Anti-biofilm and anti-adherence properties of novel cyclic dipeptides against oral pathogens

Article abstract of DOI:10.1016/j.bmc.2018.11.042

Microorganisms embedded in a biofilm are significantly more resistant to antimicrobial agents and the defences of the human immune system, than their planktonic counterpart. Consequently, compounds that can inhibit biofilm formation are of great interest for novel therapeutics. In this study, a screening approach was used to identify novel cyclic dipeptides that have anti-biofilm activity against oral pathogens. Five new active compounds were identified that prevent biofilm formation by the cariogenic bacterium Streptococcus mutans and the pathogenic fungus Candida albicans. These compounds also inhibit the adherence of microorganisms to a hydroxylapatite surface. Further investigations were conducted on these compounds to establish the structure–activity relationship, and it was deduced that the common cleft pattern is required for these molecules to act effectively against biofilms.

Full text of DOI:10.1016/j.bmc.2018.11.042

Accepted Manuscript
Anti-biofilm and anti-adherence properties of novel cyclic dipeptides against
oral pathogens
Gaëlle Simon, Christopher Bérubé, Normand Voyer, Daniel Grenier
PII:
S0968-0896(18)31654-7
https://doi.org/10.1016/j.bmc.2018.11.042
BMC 14646
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 October 2018
14 November 2018
28 November 2018
Please cite this article as: Simon, G., Bérubé, C., Voyer, N., Grenier, D., Anti-biofilm and anti-adherence properties
of novel cyclic dipeptides against oral pathogens, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/
10.1016/j.bmc.2018.11.042
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Anti-biofilm and anti-adherence properties of novel cyclic dipeptides against oral pathogens
Gaëlle Simon1,2, Christopher Bérubé2, Normand Voyer2* and Daniel Grenier1*
1 Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval,
2420, rue de la Terrasse, Québec, QC, G1V 0A6, Canada
2Département de Chimie and PROTEO, Université Laval, Québec, QC, G1V 0A6, Canada
Corresponding author: Daniel.grenier@greb.ulaval.ca and normand.voyer.@chm.ulaval.ca
*Corresponding authors contribute equally to manuscript
O
R2
O
O
H
R1
N
N
O
NH
R2
H2N
O
NH
HN
O
R1
HN
R = H or Bn
R
O
O
SPPS and peptide cyclization-
cleavage from oxime resin
Cyclic dipeptides (CDPs)
Library screening
75 cyclic dipeptides (CDPs) prepared
Identification of 5 benzylated CDPs
Biofilm formation and adherence inhibitors
Abstract:
Microorganisms embedded in a biofilm are significantly more resistant to antimicrobial agents and
the defences of the human immune system, than their planktonic counterpart. Consequently,
compounds that can inhibit biofilm formation are of great interest for novel therapeutics. In this study,
a screening approach was used to identify novel cyclic dipeptides that have anti-biofilm activity
against oral pathogens. Five new active compounds were identified that prevent biofilm formation by
the cariogenic bacterium Streptococcus mutans and the pathogenic fungus Candida albicans. These
compounds also inhibit the adherence of microorganisms to a hydroxylapatite surface. Further
investigations were conducted on these compounds to establish the structure-activity relationship,
and it was deduced that the common cleft pattern is required for these molecules to act effectively
against biofilms. To our knowledge, this is the first report of the anti-biofilm and anti-adherence
properties of synthetic cyclic dipeptides.
Keywords : cyclic dipeptides, biofilm, S. mutans, C. albicans, anti-biofilm, adherence, candidiasis,
dental caries, quorum sensing
1

1. Introduction
Numerous human infections are caused by biofilms.1 The ability of microorganisms to form biofilms
propels the prevalence, cost, and complication of healthcare-associated infections.2 Biofilms are
complex microbial communities organized in an extracellular polymeric matrix made of proteins,
polysaccharides and DNA. Their formation is a major concern in medical and oral health care, as they
allow microorganisms to become significantly more resistant to antimicrobial agents3 and the host’s
immune system.4
The oral cavity offers multiple biotic and abiotic surfaces to microorganisms to form biofilms, which
provide a challenge for therapeutic interventions as the structure of the matrix prevents efficient drug
delivery at optimal therapeutic concentrations.5 The multifactored environment provided to
polymicrobial oral biofilms promotes interspecies interactions (nutritional, adherence), and therefore
constitutes an ideal niche for harboring pathogens and favoring resistance acquisition.6 These
biofilms have been associated with the major oral infections, including dental caries7, candidiasis8,
and periodontal diseases9.
deauomnicbpsnthognvpaeifrcstaorvlhendcapuebmiltrltniasevncrieltmrcaiountrsahecirxdsleucsafibnctsiruatwo,einralytseiuhngltoainreonaletfumaoitdaveinfrodlg.ipnrtdno.bpeld!hrfadtIrteimfsnBcromhlbintpahnoeivneietuetaliydeoahvioetgastfcmroatclunitdeqfhoatilnhoesc,nhouemtiafctioanreslo-tetf,gihnvienes
Targeting virulence mechanisms to disarm pathogens in the host is considered as an alternative or
complementary approach to the use of antibiotics.10 The overall strategy is to interrupt the pathogenic
process by inhibiting specific infectious mechanisms such as colonization, invasion, and subversion
of host immune defences.11 Recent trends suggest that the development of low molecular weight
molecules that can inhibit or disperse microbial biofilms through non-microbicidal mechanisms could
work as a strategy to overcome the appearance of resistance12 and reduce side effects by limiting
damage to the beneficial microbiota. This offers an innovative strategy to fight infectious diseases.13
Cyclic dipeptides (CDPs) are common metabolites widely biosynthesized by cyclodipeptide
synthases14 or non-ribosomal peptide synthetases15 by both prokaryotic and eukaryotic cells.
Examples of CDPs that inhibit biofilm formation by Streptococcus epidermidis include cyclo-(L-
Leucyl-L-Tyrosyl) isolated from sponge-associated fungi Penicillium sp.16 and cyclo-(L-Leucyl-L-
Prolyl) isolated from Bacillus amyoliquefaciens.17 Often, CDPs exhibit other biological properties18
in addition to inhibiting biofilm formation.19 Proline residue occurs widely in antimicrobial CDPs,
and proline-containing CDPs are known to be potent chitinase inhibitors.20 Gancidin W, also known
2

as cyclo(L-Leu-L-Pro), inhibits the growth of vancomycin-resistant Enterococcus faecalis with a
minimal inhibitory concentration (MIC) of 12.5 µg/mL.21 Gancidin W is produced by the food
pathogens Cronobacter sakazakii and Bacillus cereus, and was found to regulate interspecies cross-
talking signal system between them.22 The staphylococcal quorum sensing (QS) system is affected
by Lactobacillus-derived cyclo(L-Phe-L-Pro) and cyclo(L-Tyr-L-Pro), which repress the expression
of toxic shock syndrome toxin-1 by Staphylococcus aureus23. Aromatic residues such as
phenylalanine and tyrosine are also common in biosynthesized CDPs24. Their bioactivity includes
antimicrobial and anticancer properties, as well as binding many bioreceptors.18,25 Bioactive CDPs
featuring D-amino acids were also identified26; interestingly, biofilm inhibition by D-amino acids
alone has been previously reported.27
The biological activity of CDPs can be influenced by their relative and absolute configuration; this
effect is part of what has been studied in structure-activity investigations towards designing new drugs
with the CDP scaffold.28 Numerous studies have attempted to establish guidelines or correlations
between absolute configuration and bioactivity22b,26b,29, and some have proposed that the D
configuration may improve the biological activity of CDPs26a. Nevertheless, there remains no clear
correlation between stereochemistry and biological activity, enlightening the pertinence of a
systematic structure-activity investigation when studying CDPs.
The rigid and conformationally restricted CDP heterocycle is stable to proteolysis and presents two
chiral centers whose configuration is controlled by those of the amino acids used during synthesis.
These structural characteristics have made CDPs well-recognized models for conformational
studies.30 Along with their stability and limited conformational freedom, the relative simplicity of
their synthesis has made CDPs a pharmacophore of choice for drug development.19,31
In this study, the anti-biofilm activity of 75 synthetic CDPs was assessed against oral pathogens,
allowing the identification of five novel CDPs that inhibit biofilm formation and adherence properties
by the cariogenic bacterium S. mutans and the pathogenic fungus C. albicans.
3

2. Results and discussion
2.1 Synthesis of cyclic dipeptide library
We first synthesized a library of CDPs with a variety of substituents and stereochemistries, by taking
advantage of the availability of natural and synthetic amino acids as chiral synthons. This library
facilitates the study of the structural features required for anti-biofilm activity by offering a rich
diversity of side-chain substituents and side-chain orientations. Our synthetic procedure involves the
condensation of two N-Boc amino acids with oxime resin32 and standard peptide solid-phase synthetic
strategies (Figure 1).33
R1
O
R2
O
DIC, 6-Cl-HOBt
1. 50% TFA
CH2Cl2, 30 min
H
Boc-HN
HO
COOH
Boc-HN
N
N
N
O
DMAP, DIEA
CH2Cl2/DMF, 3h, r.t.
Boc-HN
O
+
2. Boc-AA2-COOH
HCTU, 6-Cl-HOBt
R1
O
R1
N
DIEA, CH2Cl2/DMF, 3h, r.t.
1. 50% TFA
CH2Cl2, 30 min
Oxime resin
2. DIEA, AcOH
CH2Cl2, 24h, r.t.
O
O
O
O
O
O
R1
R1
R1
R1
NH
NH
R2
NH
R2
NH
HN
HN
R2
HN
HN
R2
O
O
cis-(L,L)
cis-(D,D)
trans-(L,D)
Cyclic dipeptides
Figure 1: Strategy used for synthesis of CDPs
Using this synthetic procedure, we successfully synthesized CDPs with a variety of chiral centers and
side chains: cis-(L,L) (Figure 2), cis-(D,D) (Figure 3), and trans (L,D) (Figure 4). Analogs with
methylene, methyl, isopropyl, benzyl, dibenzyl, benzyloxybenzyl, and phenol groups, as well as
tricyclic derivatives, were prepared with high yields (>90%), especially when the two constituent
amino acids had the same configuration. Yields decreased when two amino acids of different
configurations were used. Notably, in all cases, high purities (>95%) of CDPs were obtained with the
acid-catalyzed concomitant cyclization/cleavage process on an oxime resin (last step in Figure 1). A
total of 75 CDPs (1-46) were prepared and characterized by 1H and 13C NMR spectroscopy and mass
4

spectrometry analyses. The procedure is robust and efficient, allowing the quick preparation of CDPs
with high purities that could be used in the anti-biofilm bioassay.
O
O
O
O
O
O
O
O
R1
HN
NH
R2
NH
NH
NH
HN
HN
HN
R
R
R
16a : R1 = R2 = CH(CH3)2
17a : R1 = R2 = CH(CH3)CH2CH3
18a : R1 = R2 = CH2OBn
9a : R = CH(CH3)2
10a : R = CH2CH(CH3)2
11a : R = CH2Ph
1a : R = CH2CH(CH3)2
2a :R = CH2Ph
3a :R = CH2S(p-OMe)
4a :R = (CH2)2COOBn
5a : R = CH3
6a : R = CH(CH3)2
7a : R = CH2CH(CH3)2
8a : R = (CH2)2CONH2
19a : R1 = CH(CH3)2, R2 = CH2OBn
20a : R1 = CH(CH3)CH2CH3, R2 = CH2OBn
21a : R1 = R2 = CH(CH3)OBn
22a : R1 = R2 = (CH2)4NH(Cbz)
23a : R1 = R2 CH2(2-indoyl(CHO))
24a : R1 = (CH2)3(guanidyl(NO2)), R2 = (CH2)4NH(Cbz)
O
12a : R = CH2OBn
13a : R = CH2(p-NO2Ph)
14a : R = CH2(2-indoyl(CHO))
15a : R = (CH2)3(guanidyl(NO2))
O
O
R1
NH
HN
NH
NH
HN
HN
R2
R
R1
R1O
O
O
O
25a : R = CH3
35a : R1 = Bn, R2 = CH3
33a : R1 = R2 = H
26a : R = CH(CH3)2
27a : R = CH(CH3)CH2CH3
28a : R = CH2OBn
36a : R1 = Bn, R2 = CH(CH3)2
37a : R1 = Bn, R2 = CH2CH(CH3)2
38a : R1 = Bn, R2 = CH2Ph
34a : R1 = R2 = NO2
29a : R = (CH2)4NH(Cbz)
30a : R = (CH2)4NH2
31a : R = CH2Ph
39a : R1 = H, R2 = CH2Ph
40a : R1 = H, R2 = (CH2)3(guanidyl)
32a : R = CH2(2-indoyl(CHO))
O
O
O
O
OR2
NH
NH
R1
N
N
S
S
HN
N
N
N
R1O
O
41a : R1 = R2 = Bn
42a : R1 = R2 = H
O
O
O
43a : R1 = CH2CH(CH3)2
44a : R1 = CH2(p-OBn)Ph
45a
46a
Figure 2 : cis-(L,L) CDPs synthesized
5

O
O
O
O
O
O
O
O
R1
HN
R2
NH
NH
R2
NH
R2 R1
NH
HN
HN
HN
R
R1
5b : R1 = R2 = CH3
16b : R1 = R2 = CH(CH3)2
18b : R1 = R2 = CH2OBn
25b : R1 = H, R2 = CH3
28b : R1 = H, R2 = CH2OBn
9b : R = CH(CH3)2
10b : R = CH2CH(CH3)2
11b : R = Bn
33b : R1 = R2 = H
O
O
O
O
OR2
NH
NH
HN
N
HN
N
R1O
R2
R1O
O
O
35b : R1 = Bn, R2 = CH3
45b
41b : R1 = R2 = OBn
37b : R1 = Bn, R2 = CH2CH(CH3)2
38b : R1 = Bn, R2 = CH2Ph
39b : R1 = H, R2 = CH2Ph
Figure 3 : cis-(D,D) CDPs synthesized
O
O
O
O
O
R2
R1
NH
NH
NH
R1
NH
R2
HN
HN
HN
HN
R1
R2
R1
O
O
O
5c : R1 = R2 = CH3
9d : R= CH(CH3)2
11c : R = Bn
28c : R1 = H, R2 = CH2OBn
33c : R1 = R2 = H
9c : R1 = CH(CH3)2, R2= CH2CH(CH3)2
10c : R1 = R2 = CH2CH(CH3)2
18c : R1 = R2 = CH2OBn
O
O
O
O
OR2
NH
HN
NH
R2
NH
N
HN
HN
N
R1O
R2
R1O
R1O
O
O
O
O
38d : R1 = Bn, R2 = CH2Ph
36c : R1 = Bn, R2 = R1= CH(CH3)2
37c : R1 = Bn, R2 = CH2CH(CH3)2
38c : R1 = Bn, R2 = CH2Ph
41c : R1 = R2 = OBn
45c
Figure 4 : trans-(L,D) CDPs synthesized
2.2 Determination of anti-biofilm activity
The inhibition of biofilm formation was evaluated by allowing biofilm microorganism growth in flat-
bottomed 96-well plates in the presence of CDPs at concentrations up to 96 µg/mL, using optimal
culture conditions for each microorganism. After incubation, biofilm formation was quantified by
crystal violet staining.34 The initial screening for anti-biofilm activity was assessed for all molecules
using oral pathogens, including Gram-negative and Gram-positive bacteria and one fungus.
6

2.2.1 Bacterial biofilms
None of the CDPs tested up to 96 µg/mL showed inhibitory activity on biofilm formation for the
Gram-negative periodontopathogenic bacteria Fusobacterium nucleatum ATCC 25586 and
Porphyromonas gingivalis ATCC 33277 (data not shown). However, five CDPs containing aromatic
aminoacids (Figure 5) inhibited biofilm formation by the cariogenic Gram-positive bacterium S.
mutans ATCC 25175.
Figure 5. CDPs inhibiting S. mutans biofilm formation
As shown in Figure 6, compounds 38 (a,b,c) and 41 (a,b) significantly (p < .01) reduced biofilm
formation by S. mutans compared to control wells at concentrations as low as 6 to 12 µg/mL. Among
the four stereoisomers of compound 38, the cis isomers (38a, 38b) and trans cyclo(D-Phe-L-Tyr(Bn))
(38c) gave similar inhibition profiles, while no significant inhibitory activity was reported with the
trans isomer containing D-tyrosine (38d). It was observed that trans CDPs containing benzylated D-
tyrosine were less soluble in water than the other isomers, which may explain, at least in part, the fact
that 38d did not have an effect. Also, both cis isomers of benzylated tyrosine dimers (41a, 41b) led
to comparable inhibitions whereas the trans isomer (41c) was inactive. In this case, it is not possible
to attribute the inactivity of the trans isomer 41c to its reduced solubility. Other factors, such as shape
and steric hindrance, must be considered to explain the difference in activity.
7

Figure 6. Biofilm formation by S. mutans in presence of CDPs 38a, 38b, 38c (left graph) and 41a, 41b (right).
* p < 0.01
No other CDP tested inhibited biofilm formation by S. mutans, including proline containing CDPs
(43-45). Structural and activity similarities between the five polyaromatic active compounds suggest
a related mechanism of action. All CDPs inhibiting S. mutans biofilm formation (38a, 38b, 38c and
41a, 41b) contained at least one protected tyrosine residue; the other residue was either a
phenylalanine (38) or a second benzylated tyrosine (41). No activity was found in the case of a CDP
made from a tyrosine and a non-aromatic residue (35, 36, 37, 40, 44). At least one tyrosine residue
seems to be required for activity as the bis-phenylalanine CDPs (33, 34) were also found inactive.
Deprotection of the tyrosine hydroxyl groups resulted in a loss of the anti-biofilm activity against S.
mutans (39, 42), although the free bis-tyrosine CDP cyclo(L-Tyr-L-Tyr) (42a) inhibited bacterial
growth at high concentrations (MIC = 96 µg/mL, MBC > 192 µg/mL) under the biofilm assay
conditions (data not shown). A planktonic growth reduction of about 20% after 24 h of incubation
was also observed at 96 µg/mL for cis cyclo(Phe-Tyr) 39a and 39b (MIC > 192 µg/mL) without
affecting the biofilm biomass, suggesting enhanced antibacterial activity due to the free hydroxyl.
Except for compounds 39 and 42, none of the CDPs affected microbial growth. This was monitored
by measuring optical density (660 nm) prior to crystal violet staining.
Tooth decay is initiated when planktonic bacterial cells or aggregates adhere to the salivary pellicle
coating the tooth surface, leading to the formation of dental plaque or dental biofilm. Although S.
8

mutans is the primary pathogen associated with caries, initial colonizers implied in the condition also
include Candida sp, Lactobacillus sp., Actinomyces sp. and non-mutans streptococci.35 To further
investigate the effects of the bioactive CDPs (compounds 33, 38, 39, 41 and 42) against cariogenic
pathogens, anti-biofilm assays were performed against the anaerobic Gram-positive Lactobacillus
fermentum LacB1and Actinomyces naeslundii 49340.
While no inhibition of biofilm formation was observed for L. fermentum, a slight reduction of biofilm
formation by A. naeslundii was observed with compounds 38 (b,c) and 41(a,b) at concentrations of
48 µg/mL. Although the crystal violet staining method showed only a weak decrease in absorbance
(data not shown), visual inspection revealed clear evidence of biofilm reduction. The presence of
CDPs did not impede the strong adhesion of A. naeslundii colonies on the bottom of the wells but
appears to prevent further development of an homogenous biofilm (Figure 7). No effect was observed
this time with compound cyclo(L-Phe-L-Tyr-Bn) (38a) at 96 µg/mL. This result illustrates the
importance of systematic structure-activity investigation when studying CDPs or any other class of
compounds. As was the case with the other Gram-positive bacterium S. mutans, the analogs with free
hydroxyls (39a, 39b, 42) did not show anti-biofilm activity against A. naeslundii.
Figure 7. Effect of CDPs 38 (b,c) and 41 (a,b) on A. naeslundii biofilm.
2.2.2 C. albicans biofilm
Screening of the complete library for the ability to inhibit biofilm formation by the fungus C. albicans
ATCC 28366 led to the identification of the same five active compounds. Inhibition of biofilm
9

formation by 38(a,b,c) and 41(a,b) was observed at comparable concentrations (24 to 48 µg/mL) for
all active CDPs (Figure 8). No significant reduction of fungal growth was observed.
Figure 8. Biofilm formation by C. albicans in presence of CDPs 38a, 38b, 38c (left) and 41a, 41b (right). * p
< 0.01
Comparable patterns of inhibitory activity were observed with the compounds that share structural
similarities. Biofilm formation was reduced in a dose-dependent manner by the five CDPs at
concentrations slightly higher than those required to inhibit the formation of biofilms by S. mutans.
Interestingly, different results were obtained with the analogs that contained deprotected tyrosine
residues. The analogs cyclo(L-Phe-L-Tyr) (39a), cyclo(D-Phe-D-Tyr) (39b), and cyclo(L-Tyr-L-Tyr)
(42a) (Figure 9) exhibited an anti-biofilm activity comparable to the benzylated CDPs, without
affecting fungal growth (Figure 10).
Figure 9. Naturally occurring CDPs inhibiting C. albicans biofilm formation.
10

Figure 10. Biofilm formation by C. albicans in presence of CDPs 39a, 39b, 42.
Isolation and evaluation of biological activities of these three CDPs have previously been reported in
the literature. The (L,L)-CDPs 39a and 42 are naturally produced by cyclodipeptide synthases24a
found in different families of actinobacteria.36 Both compounds (39a, 42) were suggested as potent
therapeutic agents for the treatment for congestive heart failure, as they respectively increase or
decrease coronary blood flow and cardiac output in rats via modulation of calcium channels.37 In an
independent study, 39a and 42 were also found to bind µ-opioid receptors, and cyclo(L-Phe-L-Tyr)
(39a) showed further anticancer activity, inhibiting the growth of tumor cell lines at 2.5mM.38 More
recently, cyclo(D-Phe-D-Tyr) (39b) was isolated from Bacillus sp. and reported with anticancer,
antioxidant and antimicrobial activities.26b Assays were performed for 39a and 39b for comparison
purposes. The (L,L)-isomer (39a) exhibited greater reducing potential but weaker inhibition of A549
cancer cells growth (IC50 value: 10ꢀμM) than the (D,D) (39b). No cytotoxicity against normal human
fibroblast was observed in any case. Antibacterial activity assessed by disk diffusion assay resulted
in lower MICs for 39b compared to its enantiomer 39a. To the best of our knowledge, this is the first
study to report an anti-biofilm activity for these compounds (39, 42). There was no significant
difference in anti-biofilm activity between the (L,L) and (D,D) isomers.
The screening of a library of CDPs as potential inhibitors of biofilm formation by oral pathogens
allowed the identification of five novel active molecules, 38(a,b,c) and 41(a,b). These CDPs contain
11

benzyl substituents as lateral protecting groups of the tyrosine residues and can be prepared with a
simple two-step synthesis. Thus, benzylation of tyrosine from natural CDPs led to an increase of anti-
biofilm activity.
2.2.3 Specific inhibition of cariogenic biofilms by CDPs
Affecting approximately 2.4 billion people worldwide39, tooth decay is the most prevalent disease in
41
humans globally40 and still the most common chronic disease in children in the USA. Its prevention
is a major public health issue and its treatment remains as an important challenge, as therapeutic
agents are quickly cleared from the mouth and many drugs that proved efficient in vitro performed
poorly against oral biofilms.42 Inhibition of biofilm formation by Candida albicans and Streptococcus
mutans is of particular interest in the context of dental caries, where both microorganisms play a
causal role and benefit of a symbiotic enhancement of virulence. Although the fungal participation in
decay development via symbiotic interactions with cariogenic bacteria has been extensively
demonstrated43, further studies suggest that acids production by C. albicans alone may be sufficient
to cause advanced occlusal caries.44 Plaque and salivary carriage of C. albicans and S. mutans
together were linked to the prevalence and severity of early-childhood caries (ECC).45 The
comorbidities that accompany ECC are a burden to communities, health care systems and children
development that can lead in severe cases to serious disabilities or even death46. C. albicans is also
an opportunistic oral pathogen causing denture stomatitis, a common form of candidiasis affecting
denture wearers and associated with inflammation of the oral mucosa. Initial adhesion and biofilm
formation are key steps in denture stomatitis.47
The reported inhibition of biofilm formation of these two species by the five CDPs identified in this
study is a significant finding that will help to shed light on the cross-kingdom interactions and
mechanisms implied in oral biofilms and further treatment of caries. Altogether, the absence of
antibacterial activity and the specific inhibition of biofilm of cariogenic pathogens suggest a quorum
sensing mediated mode of action. It is likely that pathogens that share the same environment as S.
mutans and A. naeslundii will develop common communication pathways and can therefore be
affected by the same QS perturbators. This also applies in the context of the symbiotic relationship
between C. albicans and oral streptococci, which is also known for its cross-kingdom interactions.48
12

2.3 Biofilm disruption
The above results were obtained when growing microorganisms in the presence of CDPs. Therefore,
the active CDPs identified inhibit the formation of biofilm. We then tested the ability of the active
CDPs to disrupt a pre-formed biofilm (24 h), as determined by crystal violet staining. No significant
reduction in biofilm biomass was observed following a 12 h exposure of the biofilm with 96 µg/mL
of CDPs in PBS (data not shown).
2.4 Antimicrobial susceptibility assay
Antimicrobial susceptibility was assessed by a microdilution method according to the
recommendations of the Clinical and Laboratory Standards Institute (Mueller Hinton Broth, 5x105
CFU/mL) for compounds 33, 38, 39, 41 and 42 against non-cariogenic pathogens. None of the CDPs
inhibited the growth of Escherichia coli ATCC 15939, Pseudomonas aeruginosa ATCC 10145,
Staphylococcus aureus ATCC 6538 or Listeria monocytogenes WSLC 1001 within the range of
antibiofilm concentrations (MIC > 192 µg/mL). These data disagree with those of a previous study
that reported an antibacterial activity of cyclo(D-Phe-D-Tyr) (39b) against S. aureus, E. coli and P.
aeruginosa with MICs of 16, 64 and 32 µg/mL, respectively 26b. This may be explained by the use of
different strains and antibacterial methods.
2.5 Effects of CDPs on adherence of S. mutans and C. albicans to an hydroxylapatite surface
Biofilm formation initially involves adherence of microorganisms to a surface. The initial adherence
is modulated by bacterial adhesins but also by other non-specific, physicochemical interactions. It
was therefore of interest to verify the effects of CDPs on the adherence of S. mutans and C. albicans
to a surface of hydroxylapatite that mimics the tooth enamel.49
13

Figure 11. Effect of CDPs 38 (a,b,c) and 41 (a,b) on the adherence of S. mutans to an hydroxylapatite surface.
* p < 0.01
All five CDPs that possessed anti-biofilm activity 38(a,b,c) and 41(a,b) reduced the adherence of S.
mutans (Figure 11) and C. albicans (Figure 12) to hydroxylapatite-coated wells of a microplate at
comparable concentrations.
Figure 12. Effect of CDPs 38 (a,b,c) and 41 (a,b) on the adherence of C. albicans to an hydroxylapatite surface.
* p < 0.01
Moreover, reduction of adherence was observed for both microorganisms by the previously inactive
trans isomer cyclo(L-Tyr-(Bn)-D-Tyr-(Bn)) (41c) at high concentrations. The adherence of S. mutans
14

and C. albicans to the hydroxylapatite-coated surface was significantly reduced (p < .01) to (82±3)%
and (53±12)% respectively in the presence of 41c at 96 µg/mL (data not shown). No significant effect
was measured at lower concentrations. The tyrosine dimer 42a was also found to reduce the adherence
of C. albicans by 20%, but did not attenuate the adherence of S. mutans. This is consistent with the
specific inhibition of fungal biofilms by 42a. On the other hand, compounds 39a and 39b had greater
inhibitory activity against fungal biofilm than 42a, while it did not prevent C. albicans from adhering
to the hydroxylapatite-coated surface. Again, this supports the idea that inhibition of initial adhesion
to a surface is only one of the underlying mechanisms that may be associated with the antibiofilm
properties of CDPs.
3. Conclusion
Screening of a library of 75 CDPs allowed the identification of five novel CDPs (39a, 39b, 39c, 41a,
41b), which significantly inhibit the formation of biofilms of both S. mutans (6 to 12 µg/mL) and C.
albicans (24 to 48 µg/mL). Only CDPs possessing two aromatic amino acids were active. Further
studies will aim at determining whether the anti-biofilm activity of these new active compounds may
involve QS perturbation. Adherence of both microorganisms to an hydroxylapatite surface was
reduced in the presence of these CDPs at concentrations preventing biofilm formation. Moreover, it
was found that compounds 39(b,c) and 42(a,b) reduce biofilm formation by a second cariogenic
bacterium A. aneslundii. The specific effect on cariogenic pathogens behavior and the absence of
antimicrobial activity of these five new synthetic CDPs makes them promising compounds in the
context of dental caries, a condition promoted by the dense biofilm formed by cariogenic
microorganisms. The ability to affect both streptococcal and fungal biofilm formation highlights the
pertinence of targeting shared signalling pathways, especially when assessing polymicrobial diseases
such as ECC. All the above results reported constitute the first report on these CDPs and their
biological properties.
Although the isomers of the (D,D) configuration showed slightly higher anti-biofilm properties, the
similar activity profiles of these molecules, which share structural features, suggest a common mode
of action. The structure-activity relationship was evaluated and suggests that the common pattern
cyclo(Phe-Tyr-Bn) under particular stereochemical conditions is required for activity and should be
conserved in further analog development or action mechanism investigations. The benzyl substituent
15

improves bioactivity and is essential for inhibiting the biofilm formed by S. mutans and A. naeslundii.
The non-benzylated natural analogs 39(a,b) and 42 only affected fungal biofilm formation and did
not (39) or did only slightly (42) reduce fungal adherence to hydroxylapatite. The inhibition of fungal
biofilm demonstrated in this study constitutes the groundwork of our antibiofilm agent development
in our laboratories.
4. Experimental
4.1 General information
Oxime resin, coupling reagents and N-Boc-protected amino acids were purchased from Matrix
Innovation (Québec City, QC, Canada) and GL Shanghai (Shanghai Shi, Chine). Resin with a
substitution level of 1.12 mmol/g of oxime group was used. All chemicals were purchased from
commercial sources and used directly, unless indicated otherwise. Nuclear magnetic resonance
(NMR) spectra were recorded using Varian Inova 400 MHz and NMR Agilent DD2 500 MHz
spectrometers. The coupling constants are reported in hertz (Hz). Chemical shifts are reported in parts
per million downfield from TMS. Splitting patterns are designated as s (singlet), d (doublet), dd
(doublet of doublets), t (triplet), q (quartet), brs (broad singlet), brt (broad triplet), brq (broad
quadruplet) and m (multiplet). Mass spectra were obtained on an Agilent 6210 LC Time of Flight
Mass Spectrometer in direct injection mode. Optical density and absorbance values for antimicrobial
susceptibility and biofilm formation assays were obtained using a BIO-RAD xMark Microplate
spectrophotometer.
4.2 Preparation of CDPs (1-75)
4.2.1. Coupling of the first N-Boc protected -amino acid on oxime resin
A desired quantity of oxime resin (1.12 mmol/g) was added to a peptide synthesis vessel. The resin
was treated three times with CH2Cl2. Amino acid (3.0 equiv) and HOBt (3.0 equiv) were dissolved
in DMF in a 100 mL flask and the mixture was stirred for few minutes at 0°C. DIC (3.0 equiv), DIEA
(3.0 equiv) and DMAP (0.1 equiv) were added and the mixture was introduced into the peptide
synthesis vessel and stirred mechanically for 3 h. The mixture was filtered under vacuum and the
16

resin was washed [DMF (3 x 100 mL), MeOH (3 x 100 mL), DMF (3 x 100 mL), MeOH (3 x 100
mL)] and dried under reduced pressure.
4.2.2 Acetylation of unreacted sites on oxime resin
The resin was treated three times with DMF (3 x 50 mL). A solution of 50% (v/v) DMF/acetic
anhydride (80 mL) and DIEA (1 mL) were added to the peptide synthesis vessel and shaken for 1
hour. Then, the mixture was filtered under vacuum and the resin was washed [DMF (3 x 100 mL),
MeOH (3 x 100 mL), DMF (3 x 100 mL), MeOH (3 x 100 mL)] and dried under reduced pressure.
4.2.3 Removal of the N-Boc protecting group
The resin was treated three times with CH2Cl2 (100 mL). A 50% v/v solution of trifluoroacetic acid
(TFA) in CH2Cl2 was added to the peptide synthesis vessel and shaken for 30 minutes. Then the
mixture was filtered under vacuum and the resin was washed with DMF (3 x 100 mL), MeOH (3 x
100 mL), DMF (3 x 100 mL), MeOH (3 x 100 mL) and with a solution of 10% v/v DIEA in CH2Cl2
(100 mL).
4.2.4 Coupling of the second N-Boc protected -amino acid
The amino acid (3.0 equiv) was dissolved in DMF. The solution was cooled to 0°C, then HBTU (3.0
equiv) and HOBt (3.0 equiv) were added. The mixture was poured into the peptide synthesis vessel
in which the resin had been previously treated with CH2Cl2. DIEA (6.0 equiv) was also added to the
vessel and the mixture was shaken for 3 h. After filtration under vacuum, the resin was washed [DMF
(3 x 100 mL), MeOH (3 x 100 mL), DMF (3 x 100 mL) and MeOH (3 x 100 mL)] and dried under
reduce pressure. The Kaiser ninhydrin test was performed to monitor the efficiency of the coupling,
and the coupling procedure was repeated as needed.
4.2.5. Cyclization/cleavage from the resin
First, the N-Boc group was removed using the procedure described in (4.2.3), but without the 10%
v/v DIEA/CH2Cl2 washing step. After drying, CH2Cl2 and DIEA (2.5 equiv) were added to the
peptide synthesis vessel and the mixture was shaken for 2 min. Acetic acid (5.0 equiv) was then added
and the content was shaken for 24 h. Then the filtrate was collected and the resin was rinsed several
times with CH2Cl2 and MeOH. All filtrates were combined and evaporated, and the resulting solid
was dissolved in CH2Cl2. Amberlite IR-120 was introduced to the solution to remove the remaining
17

traces of DIEA. The mixture was stirred for a few minutes and filtered. The filtrate was evaporated
to give compounds 1 to 46. Trituration in a minimum of cold ether was performed and led to desired
compounds with satisfying purity.
4.3 Characterization of CDPs 1-46
For characterization of previously reported cyclic dipeptides (1-40, 43-75), see references33b,33c
1
4.3.1. (3R,6R)-3,6-bis(ρ-(benzyloxy)benzyl)piperazine-2,5-dione (41b). White powder; H NMR
(400 MHz, DMSO-d6): δ = 2.13 (dd, J = 13.9, 6.0 Hz, 2H), 2.47 (dd, J = 9.7, 4.7 Hz, 2H), 3.83-3.89
(m, 2H), 4.99 (s, 4H), 6.87 (d, J = 9.3 Hz, 2H), 6.89 (d, J = 9.3 Hz, 2H), 7.21-7.33 (m, 10H), 7.83 (s,
2H); 13C NMR (100 MHz, DMSO-d6): δ = 39.2, 56.2, 67.7, 115.2, 128.1, 128.4, 129.0, 129.3, 131.5,
137.8, 157.8, 167.0, HRMS (ESI-TOF, m/z) calcd for C32H31N2O4 (M+H)+ = 507.2278, found
507.2286.
4.3.2. (3S,6S)-3,6-bis(ρ-(benzyloxy)benzyl)piperazine-2,5-dione (41c). White powder; 1H NMR (400
MHz, DMSO-d6): δ = 2.13 (dd, J = 13.9, 6.0 Hz, 2H), 2.47 (dd, J = 9.7, 4.7 Hz, 2H), 3.83-3.89 (m,
2H), 4.99 (s, 4H), 6.85 (d, J = 9.3 Hz, 2H), 6.89 (d, J = 9.3 Hz, 2H), 7.21-7.33 (m, 10H), 7.83 (s, 2H);
13C NMR (100 MHz, DMSO-d6): δ = 39.2, 56.2, 67.7, 115.2, 128.1, 128.4, 129.0, 129.3, 131.5,
137.8, 157.8, 167.0; HRMS (ESI-TOF, m/z) calcd for C32H31N2O4 (M+H)+ = 507.2278, found
507.2281.
1
4.3.3. (3S,6S)-3,6-bis(4-hydroxybenzyl)piperazine-2,5-dione (42a); White powder; H-NMR (400
MHz, DMSO-d6): δ = 2.55–2.60 (dd, J = 8.7, 2.9 Hz, 2H), 2.86–2.91 (dd, J = 8.5, 2.0 Hz, 2H), 3.31
(br, 2H), 6.58–6.61 (d, J = 5.0 Hz, 4H), 6.90–6.93 (d, J = 5.2 Hz, 4H), 7.90 (br, 2H), 9.18 (br, 2H). 13C
NMR (101 MHz, DMSO-d6): δ = 37.0, 55.0, 114.8, 125.6, 131.0, 156.2, 167.1. HRMS (ESI-TOF,
m/z): calcd for C18H19N2O4 (M+H)+ = 327.1339; found 327.1328.
4.4 Anti-biofilm activity
4.4.1. Inhibition of biofilm formation
Inhibition of biofilm formation was assessed using the crystal violet staining method following
microbial growth (24 h) in tissue culture-treated 96-well flat-bottom microplates as previously
18

described.34 Two-fold serial dilutions of CDP solutions were made in the appropriate culture medium
for each microorganism (Table 1).
Table 1. Microorganisms and growth conditions for anti-biofilm activity testing.
Microorganism
Culture Broth
Atmosphere
Candida albicans
ATCC 28366
Sabouraud, pH 7.0
Aerobic
Streptococcus mutans
ATCC 25175
Castenholz tryptone-yeast extract (TYE) +
glucose (0.5%), pH 7.2
Aerobic
Actinomyces naeslundii
ATCC 49340
Tryptic Soy Broth (TSB)
Anaerobic
Anaerobic
Lactobacillus fermentum
LacB1
Lactobacillus Broth De Man, Rogosa and
Sharpe (MRS)
Fusobacterium nucleatum
ATCC 25586
Porphyromonas gingivalis
Todd Hewitt Broth + 0.001% hemin and
0.0001% vitamin K
Anaerobic
ATCC 33277
19

Fresh cultures of microorganisms were added to obtain a final optical density of 0.1 at 660 nm in each
well. The plates were incubated at 37°C for 24 h to allow biofilm formation. After recording the
optical density at 660 nm (OD660) to monitor microbial growth, the culture medium was removed
from the wells, which were then rinsed with water and the biofilm was stained with 100 µL of an
aqueous solution of crystal violet (0.001%) for 15 minutes. The wells were rinsed again with water
after dye removal and allowed to dry. Ethanol (75%) was added (100 µL) to each well to release the
dye, and the biofilm was quantified by measuring the absorbance at 550 nm. Control wells containing
no microorganisms were included for each concentration of CDP (blank(x)) as well as controls
containing only culture medium (blank(0)). The biofilm formation percentage (%) was calculated for
each concentration (x) by reporting the absorbance A (550 nm) as a percentage of the absorbance of
wells containing microorganisms in culture broth (A100% growth) after subtraction of blanks, as follows:
[
]
[
]
퐴(푥) ‒ 퐴푏푙푎푛푘 (푥)
퐵푖표푓푖푙푚 푓표푟푚푎푡푖표푛 (%) =
∗ 100%
(
[
)
]
][
퐴100% 푔푟표푤푡ℎ 퐴푏푙푎푛푘 (0)
A compound was found to possess inhibitory properties on biofilm formation when a significant
reduction in biofilm formation was observed at concentrations not affecting microbial growth. The
results are a mean of at least two independent experiments performed in triplicate.
4.4.2. Disruption of pre-formed biofilms
Microorganisms were cultivated in a 96-well microplate in the absence of CDPs. The culture medium
and free-floating bacteria were removed, and the biofilm was rinsed once with PBS. Two-fold serial
dilutions of CDPs in PBS were added to the pre-formed mature biofilms. Microplates were further
incubated for 12 h prior to wash wells and to quantify the biofilm by crystal violet staining as above.
Biofilms treated with PBS were used as controls.
4.5 Determination of minimal inhibitory concentration
20

The antimicrobial susceptibility of microorganisms was assessed using the microdilution method as
described by the Clinical and Laboratory Standards Institute (2012).50 Two-fold serial dilutions of
the test compounds in Mueller-Hinton Broth (MHB) were prepared in sterile 96-well plates to obtain
final concentrations of CDPs ranging from 0 to 96 µg/mL. Fresh cultures of microorganisms diluted
in Mueller Hinton Broth were added to the plates to obtain a final concentration of 5 x 105 CFU/mL.
The microbial concentration was adjusted to an OD660 previously determined by plate count.
Experiments included positive control wells containing no CDP (100% growth) and blanks containing
no microorganism for every CDP concentration (0% growth). Ampicillin was used as a reference
antibiotic. Plates were incubated aerobically for 24 h at 37°C prior to recording microbial growth.
The minimal inhibitory concentration (MIC) was defined as the lowest concentration of the test
compound that completely inhibited microbial growth as determined visually and by recording the
OD660.
4.6 Adherence to a hydroxylapatite surface
To determine the effect of CDPs on the adherence properties of the biofilms, microorganisms were
labeled with fluorescein isothiocyanate (FITC). Briefly, overnight cultures (10 mL) of
microorganisms were centrifuged at 7000 x g for 10 min, and the cells were suspended in 12 mL of
0.5 M NaHCO3 (pH 8) containing 0.03 mg/mL of FITC. The microbial suspensions were incubated
in the dark at 37°C for 30 min with constant shaking. Microorganisms were then washed three times
by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS.
Hydroxylapatite-coated wells of 96-well microplates (black wall, black bottom) were prepared
according to the procedure described by Shahzad et al. (2015).49 FITC-labeled cells of S. mutans
ATCC 25175 or C. albicans ATCC 28366 (100 µL; OD660ꢀ=ꢀ0.5) were added to the wells, along with
CDPs at concentrations ranging from 96 to 3 µg/mL. The plate was incubated for a further 1 h at
37°C under low agitation. Unbound microorganisms were removed by aspiration, and the wells were
washed twice with PBS. Relative fluorescence units (RFU; excitation wavelength 495ꢀnm; emission
wavelength 525ꢀnm) corresponding to the level of microbial adherence were determined using a
Synergy 2 microplate reader. Control wells without CDPs were used to determine 100% adherence
values, while wells without microorganisms were used as controls to measure basal autofluorescence.
21

Relative fluorescence corresponds to the level of bacterial or fungal adherence. In a second
hydroxylapatite-coated microplate, the fluorescence values of the wells containing a solution of FITC
(0.5 µg/mL in PBS) in the absence or presence of CDPs were compared as a control experiment to
detect possible fluorescence quenching by the dipeptides. The results are a mean of two separate
experiments performed in triplicate.
Acknowledgments
This work was supported by the NSERC of Canada, the FRQNT of Quebec, PROTEO and Université
Laval. The authors would like to thank Geneviève Lebel and Jabrane Azelmat for their useful advice.
Declaration of interest
The authors declare that there is no conflict of interest regarding the publication of this article.
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