Synthesis, physicochemical, and biological activities of novel N-acyl tyrosine monomeric and Gemini surfactants in single and SDS/CTAB–mixed micellar system

Article abstract of DOI:10.1002/poc.3675

A series of single-chained N-acyl tyrosine surfactants with varying chain lengths (C₁₀-C₁₈) and degree of unsaturation, as well as an N-acyl Gemini tyrosine surfactant with chain length C₁₂, were synthesized, and the struc

Full text of DOI:10.1002/poc.3675

Received: 21 May 2016
DOI 10.1002/poc.3675
Revised: 27 September 2016
Accepted: 7 November 2016
R E S E A R C H A R T I C L E
Synthesis, physicochemical, and biological activities of novel
N‐acyl tyrosine monomeric and Gemini surfactants in single and
SDS/CTAB–mixed micellar system
Nausheen Joondan1 | Sabina Jhaumeer‐Laulloo1 | Prakashanand Caumul1 | Matthew Akerman2
1 Department of Chemistry, Faculty of Science,
Abstract
University of Mauritius, Réduit, Mauritius
2 University of KwaZulu‐Natal, Pietermaritzburg,
South Africa
A series of single‐chained N‐acyl tyrosine surfactants with varying chain lengths
(C10‐C18) and degree of unsaturation, as well as an N‐acyl Gemini tyrosine surfac-
tant with chain length C12, were synthesized, and the structures were confirmed
using spectral analysis. The effect of chain length and level of unsaturation on the
physicochemical and antibacterial properties of the N‐acyl tyrosine surfactants was
evaluated. The C12 derivative displayed the optimum antibacterial activity among
the single chain surfactants, and the presence of double bond in the oleoyl derivative
enhanced the antibacterial activity over its saturated analogue. The N‐acyl Gemini
surfactant displayed the highest antibacterial activity among the series and also
showed greater micelle forming ability than its single chain analogue. Mixed micel-
lar behavior of the N‐acyl Gemini surfactant with conventional cationic (CTAB) and
anionic (SDS) surfactants in aqueous solution was studied. The negative value of the
interaction parameter β12 observed for the N‐acyl Gemini in binary mixture with
CTAB surfactant indicated a synergistic interaction within the mixed micellar sys-
tem. However, the binary mixture with SDS displayed antagonistic behavior. The
binary mixture of N‐acyl Gemini surfactant with CTAB displayed better antibacte-
rial activity and foaming properties than with SDS mixtures. Optimum antibacterial
activity was observed for N‐acyl Gemini surfactant with mole ratio 0.4 to 0.6 in the
CTAB binary mixture, at which the lowest ocular irritation index was observed.
Overall, the study showed that the Gemini surfactant in combination with the con-
ventional surfactant CTAB can be used as potential ingredients in detergent and
pharmaceutical formulations.
Correspondence
Sabina Jhaumeer‐Laulloo, Department of
Chemistry, Faculty of Science, University of
Mauritius, Réduit, Mauritius.
Email: sabina@uom.ac.mu
KEYWORDS
critical micelle concentration, mixed micellar system, biological activity, N‐acyl
tyrosine surfactants
1
|
INTRODUCTION
Among the amino acid surfactants, the N‐acyl amino acid
derivatives are by far the most widely used in consumer
The strong demand towards the production of more
environmentally benign chemicals has led to the use of
greener alternatives to replace petroleum‐based chemicals
for the preparation of surfactants.[1] Amino acid–based
surfactants have emerged as ecological, biocompatible, and
renewable amphiphiles that have been applied in wide
areas.[2–4]
product formulations because of their low toxicity and hypoal-
lergenic, low irritancy, and high biodegradability.[5,6] In the
N‐acyl derivatives, the amide bonds are known to act as both
hydrogen bond donors (the N–H moiety) and hydrogen bond
acceptors (the C=O group) in intramolecular and intermolecu-
lar interactions during self‐assembly in bulk and at surfaces to
form various highly organized nanometer‐scale structures.[7]
J Phys Org Chem 2016; 1–13
wileyonlinelibrary.com/journal/poc
Copyright © 2016 John Wiley & Sons, Ltd.
1

2
JOONDAN ET AL.
This class of surfactants has been thoroughly investigated with
respect to dermatological properties and is generally regarded
as safe with respect to skin irritation.[8] Moreover, N‐acyl
amino acid surfactants possess excellent physicochemical
and antimicrobial activities, which render them valuable as
additives in the formulation of food, detergents, cosmetics,
personal care, and pharmaceutical products.[9,10]
Chemika (India) was used for column chromatography.
Mueller Hinton broth was obtained from Oxoid Ltd
(United Kingdom). The different bacterial strains were
obtained from Microbiologics (St. Cloud, Minnesota) and
Oxoid Ltd (United Kingdom).
The 1H NMR and 13C NMR spectra were recorded at 250
and 62.9 MHz on a Bruker electro spin nuclear magnetic res-
onance spectrometer using CDCl3, D2O, and DMSO‐d6 as
solvents. Infrared (IR) spectra were recorded on a Bruker
Alpha Fourier transform infrared spectrometer. Fluorescence
intensities were recorded on an LS 55 Perkin Elmer fluores-
cence spectrophotometer. Conductivity measurements were
made using a Jenway 4320 conductivity meter. Elemental
analysis was determined from a Eurovector EA3000 Elemen-
tal analyzer.
The X‐ray data of compound 1 was recorded on a Bruker
Apex Duo diffractometer equipped with an Oxford Instrument
Cryojet operating at 100(2) K and an Incoatec microsource
operating at 30‐W power. The data were collected with MoKα
(λ = 0.71073 Å) radiation at a crystal‐to‐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 APEX2.16 The data were reduced
with the program SAINT using outlier rejection, scan speed
scaling, and standard Lorentz and polarization correction fac-
tors. A SADABS semiempirical multi‐scan absorption correc-
tion was applied to the data. Direct methods, SHELX‐2014[17]
and WinGX,[18] were used to solve the data. All hydrogen
atoms were included as idealized contributors in the least
squares process. Their positions were calculated using a stan-
dard riding model with C–Haromatic distances of 0.93 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
O–H and N–H atoms were located in the difference density
map and refined isotropically. Crystal and structure refine-
ment data of compound 1 are given in Table 1.
The chemical and enzymatic syntheses of single chain and
Gemini N‐acyl surfactants derived from various amino acids
have been reported in view of systematically producing surfac-
tants with varying head groups. Sreenu et al[11] reported the
synthesis, surface, and micellar properties of N‐oleoyl surfac-
tants derived from isoleucine and proline, and they were found
to exhibit good surface tension reduction, emulsion stability,
and calcium tolerance compared to conventional surfactants.
N‐decanoyl amino acid surfactants derived from leucine,
methionine, serine, and proline were reported to exhibit favor-
able toxicity profiles.[12] N‐acyl amino acids derived from the
aromatic amino acid phenylalanine have also been reported,
and these have been known to possess unique properties com-
pared to other N‐acyl amino acid surfactants.[13,14] From our
previous studies, it was found that the O‐alkyl esters of tyro-
sine showed enhanced physicochemical and antibacterial
activities because of the presence of the phenolic group that
enhances micellar formation and causes greater interaction
with bacterial membrane.[15] However, to the best of our
knowledge, there is no report on the systematic study of N‐acyl
derivatives derived from tyrosine.
This study reports the synthesis, physicochemical, and
antibacterial activities of a series of N‐acyl surfactants
derived from tyrosine. The effect of chain lengths and pres-
ence of unsaturation of the fatty acid chain on the micellar
and antibacterial properties were investigated. Mixed surfac-
tant systems have been reported to have better properties than
single surfactant systems. With this view, the physicochemi-
cal and biological activities of the N‐acyl tyrosine surfactants
in both single and mixed surfactant systems with conven-
tional cetyl trimethyl ammonium bromide (CTAB) and
sodium dodecyl sulfate (SDS) were evaluated. To be able to
study their effectiveness as potential ingredients in deter-
gents, the foaming properties as well as the ocular irritancy
of the CTAB/SDS‐N‐acyl tyrosine surfactant mixtures were
investigated.
2.2 | Synthesis and characterization
N‐acyl tyrosine surfactants were synthesized by the reaction
of the tyrosine ester with selected fatty acid (butanoic,
decanoic, dodecanoic, tetradecanoic, palmitic, stearic, and
oleic acids) chlorides of varying chain lengths. The corre-
sponding fatty acid chloride (1.5 eq) was added to a solution
of L‐Tyrosine ester (4.31 mmol) and triethylamine (3.2 mL)
in tetrahydrofuran (70 mL) followed by a catalytic amount
of 4‐dimethylaminopyridine. The mixture was refluxed for
18 hours and then quenched with water (100 mL). The
organic phase was extracted with ethyl acetate. The combined
organic extracts were washed with sodium bicarbonate (5%,
50 mL) and dried over anhydrous magnesium sulfate, and
the solvent was removed in vacuo. The crude product was
washed with excess ether to give the corresponding N‐acyl
derivative as white solid.
2
| MATERIALS AND METHODS
2.1 | Chemicals and instrumentation
L‐Tyrosine, decanoic acid, dodecanoic acid, tetradecanoic
acid, palmitic acid, stearic acid, cetyl pyridinium chloride
(CPC), and the fluorescence probe pyrene were purchased
from Sigma‐Aldrich (St. Louis, USA). Oleic acid and
CTAB were obtained from BDH Laboratory Supplies
(England). Silica gel (60‐120 mesh) obtained from Alpha

JOONDAN ET AL.
3
TABLE 1 Crystal and structure refinement data of compound 1
Crystal Data
Compound 1
Chemical formula
C14H19NO4
Molar mass (g mol−1
)
265.30
Crystal system, space group
Temperature (K)
a, b, c (Å)
Orthorhombic, P212121
100(2)
8.6205(8), 14.9938(12), 22.1253(18)
α, β, γ (°)
α = β = γ = 90
V (Å3)
2859.8(4)
Z
8
Radiation type
MoKα
μ (mm−1
)
0.09
Crystal size (mm)
0.39 × 0.28 × 0.11
Data Collection
Diffractometer
Bruker Apex Duo CCD diffractometer
Multi‐scan, SADABS, Bruker 2012
0.679, 0.745
Absorption correction
Tmin, Tmax
No. of measured, independent, and observed [I > 2σ(I)] reflections
18931, 5710, 5483
Rint
0.021
Refinement
R[F2 > 2σ(F2)], wR(F2), S
No. of reflections
No. of parameters
No. of restraints
H‐atom treatment
0.029, 0.072, 1.06
5710
364
0
H atoms treated by a mixture of independent and constrained refinement
Δρmax, Δρmin (e Å−3
)
0.17, −0.15
Absolute structure
Refined as an inversion twin
0.2(8)
Absolute structure parameter
2.2.1
|
N‐butyroyl L‐tyrosine methyl ester 1
(CH), 115.5, 127.2, 130.3, 155.4 (C6H4), 172.4, 173.3
(C=O).
Yield: 63%.1H NMR (CDCl3), δ (ppm): 0.87 (t, J 7 Hz, 3H,
COCH2CH2CH3), 1.59 (m, 2H, COCH2CH2CH3), 2.15 (m,
2H, COCH2CH2CH3), 3.00 (2H, m, CH2Ph), 3.70 (s, 3H,
OCH3), 4.85 (m, 1H, CH), 5.98 (d, J 8 Hz, 1H, N–H), 6.73
(d, J 8 Hz, 2H, Ph), 6.92 (d, J 8 Hz, 2H, Ph). 13C NMR
2.2.3
| N‐dodecanoyl L‐tyrosine methyl ester 3
Yield: 73%. Elem Anal Found: C, 69.22; H, 10.46; N, 3.94;
Calcd for C22H35NO4: C, 69.49; H, 9.84; N, 3.71. GC‐MS,
(CDCl3),
δ
(ppm): 13.7 (COCH2CH2CH3), 19.0
1
m/z: 377.14. IR, νmax (cm−1): 3334, 1749, 1646. H NMR
(COCH2CH2CH3), 37.2 (COCH2CH2CH3), 38.4 (CH2Ph),
52.4 (OCH3), 53.1 (CH), 115.6, 127.0, 130.3, 155.6
(C6H4), 172.4, 173.1(C=O).
(CDCl3), δ (ppm): 0.89 (t, J 7 Hz, 3H, CO(CH2)10CH3), 1.27
(m, 16H, COCH2CH2(CH2)8CH3), 1.60 (m, 2H,
COCH2CH2(CH2)8CH3), 2.19 (m, 2H, COCH2(CH2)9CH3),
3.06 (dd, J 14 Hz, J 7 Hz, 1H, CHHPh), 3.12 (dd, J 14 Hz,
J 7 Hz, 1H, CHHPh), 3.75 (s, 3H, OCH3), 4.88 (m, 1H, CH),
5.92 (d,J 8 Hz, 1H, NH), 6.77 (d, J 8 Hz, 2H, Ph), 6.98 (d,
J 8 Hz, 2H, Ph). 13C NMR (CDCl3), δ (ppm): 14.1 (CO
(CH2)10CH3), 22.7 to 31.9 (COCH2(CH2)9CH3), 36.6
(COCH2(CH2)9CH3), 37.3 (CH2Ph), 52.4 (OCH3), 53.1
(CH), 115.5, 127.3, 130.3, 155.3 (C6H4), 172.4, 173.2 (C=O).
2.2.2
| N‐decanoyl L‐tyrosine methyl ester 2
Yield: 68%. Elem Anal Found: C, 67.14; H, 8.98; N, 3.70;
Calcd for C20H31NO4: C, 67.74; H, 8.94; N, 4.01. GC‐MS,
m/z: 349.93. IR, νmax (cm−1): 3333, 1749, 1646. H NMR
1
(CDCl3), δ (ppm): 0.89 (t, J 7 Hz, 3H, CO(CH2)8CH3),
1.27 (m, 12H, COCH2CH2(CH2)6CH3), 1.62 (m, 2H,
COCH2CH2(CH2)6CH3), 2.19 (m, 2H, COCH2(CH2)7CH3),
3.03 (dd, J 14 Hz, J 7 Hz, 1H, CHHPh), 3.05 (dd, J 14 Hz,
J 7 Hz, 1H, CHHPh), 3.74 (s, 3H, OCH3), 4.89 (m, 1H,
CH), 5.97 (d, J 8 Hz, 1H, N–H), 6.76 (d, J 8 Hz, 2H, Ph),
6.96 (d, J 8 Hz, 2H, Ph). 13C NMR (CDCl3), δ (ppm): 14.1
(CO(CH2)8CH3), 22.7 to 31.9 (COCH2(CH2)7CH3), 36.6
(COCH2(CH2)7CH3), 37.3 (CH2Ph), 52.4 (OCH3), 53.1
2.2.4
| N‐tetradecanoyl L‐tyrosine methyl ester 4
Yield: 54%. Elem Anal Found: C, 72.40; H, 11.87; N,
2.88; Calcd for C24H39NO4: C, 71.97; H, 11.69; N,
2.45. IR, νmax (cm−1): 3376, 2949, 1736. 1H NMR
(CDCl3), δ (ppm): 0.89 (t, J 7 Hz, 3H, CO(CH2)12CH3), 1.27
(m, 20H, COCH2CH2(CH2)10CH3), 1.63 (m, 2H,

4
JOONDAN ET AL.
COCH2CH2(CH2)10CH3), 2.22 (m, 2H, COCH2(CH2)11CH3),
3.07 (m, 2H, CH2Ph), 3.74 (s, 3H, OCH3), 4.89 (m, 1H, CH),
5.93 (d, J 8 Hz, 1H, NH), 6.77 (d, J 8 Hz, 2H, Ph), 6.99 (d,
J 8 Hz, 2H, Ph). 13C NMR (CDCl3), δ (ppm): 14.1 (CO(CH2)
12CH3), 22.7 to 31.9 (COCH2(CH2)11CH3), 36.6 (COCH2
(CH2)7CH3), 37.3 (CH2Ph), 52.4 (OCH3), 52.9 (CH), 115.6,
127.5, 130.3, 155.3 (C6H4), 172.4, 173.0 (C=O).
2.2.8 | N‐dodecanoyl L‐tyrosine dodecyl ester 8
Yield: 77%. Elem Anal Found: C, 73.425; H, 10.60;
N, 4.82; Calcd for C33H57NO4: C, 73.53; H, 10.80;
N, 4.63. ESI‐MS, m/z: 554.42 (M+ Na+). IR, νmax (cm−1):
3376, 2949, 1736. 1H NMR (DMSO),
δ
(ppm):
0.85 (m, 3H, CO(CH2)10CH3), 0.85 (m, 3H, O(CH2)11CH3),
1.23 (m, 16H, COCH2CH2(CH2)8CH3), 1.23 (m, 18H,
OCH2CH2(CH2)9CH3), 1.47 (m, 2H, COCH2CH2(CH2)
8CH3), 1.47 (m, 2H, OCH2CH2(CH2)9CH3), 2.03 (m, 2H,
COCH2(CH2)9CH3), 2.78 (m, 2H, CH2Ph), 3.96 (t, J
7 Hz, 3H, OCH2(CH2)10CH3), 4.32 (m, 1H, CH), 6.63 (d,
J 8 Hz, 2H, Ph), 6.98 (d, J 8 Hz, 2H, Ph), 8.15 (d, J
8 Hz, 1H, NH), 9.25 (s, 1H, OH). 13C NMR (CDCl3), δ
(ppm): 14.1 (CO(CH2)10CH3), 14.1 (O(CH2)11CH3), 22.7 to
31.9 (COCH2(CH2)9CH3), 22.7 to 31.9 (OCH2(CH2)10CH3),
35.5 (CH2Ph), 36.5 (COCH2(CH2)9CH3), 54.5 (CH), 64.8
(OCH2(CH2)10CH3), 115.5, 127.8, 130.4, 156.5 (C6H4),
172.4, 173.7 (C=O).
2.2.5
| N‐palmitoyl L‐tyrosine methyl ester 5
Yield: 71%. Elem Anal Found: C, 72.16; H, 10.87; N,
4.20; Calcd for C26H43NO4: C, 72.02; H, 10.00; N, 3.93.
IR, νmax (cm−1): 3376, 2949, 1736. 1H NMR (CDCl3),
δ (ppm): 0.89 (t, J 7 Hz, 3H, CO(CH2)14CH3), 1.26
(m, 24H, COCH2CH2(CH2)12CH3), 1.57 (m, 2H,
COCH2CH2(CH2)12CH3), 2.19 (m, 2H, COCH2(CH2)
13CH3), 3.04 (m, 2H, CH2Ph), 3.75 (s, 3H, OCH3), 4.87 (m,
1H, CH), 5.90 (d, J 8 Hz, 1H, NH), 6.73 (d, J 8 Hz, 2H, Ph),
6.97 (d, J 8 Hz, 2H, Ph). 13C NMR (CDCl3), δ (ppm): 14.1
(CO(CH2)12CH3), 22.7 to 31.9 (COCH2(CH2)11CH3), 36.6
(COCH2(CH2)7CH3), 37.2 (CH2Ph), 52.4 (OCH3), 53.1
(CH), 115.5, 127.6, 130.4, 155.0 (C6H4), 172.4, 172.9 (C=O).
2.3 | Critical micelle concentration determination
2.3.1
| Critical micelle concentration determination of 2‐8
Pyrene was used as a fluorescence probe to determine the
critical micelle concentration (CMC) of the N‐acyl tyrosine
surfactants (compounds 2‐8) in aqueous solution at 25°C.
Stock solution of pyrene in methanol (10 μL, 0.1mM) was
transferred into vials. After evaporating the methanol, surfac-
tant solutions (3 mL) of varying concentrations were added to
the vials to give a final concentration of 1.6μM of pyrene in
each vial. Fluorescence spectra of pyrene were recorded over
the spectral range 350 to 450 nm. The excitation wavelength
was kept at 334 nm, and the emission was recorded at 373 (I1)
and 384 (I3) nm. The ratio of the intensities of the first and
third vibronic peaks in the fluorescence spectrum of pyrene
(I1/I3) was recorded as a function of the N‐acyl surfactant
concentrations to determine the CMCs.
2.2.6
| N‐stearoyl L‐tyrosine methyl ester 6
Yield: 65%. Elem Anal Found: C, 71.95; H, 11.91; N,
3.20; Calcd for C28H47NO4: C, 72.34; H, 11.26; N, 3.03.
IR, νmax (cm−1): 3376, 2949, 1736 1H NMR
(CDCl3), δ (ppm): 0.89 (m, 3H, CO(CH2)16CH3), 1.26
(m, 28H, COCH2CH2(CH2)14CH3), 1.61 (m, 2H,
COCH2CH2(CH2)14CH3), 2.19 (m, 2H, COCH2(CH2)
15CH3), 3.05 (m, 2H, CH2Ph), 3.74 (s, 3H, OCH3), 4.87 (m,
1H, CH), 5.97 (d, J 8 Hz,1H, NH), 6.74 (d, J 8 Hz, 2H, Ph),
6.95 (d, J 8 Hz, 2H, Ph). 13C NMR (CDCl3), δ (ppm): 14.1
(CO(CH2)16CH3), 22.7 to 31.9 (COCH2(CH2)15CH3), 36.6
(COCH2(CH2)15CH3), 37.2 (CH2Ph), 52.4 (OCH3), 53.1
(CH), 115.5, 127.5, 130.4, 155.1 (C6H4), 172.4, 173.0
(C=O).
2.3.2
| Critical micelle concentration determination of
SDS/CTAB–8 mixed system.
Mixed SDS‐8 systems with varying mole fractions of 8 (0,
0.2, 0.4, 0.6, 0.8, 1) were prepared by mixing precalculated
volumes of the stock solutions of SDS and 8 in water, and
the solutions were stirred for 1 hour. Mixed CTAB‐8 systems
were also prepared in a similar way, and the mole fraction of
8 in the mixed solution was expressed as
2.2.7
| N‐oleoyl L‐tyrosine methyl ester 7
Yield: 78%. IR, νmax (cm−1): 3376, 2949, 1736. H NMR
(CDCl3), δ (ppm): 0.86 (m, 3H, CO(CH2)7–CH=CH–(CH2)
7CH3), 1.25 (m, 20H, COCH2CH2(CH2)4CH2–CH=CH–CH2
(CH2)6CH3), 1.54 (m, 2H, COCH2CH2(CH2)4CH2–
CH=CH–CH2(CH2)6CH3), 2.16 (m, 2H, COCH2(CH2)6–
CH=CH–(CH2)7CH3), 3.00 (m, 2H, CH2Ph), 3.71 (s, 3H,
OCH3), 4.86 (m, 1H, CH), 5.34 (m, 2H, CO(CH2)7–
CH=CH–(CH2)7CH3), 5.96 (d, J 8 Hz, 1H, NH), 6.73 (d, J
8 Hz, 2H, Ph), 6.93 (d, J 8 Hz, 2H, Ph). 13C NMR (CDCl3), δ
(ppm): 14.1 (CO(CH2)7–CH=CH–(CH2)7CH3), 22.7 to 31.9
(COCH2(CH2)6–CH=CH–(CH2)7CH3), 36.6 (COCH2(CH2)
6–CH=CH–(CH2)7CH3), 37.3 (CH2Ph), 52.4 (OCH3), 53.2
(CH), 115.6, 127.0, 130.3, 155.6 (C6H4), 129.8, 130.0 (CO
(CH2)7–CH=CH–(CH2)7CH3), 172.4, 173.3 (C=O).
1
½8ꢀ
α8 ¼
½8ꢀ þ ½SDS=CTABꢀ
where [8], [SDS], and [CTAB] are the concentrations of the
Gemini surfactant 8, SDS, and CTAB in the mixed solutions,
respectively.
The CMC of the different mixed systems was determined
via conductivity measurements by adding successive

JOONDAN ET AL.
5
amounts of the stock solutions to deionized water in the form
of a titration.
cylinder with a stopper, and the height of the foam formed
was measured. The different SDS/CTAB–8 (α8 = 0, 0.2,
0.4, 0.6, 0.8, and 1) mixed systems (5 mL, 500mM) were
shaken vigorously for 10 seconds in a calibrated 10‐mL
glass cylinder, and the height of the foam was recorded.
All the measurements were performed at 25°C in triplicate,
and the results were reported as the mean value standard
deviation.
2.4 | Aggregation number
Pyrene solution in methanol (10 μL, 0.1mM) was pipetted
into 4‐mL vials. After evaporating the methanol, 10 μL of
aqueous quencher solution, CPC of varying concentration
(0M‐0.3M) was pipetted into the pyrene followed by the
surfactant (2‐8) solution (3 mL), with concentration 5 times
above their respective CMC values. The fluorescence
intensities of pyrene in the different solutions were then
measured at 373 nm.
3
| RESULTS AND DISCUSSION
3.1 | Synthesis and characterization
A series of N‐acyl surfactants derived from L‐Tyrosine were
synthesized by the condensation of tyrosine ester with fatty
acid chlorides of varying chain lengths (C4 and C8‐C18)
(Figure 1).
2.5 | Krafft temperature
To measure the Krafft temperature of the CTAB‐8 system,
0.01‐M solutions of varying mole fractions of surfactant 8
(0, 0.2, 0.4, 0.6, 0.8) were prepared in deionized water and
placed in the refrigerator for 24 hours at 4°C. The conductiv-
ity was noted as the temperature of the solution was raised
under gentle stirring.
In previous reports, the N‐acyl amino acid surfactants
were synthesized by the Schotten‐Baumann reaction,
whereby NaOH in tetrahydrofuran/water was used.[20] In this
study, a milder base (triethylamine) was used for the
synthesis of the N‐acyl tyrosine surfactants because of the
presence of the ester moiety that is sensitive to hydrolysis.
All the N‐acyl tyrosine surfactants were solids at room
temperature, and the chemical structures were confirmed by
1H NMR, 13C NMR, IR spectra, GC‐MS, MS, and elemental
analysis.
The presence of the new carbonyl signal at δ 172.7 to
173.7 ppm in the 13C NMR and at 1645 to 1647 cm−1 in the
IR spectra confirmed the formation of the N‐acyl surfactants.
To study the effect of the presence of an unsaturated sys-
tem in the hydrophobic tail of the N‐acyl surfactants, tyrosine
methyl ester was made to react with oleoyl chloride, leading
to the formation of N‐oleoyl tyrosine methyl ester 7. The
presence of the unsaturated system was confirmed by a mul-
tiplet at δ 5.34 ppm in the 1H NMR spectrum corresponding
to the 2 olefinic protons.
In view of producing Gemini‐like amphiphiles, N‐acyla-
tion was performed on a longer chain tyrosine ester, to pro-
duce an amphiphile with a double tail system having chain
length C12 at both the N‐acyl and O‐ester moieties (8).
The surfactant 8 (Figure 2) was found to possess the
molecular formula C33H57NO4. The 13C NMR spectrum
showed 18 signals, 4 from which correspond to the aromatic
segment. The 2 signals in the 13C NMR with the highest
chemical shift, 172.4 and 172.7 ppm, correspond to the
carbonyl groups C‐13 and C‐16, respectively. In the aromatic
segment, magnetically equivalent carbon atoms C‐30 and
C‐34 appear at δ 130.4 ppm, while C‐31 and C‐33 appear at
δ 115.4 ppm in the 13C NMR spectrum. These assignments
were confirmed by bidimentional techniques (Homonuclear
correlation spectroscopy (COSY), Heteronuclear single‐
quantum correlation spectroscopy (HSQC) and Heteronuclear
multiple‐bond correlation spectroscopy (HMBC)).The
selected data are shown in Table 2.
2.6 | Antibacterial activity
The antibacterial activities were determined against 3 gram‐
positive strains, namely, Staphylococcus aureus (ATCC
25923), Staphylococcus epidermidis (ATCC 12228), and
Bacillus cereus (ATCC 11778, ATCC 10876), and 3 gram‐
negative strains, namely, Klebsiella pneumoniae (ATCC
13883), Escherichia coli (ATCC 22922), and Salmonella
typhimurium (ATCC 14028), using the broth dilution
method.[19] The antibacterial activity was expressed as the
minimum inhibitory concentration (MIC) that was defined
as the lowest concentration that inhibits the growth of bacte-
ria. Cetyl trimethyl ammonium bromide and sodium dodecyl
sulfate were used as positive controls. The antibacterial activ-
ities of the different SDS/CTAB–8 mixed micelle systems
(α8 = 0, 0.2, 0.4, 0.6, and 0.8) were determined starting from
solutions with a total surfactant concentration (CT) of 0.05M
([8] + [SDS] or [CTAB]).
All wells were inoculated with 50 μL of a bacterial suspen-
sion adjusted to 0.5 McFarland in physiological solution.
Microplates were covered and incubated for 24 hours at 37°
C. The MIC of the surfactants was detected following addition
of 20‐μL iodonitrotetrazolium chloride (0.4 mg mL−1) and
incubation at 37°C for 30 minutes. Viable microorganisms
reduced the yellow dye to a pink color. Minimum inhibitory
concentration was defined as the lowest sample concentration
that prevented this change and exhibited complete inhibition
of bacterial growth.
2.7 | Foamability measurements
The surfactant solutions (20 mL, 0.1 wt%) were shaken vig-
orously for 10 seconds in a calibrated 100‐mL glass

6
JOONDAN ET AL.
FIGURE 1 Synthesis of N‐acyl tyrosine surfactants 1‐8
The key structural difference between the 2 molecules in
the asymmetric unit is the relationship between the amide N
atom and ester groups. In one molecule (as shown in
Figure 3), the carbonyl oxygen atom is trans to the amide N
atom and in the second molecule the geometry is cis. By
inspection, it would appear that neither geometry leads to sig-
nificant steric strain within the molecule and therefore, the
energy difference between the 2 geometries is likely to be
modest.
The mean C=O bond length of the amide group measures
1.214(3), this coupled with the N–C–O and C–C–O bond
lengths which average 121.0(3)° and 122.5(3)° illustrate the
sp2 hybridized nature of the amide carbon atom.
FIGURE 2 Labelled structure of Gemini surfactant 8
The molecule exhibits a number of intermolecular
hydrogen bonds. Firstly, the 2 molecules A and B in the
asymmetric unit are linked through a hydrogen bond between
the hydroxy groups of the 2 adjacent molecules. One hydroxy
group acts as the H‐bond donor and the other as the H‐bond
3.1.1
| X‐ray structure of compound 1
Compound 1 crystallized in the orthorhombic space group
P212121 with 2 molecules linked through a hydrogen bond
in the asymmetric unit (ie, Z = 8) as shown in Figure 3.
TABLE 2 Selected spectral data of compound 8
DEPT (Distortionless enhancement
Atom
δ (1H) ppm
1H–1H COSY
δ (13C) ppm
by polarization transfer)
HMBC
12
13
14
15
16
17
28
29
30
31
32
33
34
3.96 (t, 7 Hz, 2H)
11
64.8
172.4
54.5
CH2
13b
4.32 (m, 1H)
15, 28
14
CH
13a
16a
15a, 17a
16a
8.17 (d, 7.7 Hz, N–H)
172.7
35.5
2.02 (t, 7.5 Hz, 2H)
18
14
CH2
CH2
C
2.78 (dd, J 14 Hz, J 7 Hz, 1H, CH2Ph)
36.5
29a, 30b, 34b
127.8
130.4
115.4
156.5
115.4
130.4
6.98 (d, J 8 Hz, 1H, Ph)
6.63 (d, J 8 Hz, 1H, Ph),
31
30
CH
CH
C
29a, 31a, 33a, 32b
30a, 32b, 33b
6.63 (d, J 8 Hz, 1H, Ph),
6.98 (d, J 8 Hz, 1H, Ph)
34
33
CH
CH
31b, 32a, 34a
29a, 33a, 30b, 32b
a2‐bond correlation.
b3‐bond correlation.

JOONDAN ET AL.
7
TABLE 3 Hydrogen bond parameters for compound 1
Bond
D–H (Å)
H···A (Å)
D···A (Å)
D–H···A (°)
O5–H103···O1
O1–H104···O6
N1–H101···O5
N2–H102···O2
0.87(3)
0.80(4)
0.87(2)
0.89(2)
1.78(3)
2.60(4)
2.06(3)
1.98(2)
2.649(2)
3.384(3)
2.922(2)
2.859(2)
173(3)
179(3)
171(2)
169(5)
The hydrogen bond distances summarized in Table 2 are
significantly shorter than the sum of the van der Waals radii.
The D···A bond lengths are shorter than the sum of the van
der Waals radii by 0.661 to 1.010 Å; this is a significant dif-
ference when compared to previously reported hydrogen
bonds[21] and suggests that the interaction is likely strong.
This is further supported by the fact that the hydrogen bond
angles do not deviate significantly from the ideal angle of
180°. The 4 hydrogen bonds stabilize a 3‐dimensional supra-
molecular structure that packs in alternating layers of mole-
cules A and B as shown in Figure 5.
FIGURE 3 Displacement ellipsoid plot (50% probability) of compound 1.
For clarity, a single molecule from the asymmetric unit has been shown.
Hydrogen atoms are drawn as spheres of arbitrary radius
3.2 | Critical micelle concentration and aggregation
number of 2‐8
acceptor. In addition to this hydrogen bond, there are 3 addi-
tional hydrogen bonds. One of the amide NH groups is
hydrogen‐bonded to the amide carbonyl oxygen of an adja-
cent molecule and the other to one of the hydroxy groups.
The final hydrogen bond is between one of the hydroxy
groups and the amide carbonyl of an adjacent molecule. Each
of the hydroxy groups therefore acts as both an H‐bond
acceptor and an H‐bond donor; the oxygen atoms of the ester
groups are not involved in H‐bonds. These interactions link 5
molecules, as shown in Figure 4. The hydrogen bond param-
eters are summarized in Table 3.
The critical micelle concentration of the synthesized N‐acyl
tyrosine surfactants 2‐8 was determined by fluorescence
study using pyrene as probe. Pyrene molecules are highly
hydrophobic, and in micellar solution, they are preferentially
solubilized within the hydrophobic interior of the aggregates
and are strongly distributed into the micelle as soon as they
form.[22]This causes an abrupt decrease in the I1/I3 ratio at
the onset of micellar formation when a surfactant is added
to an aqueous solution of pyrene. Figure 6 represents the
fluorescence spectrum of pyrene in the presence of the
N‐acyl tyrosine surfactant 3.
The micellar aggregation number (Nss) of the N‐acyl
tyrosine surfactants 2‐8 was determined by steady‐state fluo-
rescence quenching technique, using pyrene as probe and
FIGURE 4 Hydrogen bonding of compound 1. The 4 hydrogen bonds
stabilize a 3‐dimensional supramolecular structure. Hydrogen bonds are
shown as dashed purple tubes
FIGURE 5 Alternating layers of molecules A and B in the 3‐dimensional
supramolecular structure supported by extensive hydrogen bonding

8
JOONDAN ET AL.
its analogous saturated derivative 6.[23] This is due to the
increase in the bulkiness and rigidity of the chain causing a
loss in the degree of rotational freedom, which interferes with
the tight packing of the tails in the micelle.[24] The increase in
CMC may also be due to the decrease in hydrophobicity of
the chain because of the delocalized π electrons that causes
an increase in the polarizability of the chain and hence
decreases the ease of micellization.
The presence of a second hydrophobic chain in the Gem-
ini surfactant 8 causes a decrease in the CMC of the surfac-
tant compared to its single chain analogue 3, because of an
increase in hydrophobic character of the surfactant that
enhances micellar formation.
Mixed surfactant systems are known to influence physical
properties of the individual components, and these systems
are encountered in several applications.[25,26] Mixed surfac-
tant systems in water undergo several physicochemical
changes because of the interaction between amphiphiles and
enhance the interfacial and micellar properties.[27] In this
study, the use of 8 in mixed micelle solutions with the con-
ventional anionic surfactant SDS as well as the cationic sur-
factant CTAB has been investigated. The CMC values of
the different binary combinations of SDS/CTAB and 8 are
presented in Table 5.
The CMC values of the binary mixtures were lower com-
pared to the CMC of the pure SDS or CTAB solution, sug-
gesting that micellar formation is more favored in the mixed
micellar system. This is mainly due to the electronic charge
screening of the anionic head group of SDS and cationic moi-
ety of CTAB by the nonionic molecules of 8, causing less
repulsion between the molecules.
FIGURE 6 CMC determination of N‐decanoyl tyrosine methyl ester (3).
CMC indicates ritical micelle concentration
CPC as quencher. The aggregation numbers were determined
by using Equations 1 and 2.
ꢀ
ꢁ
C−CMC
½Micelleꢀ
NSS
¼
(1)
(2)
ꢀ
ꢁ
I0
IQ
½Qꢀ
½Micelleꢀ
¼ exp
where I0 and IQ are the fluorescence intensities in the absence
and presence of CPC, respectively, C is the concentration of
surfactant added, and [Q] is the concentration of the quencher
CPC. The concentration of micelles [Micelle] was calculated
from the plot of ln (I0/IQ) versus [Q] and replaced in Equation
1 to determine Nss.
The ideal CMC of the SDS/CTAB–8 binary mixture can
be predicted by the Clint equation[28] (Equation 3):
The CMC and the aggregation numbers (Nss) of the
N‐acyl surfactants are shown in Table 4.
1
α8
ð1−α8Þ
¼
þ
(3)
cmcꢁ cmc8 cmcSDS=CTAB
As expected, increasing the chain length of the N‐acyl
tyrosine surfactants causes a decrease in the CMC and an
increase in aggregation number because of the greater hydro-
phobicity of the molecules. The presence of an unsaturated
system in the alkyl chain of 7 was found to increase the
CMC of the surfactant as previously reported, compared to
where α8 is the mole fraction of the 8 in the mixture, cmc8
and cmcSDS/CTAB correspond to the critical micelle concen-
trations of pure components 8 and SDS or CTAB, respec-
tively, and cmc* is the value under ideal mixing.
TABLE 4 Critical micelle concentration and aggregation number of N‐acyl
tyrosine surfactants
TABLE 5 CMC of SDS/CTAB–8 mixed system
Mole Fraction
Chain Length:
Double Bonds
CMC Value
Aggregation
Number (Nss)
Compound
(μM)
Surfactant 8
SDS
CMC (μM)
CTAB
CMC (μM)
2
3
4
5
6
7
8
10:0
12:0
14:0
16:0
18:0
18:1
12:0
203
117
25
6
13
17
18
22
12
20
0
1
8300
462
246
113
95
1
917
148
77
0.2
0.4
0.6
0.8
1
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
18
39
10
19
22
50
50
50
Abbreviations: CMC, critical micelle concentration; CTAB, cetyl trimethyl
ammonium bromide; SDS, sodium dodecyl sulfate.
Abbreviation: CMC, critical micelle concentration.

JOONDAN ET AL.
9
FIGURE 7 Variation of CMC with mole fraction of surfactant 8 in (A) SDS and (B) CTAB mixed systems. CMC indicates critical micelle concentration;
CTAB, cetyl trimethyl ammonium bromide; SDS, sodium dodecyl sulfate
The micellar molar fraction of 8 in the ideal state χidwas
From the equation, it is possible to obtain the molar frac-
8
tion of 8 in the mixed micelle, χ8 as a function of the surfac-
tant molar fraction in bulk.
evaluated according to Equation 4, assuming binary ideal
mixture.
The amount of surfactant 8 in the mixed micelle is supe-
rior to the value predicted for ideal mixing (Equation 3), sug-
gesting that the surfactant 8 becomes more prominent in the
mixed micelle become richer with than with CTAB or SDS.
A negative β12 value accounts for synergism while a pos-
itive value indicates antagonism behavior for the mixed
micelle formation.[29] In the SDS‐8 system, positive β12
values (0.57 to 8.85) were obtained from the different mole
ratios studied that indicates the antagonistic behavior within
the mixed micelle. However, for CTAB‐8 system, negative
β12 values (−3.30 to −15.33) were obtained that confirms
synergistic effect within the binary system.
α8cmcSDS=CTAB
χi8d
¼
:
(4)
α8cmcSDS=CTAB þ ð1−α8Þcmc8
The experimental value and theoretical value predicted by
the Clint equation for the CMC of each of the surfactant
binary mixtures are shown in Figure 7.
The experimental CMC values of 8 in a mixture of SDS
and CTAB showed a slight deviation from the values predicted
by the Clint model as can be seen from Figure 6, suggesting
nonideal behavior in the mixed micellar system. The CMC
values obtained for the SDS‐8 binary system were slightly
higher than those predicted by the Clint model that suggested
antagonistic behavior in the binary system. However, in the
case of CTAB‐surfactant binary mixture, the experimental
CMC was lower than those predicted by the Clint model sug-
gesting synergism in the mixed micelle formation.
The extent of nonideality of surfactant interactions is usu-
ally evaluated using the regular solution theory that includes
an interaction parameter, β12, to characterize the interactions
between the 2 components within the mixed micelles. This
parameter is related to the activity coefficients, f, of the sur-
factants within the micelle, according to
3.3 | Krafft temperature
The micelle forming ability of surfactants is responsible for
their surface active and detergent properties, rendering them
useful in many applications. However, surfactants exhibit
their micellar properties only above a certain temperature
called the Krafft temperature (TK), which is defined as the
minimum temperature above which the surfactants are able
to form micelles in aqueous solution.[30] Therefore, it is
essential to lower the TK of surfactants inferior to room tem-
perature to be able to render them practical for pharmaceuti-
cal and industrial use.[31] In this study, the TK of the
synergistic CTAB‐8 mixtures with varying mole fractions of
8 was studied. The TK of the mixtures was taken by the abrupt
change in conductivity versus temperature plot (Figure 8).
In pure water, the TK value obtained for CTAB was found
to be 22°C that is comparable to that reported in previous
report.[31] The TK of pure surfactant 8 could not be obtained
since it is uncharged and therefore it does not have a conduc-
tivity value. Usually, addition of electrolytes or surfactants
that lower the CMCs of surfactants tends to increase their
TK rendering them impractical for many applications.[32]
However, in this study, addition of surfactant 8 lowered the
TK of CTAB. Increase in mole fraction of surfactant 8 from
2
f 1 ¼ expβ12 ð1−χ8Þ ;
f 2 ¼ expβ12 χ8;
(5)
(6)
where χ8, the molar fraction of 8 in the mixed micelle, can be
obtained by solving the following equation iteratively:
χ28 lnðα8cmc=χ8cmc8Þ
ð1−χ8Þ ln½ð1−α8Þcmc=ð1−χ8ÞcmcSDS
¼ 1:
(7)
2
The interaction parameter β12 can then be evaluated from
lnðα8cmc=χ8cmc8Þ
(8)
β12
¼
:
2
ð1−χ8Þ

10
JOONDAN ET AL.
be associated with their intercalation into target cell mem-
brane that causes disturbance in some membrane processes
leading to cell death.
Presence of a cis double bond in 7 enhanced the anti-
bacterial activity over 6, which is the saturated analogue
of 7 with the same number of carbon atom (C18). The
enhanced antibacterial activity of compound 7 can be attrib-
uted to its better solubility compared to the saturated
stearoyl analogue and is more likely to possess the right
lipophilic/hydrophilic balance that allows the molecule to
disrupt the cell membrane of the microorganism as sug-
gested by Birnie et al.[33]
FIGURE 8 Krafft temperature of cetyl trimethyl ammonium bromide–8
system with α8 = 0.2. The arrow indicates the Krafft temperature
The Gemini surfactant 8 that consists of 2 alkyl chains
with 12 carbon atoms displayed enhanced antibacterial
activity because of an increase in the hydrophobic interac-
tion between the surfactant molecule and the bacterial mem-
brane. The surfactant 8 showed the best activity towards B
cereus among the gram‐positive bacteria tested while K
pneumonia was the most susceptible among the gram‐
negative strains.
α8 = 0 to α8 = 0.8 gradually decreased the TK from 22°C to
12°C (Figure 9) that might be due to the increase in syner-
gism in the mixed micelle that facilitates micelle formation
at a lower temperature.
Subsequently, the antibacterial activity of the binary mix-
ture of 8, with conventional anionic and cationic surfactants
SDS and CTAB, respectively, was evaluated against B cereus
and K pneumonia. The results are shown in Table 7.
Overall, the CTAB‐8 binary mixture showed better anti-
bacterial activity compared to that of the SDS‐8 mixture.
Sodium dodecyl sulfate and 8 exhibited comparable antimi-
crobial properties against the bacteria tested. It was found that
for the SDS‐8 binary mixture, an increase in mole fraction of
8 from 0 to 0.2 increases the antibacterial activity and further
increase in α8 causes a decrease in the antibacterial activity.
In the case of CTAB‐8 binary mixture, an increase in the
mole fraction of 8 increases the antibacterial activity up to
α8 = 0.6 and then decreases with further increase in mole
fraction of 8.
3.4 | Antibacterial activity
The antibacterial activity of the N‐acyl surfactants was deter-
mined, and the MICs are given in Table 6.
Overall, the N‐acyl surfactants displayed moderate to
good antibacterial activity. Among the straight chain surfac-
tants tested, the C12 derivative (3) displayed the best activity
among the series. The mechanism of activity is suggested to
To be able to evaluate the activity of combinations of
agents, fractional inhibitory concentration (FIC) indices were
calculated using the following formula:
*
FIC = (MIC8 /MIC8 alone) + (MICSDS or CTAB* in com-
bination/MICSDS or CTAB),
where MIC8* and MICSDS or CTAB* are the MICs of 8 and
SDS/CTAB in combination, respectively; MIC8 and MICSDS
FIGURE 9 Effect of surfactant 8 on TK of cetyl trimethyl ammonium
bromide–8 mixture
are the MICs of 8 and SDS/CTAB, respectively.
or CTAB
TABLE 6 Minimum inhibitory concentration of the N‐acyl Tyrosine surfactants
MIC (mM)
Microorganisms
2
3
4
5
6
7
8
CTAB
Gram positive
S aureus
6.25
6.25
1.56
0.78
1.56
6.25
6.25
6.25
12.5
12.5
12.5
12.5
12.5
12.5
12.5
1.17
1.56
3.13
1.56
3.13
3.13
0.78
0.78
0.15
3.13
6.25
6.25
13.39
3.35
3.35
53.5
62.5
53.5
S epidermidis
B cereus
9.38
12.5
6.25
3.13
6.25
9.38
Gram negative
K pneumoniae
P aeruginosa
S typhimurium
6.25
9.38
12.5
6.25
12.5
9.38
6.25
9.38
12.5
Abbreviation: CTAB, cetyl trimethyl ammonium bromide; MIC, minimum inhibitory concentration.

JOONDAN ET AL.
11
TABLE 7 Antibacterial activity of surfactant 8 in SDS and CTAB mixtures
MIC (mM)
Mole Fraction Surfactant 8
0
0.2
0.4
0.6
0.8
1
B cereus
SDS
CTAB
0.195
0.003
0.097
0.003
0.293
0.002
0.390
0.0015
0.390
0.0183
0.15
0.15
K pneumoniae
SDS
CTAB
1.56
0.037
0.39
0.048
0.59
0.073
0.78
0.39
3.12
3.12
3.13
3.12
Abbreviations: CTAB, cetyl trimethyl ammonium bromide; MIC, minimum inhibitory concentration; SDS, sodium dodecyl sulfate.
The interpretation made was as follows: for synergy
FIC ≤ 0.5; partial synergy FIC > 0.5 but <1; and additive
FIC = 1.0 and antagonistic when values were >1.0.[34,35]
The classification of surfactant 8 in the different surfactant
mixtures is shown in Table 8.
From the FIC values with respect to B cereus, it was
found that in the SDS mixture, 8 has an additive effect at
mole fraction 0.2 and displayed antagonistic effect with fur-
ther increase in mole fraction. The CTAB‐8 mixtures showed
additive behavior at mole fraction α8 = 0.2, displaying partial
synergism to synergistic behavior with increasing mole frac-
tion of 8 up to 0.6. However, further increase in α8 resulted in
antagonistic behavior. In the case of K pneumonia, the SDS
mixture showed synergism to partial synergism at mole frac-
tion α8 = 0.2 to 0.6 and showed antagonism at mole fraction
α8 = 0.8. The CTAB mixtures showed antagonistic effect
against K pneumonia.
FIGURE 10 Foaming ability of surfactant 8 in CTAB and SDS mixtures.
CTAB indicates cetyl trimethyl ammonium bromide; SDS, sodium dodecyl
sulfate
of the mixture compared to surfactant 8‐SDS mixture that
displays antagonistic behavior. Hence, it may be assumed that
in this case, synergism plays an important role in the foaming
ability of the mixed surfactant systems.
3.5 | Foaming ability
Foaming is a special feature of surfactants that makes them
useful in different applications, such as household detergents
and cosmetics. The N‐acyl tyrosine surfactants were found to
possess promising antibacterial activity as single or mixed
systems. In view of investigating their potential use in deter-
gent formulations, their foamability was investigated. In gen-
eral, the N‐acyl tyrosine surfactants were found to have very
poor foaming ability.
Addition of SDS or CTAB to an aqueous solution of 8
was found to enhance the foaming ability of the solution
(Figure 10). Surfactant 8‐CTAB mixtures showed better
foaming ability compared to surfactant 8‐SDS mixtures. This
may be due to the synergistic behavior of the surfactant
8‐CTAB mixed system, which enhances the foaming ability
3.6 | Hemolytic activity and ocular irritation
The red blood cell assay is a renowned method used for the
estimation of potential irritation of surfactants and deter-
gents. This method has been known to provide reliable
results on the in vitro effects of test substances without
involving the use of animal testings. The red blood cell
assay is based on the degree of hemolysis and cell protein
denaturation caused by the surfactants and the ratio of both
parameters.[36]
The binary mixture of the surfactant 8, with conven-
tional anionic and cationic surfactants SDS and CTAB,
respectively, was found to possess interesting physicochem-
ical and antibacterial activities. To be able to evaluate the
TABLE 8 FIC indices
FIC
FIC
Mole
Fraction
of 8
FIC (B cereus)
in Combination
with CTAB
(K pneumoniae) in
Combination with
CTAB
FIC (B cereus)
in Combination
with SDS
(K pneumoniae) in
Combination
with SDS
Inference
Inference
Inference
Inference
0.2
0.4
1.00
0.68
Additive
1.31
1.99
Antagonistic
Antagonistic
1.1
3.5
Additive
0.37
0.57
Synergistic
Partial
Antagonistic
Partial
synergy
synergy
0.6
0.8
0.50
6.20
Synergistic
10.7
85.5
Antagonistic
Antagonistic
4.6
4.6
Antagonistic
Antagonistic
0.75
2.00
Partial
synergy
Antagonistic
Antagonistic
Abbreviation: CTAB, cetyl trimethyl ammonium bromide; FIC, fractional inhibitory concentration; SDS, sodium dodecyl sulfate.

12
JOONDAN ET AL.
TABLE 9 Hemolytic activity and in vitro classification of compound 8 in SDS and CTAB surfactant mixtures
L/D
Ratio
In Vitro
Classification
L/D
Ratio
In Vitro
Classification
αcompound 8
HC50 in CTAB (μg mL−1
)
DI %
HC50 in SDS (μg mL−1
)
DI %
0
1.82
191
22
95
0.08
2
Irritant
43.6
203
100
16.5
70.2
23.3
65.0
62
0.4
Irritant
0.2
0.4
0.6
0.8
1
Moderate irritant
Moderate irritant
Moderate irritant
Moderate irritant
Moderate irritant
12.3
4.94
15.0
5.47
2.57
Weak irritant
Moderate irritant
Weak irritant
Moderate irritant
Moderate irritant
300
71.7
25
4
347
233
9.32
3.16
2.57
350
215
68
356
159.3
62
159.3
Abbreviation: CTAB, cetyl trimethyl ammonium bromide; SDS, sodium dodecyl sulfate.
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4
| CONCLUSION
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N‐acyl surfactants with varying chain length were success-
fully synthesized in good yields. Increasing the chain length
of the hydrophobic portion of the surfactants favors micellar
formation while the presence of an unsaturated system was
found to decrease the ease of micellization. The N‐acyl tyro-
sine surfactants showed moderate to good antibacterial activ-
ity that was affected by both chain length and level of
unsaturation of the hydrophobic tail. Among the single chain
N‐acyl surfactants, the C12 derivative (surfactant 3) displayed
the highest antibacterial activity. The presence of double
bond in the oleoyl derivative enhanced the antibacterial activ-
ity over its saturated analogue. The Gemini surfactant 8
displayed the highest activity among all the compound tested.
The study of binary mixtures of compound 8 with conven-
tional surfactants shows that surfactant 8 displayed antagonis-
tic behavior in SDS and synergistic behavior in CTAB mixed
micellar systems. The synergistic CTAB‐8 mixtures
displayed better antibacterial activity and foaming properties
than the SDS‐8 mixtures. The CTAB‐8 mixture with α8 = 0.4
to 0.6 showed the highest antibacterial activity and lowest
ocular irritation index that makes it a good candidate in its
use as detergents and antiseptic.
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