis of N-acyl homoserine
lactones (AHLs), molecules integral to bacterial quorum sensing.
The catalysts afford rate enhancement of up to 10 times the control
in CD3CN. Mild catalysis in other polar aprotic solvents is still
observed, while the activity is attenuated in polar protic solvents.
Translating well-established organic reactions to physiological
conditions is one approach for solving contemporary problems
in biology. Bacterial virulence mediated by a density-dependent
communication pathway known as quorum sensing (QS) is one
such problem.1 For instance, QS pathways in P.aeruginosa
and B. cepacia are responsible for opportunistic infections in
immunocompromised patients.2
In most gram-negative bacteria, N-acyl homoserine lactones
(AHLs) are integral to this multicellular communication network
(Scheme 1).3 Efforts to inhibit QS have focused on small molecule
inhibitors of the AHL synthases and receptors, while methods of
disabling the AHL messenger directly have been less extensively
investigated.4–6 In fact, beyond the physiological degradation of
the AHLs, their fundamental reactivity is not well documented.
Scheme 2 The proposed mode of activation of an AHL through H-bonding with
a thiourea catalyst.
To begin addressing this deficiency, we sought to determine the
susceptibility of AHLs to nucleophilic ring opening, specifically by
amines to form stable amides (eqn (1)).
Traditionally, reactions of amines with butyrolactones are
sluggish and require a large excess of the nucleophile and/or
heat.7 The recalcitrant nature of this simple acyl transfer
reaction offered the opportunity to develop catalysts for the
aminolysis of AHLs. Use of thiourea organocatalysts appeared
fitting as they have proven effective in the supramolecular
activation of esters (Scheme 2).8 This H-bond activation mecha-
nism has been showcased in a variety of classic organic
methodologies, including the polymerization of cyclic esters.9
Of relevance to potential biological applications are examples
of thiourea catalysis in water.10 In this communication, the
utility of thiourea catalysts for the aminolysis of AHLs is
assessed to determine if these catalysts can be utilized for
irreversible AHL derivitization.
Scheme 1 AHLs implicated in quorum sensing; sites for interspecies variability
include the length of the N-acyl chain (red) and the oxidation state (blue). The
C6-AHL 3, the C4-AHL 4, and 3-oxo-C12-AHL 5 were each tested for amenability to
thiourea catalysis.
a Department of Chemistry, University of North Carolina at Chapel Hill,
Campus Box 3290, Caudill and Kenan Laboratories, Chapel Hill, NC 27599-3290,
USA. E-mail: mgagne@unc.edu; Fax: +1 919-962-2388; Tel: +1 919-962-0278
b US Army Research Office, P.O. Box 12211, Research Triangle Park, NC 27709, USA
† Electronic supplementary information (ESI) available: Synthetic procedures,
1H and 13C NMR spectra, and catalyst screening procedure. See DOI: 10.1039/
c3cc00268c
(1)
To evaluate the ability of thiourea organocatalysts to
accelerate the aminolysis of AHLs, we synthesized a small
library of catalysts (Table 1). The thioureas were assembled
c
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Table 1 Yields for the synthesis of each thiourea catalyst and relative rate
constants for 10 mol% of the catalysts in aminolysis of the C6-AHL with
piperidinea
was observed, supporting an AHL–catalyst interaction. The
entire library of thioureas proved active in accelerating the
aminolysis of the AHL. The monofunctional and bifunctional
thioureas enhanced the nucleophilic ring opening up to 5.6
times the background reaction while the dithioureas reached a
rate 10-fold faster than the control. Notably, the bifunctional
catalysts did not provide any noticeable advantage compared to
the monofunctional catalysts. This lack of secondary activation
may be due to an inherent weakness of the anticipated N–HÁ Á ÁN
bonding interaction between the amine nucleophile and the
nitrogen containing appendages of the catalysts.
Given the structural variety in the AHL family, two other
AHLs were tested. The C4-AHL (4) and 3-oxo-C12-AHL (5) are
both utilized by the human pathogen P.aeruginosa, but vary in
alkyl chain length; the 3-oxo-C12-AHL is also oxidized at C3 of
the alkyl chain (Scheme 1).3 Both AHLs were screened in
CD3CN in the presence of catalyst 2a and our best performing
catalyst 2h (10 mol%) (Table 2). The C4-AHL performed
comparably to the C6-AHL. On the other hand, the barely
soluble 3-oxo-C12-AHL was less amenable to thiourea catalysis
with 2a and 2h, displaying a rate increase only 2.4 and 4.0 times
the control, respectively. Though lipophilic interactions could
be leading to substrate aggregation in the markedly nonpolar
3-oxo-C12-AHL, the decrease in relative rate may also be
attributed to the extra carbonyl moiety’s competitive affinity
Catalyst
2a
R
Yield (%) krel CD3CN krel DMF-d7
4.5
5.6
4.3
4.4
3.4
3.6
3.4
3.3
90
84
82
87
2b
2c
2d
2e
38
11
5.5
4.5
3.2
—
2f
2g
79
83
7.9
5.2
5.3
2h
10
a
For further details on the screening procedure, see ESI. The reactions
were monitored until 50% conversion (t50) was reached. See Table 3 for
the t50 values of the uncatalyzed reaction.
through the reaction of a primary amine with 3,5-bis(trifluoro- for the thiourea.
methyl) phenylisothiocyanate (1) to form 2 (eqn (2)). The
The effect of solvent on catalysis and thermal background
electron withdrawing ability of the 3,5-bis(trifluoromethyl) reactivity was also assessed (Table 3). In general, more polar
phenyl functionality is known to enhance the H-bond donating solvents decelerated the aminolysis rate (entries 1–5), though
ability and consequent catalytic potency of the thiourea.11 Eight catalytic rate accelerations were still observed. Catalyst 2h was
potential organocatalysts were synthesized; monofunctional quite effective in DMF-d7 (entry 4), a solvent that we expected
catalysts 2a and 2b from simple alkyl amines, and bifunctional might deactivate the catalyst by competitive H-bonding.
catalysts 2c–2f from alkylated diamines and histamine. The
To progress closer to physiological conditions, a variety of
scope of bifunctional catalysts ranged from catalysts hypo- protic co-solvents (10% v/v) were tested. These protic additives
thesized to assist in nucleophile activation by H-bonding
interactions (2c and 2d) to those conceivably providing anionic
Table 2 Relative rates of aminolysis of different AHLs in CD3CNa
transition state stabilization (2f).9,12 The reaction of simple
diamines with 1 yielded dithiourea compounds 2g and 2h.
AHL
krel (2a)
krel (2h)
C6-AHL (3)
C4-AHL (4)
3-oxo-C12-AHL (5)
4.5
4.1
2.7
10
7.3
4.0
a
With piperidine as the amine nucleophile in 20-fold excess.
(2)
Table 3 Relative rates of C6-AHL aminolysis in the presence of catalysts 2a and
2h in aprotic solvents and protic solvent mixturesa
t50 uncat (Â103 s) krel (2a) krel (2h)
b
Entry Solvent (% v/v)
1
2
3
4
5
6
7
8
9
C6D6
CD2Cl2
CD3CN
DMF-d7
5.1
3.6
2.0
1.0
4.5
3.4
1.9
0.9
1.3
1.1
1.0
4.3
1.1
10
5.3
2.2
1.5
1.0
1.0
0.9
35.5
100.7
123.2
14.3
21.0
9.7
(3)
DMSO-d6
10% MeOH-d4–CD3CN
10% D2O–DMSO-d6
10% TFE-d3–CD3CN
10% D2O–CD3CN
The catalysts were then screened for activity in a C6-AHL (3)
(50 mM) and piperidine (20 eq.) control reaction (eqn 3).‡ AHL
conversion in each catalytic trial was monitored by 1H NMR
spectroscopy in CD3CN. Prior to the addition of piperidine,
a slight downfield shift in the N–H resonances of the thiourea
4.2
a
b
With piperidine as the amine nucleophile in 20-fold excess.
t50 represents the reaction time to reach 50% conversion.
c
2056 Chem. Commun., 2013, 49, 2055--2057
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‡ Piperidine was chosen during a screen of several amines for reaction
with C6-AHL. The timescale of the reaction was appropriate for
monitoring aminolysis by 1H NMR.
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c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 2055--2057 2057