Cationic surfactants from lysine: Synthesis, micellization and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2008.11.003

Biocompatible cationic surfactants from the amino acid lysine (hydrochloride salts of N^ε-lauroyl lysine methyl ester, N^ε-myristoyl lysine methyl ester and N^ε-palmitoyl lysine methyl ester) have been prepared in high yields

Full text of DOI:10.1016/j.ejmech.2008.11.003

European Journal of Medicinal Chemistry 44 (2009) 1884–1892
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Cationic surfactants from lysine: Synthesis, micellization and biological evaluation
a
,
a
a
a
b
*
´
´
Lourdes Perez , Aurora Pinazo , M. Teresa Garcıa , Marina Lozano , Angeles Manresa ,
Marta Angelet a, M. Pilar Vinardell c, Montse Mitjans c, Ramon Pons a, M. Rosa Infante a
a Departamento de Tecnolog´ıa de Tensioactivos, Instituto de Qu´ımica Avanzada de Catalun˜a, CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain
b
`
Laboratori de Microbiologia, Facultat de Farmacia, Universitat de Barcelona, Joan XXIII s/n, 08028 Barcelona, Spain
c
`
Departament de Fisiologia, Facultat de Farmacia, Universitat de Barcelona, Joan XXIII s/n, 08028 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Biocompatible cationic surfactants from the amino acid lysine (hydrochloride salts of N3-lauroyl lysine
methyl ester, N3-myristoyl lysine methyl ester and N3-palmitoyl lysine methyl ester) have been prepared
Article history:
Received 1 October 2008
Received in revised form
3 November 2008
in high yields by lysine acylation in
3 position with three natural saturated fatty acids. The micellization
process of these surfactants has been studied using the PGSE-NMR technique. The compounds were
tested as antimicrobial agents against Gram-positive and Gram-negative bacteria. The surfactants show
moderate antimicrobial activity against the Gram-positive bacteria but Gram-negative bacteria are
resistant to these surfactants in the concentration range tested. The haemolytic activity is considerably
lower than those reported for other cationic Na-acyl amino acid analogues. The acute toxicity against
Daphnia magna and biodegradability was studied. The toxicity is clearly lower than that reported for
conventional cationic surfactants from quaternary ammonium and the three surfactants from lysine can
be classified as ready biodegradable surfactants.
Accepted 6 November 2008
Available online 14 November 2008
Keywords:
Lysine
Surfactants
Haemolysis
Antimicrobial properties
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
acid surfactants have aroused considerable interest. Nowadays,
cationic surfactants are being tested in new biomedical applica-
Although surfactants are widely used in both consumer and
industrial applications, they can adversely affect the environment.
Therefore, there is a growing demand for mild, biodegradable and
nontoxic products that are made from natural raw materials.
Moreover, there is also a demand for multifunctional compounds in
order to reduce cost and to reduce the amount of chemicals added
to wastewater. Accordingly, preparation of surfactants which mimic
the structure of natural compounds such as lipoaminoacids,
phospholipids and glycerolipids has assumed a new importance
because of their unique physicochemical and biological properties
[1]. Amino acid based surfactants with one or two hydrocarbon tails
have been described in the literature [2,3] particularly salts of long
chain Na-acyl amino acids that are currently used as detergents,
foaming agents and shampoos because they are mild, non-irritating
to the human skin and highly biodegradable [4].
tions such as drug delivery systems in cationic vesicles. The use of
cationic amphiphiles for mediating DNA transfection has become
widespread ever since it was shown that cationic lipids, known as
cytofectins, are capable of delivering functional genes [12]. Cationic
amino acid based surfactants are good candidates to act as trans-
fection agents given their low toxicity, high biodegradability and
their structural characteristics suitable for DNA transfection. Some
surfactants based on dibasic amino acids have been considered for
interaction with DNA [13,14] and the most promising results have
been obtained with lysine and arginine [15]. In this field it is
necessary to strike a balance between antimicrobial activity on the
one hand and low toxicity and efficient biodegradability on the
other. For these uses, it would be suitable the use of cationic
surfactants with low antimicrobial activity because typically they
present high biodegradability and less toxicity. For all these
reasons, the knowledge of the aggregation behaviour as well as the
biological properties (in particular biodegradation and cytotoxicity)
is essential to establish structure/activity relationships.
In the last two decades, our group has published a number of
papers addressing the synthesis and properties of biocompatible
cationic amino acid based surfactants of different structures [5–11].
These surfactants show a low toxicity profile and an antimicrobial
activity similar to those of conventional cationic surfactants. From
the health and the environmental points of view, cationic amino
In this paper, we describe the chemical synthesis and charac-
terization of the hydrochloride salts of N3-acyl lysine methyl ester
with alkyl chain lengths of 12 (LKM), 14 (MKM) and 16 (PKM)
carbon atoms (Fig. 1). These amino acid based cationic surfactants
have a hydrophobic chain attached to the
lysine through an amide bond. We focus on the systematic study of
3-amino group of the
* Corresponding author. Tel.: þ34 93 4006164; fax: þ34 93 2045904.
´
E-mail address: lpmste@cid.csic.es (L. Perez).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.11.003

L. Pe´rez et al. / European Journal of Medicinal Chemistry 44 (2009) 1884–1892
1885
compounds was carried out by silica gel chromatography and
satisfactory HPLC analyses were obtained for these materials
yielding FABMS and NMR spectra, which were consistent with the
target compounds. (3) Deprotection of the Cbz group by catalytic
hydrogenation of the corresponding N3-acyl-Na-Cbz-lysine methyl
ester using Pd over charcoal. The reaction was carried out by
controlling the pH to prevent the hydrolysis of the ester linkage
present in these compounds. Pure compounds were obtained after
several crystallisations in methanol/acetonitrile. The chemical
structure of these compounds was checked by NMR. The proton
and carbon NMR spectra were in concordance with the proposed
structure. Elemental analyses for the derivatives were in good
agreement with the calculated ones.
CH3
O
O
H
NH3+Cl-
O
3. Pharmacology
3.1. Antimicrobial activity
N
H
n
Antimicrobial tests were performed using bacteria and fungi
which are stored in our laboratory. Microorganisms were Bacillus
cereus var. mycoides, Enterococcus hirae, Micrococcus luteus,
Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermis,
Mycobacterium phlei, Candida albicans, Klebsiella pneumoniae,
Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa,
Bordetella bronchiseptica, Serratia marcescens, Enterobacter aerogenes.
Fig. 1. Molecular structure of the N3-acyl lysine methyl ester surfactants: n ¼ 10 LKM,
n ¼ 12 MKM and n ¼ 14 PKM.
biological and surface properties of these lysine derivatives in order
to determine their possible use in new biomedical applications.
In the relevant literature we can find several works concerning
the synthesis and properties of Na-acyl lysine [16] derivatives but
the studies referring to N3-acyl lysine compounds [17–19] are
scarce and none of these works reported a systematic study on the
biocompatibility and micellization process of these compounds.
3.2. Haemolytic activity
The determination of the haemolytic activity was made on
human red blood cells obtained from healthy donors by veni-
puncture (Blood Bank of Hospital Vall d’Hebron, Barcelona, Spain),
following the ethical guidelines of the Hospital, and collected in
citrated tubes.
2. Chemistry
With the aim to systematically study the influence of the alkyl
chain length on the properties of long chain N3-acyl lysine methyl
ester salts we synthesized at multigram scale three pure homo-
logues with alkyl chain of 12 (LKM), 14 (MKM) and 16 (PKM) carbon
atoms (Fig. 1). The procedure used for the synthesis of these
compounds was easy and very efficient. It consisted of three steps
(Fig. 2) using the Na-Cbz-lysine$HCl as starting material. (1) Prep-
4. Results and discussion
4.1. PGSE-NMR results
PGSE-NMR was used to determine self-diffusion coefficients
[20] in aqueous solutions of LKM and MKM at 25 ꢀC. The PKM has
been studied at 50 ꢀC owing to the very low solubility of this
surfactant at 25 ꢀC.
The micellization process for each surfactant was followed by
measuring samples with a surfactant concentration below CMC and
above the CMC (Fig. 3). For all the systems considered, the intra-
diffusion coefficient of the surfactant ion, D, plotted as a function of
aration of the Na-Cbz-lysine methyl ester HCl salt (
group protection). (2) Synthesis of N3-acyl-Na-Cbz-lysine methyl
ester by acylation of
-amino group of the Na-Cbz-lysine methyl
a-carboxylic
3
ester HCl salt with the corresponding long chain acid chloride. The
reaction progress was monitored by HPLC, after 4 h a 92% conver-
sion of the starting reagent was obtained. The isolation of pure
O
O
H
N
MeOH
SOCl2
H
N
O
O
+
+
HCl
SO2
OH
O
O
O
NH3+Cl-
NH3+Cl-
N -Cbz-Lysine HCl
N -Cbz-Lysine methyl ester HCl
O
O
O
H
O
H
N
+H3N
Cl-
O
N
O
O
O
O
Pd/C
H2
Pyr
O
+
O
Cl
n
O
O
Me(OH)/HCl
N
+
NH3 Cl-
n
N
H
n
H
N -Cbz-Lysine methyl ester HCl
N -Cbz-N -acyl-Lysine methyl ester
Fig. 2. Synthetic procedure used to obtain the lysine based surfactants.
N -acyl-Lysine methyl ester HCl

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L. Pe´rez et al. / European Journal of Medicinal Chemistry 44 (2009) 1884–1892
A
4,0
3,5
3,0
2,5
2,0
1,5
1,0
0,5
1
10
Concentration (mM)
B
6
5
4
3
2
1
1
concentration (mM)
Fig. 3. Diffusion coefficients versus concentration. (A) LKM (-) and MKM (;) at 25 ꢀC, (B) PKM at 50 ꢀC.
the surfactant molarity shows a change of slope at the onset of
micellization, allowing for the determination of CMC. The moni-
toring of the association process is based on the difference in the
self-diffusion coefficient for the surfactant monomers and micelles.
The association process of these surfactants is reflected in a sensi-
tive manner by the decrease of their self-diffusion coefficients. The
effective translational mobility of the surfactant is considerably
reduced because they diffuse in the form of an aggregate. Moreover,
the proton spectra at high concentrations show typical high-reso-
lution features with Lorentzian bandshapes, which indicates the
presence of spherical micelles. Large aggregates give rise to proton
spectra with little structure and broadened signals [21]. From the
chemical structures of these surfactants, the formation of spherical
micelles is expected. The aggregate type formed by the surfactants
depends on the surfactant packing parameter P ¼ v/al were v and l
are the volume and the length of the surfactant alkyl chain and a is
the surface area occupied by the head group [22]. Surfactants with
P < 1/3 give rise to spherical micelles. The N3-lysine derivatives
have one polar head and one hydrophobic chain, these compounds
usually present conical shape with P < 1/3, therefore they form
spherical micelles. Studies carried out with one cationic surfactant
from arginine, the Na-lauroyl-arginine methyl ester, LAM (Fig. 4),
surfactant with one cationic head group and the same alkyl chain,
demonstrate that this compound forms spherical micelles [23,24].
Given the rapid exchange between free and micellized surfac-
tant molecules, in the micellar composition range the experimental
surfactant intradiffusion coefficient is a mean value between that of
free monomers, Dfree, and that of the micellized molecules, Dmic
.
This can be described using the two-site exchange model [23].
Dobs ¼ pmicDmic þ ð1 ꢁ pmicÞDfree
(1)
where pmic ¼ (c ꢁ CMC)/c is the fraction of micellized surfactant and
Dobs, Dmic and Dmon are the observed mean diffusion coefficient, the
self-diffusion coefficient of the micelles and the monomers,
respectively. Table 1 shows the Dmic, the Dmon and the CMC for the
CH3
O
CH3
O
O
H
O
O
H
CH3
O
10
NH
CH3
10
NH
H2N
Cl-
NH
LLM
LAM
+Cl-
NH3
H2N
Fig. 4. Molecular structure of the Na-lauroyl lysine methyl ester (LLM) and Na-lauroyl-
arginine methyl ester (LAM).

L. Pe´rez et al. / European Journal of Medicinal Chemistry 44 (2009) 1884–1892
1887
Table 1
that this compound forms large micelles. For this surfactant, post-
micellar concentrations are near the two phases region boundary
(micellar solution and hydrated solid), because of that, higher
interaction between micelles and micelle growth is expected, some
micelles can diffuse together giving rise to lower micellar diffusion
coefficients and consequently the calculated hydrodynamic radius
is bigger than expected.
Dmic, Dmon and the CMC for the N3-acyl lysine surfactants.
Surfactant
CMC (mM)
Dmon ꢂ 1010 (m2 sꢁ1
)
Dmic ꢂ 1010 (m2 sꢁ1
)
Rh (nm)
LKM
MKM
PKMa
5.5
1.6
0.6
3.9
3.3
5.8
1.03
0.37
1.4
1.9
5.4
2.6
a
Diffusion coefficients determined at 50 ꢀC.
The micellar parameters obtained for LKM (CMC, Dmic, Rh) are
similar to those obtained for LAM (arginine derivative with the
same number of carbon atoms in the hydrophobic part [23]). The
results indicate that the cationic charge type in these ionic amino
acid surfactants does not affect the micellization process; this
process is mainly controlled by the hydrophobic part.
two surfactants obtained by fitting Eq. (1) to the descending parts
of the experimental curves (Fig. 3). This table also shows the
micellar parameters obtained for PKM at 50 ꢀC.
Micellization (or CMC value) of these surfactants from lysine
takes place at 5.5 mM for the LKM, and 1.6 mM for MKM. As
expected, the CMC decreases when the alkyl chain increases as
a consequence of the higher hydrophobic content of the molecule.
The CMC of the PKM at 50 ꢀC is 0.60 mM.
At constant temperature, for homologous straight-chain ionic
surfactants in aqueous medium, the relation between the CMC and
the number of carbon atoms N in the hydrophobic chain takes the
form [25]
4.2. Antimicrobial activity
Minimum inhibitory concentration (MIC) values for LKM, MKM
and PKM compounds are summarised in Table 2. Due to the low
solubility of MKM and PKM, they were dissolved in the minimum
possible amount of dimethylformamide (DMF) and then diluted in
the medium to the testing concentration [26].
To ensure that the solvent had not effect on the microorganism
growth, a control test was performed with the test medium sup-
plemented with DMF as the same concentration used in the
experiments. For the sake of comparison, the MICs of two amino
acid cationic surfactants, one from arginine, LAM (Na-lauroyl-
arginine methyl ester hydrochloride) and the other from lysine LLM
[9] (Fig. 4) (Na-lauroyl lysine methyl ester hydrochloride) have been
assessed. In these surfactants (LAM, LLM), the alkyl chain is linked
log CMC ¼ A ꢁ BN
where A and B are constants that depend on the molecular struc-
ture of the surfactant.
From the CMC values of the LKM and MKM it is possible to
estimate the log CMC ¼ A ꢁ BN relation corresponding to these
analogues at 25 ꢀC (Fig. 5). Considering this relation the expected
CMC of the PKM at 25 ꢀC would be 0.48 mM. This value is slightly
lower than those obtained at 50 ꢀC (0.6 mM). This fact indicates
that the temperature induces only small changes in the CMC of this
compound. Usually, in cationic surfactants the CMC slightly
increases with the temperature.
(2)
to the
a
-amino group of the corresponding amino acid and the
-amino group (Fig. 4, Table 2). Table 2
positive charge lies in the
3
also includes the MIC values corresponding to the hexadecyl tri-
methyl ammonium bromide [27], a classical antimicrobial surfac-
tant agent. The three N3-acyl lysine methyl ester hydrochlorides
show antimicrobial activity against a wide range of Gram-positive
bacteria. The activity against Gram-positive bacteria is similar to
that shown by the LLM and LAM. Compared with the classical
quaternary ammonium surfactants (QAS) [27,28] these substances
show a moderate activity level against bacteria with MIC values of
From the self-diffusion coefficient of the micellized surfactant,
the hydrodynamic radius (Rh) (Table 1) can be calculated using the
Stokes–Einstein equation.
Dmic ¼ KBT=6Rhph
where KB is the Boltzmann constant, T is the temperature,
(3)
h
is the
150–350 mM. However the use of QAS in some fields is limited due
viscosity of the solvent and Rh is the hydrodynamic radius of the
aggregates.
Similar micellar sizes have been obtained for LKM and PKM but
the big value of hydrodynamic radius obtained for MKM suggests
to the developed microbial resistance against QAS [29], their high
acute toxicity and low biodegradability [30]. For medical applica-
tions, the use of cationic surfactants with low antimicrobial activity
would be necessary because they use to present high biodegrad-
ability and less toxicity.
In general, the antimicrobial activity of the surfactants depends
on the alkyl chain length, however the correlation between the
alkyl chain length and the microbial activity is not linear. For the
long chain N3-acyl lysine methyl ester series under study the chain
length does not affect the antimicrobial properties. The only
difference is that the LKM presents activity against C. albicans and
K. pneumoniae whereas the MKM and PKM are not active against
these bacteria at the highest concentration tested.
On the other hand, Gram-negative bacteria (except E. coli) are
resistant to these surfactants in the concentration range tested. By
contrast, LAM and LLM surfactants present a similar activity against
both Gram-positive and Gram-negative bacteria. Other cationic
lysine derivatives also show a similar activity against Gram-positive
and Gram-negative bacteria [31,32]. Gram-negative bacteria are
generally more resistant to antimicrobial agents than are Gram-
positive bacteria. This can be explained by the different cell
membrane structure of the two bacterial types. The external layer
of the outer membrane of the Gram-negative bacteria is almost
entirely composed of lipopolysaccharides and proteins that restrict
the entrance of biocides and amphiphilic compounds [33]. The
perturbation of this outer membrane requires a fine tuning of the
1
12
13
Carbon atoms in the alkyl chain
14
15
16
Fig. 5. CMC versus carbon atoms in the hydrophobic chain for the N3-acyl lysine
surfactants.

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L. Pe´rez et al. / European Journal of Medicinal Chemistry 44 (2009) 1884–1892
Table 2
MIC values (
m
M) for compounds LKM, MKM, PKM, LLM (Na-lauroyl lysine methyl ester), LAM (Na-lauroyl-arginine methyl ester) and hexadecyl trimethyl ammonium bromide
(HTAB).
Microorganism
Gram positives
MIC mM (mg/L)
LKM
MKM
PKM
LLM
LAM
HTAB
Bacillus cereus var. mycoides
Enterococcus hirae
Micrococcus luteus
Staphylococcus aureus
Bacillus subtilis
Staphylococcus epidermis
Mycobacterium phlei
Candida albicans
169 (64)
338 (128)
338 (128)
338 (128)
338 (128)
169 (64)
169 (64)
169 (64)
157 (64)
157 (64)
157 (64)
157 (64)
157 (64)
157 (64)
78 (32)
R
148 (64)
74 (32)
74 (32)
148 (64)
148 (64)
148 (64)
148 (64)
R
–
–
39 (16)
39 (16)
39 (16)
39 (16)
157 (64)
20 (8)
–
–
11 (4)
44 (16)
–
44 (16)
10 (4)
–
–
85 (32)
42 (16)
42 (16)
–
–
39 (16)
157 (64)
Gram negatives
Klebsiella pneumoniae
Escherichia coli
Salmonella typhimurium
Pseudomonas aeruginosa
Bordetella bronchiseptica
Serratia marcescens
338 (128)
169 (64)
R
R
R
R
R
R
R
R
85 (32)
42 (16)
85 (32)
169 (64)
42 (16)
338 (128)
–
79 (32)
157 (64)
157 (64)
157 (64)
157 (64)
315 (128)
157 (64)
44 (16)
44 (16)
–
–
20 (8)
–
–
157 (64)
R
R
R
R
R
R
R
R
R
R
Enterobacter aerogenes
R: resistant microorganism at the highest concentration tested (128 mg/mL); –: not available result.
hydrophobic–hydrophilic balance of the surfactants. The difference
between LKM, LLM and LAM lies in the polar group. The polar group
of LKM consists of the lysine amino acid with a positive charge in
magna 24-h immobilisation tests [38] (IC50) of the investigated
surfactants are given in Table 3. The lower the IC50 value, the higher
the toxicity of the compound. PKM has not been tested owing to the
very low solubility of this surfactant in the medium.
the
a
-amino group (pKa ¼ 8.9). The polar group of the LLM is based
on the lysine amino acid with a positive charge in the
3
-amino
The acute toxicity of the cationic surfactants from lysine was
clearly lower than the toxicity reported for conventional mono-
quats [30], and even lower than that reported for other environ-
mentally friendly cationic surfactant molecules such as arginine
based Gemini cationic surfactants [39] and single chain arginine
surfactants [40] with two cationic charges in the head group. Toxic
activity is comparable to that shown by LAM (Table 3). Our data
indicated that LKM exhibited lower aquatic toxicity than the MKM.
Concerning the effect of the alkyl chain length, higher toxicity
would be expected with greater hydrophobicity in the molecule, as
reported for anionic surfactants on D. magna [41] and for alcohol
ethoxylated surfactants on Photobacterium phosphoreum [42] and
on D. magna [43]. This could explain the increase in toxicity
between these two compounds.
group (pKa ¼ 10.5) and the polar head in the LAM consists of the
arginine amino acid with a positive group in the guanidine function
(pKa ¼ 12.4). The main difference between these compounds is the
pKa associated to the protonated amino group on the polar heads.
The pKa of the protonated guanidine group of the arginine and
protonated
3-amino group of the lysine is considerably higher than
that of the protonated
a
-amino group of the lysine. Because the pKa
can be affected by aggregation, we have calculated the pKa of the
LKM and MKM at concentration values that show antimicrobial
activity [34] from pH measurements.
The apparent pKa values for these surfactants at these concen-
trations are similar to the pKa reported in the literature for the a-
amino group of the lysine (pKa ¼ 8.2 for LKM and pKa ¼ 7.8 for
MKM). At pH equal to the pKa, the amino acid will be 50%
protonated. This means that the average charge will be 0.5. As the
pH is decreased more than 2 units from the point of pH ¼ pKa it can
be considered that the amino acid is 100% protonated. This would
mean that the average charge is 1. Given that the pKa of the N3-
lysine derivatives is around 8 the average charge of the compound
could be lower than 1 at the pH of the test medium (pH ¼ 6), and
hence not suitable for disrupting the membrane of the Gram-
negative bacteria. These results allow us to state that the antimi-
crobial properties of pH-sensitive amino acid surfactants correlate
with the pH of the medium.
As for the effect of the compound net charge on its bactericidal
activity, numerous studies show that the electrostatic interactions
play a key role in the action of cationic systems, and that a decrease
in the charge density of the cationic compound results in a reduc-
tion in adsorption and bactericidal effects [35,36]. Accordingly, the
antibacterial activity varies with the pH in systems in which the net
charge depends on the pH. For example, a decrease of pH increases
the antimicrobial activity of chitosan and some peptides [37].
4.4. Biodegradability assessment
Biodegradation is the most important mechanism for the irre-
versible removal of chemicals from the aquatic and terrestrial
environments. It may be defined as the destruction of chemical
compounds by the biological action of living organisms. The
biodegradability of these surfactants was evaluated by applying the
Modified Screening Test [44]. In this test, the ultimate biodegra-
dation or mineralization of the surfactants (i.e., the microbial
transformation of the parent chemical into inorganic final products
of the degradation process, such as carbon dioxide, water, and
assimilated biomass) was evaluated. In the course of the biodeg-
radation test, the DOC (dissolved organic carbon) concentrations
were determined at the beginning and at regular time intervals for
a 28-day period. Biodegradation was stated as the percentage of
DOC removal within 28 days (Fig. 6).
Table 3
Aquatic toxicity values on Daphnia magna for the lysine derivative surfactants and
for the LAM.
4.3. Aquatic toxicity assessment
IC50 (mM)
It has been shown that the toxicity of surfactants against the
aquatic species is caused by the ability of the monomers to disrupt
Mean value
95% Confidence range
LAM
LKM
MKM
36
32
7.3
(27–54)
26–53
2–29
the integral membrane by
phenomenon at the cell membrane/water interface in a way similar
to that of the antimicrobial mode of action. The results of Daphnia
a hydrophobic/ionic adsorption

L. Pe´rez et al. / European Journal of Medicinal Chemistry 44 (2009) 1884–1892
1889
Table 4
100
80
60
40
20
0
Haemolytic assessment of the N3-acyl lysine surfactants and Na-acyl arginine methyl
ester (LAM).
Compound
HC50 (mM)
LAM
LLM
LKM
MKM
PKM
150
199
391
1232
1454
ester series showed significantly lower haemolytic activity than the
Na-acyl lysine and Na-acyl arginine compounds. The positive charge
nature could affect the capability of these surfactants to disrupt the
fragile cellular surface of the erythrocytes. Surfactant intercalation
into membrane leads to changes in the membrane molecular orga-
nization and increase of membrane permeability that concludes with
cell lyses [49]. However, haemolysis depends upon the adsorption of
the surfactant to components of the membrane surface which is
influenced by electrostatic attraction between the surfactant mole-
cules and membrane components, among other factors. In this sense
Shalel et al. [49], demonstrated that the ionic strength of the solution
diminishes the effects of the erythrocyte negative surface charge at
physiological pH and thus decreases the rate and amount of surfac-
tant incorporated into the cell membrane, and consequently reduces
the membrane permeability. The decrease of the haemolytic activity
could be associated with the decrease of the antimicrobial activity of
0
7
14
time (days)
21
28
Fig. 6. Biodegradation level obtained for the LKM (A), MKM (:) and sodium dodecyl
sulphate (-).
The biodegradation curve obtained for PKM was removed from
Fig. 6 because inconsistent results were obtained probably due to
the low solubility of this compound in the test medium. Biodeg-
radation percentages of approximately 75% at day 28, clearly
exceed the specified biodegradation pass level in this test (70%),
allowing to classify LKM and MKM as readily biodegradable
compounds. The conditions in this test are so stringent (relatively
low density of not preadapted bacteria, relatively short duration,
and absence of other sources of organic carbon) that chemicals
exceeding the specified biodegradation level will rapidly and
completely biodegrade in an aquatic environment under aerobic
conditions. These good results contrast with the low biodegrada-
tion level that usually reaches the cationic surfactants from
quaternary ammonium [45] and are similar to the findings on
cationic surfactants from arginine amino acids [46].
It should also be noted that these compounds reach the
biodegradation level in half the time proposed in the test. In fact,
this chemical structure has been designed to obtain ready biode-
gradable compounds. One of the strategies used by the bacteria to
access the carbon in surfactants is the initial separation of the
hydrophile from the hydrophobe (hydrophile attack). The amide
linkage between the alkyl chain and the lysine is readily attacked by
the microorganisms and then the fatty acids follow the pathway of
these surfactants. The change of the positive charge from the
group to the -amino group in the amino acid belongs to compounds
3-amino
a
with moderate antimicrobial activity and lower cellular toxicity.
The haemolytic activity of these lysine surfactants decreases
with increasing length of the hydrophobic tail. This behaviour
contrasts with those described for the cationic surfactants in witch
the haemolytic activity typically increases with increasing chain
length [50]. The decrease of the haemolytic activity with increasing
of the alkyl chain has been reported for partially fluorinated pyr-
idinium bromides [51]. On the other hand the haemolytic activity
showed for these lysine derivatives is clearly lower than those
found for classical cationic quaternary ammonium surfactants
as alkyltrimethylammonium salts [50], CTAB (hexadecyltrime-
thylammonium bromide) and DODAB (dioctadecyldimethyl
ammonium bromide) and for new Gemini QACs [52] (benzalko-
nium chloride) and partially fluorinated pyridinium bromides [53].
These results showed that the erythrocytes-disrupting ability of
these surfactants is correlated to the cationic charge type and to the
alkyl chain length.
For these lysine surfactants the HC50 decreases linearly as the
CMC increases. For LKM haemolysis occurs at monomer concen-
tration, whereas MKM induces erythrocyte lysis at concentrations
closer to its CMC and PKM at micellar concentrations. Different
studies were performed to demonstrate a positive correlation
between haemolytic action and the CMC of surfactants; however,
no clear conclusions were reported in the literature. In the case of
non-ionic surfactants, it has been pointed out that hydrophobic
interaction and micellization are the main driving forces in the
induction of haemolysis [54]. In a similar way, Gemini surfactants
[55] or antipsychotic drugs [56] presented good correlation
between haemolytic effects and CMC. In contrast, this good rela-
tionship cannot be established for different chemicals with
amphiphile properties [56,57].
chain-shortening through fatty acid
b-oxidation [47] and the
microorganisms completely degrade the naturally occurring lysine.
On the other hand, the resistance of Gram-negative bacteria to
these surfactants could facilitate the biodegradability process of
these molecules. The alkyl chain length does not affect the
biodegradation level, even the surfactant with 14 carbon atoms in
the molecule (which has a high hydrophobic character) exceeds the
biodegradation level in 15 days.
4.5. Haemolytic assessments
Haemolysis by surfactants is a process of great fundamental and
practical importance. The human erythrocyte lacks internal
organelles and since it is the simplest cellular model obtainable, it is
the most popular cell membrane system to study the surfactant–
membrane interaction [48]. For all these reasons it is adopted as
a convenient model system. In addition, the potential uses of
surfactants as drug delivery systems make of great importance the
evaluation of haemolysis.
5. Conclusions
Evaluation of the concentration that induces the haemolysis of
50% of the erythrocytes (HC50) was determined and quantified from
plots of percentage haemolysis as a function of amphiphile concen-
tration (Table 4). Interestingly, the long chain N3-acyl lysine methyl
This work combines the study of the biological and physico-
chemical properties of the N3-acyl lysine surfactants. The results
obtained have been compared with those reported for Na-acyl
amino acid surfactants with the aim of knowing how the cationic

1890
L. Pe´rez et al. / European Journal of Medicinal Chemistry 44 (2009) 1884–1892
charge type affects the behaviour of these kinds of compounds.
Satisfactory synthesis for the preparation of long chain N3 acyl
lysine methyl ester hydrochloride salts as surfactants was carried
out in a three-step procedure. These compounds act as moderate
antimicrobial agents against Gram-positive bacteria. The haemo-
lytic activity is considerably lower than those reported for other
cationic surfactants, Na-amino acid or ammonium quaternary type.
As regards the aquatic toxicity, they present a good behaviour,
moderate toxicity and high biodegradation level. The surfactants
form micelles at millimolar concentrations. The results clearly
show that the positive charge nature affects the antimicrobial
properties as well as the haemolysis activity but not the biode-
gradability and micellization parameters. Taking into account the
high biodegradation level and the low haemolytic activity of these
compounds could be considered safe surfactants in relation to the
cell of the human body. These properties make the compounds
suitable candidates for biological applications such as the
construction of cationic synthetic delivery systems.
Methanol, excess thionyl chloride and chloride acid formed
during the reaction were eliminated by successive vacuum evapo-
rations. After lyophilisation a white solid corresponding to the title
product was obtained.
Analytical data and spectral assignments for Na-Cbz-lysine
methyl ester$HCl.
Yield: 97%. HPLC, tr ¼ 5.88 min. MW 330.5. Elem. Analy. Found:
C, 51.13; H, 6.89; N, 8.11; Calcd. for C15H23O4N2Cl$1H2O: C, 51.65; H,
7.17; N, 8.03. 1H NMR: dH (CD3OD): 1.42–1.89 [m, 6H, 3CH2 side
chain of lysine group], 2.85 [t, 2H (–CH2–NH2), 3.74 [s, 3H (–COO–
CH3)], 4.21 [m, 1H (–NH–CH–COO–)], 5.17 [s, 2H (Cbz–CH2–)], 7.25–
7.31 [m, 5H of the Cbz group (Z)].
13C NMR: dC (CD3OD): 23.83, 28.60, 32.05 [–CH2– side chain of
lysine], 40.49 [–CH2–NH2], 52.75 [–COO–CH3], 55.12 [–NH–CH–
COO–], 67.67 [Cbz–CH2–], 128.81–129.48 [6C of the Z group (Cbz)],
158.69 [COO–NH of the Z group (Cbz)], 174.38 [–COO–CH3].
6.2.2. Synthesis of N3-acyl-Na-Cbz-lysine methyl ester
A solution of pyridine (30 mL) containing 0.015 mol (5 g) of Na-
Cbz-lysine methyl ester$HCl was placed in a round-bottomed flask.
To this solution, 0.015 mol of the corresponding acyl chloride was
dropwise added and stirring continued at room temperature for
4 h. After completion of the reaction, pyridine was removed by
vacuum pump. The resulting solid was dissolved in methanol
(25 mL) and extracted three times with petroleum ether to remove
the corresponding fatty acid excess. The solvent was evaporated
and the solid was dissolved in chloroform (25 mL). Thereafter, this
solution was shaken with water to eliminate water-soluble impu-
rities. Silica acid flash chromatography: 100 mL of silica (Chromagel
60A CC, 70–230) was packed into a flash chromatography column.
Then 3 g of the N3-acyl-Na-Cbz-lysine methyl ester dissolved in
chloroform was loaded into the column and was eluted from
chloroform to chloroform/methanol. The fractions containing the
pure target products were pooled. The identification of the prod-
6. Experimental protocols
6.1. Chemistry
All solvents were reagent grade and were used without further
purification. The acyl chlorides with 12, 14 and 16 carbon atoms
were from Fluka. The progress of the reactions was monitored by
HPLC, model Merck-Hitachi D-2500 using UV–VIS detector L-4250
at 215 nm. A Lichrospher 100 CN (propylcyano) 5-
m
m, 250 ꢂ 4 mm
column was used. A gradient elution profile was employed from the
initial solvent composition of A/B 75/25 (by volume), changing
during 24 min to a final composition of 5/95 where solvent A is 0.1%
(vol/vol) trifluoro-acetic acid (TFA) in H2O and solvent B is 0.085% of
TFA in H2O/CH3CN 1:4. The flow-rate through the column was
1.0 mL minꢁ1
.
The structures of the pure compounds were checked by 1H and
13C nuclear magnetic resonance (NMR) analyses which were
recorded on a Varian spectrometer at 499.803 (1H) and 125.233
ucts was carried out by HPLC, Elemental analysis, HNMR and
1
13
CNMR.
(
13C) MHz, respectively, using the deuterium signal of the solvent as
6.2.2.1. Analytical data and spectral assignments for N3-acyl-Na-Cbz-
lysine methyl ester
the lock. Chemical shifts (d) are reported in parts per million (ppm)
downfield from tetramethylsilane (TMS). All measurements were
carried out on 0.6 mL samples in 5 mm tubes using a 5 mm indirect
broadband probe. 13C NMR spectra were recorded under composite
decoupling to eliminate 13C–1H coupling. The distortionless
enhancement by polarization transfer spectra (Dept) were run in
a standard way to separate the CH/CH3 and CH2 lines phased up and
down, respectively.
Mass spectroscopy (MS) spectra with fast atom bombardment
(FAB) or electrospray techniques were carried out with a VG-
QUATTRO from Fisons Instruments. Elemental analysis of the final
compounds was also achieved.
6.2.2.1.1. N3-lauroyl-Na-Cbz-lysine methyl ester. Yield: 91%.
HPLC, tr ¼ 19.55 min. MW 476.29. ESI-MS, m/z ¼ 477.34 corre-
sponding to (M þ H)þ.
6.2.2.1.2. N3-myristoyl-Na-Cbz-lysine methyl ester. Yield: 93%.
HPLC, tr ¼ 20.34 min. MW 504.29. ESI-MS, m/z ¼ 505.36 corre-
sponding to (M þ H)þ.
6.2.2.1.3. N3-palmitoyl-Na-Cbz-lysine methyl ester. Yield: 90%.
HPLC, tr ¼ 20.72 min. MW 532.29. ESI-MS, m/z ¼ 533.39 corre-
sponding to (M þ H)þ.
6.2.2.1.4. 1H NMR and 13C NMR resonances of the N3-acyl-Na-Cbz-
lysine methyl ester compounds. 1H NMR: dH (DMSO-d6), 0.84 [t, 3H,
CH3 alkyl chain], 1.22–1.63 [m, CH2 alkyl chain, 2CH2 side chain of
lysine group], 2.00 [t, 2H (–CH2–CO–NH– alkyl chain)], 2.98 [m, 2H
(–CO–NH–CH2– side chain of lysine group)], 3.61 [s, 3H (–CH3 lysine
group)], 3.99 [m, 1H (–NH–CH–COO–)], 5.02 [s, 2H (Cbz–CH2–O–)],
7.29–7.36 [m, 5H (Z group)].
6.2. General procedure synthesis of long chain N3-acyl lysine methyl
ester hydrochloride salts
The synthesis of LKM, MKM, and PKM has been carried out
following a three-step procedure starting from the commercial Na-
Cbz-LysOH$HCl (Na-bencyloxycarbonyl-LysOH$HCl) (Fig. 2).
13C NMR: dH (DMSO-d6), 13.7 [CH3– alkyl chain], 21.9, 22.7, 25.1,
28.5, 28.6, 28.7, 28.8, 28.8, 30.2, 31.1 [–CH2– alkyl chain and side
chain of lysine group], 35.3 [–CO–CH2–CH2– alkyl chain], 37.9
[–CH2–NH–CO lysine group], 51.6 [–CH–COO–], 53.8 [–COO–CH3],
65.4 [Cbz–CH2–], 127.5, 127.6, 128.2 [–CH– Z group], 171.8 [–CONH–
], 172.7 [–COO–].
6.2.1. Synthesis of Na-Cbz-lysine methyl ester$HCl (
a-carboxylic
group protection)
Methanol (400 mL) was cooled to ꢁ80 ꢀC using acetone/solid
carbon dioxide, then, Na-Cbz-LysOH$HCl (25 g, 0.089 mol) was
added. Thereafter, 19.5 mL of thionyl chloride (0.027 mol) was
dropwise added. The mixture was stirred at room temperature for
20 h. The end of the reaction was checked by HPLC with the
decrease in the peak area corresponding to Na-Cbz-LysOH$HCl.
6.2.3. Synthesis of long chain N3 acyl lysine methyl ester
hydrochloride salt
The target compounds were obtained by hydrogenation of
the corresponding pure N3-acyl-Na-Cbz-lysine methyl ester

L. Pe´rez et al. / European Journal of Medicinal Chemistry 44 (2009) 1884–1892
1891
(0.002 mol) in 30 mL methanol/HCl (HCl moles/lysine derivative
moles ¼ 1.3) using Pd in activated charcoal (10% Pd) as catalyst. The
reaction was carried out at atmospheric pressure. The mixture was
stirred for 30 min. Given that HCL is present in the reaction
medium, the final surfactants were obtained as HCl salts. At the end
of the reaction, the catalyst was filtered off on celite. The solvent
was evaporated under reduced pressure. Pure compounds were
obtained by several crystallizations from methanol/acetone.
Â
Ã
2
ðꢁ
g
G Þ Dð
d D
d
ꢁ =3Þ
I ¼ I0e
(4)
In Eq. (4), I is the measured signal intensity, I0 is the eco intensity in
the absence of field gradient pulses,
gradients in the pulse sequence, is the magnetogyric ratio, G is the
field gradient strength and is the duration of the gradient pulse.
The experiment has been carried out by varying the gradient
strength G. Typically, a value of 50–250 ms is used for , 5–10 ms is
used for
and G is varied from 0.03 to 0.33 T mꢁ1 in 15–20 steps.
The combination of G, and was chosen to generally obtain 90–
D is the time between the two
g
d
D
6.2.3.1. Analytical data and spectral assignments for N3-acyl lysine
methyl ester HCl salts
d
d
D
6.2.3.1.1. N3-lauroyl lysine methyl ester hydrochloride salt. Yield
70%. HPLC, tr ¼ 14.8 min. MW 378.45: ESI-MS, m/z: 343.2 (Mþ
without the Clꢁ). Elem. Analy. Found: C, 57.02; H, 11.20; N, 7.27;
Calcd. for C19H39O3N2Cl$1.2H2O: C, 56.99; H, 10.35; N, 6.99. 1H
NMR: dH (CD3OD) 0.89 [t, 3H, (CH3 alkyl chain)], 1.29 [s, 16H, 8CH2
alkyl chain], 1.50–1.60 [m, 6H, (CH2–CH2–CO–NH–), (–CH2–CH2–
CH2–CH2–NH–)], 1.92 [m, 2H, (–CH2–CH2–CH2–CH2–NH–)], 2.18 [t,
2H (–CH2–CO–NH–)], 3.20 [t, 2H (–CH2–CH2–CH2–CH2–NH–)], 3.8
[s, 3H, (–COO–CH3)], 4.0 [m, 1H (–NH–CH–COOCH3)].
95% total signal attenuation throughout the experiment. The cali-
bration of the gradient strength was performed using heavy water
with trace amounts of light water (DHDO) [58] 18.72 ꢂ 10ꢁ10 m2 sꢁ1
.
The intradiffusion coefficients were calculated by following the
signal intensities of the CH2 groups in the alkyl chain.
6.4. Antimicrobial activity
The compounds tested were dissolved in nutrient agar medium
13C NMR: dC (CD3OD), 14.4 [CH3–, alkyl chain], 23.3, 23.7, 27.0,
29.9, 30.3, 30.4, 30.6, 30.7, 31.1, 33.0, [–CH2– alkyl chain and side
chain of lysine group] 37.1, [–CO–CH2–CH2– alkyl chain], 39.6
[–CH2–NH–CO– lysine group] 53.6 (–CH–COO–), 53.8 (–COO–CH3),
171.0 (–COO–), 176.4(–COHN–).
in the concentration range of 0.1–256 mg/mL. Then 10 mL of
a nutrient broth starter culture of the each bacterial strain was
added to achieve final inoculums of ca. 5 ꢂ10ꢁ4–5 ꢂ10ꢁ5 colony
forming units per mL. The cultures were incubated overnight at
37 ꢀC. Nutrient broth medium without the compound served as
control. The growth of the microorganisms was determined visu-
ally after incubation for 24 h at 37 ꢀC. The development of turbidity
in an inoculated medium is a function of growth. A rise in turbidity
reflects the increase in both mass and cell number. Changes in
turbidity were correlated with changes in cell numbers. The lowest
concentration of antimicrobial agent at which no visible turbidity
was observed was taken as the minimum inhibitory concentration.
6.2.3.1.2. N3-myristoyl lysine methyl ester hydrochloride salt.-
Yield 75%. HPLC, tr ¼ 15.4 min. MW 406.66: ESI-MS, m/z: 371.5 (Mþ
without the Clꢁ). Elem. Analy. Found: C, 61.77; H, 10.78; N, 6.73;
Calcd. for C21H43O3N2Cl: C, 62.02; H, 10.56; N, 6.88. 1H NMR: dH
(CD3OD), 0.84 [t, 3H, (CH3 alkyl chain)], 1.26 [s, 20H, 10CH2 alkyl
chain], 1.36–1.48 [m, 6H, (CH2–CH2–CO–NH–), (–CH2–CH2–CH2–
CH2–NH–)], 1.73 [m, 2H, (–CH2–CH2–CH2–CH2–NH–)], 2.03 [t, 2H
(–CH2–CO–NH–)], 3.17 [t, (–CH2–CH2–CH2–CH2–NH–)], 3.8 [s, 3H,
(–COO–CH3)], 4.0 [m, 1H (–NH–CH–COO)].
6.5. Aquatic toxicity tests
13C NMR: dC (CD3OD), 14.4 [CH3–, alkyl chain], 23.2, 23.7, 27.0,
29.9, 30.3, 30.4, 30.5, 30.6, 30.7, 30.8, 31.1, 33.1 [–CH2– alkyl chain
and side chain of lysine group] 37.1, [–CO–CH2–CH2– alkyl chain],
39.6 [–CH2–NH–CO– lysine group] 53.6 (–CH–COO–), 53.8 (–COO–
CH3), 171.04 (–COO–), 176.4(–COHN–).
To determine aquatic toxicity, D. magna acute toxicity tests [38]
were carried out. D. magna, laboratory bred, not more than 24 h old
were used in this test, where the swimming incapability is the end
point. The pH of the medium was 8.0 and the total hardness was
250 mg/L (as CaCO3), with a Ca/Mg ratio of 4/1. Tests were per-
formed in the dark at 20 ꢀC. Twenty Daphnia, divided into four
groups of five animals each, were used at each test concentration.
For each surfactant, ten concentrations in a geometric series were
tested in the concentration range first established in a preliminary
test. The percentage immobility at 24 h was plotted against
concentration on logarithmic-probability paper. Normal statistical
procedures were then employed to calculate the IC50 and to
determine the 95% confidence ranges for the calculated IC50 values.
6.2.3.1.3. N3-palmitoyl lysine methyl ester hydrochloride salt.-
Yield 82%. HPLC, tr ¼ 18.07 min. MW 434.66. ESI-MS, m/z: 399.35
(Mþ without the Clꢁ). Elem. Analy. Found: C, 63.61; H, 11.20; N,
6.37; Cl, 8.27; Calcd. for C23H47O3N2Cl: C, 63.49; H, 10.81; N, 6.44;
Cl, 8.05. 1H NMR: dH (CD3OD), 0.83 [t, 3H, CH3 alkyl chain], 1.214
[s, 22H, 12CH2 alkyl chain], 1.34–1.43 [m, 6H, (–CH2–CH2–CH2–
CO–NH), (–CH2–CH2–CH2–CH2–NH–)], 1.70 [m, 2H (–CH2–CH2–
CH2–CH2–NH–)], 2.00 [t, 2H (–CH2–CO–NH–)], 2.98 [m, 2H
(–CH2–CH2–CH2–CH2–NH–)], 3.69 [s, 3H, (–COO–CH3)], 3.80 [m,
1H (–NH–CH–COO)].
13C NMR: dH (CD3OD), 14.46 [CH3– alkyl chain], 23.2, 23.7, 27.1,
29.9, 30.3, 30.4, 30.5, 30.6, 30.7, 30.8, 31.1, 33.1 [–CH2– alkyl chain
and side chain of lysine group], 37.0 [–CO–CH2–CH2– alkyl chain],
39.8 [–CH2–NH–CO– lysine group] 53.6 [–CH–COO–], 53.8 [–COO–
CH3–], 170.9 [–COO–], 176.6 [–CONH–].
6.6. Biodegradation test
The Modified Screening Test [44] described in the Organization
for the Economic Cooperation and Development (OECD) Guidelines
was applied to determine the biodegradation. A mixture of equal
volumes of secondary effluent from a wastewater treatment plant
and garden soil aqueous suspension was used as inoculums for the
biodegradation test.
6.3. PGSE-NMR
The pulsed gradient stimulated echo experiment was used to
determine the self-diffusion coefficients of the surfactants at 25
(ꢃ0.5) ꢀC. The 1H NMR measurements were obtained on a Varian
Inova 500-MHz spectrometer equipped with a standard 5-mm
indirect detection, pulse field gradient (PFG) probe. The combina-
tion provides a z gradient strength up to 0.33 T mꢁ1 (33 G cmꢁ1).
All the NMR signals give rise to single exponential decays in the
diffusion experiment, and the diffusion coefficient, D, is obtained by
fitting the following equation to the NMR data.
6.7. Haemolytic activity
Erythrocytes were washed three times in phosphate buffer
isotonic saline (PBS), containing 22.2 mmol/L Na2HPO4, 5.6 mmol/L
KH2PO4, 123.3 mmol/L NaCl, Glucose 10.0 mmol/L in distilled water
(pH ¼ 7.4). The erythrocytes were then suspended in PBS at a cell
density of 8 ꢂ 109 cell/mL.

1892
L. Pe´rez et al. / European Journal of Medicinal Chemistry 44 (2009) 1884–1892
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A series of different volumes of surfactant solution (1 mg/mL),
ranging from 10 to 80
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aliquot of 25 L of erythrocyte suspension was added to each tube.
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shaking conditions. Following incubation, the tubes were centri-
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