Polyhydroxyalkanoate-based 3-hydroxyoctanoic acid and its derivatives as a platform of bioactive compounds

Article abstract of DOI:10.1007/s00253-015-6984-4

A library of 18 different compounds was synthesized starting from (R)-3-hydroxyoctanoic acid which is derived from the bacterial polymer polyhydroxyalkanoate (PHA). Ten derivatives, including halo and unsaturated methyl and benzyl esters, were synthesized and characterized for the first time. Given that (R)-3-hydroxyalkanoic acids are known to have biological activity, the new compounds were evaluated for antimicrobial activity and in vitro antiproliferative effect with mammalian cell lines. The presence of the carboxylic group was essential for the antimicrobial activity, with minimal inhibitory concentrations against a panel of bacteria (Gram-positive and Gram-negative) and fungi (Candida albicans and Microsporum gypseum) in the range 2.87.0 mM and 0.16.3 mM, respectively. 3-Halogenated octanoic acids exhibited the ability to inhibit C. albicans hyphae formation. In addition, (R)-3-hydroxyoctanoic and (E)-oct-2-enoic acids inhibited quorum sensing-regulated pyocyanin production in the opportunistic pathogen Pseudomonas aeruginosa PAO1. Generally, derivatives did not inhibit mammalian cell proliferation even at 3-mM concentrations, while only (E)-oct-2-enoic and 3-oxooctanoic acid had IC₅₀ values of 1.7 and 1.6 mM with the human lung fibroblast cell line.

Full text of DOI:10.1007/s00253-015-6984-4

Appl Microbiol Biotechnol
DOI 10.1007/s00253-015-6984-4
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Polyhydroxyalkanoate-based 3-hydroxyoctanoic acid
and its derivatives as a platform of bioactive compounds
1
,2
2
1
1
Jelena Radivojevic & Sanja Skaro & Lidija Senerovic & Branka Vasiljevic
&
3
,5
3
2
2
Maciej Guzik & Shane T. Kenny & Veselin Maslak & Jasmina Nikodinovic-Runic
Kevin E. O’Connor
&
3
,4
Received: 7 July 2015 /Revised: 24 August 2015 /Accepted: 2 September 2015
Springer-Verlag Berlin Heidelberg 2015
#
Abstract A library of 18 different compounds was synthe-
sized starting from (R)-3-hydroxyoctanoic acid which is de-
rived from the bacterial polymer polyhydroxyalkanoate
exhibited the ability to inhibit C. albicans hyphae formation.
In addition, (R)-3-hydroxyoctanoic and (E)-oct-2-enoic acids
inhibited quorum sensing-regulated pyocyanin production in
the opportunistic pathogen Pseudomonas aeruginosa PAO1.
Generally, derivatives did not inhibit mammalian cell prolif-
eration even at 3-mM concentrations, while only (E)-oct-2-
enoic and 3-oxooctanoic acid had IC50 values of 1.7 and
1.6 mM with the human lung fibroblast cell line.
(
PHA). Ten derivatives, including halo and unsaturated meth-
yl and benzyl esters, were synthesized and characterized for
the first time. Given that (R)-3-hydroxyalkanoic acids are
known to have biological activity, the new compounds were
evaluated for antimicrobial activity and in vitro antiprolifera-
tive effect with mammalian cell lines. The presence of the
carboxylic group was essential for the antimicrobial activity,
with minimal inhibitory concentrations against a panel of bac-
teria (Gram-positive and Gram-negative) and fungi (Candida
albicans and Microsporum gypseum) in the range 2.8–7.0 mM
and 0.1–6.3 mM, respectively. 3-Halogenated octanoic acids
.
Keywords Polyhydroxyalkanoate (R)-3-Hydroxyalkanoic
.
.
acids Antimicrobial activity Chemical derivatization
Introduction
Bio-based production of chemical building blocks from re-
newable resources is an attractive alternative to using finite
petroleum-based resources. Polyhydroxyalkanoates (PHAs)
are a family of natural polyesters, usually produced by pro-
karyotes as cytoplasmatic water-insoluble storage compounds
of carbon and energy (Fig. 1a). They are biodegradable poly-
mers consisting of usually chiral hydroxyalkanoic acids
(Fig. 1b) (Chen 2009; Steinbüchel and Valentin 1995).
PHAs can be made from renewable biological resources such
as glucose, glycerol, and fatty acids (Follonier et al. 2014;
Nikodinovic-Runic et al. 2013). PHAs have a number of uses
ranging from compostable packaging materials or as materials
for medical applications, such as drug delivery (Chen 2011;
Chen and Wu 2005).
PHAs are classified into two major subdivisions by the
carbon chain length of their monomeric units (Fig. 1b), i.e.,
short-chain-length-3-hydroxyalkanoates (scl-3HA) have 3–5
c a r b o n a t o m s a n d m e d i u m - c h a i n - l e n g t h - 3 -
hydroxyalkanoates (mcl-3HA) with 6–16 carbon atoms
(Chen 2009). PHAs are optically active polymers containing
Electronic supplementary material The online version of this article
(doi:10.1007/s00253-015-6984-4) contains supplementary material,
which is available to authorized users.
*
*
Jasmina Nikodinovic-Runic
jasmina.nikodinovic@imgge.bg.ac.rs
Kevin E. O’Connor
Kevin.oconnor@ucd.ie
1
Institute of Molecular Genetics and Genetic Engineering, University
of Belgrade, Vojvode Stepe 444a, P.O. Box 23, Belgrade 11010,
Serbia
2
3
Faculty of Chemistry, University of Belgrade, Studentski trg 12-16,
Belgrade, Serbia
Bioplastech, NovaUCD, Belfield Innovation Park, Belfield, Dublin
4
, Ireland
4
5
University College Dublin, Belfield, Dublin 4, Ireland
Present address: Jerzy Haber Institute of Catalysis and Surface
Chemistry, Polish Academy of Sciences, Niezapominajek 8,
3
0-239 Cracow, Poland

Appl Microbiol Biotechnol
Sharpless’ asymmetric epoxidation and hydroxylation or
through Brown’s asymmetric allylboration (Brown and
Ramachandran 1991). Products often have lower enantiomer-
ic excesses than those obtained by biosynthetic processes (Lee
et al. 1999).
It has been anticipated that chiral R-HAs are attractive com-
pounds with high application potential (antibacterial, antiviral,
synthons for organic synthesis, biofuels) (Ren et al. 2010). R-
HAs have a similar structure to fatty acids which have been
known as antimicrobial agents for more than 80 years
(Desbois and Smith 2010). However, only few of these com-
pounds are commercially available; thus, they have not yet
been assessed for synthetic chemical modification and biolog-
ical activity. Biotechnologically produced PHA can be tai-
lored for their monomer composition by controlling the sub-
strate feed which makes them an excellent target for further
derivatizations. Therefore, we have used biotechnologically
produced mcl-PHA consisting of predominantly (R)-3-
hydroxyoctanoic acid monomers (Elbahloul and Steinbuchel
Fig. 1 Polyhydroxyalkanoates (PHA). a As intracellular bacterial carbon
and energy storage material of Pseudomonas putida. b General structural
formula
2009) and utilized it as a starting material for synthetic routes
to generate a library of derivatives for biological activity
evaluation.
mostly the (R)-enantiomer of 3-hydroxyalkanoates. Various
(R)-3-hydroxyalkanoic acids have been reported to constitute
Materials and methods
PHA polymers, with diverse functional side groups such as
unsaturated bonds, ester, cyano, amino, halogen, and phenyl,
which greatly expand their potential applications (Steinbüchel
and Valentin 1995, Grage et al. 2009). Enantiomerically pure
Materials, procedures for compound synthesis,
and analytical methods
(
R)-3-hydroxyalkanoic acids (3R-HAs) can be conveniently
All chemical reagents were obtained from Sigma–Aldrich
(Germany). Ethyl acetate, dichloromethane, and other sol-
vents were of HPLC reagent grade and purchased from
Fisher Scientific (Hampton, NH, USA).
prepared by depolymerizing the biosynthesized PHA either
enzymatically (Lee et al. 1999, Ren et al. 2005; Ren et al.
2
007) or chemically (de Roo et al. 2002). After chemical deg-
radation by acid methanolysis, the obtained (R)-3-
hydroxyalkanoyl methyl esters are saponified to yield the cor-
responding (R)-3-hydroxyalkanoic acids (Ruth et al. 2007).
Therefore, PHAs can be considered as a source of chiral com-
pounds that have great potential as valuable synthons for the
chemical and pharmaceutical industry (Ren et al. 2010;
Tortajada et al. 2013).
There are several methods of chemical synthesis of hydrox-
ylated alkanoic acids (R-HAs). General drawbacks of these
reactions include limited amounts and scalability; the require-
ment of often expensive, chiral metal-complex catalysts; the
contamination of the end product with catalysts; and the high
price of pure substrates (Ren et al. 2010). They can be chem-
ically synthesized by enantioselective reduction of the corre-
sponding 3-keto acids (Noyori et al. 2004). For certain prod-
ucts, this process requires the synthesis of precursor molecules
at an elevated pressure of 100 atm and in the presence of chiral
catalysts, which makes the synthetic procedure difficult (Case-
Green et al. 2008; Wang et al. 1999). Other synthetic ap-
proaches include stereoselective functionalization through
The enantiomeric excess was determined by HPLC
(Agilent Technologies, HP1100) with a Chiralpak ID column
(Chiral Technologies Europe, Cedex, France) at 210 nm for all
of the samples. NMR spectra were recorded on a Varian
1
13
Gemini 200 ( H NMR at 200 MHz, C NMR at 50 MHz,
for samples in deuterated chloroform) and on Bruker Avance
1
13
III 500 ( H NMR at 500 MHz, C NMR at 125 MHz).
Chemical shifts are expressed in ppm (δ) using
tetramethylsilane as an internal standard, and coupling con-
stants (J) are in hertz. IR spectra were recorded on a Nicolet
6700 FT instrument and are expressed in per centimeter. Mass
spectra were obtained on Agilent Technologies 6210 TOF LC/
MS instrument (LC: series 1200). Optical rotations were de-
termined by a Rudolph Research Analytical AUTOPOL IV
automatic polarimeter (10 cm, 1-ml cell).
Methyl 3-hydroxyoctanoate (1) (Hon et al. 2005) Poly-(R)-
3-hydroxyoctanoic acid (500 mg, 3.12 mmol, 1 eq) was
placed in methanol (20 ml), and sulfuric acid (1 ml,
18.8 mmol, 6 eq) was added. After 3 h of stirring at room

Appl Microbiol Biotechnol
temperature, methanol was removed under reduced pressure
and the residue was dissolved in dichloromethane, washed
Chemical synthesis of methyl 3-hydroxyoctanoate (1r)
(Hon et al. 2005) A suspension of the activated zinc dust
(2.28 g, 35 mmol) in 10 ml of anhydrous benzene was heated
up to reflux for 10 min. To the refluxing suspension, a mixture
of n-hexanal (4 ml, 33 mmol) and methyl bromoacetate
(5.54 ml, 35 mmol) in 60 ml of benzene was slowly added
over a period of 1 h. After 2 h, the reaction mixture was cooled
to 0 °C. 1 M HCl was added to acidify the reaction mixture
which was extracted with ether (40 ml × 3). The combined
with saturated aqueous NaHCO solution and then with brine,
3
and dried over anhydrous MgSO . The solvent was removed
4
in vacuum and the crude product was purified by dry flash
chromatography (chloroform/ethyl acetate = 9/1 to 8/2) to
yield a 450-mg (90 %) pure product, as colorless oil.
1
[
α] = −28 (c 1, CHCl ); ee >99 %; H NMR (200 MHz,
D
3
CDCl ): δ 4.15–3.89 (m, 1 H), 3.72 (s, 3 H), 2.90 (d,
3
J = 4.0 Hz, 1 H), 2.58–2.34 (m, 2 H), 1.71–1.30 (m, 8 H),
organic phase was dried (anhydrous MgSO ) and the solvent
4
1
3
0
6
2
.89 (t, J = 6.3 Hz, 3 H). C NMR (50 MHz, CDCl ): δ 173.5,
7.9, 51.7, 41.1, 36.4, 31.6, 25.1, 22.5, 13.9. IR (ATR): 3457,
958, 2933, 2865, 1741, 1441, 1171.
was removed under reduced pressure. The crude product was
purified on a silica gel column (petrol ether/ethyl acetate = 9/
1) to give the desired β-hydroxy ester 1r (6.4 g) in 80 % yield
as a colorless oil.
3
Benzyl 3-hydroxyoctanoate (2) (Smith et al. 2011) To a
solution of 3-hydroxyoctanoic acid (1.25 mmol, 1 eq) and
Na CO (3.75 mmol, 3 eq) in dimethylformamide (2 ml),
benzyl-bromide (1.4 mmol, 1.1 eq) was added dropwise at
room temperature. The resulting mixture was stirred for
General procedure for 3-chloro derivative preparation
2
3
(
Takashi et al. 2003) To a solution of 3-hydroxyester (1 eq,
.575 mmol) and triethylamine (0.15 eq, 0.086 mmol) in
diisopropyl ether (4 ml), one half of a solution of SOCl2
1.175 eq, 0.675 mmol) in diisopropyl ether (5 ml) was
0
2
4 h, and the reaction was monitored by thin-layer chroma-
(
tography (TLC) (chloroform/ethyl acetate = 9/1) until full
conversion of the acid was achieved. Water was added and
the aqueous phase was extracted three times with diethyl
ether, washed with brine, and dried over anhydrous
added dropwise at −10 °C. The resulting mixture was
then stirred for 10 min at −10 °C and then allowed to
warm to room temperature. The second half of the
SOCl2 solution (5 ml) was then added dropwise to a
reaction mixture and heated to 75 °C until full conver-
sion of the alcohol. After cooling to room temperature,
the reaction mixture was neutralized with saturated
MgSO . After removal of the solvent under reduced
4
pressure, the crude product was purified by dry flash
chromatography on silica gel (chloroform/ethyl ace-
tate = 9/1), and 188 mg (60 %) of product 2 as yellow
aqueous NaHCO solution. The aqueous phase was ex-
3
1
oil was yielded. [α]D = −20 (c 1, CHCl ); ee 75 %; H
3
tracted with CH C1 (3 × 10 ml) and washed with brine
2
2
NMR (200 Hz, CDCl ): δ 7.36 (s, 5 H), 5.15 (s, 2 H),
3
and dried over anhydrous MgSO . After removal of the
4
4
2
3
1
1
1
.12–3.92 (m, 1 H), 2.87 (d, J = 4.1 Hz, 1 H), 2.66–
.34 (m, 2 H), 1.70–1.23 (m, 8 H), 0.88 (t, J = 6.4 Hz,
solvent under reduced pressure, the crude product was
purified by dry flash chromatography on silica gel (pet-
rol ether/ethyl acetate = 95/5).
1
3
H). C NMR (50 Hz, CDCl ): 172.9, 135.6, 128.6,
3
28.4, 128.3, 68.0, 66.4, 41.3, 36.4, 31.6, 25.1, 22.5,
3.9. IR (ATR): 3448, 2957, 2932, 2860, 1737, 1459,
168.
Methyl 3-chlorooctanoate (4) Yield 51 mg (46 %), yellow
1
oil. [α] = −16 (c 1, CHCl ); ee 22 %; H NMR (200 MHz,
D
3
CDCl ): δ 4.3 (quint, J = 7 Hz, 1 H), 3.73 (s, 3 H), 2.75 (d,
3
3
-Hydroxyoctanoic acid (3) (Brandl et al. 1988; de Rijk
J = 6.8 Hz, 2 H), 1.84–1.21 (m, 8 H), 0.90 (t, J = 6.5 Hz, 3 H).
et al. 2001) To a solution of methyl 3-hydroxyoctanoate
1
3
C NMR (50 Hz, CDCl ): δ 171.1, 57.9, 51.9, 43.5, 37.9,
3
(
(
90 mg, 0.516 mmol) in THF (2.7 ml) and methanol
1.8 ml), a water solution of LiOH 2.5 M (0.9 ml) was care-
3
1
1.1, 25.9, 22.4, 13.9. IR (ATR): 2959, 2933, 2865, 1745,
463, 1169. HRMS (ESI): calculated for C H O Cl (M +
9
17 2
fully added. The reaction mixture was stirred for 24 h at room
temperature. After 24 h, the pH was adjusted to 2 with 1 M
HCl, and the resulting solution was washed three times with
ethyl acetate (10 ml × 3) followed by washing with brine and
+
NH ) 210.12553, found 210.12571.
4
Benzyl 3-chlorooctanoate (5) Yield 87 mg (56 %), yellow
1
drying over anhydrous MgSO . After removing the solvent
oil. [α] = −9 (c 1, CHCl ); ee 17 %; H NMR (200 MHz,
4
D
3
under reduced pressure, pure compound 3 was obtained
CDCl ): δ 7.37 (s, 5 H), 5.17 (s, 2 H), 4.32 (quint, J = 7 Hz,
3
1
(
(
(
79 mg, 96 %). [α] = −20 (c 1, CHCl ); ee 99 %; H NMR
1 H), 2.86–2.73 (m, 2 H), 1.85–1.10 (m, 8 H), 0.89 (t,
D
3
1
3
200 MHz, CDCl ): δ 5.57 (bs, 2 H), 4.04 (m, 1 H), 2.73–2.27
J = 6.6 Hz, 3 H). C NMR (50 MHz, CDCl ): δ 170.1,
3
3
1
3
m, 2 H), 1.75–1.11 (m, 8 H), 0.89 (t, J = 6.3 Hz, 3 H).
C
135.8, 128.6, 128.4, 128.3, 66.6, 57.9, 43.6, 37.9, 31.1,
25.9, 22.4, 13.9. IR (ATR): 3066, 3034, 2956, 2931, 2861,
1741, 1458, 1159. HRMS (ESI): calculated for C H O Cl
NMR (200 MHz, CDCl ): δ 177.6, 68.1, 41.0, 36.4, 31.6,
3
2
1
5.1, 22.5, 13.9. IR (ATR): 3396, 2931, 2956, 2862, 1714,
411, 1174.
1
5 21 2
+
(M + NH ) 286.15683, found 286.15665.
4

Appl Microbiol Biotechnol
1
3
General procedure for 3-bromo derivatives (Anessian
et al. 1987; Desmaris et al. 2003) To a solution of 3-
hydroxy ester (1 eq, 0.2 mmol) and carbon tetrabromide
J = 6.5 Hz, 3 H). C NMR (50 MHz, CDCl ): δ 170.7 (d,
3
J = 5.9 Hz), 90.4 (d, J = 158.1 Hz), 51.8 (d, J = 6.2 Hz), 40.2
(d, J = 24.2 Hz), 34.8 (d, J = 20.5 Hz), 31.4, 24.4 (d,
J = 4.0 Hz), 22.4, 13.9. IR (ATR): 2956, 2865, 1744, 1439,
1172.
(
1.6 eq, 0.32 mmol) in dichloromethane (1 ml) at 0 °C,
triphenylphosphine (1.13 eq, 0.225 mmol) was added. The
mixture was stirred for 4 h at room temperature. After removal
of the solvent under reduced pressure, the product was puri-
fied by dry flash chromatography on silica gel in gradient
Benzyl 3-fluorooctanoate (11) Yield 40 mg (60 %), colorless
oil. [α] = −3 (c 1, CHCl ); ee 65 %; H NMR (200 MHz,
CDCl ): δ 7.36 (s, 5 H), 5.16 (s, 2 H), 5.08 (ddd, J = 12.5, 7.8,
1
D
3
(
petrol ether/ethyl acetate = 95/5 to 8/2).
3
4
2
.7 Hz, 1 H), 4.83 (dt, J = 12.4, 4.2 Hz, 2 H), 2.95–2.27 (m,
H), 1.88–1.14 (m, 6 H), 0.89 (t, J = 6.5 Hz, 3 H). C NMR
Methyl 3-bromooctanoate (7) Yield 15 mg (32 %), yellow
13
1
1
oil. [α] = +5 (c 1, CHCl ); ee 16 %; HNMR (200 Hz): H
D
3
(50 MHz, CDCl ): δ 170.2 (d, J = 5.6 Hz), 135.7, 128.6,
3
NMR (200 MHz, CDCl ): δ 4.34 (quint, J = 7 Hz, 1 H), 3.72
3
128.3, 128.2, 90.4 (d, J = 169.7 Hz), 66.6, 40.4 (d,
J = 24.1 Hz), 34.8 (d, J = 20.5 Hz), 31.4, 24.4 (d,
J = 4.3 Hz), 22.4, 13.9. IR (ATR): 3066, 3033, 2955, 2933,
(
(
s, 3 H), 2.90 (d, J = 7 Hz, 2 H), 1.83–1.77 (m, 2 H), 1.63–1.16
m, 6 H), 0.89 (t, J = 6.6 Hz, 3 H). C NMR (50 Hz, CDCl3):
1
3
δ 170.9, 51.9, 50.0, 44.1, 38.6, 30.9, 27.1, 22.4, 13.9. IR
ATR): 2957, 2932, 2860, 1746, 1438, 1157. HRMS (ESI):
2
864, 1741, 1458, 1169. HRMS (ESI): calculated for
(
+
C H O F (M + NH ) 270.18638, found 270.18630.
1
5
21
2
4
+
calculated for C H O Br (M + NH ) 254.07502, found
9
17
2
4
2
54.07547.
Methyl (E)-oct-2-enoate (13) (El-Batta et al. 2007) Olefinic
derivatives were obtained as by-product in 3-fluoro derivative
synthesis. Yield 5 mg (15 %), colorless oil. H NMR
Benzyl 3-bromooctanoate (8) Yield 60 mg (95 %), yellow
1
1
oil. [α] = 0 (c 1, CHCl ); ee 1 %; H NMR (200 MHz,
D
3
(
200 MHz, CDCl ): δ 6.98 (dt, J = 15.6, 7.0 Hz, 1 H), 5.82
3
CDCl ): δ 7.36 (s, 5 H), 5.16 (s, 2 H), 4.36 (quint, 7 Hz,
3
(dt, J = 15.6, 1.5 Hz, 1 H), 3.73 (s, 3 H), 2.20 (dq, J = 7,
1
1
H), 2.95 (d, J = 7 Hz, 2 H), 1.83 (dt, J = 8.7, 7.0 Hz, 2 H),
.72–1.08 (m, 6 H), 0.89 (t, J = 6.6 Hz, 3 H). C NMR
1
.5 Hz, 2 H), 1.46–1.26 (m, 6 H), 0.90 (t, J = 6 Hz, 3 H).
1
3
1
3
C NMR (50 MHz, CDCl ): δ 167.1, 149.8, 120.7, 51.2,
3
(
50 Hz, CDCl ): 170.2, 135.5, 128.6, 128.3, 66.7, 49.9,
3
3
1
2.1, 31.2, 27.6, 22.3, 13.8. IR (ATR): 2930, 2956, 2860,
727, 1437, 1271.
4
3
4.3, 38.6, 30.9, 27.0, 22.4, 13.9. IR (ATR): 3729, 3065,
034, 2956, 2930, 2861, 1740, 1457, 1151. HRMS (ESI):
+
calculated for C H O Br (M + NH ) 330.10632, found
1
5
21
2
4
Benzyl (E)-oct-2-enoate (14) Yield 6 mg (10 %), colorless
3
30.10688.
1
oil. H NMR (200 MHz, CDCl ): δ 7.36 (s, 5 H), 7.02 (dt,
3
J = 15.5, 6.9 Hz, 1 H), 5.86 (d, J = 15.6 Hz, 1 H), 5.14 (d,
J = 11.8 Hz, 9 H), 2.55–2.06 (m, 1 H), 1.86–1.08 (m, 8 H),
General procedure for 3-fluoro derivatives (Delgado et al.
1
991) To a cold solution (−70 °C) of 3-hydroxyester (1 eq,
1
3
0
1
2
1
.88 (t, J = 6.4 Hz, 3 H). C NMR (50 MHz, CDCl ): δ 166.6,
0
.24 mmol) in anhydrous CH Cl (2 ml), diethylaminosulfur
3
2
2
50.2, 136.2, 128.5, 128.2, 128.1, 120.8, 65.9, 32.2, 31.2,
7.6, 22.4, 13.9. IR (ATR): 2929, 2956, 3034, 3064, 2860,
723, 1457, 1167. HRMS (ESI): calculated for C H O
trifluoride (DAST) (1.1 eq, 0.264 mmol) was added under a
stream of argon. After 20 min of stirring at −70 °C, the reac-
tion was allowed to warm to room temperature and quenched
15 20
2
+
(
M + NH ) 250.18016, found 250.18019.
with saturated solution of NaHCO and water. The aqueous
4
3
phase was extracted with dichloromethane (3 × 5 ml), washed
with brine and dried over anhydrous MgSO . The residue was
General procedure for 3-oxo derivatives (Collins et al.
1968) A solution of Collins reagent was prepared from
chromium(VI) oxide (6 eq, 2 mmol) and pyridine (12 eq,
4 mmol) in dichloromethane. The mixture was stirred at room
temperature under argon for 30 min; then, a solution of 3-
hydroxyester compound (1 eq, 0.34 mmol) in dichlorometh-
ane was added and stirring was continued for 12 h at room
temperature. The reaction was monitored by TLC (petrol
ether/ethyl acetate = 9/1). Ether was added and the residue
from the Collins reagent was washed three times with ether.
The combined ether layers were filtered through a 1-cm col-
umn of silica gel, and the solvent was evaporated under re-
duced pressure and the residue purified by dry flash chroma-
tography (petrol ether/ethyl acetate = 9/1).
4
purified by dry flash chromatography (petrol ether/ethyl ace-
tate = 95/5) to yield fluoro ester, containing olefinic by-prod-
ucts. These were efficiently removed by ozonolysis of the
crude material in CH Cl at −15 °C followed by treatment
2
2
with 0.3 M periodic acid in THF for 1 h at room temperature.
The solvent was removed under reduced pressure and the
product was purified again by dry flash chromatography (pet-
rol ether/ethyl acetate = 9/1).
Methyl 3-fluorooctanoate (10) Yield 17 mg, (40 %), color-
1
less oil. [α] = −5 (c 1, CHCl ); ee 19 %; H NMR (200 MHz,
D
3
CDCl ): δ 5.12–4.99 (m, 1 H), 4.88–4.75 (m, 1 H), 3.72 (s,
3
3
H), 2.86–2.32 (m, 2 H), 1.88–1.14 (m, 8 H), 0.90 (t,

Appl Microbiol Biotechnol
Methyl 3-oxooctanoate (16) (Balducci et al. 2011; Mordant
et al. 2004) Yield 20 mg (35 %), colorless oil. H NMR
(E)-Oct-2-enoic acid (15) (SDBS Web 2014) Yield 61 mg
1
1
(100 %), colorless oil. H NMR (200 MHz, CDCl ): δ 11.77–
3
(
200 MHz, CDCl ): δ 3.74 (s, 3 H), 3.45 (s, 2 H), 2.53 (t,
8.85 (bs, 1 H), 7.09 (dt, J = 15.6, 7.0 Hz, 1 H), 5.82 (dt,
J = 15.6, 1.5 Hz, 1 H), 2.23 (dq, J = 7, 1.5 Hz, 2 H), 1.68–
1.15 (m, 6 H), 0.90 (t, J = 6.5 Hz, 3 H). C NMR (50 MHz,
3
J = 7.3 Hz, 2 H), 1.68–0.95 (m, 6 H), 0.89 (t, J = 6.6 Hz, 3 H).
1
3
13
C NMR (50 MHz, CDCl ): δ 202.9, 167.7, 52.3, 48.9, 43.0,
3
1
3
2
1.1, 23.1, 22.3, 13.8. H NMR: δ 12.03 (s, 1 H). IR (ATR):
CDCl ): δ 172.4, 152.6, 120.6, 34.1, 32.2, 31.3, 27.5, 22.4,
3
956, 2932, 2864, 1748, 1717, 1440, 1240, 1320, 1159.
13.9. *IR (ATR): 2931, 2959, 2860, 2674, 1699, 1421, 1285.
*cis isomer in trace.
Benzyl 3-oxooctanoate (17) (Balducci et al. 2011) Yield
1
6
7
2
7.5 mg (80 %), colorless oil. H NMR (200 MHz, CDCl ):
3
3
-Oxooctanoic acid (18) (Giddens et al. 2008) Yield 63 mg
.36 (s, 5 H), 5.18 (s, 2 H), 3.48 (s, 2 H), 2.50 (t, J = 7.3 Hz,
H), 1.58 (dt, J = 14.4, 7.3 Hz, 2 H), 1.43–1.07 (m, 4 H), 0.87
t, J = 6.6 Hz, 3 H). C NMR (50 MHz, CDCl ): δ 202.7,
1
(99 %), white powder. H NMR (200 MHz, CDCl ): δ 6.97 (s,
3
1
0
6
2
H), 4.03 (s, 1 H), 2.72–2.23 (m, 2 H), 1.78–1.11 (m, 8 H),
1
3
(
13
3
.89 (t, J = 6.3 Hz, 3 H). C NMR (50 MHz, CDCl ): δ 177.5,
3
1
2
2
67.1, 135.3, 128.6, 128.4, 128.3, 67.0, 49.2, 42.9, 31.1, 23.0,
2.3, 13.8. enol: H NMR: δ 12.02 (s, 1 H). IR (ATR): 2957,
931, 2866, 1744, 1717, 1646, 1458.
8.2, 41.3, 36.4, 31.6, 25.1, 22.5, 13.9. IR (ATR): 3536, 2955,
926, 2857, 1771, 1681, 1440.
1
In vitro antimicrobial and antiproliferative assays
General procedure for deprotection of benzyl esters The
benzyl ester was dissolved in MeOH, and a catalytic amount
of 10 % Pd/C was added. The apparatus was evacuated and
To test the antimicrobial properties of the synthesized deriva-
tive, a range of bacterial strains from the National Collection
of Type Cultures (NCTC) and the American Type Culture
Collection (ATCC) including Escherichia coli NCTC 9001,
Salmonella typhimurium NCTC 12023, Staphylococcus
aureus NCTC 6571, Listeria monocytogenes NCTC 11994,
Pseudomonas aeruginosa PAO1 NCTC 10332, Candida
albicans ATCC 10231, and Micrococcus gypseum
ATCC24102 in previously described standard broth micro
dilution assays (Casey et al. 2004) defined by the National
Committee for Clinical Laboratory Standards (M07-A8) for
bacteria and standards of the European Committee on
Antimicrobial Susceptibility Testing (EDef7.1.).
flushed with H . The mixture was stirred in an atmosphere of
2
H (50 psi) for 8 h. The reaction mixture was filtered through
2
Celite, and the solvent was removed under reduced pressure to
yield quantitatively carboxylic acid.
3
-Chlorooctanoic acid (6) Yield 66 mg (100 %), yellow oil.
1
[α] = −4.5 (c 1, CHCl ); ee 27 %; H NMR (200 MHz,
D
3
CDCl ): δ 10.53 (bs, 1 H), 4.35–4.08 (m, 1 H), 2.81 (d,
3
J = 6.9 Hz, 2 H), 1.91–1.12 (m, 8 H), 0.90 (t, J = 6.5 Hz,
1
3
3
3
1
H). C NMR (50 MHz, CDCl ): δ 176.5, 57.3, 43.4, 37.9,
3
1.1, 25.9, 22.4, 13.9. IR (ATR): 2932, 2958, 2861, 1716,
286, 1433. HRMS (ESI): calculated for C H O Cl (M +
The antiproliferative activity of the compounds was
assessed against the human lung fibroblast MRC5 cell line
obtained from ATCC and melanoma cell A375 using MTT
8
15 2
+
NH ) 196.10988, found 196.11039.
4
(
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
3
-Bromooctanoic acid (9) Yield 71 mg (100 %), yellow oil.
1
D 3 3
mide) reduction assay (Hansen et al. 1989). The MTT assay
was performed after 48 h of treatment with compounds three
times in three replicates in comparison to control (DMSO-
treated cells) that were arbitrarily set to 100 %.
[α] = 0 (c 1, CHCl ); ee 23 %; H NMR (200 MHz, CDCl ):
δ 10.70 (bs, 1 H), 4.54–4.14 (m, 1 H), 2.96 (d, J = 6.9 Hz,
2
1
3
H), 1.95–1.15 (m, 8 H), 0.90 (t, J = 6.6 Hz, 3 H). C NMR
(
50 MHz, CDCl ): δ 176.6, 49.1, 44.1, 38.6, 30.9, 27.1, 22.4,
3
1
3.9. IR (ATR): 2930, 2958, 2860, 1715, 1430, 1282. HRMS
+
(
ESI): calculated for C H O Br (M + NH ) 240.05937,
8 15 2 4
Effects of PHA monomer derivative alkanoic acids
on Candida yeast–hyphal transition
found 240.05985.
3
-Fluorooctanoic acid (12) Yield 64 mg (100 %), white pow-
Morphological changes of C. albicans in the presence and
absence of the alkanoic acids (3, 6, 9, 12, 15, and 18) were
observed upon C. albicans growth on Spider medium as pre-
viously described (Shafreen et al. 2014). The plates were sup-
plemented with sub-minimum inhibitory concentrations
(MICs) of each derivative, as well as with C8, and incubated
at 37 °C for 36 h. The morphology of C. albicans colony
formed on the Spider medium was visualized using a light
microscope (CKX41; Olympus, Tokyo, Japan).
1
der. [α] = −3 (c 1, CHCl ); ee 66 %; H NMR (500 MHz,
CDCl ): δ 9.05 (bs, 1 H), 5.00–4.85 (m, 1 H), 2.82–2.39 (m,
2
D
3
3
1
3
H), 1.81–1.18 (m, 8 H), 0.90 (t, J = 6.9 Hz, 3 H). C NMR
(
125 MHz, CDCl ): δ 176.3 (d, J = 5.0 Hz), 90.2 (d,
3
J = 169.5 Hz), 40.3 (d, J = 24.1 Hz), 34.8 (d, J = 20.4 Hz),
3
2
1.4, 24.5 (d, J = 4.3 Hz), 22.5, 13.9. IR (ATR): 2932, 2957,
862, 1716, 1433, 1266. HRMS (ESI): calculated for
+
C H O F (M + NH ) 180.13943, found 180.13985.
8
15
2
4

Appl Microbiol Biotechnol
Effects of PHA monomer derivative carboxylic acids
on pyocyanin production by P. aeruginosa PAO1
methyl ester (1) obtained via acid methanolysis of biotechno-
logically produced mcl-PHA containing up to 97 % (R)-3-
hydroxyoctanoic acid monomers (de Roo et al. 2002;
Elbahloul and Steinbuchel 2009) were synthesized due to
the importance of the halogen moiety in the currently used
drugs. Spectral data of all compounds are given as supporting
information (Supporting File S1).
To test the effect of derivatives 3, 6, 9, 12, 15, and 18 on
pyocyanin production in P. aeruginosa PAO1, a slightly mod-
ified protocol of O’Loughlin et al. was used (O’Loughlin et al.
2
013). P. aeruginosa PAO1 was grown in Kings A medium
(
1.5 % (v/v) glycerol, 20 g/l peptone, 1.64 g/l MgCl , 10 g/l
Functionalization of the (R)-3-hydroxyoctanoic acid was
not possible without protection of the free carboxylic group;
therefore, all reactions were performed using methyl and ben-
zyl esters. The methyl ester was hydrolyzed to generate 3-
hydroxyoctanoic acid which was converted to the benzyl ester
form which proved suitable for the preparation of derivatives
as depicted in Fig. 3. In this way, the importance of the free
carboxylic group for biological activity could be determined
by direct comparison with biological activity of the generated
esters. All chemical reactions used during this study were
based on previously reported procedures (Albert et al. 2002;
Anessian et al. 1987; Collins et al. 1968; Delgado et al. 1991;
Takashi et al. 2003) with noted adaptations made to ensure
successful reactions with 3-hydroxyoctanoyl esters derived
from PHA as outlined in Fig. 4.
2
K SO ) at 37 °C for 24 h. An overnight P. aeruginosa PAO1
culture was diluted 1:1000 into 5 ml Kings A medium and,
after addition of derivatives and C8 to a final concentration of
2
4
0
.06 mM or an equivalent amount of DMSO as control, cul-
tured for another 24 h at 37 °C with shaking. A culture aliquot
1 ml) was harvested at 14,000 rpm for 20 min, and the su-
(
pernatant was analyzed for pyocyanin on a UV–vis spectro-
photometer Ultrospec 3300pro (Amersham Biosciences,
Piscataway, NJ, USA) at 695 nm. All experiments were per-
formed in triplicate and repeated at least three times.
Results
In order to compare the results that were obtained by de-
rivatization of biological (R)-3-hydroxyoctanoate, we synthe-
sized racemic methyl 3-hydroxyoctanoate (1r) by the
Reformatsky reaction with a good product yield of 80 % using
a previously described procedure (Hon et al. 2005). Recently,
the use of this reaction for the related purpose of producing
methyl and ethyl esters was published with similar reaction
yields (Sailer et al. 2015). We used 1r to produce all other
racemic derivatives from the library applying the same proce-
dures applied on 1. In some instances such as 2r, 4r, 7r, and
Synthesis and characterization of the library
of compounds based on hydroxyoctanoic acid
A library containing 18 compounds has been generated using
(R)-3-hydroxyoctanoic acid monomers derived from a bacte-
rial polyester PHA, as the starting material (Fig. 2).
Predominantly halogen derivatives of (R)-3-hydroxyoctanoic
11r, product yields were between 5 and 13 % better in com-
parison to yields when 1 was used as the starting material
(Table 1).
Chloro derivatives (4 and 5) were made in a substitution
reaction using thionyl chloride and triethylamine in
diisopropyl ether with heating at 75 °C overnight. A signifi-
cantly reduced ee was observed for all products synthesized
under these conditions (ee from 17 to 27 %). In an attempt to
improve enantioselectivity, we have performed reactions at
lower temperatures (4 and 25 °C); however, reduction of the
reaction temperature resulted in very poor product yields and
no improvement of the enantioselectivity (results not shown).
Bromo derivatives (7 and 8) were made from 1 and 2 in
reactions with carbon tetrabromide and triphenylphospine in
dichloromethane (Fig. 4). Under these reaction conditions, all
obtained bromo derivatives were racemic. According to the
literature, this type of substitution results in an inversion of the
configuration, which is probably the reason for complete ra-
cemization of the optically pure starting (R)-3-
hydroxyoctanoic acid (Anessian et al. 1987; Desmaris et al.
2003).
Fig. 2 Library of compounds (1–18) based on (R)-3-hydroxyoctanoic
acid derived from bacterial polyester polyhydroxyoctanoate

Appl Microbiol Biotechnol
Fig. 3 PHA monomer synthesis and esterification
To produce fluorinated compounds, we used DAST at
70 °C in dichloromethane (Fig. 4). In this conversion, the
Antimicrobial properties All of the synthesized compounds
were evaluated for their activity against five bacterial strains,
including E. coli, S. typhimurium, S. aureus,
L. monocytogenes, and P. aeruginosa PAO1, and two fungal
species, C. albicans and Microsporum gypseum. Their antimi-
crobial activity was compared with the antibacterial and anti-
fungal properties of octanoic acid (C8). MIC values for
alkanoic acids were between 2.7 and 7.0 mM for bacterial
strains, while a higher activity was detected against fungi with
MIC values between 0.15 and 6.32 mM (Table 2). Generally,
methyl and benzyl esters did not exhibit strong antimicrobial
properties, with MIC values between 10 and 14 mM (results
not shown).
Antibacterial activity was test strain dependant, and all hal-
ogen derivatives were up to twofold more active than the (R)-
3-hydroxyoctanoic acid (3) but exhibit the same or lower ac-
tivity in comparison to fatty acid C8. Chloro derivative 6 was
the most active against S. aureus and L. monocytogenes
(Table 2). Derivative 18 is structurally very similar to the
starting monomer and it exhibited almost identical antimicro-
bial activity (Fig. 2, Table 2).
−
yield of methyl ester (∼40 %) was obtained, whereas the yield
of benzyl ester was higher (60 %), which has previously been
described for similar compounds. We also obtained an olefine
as a by-product in fluorination reaction (13 and 14) which was
isolated and characterized. Compounds 11 and 12 (Table 1,
entries XI and XII) had higher enantiomeric excess than other
halo derivatives (ee 65 %) which may be due to the reaction
conditions that included a low temperature of −70 °C
(
Table 1).
Collins oxidation with chromium(VI) oxide in dichloro-
methane (Collins et al. 1968) was used for the synthesis of
derivatives 16 and 17 which are important organic compounds
with a wide range of applications. All alkanoic acids from the
library (3, 6, 9, 12, 15, and 18) were produced from the cor-
responding benzyl esters in the reaction of deprotection using
a catalytic amount of Pd/C in an atmosphere of hydrogen
(
50 psi). The reactions provided mild conditions, stoichiomet-
ric conversion, and no by-product generation.
We have generated 17 derivatives in a few steps with var-
ious yields (32–100 %). While the structures of the 3-
hydroxyoctanoic acid-based derivatives are simple, there are
ten derivatives, in the current study, which were completely
characterized for the first time (4–12 and 14). Derivative 12
was previously reported but not characterized (Watanabe et al.
In general, the compounds investigated showed higher an-
timicrobial potential towards fungal than bacterial strains
(Table 2). In particular, all halogenated acids (6, 9, and 12)
showed promising antifungal activity against C. albicans and
M. gypseum (Table 2). MIC values for planktonic growth were
determined in standard Sabouraud dextrose broth. Compound
1
989; Watanabe et al. 1983).
12 inhibited fungal growth at concentrations tenfold lower
than the starting (R)-3-hydroxyoctanoic acid (3) and approxi-
mately fivefold lower in comparison to octanoic acid.
Derivatives 6 and 9 showed sevenfold and ninefold lower
MIC values in comparison to derivative 3, respectively.
In addition, derivatives 6, 9, and 12 and octanoic acid
(C8), at sub-MICs (75 % of the MIC value determined
for the planktonic growth) showed complete inhibition
Biological activity of the library of compounds based
on hydroxyoctanoic acid
The library of 3-substituted alkanoic acids and esters generat-
ed from PHA-derived (R)-3-hydroxyoctanoic acid (Fig. 2)
was assessed for antimicrobial and antiproliferative properties.
Fig. 4 Chemical synthesis of halogenated, keto, and unsaturated derivatives of octanoic acid

Appl Microbiol Biotechnol
Table 1 Comparative synthesis of β-hydroxyoctanoic acid derivatives
1–18)
(
Entry Compound Reaction time (h) Isolated yield (%) ee (%)
I
1
3.0
3.5
24
90
>99
6
I′
1r
2
80
II
60
75
1
II′
2r
3
24
65
III
24
96
99
9
III′
IV
3r
4
24
96
0.5
0.5
20
46
22
35
17
13
27
12
16
2
IV′
V
4r
5
56
56
V′
5r
6
20
64
VI
8.0
8.0
4.0
4.0
4.0
4.0
8.0
8.0
0.5
0.5
0.5
0.5
8.0
8.0
0.5
0.5
8.0
20
100
100
32
VI′
VII
VII′
VIII
VIII′
IX
6r
7
7r
8
45
95
1
8r
9
97
1
100
100
40
23
5
IX′
X
9r
10
10r
11
11r
12
12r
13
14
15
16
17
19
8
X′
46
XI
60
65
9
XI′
XII
XII′
XIII
XIV
XV
XVI
XVII
71
100
100
15
66
8
–
Fig. 5 C. albicans spread on Spider medium in the presence of
derivatives: a DMSO control, b octanoic acid (C8), c 3, d 6, e 9, f 12, g
15, and h 18
10
–
100
35
–
–
20
80
–
acid (3) reduced hyphae formation to a lesser extent in
comparison to octanoic acid, while 15 and 18 appeared
to have a positive effect on the hyphae formation at the
tested concentrations. The transition from yeast to hy-
phal growth is important for Candida biofilm formation,
so molecules that interfere with this process are highly
XVIII 18
8.0
80
–
Italicized entries denote chemically synthesized racemic derivatives
of C. albicans filamentous growth on the hyphal-
inducing Spider medium (Fig. 5). (R)-3-hydroxyoctanoic
Table 2 Antimicrobial activity of carboxylic acids generated in this study expressed as minimal inhibitory concentration (MIC) in mM
Compound
E. coli
S. typhimurium
S. aureus
L. monocytogenes
C. albicans
M. gypseum
NCTC 9001
NCTC 120023
NCTC 6571
NCTC 11994
ATCC10231
ATCC24102
3
6
9
6.24
n.a.
6.24
5.60
4.48
6.16
7.02
6.32
3.46
6.24
2.79
4.48
6.16
7.02
6.32
3.46
6.24
2.79
4.48
n.a.
1.55
0.22
0.18
0.15
3.52
6.32
0.69
1.56
1.12
0.90
0.60
3.52
3.16
1.73
4.48
6.16
7.02
6.32
3.46
1
1
1
2
5
8
7.02
n.a.
C8
3.46
n.a. no activity under conditions tested

Appl Microbiol Biotechnol
sought after as possible treatment options according to
Shafreen et al. (2014).
Pyocyanin production As none of the compounds showed
significant activity against P. aeruginosa PAO1, except
for C8 with a relatively high MIC of 6.9 mM, we de-
cided to assess the effect of acids generated in this
study on pyocyanin production (Fig. 6). Pyocyanin is
a blue green redox-active secondary metabolite that is
produced by P. aeruginosa under quorum sensing regu-
lation and one of the factors contributing to virulence of
this strain (Lau et al. 2004; Whooley and McLoughlin
1
982). It was evident that even at concentrations as low
−
1
as 0.06 mM (between 5 and 10 μg mL ), 3 and 15
significantly inhibited pyocyanin production in
P. aeruginosa PAO1, while 9 and C8 stimulated pyocy-
anin production in the same strain (Fig. 6).
Fig. 7 In vitro antiproliferative effect of carboxylic acids on human
fibroblasts following 48 h of exposure at a range of concentrations
(white bar 0.3 mM, gray bar 1.5 mM, and black bar 3 mM)
Discussion
In vitro antiproliferative effect of 3-substituted alkanoic
acids The cytotoxicity of 3-substituted alkanoic acids 3, 6,
Various free fatty acids are attracting attention as potential
therapeutic antimicrobial agents due to their potency, broad
spectrum of activity, non-specific mode of action, and lack
of classical resistance mechanisms against the actions of these
compounds. Their antibacterial properties have been known
for a while (Desbois and Lawlor 2013; Desbois and Smith
9
, 12, 15, 18, and C8 which showed antimicrobial activity
Table 2) was carried out by determining antiproliferative ac-
tivity with human fibroblasts in a concentration range
.5–9 mM. No observable antiproliferative effect was
(
0
detected against human fibroblasts after 48 h of treat-
ment using all compounds at 1.5 mM concentration
2010). Hydroxylated alkanoic acids are also gaining increased
(
Fig. 7). Compounds 3, 6, and 9 showed no cytotoxicity
attention in terms of potential industrial applications (Ren
et al. 2010).
at 1.5 or 3 mM, nor at 9 mM (results not shown).
Compounds 15 and 18 caused about 50 % inhibition
of cell proliferation at 1.5 mM while 12 was toxic at
Taking an advantage of the controllability of monomer
composition during biotechnological production of mcl-
PHA, we have decided to gain access to valuable (R)-3-
hydroxyoctanoic acid monomers starting with a biological
polymer that contains 97 % (R)-3-hydroxyoctanoic acid
monomers (Elbahloul and Steinbuchel 2009). Due to the fact
that 20 % of all pharmaceutical small-molecule drugs are ha-
logenated (Marris 2006), we decided to synthesize halogen
derivatives of a (R)-3-hydroxyoctanoic monomer (1)
3
mM, inhibiting proliferation of 40 % of human fibro-
blasts (Fig. 7). The low cytotoxicity of 3 is in accor-
dance with our previous study where IC50 values of (R)-
3
-hydroxydecanoic acid, as well as decanoic acid (C10),
against several cell lines were determined to be above
−
1
3
mM (500 μg ml ) (O’Connor et al. 2013).
(Fig. 2). Halogen derivatives were obtained in good yields in
single-step reactions (Figs. 3 and 4). Previous attempts to in-
troduce chlorine into the structure of PHA meant feeding bac-
teria with 1-chlorooctane to obtain copolymers with a small
amount of this halogen (Doi and Abe 1990). Arkin et al. de-
scribed chlorination of PHA by using an unsaturated polymer
and passing by chlorine gas through their solutions (Arkin
and Hazer 2002). We have recently made 3-
chlorodecanoic acid from (R)-3-hydroxydecanoate de-
rived from the PHA applying procedure similar to one
described herein (Szwej et al. 2015). On the other side,
production of methyl 3-fluorohexadecanoate was de-
scribed through a three-step synthesis by acylation of
Meldrum’s acid as the starting molecule (Delgado
Fig. 6 Pyocyanin production by Pseudomonas aeruginosa PAO1 in
response to carboxylic acids generated in this study. Star denotes
statistically significant value (p < 0.05). Values are given relative to a
control where cells are grown on Kings A medium with DMSO

Appl Microbiol Biotechnol
et al. 1991). In the procedure described here, we obtained
L. monocytogenes, and S. typhimurium with MICs ranging
from 12.5 to 25 mg/ml (Allen et al. 2012), which was much
higher than MICs determined in the current study for individ-
ual 3-substituted compounds (Table 2).
The introduction of a trans double bond (15) resulted
in decreased antimicrobial activity twofold in compari-
son to octanoic acid. This is in agreement with the
previous studies that the geometry of the double bond
is important for the activity of unsaturated fatty acids. It
has been reported that the addition of a cis double bond
increased the activity of all fatty acids tested (C14, C16,
and C18), where the trans isomer showed no activity
(Kabara et al. 1972).
Due to the fact that the MIC values were not high against
bacterial strains, we decided to test their potential to interfere
with cell-to-cell signaling communication systems that control
virulence in many pathogenic bacterial strains. Small mole-
cules with the ability to interfere with pathogen communica-
tion (i.e., quorum sensing) are coming into focus as promising
new treatment options, especially as pathogens become resis-
tant to antibiotics and other drugs (Kalia and Purohit 2011).
Little is known about the effect of C8 or similar fatty acids on
quorum sensing in P. aeruginosa; however, it has been report-
ed that cis-2-dodecenoic acid could modulate P. aeruginosa
virulence through interference with quorum sensing and type
III secretion system (Deng et al. 2013). On the other hand,
longer chain fatty acids such as linoleic, oleic, and palmitic
have been shown to inhibit quorum sensing in Vibrio harveyi
BB170 and as such offer a means to control foodborne path-
ogens and reduce microbial spoilage (Widmer et al. 2007).
Therefore, derivative 3, as well as 15, that showed significant
inhibition of pyocyanin production (Fig. 6) could be further
examined for quorum quenching or inhibitory ability. On the
other side, an important link has been made between pyocya-
nin produced by P. aeruginosa and its activity on Candida
morphology in mixed infections. At high concentrations, pyo-
cyanin and its metabolites are fungicidal, while at subinhibi-
tory concentrations, it inhibits filamentation and biofilm for-
mation (Gibson et al. 2009; Morales et al. 2013). Taken to-
gether, derivative 9, as well as octanoic acid, may be suitable
for further development in mixed Pseudomonas–Candida in-
fections (Table 2, Fig. 6).
1
2 in a one-step process using (R)-3-hydroxyoctanoate.
β-Keto esters, such as derivatives 16 and 17, have been one
of the most important intermediates in organic synthesis and
have been used in the synthesis of naturally occurring
enantiopure bioactive compounds (Case-Green et al. 2008;
Giddens et al. 2008). Synthesis of β-keto esters via acetoacetic
esters, via mixed malonic esters, from Meldrum’s acid was
reported that included a three-step synthesis using fatty acids
as the starting material (Brinkerhoff et al. 2014). Using our
described approach (Fig. 4), we have obtained products in just
one step with excellent yields (up to 80 %), indicating a con-
venient way of obtaining these important compounds.
Overall, using known chemical transformations, we have
shown that mcl-PHA was a suitable starting material for the
straightforward generation of different small molecules with
good yields (Table 1).
The antibacterial properties of the compounds generated
were not high in comparison to commercially available anti-
biotics, such as gentamicin which has MIC values in a range
of 4 μM concentration; it was evident that the free carboxylic
group was essential for the antimicrobial activity of the com-
pounds (Table 2). This was in an agreement with the study of
Kabara et al. that showed the free carboxyl group was neces-
sary for the activity of lauric acid (Kabara et al. 1972). It has
been proposed that the prime target of free fatty acid (FFA)
action is the cell membrane, where FFAs disrupt the electron
transport chain and oxidative phosphorylation. Besides inter-
fering with cellular energy production, FFA may also inhibit
other metabolic enzyme activities (such as enoyl-ACP reduc-
tase), impair nutrient uptake, be involved in the generation of
peroxidation and auto-oxidation degradation products, or
cause direct lysis of bacterial cells (Desbois and Lawlor
2
013; Desbois and Smith 2010).
There was no observable difference in MIC values when
(
(
R,S)-3-hydroxyoctanoic acid (3r) was used in comparison to
R)-3-hydroxyoctanoic acid (data not shown). This is in slight
disagreement with the previous study of Ruth et al. when
commercially available (R,S)-3-hydroxyoctanoic acid exhibit-
ed slightly lower antibacterial activity to (R)-enantiomer
against a range of Listeria species (Ruth et al. 2007).
However, in the same study, no activity of C8 was reported,
which is in disagreement with our current study and other
works reporting the antibacterial properties of free fatty acids
including octanoic acid (Marounek et al. 2003; Skrivanova
et al. 2004). Previously, R-3-hydroxyoctanoic acid (3), (R)-
Given that antibacterial MIC values of 3-substituted
alkanoic acids were between 2.8 and 7 mM, the gener-
ally low antiproliferative activity does not make them
suitable targets for direct antibacterial applications. The
MIC value for 6, 9, and 12 was between 0.15 and
0.22 mM with C. albicans ATCC10231 (Table 2),
which is an order of magnitude lower than the concen-
tration at which no cytotoxicity is observed with human
fibroblasts for 6 and 9 and some cytotoxicy for 12
(Fig. 7) which is positive for the development of these
compounds as antifungal agents.
3
-hydroxy-8-nonenoic acid, and (R)-3-hydroxy-10-
undecenoic acid were shown to inhibit the growth of
Listeria species and S. aureus with a MIC of 1–5 mM while
(
R)-3-hydroxyphenylalkanoates also inhibited bacterial
growth with MICs of 3–6 mM (Ruth et al. 2007; Sandoval
et al. 2005). According to Allen et al., a mixture of 3-HAs
exhibited antimicrobial activity against E. coli O157:H7,

Appl Microbiol Biotechnol
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We have shown that PHA can be a suitable source of valu-
able synthons that could be further derivatized using simple
chemical transformations. Derivatives containing a carboxylic
group exhibited low antibacterial and higher antifungal prop-
erties that could be further developed, considering the absence
of in vitro toxicity. Furthermore, these compounds appear to
be affecting bacterial and fungal signaling molecules.
Acknowledgments This work has been financially supported by the
Ministry of Education and Science, Republic of Serbia, under Grant
No. 173048. JR was partially supported by research project BP2013
(Bioplastech Ltd., Dublin, Ireland).
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Conflict of interest The authors declare that they have no competing
interests.
Deng Y, Boon C, Chen S, Lim A, Zhang L-H (2013) Cis-2-dodecenoic
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Compliance with ethical standards
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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