Phosphonium pillar[5]arenes as a new class of efficient biofilm inhibitors: Importance of charge cooperativity and the pillar platform

Article abstract of DOI:10.1039/c6cc05170g

Biofilm formation, which frequently occurs in microbial infections and often reduces the efficacy of antibiotics, also perturbs many industrial and domestic processes. We found that a new class of water soluble pillar[5]arenes bearing phosphonium moieties

Full text of DOI:10.1039/c6cc05170g

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Phosphonium pillar[5]arenes as a new class of
efficient biofilm inhibitors: importance of charge
cooperativity and the pillar platform†
Cite this:DOI: 10.1039/c6cc05170g
Received 21st June 2016,
Accepted 2nd August 2016
Roymon Joseph,‡ Dana Kaizerman,‡ Ido M. Herzog, Maya Hadar, Mark Feldman,
Micha Fridman and Yoram Cohen*
DOI: 10.1039/c6cc05170g
www.rsc.org/chemcomm
Biofilm formation, which frequently occurs in microbial infections
Among various interactions responsible for the formation of
and often reduces the efficacy of antibiotics, also perturbs many biofilms, electrostatic interactions are considered as one of the
industrial and domestic processes. We found that a new class of earliest forces influencing the adherence of bacterial cells to
6
water soluble pillar[5]arenes bearing phosphonium moieties (1, 2) surfaces. The outer surfaces of biofilms consist of an anionic
and their respective ammonium analogues (3, 4) inhibit biofilm matrix, and disruption of this matrix is thought to be an
formation with IC50 values in the range of 0.67–1.66 lM. These effective approach for preventing biofilm formation. Cationic
compounds have no antimicrobial activity, do not damage red amphiphiles therefore appear to be attractive candidates to
blood cell membranes, and do not affect mammalian cell viability inhibit early-stage biofilm formation by preventing adhesion of
7
in culture. Comparison of the antibiofilm activities of the phosphonium- the bacteria to a surface.
decorated pillar[5]arene derivatives 1 and 2 with their respective
In recent years a few inhibitors of biofilm formation based
8
ammonium counterparts 3 and 4 and their monomers 5 and 6, on cationic amphiphiles have been reported. Among the cationic
demonstrate that while positive charges, charge cooperativity and amphiphiles derived from quaternary ammonium and phos-
the pillararene platform are essential for the observed antibiofilm phonium salts, the later ones display increased antimicrobial
9
activity the nature of the charges is not.
properties compared to their ammonium counterparts. For
example, Endo and co-workers demonstrated the antimicrobial
According to the reports from the National Institutes of Health, properties of a series of phosphonium salts against 11 strains of
about 65% of infections treated in the developed world involve microorganisms including methicillin-resistant Staphylococcus
1
9a
microbial biofilms. Biofilm-associated diseases in humans aureus (MRSA). In recent years, compounds with phosphonium
include lung infections, ear infections, urinary and gastroin- moieties have been used in various biomedical applications and
10
testinal tract infections, chronic and burn wound infections, in water treatment including for antifouling purposes. Most of
2
nosocomial, catheter-related, and dental infections. The for- the aforementioned studies report use of phosphonium salts as
mation of biofilms on biomedical devices, surgical implants, biocides. The exception is a recent report by Fern ´a ndez and
urinary tract catheters, and contact lenses, dramatically increases co-workers describing the antifouling properties of several alkyl-
the chances of introducing persistent infections into the human triphenylphosphonium salts and their abilities to act as non-toxic
2
11
body. Biofilm growth also has detrimental effects in industrial quorum sensing disruptors. Indeed, Melander and co-workers
and domestic domains resulting in high costs associated with pointed out that it is extremely important to develop antibiofilm
3
cleaning and maintenance. In addition, bacteria in biofilms are agents operating via non-biocidal mechanisms for several reasons,
significantly more resistant to antibiotics than are bacteria grown the most important one is avoiding resistance development.
in suspension. Since prevention of biofilm formation could
12
4
Very recently, we found that ammonium and methyl imida-
dramatically reduce effects of infectious diseases and the cost of zolium cationic pillar[n]arenes are effective inhibitors of biofilm
industrial processes there is a great demand for molecules that formation by several strains of Gram-positive bacteria. Interest-
5
will effectively inhibit biofilm formation.
ingly, we observed that this new class of antibiofilm agents
shows no antimicrobial activity and no effect on bacterial growth
and causes no damage to red blood cells or toxicity to human
cells in culture. Therefore in the present work, we prepared a
series of phosphonium and ammonium decorated pillar[5]arenes
School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences,
Tel Aviv University, Tel Aviv 69978, Israel. E-mail: ycohen@post.tau.ac.il
13
†
Electronic supplementary information (ESI) available: Experimental procedures,
spectroscopic characterization data, and detailed protocols for the biological assays.
See DOI: 10.1039/c6cc05170g
(1–4, Scheme 1) and their respective monomers (5 and 6, Scheme 1)
‡
These authors contributed equally.
and studied their anti-biofilm activity with the aim of evaluating the
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a
Table 1 Inhibition of biofilm formation: MBIC50 values
b
À1
MBIC50 in mM (mg mL
)
Compounds
S. aureus ATCC 33592
E. faecalis ATCC 29212
1
2
3
4
5
6
1.55 (4)
1.33 (4)
1.66 (4)
0.71 (2)
4317 (160)
4340 (160)
1.55 (4)
0.67 (2)
1.66 (4)
1.41 (4)
4317 (160)
4340 (160)
Scheme 1 Schematic representation of cationic pillar[5]arene conjugates
(1–4) and monomers (5 and 6).
a
Compounds were evaluated using the double-dilution method. Each
value is the mean of at least three independent experiments that
b
included five replicates at each concentration. Values in parenthesis
are of MBIC50 in mg mL
effect of (i) the nature of the positive charges, (ii) the cooperativity of
the overall positive charges, and (iii) the pillar[n]arene platform on
the observed anti-biofilm activity.
À1
.
1
4a
Pillar[n]arenes, first reported in 2008,
have symmetrical
All the reported cationic pillar[5]arene derivatives exhibited
cylindrical structures and relatively large free volumes. potent inhibition of biofilm formation against the two tested Gram-
Pillar[n]arenes can be obtained and functionalized by simple positive pathogens. The MBIC50 values of the deca-phosphonium
and high yield synthesis routes making them a versatile macro- pillar[5]arenes 1 and 2 were found to be in the range of 0.67–1.55 mM
1
4,15
cycles for various applications.
These macrocycles possess for both of the tested strains. The corresponding deca-ammonium
host–guest properties owing to their p-electron rich cavity and pillar[5]arene analogues 3 and 4 showed a similar range of MBIC50
crown ether-like arrangement of oxygen atoms at both rims. values, 0.71–1.66 mM. These results indicate that replacement of the
Hence, in recent years, pillar[n]arenes have been used in host– ammonium cations by phosphonium cations does not significantly
guest chemistry and as sensors and were used to construct affect the inhibition of biofilm formation by cationic pillararenes.
supramolecular polymers, interlocked molecules, and hybrid Thus, the positive charges are essential for the observed anti-biofilm
1
4,15
16a
biomolecular materials.
More recently a phosphonium and activity; however, the nature of the charges has a marginal effect.
16b,c
several other phosphorus
containing pillararenes were reported. Note that in our previous study we showed that a negatively charged
However, despite the significant attention that pillararenes have deca-carboxylate derivative of pillar[5]arene does not significantly
1
3
received from the chemical community, their biological activity is inhibit biofilm formation.
only recently starting to be unravelled.
13,17
To evaluate the effect of hydrophobicity on the biofilm inhibi-
The water-soluble cationic pillar[5]arene derivatives used in tion activity, we compared compounds 2 and 4, in which the
this study were synthesized by a four-step process (see Scheme S1 in ammonium or phosphonium cations are attached to triethyl
the ESI†). Briefly, in the first step commercially available moieties, to compounds 1 and 3, which carry trimethyl moieties.
hydroquinone was alkylated with 1,3-dibromopropane using Despite the fact that compounds 2 and 4 have 30 more carbon
potassium carbonate in acetone to afford the monomer 1a atoms than do compounds 1 and 3, their MBIC50 values did not
(Scheme S1 in ESI†). The functionalized pillar[5]arene 1b was significantly differ (Table 1 and Fig. 1a, b). In addition, we found
obtained by the cyclization of monomer 1a with paraformalde- that the dose response for the tested pillar[5]arene derivatives 1–4
hyde and boron trifluoride diethyletherate in dichloroethane. (Fig. 1a and b) were also very similar, further corroborating the fact
Reaction of 1b with an excess of trimethylphosphine in ethanol that pillararenes 2 and 4 are as effective as 1 and 3 in preventing
under reflux gave the water-soluble compound 1 (Scheme 1). A biofilm formation by the two tested strains.
similar procedure was followed to obtain pillar[5]arene deriva-
To understand the cumulative effect of the positive charges and
tives 2, 3, and 4 by reaction with excess of triethylphosphine, the advantage of clustering these charges on a pillararene scaffold,
trimethylamine, and triethylamine, respectively (Scheme 1 and we synthesized two monomers, 5 and 6, corresponding to the
Scheme S1, ESI†). The control monomers 5 and 6 were synthe- repeating units of pillar[5]arenes 1 and 3, respectively. Compounds
sized by reacting 1a with excess trimethylphosphine and trimethyl- 5 and 6 were also tested for their biofilm inhibition properties
1
amine, respectively. All the compounds were characterized by H towards the two bacterial strains. Monomers 5 and 6 were tested at
13
and C NMR and high-resolution mass spectroscopy (HRMS). B5-fold higher concentrations than were compounds 1–4 such
Detailed synthetic procedures and the characterization data of that the numbers of charges and ionic strengths of the tested
compounds 1–6 are presented in the ESI† (see Fig. S1–S8).
solutions were comparable. Up to 317 mM of 5 and 340 mM of 6
À1
The effects of compounds 1–4 were evaluated on biofilm for- (160 mg mL of 5 and 6), neither 5 nor 6 caused a measurable
mation by two clinically important Gram-positive bacterial strains, inhibition of biofilm formation (Fig. 1c), suggesting that in these
S. aureus ATCC 33592 and Enterococcus faecalis ATCC 29212. Inhibi- anti-biofilm agents the cumulative charge organization on the
tion of biofilm formation was determined using the crystal violet pillar[5]arene scaffold is a crucial factor for the observed activity.
18
19
staining assay. The minimal concentration at which at least 50% This may be the manifestation of the multivalency effect.
reduction in biofilm formation compared to untreated cells (MBIC50 Many cationic amphiphiles act as antimicrobial agents that
was determined, and the results are summarized in Table 1. The kill bacteria.
dose responses are presented in Fig. 1 (see also Fig. S9–S11 in ESI†). effect of compounds 1–4 on biofilm formation originated from
)
9
,20
Therefore, to evaluate whether the inhibiting
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phosphonium and ammonium pillar[5]arene derivatives was
evaluated by incubation for 4 hours in solutions at different pH
values. Thereafter the materials were freeze dried, inspected by
1
H-NMR and tested for their biofilm inhibition properties. No
1
significant decomposition was observed in the H-NMR spectra
recorded after exposure to acidic or alkaline pH (see Fig. S11 in
ESI†). More importantly the anti-biofilm activities of compounds
2
and 4 remained unchanged after these exposures as seen in
Fig. S12 and Table S1 (ESI†).
Finally, it is well established that many cationic amphiphiles
disrupt mammalian cell membranes, which limits potential for
8
b
clinical utility. We therefore determined the haemolytic effect
of pillar[5]arenes 1 and 2 on rat red blood cells (RBCs). Up to a
concentration of 85 mM, none of the phosphonium-decorated
pillar[5]arenes caused measurable haemolysis of RBCs. In addi-
tion, compounds 1–3 were found to have no effect on mammalian
À1
cell viability up to a concentration of 128 mg mL as shown in
Fig. S13 (see ESI†). These findings are in accordance to our
1
3
previous results.
To conclude, we synthesized four water-soluble cationic
pillar[5]arenes capable of inhibiting biofilm formation at sub
mM concentrations without affecting the tested bacterial cell
and mammalian cell viability or causing measurable damage to the
membranes of mammalian RBCs. The phosphonium-decorated
pillar[5]arenes showed similar potencies as inhibitors of biofilm
formation as their corresponding ammonium analogues, demon-
strating that the number of positively charged groups and not
their chemical identity are key to their antibiofilm activity. The
pillararene platform appears to be important and positive charges
operating cooperatively are needed for effective antibiofilm activity
as shown by our finding that the respective cationic monomers
were completely inactive. We also demonstrated that the reported
cationic pillar[5]arene derivatives retained their antibiofilm
capability even after 4 hours of exposure to acidic or alkaline
pH. More studies are undergoing in our laboratory to under-
stand the mechanistic aspects and the role of host–guest
properties of pillar[n]arene derivatives in the inhibition of
biofilm formation.
Fig. 1 (a and b) Biofilm formation by (a) S. aureus ATCC 33592 (MRSA) and
We gratefully acknowledge The Israel Science Foundation
(
b) E. faecalis ATCC 29212 evaluated using the double-dilution method
(ISF, Grant 804/13 to YC). R. J. Acknowledges funding by the
with starter inoculum of 1 : 100 (OD600 = 0.01) in the presence of
Post-Doctoral Program for outstanding Post-Doctoral Researchers
compounds 1–4. (c) Biofilm formation in the presence of compounds
5
and 6. Concentration ranges of the tested compounds were: (1) from China and India (PBC program).
0
.19–12.40, (2) 0.17–10.66, (3) 0.21–13.27, (4) 0.18–11.30, (5) 4.95–317
and (6) 5.3—340 mM.
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