Synthesis, micellisation and interaction of novel quaternary ammonium compounds derived from L-Phenylalanine with 1,2-dipalmitoyl-sn-glycero- 3-phosphocholine as model membrane in relation to their antibacterial activity, and their selectivity over human red blood cells

Article abstract of DOI:10.1016/j.bioorg.2015.01.001

A series of quaternary ammonium compounds (QUATS) derived from L-Phenylalanine have been synthesized and their antibacterial efficiencies were determined against various strains of Gram-positive and Gram-negative bacteria. The antibacterial activity increased with increasing chain length, exhibiting a cut-off effect at C₁₄ for Gram-positive and C₁₂ for Gram-negative bacteria. The L-Phenylalanine QUATS displayed enhanced antibacterial properties with a higher cut-off point compared to their corresponding L-Phenylalanine ester hydrochlorides. The CMC was correlated with the MIC, inferring that micellar activity contributes to the cut-off effect in antibacterial activity. The hemolytic activities (HC₅₀) of the QUATS against human red blood cells were also determined to illustrate the selectivity of these QUATS for bacterial over mammalian cells. In general, the MIC was lower than the HC₅₀, and assessment of the micellar contribution to the antibacterial and hemolytic evaluation in TBS as a common medium confirmed that these QUATS can act as antibacterial, yet non-toxic molecules at their monomer concentrations. The interaction of the QUATS with the phospholipid vesicles (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) in the presence of 1-anilino-8-naphthalene sulfonate (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescence probes showed that the presence of the quaternary ammonium moiety causes an increase in hydrophobic interactions, thus causing an increase in antibacterial activity.

Full text of DOI:10.1016/j.bioorg.2015.01.001

Bioorganic Chemistry 58 (2015) 117–129
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, micellisation and interaction of novel quaternary ammonium
compounds derived from L-Phenylalanine with 1,2-dipalmitoyl-sn-
glycero-3-phosphocholine as model membrane in relation to their
antibacterial activity, and their selectivity over human red blood cells
Nausheen Joondan a, Prakashanand Caumul a, Matthew Akerman b, Sabina Jhaumeer-Laulloo a,
⇑
a Department of Chemistry, Faculty of Science, University of Mauritius, Réduit, Mauritius
b University of KwaZulu-Natal, Private Bag X01, Scottsville, Pietermaritzburg 3209, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 June 2014
Available online 12 January 2015
A series of quaternary ammonium compounds (QUATS) derived from
sized and their antibacterial efficiencies were determined against various strains of Gram-positive and
Gram-negative bacteria. The antibacterial activity increased with increasing chain length, exhibiting a
L
-Phenylalanine have been synthe-
cut-off effect at C14 for Gram-positive and C12 for Gram-negative bacteria. The
displayed enhanced antibacterial properties with a higher cut-off point compared to their corresponding
-Phenylalanine ester hydrochlorides. The CMC was correlated with the MIC, inferring that micellar activ-
L-Phenylalanine QUATS
Keywords:
L
-Phenylalanine QUATS
L
Critical micelle concentration
Antibacterial activity
Phospholipid
ity contributes to the cut-off effect in antibacterial activity. The hemolytic activities (HC50) of the QUATS
against human red blood cells were also determined to illustrate the selectivity of these QUATS for bac-
terial over mammalian cells. In general, the MIC was lower than the HC50, and assessment of the micellar
contribution to the antibacterial and hemolytic evaluation in TBS as a common medium confirmed that
these QUATS can act as antibacterial, yet non-toxic molecules at their monomer concentrations. The
interaction of the QUATS with the phospholipid vesicles (1,2-dipalmitoyl-sn-glycero-3-phosphocholine,
DPPC) in the presence of 1-anilino-8-naphthalene sulfonate (ANS) and 1,6-diphenyl-1,3,5-hexatriene
(DPH) as fluorescence probes showed that the presence of the quaternary ammonium moiety causes
an increase in hydrophobic interactions, thus causing an increase in antibacterial activity.
Ó 2015 Elsevier Inc. All rights reserved.
Hemolytic activity
1. Introduction
against microorganisms [7–10]. Head group architecture also pro-
foundly affects antimicrobial activity, as the number, positioning
In recent years, quaternary ammonium compounds (QUATS)
have been widely used as biocides [1,2]. These compounds were
found to exert antimicrobial activities against both Gram-positive
and Gram-negative bacteria as well as against some pathogenic
species of fungus and protozoa [3,4]. However, various bacteria
have been found to develop resistance towards some QUATS deriv-
atives, rendering these compounds ineffective as antibacterial
agents [5]. Moreover, QUATS in general have been found to have
toxic effects towards mammalian cells, thus some of them are
acceptable only for topical applications as they are considered
too toxic for systemic applications [6]. This brings the necessity
for the search of new and more effective biocides.
and nature of cationic groups dictate the interaction with the cell
surface [11–13]. Surfactants with large head groups and a single
hydrophobic tail have been reported to have significant membrane
lysing potential, allowing more efficient membrane disruption
[14].
QUATS derived from amino acids have attracted much interest
due to their lower toxicity profile. In literature, there are several
works concerning the synthesis of cationic surfactants derived
from amino acids such as lysine and arginine, but there are very
few studies on QUATS derived from aromatic amino acids [15–18].
Surfactants containing phenylalanine residues have also been
found to possess good antimicrobial activities with optimal effec-
tiveness at some intermediate chain length, called the cut-off
point, above and below which activity decreases [19]. In our previ-
ous study, we reported the effect of chain length on the bacterial
A
number of studies have demonstrated the relationship
between surfactant hydrophobic chain lengths and its activity
activity of a series of the hydrochloride salts of
esters [20] (Fig. 1).
L-Phenylalanine
⇑
Corresponding author.
E-mail address: sabina@uom.ac.mu (S. Jhaumeer-Laulloo).
http://dx.doi.org/10.1016/j.bioorg.2015.01.001
0045-2068/Ó 2015 Elsevier Inc. All rights reserved.

118
N. Joondan et al. / Bioorganic Chemistry 58 (2015) 117–129
This study is therefore intended to develop novel
L
-Phenylala-
sharp change in conductivity values as concentrations are
increased.
nine QUATS esters by prompting the amino moiety with alkyl
groups to investigate the effect of the quaternary ammonium moi-
ety on the biocidal activities. In this paper, we report the synthesis,
CMC and antibacterial activity of a series of novel quaternary
4. Pharmacology
ammonium surfactant analogues derived from
with varying O-alkyl ester chain length. The biological efficiencies
of these quaternary ammonium surfactants of -Phenylalanine
esters were determined by bacterial growth inhibition assays.
The hemolytic activity against red blood cells (RBCs) was under-
taken to provide selectivity of these QUATS for bacterial over
mammalian cells. In order to study the behaviour of the polar
cationic head and lipophilic moiety of these QUATS with the
phospholipid layer of the bacterial cell wall, the binding studies
L-Phenylalanine
4.1. Antimicrobial activity
L
Antimicrobial tests were performed against five Gram-positive
bacteria Staphylococcus aureus (ATCC 29213, ATCC 25923), Staphy-
lococcus epidermidis (ATCC 12228) and Bacillus cereus (ATCC 11778,
ATCC 10876) and four Gram-negative bacteria Escherichia coli
(ATCC 22922), Pseudomonas aeruginosa (ATCC 27853), Salmonella
typhimurium (ATCC 14028) and Klebsiella pneumoniae (ATCC
13883).
of selected L-Phenylalanine esters with 1,2-dipalmitoyl-sn-glyce-
ro-3-phosphocholine (DPPC) as model phospholipid membrane
using 1-anilino-8-naphthalenesulfonate (ANS) and 1,6-diphenyl-
1,3-5-hexatriene (DPH) as fluorescence probes were also undertaken.
4.2. Hemolytic activity
The determination of the hemolytic activity was made on
human red blood cells obtained from a healthy donor by veni-
puncture and collected in sodium-EDTA coated tubes.
2. Chemistry
With the aim to systematically study the influence of the
quaternary ammonium moiety on the biological properties of the
4.3. Phospholipid binding assay
L
-Phenylalanine QUATS,
with methyl and ethyl groups. In this series, the quaternary ammo-
nium moiety of -Phenylalanine QUATS represent the polar head
L-Phenylalanine esters were quaternised
Binding studies of the C12, C14 and C16 derivatives of L-Phenylal-
anine QUATS (6b–8b) with phospholipid 1,2-dipalmitoyl-sn-glycero-
3-phosphocholine (DPPC) were carried out in TBS (0.01 M, pH
L
group while the alkyl ester chain represents the hydrophobic tail
of the QUAT molecules (Fig. 2).
7.0) by
described previously [20].
a fluorescence probe technique using the protocol
L
-Phenylalanine QUATS of varying chain lengths were obtained
in moderate yields and were soluble in water. The 1H and 13C NMR
5. Experimental protocol
spectra were concordant with the expected structures.
5.1. Chemistry
3. Critical micellar concentration (CMC)
L
-Phenylalanine was obtained from HiMedia Laboratories
(India). The fluorescence probes, 1-anilino-8-naphthalene sulfo-
nate (ammonium salt; ANS) and 1,6-diphenyl-1,3,5-hexatriene
(DPH), Tris Buffered saline (0.05 M, pH 7.4), bromoethane, decan-
1-ol, dodecan-1ol, tetradecan-1-ol, hexadecan-1-ol, octadecan-1-ol
and eicosan-1-ol, 0.65 mM phosphorus standard solution,
ammonium molybdate(VI) tetrahydrate, hydrogen peroxide (30%)
and streptomycin were obtained from Sigma–Aldrich (St. Louis,
USA). p-Toluene sulfonic acid (PTSA) was obtained from Merck
(Germany). Silica gel (60–120 Mesh) obtained from Alpha Chemika
(India) was used for column chromatography. Mueller Hinton agar
and Mueller Hinton broth were obtained from Oxoid Ltd. (United
Kingdom). Cetyl trimethyl ammonium bromide (CTAB) was
Selected CMCs were determined in deionised water/Tris buf-
fered saline (TBS) solution (0.05 M, pH 7.4) by using measured con-
ductivity values as
a
function of
L-Phenylalanine QUATS
concentration at 25 °C. The CMCs were determined by plotting
the conductivity values against the concentration of the
compounds in aqueous solutions. The CMCs were noted by the
O
OCnH2n+1
n=1-4, 8, 10, 12, 14, 16, 18, 20
NH
3 Cl
obtained from BDH Laboratory Supplies (England). L-Ascorbic acid
Fig. 1. Hydrochloride salts of
L-Phenylalanine esters.
was obtained from s.d fine chemicals (India). The synthetic 1,2-
dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was obtained
from Avanti Polarlipids, Inc (USA). The different bacterial strains
were obtained from MicrobiologicsÒ (St. Cloud, MN, USA) and
Oxoid Ltd. (United Kingdom).
O
O
n
1H NMR and 13C NMR spectra were recorded at 250 MHz and
62.9 MHz on a Bruker electro spin NMR spectrometer using CDCl3,
D2O and DMSO-d6 as solvents. Mass spectra were recorded on a
TOF mass spectrometer with electrospray ionization in the positive
mode. Fluorescence intensities were recorded on a LS 55 Perkin
Elmer fluorescence spectrophotometer. UV absorbances were
recorded on a Biochrom Libra S22 UV–VIS spectrophotometer.
Conductivity measurements were made using a Jenway 4320 con-
ductivity meter and sonification was carried out using a Soniprep
150 sonifier. The clogP values were predicted by Chemdraw ultra
8.0.
X-
N
R= CH3, C2H5
X= Br, I
R
Compound
1b
0
2b
1
3b
2
4c
3
5b
9
6b
11
7b
13
8b
15
n
R
X
C2H5 C2H5 C2H5 CH3 CH3 CH3 CH3 CH3
Br Br Br
Fig. 2. Molecular structure of
I
I
I
I
I
The X-ray data were recorded on a Bruker Apex Duo diffractom-
eter equipped with an Oxford Instruments Cryojet operating at
L
-Phenylalanine QUATS.

N. Joondan et al. / Bioorganic Chemistry 58 (2015) 117–129
119
Table 1
Crystal data and structure refinement details for 1b.
Crystal data
1b
Chemical formula
C
16H26BrNO2ꢀ2H2O
Molar mass (g molꢁ1
)
362.29
Crystal system, space group
Temperature (K)
a, b, c (Å)
Monoclinic, P21
100(2)
10.0997(5), 10.8334(6), 16.8425(9)
a
, b,
c
(°)
a = c = 90, b = 104.852(2)
1781.24(16)
V (Å3)
Z
4
Radiation type
MoKa
2.32
l
(mm–1
)
Crystal size (mm)
0.52 ꢂ 0.20 ꢂ 0.11
Data collection
Diffractometer
Absorption correction
Tmin, Tmax
Bruker Apex Duo CCD diffractometer
Multi-scan, SADABS, Bruker 2012
0.379, 0.785
No. of Measured, independent and observed [I > 2
r(I)] reflections
14068, 6937, 6561
Rint
0.023
Refinement
R[F2 > 2
r
(F2)], wR(F2), S
0.022, 0.045, 0.84
No. of reflections
No. of parameters
No. of restraints
H-atom treatment
6937
403
0
H atoms treated by a mixture of independent and constrained refinement.
D
qmax
,
D
qmin (e Å–3
)
0.29, ꢁ0.20
Absolute structure
Flack parameter
Flack, H.D. (1983), ActaCryst. A39, 876–881
0.011(4)
100(2) K and an Incoatecmicrosource operating at 30 W power.
Crystal and structure refinement data are given in Table 1.
Selected bond lengths and angles are given in Table 2. The data
5.1.1. General synthesis of N,N,N-triethyl ammonium
esters (1b–3b)
L-Phenylalanine
L
-Phenylalanine esters were synthesized according to a modi-
were collected with Mo K
a
(k = 0.71073 Å) radiation at a crystal-to-
fied procedure [24]. L-Phenylalanine (1.00 g, 6 mmol) was stirred
detector distance of 50 mm. The data collection was performed
using omega and phi scans with exposures taken at 30 W X-ray
power and 0.50° frame widths using APEX2 [21]. The data were
reduced with the programme SAINT [21] using outlier rejection,
scan speed scaling, as well as standard Lorentz and polarisation
correction factors. A SADABS semi-empirical multi-scan absorption
correction [21] was applied to the data. Direct methods, SHELXS-97
[22] and WinGX [23] were used to solve the data. All non-hydrogen
atoms were located in the difference density map and refined
anisotropically with SHELXL-97 [22]. All hydrogen atoms were
included as idealized contributors in the least squares process.
Their positions were calculated using a standard riding model with
C–Haromatic distances of 0.95 Å and Uiso = 1.2 Ueq, C–Hmethine dis-
tances of 1.00 Å and Uiso = 1.2 Ueq, C–Hmethylene distances of 0.99
Å and Uiso = 1.2 Ueq and C–Hmethyl distances of 0.98 Å and Uiso = 1.5
Ueq. The hydrogen atoms of the water solvate molecules were
located in the difference density map, and refined isotropically.
with the corresponding alcohol (15 mL) at 0 °C. Thionyl chloride
(1 mL, 13.8 mmol) was added dropwise and the reaction mixture
was stirred at room temperature for 48 h. Excess alcohol was evap-
orated in vacuo and the residue washed with diethyl ether to yield
the compound as a white solid.
Bromoethane (4 mL) was added to a mixture of L-Phenylalanine
ester (3 mmol) and K2CO3 (1.2 g) in acetonitrile (10 mL) and the
mixture was heated to 90 °C in a sealed tube for 18 h. The reaction
mixture was filtered to remove excess K2CO3 and the excess sol-
vent was then removed under vacuo. Diethyl ether was added
and the mixture was filtered. The residue was collected and
washed with ether to yield the N,N,N-triethyl derivative as a white
solid, which was recrystallised in acetonitrile. Evaporation of the
filtrate yielded the N,N-diethyl derivative as a colourless oil.
5.1.2. General synthesis of N,N-diethyl,N-methyl phenylalanine
esters(4c, 5b–8b)
The C10–C16 esters of L-Phenylalanine were synthesized using
the modified procedure reported by Bazcko et al. [25]. A mixture
of the amino acid (12.1 mmol), PTSA (2.30 g, 12.1 mmol) and alco-
hol (14.5 mmol) in toluene (100 mL) was heated for 48 h instead of
24 h using a Dean–Stark apparatus. The crude products were puri-
fied by column chromatography on silica gel with EtOAC/Hexane
(1:2) to afford the corresponding esters as oils.
Table 2
Selected bond lengths (Å) and bond angles (°) of 1ba.
Bond
Molecule A
Molecule B
The esters were then converted to the N,N-diethyl derivatives
using EtBr/K2CO3 in a sealed tube at 90 °C. The QUATS (5b–8b) were
then synthesised by heating the corresponding N,N-diethyl deriva-
tives with excess MeI in a sealed tube at 90 °C for 18 h. The solvent
was removed under vacuo and on addition of diethyl ether the
desired quaternary ammonium compound precipitated out.
b
N–Cmethylene
N–C7
C15–O1
C15–O2
C16–O2
1.53(5)
1.54(3)
1.21(3)
1.33(2)
1.47(2)
1.53(4)
1.54(3)
1.21(2)
1.33(2)
1.46(2)
Angle
C7–N1–Cmethylene
O1–C15–O2
C15–O2–C16
108.4(2)
124.4(2)
115.4(2)
110.1(2)
124.2(2)
115.5(2)
5.1.3. Analytical data and spectral assignments of N,N,N-trialkyl
ammonium L-Phenylalanine esters
5.1.3.1. Compound 1a. Yield: 63%. 1H NMR: dH (CDCl3), 1.16 (t, 6H,
a
Standard uncertainties given in parentheses.
Mean of the three N–Cmethylene bonds per molecule.
b
N-CH2CH3), 2.87 (m, 2H, N-CH2CH3), 2.98 (m, 2H, N-CH2CH3),

120
N. Joondan et al. / Bioorganic Chemistry 58 (2015) 117–129
3.02 (m, 1H, CHCHPh), 3.05 (m, 1H, CHCHPh), 3.25 (s, 3H, CH3),
4.04 (m, 1H, CH), 7.31–7.43 (m, 5H, Ph).
CHCHPh), 3.10 (t, 1H, CH), 3.99 (t, 2H, OCH2CH2CH2CH3), 7.13–
7.22 (m, 5H, Ph).
13C NMR: dC (CDCl3), 14.1 (N-CH2CH3), 36.2 (CH2Ph), 44.5 (N-
CH2CH3), 51.4 (OCH3), 65.0 (CH), 126.0–139.1 (C6H4), 173.0 (C@O).
13C NMR: dC (CDCl3), 13.6 (OCH2CH2CH2CH3), 13.9 (N-CH2CH3),
19.1 (O CH2CH2CH2CH3), 30.7 (OCH2CH2CH2CH3), 36.5 (CH2Ph),
44.6 (N-CH2CH3), 63.9 (CH), 65.1 (OCH2CH2CH2CH3), 126.2–138.8
(C6H4), 172.8 (C@O).
5.1.3.2. Compound 1b. Yield: 35%. M.p: 89 °C. MW: 343.1964: ESI-
MS, m/z: 264.1961 (M+ without the Br-). Elem. Anal. Found: C,
55.65; H, 7.80; N, 4.45; Calcd. for C16H26NO2Br: C, 55.82; H, 7.61;
N, 4.07. 1H NMR: dH (CDCl3), 1.42 (t, 9H, N-CH2CH3), 3.24 (m, 1H,
CHCHPh), 3.38 (m, 1H, CHCHPh), 3.49 (s, 3H, CH3), 3.66 (m, 6H,
N-CH2CH3), 4.36 (m, 1H, CH), 7.25–7.40 (m, 5H, Ph).
5.1.3.10. Compound 4c. Yield: 52%. 1H NMR: dH (CDCl3), 0.94 (t, 3H,
OCH2CH2CH2CH3), 1.02 (m, 2H, OCH2CH2CH2CH3), 1.21 (m, 2H,
OCH2CH2CH2CH3), 1.27 (m, 6H, N-CH2CH3), 3.21 (m, 1H, CHCHPh),
3.48 (s, N-CH3), 3.62 (m, 1H, CHCHPh), 3.67 (m, 2H, N-CH2CH3),
3.74 (m, 2H, N-CH2CH3), 3.96 (m, 2H, OCH2), 4.27 (m, 1H, CH),
7.17–7.25 (m, 5H, Ph).
13C NMR: dC (CDCl3), 11.3 (N-CH2CH3), 35.4 (CH2Ph), 56.4
(OCH3), 58.0 (N-CH2CH3), 75.3 (CH), 130.9–136.1 (C6H4), 171.0
(C@O).
13C NMR: dC (CDCl3), 9.2 (N-CH2CH3), 13.2 (OCH2CH2CH2CH3),
18.4 (O CH2CH2CH2CH3), 29.7 (CH2Ph), 33.3 (OCH2CH2CH2CH3),
46.7 (N-CH3), 57.3 (N-CH2CH3), 67.2 (OCH2CH2CH2CH3), 72.9
(CH), 128.1–132.1 (C6H4), 166.3 (C@O).
5.1.3.3. Compound 1c. Yield: 88%. 1H NMR: dH (CDCl3), 3.30 (m, 1H,
CHCHPh), 3.36 (s, 9H, N-CH3), 3.61 (s, 3H, OCH3), 3.72 (m, 1H,
CHCHPh), 4.54 (m, 1H, CH), 7.30–7.43 (m, 5H, Ph).
13C NMR: dC (CDCl3), 32.6 (CH2Ph), 52.6 (NCH3), 53.6 (OCH3),
75.6 (CH), 128.1–132.9 (C6H4), 167.8 (C@O).
5.1.3.11. Compound 5a. Yield: 76%. 1H NMR: dH (CDCl3), 0.89 (t, 3H,
O(CH2)9CH3), 1.03 (t, 6H, N-CH2CH3), 1.55 (m, 14H, OCH2CH2
(CH2)7CH3), 1.62 (m, 2H, OCH2CH2(CH2)7CH3), 2.70 (m, 2H,
NCH2CH3), 2.76 (m, 2H, NCH2CH3), 2.81 (m, 1H, CHCHPh), 2.89
(m, 1H, CHCHPh), 3.06 (m, 1H, CH), 3.59 (m, 2H, OCH2(CH2)8CH3),
7.15–7.27 (m, 5H, Ph).
5.1.3.4. Compound 1d. Yield: 38%. 1H NMR: dH (CDCl3), 1.28 (t, 9H,
N-CH2CH3), 3.27 (m, 6H, N-CH2CH3), 7.37–7.65 (m, 5H, Ph).
13C NMR: dC (CDCl3), 9.6 (N-CH2CH3), 30.4 (CH2Ph), 55.5 (N-CH2-
CH3), 60.1 (CH), 130.2–138.8 (C6H4), 168.4 (C@O).
13C NMR: dC (CDCl3), 13.8 (N-CH2CH3), 14.1 (O(CH2)9CH3), 22.7–
31.9 (OCH2(CH2)8CH3), 36.4 (CH2Ph), 44.5 (N-CH2CH3), 55.7
(OCH2(CH2)8CH3), 65.1 (CH), 126.2–138.8 (C6H4), 172.8 (C@O).
5.1.3.5. Compound 2a. Yield: 68%. 1H NMR: dH (CDCl3), 1.01 (t, 6H,
N-CH2CH3), 1.13 (t, 3H, OCH2CH3), 2.54 (m, 2H, N-CH2CH3), 2.76
(m, 2H, N-CH2CH3), 2.85 (m, 1H, CHCHPh), 2.90 (m, 1H, CHCHPh),
3.59 (t, 1H, CH), 4.01 (m, 2H, O-CH2CH3), 7.31–7.43 (m, 5H, Ph).
5.1.3.6. 13C NMR: dC (CDCl3), 13.8 (N-CH2CH3), 14.1(OCH2CH3),
35.3 (CH2Ph), 44.5 (N-CH2CH3), 60.0 (OCH2CH3), 65.1 (CH),
126.2–138.8 (C6H4), 172.6 (C@O).
5.1.3.12. Compound 5b. Yield: 65%. M.p: 129 °C. Elem. Anal. Found:
C, 57.30; H, 8.34; N, 3.43; Calcd. for C24H42NO2I: C, 57.25; H, 8.41;
N, 2.78. 1H NMR: dH (CDCl3), 0.86 (m, 3H, O(CH2)9CH3), 1.24 (m,
14H, OCH2CH2(CH2)7CH3), 1.48 (m, 6H, N-CH2CH3), 1.89 (m, 2H,
OCH2CH2(CH2)7CH3), 3.22 (m, 1H, CHCHPh), 3.47 (s, 3H, N-CH3),
3.73 (m, 2H, NCH2CH3), 3.88 (m, 2H, NCH2CH3), 3.95 (m, 1H,
CHCHPh), 4.03 (m, 2H, OCH2(CH2)8CH3), 4.24 (m, 1H, CH), 7.24–
7.30 (m, 5H, Ph).
5.1.3.6. Compound 2b. Yield: 28%. M.p: 90 °C. Elem. Anal. Found: C,
56.23; H, 8.51; N, 4.90; Calcd. for C17H28NO2Br: C, 56.98; H, 7.88; N,
3.91. 1H NMR: dH (CDCl3), 0.96 (t, 3H, OCH2CH3), 1.44 (t, 9H, N-CH2-
CH3), 3.28 (m, 1H, CHCHPh), 3.38 (m, 1H, CHCHPh), 3.67 (m, 6H, N-
CH2CH3), 4.03 (m, 2H, OCH2CH3), 4.34 (m, 1H, CH), 7.26–7.41 (m,
5H, Ph).
13C NMR: dC (CDCl3), 8.9 (N-CH2CH3), 14.1 (O(CH2)9CH3), 22.7–
31.9 (OCH2(CH2)8CH3), 32.8 (CH2Ph), 46.7 (N-CH3), 57.3 (N-CH2-
CH3), 64.1 (OCH2(CH2)8CH3), 72.1 (CH), 128.2–132.1 (C6H4), 166.6
(C@O).
13C NMR: dC (CDCl3), 8.20 (N-CH2CH3), 13.2 (OCH2CH3), 32.9
(CH2Ph), 55.1 (N-CH2CH3), 63.8 (OCH2CH3), 72.3 (CH), 128.1–
133.6 (C6H4), 167.7 (C@O).
5.1.3.13. Compound 6a. Yield: 70%. 1H NMR: dH (CDCl3), 0.89 (t, 3H,
O(CH2)11CH3), 1.03 (t, 6H, N-CH2CH3), 1.52 (m, 18H, OCH2CH2
(CH2)9CH3), 1.57 (m, 2H, OCH2CH2(CH2)9CH3), 2.71 (m, 2H,
NCH2CH3), 2.79 (m, 2H, NCH2CH3), 2.82 (m, 1H, CHCHPh), 2.87
(m, 1H, CHCHPh), 3.09 (m, 1H, CH), 3.94 (m, 2H, OCH2(CH2)10CH3),
7.15–7.25 (m, 5H, Ph).
5.1.3.7. Compound 3a. Yield: 76%. 1H NMR: dH (CDCl3), 0.85 (t, 3H,
OCH2CH2CH3), 1.00 (t, 6H, N-CH2CH3), 1.57 (m, 2H, OCH2CH2CH3),
2.52 (m, 2H, N-CH2CH3), 2.76 (m, 2H, N-CH2CH3), 2.87 (m, 1H,
CHCHPh), 3.05 (m, 1H, CHCHPh), 3.60 (m, 1H, CH), 4.01 (t, 2H, O-
CH2 CH2CH3), 7.31–7.43 (m, 5H, Ph).
13C NMR: dC (CDCl3), 13.8 (N-CH2CH3), 14.1 (O(CH2)11CH3),
22.7–31.9 (OCH2(CH2)10CH3), 36.4 (CH2Ph), 44.5 (N-CH2CH3), 55.7
(OCH2(CH2)10CH3), 65.1 (CH), 126.2–138.8 (C6H4), 172.8 (C@O).
13C NMR: dC (CDCl3), 13.8 (N-CH2CH3), 14.1(OCH2CH3), 35.3
(CH2Ph), 44.5 (N-CH2CH3), 60.0 (OCH2CH3), 65.1 (CH), 126.2–
138.8 (C6H4), 172.6 (C@O).
5.1.3.14. Compound 6b. Yield: 43%. M.p: 132 °C. Elem. Anal. Found:
C, 55.65; H, 7.80; N, 4.45; Calcd. for C26H46NO2I: C, 55.82; H, 7.61;
N, 4.07. 1H NMR: dH (CDCl3), 0.88 (m, 3H, O(CH2)11CH3), 1.26 (m,
18H, OCH2CH2(CH2)9CH3), 1.51 (m, 6H, N-CH2CH3), 1.78 (m, 2H,
OCH2CH2(CH2)9CH3), 3.08 (m, 1H, CHCHPh), 3.23 (m, 1H, CHCHPh),
3.48 (s, 3H, N-CH3), 3.66 (m, 2H, NCH2CH3), 3.72 (m, 2H, NCH2CH3),
3.99 (m, 2H, OCH2(CH2)10CH3), 4.26 (m, 1H, CH), 7.21–7.34 (m, 5H,
Ph).
5.1.3.8. Compound 3b. Yield: 6%. M.p: 92 °C. 1H NMR: dH (CDCl3),
0.49 (t, 3H, OCH2CH2CH3), 1.26 (t, 9H, N-CH2CH3), 1.35 (m, 2H,
OCH2CH2CH3), 3.13 (m, 1H, CHCHPh), 3.25 (m, 1H, CHCHPh), 3.50
(m, 6H, N-CH2CH3), 3.78 (m, 2H, OCH2CH2CH3), 4.22 (m, 1H, CH),
7.10–7.26 (m, 5H, Ph).
13C NMR: dC (CDCl3), 10.5 (OCH2CH2CH3), 13.9 (N-CH2CH3), 22.0
(OCH2CH2CH3), 36.5 (CH2Ph), 44.6 (N-CH2CH3), 65.1 (CH), 65.7
(OCH2CH2CH3), 126.2–138.8 (C6H4), 172.8 (C@O).
13C NMR: dC (CDCl3), 9.2 (N-CH2CH3), 14.4 (O(CH2)11CH3), 23.0–
31.2 (OCH2(CH2)10CH3), 33.6 (CH2Ph), 46.8 (N-CH3), 57.7 (N-CH2-
CH3), 67.4 (OCH2(CH2)10CH3), 72.9 (CH), 128.5–132.3 (C6H4),
166.8 (C@O).
5.1.3.9. Compound 4a. Yield: 78%. 1H NMR: dH (CDCl3), 1.00 (t, 3H,
OCH2CH2CH2CH3), 1.03 (t, 6H, N-CH2CH3), 1.25 (m, 2H, OCH2CH2-
CH2CH3), 1.49 (m, 2H, OCH2CH2CH2CH3), 2.56 (m, 2H, N-CH2CH3),
2.73 (m, 2H, N-CH2CH3), 2.79 (m, 1H, CHCHPh), 2.87 (m, 1H,
5.1.3.15. Compound 7a. Yield: 78%. 1H NMR: dH (CDCl3), 0.86 (t, 3H,
O(CH2)13CH3), 1.03 (t, 6H, N-CH2CH3), 1.49 (m, 22H, OCH2CH2

N. Joondan et al. / Bioorganic Chemistry 58 (2015) 117–129
121
(CH2)11CH3), 1.54 (m, 2H, OCH2CH2(CH2)11CH3), 2.54 (m, 2H,
NCH2CH3), 2.76 (m, 2H, NCH2CH3), 2.82 (m, 1H, CHCHPh), 2.87
(m, 1H, CHCHPh), 3.09 (m, 1H, CH), 3.94 (m, 2H, OCH2(CH2)12CH3),
7.16–7.25 (m, 5H, Ph).
centrifuged (5 min, 3000 rpm). The hemolysis degree was deter-
mined by comparing the absorbance (540 nm) of the supernatant
with that of the control samples which are totally hydrolysed with
distilled water.
13C NMR: dC (CDCl3), 13.8 (N-CH2CH3), 14.1 (O(CH2)13CH3),
22.7–31.9 (OCH2(CH2)12CH3), 36.4 (CH2Ph), 44.6 (N-CH2CH3), 63.0
(OCH2(CH2)12CH3), 65.1 (CH), 126.2–138.8 (C6H4), 172.8 (C@O).
5.4. Phospholipid binding studies
5.4.1. 1-Anilino-8-naphthalene sulfonate (ANS)
5.1.3.16. Compound 7b. Yield: 42%. M.p: 152 °C. MW 559.38: ESI-
MS, m/z: 432.38 (M+ without the Iꢁ) Elem. Anal. Found: C, 60.37;
H, 8.65; N, 2.97; Calcd. for C28H50NO2I: C, 60.10; H, 9.01; N, 2.50.
1H NMR: dH (CDCl3), 0.86 (m, 3H, O(CH2)13CH3), 1.24 (m, 22H,
OCH2CH2(CH2)11CH3), 1.52 (m, 6H, N-CH2CH3), 1.75 (m, 2H, OCH2-
CH2(CH2)11CH3), 2.77(m, 1H, CHCHPh), 3.15 (m, 1H, CHCHPh), 3.46
(s, 3H, N-CH3), 3.70 (m, 2H, NCH2CH3), 3.84 (m, 2H, NCH2CH3), 3.96
(m, 2H, OCH2(CH2)12CH3), 4.26 (m, 1H, CH), 7.25–7.27 (m, 5H, Ph).
13C NMR: dC (CDCl3), 9.1 (N-CH2CH3), 14.2 (O(CH2)13CH3), 22.7–
32.0 (OCH2(CH2)12CH3), 33.3 (CH2Ph), 46.7 (N-CH3), 57.2 (N-CH2-
CH3), 67.3 (OCH2(CH2)12CH3), 72.7 (CH), 128.3–132.1 (C6H4),
166.7 (C@O).
Fluorometric titrations of solutions containing 5 ꢂ 10ꢁ4
M
QUATS (6b, 7b, 8b) and phospholipid (1 ꢂ 10ꢁ3 M) with stock solu-
tions of ANS were carried out. Phospholipid vesicles were prepared
using the method described previously [20]. The excitation and
emission wavelength were 380 nm and 480 nm respectively. The
slope of the linear plot was taken to be the emission coefficient of
the ternary ANS complex (At). For the quantitative determination
of the binding of ANS to lipid in the presence of the QUATS, a solution
containing 2 ꢂ 10ꢁ5 M lipid and 5 ꢂ 10ꢁ4 M QUAT was titrated with
stock solution of ANS and the fluorescence of each titration incre-
ment was measured. The concentrations of the bound ANS, Xb, in
these solutions were calculated according to the Eq. (1). The free
and bound concentrations of ANS corresponding to each total ANS
concentration along the titration curve were calculated.
5.1.3.17. Compound 8a. Yield: 73%. 1H NMR: dH (CDCl3), 0.99 (t, 3H,
O(CH2)15CH3), 1.23 (t, 6H, N-CH2CH3), 1.48 (m, 26H, OCH2CH2
(CH2)13CH3), 1.55 (m, 2H, OCH2CH2(CH2)13CH3), 2.56 (m, 2H,
NCH2CH3), 2.73 (m, 2H, NCH2CH3), 2.78 (m, 1H, CHCHPh), 2.86
(m, 1H, CHCHPh), 3.06 (m, 1H, CH), 3.94 (m, 2H, OCH2(CH2)14CH3),
7.14–7.26 (m, 5H, Ph).
Xb ¼ ðFb ꢁ F0Þ=ðAt ꢁ A0Þ
ð1Þ
Fb and F0 are the fluorescence intensities for solutions with and
without lipid, while At and A0 are the emission coefficients for the
lipid-bound and free ANS respectively. From the value of Xb, the
concentrations of free and bound ANS were calculated for each
solution and the data were then treated using the Scatchard equa-
tion (Eq. (2)). The binding constant and the binding capacity
between ANS and DPPC vesicles were determined using this equa-
tion, assuming equal binding strength for all binding sites.
5.1.3.18. Compound 8b. Yield: 65%. M.p: 164 °C. Elem. Anal. Found:
C, 60.65; H, 9.46; N, 2.29; Calcd. for C30H54NO2I: C, 61.31; H, 9.26;
N, 2.38. 1H NMR: dH (CDCl3), 0.83 (m, 3H, O(CH2)15CH3), 1.24 (m,
26H, OCH2CH2(CH2)13CH3), 1.53 (m, 6H, N-CH2CH3), 1.75 (m, 2H,
OCH2CH2(CH2)13CH3), 2.77 (m, 1H, CHCHPh), 3.16 (m, 1H,
CHCHPh), 3.49 (s, 3H, N-CH3), 3.67 (m, 2H, NCH2CH3), 3.73 (m,
2H, NCH2CH3), 3.95 (m, 2H, OCH2(CH2)14CH3), 4.26 (m, 1H, CH),
7.24–7.28 (m, 5H, Ph).
m
½Pꢃ
¼ nK ¼
mK
ð2Þ
where
m is the number of moles of bound probe per mol of lipid; [P]
13C NMR: dC (CDCl3), 9.1 (N-CH2CH3), 14.2 (O(CH2)15CH3), 22.7–
32.0 (OCH2(CH2)14CH3), 33.3 (CH2Ph), 46.7 (N-CH3), 57.2 (N-CH2-
CH3), 67.3 (OCH2(CH2)14CH3), 72.7 (CH), 128.3–132.1 (C6H4),
166.7 (C@O).
is the free concentration of the probe, ANS; n is the maximum value
of which indicates the binding capacity of the lipid for ANS; and K
m
is the binding constant. Previous studies carried out on the binding
of ANS to DPPC, the emission coefficients of free ANS, A0 and that of
lipid bound ANS, Ab were found to be 1 ꢂ 107 and 5 ꢂ 108 respec-
tively [34]. The binding capacity of the lipid, n and the binding con-
stant, K were found to be 0.0016 and 2 ꢂ 106 Mꢁ1 respectively
5.2. Antimicrobial activity
A growth inhibition assay was performed using the Kirby-Bauer
disc diffusion method [26] using Muellor Hinton agar and Muellor
Hinton broth (full strength) for bacterial culture. Minimum Inhib-
itory Concentrations (MICs) of the synthesized compounds were
determined by a dilution assay. The stock solutions of the com-
pounds were prepared in distilled water/TBS (0.05 M, pH 7.4)
5.4.2. DPH (1,6-diphenyl-1,3,5-hexatriene)
For the competitive binding studies, mixtures containing DPPC
(2 ꢂ 10ꢁ5 M), and DPH were titrated against varying concentra-
tions of the QUATS, and the quenching of the fluorescence were
recorded as a function of QUATS concentrations. The binding of
the QUATS to DPPC were determined using Eq. (3). This equation
was derived from the competitive binding mechanism assuming
that the QUATS competes for n1 of the n probe binding sites with
an intrinsic binding strength of K1.
and were serially diluted with concentration of the
L-Phenylalanine
QUATS ranging from 1000 g/mL to 1 g/mL. 10 L of these sample
l
l
l
solutions were pipetted onto the discs and allowed to incubate at
37 °C for 24 h. The MICs were determined by measuring the diam-
eter of the zone of inhibition in mm. CTAB which is commonly use
as an antiseptic was used as a positive control using the same con-
centration of the tested compounds. The lowest concentration that
prevented colony formation was defined as the MIC.
ðn ꢁ m1
Þ
m1
m1Þ½Dꢃ
¼ n1K1
ꢁ
m1K1
ð3Þ
ðn ꢁ mꢄ
ꢁ
where m⁄ is the ratio of bound-probe per total lipid concentration in
the presence of the QUATS; 1 is the ratio of bound QUATS per total
lipid concentration; n1 is the maximum value of 1 which indicates
m
m
5.3. Hemolytic activity
the probe binding sites which may be replaced by QUATS; [D] is the
free QUATS concentration; K1 is the binding constant for the
QUATS-lipid interaction; and m1 was obtained from Eq. (4), using
n and K values from Eq. (2).
Erythrocytes were suspended in saline buffer/TBS (0.05 M, pH
7.4). 0.5 mL of a series of surfactant solutions with concentration
ranging from 1 mg/mL to 0.01 mg/mL were placed in centrifuge tube
mꢄ
containing saline buffer/TBS. An aliquot of 50 lL of erythrocyte
suspension were added to each tube and were incubated for
30 min at room temperature. Following incubation, the tubes were
m1 ¼ n ꢁ mꢄ
ꢁ
ð4Þ
½PꢃꢄK

122
N. Joondan et al. / Bioorganic Chemistry 58 (2015) 117–129
[P]⁄ represents the free concentration of the probe in the presence
of the QUATS. [D] was taken to be the difference between the total
concentration of the QUATS and the product of m1 and total lipid
concentration.
ester hydrochloride was first made to react with TEA, to produce
the free amine which was then reacted with EtI. However, the
desired product was not formed and this may be due to the
absence of base in the reaction medium during quaternisation.
The reaction of a primary amine with an alkyl halide involves the
liberation of a hydrohalic acid which combines with amines to
form a mixture of amine hydrohalide salts [31]. Consequently, very
low concentration of the free amine remained for subsequent
alkylation.
6. Results and discussion
6.1. Synthesis of quaternary ammonium compounds derived from
Using EtBr in acetonitrile at 90 °C and K2CO3 as base in a sealed
tube gave a mixture of the desired product 1b together with the
dialkylated product 1a as the major product. The structure of com-
pound 1b was confirmed by spectral data and X-ray crystallogra-
phy. From the 1H NMR spectrum the presence of the three ethyl
groups in 1b were confirmed by the triplet (9 H) at 1.42 and a mul-
tiplet of 6 H at 3.66 ppm. The structure of 1a was confirmed by
L
-Phenylalanine esters
L
-Phenylalanine QUATS were synthesised by quaternising the
amino group of -Phenylalanine esters with alkyl halides. The
method was standardised using -Phenylalanine methyl ester as
precursor. A number of methods for quaternising primary and sec-
ondary amines involving the use of alkylating agents in the pres-
ence of strong inorganic bases have been reported [27,28]. Most
of these reactions often involve prolonged reaction times and heat
which are conducive to undesirable side reactions [29]. Several
L
L
spectral data. The N-ethyl groups appeared as
a triplet at
1.16 ppm corresponding to two CH3 protons and 2 multiplets at
2.87–2.98 ppm N-CH2 moieties.
Different phenylalanine esters with alkyl chain lengths varying
from C1 to C4 were quaternised using EtBr/K2CO3 in a sealed tube at
90 °C (Scheme 2).
attempts were made in order to quaternise
ester using ethyl groups to yield compound 1b (Scheme 1).
Reaction of -Phenylalanine methyl ester with MeI in the pres-
L-Phenylalanine methyl
L
An increase of the alkyl ester chain length from methyl to pro-
pyl ester caused a decrease in the yield of the quaternary ammo-
nium compounds and an increase in the diethyl derivative. In the
case of the butyl ester, the diethyl derivative 4a was formed as
the only product. An increase in the alkyl chain length of the esters
rendered quaternisation more difficult, possibly due to steric hin-
drance. The bulky ethyl groups rendered quaternisation unfavour-
able with phenylalanine esters of chain length >3. Reaction of 4a
with excess bromoethane did not give the desired triethyl deriva-
tive. When compound 4a was reacted with excess MeI in a sealed
tube, the quaternary ammonium compound 4c was formed as an
ence of NaHCO3 yielded the N,N,N-trimethyl phenylalanine methyl
ester 1c derivative in 88% yield. However, when the same reaction
was repeated using ethyl iodide, only the dialkylated product 1a
was formed rather than the N,N,N-triethyl derivative. Heating the
reaction to 90 °C in a sealed tube for 18 h yielded only the dialky-
lated derivative 1a. When EtI/NaOH was employed, the amino
group was successfully quaternised but the ester moiety hydroly-
sed to the carboxylate salt 1d. Triethylamine (TEA), a milder base
was then employed. However, as it was envisaged that the forma-
tion of the quaternised base rendered the isolation of the desired
quaternary ammonium compound difficult [30], the phenylalanine
O
O
O
O
N
I-
N
1c
1a
(i)
(ii), (iii)
O
O
Cl-
NH3
(iv)
(vi)
(v)
O
O
O
O
O
O
Na+
No reaction
+
Br-
I-
N
N
N
1a
1b
1d
(i) NaHCO3/MeI, MeOH, RT, (ii) NaHCO3/EtI, EtOH, RT, (iii) NaHCO3/ EtBr, 90°C, sealed tube, (iv)
NaOH/EtI, 90°C, sealed tube, (v) TEA/ EtI, 90°C, sealed tube, (vi) K2CO3/EtBr, 90°C, sealed tube.
Scheme 1. Synthesis attempts of N,N,N-triethyl phenylalanine methyl ester.

N. Joondan et al. / Bioorganic Chemistry 58 (2015) 117–129
123
O
O
O
O
O
n
n
O
n
Cl-
NH3
N
Br-
+
N
1a-4a; n=0-3
1b-4b; n=0-3
n
Compound %Yield
number
Compound
Number
%Yield
0
1
2
3
1b
35
28
6
1a
2a
3a
4a
63
68
76
78
2b
3b
4b
0
Scheme 2. Quaternisation attempts of esters with varying chain length.
O
n
O
I-
N
n=9,11,13,15
Fig. 3. General structure of N,N-Diethyl,N-methyl L-Phenylalanine esters.
oil in moderate yield. Higher homologues of the L-Phenylalanine
QUAT derivatives (n = C10–C16) were prepared by alkylating the
amino group with ethyl and methyl groups. The products were
obtained in moderate yield (Fig. 3).
Fig. 4. Fully labelled thermal ellipsoid plot of 1b shown at 50% probability. Since
the geometrical parameters of molecules A and B differ only marginally, molecule A
is shown as being representative of both molecules. Water molecules have been
omitted for clarity. Hydrogen atoms have been rendered as spheres of arbitrary
radius.
6.2. X-ray analysis of compound 1b
The compound 1b crystallised in the monoclinic space group
P21 with two independent cations and the associated bromide
anions in the asymmetric unit, Z = 4. The compound crystallised
as the dihydrate (one water molecule per cation, two per asym-
metric unit) (Fig. 4).
acceptors in all cases. Although hydrogen bond length does not
necessarily correlate linearly to bond strength due to packing con-
straints in the lattice [34], these hydrogen bonds are all signifi-
cantly shorter than the sum of the van der Waals radii of the
interacting atoms. This coupled with the fact that the hydrogen
bond angles do not deviate significantly from the optimum bond
angle suggests that these are likely to be moderately strong inter-
actions. There are few significant interactions linking the cations
into an extended supramolecular structure. The most notable is a
Selected bond lengths and bond angles are summarised below
in Table 2.
The data in Table 2 illustrate the similarity of the two cations in
the asymmetric unit. The most notable difference between the two
structures is the C7–C8–C9–C14 torsion angles which measure
76.8(2)° and 70.0(2)° for molecules A and B, respectively. Although
there is a moderate difference between the C7–N1–Cmethylene bond
angles of molecules A and B, both illustrate the sp3 hybridisation of
the quaternary nitrogen atoms. A Mogul structural search [32]
shows that the bond lengths and bond angles of compound com-
pare favourably to those of related structures in the Cambridge
Structural Database [33].
The three-dimensional structure of 1b is interesting in that the
cations pack such that there are channels running through the lat-
tice (Fig. 5a). The channels are co-linear with the b-axis. In the
present structure these channels are occupied by the bromide
counter ions and the solvent molecules. These bromide ions and
water molecules are linked through extensive hydrogen bonding;
refer to Fig. 5b and Table 3. The water molecules act as the hydro-
gen bond donors and the bromide ions as the hydrogen bond
weak C–Hꢀ ꢀ ꢀ
p interaction between the methine C–H and the car-
bonyl oxygen atom of an adjacent molecule.
6.3. CMC (Critical micelle concentration)
The CMCs of the C10–C16 of the L-Phenylalanine QUATS were
determined in aqueous solution by measuring the conductivity
values of different concentrations of surfactant solutions (Table 4).
The CMC was estimated from the breakpoint in the conductivity
versus surfactant concentration plots. As expected, the CMC
decreases with increasing alkyl chain as a consequence of a higher
hydrophobic content of the molecule which involves more interac-
tions between the chains, favouring micellar formation (Fig. 6).
Considering the Kleven’s equation for homologous straight
chain ionic surfactants in aqueous medium [35], the relation

124
N. Joondan et al. / Bioorganic Chemistry 58 (2015) 117–129
Fig. 6. CMC versus chain length of L-Phenylalanine QUATS.
Table 5
MIC values for compounds 5b, 6b, 7b and 8b and CTAB.
Microorganisms
MIC (
l
M)
6b 7b
6.6 3.6 8.5 8.2
5b
8b
CTAB
Gram-positive S. aureus (ATCC 29213)
S. aureus (ATCC 25923)
94
30
20
131
7.5 3.6 12
5.7 4.5 12
9
8.2
2.7
27
S. epidermidis (ATCC 12228)
B. cereus (ATCC 10876)
Fig. 5. (a) Partial space-filling model illustrating the channels in the lattice of 1b,
viewed down the b-axis. The cations have been rendered as space filling models, the
bromide ions and water molecules as ball and stick models; (b) Hydrogen bonding
(illustrated as broken purple tubes) between the bromide ions and water molecules
within the channels.
5.4 17
B. cereus (ATCC 11778)
150 15 5.4 17
27
Gram-negative K. pneumoniae (ATCC 13883) 139 19 89
170 27
S. typhimurium (ATCC 14028) 198 94 178 425 13700
E. coli (ATCC 22922)
99
19 178 340 2200
P. aeruginosa (ATCC 27853)
397 94 268 425 1370
Table 3
Summary of hydrogen bond lengths (Å) and bond angles (°) of 1b.
Hydrogen Bond
D–H
Hꢀ ꢀ ꢀA
Dꢀ ꢀ ꢀA
D–Hꢀ ꢀ ꢀA
6.4. Antibacterial activity
The antibacterial activity of the synthesised
QUATS was determined by their minimum inhibitory concentra-
tion (MIC) values. The results are summarised in Table 5.
O1s–H1sꢀ ꢀ ꢀBr2
O1s–H2sꢀ ꢀ ꢀBr2
O2s–H3sꢀ ꢀ ꢀBr1
O2s–H4sꢀ ꢀ ꢀBr1
0.75(2)
0.79(3)
0.88(4)
0.78(4)
2.67(2)
2.60(3)
2.49(4)
2.63(4)
3.41(2)
3.38(2)
3.31(2)
3.40(2)
168(2)
171(3)
156(3)
170(3)
L
-Phenylalanine
The antibacterial effects were clearly strain-dependant. The
compounds (5b-8b) showed better activity against Gram-positive
than Gram-negative strains. An increase in activity against Gram-
positive bacteria was observed with increasing chain length which
then tails off, exhibiting a cut-off effect at C14. Lukac et al. reported
the antibacterial activity of a series of quaternary ammonium
compounds derived from phenylalanine, namely N-alkyl-N,N-
dimethyl-(1-hydroxy-3-phenylpropyl)-2-ammonium bromides
[19]. The phenylalanine QUATS (5b–8b) displayed relatively better
antibacterial activity against S. aureus compared to the phenylala-
nine derivatives reported by Lukac et al. However, in the case of E.
coli, the phenylalanine QUATS were found to be less active. The
phenylalanine QUATS displayed a cut-off point at C14 and C12 with
respect to Gram-positive and Gram-negative bacteria respectively,
whereas the phenylalanine quaternary derivatives reported by
Lukac et al. showed a cut-off effect at C14 for both Gram-negative
and Gram-positive strains.
Table 4
CMC of the homologous series of L-Phenylalanine QUATS.
Compound
Chain length
CMC value (lM)
5b
6b
7b
8b
10
12
14
16
455
74.1
5.66
1.96
between log CMC (lM) and the number of carbon atoms n in the
hydrophobic chain of the homologous series of
QUATS at 25 °C, is given in Eq. (5)
L-Phenylalanine
log CMC ¼ 6:7325 ꢁ 0:4107n
ð5Þ
The increase in antibacterial activity with respect to chain
length might be attributed to an increase in hydrophobic interac-
tions between the surfactant chain and those of the neighbouring
lipid molecule of the bacterial membrane, which in turn pulls the
surfactant molecule further into the membrane. These factors
including the interaction of the quaternary ammonium moiety of
the surfactant and the polar head of lipid tend to destabilise the
membrane causing lysis. However, a further increase in chain
length (>C14) causes a decrease in antibacterial activity, a phenom-
enon referred to as the cut-off effect. Janoff and Pringle associated
this effect with a decrease in solubility of the surfactant with chain
length [39,40]. Lobecce et al. and Sarapuk et al. stated the possibil-
ity of a sewing of the lipid bilayer caused by the incorporation of
The CMC of the QUATS were found to be much lower compared
to those previously reported for the hydrochloride salts of -Phen-
L
ylalanine ester series. This is in accordance with previous studies
whereby it was observed that surfactants with a more hydrophobic
head group present lower CMC values [36]. Ionic surfactants have
higher CMC values compared to non-ionic ones due to repulsion
between their ionic groups. However, the head group repulsion is
counteracted when there is a hydrophobic attraction between
the head groups [37,38]. In this case the additional alkyl groups
attached to the nitrogen moiety of phenylalanine screens the elec-
trostatic head-group repulsion and strongly facilitates the surfac-
tants self assembly, giving rise to a much lower CMC value
compared to the hydrochloride salts of phenylalanine esters.

N. Joondan et al. / Bioorganic Chemistry 58 (2015) 117–129
125
The MIC of the quaternary ammonium surfactants were corre-
lated with their CMCs in order to relate their activities with their
micellar forming abilities (Figs. 7a and 7b).
Comparing the CMC and the MIC, it was found that these 2
parameters intersect each other at the cutoff point, as previously
observed in the case of L-Phenylalanine ester hydrochloride series.
In the case of Gram-positive bacteria, the MIC of the C10–C14 was
found to be below the CMC, while that of the C16 derivatives was
above the CMC. The CMC line also intersects the MIC at the cut-
off point in the case of Gram-negative bacteria. This could be
explained by the fact that the antibacterial activities of these sur-
factants are primarily due to their presence as monomers and
not to that of their micellar form, which was consistent to what
we have reported previously [20].
6.5. Phospholipid binding assay
Fig. 7a. Correlation between CMC and MIC with respect to Gram-positive bacteria.
The phospholipid molecules present in bacterial membranes
seem to be the target for quaternary ammonium surfactants. Phos-
phatidyl choline (PC) is the major membrane-forming phospho-
lipid in cells. Although many prokaryotes lack PC, it can be found
in significant amounts in membranes of rather diverse bacteria
[44]. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) is con-
sidered to be a simple and well characterized model which has sev-
eral advantages over more complex model systems [45]. DPPC has
been used as model membrane to provide valuable information for
understanding the mechanism of action of surfactant molecules as
antibacterial agents [15,45,46]. Therefore, to provide more insight
on the mechanism of the synthesised L-Phenylalanine QUATS on
antibacterial activity, the binding studies of phospholipid (DPPC)
with the C12, C14 and C16 QUAT derivatives were carried out using
the fluorescence probe technique. These QUATS (6b–8b) were cho-
sen in order to relate their binding ability with phospholipid and
their antibacterial activity. 1-Anilino-8-naphthalenesulfonate
(ANS) and 1,6-diphenyl-1,3-5-hexatriene (DPH) were used as fluo-
rescent probes.
ANS shows enhanced fluorescence intensity at 480 nm (excited
at 380 nm) when bound to DPPC. ANS bind to phospholipid mole-
cules in such a way that the anionic sulfonate group is orientated
towards the hydrophilic head group of the lipid and the addition
of cationic species capable to bind with the phospholipid molecule
was found to bring about more fluorescence enhancement of the
lipid-bound ANS [20].
The fluorescence of the DPPC bound ANS was further increased
by the addition of the synthesized QUATS, suggesting the forma-
tion of QUATS–lipid–ANS complex in the ternary system.
From previous studies, the binding capacity of DPPC for ANS, n
Fig. 7b. Correlation between CMC and MIC with respect to Gram-negative bacteria.
the surfactant molecule, stabilising it and making it more resistant
to lysis [41,42].
The presence of the quaternary ammonium moiety was found
to enhance the antibacterial activity of the L-Phenylalanine QUATS
as compared to the hydrochloride salt due to the additional hydro-
phobic interactions between the alkyl groups attached to the nitro-
gen atom with the lipid of the bacterial membrane, and hence
aiding to disruption. The cut-off effect was observed at a higher
and the binding constant,
K were found to be 0.0016 and
2 ꢂ 106 Mꢁ1 respectively. The value of Ab was found to be
5 ꢂ 108. Addition of the QUATS (6b–8b) to the ANS–DPPC system
enhanced the fluorescence intensity of DPPC bound ANS in the
order of 7b > 6b > 8b (Fig. 8).
chain length (C14) for the
the hydrochloride salt of
L
-Phenylalanine QUATS compared to
L
-Phenylalanine esters (C12) which we
The binding constants (K) and the binding capacity (n) in the
presence of the QUATS were obtained from Eq. (2). A plot that illus-
trates the binding of ANS to DPPC in the presence of 7b is shown in
Fig. 9.
have reported earlier [20]. The increase in hydrophobic interaction
caused by the alkyl groups attached to the nitrogen might be
destabilising the membrane much more than the NH+3, hence lead-
ing to a greater free volume in the membrane. The free volume can
therefore accommodate a longer chain length (C14).
The compounds exhibited a strain-dependant action in which
the Gram-negative species were more resistant than the Gram-
positive bacteria. This was also observed with many other surfac-
tants and is attributed to the presence of the cell wall lipopolysac-
charide (LPS) and the outer membrane, two structures both absent
in Gram-positive bacteria which could prevent the active com-
pounds to reach the cytoplasmic membrane of the Gram-negative
species [43].
Table 6 summarises the binding constant and binding capacity
of the ANS–DPPC complex in the presence of the QUATS.
In general, the binding constant in the presence of the QUATS
were greater than that of the binary ANS–DPPC system, suggesting
that these surfactant–lipid binding induces a secondary binding
between the probe and the lipid. The binding constant, K is higher
than that previously observed with the phenylalanine ester hydro-
chloride derivatives showing that the QUATS have higher ionic
attraction for the phospholipid molecule. The C14 derivative 7b
showed the highest binding constant and the highest binding

126
N. Joondan et al. / Bioorganic Chemistry 58 (2015) 117–129
220.6
200
D
150
100
C
B
A
50
Fig. 10. Binding of 7b to DPPC measured by using DPH as probe (Eq. (4)).
8.5
400.0
450
500
nm
550
593.5
through hydrophobic interactions are able to quench the fluores-
cence of the lipid–DPH solution. From previous studies on the
binding of DPH to DPPC, the value of the binding constant K, and
Wavelength
Fig. 8. Fluorescence intensity spectra solutions containing 5 ꢂ 10ꢁ8 M ANS and
1 ꢂ 10ꢁ3 M DPPC; A: no QUATS, B: 5 ꢂ 10ꢁ4 M of 8b, C: 5 ꢂ 10ꢁ4 M of 6b, D:
5 ꢂ 10ꢁ4 M of 7b.
the binding capacity
n from Eq. (2) were calculated to be
3 ꢂ 106 Mꢁ1 and 0.0323 respectively, which indicates a strong
hydrophobic interaction between the probe and the lipid [20].
The L-Phenylalanine QUATS 6b–8b were able to quench the
fluorescence of DPPC–DPH solution indicating a binding displace-
ment of DPH by these surfactants which binds in close proximity
to the DPH binding sites. The binding constant K1 and binding
capacity n1 were obtained from Eq. (3). A plot that illustrates the
competitive binding between 7b and DPH to DPPC (Eq. (4)) is
shown in Fig. 10.
The binding constant and binding capacity is much lower in the
presence of the surfactants 6b–8b compared to the binary DPH–
DPPC (Table 7).
The surfactants were able to quench the fluorescence of the bin-
ary DPH–DPPC showing that these surfactants interact with the
phospholipid via hydrophobic interactions in the order
7b > 6b > 8b (Fig. 11).
Fig. 9. Scatchard plot (Eq. (1)) for the binding of ANS to DPPC (2 ꢂ 10ꢁ5 M) at 25 °C
The C14 derivative 7b showed the lowest binding constant and
binding capacity showing that this particular QUAT derivative
was able to interact more with the lipid, and therefore was able
to quench the fluorescence of DPH bound lipid much more than
the other QUATS. The binding of DPH to DPPC was also determined
in the presence of CTAB and streptomycin. The results showed that
K1 value decreases in the presence of CTAB, indicating that this sur-
factant was able to bind with DPPC by competing with DPH bind-
ing site, in the same manner to that of the phenylalanine QUATS.
However, the phenylalanine QUATS were able to compete with
DPPC much more than CTAB, due to a higher clogP value (clogP
CTAB: 2.68; clogP QUATS > 4). This implies that the phenyl groups
in these amino acid surfactants render the QUATS more lipophilic
and contribute to a certain extent to the hydrophobic interactions
between the surfactant and the lipid. However, streptomycin did
not show any significant binding displacement of the DPH-DPPC
complex, showing that this antibiotic does not bind to DPPC via
in the presence of 7b;
m is the number of moles of bound ANS per mol of lipid; [P] is
the free concentration of ANS.
Table 6
Effect of the different L-Phenylalanine QUATS on the binding of binary ANS-DPPC
complex.
Compound
Chain length
At/Ab
K (Mꢁ1
)
n
6b
7b
8b
CTAB
12
14
16
16
–
4
4
4
4
4
–
8.0 ꢂ 107
1.0 ꢂ 108
3.0 ꢂ 107
6.0 ꢂ 107
9.0 ꢂ 106
2.0 ꢂ 106
0.001
0.004
0.002
0.001
0.002
0.002
Streptomycin
Controla
–
a
Binary system consisting of ANS and DPPC in the absence of the QUATS.
capacity. The binding of ANS to DPPC were also determined in the
presence of a cationic surfactant CTAB, and a clinical antibiotic
streptomycin, with no surfactant-like structure. CTAB was found
to enhance the binding between ANS and DPPC showing that CTAB
also binds to the phospholipid via electrostatic interaction. How-
ever, no significant binding enhancement between ANS to DPPC
was observed in the presence of streptomycin, showing that this
antibiotic has a low tendency to bind to negatively charged
phospholipids.
DPH was used as probe to determine the hydrophobic interac-
tion between the QUATS and the phospholipid. DPPC bound DPH
shows enhanced fluorescence intensity at 440 nm (excited at
368 nm) and compounds capable to bind with the phospholipid
Table 7
Effect of the different L-Phenylalanine QUATS on the binding of binary DPH-DPPC
complex.
Compound
Chain length
K1 (Mꢁ1
)
n1
6b
7b
8b
CTAB
12
14
16
16
16
–
3.6 ꢂ 104
6.5 ꢂ 103
4.2 ꢂ 105
1.5 ꢂ 105
3.0 ꢂ 106
3.0 ꢂ 106
0.023
0.010
0.013
0.014
0.015
0.032
Streptomycin
acontrol
a
Binary system consisting of DPH and DPPC in the absence of the QUATS.

N. Joondan et al. / Bioorganic Chemistry 58 (2015) 117–129
127
Table 8
750.8
700
A
B
Hemolytic activity of the different L-Phenylalanine QUATS.
Compound
HC50 (lM)
600
500
400
5b
6b
7b
8b
120
75
35
17
5.5
CTAB
300
200
100
3.8
C
Because the human red blood cell lacks internal organelles, the
only way for the surfactant molecule to interact with it is via the
cell membrane [50]. However, the lysis process is still unclear
despite the numerous advances made in understanding the mech-
anism. The most accepted hypothesis is that surfactants are inter-
calated into the lipid bilayer of the cell membrane of the red blood
cells, such that the hydrophobic chain lies within the hydrophobic
core region of the bilayer while the hydrophilic head is located at
the polar region of the membrane. Surfactant intercalation into
membrane leads to changes in the membrane permeability that
concludes with cell lysis [51]. Hemolysis also depends on the
adsorption of the surfactant to components of the membrane sur-
face which is influenced by electrostatic interaction [17].
D
400.0
450
500
nm
Wavelength
550
593.5
Fig. 11. Fluorescence spectra of solutions containing 5 ꢂ 10ꢁ8 M DPH and 2 ꢂ 10ꢁ5
M DPPC in the presence and absence of QUATS. A: no QUATS, B: 5 ꢂ 10ꢁ5 M of 8b, C:
5 ꢂ 10ꢁ5 M of 6b and D: 5 ꢂ 10ꢁ5 M of 7b.
749.2
A
700
For these L-Phenylalanine QUATS, the HC50 was found to be
600
B
above the CMC, except for 5b, showing that hemolysis is induced
above the micelle concentration rather than at monomer concen-
tration. Different studies were performed to demonstrate a positive
correlation between HC50 and CMC of surfactant; however, no clear
conclusions were reported in the literature [52].
The selectivity of the QUATS for bacterial cell over mammalian
cell was calculated by the ratio of their HC50 to their MIC values
with respect to all the bacterial strains tested. The results are sum-
marised in Table 9.
500
400
300
200
C
100
In general, the results showed a good selectivity of the com-
pounds for bacterial cells. A relatively better selectivity was
observed for Gram-positive bacteria. In this case, the MIC values
3.8
400.0
450
500
nm
550
593.5
of the L-Phenylalanine QUATS were much lower compared to their
Wavelength
HC50 values. The antibacterial activities of these QUATS were found
to increase at monomer concentration whereas the hemolytic
activity occurs mostly at micellar concentration. In this way, these
surfactants can act as antibacterials, yet non toxic compounds at a
concentration lower than their respective CMCs, hence providing a
good selectivity for bacterial cells.
Fig. 12. Fluorescence spectra of solutions containing 5 ꢂ 10ꢁ8 M DPH and 2 ꢂ 10ꢁ5
M DPPC in the presence and absence of QUATS. A: no QUATS, B: 5 ꢂ 10ꢁ5 M of C14
L
-Phenylalanine ester hydrochloride, C: 5 ꢂ 10ꢁ5 M of C14
L-Phenylalanine QUATS.
hydrophobic interactions. It was observed that the
QUATS were able to quench the fluorescence of DPPC-DPH solution
much more than the -Phenylalanine ester hydrochloride (Fig. 12)
L-Phenylalanine
L
6.7. Comparing the CMC, antibacterial and hemolytic activity of the
indicating a greater hydrophobic interactions between the QUATS
and the phospholipids.
L-Phenylalanine QUATS in the same medium
Maisuria et al. studied the antibacterial activity of di- and tri-
carboxyl dendritic amphiphiles in brain–heart infusion broth using
the broth dilution method [53]. To assess the potential safety of
6.6. Hemolytic activity
Hemolysis by surfactant molecules is a process of great funda-
mental and practical importance [47]. Hemolytic activity of surfac-
tants can also be used as a mean for their toxicological assessment
[48]. Evaluation of the concentration that induces the hemolysis of
Table 9
Selectivities of L-Phenylalanine QUATS for bacterial cells over mammalian cells.
Microorganisms
Gram-positives
Selectivity HC50/MIC
50% of the erythrocytes was determined. The L-Phenylalanine
5b
6b
7b
8b
QUATS series showed moderate hemolytic activity with an
increase in activity with increasing chain length of the hydropho-
bic tail (Table 8).
This behaviour is in line with those described for typical cat-
ionic surfactant [49]. The hemolytic activity is lower than those
found for CTAB. Comparing the CMC with the hemolytic activity,
the HC50 was found to be higher than the respective CMC value
of the QUATS, except for 5b, whereby the HC50 value was much
below its CMC value.
S. aureus (ATCC 29213)
S. aureus (ATCC 25923)
S. epidermidis (ATCC 12228)
B. cereus (ATCC 10876)
B. cereus (ATCC 11778)
1.3
4
4
0.9
0.8
11
10
13
8
10
10
8
6.7
6.7
2
1.4
1.4
1
5
1
Gram-negatives
K. pneumoniae (ATCC 13883)
S. typhimurium (ATCC 14028)
E. coli (ATCC 22922)
0.9
0.3
0.6
1.2
4
0.4
0.1
0.2
0.2
0.1
0.8
0.8
4
0.04
0.04
0.05
P. aeruginosa (ATCC 27853)

128
N. Joondan et al. / Bioorganic Chemistry 58 (2015) 117–129
Table 10
micellar forms, while the monomers were responsible for their
antibacterial effects (Fig. 13).
CMC, antibacterial and hemolytic activity of phenylalanine QUATS in TBS.
Compound CMC
M)
MIC (
l
M)
HC50
M)
(
l
(
l
7. Conclusion
S. aureus (ATCC
S. epidermidis (ATCC
25923)
12228)
The synthesised L-Phenylalanine QUATS were found to display
5b
6b
7b
8b
307
28.3
3.7
1.0
720
99
19
3.6
17
28
20
15
3.6
170
28
152
92
71
66
9.2
good antibacterial activities, which were found to increase up to
a certain chain length, above and below which activity decreases,
exhibiting a cut-off effect. For Gram-positive bacteria, the cut-off
point was observed at C14 while in the case of Gram-negative bac-
teria, the cut-off point was observed at a lower chain length (C12).
This difference between the two types of bacterial strains may be
accounted by the difference in their cell wall structures. The
CTAB
using these dendritic amphiphiles as drugs, measurements of
micellar and hemolytic properties were conducted in the same
medium (full-strength BHIB) that was used for antibacterial activ-
ity. In this study, the micellar, hemolytic and antibacterial data
reported were intercorrelated to provide a relationship between
micellization and biological properties. In view of providing a fair
assessment of the relationship between CMC, MIC and HC50, a com-
mon medium was chosen to measure all three parameters. As
hemolysis could not be measured in distilled water due to lysis
of red blood cells, measurements of all the three properties were
made in TBS solution. TBS was chosen in order to emulate physio-
logical conditions with a slightly alkaline pH (7.4) and an isotonic
NaCl concentration (150 mM or 0.9% NaCl). The disc diffusion
method was used for antibacterial evaluation of the phenylalanine
QUATS where solutions of varying concentration of the compound
dissolved in TBS. Table 10 summarises the CMC, MIC (with respect
to two selected Gram-positive bacteria) and hemolytic activity of
the phenylalanine QUATS.
L
-Phenylalanine QUATS displayed relatively better antibacterial
property with a higher cut-off point compared to the ester hydro-
chlorides. The study showed the monomers of the QUAT deriva-
tives were responsible for their antibacterial activities while their
hemolytic activities were primarily governed by their micellar
form. The use of TBS as common medium showed similar interre-
lationship between micellisation, antibacterial and hemolytic
activities. The binding studies carried out with phospholipid dem-
onstrate that the QUATS bind with DPPC via both electrostatic and
hydrophobic interactions. The study shows that these interactions
have a direct effect on the antibacterial activities of the esters.
Greater hydrophobic interactions were found between the QUATS
and the phospholipid compared to the hydrochloride salt of phen-
ylalanine, showing that the presence of the quaternary ammonium
moiety accounts for further interactions between the surfactant
and the lipid, giving rise to a better antibacterial activity. This
study has given insights regarding the mode of action of the QUATS
as antibacterial agents, which includes their interaction with
phospholipid molecules in the bacterial cell membrane, giving rise
to cell lysis.
The change in the ionic strength of TBS slightly changed the val-
ues of the different properties measured. The CMC of the phenylal-
anine QUATS in TBS were found to be lower than those measured
in deionised water which is due to the effect of the dissolved NaCl
that facilitates micellar formation. Similar effect was observed for
CTAB (CMC in water: 1050 lM). The HC50 values of the QUATS in
Appendix A. Supplementary material
TBS were larger than those observed in saline solution, indicating
that these compounds have a lower tendency to lyse red blood
cells under these conditions. The MIC values of the QUATS dis-
solved in TBS with respect to S. aureus (ATCC 25923) and S. epide-
rmidis (ATCC 12228) were found to be generally higher than those
observed for solutions of the QUATS dissolved in water. This might
be attributed to a lower solubility of these compounds in TBS than
in water. However, the MIC values of 7b in both water and TBS
were comparable. The relationship between the CMC, MIC and
HC50 in TBS were comparable to the results obtained when these
parameters were determined in different media, which showed
that in most cases, hemolytic activities were governed by their
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2015.01.
001.
References
[1] X. Domingo, in: E.G. Lomax (Ed.), Amphoteric Surfactants, Marcel Dekker Inc.,
New York, 1996.
[2] B. Bogumil, Z. Dega-Szafran, I. Mirska, Pol. J. Microbiol. 59 (1) (2010) 49–53.
_
[3] E. Woz´niak, A. Morzymas, A. Czarny, B. Kocie˛ba, B. Rózycka-Roszak, Z. Dega-
Szafran, E. Dulewicz, M. Petryna, Z. Naturforsch. 59c (2004) 782–786.
[4] A.N. Petrocci, in: S.S. Block (Ed.), Disinfection, Sterilization and Preservation,
Lea & Febiger, Philadelphia, 1983.
[5] J.N. Mbithi, V.S. Springthorpe, S.A. Sattar, Appl. Environ. Microbiol. 56 (1990)
3601.
[6] T. Thorsteinsson, M. Masson, K.G. Kristinsson, M.A. Hjalmarsdottir, H.
Hilmarsson, T. Loftsson, J. Med. Chem. 46 (2003) 4173–4181.
[7] M. Lindstedt, S. Allenmark, R.A. Thompson, L. Edebo, Antimicrob. Agents
Chemother. 34 (1990) 1949–1954.
[8] C.R. Birnie, D. Malamud, R.L. Schnaare, Antimicrob. Agents Chemother. 44
(2000) 2514–2517.
[9] A. Laatiris, M. El Achouri, M.R. Infante, Y. Bensouda, Microbiol. Res. 163 (2008)
645–650.
[10] P. Gilbert, L.E. Moore, J. Appl. Microbiol. 99 (2005) 703–715.
[11] S. Roy, P.K. Das, Biotechnol. Bioeng. 100 (2008) 756–764.
[12] J. Pernak, M. Branicka, J. Surfact. Deterg. 6 (2003) 119–123.
[13] R.N. Mitra, A. Shorne, P. Paul, P.K. Das, Org. Biomol. Chem. 7 (2009) 531–536.
[14] J. Haldar, P. Kondaiah, S. Bhattacharya, J. Med. Chem. 48 (2005) 3823–3831.
[15] N. Lozano, L. Perez, R. Pons, J.R. Luque-Ortega, M. Fernandez-Reyes, L. Rivas, A.
Pinazo, Colloid Surf. A: Physicochem. Eng. Aspect 319 (2008) 196–203.
[16] L. Perez, M.R. Infante, R. Pons, M.C. Moran, M.P. Vinardell, M. Mitjans, A.
Pinazo, Colloids Surf. B 35 (2004) 235.
[17] L. Perez, A. Pinazo, M. Tereza Garcia, M. Lozano, A. Manresa, M. Angelet, M.P.
Vinardell, M. Mitjans, R. Pons, M.R. Infante, Eur. J. Med. Chem. 44 (2009) 1884–
1892.
Fig. 13. Comparison of CMC, antibacterial and hemolytic activities in TBS.
[18] H. Tan, H. Xiao, Tetrahedron Lett. 49 (2008) 1759–1761.

N. Joondan et al. / Bioorganic Chemistry 58 (2015) 117–129
129
[19] M. Lukac, I. Lacko, M. Bukovsky, Z. Kyselova, J. Karlovsta, B. Horvath, F.
Devinsky, Cent. Eur. J. Chem. 8 (2010) 194–201.
[37] L. Goracci, R. Germani, G. Savelli, D.M. Bassani, ChemBioChem 6 (2005) 197–
203.
[20] N. Joondan, S. Jhaumeer-Laulloo, P. Caumul, Microbiol. Res. 169 (2014) 675–
685.
[38] V. Jadhav, S. Maiti, A. Dasgupta, P.K. Das, R.S. Dias, M.G. Miguel, B. Lindman,
Biomacromolecules 9 (2008) 1852–1859.
[21] Bruker APEX2, SAINT and SADABS, Bruker AXS Inc., Madison, Wisconsin., USA,
2012.
[22] G.M. Sheldrick, Acta Cryst. A64 (2008) 112–122.
[23] L.J. Farrugia, J. Appl. Cryst. 45 (2012) 849–854.
[24] R. Vijay, S. Angayarkann, G. Baskar, Colloid Surf. A: Physicochem. Eng. Aspect
317 (2008) 643–649.
[39] A. Janoff, M. Pringle, K. Miller, Biochim. Biophys. Acta 649 (1981) 125–128.
[40] M. Pringle, K. Brown, K. Miller, Mol. Pharmacol. 19 (1981) 49–55.
[41] L. Lobbecke, G. Cevc, Biochim. Biophys. Acta 1237 (1995) 59–69.
[42] J. Sarapuk, K. Kubica, Cell. Mol. Biol. Lett. 3 (1998) 261–269.
[43] N. Campbell, Biology, third ed., Benjamin Cummings Publishing, Redwood
City., Calif, 1993.
[25] K. Bazco, C. Larpent, P. Lesot, Tetrahedron: Asymmet. 15 (2004) 971–982.
[26] A.W. Bauer, W.M.M. Kirby, J.C. Sherris, M. Turck, Am. J. Clin. Pathol. 45 (1966)
493–496.
[27] J. Goerdeler, Mouben-Weyl, E. Muller. Georg Thieme Verlag, Stuttgart, BRD XI/
12 (1958) 587–640.
[28] C. Francis, M. Chen, N. Leo Benoiton, Can. J. Chem. 54 (1976) 3310.
[29] C.A. Hurley, J.B. Wong, H.C. Hailes, A.B. Taber, J. Org. Chem. 69 (2004) 980–983.
[30] H.Z. Sommer, L.L Jackson, Edgewood Arsenal Technical Report. EATR 4311, vol.
7, 1969.
[44] F. Martinez-Morales, M. Schobert, I.M. Lopez-Lara, O. Geiser, Microbiology 149
(2003) 3461–3471.
[45] J.A.S. Almeida, M.C. Moran, M.R. Infante, A.A.C.C. Pais, ARKIVOC v (2010) 34–
50.
[46] J.A. Castillo, A. Pinazo, J. Carilla, M.R. Infante, M.A. Alsina, I. Haro, P. Clapes,
Langmuir 20 (2009) 3379–3387.
[47] P. Maher, S.J. Singer, Biochemistry 23 (1984) 232–240.
[48] T. Eren, A. Som, J.R. Rennie, C.F. Nelson, Y. Urgina, K. Nusslein, E.B. Coughlin,
G.N. Tew, Chem. Phys. 209 (2008) 516–524.
[31] P. Karrer, Organic Chemistry, fourth ed., Elsevier, Amsterdam, The
Netherlands, 1950.
[32] I.J. Bruno, J.C. Cole, M. Kessler, J. Luo, W.D.S. Motherwell, L.H. Purkiss, B.R.
Smith, R. Taylor, R.I. Cooper, S.E. Harris, A.G. Orpen, J. Chem. Inf. Comput. Sci.
44 (2004) 2133–2144.
[49] B. Isomaa, Biochem. Pharmacol. 28 (1979) 975–980.
[50] S. Svetina, D. Kuzman, R.E. Waugh, P. Ziherl, B. Zeks, Bioelectrochemistry 62
(2004) 107–113.
[51] S. Shalel, S. Streichman, A. Marmur, J. Colloid Interf. Sci. A 252 (2002) 66–76.
[52] L. Sanchez, V. Martinez, M.R. Infante, M. Mitjans, M.P. Vinardell, Toxicol. Lett.
169 (2007) 177–184.
[33] F.H. Allen, Acta Cryst. B58 (2002) 380–388.
[34] T. Steiner, Chem. Commun. 8 (1997) 727–734.
[35] H.B. Klevens, J. Am. Oil Chem. Soc. 30 (1953) 74–80.
[36] M. Rosen, Surfactants and Interfacial Phenomena, third ed., John Wiley & Sons.,
Inc., Hobaken, 2004.
[53] B.B. Maisuria, M.L. Actis, S.N. Hardrict, J.O. Falkinham III, M.F. Cole, R.L. Cihlar,
S.M. Peters, R.V. Macri, E.W. Sugandhi, A.A. Williams, M.A. Poppe, A.R. Esker,
R.D. Gandour, Bioorg. Med. Chem. 19 (2011) 2918–2926.