Antibacterial activities of fluorescent nano assembled triphenylamine phosphonium ionic liquids

Article abstract of DOI:10.1016/j.bmcl.2016.05.055

Staphylococcus aureus, a Gram positive coccal bacterium is a major cause of nosocomial infection. We report the synthesis of new triphenylamine phosphonium ionic liquids which are able to self-assemble into multiwall nanoassemblies and to reveal a strong

Full text of DOI:10.1016/j.bmcl.2016.05.055

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antibacterial activities of fluorescent nano assembled
triphenylamine phosphonium ionic liquids
Frédéric Brunel a, Christelle Lautard b, Frédéric Garzino a, Suzanne Giorgio a, Jean M. Raimundo a,
,
⇑
Jean M. Bolla b, , Michel Camplo a,
⇑
⇑
a Aix-Marseille Université, CNRS, Centre Interdisciplinaire de Nanoscience de Marseille, UMR 7325, UMR 7325163, avenue de Luminy, 13288 Marseille, France
b Aix-Marseille Université, IRBA, Transporteurs Membranaires, Chimiorésistance et Drug-Design (TMCD2), UMR-MD1, 27 boulevard Jean Moulin, 13385 Marseille cedex 05, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Staphylococcus aureus, a Gram positive coccal bacterium is a major cause of nosocomial infection. We
report the synthesis of new triphenylamine phosphonium ionic liquids which are able to self-assemble
into multiwall nanoassemblies and to reveal a strong bactericidal activity (MIC = 0.5 mg/L) for Gram
positive bacteria (including resistant strains) comparable to that of standard antibiotics. Time kill,
metabolism and fluorescence confocal microscopy studies show a quasi-instantaneously penetration of
the nanoassemblies inside the bacteria resulting of a rapid blocking (30 min) of their proliferation. As
confirmed by rezasurin reduction monitoring, these compounds strongly affect the bacterial metabolism
and a Gram positive versus Gram negative selectivity is clearly observed. These fluorescent phosphonium
ionic liquid might constitute a useful tool for both translocation studies and to tackle infectious diseases
related to the field of implantology.
Received 21 April 2016
Revised 17 May 2016
Accepted 19 May 2016
Available online xxxx
Keywords:
Triphenylamine
Phosphonium ionic liquids
Fluorescent probes
Antimicrobial
Ó 2016 Elsevier Ltd. All rights reserved.
Staphylococcus aureus
Staphylococcus aureus is a threatening pathogen and is a major
cause of human infections, while 30% of the human population is
commensally colonized with the bacterium. Infections caused by
this pathogen including bacteremia and infective endocarditis
(IE) as well as osteoarticular, skin and soft tissue, pleuropul-
monary, and device-related infections.1 S. aureus, in addition to
be responsible for many hospital’s acquired infections,2 belongs
to the ESKAPE group that includes major human pathogens with
multi-drug resistance.3 Despite recent findings,4 there is still an
urgent need to discover new molecules to fight against these par-
ticularly adaptive pathogens which are continuously developing
mechanisms of resistance.5 In this context, the aim of our studies
is the design of new ionic liquid and to evaluate the antibacterial
potential of new phosphonium ionic liquids (PILs) derived from
triphenylamine (TPA). Phosphonium ionic liquids are by far the
less studied ILs in comparison to nitrogen ionic liquids such as
alkylammonium or imidazolium salts. As a result, few examples
of antibacterial activity have been reported in the literature.6–8
Kanazawa et al.8 have shown that phosphonium salts provide an
advantage over the corresponding ammonium salts in bactericidal
activity and killing rate particularly against S. aureus. However, the
mechanism of action of phosphonium derivatives remained
unclear and the conjugation of a fluorescent probe like TPA with
PILs could provide a useful tool for bacteria localization of these
new TPA phosphonium conjugated salts. Indeed, TPA group has
been already used as fluorescent sensors or dyes9,10 but to our
knowledge only one example concerning a TPA coumarin deriva-
tive reports moderate activity against S. aureus.11 TPA/PIL’s deriva-
tives described in this Letter are amphiphilic molecules susceptible
to self-assembled into nanoassemblies and consequently they
might represent a valuable advantage for the crossing of outer
and/or inner bacterial membranes. In this Letter, we demonstrate
the remarkable antibacterial activity of TPA/PIL’s over a wide range
of bacteria strains particularly against ESKAPE’s Gram positive bac-
teria S. aureus and Enterococcus faecium as well as S. aureus resis-
tant strains with a MIC comparable to that of the best known
antibiotics.12 Those TPA/PILs also display improved MIC compared
to benzalkonium chlorides (BAC),13 cationic surfactants which are
the standard of comparison for antimicrobial ILs. Fluorescent
microscopy of the nanoassemblies formed provides evidence of a
Abbreviations: ESKAPE, Enterococcus faecium, Staphylococcus aureus, Klebsiella
species, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter
species; IL’s, ionic liquids; MIC, minimal inhibitory concentration; TBAF, tetrabuty-
lamonium fluoride; TEM, transmission electronic microscopy; TPA, triphenylamine;
PIL’s, phosphonium ionic liquids.
⇑
Corresponding authors.
E-mail addresses: jean-manuel.raimundo@univ-amu.fr (J.M. Raimundo), jean-
michel.bolla@univ-amu.fr (J.M. Bolla), michel.camplo@univ-amu.fr (M. Camplo).
http://dx.doi.org/10.1016/j.bmcl.2016.05.055
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Brunel, F.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.055

2
F. Brunel et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
quasi-instantaneously insertion onto the bacteria resulting of a
time kill inferior to that of 1 h.
PIL’s 12–14 as well as TPA alone didn’t show any antimicrobial
activity no matter the counterion nature. Moreover, compound
15 possessing an ether function similar to that of the active com-
pounds 9–11 was found inactive.
The target molecule 9, 10 and 11 can be easily synthesized via a
5 to 6 steps sequence (Scheme 1). Firstly, a Vilsmeier–Hack formy-
lation of commercial TPA affords the aldehyde 1 in good yield
(77%, see SI).14 Compound 1 was then successfully reduced with
NaBH4 in alcohol 2 in quantitative yield.15 The conversion of 2 in
corresponding sodium alkoxide using NaH allowed the Williamson
etherification by reaction with previously prepared silylated bro-
mohexanol 3 to give 4 (see SI). Protecting group was subsequently
removed with TBAF and the corresponding alcohol 5 was obtained
in quantitative yield. The following bromination reaction of 5 was
the tricky step of the synthesis. Among the several attempts
performed including Appel reactions, NBS/PPh3 in DCM appeared
to be the most reactive bromination agent affording 6 in 61% yield.
Lastly, in order to optimize yield and purity of final ionic liquid 9,
we focused on a fast, clean and safe solvent free microwave activa-
tion (200 W). Under these conditions, final compound 9 was
obtained in excellent yield (93%) within two hours. Compound
10 was obtained in 95% yield from 5 by treatment under Appel
conditions to give the corresponding chlorinated compound 7 in
65% yield followed by a nucleophilic substitution under micro-
waves. The last iodide salt 11 was obtained in 95% yield by
chloride-iodide exchange on 8 followed by substitution under
microwaves.
In order to complete our studies, the control’s phosphonium
salts described in Figure 1 were prepared through trivial syntheses
detailed in the experimental part. Note that compounds 12 and 13
have been synthesized according to previous literature reported
procedures.16
In order to prepare NAs from 9 a nano-precipitation method
was used.17 Compound 9 was solubilized into THF and the solution
was added to pure water under stirring. The nano-structures were
formed spontaneously. Following the removal of THF, a well-
dispersed colloid of 9 NAs was thus obtained. The sample was
characterized by TEM according to a known procedure18 and
nanoparticles inferior to that of 100 nm were observed (Fig. 1a),
Figure 2b showing a single multiwall nanoassembly.
This suggest that the unique combination of both TPA and tet-
raalkyl phosphonium part is determinant for the anti-bacterial
activity. In addition, we also tested the activity of compounds on
a strain of S. aureus overproducing the NorA efflux pump responsi-
ble for a multi-drug resistant phenotype.19 The same efficacy was
observed on the NorA overproducer SA1199B and its isogenic par-
ent SA1199, suggesting that the compounds were not efflux-pump
substrates. No activity however, was found on Gram negatives bac-
teria. The discrimination of activity between Gram positive and
Gram negative bacteria strains could be explained by considering
the structural diversity of the bacterial cell envelope. Whereas
Gram positive bacteria have a cytoplasmic membrane recovered
by a thick layer of peptidoglycan, Gram negative bacteria are sur-
rounded by additional hydrophobic membrane that may be less
susceptible to be crossed by the TPA/PIL’s compounds. As described
here, there is no significant difference in the activity examined for
the three compounds 9, 10 and 11 considered above. They share
the same molecular scaffold and only differ by the presence of a
different counterion (BrÀ, ClÀ, IÀ respectively). Thus, we selected,
one of them, compound 9 bearing a bromide ion for further studies.
Compound 9 showed a complete inhibition of growth of S. aureus
(strain CIP 7625) as shown in Figure 3A. This inhibition
corresponds to a strong bactericidal activity (Fig. 3B) with a
>3log10 CFU/mL inoculum reduction during the first 30 min. A sec-
ond period of inoculum decrease is observed with a smaller slope
after a 30 min time lag. Together, these two periods correspond
to a >4log10 CFU/mL inoculum reduction during the first 2 h of
incubation with 9 at 2 mg/L (MIC Â 4 for this strain). We then were
wondering if the bactericidal activity observed resulted from a
rapid inhibition of metabolic activity (Fig. 3C). The dashed line
curve shows the reduction rate of resazurin in resorufin after treat-
ment for 15 min of bacteria with 9 in the same amount as in A and
B. Compared to the untreated control (solid line), only a weak
reduction rate is observed in the treated cells, demonstrating a
strong inhibition of the bacterial metabolism.
The Minimal Inhibitory Concentration (MIC) of each compound
was determined against a series of strains representative of the
ESKAPE pathogens3 and are summarized in Table 1. While TPA
and compounds 12–15 did not show antimicrobial activity in a
range of concentration up to 64 mg/L, three compounds, 9, 10
and 11 exhibited a significant activity against the Gram positive
Staphylococcus aureus (MIC = 0.5–1 mg/L), and the difficult-to-treat
Enterococci (MIC = 1–2 mg/L). It is noteworthy that the tetraalkyl-
This remarkable results encouraged us to undertake further
experiments taking advantage of the intrinsic fluorescence of the
TPA moiety of 9. Intra-cellular accumulation of a drug is a key step
in biocides mode of action. This process can be impaired by envel-
ope impermeability and/or efflux transport. Together these
processes can drive resistance of the bacteria by strongly decreas-
ing the intra-cellular concentration of the drug, and consequently
O
OTBDMS
O
OH
O
OH
4
4
(b)
(c)
(a)
N
N
N
N
1
2
4
5
O
X
4
(d)
(e)
(g)
(g)
9 (93%), X = P+Bu3 Br-
10
6
, X= Br
(87%), X = P+Bu3 Cl-
11 (95%), X = P+Bu3 I-
7
, X= Cl
N
(g)
(f)
8, X= I
Scheme 1. Synthesis of the target compounds 9, 10 and 11. Reagents and conditions: (a) NaBH4, ethanol/chloroform, RT (quant.); (b) NaH, 18-crown-6, 78 °C and Br
(CH2)6OTBDMS 3 (86%); (c) TBAF, THF, RT (quant.); (d) NBS, PPh3, DCM, 0 °C to RT (61%); (e) CCl4, P(Ph)3, 0 °C to reflux (65%); (f) NaI, acetone, reflux (95%) (g) PBu3,
microwaves (200 W), 140 °C.
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F. Brunel et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Br
Cl
Br
I
P
P
P
P
O
12
13
15
14
Figure 1. Structure of phosphonium salts 12–15.
(a)
(b)
Figure 2. (a) TEM image of compound 9. (b) Enlargement of a single nanoassembly (distance between two arrows or walls: 2–2.6 nm).
Table 1
Minimal Inhibitory Concentration (MIC) (mg/L) of PIL’s and TPA PIL’s on ESKAPE bacteria
Compounds
20477
CIP 7625
CIP 82.91
ATCC 19606
CIP 100720
ATCC 13048
CIP 54.8
SA1199
SA1199B
E. faecium
S. aureus
K. pneumoniae
A. baumannii
P. aeruginosa
E. aerogenes
E. coli
S. aureus
S. aureus
9
1
1
2
>64
>64
>64
>64
>64
0.5
1
1
>64
>64
>64
>64
>64
8
16
16
>64
>64
>64
>64
>64
16
16
16
>64
>64
>64
>64
>64
>64
32
32
>64
>64
>64
>64
>64
32
16
32
>64
>64
>64
>64
>64
16
16
16
>64
>64
>64
>64
>64
0.5
0.5
1
>64
>64
>64
>64
>64
0.5
0.5
1
>64
>64
>64
>64
>64
10
11
12
13
14
15
TPA
Figure 3. Mode of action of compound 9. Proliferation assay assessed by absorbance at 600 nm (A) viability assays assessed by CFU counts (B) and metabolic activity assay
based on resazurin reduction monitoring (C) of S. aureus (CIP7625) with (dashed lines) or without (solid lines) compound 9 at 2 mg/L (MIC Â 4).
abolish the inhibition of the target. The balance of these two pro-
cesses can be observed by following the kinetics of accumulation
of the molecule in bacteria. As shown on Figure 4, a fluorescence
was observed into the bacteria from about 1.7 s of incubation,
and this fluorescence reach a maximum at about 35 s. Phase
contrast allow us to realize that all the bacteria accumulate the
compound. This showed that 9 very rapidly penetrate the bacterial
envelop to reach its metabolic target.
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4
F. Brunel et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Acknowledgements
143
1.7
35.75
We are indebted to Jean-Marie Pagès, director of the UMR-MD1
A
B
for his support. We thank Pascal Weber (UMR6236, Aix Marseille
University), for his excellent technical assistance in confocal
microscopy.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.05.
055.
5 µm
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Author contributions: The manuscript was written through
contributions of all authors. All authors have given approval to
the final version of the manuscript. Frédéric Brunel and Christelle
Lautard contributed equally.
Please cite this article in press as: Brunel, F.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.055