Rhamnolipid inspired lipopeptides effective in preventing adhesion and biofilm formation of Candida albicans

Article abstract of DOI:10.1016/j.bioorg.2019.03.023

Rhamnolipids are biodegradable low toxic biosurfactants which exert antimicrobial and anti-biofilm properties. They have attracted much attention recently due to potential applications in areas of bioremediation, therapeutics, cosmetics and agriculture, however, the full potential of these versatile molecules is yet to be explored. Based on the facts that many naturally occurring lipopeptides are potent antimicrobials, our study aimed to explore the potential of replacing rhamnose in rhamnolipids with amino acids thus creating lipopeptides that would mimic or enhance properties of the parent molecule. This would allow not only for more economical and greener production but also, due to the availability of structurally different amino acids, facile manipulation of physico-chemical and biological properties. Our synthetic efforts produced a library of 43 lipopeptides revealing biologically more potent molecules. The structural changes significantly increased, in particular, anti-biofilm properties against Candida albicans, although surface activity of the parent molecule was almost completely abolished. Our findings show that the most active compounds are leucine derivatives of 3-hydroxy acids containing benzylic ester functionality. The SAR study demonstrated a further increase in activity with aliphatic chain elongation. The most promising lipopeptides 15, 23 and 36 at 12.5 μg/mL concentration allowed only 14.3%, 5.1% and 11.2% of biofilm formation, respectively after 24 h. These compounds inhibit biofilm formation by preventing adhesion of C. albicans to abiotic and biotic surfaces.

Full text of DOI:10.1016/j.bioorg.2019.03.023

Accepted Manuscript
Rhamnolipid inspired lipopeptides effective in preventing adhesion and biofilm
formation of Candida albicans
Milos Jovanovic, Jelena Radivojevic, Kevin O'Connor, Stevan Blagojevic,
Biljana Begovic, Vera Lukic, Jasmina Nikodinovic-Runic, Vladimir Savic
PII:
S0045-2068(18)31574-8
DOI:
https://doi.org/10.1016/j.bioorg.2019.03.023
YBIOO 2849
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 January 2019
22 February 2019
12 March 2019
Please cite this article as: M. Jovanovic, J. Radivojevic, K. O'Connor, S. Blagojevic, B. Begovic, V. Lukic, J.
Nikodinovic-Runic, V. Savic, Rhamnolipid inspired lipopeptides effective in preventing adhesion and biofilm
formation of Candida albicans, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.03.023
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Rhamnolipid inspired lipopeptides effective in preventing adhesion and biofilm formation of
Candida albicans
Milos Jovanovica, Jelena Radivojevicb, Kevin O'Connorc, Stevan Blagojevicd, Biljana Begovicd, Vera Lukice, Jasmina
Nikodinovic-Runicb, Vladimir Savica
a
University of Belgrade, Faculty of Pharmacy, Department of Organic Chemistry, Vojvode Stepe 450, Belgrade,
Serbia
b University of Belgrade, Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444 a, Belgrade,
Serbia
c UCD School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland
d University of Belgrade, Institute of General and Physical Chemistry, Studentski trg 12/V, Belgrade, Serbia
e University of Belgrade, School of Medicine, Institute of Forensic Medicine, Deligradska 31 a, Belgrade, Serbia
e-mail adresses
Milos Jovanovic - mjovanovic@pharmacy.bg.ac.rs
Jelena Radivojevic - jelenaradivojevic@imgge.bg.ac.rs
Kevin O'Connor - Kevin.oconnor@ucd.ie
Stevan Blagojevic - stevan.blagojevic@gmail.com
Biljana Begovic - bbegovic63@gmail.com
Vera Lukic - vera_lukic2006@yahoo.com
Jasmina Nikodinovic-Runic - jasmina.nikodinovic@imgge.bg.ac.rs
Vladimir Savic - vladimir.savic@pharmacy.bg.ac.rs
Corresponding Authors
Jasmina Nikodinovic-Runic - jasmina.nikodinovic@imgge.bg.ac.rs
Vladimir Savic- vladimir.savic@pharmacy.bg.ac.rs
Electronic supplementary information (ESI) available: Experimental procedures, structural data and copy of 1H and
13C NMR spectra of final products.

Graphical abstract
Abstract
Rhamnolipids are biodegradable low toxic biosurfactants which exert antimicrobial and anti-biofilm properties.
They have attracted much attention recently due to potential applications in areas of bioremediation,
therapeutics, cosmetics and agriculture, however, the full potential of these versatile molecules is yet to be
explored. Based on the facts that many naturally occurring lipopeptides are potent antimicrobials, our study aimed
to explore the potential of replacing rhamnose in rhamnolipids with amino acids thus creating lipopeptides that
would mimic or enhance properties of the parent molecule. This would allow not only for more economical and
greener production but also, due to the availability of structurally different amino acids, facile manipulation of
physico-chemical and biological properties.
Our synthetic efforts produced a library of 43 lipopeptides revealing biologically more potent molecules.
The structural changes significantly increased, in particular, anti-biofilm properties against Candida albicans,
although surface activity of the parent molecule was almost completely abolished. Our findings show that the most
active compounds are leucine derivatives of 3-hydroxy acids containing benzylic ester functionality. The SAR study
demonstrated the further increase in activity with aliphatic chain elongation. The most promising lipopeptides 15,
23 and 36 at 12.5 µg/mL concentration allowed only 14.3%, 5.1% and 11.2% of biofilm formation, respectively
after 24 h. These compounds inhibit biofilm formation by preventing adhesion of C. albicans to abiotic and biotic
surfaces.
Keywords: adhesion, biofilm, Candida albicans, lipopeptides, rhamnolipids.

1. Introduction
Rhamnolipids (Figure 1) are environmentally friendly, surface active molecules produced mostly by strains of
Pseudomonas aeruginosa [1]. They are composed of one or two rhamnose molecules and up to three molecules of
3-hydroxy acids. On rare occasions rhamnose moiety is esterified or the terminal hydroxy acid is methylated [2]. In
bacteria rhamnolipids fulfil a number of roles. They assist bacteria in assimilating and metabolising hydrophobic
substrates, regulate biofilm formation and are involved in making channels and pores in the structure of mature
biofilms thus allowing the inter-bacterial communication and the flow of nutrients [2-5]. As the biofilms mature,
rhamnolipids facilitate the dispersion of bacteria from the biofilm [5,6]. Rhamnolipids also have antimicrobial
properties against a large variety of Gram-positive and Gram-negative bacteria, primarily due to their ability to
penetrate biological membranes [7]. Most of the abovementioned features are a direct result of the remarkable
surfactant properties of these molecules. Rhamnolipids can reduce the surface tension of water to 25–30 mN/m,
have relatively low critical micellar concentration (between 20 and 225 mg/L) and are effective over a wide range
of pH and temperatures [8]. It should be also noted that they are biodegradable and hence potentially
environmentally benign natural products considered to be a green alternative to synthetic surfactants [9]. These
versatile molecules have a potential for application in pharmaceutical, food, cosmetic industry, bioremediation and
in protection of animals and plants against microbes [10-13]. The inefficient production of rhamnolipids in pure
form, however, significantly limits wider exploration of the chemical space defined by these molecules and their
commercial application [14]. Although several chemical synthetic pathways have been developed the routes are
not economical and require, due to nature of reacting components, many equivalents of protecting groups and
stoichiometric amount of the glycoside bond forming agent [15-17]. Furthermore, rhamnose is a relatively
expensive compound, almost twenty times more expensive than glucose. Synthesis of the other structural
component of rhamnolipids, 3-hydroxyalkanoic acid fragment with longer chain, particularly in chiral form, has
been reported in the literature but is challenging and suffers from several drawbacks [18]. To resolve these
problems, it would be necessary to replace rhamnose with other carbohydrates or to completely redesign the
rhamnolipid structure and create de novo compounds with preserved overall properties of these molecules. Our
tactic to address this setback is based on the latter approach and the results of our research are the subject of this
paper.

Figure 1. Rhamnolipids and structural variants proposed in this study (Please use colour for this figure)
2. Results and Discussion
Inspired by rhamnolipids, we embarked on the development of synthetically accessible alternatives which would
preserve or improve their biological profile or some of the key features. Having previously briefly explored the
carboxylic end of the natural molecule, which revealed the amide derivatives with improved biological profile
compared to the parent derivatives, we were particularly interested in replacing the sugar component of
rhamnolipids with amino acids and peptides (Figure 1) [19]. This approach finds its support in observation that
numerous peptides and lipopeptides have been shown to possess antimicrobial properties [20,21]. We postulated
that the availability and structural diversity of amino acids could offer an opportunity to rationally design novel
compounds and to enhance the biological activity compared to rhamnolipid derivatives. Our envisaged targets
were planned to retain 3-hydroxy acid moiety present in rhamnolipids, which is also frequent structural motif
found in numerous biologically active lipopeptides. In addition, 3-hydroxy acid building blocks are readily available
from polyhydroxyalkanoate (PHA) biopolymers [22,23].
2.1. Variation of Amino Acids
Synthesis of amino acid derivatives of rhamnolipids is outlined in Scheme 1. Prerequisite C8-β-hydroxy acid ester II,
as
a starting point of the envisaged synthetic route, was prepared by methanolysis of bacterial
polyhydroxylalkanoates (PHA) polymer I [22]. The use of PHA for monomer synthesis offers the opportunity to
incorporate microbial production in the preparation of structural components, and, thus, leads towards greener
production process. Methyl ester II was also used for the synthesis of benzyl ester IV utilising routine synthetic
procedures. The initial set of mono-amino acid derivatives was prepared using C8-β-hydroxyesters II and IV
employing EDCI as coupling reagent followed by TFA promoted deprotection to yield product VII or X. These

compounds were then utilised for the preparation of other di-, tri- and tetrapeptide derivatives. As coupling
reagent for these processes HBTU was used under basic conditions and in the presence of hydroxybenzotriazole.
Scheme 1. Synthesis of amino acid derivatives of rhamnolipids
The initial set of synthesised amino acid derivatives (1 – 23) is outlined in Figure 2. Various amino acids used in the
synthetic process were selected to cover all typical physicochemical features represented by these molecules.

7 was isolated as 9:1 mixture of diastereomers at C-2 of the amino acid (partial isomerisation under reaction conditions)
22 and 23 were isolated as 1:1 mixture of diastereomers at C-3 of the carboxylic ester (starting 3-hydroxy acid was used as racemate)
33 was isolated as 2:1 mixture of diastereomers (partial isomerisation under reaction conditions)
35 and 36 were isolated as 1:1 mixture of diastereomers at C-3 of the carboxylic ester (starting 3-hydroxy acid was used as racemate)
Figure 2. Chemical structures of the synthesised and tested compounds

In order to establish antimicrobial properties of the mono-amino acid derivatives the minimum inhibitory
concentration (MIC) against planktonic P. aeruginosa and C. albicans was determined (Table 1). In general, weak
activities were observed, without detectable effect on planktonic growth on both pathogens at concentrations as
high as 500 μg/mL, with compound 15 (MIC of 50 μg/mL, Table 1) showing the highest anti-Candida potential.
Table 1. Minimal Inhibitory concentration of tested compounds against C. albicans and P. aeruginosa
C. albicans
MIC (μg/mL)
P. aeruginosa
MIC (μg/mL)
Derivatives
amino acid
150
150
50
200
200
200
100
>500
>500
>500
>500
>500
>500
>500
5
14
15
16
17
22
23
dipeptide
60
>500
>500
100
500
500
27
28
29
31
150
100
200
50
32
200
100
>500
>500
35
36
tripeptide
80
50
100
100
41
tetrapeptide
43
We further explored the potential of synthesised mono-amino acid derivatives to inhibit biofilm formation and to
disrupt already formed biofilms. Biofilm, both bacterial and fungal, is known as one of the most common causes of
chronic infectious diseases [24]. It represents a surface-associated community of microorganisms embedded in a
polymeric matrix [25]. These communities are often comprised of various bacterial or fungal species and
sometimes even a synergistic combination of both [26]. Amongst fungal species C. albicans is the most commonly
associated with the biofilm formation. The essential step in the pathogenesis of C. albicans is its adhesion to the
host cell surface, mediated by cell walls adhesins [27]. Within biofilm organisms grow resistant to antimicrobial
therapy rendering most antifungal drugs ineffective. The toxicity of many currently used antifungal drugs further
aggravates the issue and emphasizes the need for a new class of molecules that could prevent biofilm formation
[28]. Amongst the newly discovered potential antifungal molecules, antimicrobial peptides (AMPs), and their
analogues displayed promising results [29-31]. Furthermore, many secondary metabolites including rhamnolipids
and sophorolipids, as well as some lipopeptides are known to possess antifungal properties [32-35].
Tests to explore the inhibition of biofilm formation were performed under standard conditions at four different
concentrations for both C. albicans and P. aeruginosa. Tested compounds showed little to no activity against P.
aeruginosa biofilms and were significantly more potent against C. albicans biofilm formation ability. Noteworthy is
the fact that these compounds had no ability to disrupt either Pseudomonas or Candida pre-formed biofilms
(results not shown). The results are presented as a percentage of C. albicans biofilm formed after 24h of growth in
the presence of the tested compound. For the comparison we also performed the same experiments with mono-
and di-rhamnolipids (Figure 3A). Amongst the compounds possessing C-8 carboxylic side chain the most promising
candidate was 15. While at low concentration (6.25 μg/mL; 91%) compound 15 performed comparable to mono-
and di-rhamnolipids (93%, for both), at slightly higher concentration (12.5 μg/mL) it demonstrated significantly
better properties allowing the formation of only 14% of biofilm, in comparison to untreated control. Further
increase in concentration (50 μg/mL) reduced biofilm formation to 3.5%, which was expected, as that was also the
MIC concentration against C. albicans planktonic cells for this compound (Table 1). Very noticeable was the

detrimental effect of the ester group of this compound on its properties. Replacing the benzyl ester in 15 with
methyl ester moiety (compound 2), resulted in very weak activity. One of the reasons for this striking effect might
be the outcome of changes in physicochemical properties in particular logP (calculated values: 2 logP 2.95, 15 logP
4.68)[36]. Given that cell surface hydrophobicity and hydrophobic interactions of mannoproteins play a pivotal role
in C. albicans adhesion on epithelia and plastic surfaces it is likely that more lipophilic compound would interact
more competitively with C. albicans cell wall proteins thus inhibiting their interaction with the host or plastic
surface [37,38]. The other reason for reduction in the activity of methyl ester could be its faster degradation by the
fungal esterases, as it is more likely that the sterically less demanding ester functionality is more prone to
hydrolysis by these enzymes [39].
Figure 3. Inhibition of Biofilm Formation in Candida albicans in the presence of amino acid (A) and peptide
derivatives (B). (Please use colour for this figure)
Based on the observed results, we explored the potential to influence the properties of these compounds further
by introducing additional amino acids. Therefore, a small set of dipeptide derivatives was synthesised as outlined
in Figure 2. To establish antifungal potential MICs were determined but as in case of mono amino acid derivatives
only moderate activity was observed against C. albicans and almost no or very weak activity against P. aeruginosa
(Table 1). Anti-biofilm studies of peptide derivatives produced some interesting results (Figure 3B). Amongst the
most active C-8 carboxylic derivatives were compounds 27 and 28, both possessing non-polar amino acids as
structural components. They showed better activity than mono- and di-rhamnolipids, in particular at 25 μg/mL (27
- 7% biofilm formation, 28 - 18% biofilm formation) and at 50 μg/mL concentrations (27 - 5% biofilm formation, 28
- 7% biofilm formation) while 27 was significantly more active even at 12.5 μg/mL (27 - 18% biofilm formation).
Finally, we made additional tri- and tetrapeptide derivatives as outlined in Figure 2. While some improvements
were observed MIC values confirmed a general trend of weak activity against planktonic C. albicans and P.
aeruginosa (Table 1). Interestingly, contrary to the previously discussed derivatives, compounds possessing non-
polar internal and polar terminal amino acids (41, 43) showed better potency against planktonic organisms than
compounds comprised only of nonpolar amino acids. Increasing the peptide length in general had a negative effect
on the anti-biofilm potency of the compounds. Only on rare occasions and at the highest concentration did the

synthesised tri- and tetrapeptide derivatives perform better than mono- and di-rhamnolipids (i.e leucine derivative
40).
Overall the majority of the most active compounds were benzyl esters with a noticeable predominance of non-
polar amino acid residues such as leucine, phenylalanine and methionine. In all the active compounds except 12,
the presence of leucine residue and phenyl group whether in the form of benzyl ester or as a phenyl group in
phenylalanine was conspicuous. On the other hand, only a slight change in the structure of 15 (leucine derivative)
resulted in almost complete loss of activity (16 isoleucine derivative). Some of the abovementioned structural
features are also reported in the recent work of Pierce et al. [40]. Screening the library of 20 000 small molecules
the group suggested that the most potent molecules for inhibition of biofilm formation were derivatives of
diazaspiro-decane which contained para-isopropyl benzyl side chain on the piperidine nitrogen. These results
imply that the overall physicochemical properties are not the only factor influencing the activity of these
compounds. Specific structural motifs may play an important role as well.
2.2. Variations of Chain Length of Carboxylic Acids
A further extension of the initial structure activity relationship (SAR) of the rhamnolipid inspired amino acid
derivatives focused on the carboxylic moiety. As discussed above, the initial set of compounds was based on C-8
carboxylic derivative II prepared from the depolymerisation of PHA polymer to produce a single enantiomer. To
explore the influence of this structural fragment on the biological profile of the peptide derivatives a small series of
C-10 and C-12 congeners was synthesised and explored. The C-10 and C-12 carboxylic derivatives were prepared as
previously described and were used as racemic mixtures [41]. Replacing the C-8 fragment of the most active
compounds 15 and 27 with C-10 or C-12 units afforded derivatives 22/23 and 35/36 respectively. Extending the
carboxylic chain of both mono- amino acid and dipeptide derivatives did not have a beneficial effect on the activity
against planktonic C. albicans or P. aeruginosa (Table 2). On the other hand, anti-biofilm properties improved
further for mono-amino acid derivatives 22 and 23 compared to the parent C-8 analogue 15 at all studied
concentrations (Table 2). The same trend was observed for dipeptide derivatives 35 and 36 (Table 2). It is also
worth noting that all four compounds, 22/23 and 35/36, showed better anti-biofilm properties than both mono-
and di-rhamnolipids at all studied concentrations. All C-10 and C-12 compounds, 22 (logP 5.57), 23 (logP 6.46), 35
(logP 6.3) and 36 (logP 7.2), have higher calculated logP than mono-rhamnolipid (logP 3.8) and di-rhamnolipid (logP
3.9) used for comparison. This supported our previous observation suggesting that anti-biofilm potential is likely to
be influenced, at least in part, by the lipophilicity.
Table 2. Effects of the selected amino acid derivatives on biofilm formation of C. albicans, their cytotoxic effect
given as IC50 value on healthy human fibroblasts (MRC5 cell line) and physicochemical parameters.
Biofilm formation
Concentration (μg/mL)
b
c
MICa
(μg/mL)
BIC50
(μg/mL)
IC50
Compound
C-length
LogP
(μg/mL)
6.25
91.1
56.2
40.3
98.2
63.2
42.7
12.5
14.3
10.8
5.1
38.4
39.8
11.2
25
50
C8
7.5
4.7
4.2
7.1
6.1
8.8
3.4
3.1
2.1
4.9
5.6
7.2
50
200
100
60
200
100
8
>100
20
15
>100
12
4.68
5.57
6.46
5.40
6.29
7.18
15
22
23
27
35
36
C10
C12
C8
C10
C12
6
4.5
10
6.5
4.5
8
aMinimum inhibitory concentration
bConcentration causing 50% biofilm inhibition. Results represent mean of two independant experiments done in hexaplicate, with standard
deviation between 1-5%.
cConcentration causing 50% cell killing under treatment of 48 h. Results represent mean of two independant experiments done in quadriplicate,
with standard deviation between 1-5%
Selected derivatives were found to be effective against C. albicans biofilm formation at concentrations much lower
than the ones needed to affect fungal growth (Table 2). This emphasises the importance of the anti-biofilm
potential of these compounds as they do not directly target microbial survival but rather act on properties that

influence microbial population as a whole, and as such are unlikely to cause the development of drug resistance in
C. albicans [42].
2.3. Other biological and physicochemical properties
To further evaluate the potential for their medical development, in-vitro cytotoxicity of the most potent
derivatives against healthy human lung fibroblast cell line (MRC5) has been determined (Table 2). While
compounds 15 and 27 showed no cytotoxic effects at the concentrations tested, their C-10 and C-12 analogues
22/23 and 35/36 killed almost 100% of the cells at concentrations 2-3 fold greater than their biofilm inhibition
BIC50 values (Table 2). This represents a potentially limiting factor in further exploration and development of C-10
and C-12 derivatives for biomedical applications. On the other hand, the low cytotoxicity and potent Candida anti-
biofilm activity of 15 and 27 prompted further exploration. Using GFP and RFP reporter strains of C. albicans
SC5314 we observed that both derivatives successfully prevented biofilm formation (Figure 4A) as well as adhesion
onto A549 cells monolayer (Figure 4B). At these concentrations, biofilms under treatment appeared to contain
predominantly the round yeast form of Candida in comparison to controls where an obvious network of elongated
mycelia was observed (Figure 4A). Surprisingly, 15 and 27 did not prevent hyphal formation nor reduced hyphal
length during the cell adhesion process (Figure 4B), which was also observed in-vitro on Spider medium
(Supporting Figure 1S). Taken together, these results indicate that rhamnolipid derivatives act by effectively
preventing adhesion of C. albicans to abiotic and biotic surfaces.

Figure 4. C. albicans SC5314 (GFP) biofilm formation in the presence of 15 and 27 at 12.5 µg/mL (A). Adhesion of C.
albicans SC5314 (RFP) on A549 cell monolayer (stained with DAPI) in the presence of 15 and 27 at 25 µg/mL (B).
Control represents 0.1% DMSO as vehicle solvent control and scale bars represent 10 µm. (Please use colour for
this figure)
Apart from antimicrobial properties, main features of rhamnolipids also include, surface-active properties and
biodegradability. We selected several representative molecules (amino acid derivative 11, dipeptide derivative 26,
tripeptide derivative 40, and tetrapeptide derivative 42) in order to explore the influence of replacement of the
sugar component with amino acids on the abovementioned features. Results presented in table 3 and supporting
table 1S suggest that regardless of polarity or number of amino acids in peptide chain the compounds behave in a
simillar manner. Overall the replacement of rhamnose almost completely negated the surface active properties as
the critical micellar concentrations (CMC) were significantly greater than those of rhamnolipids (Table 3). Surface

tension is existent but at this concentration (0.1%) it is rather low. On the other hand the biodegradable properties
of the parent molecule were retained as the set of studied compounds was almost completely biodegraded over
the course of 25 days.
Table 3. Critical micelle concentration, surface tension at 23.0 °C, 36.5°C and foam index of selected compounds in
comparison with commercially available rhamnolipid mixture Rha-90.
CMC (mg/L)
Foam index
0/3 min
 (mN/m)23˚C
 (mN/m) 36.5˚C
Compound
23˚C
0.1%
0.1%
Rha-90
11
175
390
420
400
315
45.5
67.6
68.5
68.0
68.2
43.2
67.5
68.0
68.0
68.0
3/3
1/0
0/0
1/0
2/0
26
40
42
These results imply that contrary to rhamnolipids and other known anti-biofilm biosurfactants such as
pseudofactin II, which act by reducing the hydrophobicity of cell surface, the molecules hereby presented may act
by a different mechanism [43-45]. The molecules likely do not target nonspecific adhesion factors such as
hydrophobicity, as they displayed anti-biofilm properties at concentrations much lower than their CMC. This is also
illustrated by the strong dependency of anti-biofilm properties on structural characteristics of the molecules (e.g.
15 and 16), thus suggesting that the more subtle interactions are at work. In this regard the mechanism is most
likely related to the interaction of the molecules with more specific targets such as cell wall proteins, namely
adhesins or mannoproteins [37,38,46].
3. Materials & Methods
3.1 Chemistry
Polyhydroxyalkanoate polymer I was purchased from Bioplastech Ltd. (Dublin, Ireland)and hydrolysed as described
by de Roo et al. [22], rhamnolipid mixture R90 was purchased from AGAE Technologies and purified as described
by Lotfabad et al. [47]. All amino acids and reagents were purchased from Acros Organics (Morris Plains, NJ, United
States), unless otherwise stated. The NMR spectra were recorded on a Bruker Ascend 400 (400 MHz) or on a
Bruker Avance III (500 MHz) spectrometer. Chemical shifts are given in parts per million (δ) downfield from
tetramethylsilane as the internal standard. Deuterochloroform was used as a solvent, unless otherwise stated.
Mass spectral data were recorded using an Agilent Technologies 6520 Q-TOF spectrometer coupled with an Agilent
1200 HPLC system or an Agilent Technologies 5975C MS system coupled with an Agilent Technologies 6890 N GC
system. Optical rotations were measured on an AUTOPOL IV Automatic Polarimeter Rudolph Research Analytical.
[α]D values are given in deg mL g−1 dm−1. IR spectra were recorded on an IR Thermo Scientific NICOLET iS10 (4950)
spectrometer. Flash chromatography employed silica gel 60 (230–400 mesh) while thin layer chromatography was
carried out using alumina plates with a 0.25 mm silica layer (Kieselgel 60 F254, Merck). Compounds were visualized
by staining with potassium permanganate solution or Dragendorff’s reagent. The solvents were purified by
distillation before use. Procedures for the preparation of lipopeptides and the corresponding structural data for
the synthesized compounds can be found in the Supporting information.
3.2. Microbial Strains and Growth Conditions.

Pseudomonas aeruginosa PAO1 NCTC 10332, Candida albicans ATCC 1023 and C. albicans SC 5314 (ATCC MYA-
2876; green fluorescence protein (GFP) and red fluorescence protein (RFP) reporter strains obtained from Prof
Bernhard Hube, Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product
Research and Infection Biology Hans Knoell Institute, Jena, Germany) were used in this study. P. aeruginosa was
grown in Luria Bertani (LB; 10 g/L peptone, 5 g/L yeast extract, 5 g/L NaCl, pH 7.2) while C. albicans strains were
grown in Sabouraud broth (SAB; 10 g/L peptone, 40 g/L dextrose, pH 5.6) on a rotary shaker at 180 rpm at 37°C.
3.3. Antimicrobial Susceptibility Tests for Planktonic Cells
The minimum inhibitory concentration (MIC) of test compounds were determined according to standard broth
microdilution assays recommended by the National Committee for Clinical Laboratory Standards (M07-A8 and
M27-A3, for bacteria and yeast respectively) [48,49]. Stock solutions of compounds were prepared in DMSO (50
mg/mL). The highest concentration used was 500 µg/mL. The inocula were 108 and 105 colony forming units
(cfu)/mL for bacteria and fungi, respectively. MIC values were read after 24 h incubation at 37°C as the lowest
concentration to exhibit an absence of growth.
3.4. Anti-Biofilm Assays
Biofilm quantification assays were performed in 96-well microtiter plate format using polystyrene flat bottom and
round bottom plates for P. aeruginosa and C. albicans, respectively and a crystal violet (CV) staining of adherent
cells. Biofilms were formed in the absence or presence (concentration range 100-1.5 µg/mL) of compounds for 24
h at 37°C for biofilm formation assay. The biofilm disruption ability was tested on developed biofilms in 96-well flat
bottom and round bottom plates for P. aeruginosa and C. albicans, respectively as described previously [50,51].
Briefly, harvested from the overnight grown P. aeruginosa and C. albicans cells were washed twice in sterile PBS
and collected by centrifugation, resuspended in RPMI-1640 with 2% glucose and adjusted to cell density of 1 × 106
cells/mL using Neubauer haemocytometer. Biofilms were formed using 100 μL of the prepared cell suspensions
into wells of a microtiter plate and incubated for 24 h at 37°C. The established biofilms were rinsed with PBS and
treated with two-fold serial dilutions of test compounds (concentration range 200-3.1 µg/mL) for 24 h at 37°C.
After incubation, biofilms were washed twice with sterile PBS and adherent cells were stained with 0.1% (v/v) CV,
the absorbance at 590 nm was read on Tecan Infinite 200 Pro multiplate reader (Tecan Group Ltd., Männedorf,
Switzerland). Biofilm formation and disruption assays were performed in six replicates and repeated three times.
Biofilm inhibition values were plotted against the log of concentration and a sigmoidal dose response curve was
calculated by non-linear regression analysis using Graphpad Prism software version 5.0 for Windows (Graphpad
Software, CA, USA). Biofilm inhibition concentrations for selected compounds are expressed as the concentration
of the compound inhibiting biofilm by 50% (BIC50). Biofilms of C. albicans SC5314 (GFP reporter strain) formed in
the presence of selected compounds were also examined using fluorescence microscope (Olympus BX51, Applied
Imaging Corp., San Jose, USA) at 20 × magnification.
3.5. C. albicans Yeast to Hyphe Transition Assay
Morphological changes of C. albicans in the presence and absence of selected compounds in subinhibitory
concentrations (MIC80; 80% of MIC value determined for the planktonic growth) was observed upon C. albicans
growth on Spider medium as previously described [52].
3.6. In-vitro cytotoxicity on Human Fibroblast Cell Line
Cytotoxicity (anti-proliferative activity) of rhamnolipids and synthetized amino- and peptide- analogues was
determined by standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay [53].
MRC5 cells (human lung fibroblast, obtained from the American Type Culture Collection) were plated in a 96-well
flat-bottom plate at a concentration of 1×104 cells per well, grown in humidified atmosphere of 95% air and 5%

CO2 at 37 °C, and maintained as monolayer cultures in RPMI-1640 medium supplemented with 100 µg/mL
streptomycin, 100 U mL-1 penicillin, and 10% (v/v) fetal bovine serum (FBS) (all from Sigma, Munich, Germany).
Assay was carried out after 48 h of cell incubation in the media containing test compounds at concentrations
ranging from 0.1 to 250 µg/mL, MTT reduction assay was carried out and cell proliferation was determined from
the absorbance at 540 nm on Tecan Infinite 200 Pro multiplate reader (Tecan Group, Männedorf, Switzerland). The
percentage viability values were plotted against the log of concentration and a sigmoidal dose response curve was
calculated by non-linear regression analysis using Graphpad Prism software version 5.0 for Windows (Graphpad
Software, CA, USA). Cytotoxicity is expressed as the concentration of the compound inhibiting growth by 50%
(IC50).
3.7. Cell Adhesion Assay
The ability of C. albicans SC5314 (RFP reporter strain) to adhere to A549 cells (human epithelial adenocarcinoma,
obtained from the American Type Culture Collection) were tested in the presence of selected compounds as
previously described by Wachtler B, et al. [54]. C. albicans cells were added to monolayer of A549 cells and co-
incubated for 1 h in serum-free RPMI-1640 medium (Gibco) in the presence and absence of selected test
compounds (at 25 µg/mL) 37°C and 5% CO2. Adherent C. albicans cells were visualized with a fluorescence
microscope (Olympus BX51, Applied Imaging Corp., San Jose, USA) at 20 × magnification.
3.8. Statistical Analysis
Results are expressed as means ± standard deviation (SD). All results were analysed by ANOVA. A P value < 0.05
was considered significant.
3.9. Conductivity and Surface Tension Measurements
Conductivity measurements were applied to determine the CMC in various combinations of surfactants. Those
measurements were carried out at 23.0 C with digital conductivity meter SensION 5 (Hach, USA) with the accuracy
 0.5 %, as well as with the 51975 conductivity probe that uses the 4ring method. The CMC values at each
surfactant formulation composition were determined by using the conventional method (Williams' method) as
well as the method proposed by Carpena at al. [55]. Surface tensions of surfactant mixtures were measured by a
stalagmometer (Traube stalagmometer Neubert Glass BN033010208), while surface tension was determined
by drop counting method. Surface tension measurements were performed at 23.0 C.
3.10. Testing of Biodegradability (Closed Bottle Test)
The Closed Bottle-Test for ultimate biodegradability determination of dishwashing liquid surfactant mixtures in
an aqueous medium primarily was established according to the standard method [SRPS] [56]. The preadapted
microorganism from the river Sava, Belgrade, Serbia was used. The aqueous medium for preparing the cultures
had a neutral pH (7.2) and contained minerals and micronutrients to support bacterial activity (2.75 g L1 NH4Cl, 1.0
1
1
1
g L K2HPO4, 0.252 g L MgSO4, 0.32 g L KCl and 0.0018 g L1 FeSO4). Tested formulations (1 5) were added to
1
the microorganism media to give a concentration of about 100 mg L . Each sample was inoculated with 1 mL of 15
1
g L suspension of aerobic microorganism. The samples in dark bottles were placed in a temperature controlled
incubator (Velp Scientifica FOC 120E, Italy), and the incubator temperature was maintained at 25 °C. BOD was
measured every five days during the analyzed time of 28 days (BOD5), by using Sensor system 6 (Velp Scientifica,
Italy).
3.11. Foam Volume Test

SRPS ISO 696: 2000 Surface active agents - Measurement of foaming power - Modified Ross-Miles method,
Institute for Standardization of Serbia. Evaluation of foam ability of surfactant mixtures was done by foam volume
measuring after free flows of 0.4 % solution. The reservoir was placed in such a way that the outlet tube was
positioned in the centre of the basin and the distance between its lower edge and bottom was 1 m.
4. Conclusion
Our synthetic efforts created a library of lipopeptides inspired by structures of naturally occurring rhamnolipids
and antimicrobial peptides. Biological and physicochemical assessment of these compounds established their
potent antibiofilm properties, in particular against C. albicans, as their most prominent feature. Overall,
lipopeptides containing benzylic esters and lipophilic amino acid residues (leucine, phenylalanine and methionine)
displayed the highest activity on the inhibition of biofilm formation of C. albicans. The length of aliphatic chain
improves the potency of compounds but also increases their in vitro cytotoxicity. The optimal number of amino
acids in the side chain was found to be one or two, as the further elongation of the peptide chain decreased their
effect on biofilm formation. Taken together these observations suggest that for anti-biofilm potential of these
compounds, the combination of specific structural motifs and lipophilicity may play more important role than their
surface activity. Evidence of successful inhibition of C. albicans biofilm formation by prevention of the adhesion to
abiotic and biotic surfaces has been provided for synthesized compounds, however, the exact mode of action still
remains to be elucidated. The compounds may have specific targets such as adhesins or mannoproteins on
Candida cell surface. The most potent amongst the synthesized compounds were able to significantly inhibit
biofilm formation whilst not affecting the fungal growth thus minimizing the chances of resistance development.
The strategy of targeting biofilm formation as a specific phenomenon may offer alternative therapeutic
opportunities. Our lipopeptide library, in that regard, represents a synthetically accessible starting point for the
development of new strategies against C. albicans infections and could represent the basis for further mechanistic
and compatibility studies that may yield compounds which completely prevent biofilm formation of C. albicans.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
Financial support from the Serbian Ministry of Education, Science and Technological Development Serbia (grants
no. 172009 and 173048) is greatly appreciated.
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Rhamnolipid inspired lipopeptides effective in preventing adhesion and biofilm formation of
Candida albicans
Milos Jovanovica, Jelena Radivojevicb, Kevin O'Connorc, Stevan Blagojevicd, Biljana Begovicd, Vera Lukice, Jasmina
Nikodinovic-Runicb, Vladimir Savica
Highlights
Substituting rhamnose in rhamnolipids with amino acids yielded 43 lipopeptides.
Their most prominent feature was anti-biofilm activity against C. albicans.
Lipopeptides inhibit biofilm formation of C. albicans by preventing its adhesion.
Compounds containing leucine residue and benzyl ester proved to be the most effective.
Compounds effectively prevent adhesion of C. albicans at sub MIC levels.