Chemo-enzymatic synthesis and antimicrobial evaluation of alkyloxy propanol amine-based cationic ether lipids

Article abstract of DOI:10.1007/s11746-012-2177-9

The present study involved the synthesis and antimicrobial evaluation of alkyloxy propanol amine-based cationic lipids N,N-dimethyl-1-octadecylamino-3- alkyloxy-2-propanol (series A, 7a-e) and N-methyl-N,N-di-(2-hydroxy-3-alkyloxy-2 propyl) octadecylamine (series B, 9a-e) and their acetylated derivatives (8a-e and 10a-e). A simple three-step chemo-enzymatic approach was employed for the synthesis of 7a-e and 9a-e in 71-80 and 67-88 % yields, respectively. The first step involved the synthesis of a series of glycidyl ethers from a series of alcohols (C₄, C₈, C₁₀, C₁₂, C ₁₄) which were opened in the second step with octadecylamine to obtain 1-octadecylamino-3-alkyloxy-2 propanol (5a-e) and N,N-di-(2-hydroxy-3- alkyloxypropyl) octadecylamine (6a-e). In the third step, alkyloxy propanolamines (5a-e, 6a-e) were quaternized using methyl iodide to yield quaternized ammonium salts. The quaternized ammonium salts were enzymatically acetylated using Candida antarctica lipase-B based immobilized enzyme Novozym 435 to obtain their acetylated derivatives. The quaternized salts as well as their acetylated derivatives were evaluated for antibacterial and antifungal properties. The title compounds were found to possess moderate to good antibacterial activities against all the studied bacterial strains namely, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella aerogenes compared to streptomycin and cetyl trimethyl ammonium bromide (CTAB). The title compounds exhibited relatively good antifungal activities against Candida albicans and no significant activities against other fungal strains namely, Saccharomyces cerevisiae, Rhizopus oryzae and Aspergillus niger when compared to amphotericin B and CTAB.

Full text of DOI:10.1007/s11746-012-2177-9

J Am Oil Chem Soc (2013) 90:437–448
DOI 10.1007/s11746-012-2177-9
ORIGINAL PAPER
Chemo-Enzymatic Synthesis and Antimicrobial Evaluation
of Alkyloxy Propanol Amine-Based Cationic Ether Lipids
•
J. R. C. Reddy B. V. S. K. Rao M. S. L. Karuna
•
•
•
K. P. Kumar U. S. N. Murthy R. B. N. Prasad
•
Received: 26 June 2012 / Revised: 27 August 2012 / Accepted: 25 October 2012 / Published online: 23 November 2012
Ó AOCS 2012
Abstract The present study involved the synthesis and
antimicrobial evaluation of alkyloxy propanol amine-based
cationic lipids N,N-dimethyl-1-octadecylamino-3-alkyloxy-
2-propanol (series A, 7a–e) and N-methyl-N,N-di-(2-
hydroxy-3-alkyloxy-2 propyl) octadecylamine (series B,
9a–e) and their acetylated derivatives (8a–e and 10a–e). A
simple three-step chemo-enzymatic approach was employed
for the synthesis of 7a–e and 9a–e in 71–80 and 67–88 %
yields, respectively. The first step involved the synthesis of a
series of glycidyl ethers from a series of alcohols (C4, C8,
C10, C12, C14) which were opened in the second step with
octadecylamine to obtain 1-octadecylamino-3-alkyloxy-2
propanol (5a–e) and N,N-di-(2-hydroxy-3-alkyloxypropyl)
octadecylamine (6a–e). In the third step, alkyloxy propa-
nolamines (5a–e, 6a–e) were quaternized using methyl
iodide to yield quaternized ammonium salts. The quatern-
ized ammonium salts were enzymatically acetylated using
Candida antarctica lipase-B based immobilized enzyme
Novozym 435 to obtain their acetylated derivatives. The
quaternized salts as well as their acetylated derivatives were
evaluated for antibacterial and antifungal properties. The
title compounds were found to possess moderate to good
antibacterial activities against all the studied bacterial
strains namely, Bacillus subtilis, Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa and Klebsiella
aerogenes compared to streptomycin and cetyl trimethyl
ammonium bromide (CTAB). The title compounds exhib-
ited relatively good antifungal activities against Candida
albicans and no significant activities against other fungal
strains namely, Saccharomyces cerevisiae, Rhizopus oryzae
and Aspergillus niger when compared to amphotericin B and
CTAB.
Keywords Antimicrobial activity Á Cationic-ether
lipids Á Chemo-enzymatic synthesis Á Quaternized salts Á
Alkyloxy-propanolamine
Introduction
Glycidyl derivatives namely acyloxy or alkyloxy propanol
amines have been widely investigated for a variety of
applications [1–8]. The presence of several key functional
groups on the glycerol backbone makes them attractive
chemical intermediates for several applications. Lipids with
ether bonds to long chain alkyl moieties in addition to ester
bonds to fatty acids are in general called as ether lipids and
are common in nature especially as membrane constituents.
In general, the ether bond is located at the sn-1 position of a
glycerol moiety, which may be part of a non-polar lipid or a
phospholipid in animal tissues or in micro-organisms.
Researchers are showing greater interest in these compounds
as ether lipids were found in cancer tissues, followed by the
discovery of distinct ether lipids such as the platelet-acti-
vating factor, with several important biological activities
[7, 8]. Pure ether lipids are rarely easy to separate from the
fully acylated forms, and often their presence in tissues is
inferred from isolation of their hydrolysis products.
J. R. C. Reddy Á B. V. S. K. Rao Á M. S. L. Karuna Á
R. B. N. Prasad (&)
Centre for Lipid Research, Indian Institute of Chemical
Technology, Hyderabad 500 007, India
e-mail: rbnprasad@iict.res.in
The existence of ether bonds which are resistant to
hydrolysis and chemically stable makes it possible to
apply this class of materials under drastic environmental
K. P. Kumar Á U. S. N. Murthy
Biology Division, Indian Institute of Chemical Technology,
Hyderabad 500 007, India
123

438
J Am Oil Chem Soc (2013) 90:437–448
conditions. Furthermore, cationic lipids are attracting a lot of
attention due to many unique applications in gene therapy,
antimicrobial properties etc. [9–14]. These lipids are com-
posed of three parts namely, a cationic head group, a lipo-
philic tail group, and a linker that tethers the hydrophilic
head group and hydrophobic tail group. In general, cationic
head groups include quaternary salt lipids, lipoamines and
combinations of both. The tail groups consist of saturated or
unsaturated alkyl chains or cholesteryl groups. The nature of
head group as well as hydrophobic tail groups are important
structural variables in a cationic lipid-based antimicrobial
agent, which governs all major applications of cationic lipid.
Another unique class of ether lipids is alkanolamines
and other derivatives. A few of such synthetic ether lipid
analogues reported are terpene glyceryl ether lipids, isos-
tearyl glyceryl ethers, 1-alkylamino-3-alkyloxy-2-propa-
nols and N,N-di-(2-hydroxy-3-alkyloxypropyl) alkyl
amines [6] with different active sites, which are responsible
for various applications such as polyfunctional fuel addi-
tives [6]. In another study, a series of alkyloxy alkylamino
propanols were synthesized from epichlorohydrin, straight
or branched chain primary and secondary alcohols and
tertiary alkyl primary amines as well as straight chain
primary amine. In addition, their sedimentation inhibition
characteristics in distillate fuels of various compositions
were studied in comparison to tertiary alkyl primary
amines as well as some known commercial additives of
similar nature [15]. 1-Alkylamino-3-alkyloxy-2-propanols,
and N,N-di-(2-hydroxy-3-alkyloxy propyl) alkyl amines
were prepared with the same chain length both for alkyl-
amino and alkyloxy groups by condensation of alkyl
glycidyl ethers with long chain alkylamines by employing
thermal and microwave-assisted methods [16]. However,
the use of alkyloxy propanolamines as antimicrobial agents
has not been studied by the researchers even though they
can be potential molecules for biological applications.
Therefore, in the present study, homologous series (A and
B) of alkyloxy propanol amine-based cationic ether-lipids
and their acetylated derivatives, of varying alkyl chain
lengths (C4, C8, C10, C12, C14) in alkyloxy group were
synthesized and evaluated for their antimicrobial activities.
Chemicals (Mumbai, India). All solvents, purchased from
Sd Fine Chemicals, were of high purity grade and were
used without further purification. The immobilized lipase
from Candida antarctica (Novozym 435) was purchased
from Novozyme A/S (Bangalore, India). The bacterial test
organisms and the fungal strains were obtained from the
Institute of Microbial Technology, Chandigarh, India. The
media was procured from M/s Himedia Laboratories
(Mumbai, India).
IR spectra were recorded on a Perkin Elmer (model:
Spectrum BX) FT-IR Spectrometer using CH2Cl2 as solvent
1
or as KBr pellets. All H-NMR spectra were recorded on a
Bruker (Wissembourg, France) ARX 400 Spectrometer
(200 MHz) with CDCl3 solvent. FAB mass spectra were
recorded on a Micromass Autospec QFPD (Manchester, UK).
Methods
Synthesis of Alkyloxy Propanol Amine-Based Cationic
Ether Lipids
A Schematic diagram illustrating the synthesis of the two
types (series A and B) of alkyloxy propanol amine-based
cationic lipids and their acetylated derivatives is presented
in Scheme 1.
A Typical Procedure for the Synthesis of n-Tetradecyl
Glycidyl Ether (3e)
The synthesis of glycidyl ethers were carried by following
the method reported by Karuna et al. [16]. Tetradecyl
glycidyl ether (3e) was prepared by stirring a suspension of
epichlorohydrin (1, 40 g, 0.44 mol), n-tetradecanol (2e,
94.2 g, 0.44 mol), potassium hydroxide pellets (56 g,
1 mol) in hexane (500 mL) at reflux temperature for 6 h
(Fig. 1). The reaction was monitored by TLC using the
solvent system hexane/ethyl acetate (90:10, v/v). At the
end of the reaction time, the reaction mixture was cooled,
filtered to remove excess potassium hydroxide. The filtrate
was further washed with water till the washings were free
from alkali. The hexane layer was passed through anhy-
drous sodium sulfate to remove traces of moisture. The
solvent was evaporated under reduced pressure. The title
product was purified using silica gel column chromatog-
raphy eluted with hexane/ethyl acetate (98:2, v/v). The
compound obtained was 64.6 g, a yield of 54.4 %.
Materials and Methods
Materials and Instruments
1H NMR (200 MHz, CDCl3) d: 0.87 (3H, Ha, t, –CH3),
1.2–1.4 [22H, Hb, m, –(CH2)11], 1.4–1.6 (2H, Hc, m,
–CH2CH2), 2.5, 2.7 (2H, Hd, He, 2 m, 2 epoxy protons),
3.05–3.1 (1H, Hf, m, 1 epoxy proton), 3.24, 3.36 (3H, Hg,
Hh, 2dd, –CH–O–CH2–), 3.5–3.6 (1H, Hi, d, –CH–O–
CH2); IR (KBr, cm-1): 1112 (C–O–C–str), 3048, 1253,
912.2 (epoxy ring).
The raw materials needed for the synthesis, such as epi-
chlorohydrin, butyl, octyl, decyl, dodecyl and tetradecyl
alcohols, n-octadecylamine, potassium hydroxide, methyl
iodide, vinyl acetate were purchased from Sd Fine Chem-
icals (Mumbai, India). Silica gel (60–120 mesh) for column
chromatography was purchased from M/s Acme Synthetic
123

J Am Oil Chem Soc (2013) 90:437–448
439
O
O
KOH/Hexane
Reflux, 6h
OH
+
O
n
n
3
Cl
1
2
Ethanol/Reflux
12 h
n = 1 (a), 5 (b), 7 (c), 9 (d), 11 (e)
4
+
N
O
N
O
n
n
H
15
15
OH
OH
5
HO
2, Alcohol;
1, Epichlorohydrin;
Glycidylether;
3,
4, Octadecylamine
6, N, N-Di (2-hydroxy-3-alkyloxypropyl)octadecylamine
O
5, 1-Octadecylamino-3-alkyloxy-2-propanol;
6
Series A
I
N
O
CH3I
+
n
H
15
N
O
n
15
OH
30 C, 18 h
OH
7
5
Novozym 435
72 h
Vinylacetate
I
+
N
O
n
15
O
8
5, 1-Octadecylamino-3-alkyloxy-2-propanol; 7, N, N Di methyl-1-octadecylamino-3-alkyloxy-2-propanol;
8, N, N Dimethyl-1-octadecylamino-2-acetyloxy-3-alkyloxy propane
O
Scheme 1 Synthetic route of N,N-dimethyl-1-octadecylamino-3-alkyloxy-2-propanol (7a–e) and N-methyl-N,N-di-(2-hydroxy-3-alkyloxy-2
propyl) octadecylamine (9a–e), and their acetylated derivatives (8a–e, 10a–e)
A Typical Procedure for the Synthesis of
1-Octadecylamino-3-tetradecyloxy-2 Propanol (5e)
and N,N-Di-(2-hydroxy-3-tetradecyloxypropyl)
Octadecylamine (6e)
clearly indicated the presence of two products due to the
formation of monomer (5e) and dimer (6e). After 12 h, the
reaction mixture was concentrated in a rotary evaporator to
get the crude product. The two pure title products 5e
(17.1 g, a yield of 31.8 %) and 6e (36.9 g, a yield of
44.8 %) were purified using silica gel column chromatog-
raphy eluted with chloroform/methanol (99:1, 98:2, v/v).
Spectral data of 5e: 1H NMR (200 MHz, CDCl3) d: 0.9 (6H,
Ha, t, –(CH2)15–CH3,–(CH2)11–CH3, 1.2–1.4 [52H, Hb, m,
–(CH2)15–CH3), –(CH2)11–CH3], 1.5–1.6 (4H, Hc, m, –HN–
CH2–CH2; –O–CH2–CH2), 2.7–2.85 (4H, Hd, m, CH2–NH–
CH2), 3.3–3.45 (4H, He, m, –CH2–O–CH2–), 3.85–3.95 (1H,
The synthesis of the title compounds was carried out by
following the method reported by Karuna et al. [16]. Tet-
radecyl glycidyl ether (3e, 25.5 g, 0.1 mol) and octadec-
ylamine (4, 26.9, 0.1 mol) were taken in ethanol (100 mL)
and the contents were refluxed over a period of 12 h. The
course of the reaction was monitored by TLC using a
solvent system of chloroform/methanol (90:10 v/v). TLC
123

440
J Am Oil Chem Soc (2013) 90:437–448
Series B
+
N
N
O
n
O
15
n
15
OH
CH3I
OH
HO
30 C, 12 h
HO
O
O
9
6
Novozym 435
120 h
Vinylacetate
+
N
O
n
15
O
O
O
O
O
10
6, N, N-Di (2-hydroxy-3-alkyloxypropyl)octadecylamine;
10, N Methyl, N, N-Di (2-acetyloxy-3-alkyloxypropyl)octadecylamine
9, N methyl, N, N-Di (2-hydroxy-3-alkyloxypropyl)octadecylamine;
Scheme 1 continued
Hf, m, –CH–(OH); IR (KBr, cm-1): 3451 (–OH, NH str); 1216,
1117 (–C–O–C–str); FABMS (m/z, rel. int.): C35H73O2N,
m/z 540 ([M ? H]?, 100); m/z 282 (C19H39O?, 25.8).
column chromatography eluted with chloroform/methanol
(98:2, v/v). As the product (more polar) and unreacted
compound have a sufficient polarity difference to differ-
entiate on the TLC and separation using silica gel column
chromatography the unreacted compounds and product
were easily separated from the crude product by eluting
chloroform/methanol (98:2, 99.5:0.5, v/v) respectively.
The compound obtained was 4.6 g, a yield of 80.3 %.
1H NMR (200 MHz, CDCl3) d: 0.9 (6H, Ha, t, –(CH2)15–
CH3,–(CH2)11–CH3, 1.2–1.4 [52H, Hb, m, –(CH2)15–CH3), –
(CH2)11–CH3], 1.5–1.8 (4H, Hc, m,–HN–CH2–CH2; –O–CH2–
CH2), 3.1–3.24 (2H, Hd, t,–?N–CH2–CH2-), 3.4–3.6 (6H, He,
m, ?N–CH2–CH(OH)–CH2–O–CH2–), 3.7 (6H, Hf, s, –CH2–
N?(CH3)2–CH2–CH(OH), 4.1–4.3 (1H, Hg, m, ?N–CH2–
CH(OH)–CH2–O–CH2–), Fig. 1a; FABMS (m/z, rel. int.):
C37H78O2N, m/z 569 ([M ? H]?, 50), Fig. 1b; IR (KBr,
cm-1): 3391 (C–OH str), 1117 (C–O–C str), Fig. 1c.
1
Spectral data of 6e: H NMR (200 MHz, CDCl3) d: 0.9
(9H, Ha, t, –(CH2)15–CH3, [–(CH2)11–CH3]2, 1.2–1.4 (74 H,
Hb, m,–(CH2)15–CH3, [–(CH2)11–CH3]2, 1.5–1.65 (6H, Hc, m,
2 X –CH2–CH2–O–, –CH2–CH2–N–), 2.5–2.6 [6H, Hd, m,–
(CH2)–N(CH2)–CH2], 3.4–3.5 [8H, He, m, 2 X CH2–O–CH2–
CH–(OH)], 3.7–3.85 (2H, Hf, m, 2 X –CH2–CH–(OH)–CH2–);
FABMS (m/z, rel. int.): C52H107O4N, m/z 811 ([M ? 2], 100),
m/z 553 (C36H74O2N?, 47.5), m/z 282 (C19H39O?, 25.8); IR
(KBr, cm-1): 3408 (C–OH str), 1112 (C–O–C–str).
A Typical Procedure for the Synthesis of N,N-Di-methyl-1-
octadecylamino-3-tetradecyloxy-2-propanol (7e)
1-Octadecylamino-3-tetradecyloxy-2-propanol (5e, 5.39 g,
0.01 mol) was taken in excess methyl iodide and stirred
magnetically at 25–30 °C for 18 h. The progress of the
reaction was monitored by TLC using a solvent system of
chloroform/methanol (90:10 v/v). The reaction was quen-
ched at 18 h and then the reaction mixture was concen-
trated in a rotary evaporator to remove unreacted methyl
iodide. The title product was separated using silica gel
A Typical Procedure for the Synthesis of N-Methyl-N,N-Di-
(2-hydroxy-3-tetradecyloxy-2-propyl) Octadecylamine (9e)
N,N-di-(2-hydroxy-3-tetradecyloxypropyl) octadecylamine
(6e, 8.1 g, 0.01 mol) was taken in excess methyl iodide and
stirred magnetically at 25–30 C over a period of 12 h. The
123

J Am Oil Chem Soc (2013) 90:437–448
441
1
Fig. 1 a H-NMR spectrum, b FAB mass spectrum, c FT-IR spectrum of N,N-di-methyl-1-octadecylamino-3-tetradecyloxy-2-propanol (7e)
progress of the reaction was monitored by TLC using a
solvent system of chloroform/methanol (90:10 v/v). The
reaction was stopped at 12 h and the reaction mixture was
concentrated in a rotary evaporator to remove excess
methyl iodide. The title product, 9e was purified using
silica gel column chromatography eluted with chloroform/
123

442
J Am Oil Chem Soc (2013) 90:437–448
Fig. 1 continued
methanol (98:2, v/v) and the unreacted compound was
eluted with the pure chloroform. The compound obtained
was 7.4 g, a yield of 88.5 %.
filtered to separate enzyme and concentrated in a rotary
evaporator to remove excess vinyl acetate. The title prod-
uct and un-acetylated compound were separated using sil-
ica gel column chromatography eluted with chloroform/
methanol (98:2, 97:3, v/v) respectively. The compound
obtained was 5 g, a yield of 80.4 %.
1H NMR (200 MHz, CDCl3) d: 0.9 (6H, Ha, t,
–(CH2)15–CH3,–(CH2)11–CH3, 1.2–1.4 [52H, Hb, m,
–(CH2)15–CH3), –(CH2)11–CH3], 1.5–1.8 (4H, Hc, m,–HN–
CH2–CH2; –O–CH2–CH2), 2.0 (3H, Hd, s, –O–CO–CH3),
3.1–3.2 (2H, He, t,–?N–CH2–CH2–), 3.4–3.6 (6H, Hf, m,
?N–CH2–CH(OH)–CH2–O–CH2–), 3.7 (6H, Hg, s,–CH2–
N?(CH3)2–CH2–CH(OH), 4.9 (1H, Hh, m, ?N–CH2–
CH(OCOCH3); IR (KBr, cm-1): 1738 (C = O str), 1217,
1116 (–C–O–C–str).
1H NMR (200 MHz, CDCl3) d: 0.9 (9H, Ha, t, –(CH2)15–
CH3, [–(CH2)11–CH3]2, 1.2–1.4 (74 H, Hb, m, –(CH2)15–
CH3, [–(CH2)11–CH3]2, 1.5–1.75 (6H, Hc, m, 2 X –CH2–
CH2–O–, –CH2–CH2–N?–), 3.3–3.6 (14H, Hd, m,–?N–
CH2–CH2–, ?N–[CH2–CH(OH)–CH2–O–CH2–]2, 3.7 (3H,
He, s,–CH2–N?(CH3)–CH2–CH(OH), 4.4 (2H, Hf, m, ?N–
[CH2–CH(OH)–CH2–O–CH2–]2, Fig. 2a; FABMS (m/z, rel.
int.): C53H110O4N, m/z 825 ([M ? H]?, 91.2), m/z 296
(C21H44?, 46), Fig. 2b; IR (KBr, cm-1): 3330 (C–OH str),
1117 (C–O–C str), Fig. 2c.
A Typical Procedure for the Synthesis of N,N-Di-methyl-1-
octadecylamino-3-tetradecyloxy-2-acetyloxy Propane (8e)
A Typical Procedure for the Synthesis of N-methyl-N,N-di-
(2-acetyloxy-3-tetradecyloxy-2-propyl)octadecylamine
(10e)
N,N-di-methyl-1-octadecylamino-3-tetradecyloxy-2-propa-
nol (7e, 5.68 g, 0.01 mol) was taken in vinyl acetate (8.6 g,
0.1 mol) and to this commercial lipase Novozym 435 (20
wt% of 7e) was added. The contents were stirred magnet-
ically at 25–30 °C for 72 h. The reaction progress was
monitored by TLC using a solvent system of chloroform/
methanol (90:10 v/v). After 72 h, the reaction mixture was
N-methyl-N,N-di-(2-hydroxy-3-tetradecyloxypropyl)
n-octadecylamine (8e, 8.24 g, 0.01 mol) was taken in vinyl
acetate (8.6 g, 0.1 mol) and to this Novozym 435 (20 wt%
of 8e) was added and stirred magnetically at 25–30 °C for
123

J Am Oil Chem Soc (2013) 90:437–448
443
Fig. 2 a 1H-NMR spectrum, b FAB mass spectrum, c FT-IR spectrum of N-methyl-N,N-di-(2-hydroxy-3-tetradecyloxy-2-propyl) octadecyl-
amine (9e)
123

444
J Am Oil Chem Soc (2013) 90:437–448
Fig. 2 continued
120 h. The reaction was monitored by TLC using solvent
system of chloroform/methanol (90:10, v/v). After 120 h,
reaction mixture was filtered to separate enzyme and con-
centrated in a rotary evaporator. The title product and
un-acetylated compound were separated using silica gel
column chromatography eluted with chloroform/methanol
(99.5:0.5, 97.5:2.5, v/v) respectively. The compound
obtained was 6.7 g,a yield of 70.3 %.
1H NMR (200 MHz, CDCl3) d: 0.9 (9H, Ha, t,–(CH2)15–
CH3, [–(CH2)11–CH3]2, 1.2–1.4 (74 H, Hb, m, –(CH2)15–
CH3, [–(CH2)11–CH3]2, 1.5–1.75 (6H, Hc, m, 2 X –CH2–
CH2–O–, –CH2–CH2–N?–), 2.2 (6H, Hd, s, 2 X –O–CO–
CH3), 3.3–3.6 (14H, He, m,–?N–CH2–CH2-, ?N–[CH2–
CH(OCOCH3)–CH2–O–CH2–]2,3.7(3H, Hf, s,–CH2–N?
(CH3)–CH2–CH(OCOCH3), 4.95 (2H, Hg, m,?N–[CH2–
CH(OCOCH3)–CH2–O–CH2–]2; IR (KBr, cm-1): 1744
(C = O str); 1230, 1121 (–C–O–C–str).
gram-positive organisms, viz. Bacillus subtilis (MTCC-
441), Staphylococcus aureus (MTCC-96), and gram-nega-
tive organisms, viz. Escherichia coli (MTCC-443), Pseu-
domonas aeruginosa (MTCC- 741) and Klebsiella
aerogenes (MTCC-39), by the well diffusion method rec-
ommended by the National Committee for Clinical Labo-
ratory (NCCL) standards [17]. Streptomycin and cetyl
trimethyl ammonium bromide (CTAB) were used as positive
controls and chloroform as a negative control for antibacte-
rial activity.
Antifungal Activity
The antifungal activities were assayed against four fungal
strains, namely Candida albicans (MTCC-227), Saccha-
romyces cerevisiae (MTCC-36), Rhizopus oryzae (MTCC-
262) and Aspergillus niger (MTCC-282), by the agar-well
diffusion method [18]. Cultures of test organisms were
maintained on potato dextrose agar (PDA) slants and sub-
cultured on petri dishes before examining for antifungal
activity employing the agar cup bioassay method. A com-
mercially prepared PDA medium (composition: potato
infusion, 200 g; dextrose, 20 g; agar, 15 g) was used in this
evaluation study. The PDA medium (39.0 g) was suspended
Biological Procedures
Antibacterial Activity
The minimum inhibitory concentration (MIC) of the various
evaluated compounds were tested against two representative
123

J Am Oil Chem Soc (2013) 90:437–448
445
in distilled water (1000 mL) and heated to boiling until it
dissolved completely (pH 5.6). The medium and the Petri
dishes were autoclaved at 120 °C at a pressure of 15 psi, for
20 min. The medium was poured into sterile petri dishes
under aseptic conditions in a laminar flow chamber. When
the medium in the plates had solidified, 0.5 mL of the test
culture was inoculated and uniformly spread over the agar
surface. Solutions were prepared at two concentrations (100
and 150 lg/mL) by dissolving the test compounds in
chloroform. After inoculation, cups were scooped out with a
6-mm sterile cork borer and the lids of the dishes were
replaced. The different concentrations (100 and 150 lg/mL)
of test solutions were added separately to each cup. The
treated and the controlled cups were kept at 27 °C for 48 h.
The diameters (mm) of the inhibition zones were then
measured. Amphotericin B and CTAB were taken as posi-
tive controls and chloroform as negative control. Three
replicates were maintained for each treatment.
quaternization reactions in both the cases did not increase
their respective yields. These synthesized compounds were
unambiguously established from the analysis of their
1
spectral data (IR, H NMR and FABMS). The presence of
1
peaks at d 3.3–3.7 in H NMR indicating –N? (CH3)2 and
–N?(CH2)2 groups confirmed the quaternization of
monomer (5a–e) and dimer (6a–e), respectively. The
quaternary compounds 7a–e, 9a–e were further acetylated
using vinyl acetate in the presence of Novozym 435 to
produce the corresponding acetylated quaternary com-
pounds 8a–e (a yield of 80–89 %) and 10a–e (a yield of
70–85 %). The presence of peak at d 2.2 in both 8a–e,
10a–e further confirmed the acetylation of the quaternized
salts. Interestingly, there was a clear difference in the
reaction periods of 7a–e (72 h) and 9a–e (120 h); this
could be due to steric hindrance in dimer.
Antibacterial Properties of Alkyloxy Propanol
Amine-Based Cationic Ether Lipids
Antimicrobial activities of alkyloxy propanol amine-based
cationic ether lipids with varied alkyl chains and head
group are reported for the first time. All the synthesized
cationic lipids exhibited good antibacterial activities at
100 lg/mL concentration against the studied strains
(Fig. 3). However, tetradecyl chain length based com-
pounds exhibited slightly poorer activity. In general, the
quaternary ammonium salts are being used as antibacterial
agents that disrupt the cell membrane through the binding
of their ammonium cations to anionic sites in the outer
layer tissue of bacteria [19]. However, in our study the
nature of the quaternary head group (dimethyl type or
monomethyl and long chain type), free secondary –OH
group, or the presence of a short chain ester group (acetyl
moiety) in place of the hydroxy group did not have a sig-
nificant influence on antimicrobial activity. This reveals
that there is no influence of acetylation of secondary –OH
on the activity. This may be due to the presence of common
long chain (octadecylamine moiety) on the amine side in
all the synthesized compounds, further it is noteworthy that
the presence of a common long chain on the amine side in
all the synthesized cationic lipids made them behave sim-
ilarly towards all the studied strains.
Results and Discussion
Synthesis of Alkyloxy Propanol Amine-based Cationic
Ether Lipids
Glycidyl ethers are versatile intermediates in organic
chemistry and their reactions with different nucleophiles
have been of extensive interest. In the present study, the
reaction of epichlorohydrin with short, medium and long
chain alcohols for 6 h (of varying C4, C8, C10, C12 and C14
alkyl chain lengths) in the presence of potassium
hydroxide was carried out in a hexane medium which
yielded the glycidyl ethers (3a–e). However, further pro-
longation of reaction up to 10 h did not increase the yield
of the products. The different glycidyl ethers prepared
were n-butyl (3a), n-octyl (3b), n-decyl (3c), n-dodecyl
(3d) and n-tetradecyl glycidyl ethers (3e) with yields in the
range of 54–57 %. The epoxy of glycidyl ethers was
opened with octadecylamine (4) in ethanol to produce
1- octadecylamino-3-alkyloxy-2-propanol (5a–e) and N,N-
di-(2-hydroxy-3-alkyloxy propyl) octadecylamine (6a–e)
with a yield of 28–41 and 41–46 %, respectively. The
structures of 5a–e, 6a–e were confirmed by 1H NMR
which showed the characteristic peaks at d 2.7–2.85 due to
the presence of NH (CH2)2 and at 2.5 due to the presence
of N (CH2)3, respectively. These compounds were then
quaternized for 18 h using methyl iodide (Scheme 1) at
30 °C to yield the corresponding quaternary compounds
(7a–e, 71–80 %; 9a–e, 67–88 % yields). However, the
reasons are not clear for longer reaction times for quat-
ernization of 5a–e (18 h) compared to 6a–e (12 h). It
should be pointed out that further prolongation of the
As a result, the present study did not reveal any pref-
erential absorption at the bacterial cell wall to disrupt the
bacterial cell membrane and further no clear trend was
observed with both the types (series A and series B) of
compounds with respect to variation in the head group and
alkyloxy moiety. Incidentally, all the studied cationic lipids
showed better activity (10–17 mm) than CTAB
(7.5–9 mm), another well known cationic lipid. However,
they exhibited inferior activity compared to streptomycin
(20–29 mm).
123

446
J Am Oil Chem Soc (2013) 90:437–448
Fig. 3 a Antibacterial activity of alkyloxy propanol amine-based
cationic ether lipids (Series A: 7a–e, 1–5; 8a–e, 6–10): 1 N,N-dimethyl-
1-octadecylamino-3-butyloxy-2-propanol, 2 N,N-dimethyl-1-octadecy-
alkyloxy propanol amine-based cationic ether lipids of (Series B: 9a–e,
1–5; 10a–e, 6–10): 1 N-methyl N,N-di-(2-hydroxy-3-butyloxypropyl)
octadecylamine, 2 N-methyl N,N-di-(2-hydroxy-3-octyloxy propyl)
octadecylamine, 3 N-methyl N,N-di-(2-hydroxy-3-decyloxy propyl)
octadecylamine, 4 N-methyl N,N-di-(2-hydroxy-3-dodecyloxy propyl)
octadecylamine, 5 N-methyl N,N-di-(2-hydroxy-3-tetradecyloxypropyl)
octadecylamine, 6 N-methyl N,N-di-(2-acetyloxy-3 butyloxy propyl)
lamino-3-octyloxy-2-propanol,
4
3 N,N-dimethyl-1-octadecylamino-3-
decyloxy-2-propanol, N,N-dimethyl-1-octadecylamino-3-dodecy-
loxy-2-propanol, 5 N,N-dimethyl-1-octadecylamino-3-tetradecyloxy-2-
propanol, 6 N,N-dimethyl-1 octadecylamino-2-acetyloxy-3-butyloxy-2-
propane, 7 N,N-dimethyl-1-octadecylamino-2-acetyloxy-3-octyloxy-2-
propane, 8 N,N-dimethyl-1-octadecylamino-2 acetyloxy-3-decyloxy-2-
propane, 9 N,N-dimethyl-1-octadecylamino-2-acetyloxy-3-dodecyloxy-
2-propane, 10 N,N-dimethyl-1-octadecylamino-2-acetyloxy-3-tetrade-
cyloxy-2-propane, 11 Streptomycin (standard), 12 Cetyl trimethyl
ammonium bromide (CTAB, standard). b Antibacterial activity of
octadecylamine,
7 N-methyl N,N-di-(2acetyloxy-3-octyloxypropyl)
octadecylamine, 8 N-methylN,N-di-(2-acetyloxy-3decyloxypropyl)octa
decylamine, 9 N-methyl N,N-di-(2acetyloxy-3–dodecyloxypropyl)octa-
decylamine, 10 N-methyl N,N-di-(2acetyloxy-3 tetradecyl oxypropyl)
octadecylamine, 11 Streptomycin (standard), 12 Cetyl trimethyl
ammonium bromide (CTAB, standard)
Fig. 4 a Antifungal activity of alkyloxy propanol amine-based cationic
ether lipids (Series A: 7a–e, 1–5; 8a–e, 6–10): 1 N,N-dimethyl-1-
octadecylamino-3-butyloxy-2-propanol, 2 N,N-dimethyl-1-octadecyla-
propanolamine-basedcationicetherlipidsof(SeriesB:9a–e, 1–5;10a–e,
6–10): 1 N-methyl N,N-di-(2-hydroxy-3-butyloxypropyl) octadecyl-
amine, 2 N-methyl N,N-di-(2-hydroxy-3-octyloxy propyl) octadecyl-
amine, 3 N-methyl N,N-di-(2-hydroxy-3-decyloxy propyl) octadecy-
lamine, 4 N-methyl N,N-di-(2-hydroxy-3-dodecyloxy propyl) octadec-
ylamine, 5 N-methyl N,N-di-(2-hydroxy-3-tetradecyloxypropyl)octa-
decylamine, 6 N-methyl N,N-di-(2-acetyloxy-3-butyloxy propyl) octa-
decylamine, 7 N-methyl N,N-di-(2acetyloxy-3-octyloxypropyl)octadec-
ylamine, 8 N-methyl N,N-di-(2-acetyloxy-3decyloxypropyl)octadecyl-
amine, 9 N-methyl N,N-di-(2acetyloxy-3–dodecyloxypropyl)octadecyl-
amine, 10 N-methyl N,N-di-(2acetyloxy-3 tetradecyl oxypropyl) octa-
decylamine, 11 Amphotericin B (standard), 12 Cetyl trimethyl ammo-
nium bromide (CTAB, standard)
mino-3-octyloxy-2-propanol,
4
3
decyloxy-2-propanol, N,N-dimethyl-1-octadecylamino-3-dodecy-
N,N-dimethyl-1-octadecylamino-3-
loxy-2-propanol, 5 N,N-dimethyl-1-octadecylamino-3-tetradecyloxy-2-
propanol, 6 N,N-dimethyl-1 octadecylamino-2-acetyloxy-3-butyloxy-2-
propane, 7 N,N-dimethyl-1-octadecylamino-2-acetyloxy-3-octyloxy-2-
propane, 8 N,N-dimethyl-1-octadecylamino-2 acetyloxy-3-decyloxy-2-
propane, 9 N,N-dimethyl-1-octadecylamino-2-acetyloxy-3-dodecyloxy-
2-propane, 10 N,N-dimethyl-1-octadecylamino-2-acetyloxy-3-tetrade-
cyloxy-2propane, 11 Amphotericin B (standard), 12 Cetyl trimethyl
ammoniumbromide(CTAB, standard). b Antifungalactivityofalkyloxy
123

J Am Oil Chem Soc (2013) 90:437–448
447
Antifungal Properties of Alkyloxy Propanol
Amine-Based Cationic Ether Lipids
CTAB. All the synthesized compounds were found to
possess moderate to good antibacterial activities. Further-
more, both the series A and B and their acetylated deriva-
tives exhibited good antifungal activity against C. albicans
compared to their activity against other fungal strains. No
difference in activity was observed with respect to acety-
lation; however, an increase in the alkyl chain to tetradecyl,
lowers both the antibacterial and antifungal activities.
The comparison of antifungal activities of all the synthe-
sized compounds with standards is depicted in Fig. 4. All
the synthesized compounds with the alkyl chain lengths
starting from C4 to C14 exhibited moderate activity against
C. albicans (10–12.5 mm); indeed, the activities in this
case are comparable to CTAB (7–8 mm) and milder than
amphotericin (24–26 mm). However, once again the same
phenomena was observed as incase of antibacterial activity,
i.e. no significant difference in antifungal activity was
found even after acetylation of both A and B series com-
pounds. Further, irrespective of variation in alkyl chain
lengths, milder activities against S. cerevisiae, R. oryzae
strains were found with all the synthesized cationic lipids.
On the whole, the synthesized cationic lipids exhibited
lower antifungal activities than corresponding antibacterial
activities. These results are in good agreement with pre-
vious results that quaternary ammonium salt-type antibac-
terial agents are in general less active against yeasts and
fungi than against bacteria since the structure of the former
is more complex than that of the latter [19–22]. The mode
of action of cationic lipids and surfactants for their anti-
fungal activity was reported by Viera and Carmona [14].
According to them, the action of the cationic detergent
(CTAB) or the bilayer-forming cationic synthetic lipid
DODAB does not involve fungus cell lysis but rather the
change of cell surface charge from negative to positive
[14]. This indicates that the compounds (series A and B)
are more specific towards bacteria in influencing mem-
brane physiology compared to fungi. The mode of action of
these lipids is not clear in the present study. However,
further studies are required with similar types of com-
pounds in order to achieve complete antimicrobial
evaluation.
References
1. Mauleon D, Dolors PM, Rosell G (1988) b-Adrenergic antago-
nists: N-alkyl and N-amidoethyl(aryloxy)propanolamines related
to propanolol. Eur J Med Chem 23:421–426
2. Macchia B, Balsamo A, Lapucci A, Machia F, Martinelli A,
Ammon HL, Prasad SM, Breschi MC, Ducci M, Martonotti E
(1987) Role of the (acyloxy) methyl moiety in eliciting the
adrenergic b-blocking activity of 3-(acyloxy) propanol amines.
J Med Chem 30:616–622
3. Reinehr D, Topfl R (1993) Phenyl alkyl glycidyl ether addition
products. US Patent 5(250):202
4. Barlow JJ, Smith LH (1991) Alkanolamine derivatives. US Patent
4(550):111
5. Kim TS, Hirao T, Ikeda I (1996) Preparation of bis-quaternary
ammonium salts from epichlorohydrin. J Am Oil Chem Soc
73:67–71
6. Satge’de Caro P, Mouloungui Z, Gaset A (1997) Synthesis of
alkyloxy (di)alkylamino propanols, hydroxy alkyloxy (di)alkyl-
oxyamino propanols, and the dimer compounds for use as fuel
additives. J Am Oil Chem Soc 74:235–240
7. Mangold HK (1988) Ether lipids: relic of the past and promise for
the future. Riv Ital Sostanze Grasse 65:349–360
8. Demopoulos CA, Pickard RN, Hanahan DJ (1979) Platelet acti-
vating factor: evidence for 1-O-alkyl-2-acetyl-sn-glyceryl-3-
phosphorylcholine as the active component (A new class of lipid
chemical mediators). J Biol Chem 254:9355–9358
9. Miller AD (1998) Cationic liposomes for gene therapy. Angew
Chem Int Ed Engl 37:1768–1785
10. Deshmukh HM, Huang L (1997) Liposome and polylysine
mediated gene transfer. New J Chem 21:113–124
11. Behr JP (1993) Synthetic gene transfer vectors. Acc Chem Res
26:274–278
12. Bhattacharya S, Mandal SS (1998) Evidence of interlipidic ion-
pairing in anion-induced DNA release from cationic amphiphile;
DNA complexes, mechanistic implications in transfection. Bio-
chemistry 37:7764–7777
13. Carmona-Ribeiro AM, Vieira DB, Nilton L (2006) Cationic
surfactants and lipids as anti-infective agents. Anti Infect Agents
Med Chem 5:33–54
14. Vieira DB, Carmona-Ribeiro AM (2006) Cationic lipids and
surfactants as antifungal agents: mode of action. J Anti microb
Chemother 58:760–767
15. Priyanka J, Anand ON (2001) Alkyloxy alkylamino propanols as
multifunctional fuel stabilizers. Petrol Sci Technol 19:411–424
16. Karuna MSL, Devi BLAP, Prasad PSS, Prasad RBN (2009)
Synthesis of sulfated sodium salts of 1-alkylamino-3 alkyloxy-2-
propanols and N,N-di-(2-hydroxy-3-alkyloxy propyl) alkylamines
as potential surfactants. J Surfact Deterg 12:117–123
17. National committee for clinical laboratory standards approved
standards (NCCLS): M2-A6 (1997) Performance standards for
antimicrobial disk susceptibility testing. NCCLS (6th edn)
Conclusion
In the present work, synthesis of a series of alkyloxy pro-
panol amine-based cationic lipids, N,N-dimethyl-1-octade-
cylamino-3-alkyloxy-2-propanol, series A (7a–e), and,
N-methyl-N,N-di-(2-hydroxy-3-alkyloxy-2 propyl) octa-
decylamine, series B (9a–e), and their acetylated derivatives
(8a–e and 10a–e) of varying alkyloxy chain lengths (C4, C8,
C10, C12, C14), in 71–80, 67–88 % yields was carried out.
The title compounds as well as their acetylated derivatives
were evaluated for antimicrobial activity against two
pathogenic strains of gram-positive bacteria, namely
B. subtilis, S. aureus and gram-negative bacteria, namely
E. coli, P. aeruginosa, and K. aerogenes. Their activities
were compared with the standards Streptomycin and
123

448
J Am Oil Chem Soc (2013) 90:437–448
18. Linday EM (1962) Practical introduction to microbiology. E. &
F.N. Spon, London, p 178
19. Yumi O, Yukishige K, Kazuo K, Toshi T, Norio Y (2008) Syn-
thesis and antibacterial activity of quaternary ammonium salt-
type antibacterial agents with a phosphate group. J Oleo Sci
57:445–452
21. Banno T, Toshima K, Kawada K, Matsumura S (2007) Synthesis
and properties of biodegradable and chemically recyclable cat-
ionic surfactants containing carbonate linkages. J Oleo Sci
56:493–499
22. Kabara JJ (ed) (1990) Science of antisepsis and bactericidal agent
p 20
20. Sun JY, Li J, Qiu XL, Quing FL (2005) Synthesis and structure-
activity relationship (SAR) of novel perfluoroalkyl-containing
quaternary ammonium salts. J Fluorine Chem 126:1425–1431
123