Cationic surfactants derived from lysine: Effects of their structure and charge type on antimicrobial and hemolytic activities

Article abstract of DOI:10.1021/jm101315k

Three different sets of cationic surfactants from lysine have been synthesized. The first group consists of three monocatenary surfactants with one lysine as the cationic polar head with one cationic charge. The second consists of three monocatenary surfactants with two amino acids as cationic polar head with two positive charges. Finally, four gemini surfactants were synthesized in which the spacer chain and the number and type of cationic charges have been regulated. The micellization process, antimicrobial activity, and hemolytic activity were evaluated. The critical micelle concentration was dependent only on the hydrophobic character of the molecules. Nevertheless, the antimicrobial and hemolytic activities were related to the structure of the compounds as well as the type of cationic charges. The most active surfactants against the bacteria were those with a cationic charge on the trimethylated amino group, whereas all of these surfactants showed low hemolytic character.

Full text of DOI:10.1021/jm101315k

J. Med. Chem. 2011, 54, 989–1002 989
DOI: 10.1021/jm101315k
Cationic Surfactants Derived from Lysine: Effects of Their Structure and Charge Type on
Antimicrobial and Hemolytic Activities
†
†
§
#
#
†
A. Colomer, A. Pinazo, M. A. Manresa, M. P. Vinardell, M. Mitjans, M. R. Infante, and L. Perez*
,†
ꢀ
†
´
Departamento de Tecnologıa Quımica y de Tensioactivos, IQAC, CSIC, C/Jordi Girona 18-26, 08034 Barcelona, Spain,
Departament de Microbiologıa, and Departament de Fisiologıa, Facultat de Farmacia, Universitat de Barcelona,
´
§
#
´
´
Avinguda Joan XIII s/n, 08028 Barcelona, Spain
Received October 13, 2010
Three different sets of cationic surfactants from lysine have been synthesized. The first group consists of
three monocatenary surfactants with one lysine as the cationic polar head with one cationic charge. The
second consists of three monocatenary surfactants with two amino acids as cationic polar head with two
positive charges. Finally, four gemini surfactants were synthesized in which the spacer chain and the
number and type of cationic charges have been regulated. The micellization process, antimicrobial
activity, and hemolytic activity were evaluated. The critical micelle concentration was dependent only
on the hydrophobic character of the molecules. Nevertheless, the antimicrobial and hemolytic activities
were related to the structure of the compounds as well as the type of cationic charges. The most active
surfactants against the bacteria were those with a cationic charge on the trimethylated amino group,
whereas all of these surfactants showed low hemolytic character.
Introduction
these surfactants are still toxic to the aquatic environment
and present problems of stability because they hydrolyze
easily in both acidic and basic conditions as well as at high
temperatures.11
Cationic surfactants have two important properties: they
are easily absorbed at solid/liquid interphases and can interact
with the cellular membranes of microorganisms. Consequently,
compounds of this type behave as good antimicrobial agents,
and for many years they have been used as disinfectants in
hospitals and in the food industry.1,2 Recently, it has been
shown that cationic amphiphiles have great potential in new
therapeutic biomedical applicationsasantimicrobialand anti-
fungal agents in human infections. They can also be used in
gene therapy (cationic vesicles made from cationic surfactants,
gemini surfactants in particular, can encapsulate RNA or
Amino acid based surfactants constitute an important class
of natural surface-active molecules of great interest to organic
and physical chemists as well as biologists with an unpredict-
able number of basic and industrial applications.12-14 Our
research group has synthesized and studied several new anti-
microbial cationic surfactants derived from amino acids with
different structures: monocatenary or single chain (one head/
one tail), gemini (two heads/two tails linked by a spacer chain),
and glycerolipid (one head/one or two tails linked together
through a glycerol skeleton).15 The presence of the cationic
guanidine group of arginine in these amphiphilic structures
gives a wide variety of compounds with a rich phase behavior
and strong antimicrobial activity. They present physicochem-
ical properties similar to those of classic cationic surfactants
but are more environmentally friendly and less toxic to the
eyes and skin than the conventional compounds.16 It has been
demonstrated that the presence of monocatenary arginine
surfactants improves the transfection efficiency when they
are conjugated to cationic liposome systems.17 Cationic ge-
mini surfactants based on arginine are extraordinarily active
at the surface.18,19 They have critical micelle concentrations
(CMCa) much lower than those of the monocatenary argi-
nine-based surfactant homologues while maintaining their
biological properties (antimicrobial activity, biodegradation,
DNA for cellular transfer),3-5 as vehicles for certain drugs6,7
,
and as modifiers of the physicochemical and biological prop-
erties of biomaterials (prosthesis and synthetic veins).8 These
types of therapeutic applications require the use of stable
surfactants under sterilization conditions that do not present
hemolytic or cytotoxic activities. Moreover, the antimicrobial
activity of amphiphilic compounds can be of great interest for
some of these applications.
Classic cationic surfactants derived from quaternary am-
monium show good antimicrobial activity and are capable of
forming vesicles to transport drugs and genetic material.9
However, these compounds present hemolytic activity;10
therefore, they are not suitable for biomedical applications.
Furthermore, their use is seriously questioned from an envi-
ronmental point of view as they are not readily biodegradable
and are toxic to aquatic organisms. The new generation
of cationic surfactants derived from quaternary ammonium,
the ester-QUATS type, improves the biodegradation, but
a Abbreviations: CMC, critical micelle concentration; Cbz, carbo-
benzyloxy, MIC, minimum inhibitory concentration ; DTAB, dodecyl-
trimethylammonium bromide; NMR, nuclear magnetic resonance;
MHB, Mueller-Hinton broth; BOP, benzotriazol-1-yl-oxytris-
(dimethylamino)phosphonium hexafluorophosphate; DABCO, 1,4-
diazabicycle(2.2.2)octane.
*Author to whom correspondence may be addressed (phone: 34 93
4006164; fax: (þ33) 34 93 2045904; e-mail: lourdes.perez@cid.csic.es).
r
2011 American Chemical Society
Published on Web 01/13/2011
pubs.acs.org/jmc

990 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Colomer et al.
Figure 1. Chemical structures of new synthetic surfactants from lysine.
and toxicity) unchanged. However, these compounds present
toxicity levels that make them unsuitable for applications in
which the compound has to be in contact with biological
systems. Therefore, it is interesting to extend this area of
research to obtain surfactants with properties similar to the
arginine derivatives but with lower toxicity against human
cells.
In this work we report the synthesis and characterization of
a new series of lysine-based surfactants designed for special
biomedical applications. The amino acid lysine, with two
amino groups of different pKa/basicity values and one car-
boxylic acid function, offers the possibility to design a wide
range of surfactant architectures, which is an excellent oppor-
tunity for tailoring the biological as well as surfactant aggre-
gation behavior for multipurpose uses.
Figure 1 shows the chemical structures and acronyms of the
synthesized surfactant molecules. We have prepared mono-
catenary and gemini lysine-based cationicsurfactantsinwhich
the nature of the head polar group, the character of the spacer,
and the type and quantity of the cationic charge in the
headgroup region have been systematically varied. The alkyl
chain has always been 12 carbon atoms except for one of the
monocatenary surfactant that contains 14 carbon atoms. The
following compounds have been prepared.
NR-lauroyl-lysine methyl ester (LLM) with one alkyl chain of
12 carbon atoms and one positive charge on the ε-amino
group of the lysine, Nε-lauroyl-lysine methyl ester (LKM) with
one alkyl chain of 12 carbon atoms and the positive charge
on the R-amino group of the lysine, and Nε-mirystoyl-NR -
trimethyl-lysine methyl ester (MKM) with one alkyl chain of
14 carbon atoms and the positive charge lying on the tri-
methylated ε-amino group.
(b) Three monocatenary surfactants with two amino acids
as the cationic polar head, two positive charges, and one alkyl
chain of 12 carbon atoms: NR-lauroylarginine-NR-lysine methyl
ester (LALM) with two positive charges, one on the guanidine
group of arginine and the other on the ε-amino group of
lysine; NR-lauroylarginine-Nε-lysine methyl ester (LAKM)
with one positive charge on the guanidine group of arginine
and the other on the R-amino group of lysine; and Nε-lauroyl-
lysine-Nε-lysine methyl ester (LKKM) in which the two
positive charges lie on the R-amino group of the lysines.
(c) Four gemini surfactants in which the spacer chain and
the number and type of cationic charges have been regulated:
Nε,Nω-bis(Nε-lauroyl-lysine) R,ω-hexylendiamide (C6(LK)2),
with two positive charges on the R-amino groups of the lysine
and a methylene-based spacer chain; NR,Nω-bis(NR-lauroyl-
lysine) R,ω-hexylendiamide (C6(LL)2), with two positive charges
on the ε-amino groups of the lysine and a methylene-based
spacer chain; Nε,Nω-bis(Nε-lauroyl-NR-trimethyl-lysine) R,
(a) Three monocatenary surfactants with one lysine as the
cationic polar head (one cationic charge) and one alkyl chain:

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 991
Scheme 1. Synthetic Pathway for the Synthesis of Single-Chain Lysine Surfactants
ω-hexylendiamide (C6(LK)2 TM), withtwo positivecharges on
the two trimethylated R-amino groups of the lysine and a
methylene-based spacer chain; and finally Nε,Nω-bis(Nε-laur-
oyl-lysine) R,ω-spermidindiamide (C7NH(LK)2), with a sper-
midine-based spacer and two positive charges on the R-amino
groups of the lysine but with a third positive charge on the
spacer chain.
These compounds may represent a step forward from a
scientific and application point of view, first, because of the
growing need for new nontoxic and biodegradable systems
and, second, because of the importance of exploring new
systems with novel properties.
acid and amino acid organic building blocks. For monocatenary
surfactants, the polar group consists of one or two positively
charged amino acids, lysine and arginine, whereas the gemini
group consists of two monocatenary NR- or Nε-lysine am-
phiphiles connected at the level of the headgroup by a spacer
chain of different ionic character. In all cases, all building
blocks are linked by amide bonds to design biodegradable
molecules that are benign to the environment.
Monocatenary N-Lauroyl-lysine Surfactants with One Po-
sitive Charge on the Polar Head. Monocatenary N-lauroyl-
lysine surfactants with one positive charge on the polar head,
LLM and LKM, were prepared using commercially available
NR-carbobenzyloxy-lysine HCl (NR-Cbz-lysine HCl) (1a)
For all of these new surfactants, we have studied the
micellization process and their antimicrobial and hemolytic
activities to determine the influence of different structural para-
meters on their physicochemical and biological properties.
3
3
or Nε-Cbz-lysine HCl (2a), respectively, as starting material.
3
Because lysine has two amino groups of similar reactivity, the
use of NR- or Nε-protected lysine is an essential requirement for
the synthesis of these compounds. A three-step procedure
has been used to obtain these compounds (Scheme 1). The
first step consisted of the preparation of the NR-Cbz-lysine
methyl ester (1b)/Nε-Cbz-lysine methyl ester (2b) HCl salts
(R-carboxylic group protection). Then, Nε-lauroyl-NR-Cbz-
lysine methyl ester (1c)/NR-lauroyl-Nε-Cbz-lysine methyl ester
(2c) was obtained by acylation of the ε/R-amino group with
lauroyl chloride. The reaction progress was monitored by
HPLC, and after 4 h, a 92% conversion of the starting
reagent was obtained. The purification of compounds was
carried out by silica gel chromatography. Satisfactory HPLC
analyses were obtained for these materials, yielding fast atom
bombardment mass spectroscopy (FABMS) and nuclear
magnetic resonance (NMR) spectra, consistent with the
target compounds. The third step involved the removal of
the Cbz group by catalytic hydrogenation of the 1c and 2c
using Pd over charcoal to give LLM and LKM, respectively.
The reaction was carried out by controlling the pH to prevent
the hydrolysis of the ester linkage present in these com-
pounds. Pure compounds were obtained after several crystal-
lizations in methanol/acetonitrile.
Results and Discussion
Synthesis. One of the most important variables in the
structure of cationic surfactants is the length of the alkyl
chain. There are numerous studies demonstrating that the
aggregation and biological properties of a surfactant are not
determined only by the hydrophobic tail but by the hydro-
philic head, including the size and the cationic charge (type,
number, and spatial positioning of the positive charge).
In this work, we have changed the position of the cationic
charge. Lysine is a dibasic amino acid consisting of two amino
groups with different basicities because the R-amino group
has a pKa =8.9 and the ε-amino group a pKa =10.5. This
feature can be used to obtain two different types of cationic
surfactants (see Figure 1): compounds with the positive
charge on the R-amino group and compounds with the
positive charge on the ε-amino group. We have prepared
monocatenary and gemini NR- and Nε-acyl lysine derivatives
with a dodecyl or tetradecyl aliphatic chain as cationic
surfactants. These compounds are made from natural fatty

992 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Colomer et al.
Scheme 2. Synthetic Pathway for the Synthesis of Single-Chain Diamino Acid Surfactants
The chemical structure of these compounds was checked
by NMR. The proton 1H and carbon 13C NMR spectra were
in concordance with the proposed structures. Elemental
analyses for the derivatives were in good agreement with
the calculated ones. MKM was obtained following the same
procedure as described for LKM. The trimethylation of MKM
(3a) was carried out using the classical reaction with dimethyl
sulfate and potassium carbonate.
was carried out by controlling the pH to prevent the hydrolysis
of the ester linkage present in these compounds. Hydrochlo-
ric acid was added to obtain the surfactants in the form of
hydrochloride salts. Typical overall yields of these products
(LALM, LAKM, LKKM) were 65-75%. The proton 1H and
carbon 13C NMR spectra of these compounds showed all peaks
corresponding to the target surfactants. The MS spectra also
agreed with the proposed structure. In addition, the results
from the combustion analysis experiments confirmed the
high purity of the compounds (on the order of 98-99%).
Gemini Surfactants with Spacer Chains of Different Catio-
nic Character. Four gemini surfactants, C6(LK)2, C6(LL)2,
C6(LL)2 TM, and C7NH(LK)2, were prepared via the syn-
thetic outline displayed in Scheme 3. Gemini C6(LK)2 and
C6(LL)2 with two amino cationic charges per molecule were
synthesized by condensation of the Cbz-protected NR/Nε-
lauroyl-lysine (LZL/LZK in Scheme 3) to both amino pri-
mary groups of the R,ε-hexylenediamine. This condensation
was carried out using BOP as coupling agent and DABCO as
base. This method was used by our group in the past to
obtain gemini surfactants from arginine with very good
yields.18 Target compounds were easy to isolate by recrys-
tallization from hot methanol (MeOH). Spermidine instead
of hexylenediamine was used as spacer chain for the pre-
paration of C7NH(LK)2, which contains three cationic
charges in the overall molecule. Removal of the Cbz protect-
ing group was achieved using the same procedure as de-
scribed above. Gemini C6(LK)2 TM with two cationic charges
of the quaternary ammonium type was obtained in good
yield by trimethylation of both NR-terminal amino groups of
the C6(LK)2 molecule with excess of dimethyl sulfate using
anhydrous potassium carbonate (K2PO3) as base. Finally,
the sulfate anion was changed by the chlorhydrate using a
cationic exchange column.
Monocatenary N-Lauroyl Surfactants with Two Positive
Charges on the Polar Head. The second group of surfactants
consisted of three cationic surfactants in which the polar
head has two positive charges provided by two basic amino
acids bonded by peptide linkages: arginine-lysine (LAKM),
arginine-lysine (LALM), and lysine-lysine (LKKM). The
general procedure for the preparation of these surfactants is
shown in Scheme 2. To prepare LALM and LAKM, the
temporary protected lauroylnitroargine (LNA) was used.
LNA was synthesized following the procedure described in
ref 18. The introduction of the strongly electronegative nitro
function decreased the basic nature of the guanidine group,
thus facilitating the incorporation of the lauroyl residue by
the formation of an amide bond without the competition of
the guanidine side chain. Compounds 4b and 5b (Scheme 2)
were obtained by condensation of LNA with the free amino
group of the Nε- or NR-Cbz-lysine methyl ester (4a and 5a,
respectively). In this reaction, the reagent benzotriazol-1-yl-
oxytris(dimethylamino)phosphonium hexafluorophosphate
(BOP) was used as condensation agent in the presence of 1,4-
diazabicycle(2.2.2)octane (DABCO) base. The formation of
LKKM was achieved using the same procedure as shown in
Scheme 2. In this case the lauroyl NR-Cbz-lysine methyl ester
(6b) was condensed to NR-Cbz-lysine methyl ester. These
reactions occurred readily in CH2Cl2 solutions at room
temperature and were essentially complete within 1 h. This
approach was chosen because it allowed us to perform the
synthesis in a short time and on a large scale. Finally, total
deprotection of the guanidine group of the arginine and the
amino groups of the lysine were carried out by removal of the
nitro and Cbz groups by means of hydrogenolysis to return
the corresponding target surfactants with yields on the order
of 95%. As we had previously observed,20 the elimination of
the Cbz group was quite rapid, but the nitro group required
more time (6 h) as well as some pressure (3 atm). The reaction
The symmetrical derivatives display simplified 1H and 13
C
NMR spectra that facilitate their interpretation. All of the
symmetrical H and C have the same chemical shift, and the
spectra are very similar to those of the corresponding single-
chain compound. Only the presence of the signals corre-
sponding to the spacer chain indicates the dimeric character
of the surfactant. The structures of all these surfactants were
also confirmed by UPLC-MS, and the purity was checked by
combustion analysis.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 993
Scheme 3. Synthetic Pathway for the Synthesis of Gemini Surfactants
Table 1. HPLC Retention Times and CMCs of Lysine Surfactants
surface tension and fluorescence measurements. Roughly,
the structure differences in the polar headgroup did not have
any notable effect on the CMC values.
surfactant
HPLC retention time (min)
CMC (mM)
LAKM
LALM
LKKM
11.4
11.4
12.8
26
25
22
As expected, diamino acid surfactants (LAKM, LALM,
LKKM) showed higher CMC values (1 order of magnitude)
than the monocatenary ones. Increasing the number of amino
acids increased the hydrophilicity of surfactants in water.
Such improvements in the solubility lowered the tendency of
surfactants to form micelles in water and increased the CMC.
In this case, neither the position of the cationic charge nor the
type of amino acids on the polar head affected the CMC value.
The micellization was mainly governed by the number of
cationic charges on the polar head as well as the length of the
alkyl chain.
LKM
LLM
MKMTM
13.4
14.8
17.7
5.5
7.2
2.2
C6(LL)2
C6(LK)2
17.6
17.8
18.7
20.5
0.74
0.50
1.9
C6NH(LK)2
C6(LK)2 TM
0.75
Critical Micelle Concentration. To investigate the effect of
the structure, number of cationic charges, and their position
on the aggregation properties, the CMC was determined for
each N-acyl-lysine-based surfactants. The conductivity of
aqueous solutions at 25 °C was measured at the adequate
concentration range. Conductivity of the aqueous solutions
increased linearly with increasing concentration up to break
points due to micellization and counterion binding that cor-
responds to the CMC of these surfactants (see Figure S1 in
the Supporting Information).
It is well known that the gemini structure increases hydro-
phobic interactions, giving rise to compounds with very low
CMC and high efficiency by reducing the surface tension of
water.24 In general, gemini surfactants show CMC values
about 1 order of magnitude lower than their corresponding
monocatenary surfactants. In the present work, the same
behavior has been observed (Table 1). C6(LL)2, C6(LK)2,
and C6(LK)2 TM have comparable CMC values 1 order of
magnitude lower than those of LKM and LLM. C7NH(LK)2
showed a higher CMC value; the third positive charge in the
spacer chain increased the hydrophilic character of the
molecule, increasing the CMC. Gemini surfactants from
arginine with the same alkyl chain and spacer lengths gave
a CMC of 0.4 mM by conductivity measurements.25 Table 1
also includes the retention time of all compounds obtained
by HPLC. The retention time is mainly affected by the
polarity of the compound. Except for C7NH(LK)2, the
CMC correlates well with the retention times. The results
indicate that the micellization process is governed by the
hydrophobic/hydrophilic balance and not by the type of
cationic charge.
Table 1 shows the CMCs obtained for each new surfac-
tant. Micellization (or CMC value) of the surfactants with
one lysine in the polar head, LKM, LLM, and MKMTM
,
took place at 7.2, 5.5, and 2.2 mM, respectively. These values
are similar to those reported for monocatenary arginine catio-
nic surfactants with the same hydrocarbon chain length21
and lower than the CMC corresponding to the commercial
12-carbon, straight-chain cationic surfactants such as dode-
cyltrimethylammonium bromide (DTAB),22 which is ap-
proximately 10 mM. The position and type of the cationic
charge do not affect significantly the concentration at which
these surfactants form molecular aggregates. Roy et al.23
prepared a series of cationic surfactants with various head-
group structures and a constant tail of 16 carbon atoms,
in terms of flexibility/rigidity, size/area, and hydrophilicity,
to investigate their effect on the CMC values obtained by
Antimicrobial Activity. Due to the positively charged polar
head, cationic surfactants can easily disturb bacterial mem-
branes and in general have antimicrobial activity.26 Bacterial cell
surface is usually negatively charged, and hence, the adsorp-
tion of cationic amphiphiles onto the negatively charged cell

994 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Colomer et al.
Table 2. MIC Values Obtained for the Monocatenary Derivatives with One or Two Amino Acids on the Polar Head at pH 7.4
one amino acid
two amino acids
LKM μM
(μg/mL)
LLM μM
(μg/mL)
MKMTM μM
(μg/mL)
LALM μM
(μg/mL)
LAKM μM
(μg/mL)
LKKM μM
(μg/mL)
Gram-positive
Bacillus subtilus
Staphylococcus
epidermidis
331 (125)
166 (63)
331 (125)
83 (31)
35 (16)
17 (8)
28 (16)
218 (125)
218 (125)
436 (250)
115 (63)
115 (63)
Micrococcus luteus
Staphylococcus aureus
Candida albicans
Gram-negative
Pseudomonas
331 (125)
331 (125)
166 (63)
83 (31)
166 (63)
166 (63)
35 (16)
35 (16)
35 (16)
28 (16)
54 (31)
218 (125)
436 (250)
436 (250)
436 (250)
115 (63)
229 (125)
Ra
R
331 (125)
R
218 (125)
R
459 (250)
aeruginosa
Klebsiella pneumoniae
Escherichia coli
331 (125)
166 (63)
331 (125)
331 (125)
278 (128)
R
218 (125)
218 (125)
R
R
459 (250)
229 (125)
a R, resistant microorganism at the highest concentration tested (250 μg/mL).
Table 3. MIC Values Obtained for Gemini Surfactants at pH 7.4
gemini surfactants
C6(LL)2 μM
(μg/mL)
C6(LK)2 μM
(μg/mL)
C7NH(LK)2 μM
(μg/mL)
C6(LK)2 TM μM
(μg/mL)
Gram-positive
B. subtilus
308 (250)
77 (63)
154 (125)
154 (125)
R
308 (250)
38 (31)
Ra
142 (125)
142 (125)
142 (125)
R
16 (15)
16 (15)
67 (60)
67 (60)
67 (60)
S. epidermidis
M. luteus
S. aureus
R
C. albicans
Gram-negative
P. aeruginosa
K. pneumoniae
E. coli
R
285 (250)
R
R
R
R
R
142 (125)
R
R
R
R
R
308 (250)
a R, resistant microorganism at the highest concentration tested (250 μg/mL).
surface is facilitated by electrostatic interactions, along with
hydrophobic interactions.27 Development of cationic am-
phiphiles having antibacterial properties with considerable
biocompatibility is of great interest in biomedical chemistry.
In this work we have studied the influence of the number
and position of cationic charges as well as the number of
alkyl chains on the bactericidal effect of cationic surfactants
from lysine. The antimicrobial activity of the lysine derivatives
was investigated by determining the minimal inhibitory con-
centration (MIC). Eight representative bacterial strains, four
Gram-positive and three Gram-negative, and one yeast were
chosen for studying antimicrobial activity in vitro. The results
of the antimicrobial tests for all lysine-synthesized deriva-
tives are reported in Tables 2 (monocatenary surfactants)
and 3 (gemini surfactants).
The lysine derivative surfactants are moderate antimicrobial
agents against Gram-positive bacteria, whereas they present
low activity against the Gram-negative ones. Gram-positive
bacteria have a rather simple cell wall composed of a pep-
tidoglycan layer, which allows amphiphiles to penetrate it
without difficulty.28 The external layer of the outer membrane
of the Gram-negative bacteria is almost entirely composed of
lipopolysaccarides and proteins that restrict the entrance of
biocides.
more active than LKM. The differences between these two
surfactants are the position of the cationic charge. LLM has
the charge on the ε-amino group, whereas LKM has the charge
on the R-amino group.
Among the monocatenary surfactants with two amino acids
on the polar head, LALM was the more active, having broad-
spectrum activity against the Gram-positive and Gram-
negative bacteria. LALM and LKKM were generally more
active than the rest of the monocatenary compounds, with
the exception of the quaternized compounds. The hydro-
philic character of these molecules increased considerably
due to the cationic charges and the higher size of the polar
group; in fact, the CMC was higher and the retention time by
HPLC smaller. As a consequence of these factors, these
molecules showed the highest solubility in pure water as well
as in the culture medium used for MIC determinations.
Haldar et al.26 found that the incorporation of multiple head
groups in cationic surfactants led to impressive antibacterial
activity. Lipo-R-peptides and their lipo-β-peptide counter-
parts with four amino acids on the polar head and one C16
lipid tail showed good antimicrobial activity.29 Moreover, it
has been also reported that polycationic neomycin-lipid
conjugates with several protonated amino groups on the
polar head displayed higher activity that the lysine deriva-
tives presented in this work.30 The adsorption of amphiphiles
with multicationic head groups onto the negatively charged
bacterial cell surface is expected to take place to a greater
extent than monocationic amphiphiles through electrostatic
interactions because of the higher charge density on multi-
head cationic surfactants.
For the monocatenary lysine derivatives, the most active
compound is by far MKMTM. This compound presents high
activity against Gram-positive microorganisms as well as
against Candida albicans. The cationic charge is situated on
the quaternized amino group, and the charge density of the
molecules does not depend on the pH. LLM was found to be

Article
It was also observed that the exchange of one of the amino
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 995
disrupting the bacterial membrane. Previous studies also
showed that neomycin B derived cationic lipids containing
a guanidinylated polycationic headgroup exhibited greater
antimicrobial potency than the neomycin B derived cationic
lipid homologues containing an amine-based polycationic
headgroup.36
acids can drastically modify antimicrobial properties (see
LAKM and LKKM), and the exchange of the position of
one of the two positive charges also affects antimicrobial
activity (see LAKM and LALM). On the other hand, in
reference to the alkyl chain length, there are numerous studies
about the influence of this parameter on antimicrobial activity.
For surfactants with the same headgroup, the optimum
antimicrobial activity is obtained for a determined alkyl
chain length, usually in the range of C12-C14. It is probable
that the optimal chain length for these diamino acid surfac-
tants is C14-C16 because the polar head volume is consider-
ably larger. For monoacyl lysine derivatives the alkyl chain
did not significantly affect antimicrobial activity,31 but
lipopeptides with two lysines as polar head and one alkyl
chain of 16 carbons atoms showed very good antimicrobial
activity against Gram-positive and Gram-negative bacteria.32
Antimicrobial activity of gemini lysine surfactants is shown
in Table 3. We found large differences in the MIC values of
these surfactants. Following the same trend as the monoacyl
derivatives, the higher activity was also obtained for the com-
pound with the positive charge on the quaternized amino
group. In fact, C6(LK)2 TM presented notable activity com-
pared with the other compounds. It can be also observed that
the gemini with the positive charges on the R-amino group
was ineffective against six of the eight bacteria tested. More-
over, this gemini surfactant did not show activity against
Gram-negative bacteria even at concentrations as high as
308 μM.
It can be stated that for the same alkyl chain length, the
gemini surfactants from lysine have lower antimicrobial activity
than the corresponding monocatenary ones. These results
contrast with those obtained for Bis(Quats) gemini surfac-
tants, which usually exhibit higher antibacterial activity than
conventional surfactants.33,34 For alkyl chains of 12 carbon
atoms, gemini surfactants from arginine had lower activity
than the corresponding monocatenary, but greater activity
was obtained with the alkyl chain of 10 carbon atoms.18 This
could indicate that for lysine gemini compounds a shorter
alkyl chain could also lead to a higher antimicrobial activity.
On the other hand, the introduction of spermidine as spacer
chain with one additional positive charge did not enhance
antimicrobial activity of these gemini surfactants.
In general, the antibacterial activity of these lysine deri-
vatives is lower than that shown by cationic surfactants from
arginine derivatives with similar chemical structures.18 These
variations could be attributed to differences between the pKa
values of the molecules. MIC values decreased with the increase
of pKa of the amino group in which the cationic charge is
situated. The headgroup charge of these surfactants is modu-
lated by the proportion of dissociation of the protonated
amino group, and this dissociation depends on the specific
architecture of the molecule. Surfactants of the NR-acyl type
have the ε-amino group protonated, and the pKa of these
molecules is around 10-12. However, surfactants of the Nε-
acyl type have the R-amino group protonated with a pKa
around 8.35 At pH equal to the pKa, the amino acid will be
50% protonated. As the pH is decreased by >2 units below
the pKa, it may be assumed that the amino acid is fully
protonated. This would mean that the average charge is 1.
Given that the pKa of the protonated R-amino group of the
Nε-lysine derivatives is around 8, the average charge of the
compound could be lower than 1 at the pH of the test medium
(pH 7.4); hence, the compounds have more difficulty in
As for the effect of the compound’s net charge on its
bactericidal activity, we have determined the antimicrobial
activity at different pH values from 5.4 to 8 (Figure 2). At pH
5.4, it is expected that all of the amino groups, the R-amino as
well as the ε-amino, were fully protonated with a net average
charge of 1. The results obtained are summarized in Tables
S1, S2, and S3 in the Supporting Information. It can be
observed that for the compounds with the charge on the
R-amino group (LKM, LAKM, and C6(LK)2) the activity
increased as the pH decreased (Figure 2). These results confirm
that the net average charge of the molecule is one of the factors
that affect antimicrobial activity. Another factor that also
affects this property is the type of cationic charge. The activity
of the chlorhydrated surfactants increased considerably as the
pH increased, but the activity obtained at the lower pH tested is
still inferior to those shown by the quaternized ones.
In summary, these studies indicate that lysine cationic
surfactants with very different structures show moderate
antimicrobial activity against tested bacteria. The position of
the positive charge, the type of cationic charge, and the number
of alkyl chains determine the extent of antimicrobial activity.
Hemolytic Activity. Hemolysis by surfactants is a process
of great importance for research and practical purposes. The
human erythrocyte lacks internal organelles and is the most
widely used cell membrane system to study surfactant-
membrane interactions.37 Surfactant intercalation into the
cell membrane leads to changes in the membrane molecular
organization and increases membrane permeability that
concludes with cell lyses. In addition, the potential use of
surfactants as drug delivery systems makes the evaluation of
hemolysis of great importance, and it is used as a measure for
cytotoxicity and a model for mammalian cells because
erythrocytes are extremely fragile.38 The polar head type
could affect the capability of surfactants to disrupt the fragile
cellular surface of the erythrocytes. However, hemolysis
depends on the adsorption of the surfactant to components
of the membrane surface, which is influenced by electrostatic
attractions between the surfactant molecules and membrane
components, among other factors.
Despite the potential use of cationic surfactants in a wide
range of pharmaceutical and biotechnological applications,
structure-membrane toxicity relationships are poorly devel-
oped. Alkyl chain length, type of alkyl chain (hydrocarbonated
or fluorinated), structure type (monocatenary or gemini
surfactants), position of the cationic charge, number of cationic
charges, counterion type, and headgroup hydrophobicity can
affect significantly the hemolytic activity of surfactants.
Nowadays, it is still not well established how these molecular
parameters determine the cytotoxicity of surfactants. More-
over, the influence of some of these parameters changes
depending on surfactant type. There are some surfactant
analogues in which the hemolysis increases when the alkyl
chain increases,39,40 whereas in other groups the hemolysis
decreases as the alkyl chain increases.41 On the other hand, in
the case of the gemini amphiphiles the role of the spacer and
its length remains unknown.
Evaluation of the concentration that induces the hemoly-
sis of 50% of the erythrocytes (HC50) was determined and

996 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Colomer et al.
Figure 2. MIC values for Gram-positive microorganisms at different pH values.
Table 4. Hemolysis of Lysine-Based Surfactants
quantified from plots of percentage hemolysis as a function
of amphiphile concentration. Table 4 shows the HC50 values
obtained for lysine derivatives. In general, these surfactants
showed significantly lower hemolytic activity than cationic
surfactants based on quaternary ammonium.42-44 The activity
of these new lysine surfactants on red blood cells was also
lower than those reported for cationic fluorinated com-
pounds in which hemolysis decreased drastically by increas-
ing the degree of fluorination and length of the hydrophobic
tail.45 The analogue derivatives with the arginine amino acid
also present higher hemolytic character.18 It has been reported
that for cationic amino acid surfactants, the cytotoxicity
changed when one amino acid was replaced by another, and
the presence of more hydrophobic groups surrounding the
positive charge gave rise to lower cytotoxicity.46
surfactant
HC50 (μM)
LAKM
LALM
LKKM
791
1353
290
LKM
LLM
MKMtrim
391
199
52
C6(LL)2
C6(LK)2
475
116
12
C7NH(LK)2
C6(LK)2trim
11
characteristics of the way in which monomers interact and
probably also of the way in which they interact with lipids in
the erythrocyte membrane. The molecular shape also deter-
mines intra- and intermolecular hydrophobic interactions. It
is well-known that the gemini structure enhances hydropho-
bic interactions, giving rise to surfactants than can aggregate
at very low concentrations. The increase of the hemolytic
Our results indicate that in general single-chain surfactants
are less hemolytic than the corresponding gemini amphiphiles.
This same trend has also been described for Bis(Quats)
gemini surfactants and their corresponding monoQuats.42,47
The molecular shape and the relationship between the hy-
drophilic and hydrophobic moieties constitute determining

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 997
character of the gemini surfactants could be due to the
increase of these interactions; in fact, it has been reported that
this type of interaction controls hemolytic activity, whereas
charge interactions have been suggested to be more impor-
tant for antibacterial activity.48 The hydrophobicity and the
capacity of forming micelles seem to influence significantly
the hemolysis of these compounds. It can be observed that
the less active compounds are the diamino acid surfactants,
whereas the most hydrophobic surfactants, the gemini, pre-
sent the highest activity against erythrocytes. However, we
have compounds with similar hydrophobicity and CMC values
(the two diamino acid surfactants or the two monocatenary
lysine derivatives) that exhibit different HC50 values. Con-
cerning the gemini surfactants, C7NH(LK)2 was the most
soluble in aqueous media and showed the highest CMC value,
but it was by far the most active against erythrocytes. This
suggests that the new cationic charge improves the interac-
tion of the compound with the membrane.
From these results, it can be stated that the number of
charges, the density of charge, and the position of the cationic
charges play also an important role in the hemolytic activity
of the compounds. LLM was nearly 2 times more potent than
LKM in causing erythrocyte lysis, and it was also more
potent in killing bacteria. Different behaviors can be observed
for gemini and diamino acid surfactants; surprisingly, in
these cases the less toxic compounds are those with the
positive charge on the R-amino group, these compounds
being the most active against bacteria. These results are in
concordance with those obtained by Sambhy et al.,49 who
reported new pyridinium polymers in which the spatial rela-
tionship between the positive charge and the pendent alkyl
chain changed drastically hemolytic activity.
In general, except for C7NH(LK)2 and C6(LK)2, the lysine
derivatives described in this work possess antibacterial ac-
tivity to Gram-positive bacteria at lower concentrations than
those in which they show erythrocyte toxicity. The safe con-
centration range is remarkably wide in the case of diamino
acid surfactants and C6(LL)2. It is worth noting that the
observed HC50 of LALM was 1385 μM, a 50-fold higher
concentration than the MICs to the Gram-positive bacteria
and a 6-fold higher concentration than the MICs against the
Gram-negative. The possible use of these compounds as
antimicrobials in clinical applications can be analyzed from
their therapeutic index, the HC50/MIC ratio. A high therapeutic
index means wide safe concentration range. The most suitable
surfactants for these applications are the diamino acid
derivatives with therapeutic index values ranging from 24 to
48. Gemini surfactants (C7NH(LK)2 and C6(LK)2) seem to
be the less appropriate to use as antimicrobial agents in
systemic applications, but some of them are still active against
bacteria at concentrations at which they do not exhibit
hemolytic activity. Nevertheless, it is noteworthy that we
have obtained very active cationic gemini surfactants with good
solubility in aqueous media and with low hemolytic character.
They can be promising compounds for biotechnological app-
lications in which active cationic compounds are required
and reducing mammalian cell toxicity is essential. The use of
very active compounds minimizes the amount of surfactant
in human cells and increases the safety of these systems.
(N-Cbz-L-lysine HCl) was obtained from Calbiochem-Nova-
3
biochem AG (Switzerland). Trifluoroacetic acid (TFA), thionyl
chloride (SOCl2), and palladium on activated charcoal (Pd/C,
10%) were supplied by Merck (Darmstadt, Germany). Deuter-
ated solvents were purchased from Eurotop. Mueller-
Hinton broth (MHB) was purchased from Difco Laboratories
(USA). Water from a Milli-Q Millipore system was used to prepare
aqueous solutions.
The progress of the reactions as well as the purity of the final
compounds was monitored by HPLC, model Merck-Hitachi
D-2500, using UV-vis detector L-4250 at 215 nm. A Lichrospher
100 CN (propylcyano) 5 μm, 250 ꢀ 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; solvent A was 0.1% (v/v) TFA in
H2O, and solvent B was 0.085% of TFA in H2O/CH3CN 1:4.
The flow rate through the column was 1.0 mL min-1
.
Methods. The structures of the pure compounds were checked
by 1H and 13C NMR analyses, which were recorded on a Varian
spectrometer at 499.803 (1H) and 125.233 (13C) MHz, respectively,
using the deuterium signal of the solvent as the lock. Chemical
shifts (δ) 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 decou-
pling to eliminate 13C-1H coupling. The distortionless enhance-
ment 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 bombard-
ment (FAB) or electrospray techniques were carried out with a
VG-QUATTRO from Fisons Instruments. Elemental analysis
of the final compounds was also achieved.
Conductivity. Conductivity was measured using an Orion
Cond. Cell 011010A with platinized platinum electrodes in
conjunction with a Thermo Orion 550A with a cell constant of
0.998 cm-1. The cell constant was calibrated periodically with
NaCl/KCl solutions of known conductivities and was used for
calculating the conductivity of the surfactant solution. The
conductivity of water was subtracted from the measured con-
ductivity of each sample. Measurements were made at increasing
concentration to minimize errors from possible contamination
from the electrode. Samples for conductivity measurements were
prepared by weight in Millipore ultrapure water.
Antimicrobial Activity. MIC Determinations. Antimicrobial
activities were determined in vitro on the basis of the MIC
values,50 defined as the lowest concentration of antimicrobial
agent that inhibits the development of visible growth after 24 h
of incubation at 37 °C. The compounds tested were dissolved in
MHB in the concentration range of 0.1-256 μg/mL, and no
precipitate was observed at the highest concentration of the
surfactant. The MHB was prepared according to the manufac-
turer’s instructions. Then 10 μL of a nutrient broth starter
culture of each bacterial strain was added to achieve final inocu-
lums of ca. 5 ꢀ 10-4-5 ꢀ 10-5 colony forming units mL-1. The
cultures were incubated overnight at 37 °C. Nutrient broth
medium without the compound served as control. The growth
of the microorganisms was determined visually 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
increases in both mass and cell number. Changes in turbidity
were correlated with changes in cell numbers. The lowest con-
centration of antimicrobial agent at which no visible turbidity
was observed was taken as the MIC.
Seven bacteria and one fungus were used for MIC determina-
tions. Gram-negative bacteria included Escherichia coli ATCC
27325, Klebsiella pneumonia ATCC 9721, and Pseudomonas aero-
ginosa ATCC 9721. Gram-positive bacteria included Bacillus
subtilis ATCC 6633, Staphylococcus aureus ATCC 25178,
Staphylococcus epidermidis ATCC 155-1, and Micrococcus
Experimental Procedures
Materials. All solvents were of reagent grade and were used
without further purification. Lauroyl and myristoyl chloride acids
were from Fluka. N-Benzyloxycarbonyl-L-lysine hydrochloride

998 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Colomer et al.
luteus ATCC 9341. The fungus used was Candida albicans
ATCC 10231.
pure target products were pooled. The identification of the
products was carried out by HPLC, elemental analysis,
1
H
Hemolytic Activity. Erythrocytes were washed three times in
phosphate buffer isotonic saline (PBS), containing 22.2 mmol/L
Na2HPO4, 5.6 mmol/L KH2PO4, and 123.3 mmol/L NaCl, in
distilled water (pH 7.4). Erythrocytes were then suspended in
PBS at a cell density of 8 ꢀ 109 cells/mL.
NMR, and 13 C NMR.
Analytical Data and Spectral Assignments for 1c. Yield, 91%;
HPLC, tr = 19.5 min; MW, 476.2 g/mol; ESI-MS, m/z 477
1
corresponding to (M þ H)þ; H NMR, δH (DMSO-d6) 0.8 [t,
3H, -CH3, alkyl chain], 1.2-1.6 [m, 24H, -CH2-, alkyl chain
and lysine lateral chain], 2.0 [t, 2H, -CH2-CONH-], 2.9
[m, 2H, -CONH-CH2-], 3.6 [s, 3H, -COO-CH3], 3.9 [m,
1H, -CH-, lysine], 5.0 [s, 2H, -CH2-, Cbz group], 7.2-7.3 [m,
5H, -CH-, aromatic ring]; 13C NMR, δH (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 lysine lateral chain],
35.3 [-CH2-CONH-], 37.9 [-CONH-CH2-], 51.6 [-COO-
CH3], 53.8 [-CH-, lysine], 67.5 [-CH2-, Cbz group], 128.7,
128.9, 129.4 [-CH-, aromatic ring], 138.1 [-C-, aromatic ring],
158.6 [-COO-, Cbz group], 171.8 [-CONH-], 172.7 [-COO-].
Analytical Data and Spectral Assignments for 2c. Yield, 89%;
HPLC, tr = 19.4 min; MW, 476.2 g/mol; ESI-MS, m/z 477
A series of different volumes of surfactant solution (1 mg/mL),
ranging from 10 to 80 μL, were placed in polystyrene tubes, and
an aliquot of 25 μL of erythrocyte suspension was added to each
tube. The tubes were incubated at room temperature with shaking
for 10 min. Following incubation, the tubes were centrifuged
(5 min at 5000 rpm). The degree of hemolysis was determined by
comparing the absorbance (540 nm) of the supernatant with that
of the control samples totally hemolyzed with distilled water.
Preparation of the Single-Chain or Monocatenary Lysine Surf-
actants LKM, LLM, and MKM (Scheme 1). (a) General Proce-
dure for the Synthesis of Nr/Nε-Cbz-lysine Methyl Ester HCl
3
(1b, 2b). MeOH (400 mL) was cooled to -80 °C using acetone/
solid carbon dioxide, and then NR/Nε-Cbz-LysOH HCl (1a, 2a)
1
corresponding to (M þ H)þ; H NMR, δH (CD3OD) 0.8 [t,
3
(25 g, 0.089 mol) was added. Thionyl chloride (19.5 mL, 0.027
mol) was added dropwise, and 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 the
starting materials. Methanol, excess thionyl chloride, and HCl
formed during the reaction were eliminated by successive va-
cuum evaporations. After lyophilization, a white solid corre-
sponding to the title product was obtained.
3H, -CH3, alkyl chain], 1.2-1.8 [m, 24H, -CH2-, alkyl chain
and lysine lateral chain], 2.2 [t, 2H -CH2-CONH-], 3.1 [m,
2H -CONH-CH2-], 3.6 [s, 3H, COO-CH3], 4.3 [m, 1H, -CH-,
lysine], 5.0 [s, 2H, CH2, Cbz group], 7.2-7.3 [m, 5H, -CH-,
aromatic ring]; 13C NMR, δH (CD3OD) 14.4 [-CH3, alkyl
chain], 23.7, 24.1, 26.9, 30.2, 30.4, 30.6, 30.7, 32.1, 33.0
[-CH2-, alkyl chain and lysine lateral chain], 36.7 [-CO-
CH2-(CH2)9-], 41.4 [-CH2-NH-CO], 52.6 [-COO-CH3 ],
53.6 [-CH-, lysine], 67.3 [-CH2-, Cbz group], 128.7, 128.9,
129.4 [-CH-, aromatic ring], 138.4 [-C- aromatic ring], 158.9
[-COO-, Cbz group], 174.3 [-CONH-], 176.5 [-COO-].
(c) General Procedure for the Synthesis of Nε-Lauroyl-lysine
(LKM) and Nr-Lauroyl-lysine (LLM) Methyl Ester Hydrochlor-
ide Salts. LKM and LLM were obtained by hydrogenation of
the corresponding pure 1c and 2c (0.002 mols) in 30 mL of
MeOH/HCl (HCl/lysine derivative moles = 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.
Analytical Data and Spectral Assignments for 1b. Yield, 97%;
HPLC, tr =5.8 min; MW, 330.5 g/mol; elem anal. found, C,
51.13; H, 6.89; N, 8.11; calcd for C15H23O4N2Cl H2O, C, 51.65;
3
H, 7.17; N, 8.03; 1H NMR, δH (CD3OD) 1.4-1.8 [m, 6H, 3 -CH2,
lateral chain of lysine], 2.8 [t, 2H, -CH2-NH3þ], 3.7 [s,
3H -COO-CH3], 4.2 [m, 1H, -CH-, lysine], 5.1 [s, 2H, -CH2-,
Cbz group], 7.2-7.3 [m, 5H, -CH-, aromatic ring]; 13C NMR,
δ
C (CD3OD), 23.8, 27.9, 32.0 [-CH2-, lateral chain of lysine],
40.4 [-CH2-NH3þ], 52.7 [-COO-CH3], 55.1 [-CH-, lysine],
67.6 [-CH2-, Cbz group], 128.8, 129.0, 129.4 [-CH-, aromatic
ring], 138.1 [-C-, aromatic ring], 158.6 [-COO-, Cbz group],
174.3 [-COO-CH3].
Analytical Data and Spectral Assignments for 2b. Yield, 97%;
HPLC, tr=5.8 min; MW, 330.5 g/mol; elem anal. found, C, 51.27;
H, 6.93; N, 8.07; calcd for C15H23O4N2Cl H2O, C, 51.65; H, 7.17;
3
1
N, 8.03; H NMR, δH (CD3OD), 1.5-1.9 [m, 6H, 3 -CH2-,
lateral chain lysine], 3.1 [t, 2H, -CONH-CH2-], 3.8 [s, 3H, -CO-
OCH3], 4.0 [t, 1H, -CH-, lysine], 5.0 [s, 2H, -CH2-, Cbz group],
7.3 [m, 5H, -CH-, aromatic ring]; 13C NMR, δC(CD3OD) 23.0,
27.9, 30.9 [-CH2-, lateral chain of lysine], 41.1 [-CONH-
CH2-], 53.6 [-COO-CH3], 53.8 [-CH-, lysine], 67.3 [-CH2-,
Cbz group], 128.7, 128.9, 129.4 [-CH-, aromatic ring], 138.3
[-C-, aromatic ring], 158.9 [-COO-, Cbz group], 170.9
[-COO-CH3].
Analytical Data and Spectral Assignments for LKM. Yield,
70%; HPLC, tr = 14.8 min; MW, 378.4 g/mol; ESI-MS, m/z
343.2 (Mþ without Cl-); elem anal. found, C, 57.02; H, 11.20; N,
7.27; calcd for C19H39O3N2Cl 1.2H2O, C, 56.99; H, 10.35; N,
3
1
6.99; H NMR, δH (CD3OD) 0.8 [t, 3H, -CH3, alkyl chain],
1.2-1.9 [m, 24H, -CH2-, alkyl chain and lysine lateral chain],
2.18 [t, 2H, -CH2-CONH-], 3.1 [t, 2H, -CONH-CH2-], 3.8
[s, 3H, -COO-CH3], 4.0 [m, 1H, -CH-, lysine]; 13C NMR, δC
(CD3OD) 14.4 [-CH3, alkyl chain], 23.2, 23.7, 27.0, 29.9, 30.3,
30.4, 30.6, 30.7, 31.1, 33.0 [-CH2-, alkyl chain and lysine
lateral chain], 37.1 [-CH2-CONH-], 39.6 [-CONH-CH2-]
53.6 [-COO-CH3], 53.8 [-CH-, lysine], 170.9 [-CONH-],
176.4 [-COO-].
(b) General Procedure for the Synthesis of Nr-Cbz-Nε-Lauroyl-
lysine Methyl Ester (1c) and Nr-Lauroyl-Nε-Cbz-lysine Methyl
Ester (2c). A solution of acetone/water (30 mL) containing 0.015
mol (5 g) of 1b/2b was placed in a round-bottom flask. To this
solution was added dropwise 0.015 mol of lauroyl chloride, and
stirring was continued at room temperature for 4 h. After
completion of the reaction, the mixture was acidified with HCl
and a white solid precipitated. The resulting solid was dissolved
in MeOH (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
(CHCl3) (25 mL). Thereafter, this solution was shaken with
water to eliminate water-soluble impurities. Finally, the solid
was purified by silica acid flash chromatography: 100 mL of
silica (Chromagel 60A CC, 70-230) was packed into a flash
chromatography column. The corresponding solid (3 g of 1c/2c)
was dissolved in CHCl3 and loaded into the column and eluted
from CHCl3 to CHCl3/MeOH. The fractions containing the
Analytical Data and Spectral Assignments for LLM. Yield,
72%; HPLC, tr = 14.8 min; MW, 378.4 g/mol; ESI-MS, m/z
343.2 (Mþ without the Cl-); elem anal. found, C, 59.30; H,
10.40; N, 7.25; calcd for C19H39O3N2Cl, C, 60.2; H, 10.37; N,
1
7.39; H NMR, δH (CD3OD) 0.8 [t, 3H, -CH3, alkyl chain],
1.2-1.9 [m, 24H, -CH2-, alkyl chain and lysine lateral chain],
2.2 [t, -CH2-CONH-], 2.9 [t, 2H, -CONH-CH2-], 3.7 [s,
3H, -COO-CH3], 4.3 [t, 1H, -CH-, lysine]; 13C NMR, δC
(CD3OD) 14.4 [-CH3, alkyl chain], 23.74, 23.8, 26.9, 27.9, 30.2,
30.4, 30.6, 30.7, 31.8, 33.8 [-CH2-, alkyl chain and lysine lateral
chain], 36.6 [-CH2-CONH-], 40.4 [-CONH-CH2-], 52.7
[-COO-CH3], 53.3 [-CH-, lysine], 173.9 [-CONH-], 176.5
[-COO-].

Article
(d) General Procedure for the Synthesis of Nε-Myristoyl-Nε-
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 999
[-COO-, Cbz group], 160.9 [-C-, guanidine], 174.0 [-NH-
CO-, lysine], 174.6 [-NH-CO, arginine], 176.3 [-COO-].
(b) General Procedure for the Synthesis of Nε-Lauroyl-lysine-
(carbobenzoxy)-Nε-lysine(carbobenzoxy) (6c). A solution of Nε-
lauroyl-lysine (6b) (20 mmol) in 50 mL of CH2Cl2 was treated
with 40 mmol of DABCO. Then, 20 mmol of NR-carbobenzox-
ylysine methyl ester was added under stirring until the com-
pounds were completely solubilized. Finally, 20 mmol of BOP
was added, and the mixture was stirred for 3 h. After complete
conversion of the starting material, the mixture was cooled to
0 °C and the precipitate was isolated by filtration. Flash
chromatography on silica gel 60, using CHCl3/MeOH as mobile
phase, afforded the pure compound, which was crystallized
from MeOH/ACN to yield the target compound 6c.
trimethyl-lysine (MKMTM). MKM was prepared using the
procedure described for the preparation of LLM and LKM.
Then, potassium carbonate was added to a solution of MKM
(0.005 mol) in methanol until pH 10. After that, dimethyl sulfate
(0.015 mol) was added dropwise, and the mixture was stirred at
room temperature until the reactants were consumed. The solvent
was evaporated and crystallized in methanol/acetone. The sulfate
salts thus obtained were treated with HCl in methanol to obtain
the corresponding chloride surfactants.
Analytical Data and Spectral Assignments for MKMTM. Yield,
70%; HPLC, tr=17.7 min; MW, 449.1 g/mol; ESI-MS, m/z 413.6
(Mþ without the Cl-); elem anal. found, C, 53.4; H, 10.0; N, 4.6;
calcd for C24H49O3N2Cl 5H2O, C, 53.4; H, 10.7; N, 5.1; 1H NMR,
3
δH (CD3OD) 0.8 [t, 3H, -CH3, alkyl chain], 1.2-1.9 [m,
24H, -CH2-, alkyl chain and lysine lateral chain], 2.2 [t, -CH2-
CONH-], 2.9 [t, 2H, -CONH-CH2-], 3.7 [s, 3H, -COO-
CH3], 4.3 [t, 1H, -CH-, lysine]; 13C NMR, δC (CD3OD) 14.4
[-CH3, alkyl chain], 23.74, 23.8, 27.0, 27.2, 30.2, 30.4, 30.6, 30.7,
33.0, 37.1 [-CH2-, alkyl chain and lysine lateral chain], 39.3
[-CH2-CONH-], 52.7 [-COO-CH3], 52.8 [-CH-, lysine],
64.1 [CH2-N-], 75.7 [-N-(CH3)3], 173.9 [-CONH-], 176.5
[-COO-].
Preparation of the Single-Chain Diamino Acid Surfactants
(Scheme 2). (a) General Procedure for the Synthesis of Nr-Laur-
oylnitroarginine-Nr-lysine(Cbz) Methyl Ester (4b) and Nr-Laur-
oylnitroarginine-Nε-lysine(Cbz) Methyl Ester (5b). A NR-laur-
oylnitroarginine (LNA) solution (20 mmol) in 50 mL of CH2Cl2
was treated with 40 mmol of DABCO. Then, 20 mmol of 4a or
5a was added under stirring until total solubilization of the
compounds was reached. Finally, 20 mmol of BOP was added,
and the mixture was stirred for 3 h. After complete conversion of
the starting LNA, the mixture was cooled to 0 °C, and the crude
precipitate was isolated by filtration. The solid was washed with
hexane and water and dried over vacuum. Flash chromatography
on silica gel 60, using CHCl3/MeOH as mobile phase, afforded
the pure compound, which was crystallized from MeOH/ACN
to yield the target compounds 4b and 5b.
Analytical Data and Spectral Assignments for 6c. Yield, 65%;
HPLC, tr=18.7 min; MW, 738.9 g/mol; ESI-MS, m/z 738.6; 1H
NMR, δH (DMSO-d6) 0.8 [t, 3H, -CH3, alkyl chain], 1.2-1.6
[m, 30H, -CH2-, alkyl chain and lysine lateral chain], 2.0 [t,
2H, -CH2-CONH-], 3.01 [m, 4H, 2 -CH2-NHCO-], 3.6 [s,
3H, -COO-CH3], 3.9 [m, 1H, -CH-, lysine], 4.0[m, 1H, -CH-,
lysine], 5.0 [m, 4H, 2 -CH2-, Cbz group], 7.3-7.4 [m, 10H, -CH-,
aromatic ring]; 13C NMR, δC(DMSO-d6) 13.7 [-CH3-, alkyl
chain], 21.9, 22.6, 22.7, 25.1, 28.4, 28.5, 28.6, 28.7, 28.8, 30.2,
31.1, 31.6 [-CH2-, alkyl chain and lysine lateral chain], 35.3
[-CH2-CONH-], 36.3 [-CONH-CH2-], 38.0 [-CONH-
CH2-], 51.6 [-COOCH3], 53.7, 54.6 [-CH-, lysine], 65.2, 65.3
[-CH2-, Cbz group], 127.4, 127.5, 127.6, 128.1, 128.2 [-CH-,
aromatic ring], 136.8, 136.9 [-C-, aromatic ring], 155.7, 156.0
[-COO-, Cbz group], 171.5, 171.7 [-CONH-], 172.7 [-COO-].
(c) General Procedure for the Synthesis of Nr-Lauroylargi-
nine-Nr-lysine Methyl Ester (LALM), Nr-Lauroylarginine-Nε-
lysine Methyl Ester (LAKM), and Nε-Lauroyl-lysine-Nε-lysine
Methyl Ester (LKKM). Compounds LALM, LAKM, and
LKKM were obtained by hydrogenation of the corresponding
pure 4b, 5b, and 6c (0.002 mol) in 30 mL of MeOH/HCl (HCl/
lysine derivative moles = 2.3) using Pd in activated charcoal
(10% Pd) as catalyst. The reaction was carried out at 2 atm.
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 MeOH/ACN.
Analytical Data and Spectral Assignments for 4b. Yield, 70%;
HPLC, tr=16.5 min; MW, 677.8 g/mol; ESI-MS, m/z 700 (M þ
1
Na); H NMR, δH (DMSO-d6) 0.8 [t, 3H -CH3, alkyl chain],
1.2-1.7 [m, 28H, -CH2, alkyl chain and lateral chain of arginine
and lysine], 2.1 [t, 2H, -CH2-CONH-], 2.9 [t, 2H, -CONH-
CH2-], 3.2 [t, 2H, -NH-CH2-, guanidine], 3.6 [s, 3H, -COO-
CH3], 4.2 [m, 1H, -CH-, arginine], 4.3 [m, -CH-, lysine], 5.0
[s, 2H, -CH2-, Cbz group], 7.2-7.3 [m, 5H, -CH-, aromatic
ring]; 13C NMR, δC(DMSO-d6) 13.6 [-CH3, alkyl chain], 21.8,
22.4, 25.0, 28.3, 28.4, 28.5, 28.7, 28.8, 29.1, 29.2, 30.3, 31.0
[-CH2-, alkyl chain and lateral chain of arginine and lysine],
35.0 [-CH2-CONH-], 38.0 [-CONH-CH2-], 40.1 [-NH-
CH2-, guanidine], 51.5 [-COO-CH3], 51.7 [-CH-, lysine],
51.9 [-CH-, arginine], 64.9 [-CH2-, Cbz group], 127.4, 127.6,
128.0 [-CH-, aromatic ring], 137.1 [-C-, Cbz group], 159.2
[-COO-, Cbz group], 160.9 [-C-, guanidine], 171.6 [-CONH-,
lysine], 172.0 [-CONH-, arginine], 172.1 [-COO-].
Analytical Data and Spectral Assignments for LALM. Yield,
91%; HPLC, tr=11.4 min; MW, 571.6 g/mol; ESI-MS, m/z 499
(Mþ without the Cl-); elem anal. found, C, 47.2; H, 9.28; N,
14.0; Cl, 11.7; calcd for C25H52Cl2N6O4 3H2O, C, 48.0; H, 9.3;
3
N, 13.5; Cl, 11.2; 1H NMR, δH (CD3OD) 0.8 [t, 3H, -CH3, alkyl
chain], 1.2-1.8 [m, 28H, -CH2-, alkyl chain and lateral chain
of arginine and lysine], 2.2 [t, 2H, -CH2-CONH-], 2.9 [m,
2H, -NH-CH2-, guanidine], 3.2 [m, 2H, þH3N-CH2-], 3.7
[s, 3H, -COO-CH3] 4.3 [m, 1H, -CH-, arginine], 4.4 [m,
1H, -CH-, lysine]; 13C NMR, δC (CD3OD) 14.4 [-CH3, alkyl
chain], 23.7, 26.2, 26.9, 27.8, 30.0, 30.3, 30.5, 30.6, 30.7, 31.6,
33.0 [-CH2-, alkyl chain and lateral chain of arginine and
lysine], 36.7 [-CH2-CONH-], 40.4 [-CH2-NH-, guanidine],
41.9 [þH3N-CH2-], 53.2 [-COO-CH3], 53.3 [-CH-, lysine],
54.4 [-CH-, arginine], 158.6 [-C-, guanidine], 173.8
[-CONH-], 174.4 [-CO-NH-], 176.5 [-COO-].
Analytical Data and Spectral Assignments for 5b. Yield, 70%;
HPLC, tr=15.6 min; MW, 677.8 g/mol; ESI-MS, m/z 700 (M þ
1
Na); H NMR, δH (CD3OD) 0.8 [t, 3H, -CH3, alkyl chain],
1.2-1.7 [m, 28H, -CH2-, alkyl chain and lateral chain of arginine
and lysine], 2.2 [t, 2H, -CH2-CONH-], 3.1 [m, 2H, -CONH-
CH2-], 3.2 [m, 2H, -NH-CH2-, guanidine], 3.7 [s, 3H, -COO-
CH3], 4.1 [m, 1H, -CH-, arginine], 4.3 [m, 1H, -CH-, lysine],
5.0 [s, 2H, -CH2-, Cbz group], 7.2-7.3 [m, 5H, -CH-, aromatic
ring]; 13C NMR, δC (CD3OD) 14.4 [-CH3, alkyl chain], 23.7,
24.1, 26.9, 29.8, 30.3, 30.4, 30.6, 30.7, 32.1, 33.0 [-CH2, alkyl
chain and lateral chain of arginine and lysine], 36.8 [-CH2-
CONH-], 39.9 [-CONH-CH2-], 41.6 [-NH-CH2-,
guanidine], 52.6 [-COO-CH3], 54.2 [-CH-, lysine], 55.4
[-CH-, arginine], 67.6 [-CH2-, Cbz group], 128.7, 128.9,
129.4 [-CH-, aromatic ring], 138.1 [-C-, Cbz group], 158.6
Analytical Data and Spectral Assignments for LAKM. Yield,
92%; HPLC, tr=11.4 min; MW, 571.6 g/mol; ESI-MS, m/z 499
(Mþ without the Cl-); elem anal. found, C, 49.0; H, 9.1; N, 13.9;
Cl, 12.3; calcd for C25H52Cl2N6O4 2H2O, C, 49.5; H, 9.2; N,
3
13.8; Cl, 11.7; 1H NMR, δH (CD3OD) 0.8 [t, 3H, -CH3, alkyl
chain], 1.2-1.9 [m, 28H, -CH2, alkyl chain and lateral chain
of arginine and lysine], 2.3 [t, 2H, -CH2-CONH-], 3.2 [m,
4H, -CH2-NH-, -þH3N-CH2], 3.8 [s, 3H, -COO-CH3],
4.0 [m, 1H, -CH-, arginine], 4.2 [-CH-, lysine]; 13C NMR,
δC(CD3OD) 14.4 [-CH3, alkyl chain], 23.0, 23.1, 23.7, 26.5,
26.9, 29.6, 29.7, 30.0, 30.3, 30.4, 30.5, 30.6, 30.7, 30.9, 31.0, 33.0
[-CH2-, alkyl chain and lateral chain of arginine and lysine],

1000 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Colomer et al.
36.8 [-CH2-CONH-], 39.6 [þH3N-CH2-], 42.0 [-CH2-
NH-], 53.6 [-COO-CH3], 53.8 [-CH-, lysine], 54.7 [-CH-,
arginine], 158.6 [-C-, arginine], 170.9 [-CONH-], 174.3
[-CONH-], 176.6 [-COO-].
Analytical Data and Spectral Assignments for 9a. Yield, 70%;
HPLC, tr =21.7 min; MW, 1005.4 g/mol; ESI-MS, m/z 1028
(M þ Na); 1HNMR, δH (DMSO-d6) 0.8[t, 6H, -CH3, alkyl chain],
1.2-1.6 [m, 56H, -CH2-, alkyl chain and lysine lateral chain],
2.1 [t, 4H, -CH2-CONH-], 3.0 [t, 8H, 2 -NH-CH2-, -CO-
NH-CH2-,-CH2-CONH-], 4.1 [m, 2H, -CH-],4.99[s,4H,2 -
CH2-, Cbz group], 7.0-7.3 [-CH-, aromatic ring]; 13C NMR,
δC(DMSO-d6) 13.6 [-CH3, alkyl chain], 21.8, 22.4, 25.0, 25.7,
28.4, 28.5, 28.7, 28.9, 31.0, 31.6 [-CH2-, alkyl chain, lysine
lateral chain, and spacer chain], 35.0 [-CH2-CONH-], 38.1
[-NH-CH2-], 40.1 [-CONH-CH2-], 52.2 [-CH-], 64.9
[-CH2-, Cbz group], 127.4, 128.0 [-CH-, aromatic ring],
137.1 [-C-, aromatic ring], 155.8 [-CO-, Cbz group], 171.3
[-CONH-], 171.8 [-CONH-].
Analytical Data and Spectral Assignments for LKKM. Yield,
91%; HPLC, tr = 12.8 min; MW, 543.6 g/mol; ESI-MS, m/z
1
472.4; H NMR, δH (CD3OD) 0.9 [t, 3H -CH3, alkyl chain],
1.2-1.9 [m, 30H, CH2-, alkyl chain and lysine lateral chain], 2.2
[t, 2H, -CH2-CONH-], 3.2 [m, 4H, 2 -CONH-CH2], 3.84 [t,
3H, -COO-CH3], 3.9, 4.0 [m, 2H, -CH-, lysine]; 13C NMR,
δC(CD3OD) 14.4 [-CH3, alkyl chain], 23.2, 23.4, 23.7, 27.1,
29.7, 30.0, 30.4, 30.6, 30.7, 31.1, 32.2, 33.0 [-CH2-, alkyl chain
and lysine lateral chain], 37.2 [-CH2-CONH-], 39.7 [-CO-
NH-CH2], 40.0 [-CONH-CH2], 53.6 [-COOCH3], 53.8,
54.45 [-CH-, lysine], 170.2 [-CONH-], 170.9 [-CONH- ],
176.4 [COO-].
Analytical Data and Spectral Assignments for 7a. Yield, 63%;
HPLC, tr =22.4 min; MW, 1034.4 g/mol; ESI-MS, m/z 1057
(M þ Na); 1HNMR, δH (DMSO-d6) 0.8[t, 6H, -CH3, alkyl chain],
1.2-1.8 [m, 54H, -CH2-, alkyl chain, lysine lateral chain, and
spacer chain], 2.1 [t, 4H, -CH2-CONH-], 2.9 [m, 4H, -CH2-
NH-CH2-, spermidine], 3.1 [t, 4H, 2 -CH2-NHCO-], 3.2
[m, 4H, CONH-CH2-(Spe)4-CH2-NHCO], 3.9 [m, 2H,
2 -CH-], 5.0 [m, 4H, -CH2-, Cbz group], 7.3 [m, 10H, -CH-,
aromatic ring]; 13C NMR, δC (DMSO-d6) 14.4 [-CH3, alkyl
chain], 23.7, 24.1, 24.3, 24.4, 27.1, 27.3, 27.5, 30.0, 30.2, 30.3,
30.4, 30.6, 30.7, 21.2, 32.4, 32.7, 33.0, 36.6 [-CH2-, alkyl chain,
lysine lateral chain, and spermidine], 37.1 [-CH2-CONH-]
39.2 [-CONH-CH2-(Spe)4-CH2-NHCO-], 39.8, 39.9 [-CH2-
NHCO-], 43.2, 45.9 [-CH2-NH-CH2-, spermidine], 56.7,
56.9 [-CH-], 65.2-67.6 [-CH2-, Cbz group], 126.9, 127.9,
128.7, 128.8, 129.0, 129.5 [-CH-, aromatic ring], 138.1 [-C-,
aromatic ring], 158.4 [-CO-, Cbz group], 175.2, 176.2 [-CO-
NH-], 176.3, 176.4 [-CH-CONH-].
Preparation of the Gemini Surfactants (Scheme 3). (a) Gen-
eral Procedure for the Synthesis of Nε-Lauroyl-Nr-carbobenzox-
ylysine (LZK) and Nr-Lauroyl-Nε-carbobenzoxylysine (LZL). In
a beaker equipped with a thermometer and a pH electrode, NR-
carbobenzoxy-lysine or Nε-carbobenzoxylysine (0.050 mmol)
was dissolved in 300 mL of water/acetone (38:62, v/v) solution.
After stirring, NaOH was added until pH 10, and the mixture
was cooled at 0 °C while lauroyl chloride (0.052 mmol) was
added dropwise. The solution was held in the pH range of 9-10
by the simultaneous addition of a 10% aqueous NaOH solution,
and the temperature was maintained below 10 °C. After com-
pletion of the addition, HCl (10%) was added until pH 2; the
resulting precipitate was filtered and washed with water until pH
7 and dried over P2O5. Then, the solid was washed with diethyl
ether and crystallized from methanol to yield the pure com-
pounds LZK and LZL.
Analytical Data and Spectral Assignments for LZK. Yield, 90%;
HPLC, tr=16.6 min; MW, 462.2 g/mol; 1H NMR, δH (CD3OD)
0.8 [t, 3H, -CH3, alkyl chain], 1.2-1.8 [m, 24H, -CH2-, alkyl
chain and lysine lateral chain], 2.1 [t, 2H, -CH2-CONH-], 3.1
[t, 2H, -CONH-CH2-], 4.1 [m, 1H, -CH-, lysine], 5.0 [s,
2H, -CH2-, Cbz group], 7.3 [m, 5H, -CH-, aromatic ring];
13C NMR, δC(CD3OD) 14.4 [-CH3, alkyl chain], 23.7, 24.2, 27.0,
29.9, 30.3, 30.4, 30.6, 30.7, 32.3, 33.0 [-CH2-, alkyl chain and
lysine lateral chain], 37.1 [-CH2-CONH-], 40.0 [-CONH-
CH2-], 55.2 [-CH-, lysine], 67.5 [-CH2-, Cbz group], 128.7,
128.9, 129.4 [-CH-, aromatic ring], 138.2 [-C-, aromatic ring],
158.6 [-CO-, Cbz group], 175.9 [-CONH-], 176.2 [COOH].
Analytical Data and Spectral Assignments for LZL. Yield, 90%;
HPLC, tr=16.6 min; MW, 462.6 g/mol; 1H NMR, δH (CD3OD)
0.9 [t, 3H, -CH3, alkyl chain], 1.2-1.8 [m, 24H, -CH2, alkyl chain
and lysine lateral chain], 2.1 [t, 2H, -CH2-CONH-], 3.2 [t,
2H, -Cbz-NH-CH2-], 4.2 [m, 1H, -CH-, lysine], 5.1 [s,
2H, -CH2-, Cbz group], 7.3 [m, 5H, -CH-, aromatic ring];
13C NMR, δC(CD3OD) 14.4 [-CH3, alkyl chain], 23.7, 24.1, 27.0,
26.9, 30.2, 30.4, 30.6, 30.7, 32.1, 33.0 [-CH2-, alkyl chain and
lysine lateral chain], 36.7 [-CH2-CONH], 41.4 [-Cbz-NH-
CH2-], 53.4 [-CH-, lysine], 67.3 [-CH2-, Cbz group], 128.7,
128.9, 129.4 [-CH-, aromatic ring], 138.4 [-C-, aromatic ring],
158.9 [-CO-, Cbz group], 175.5 [-CONH-], 176.4 [-COOH].
(b) General Procedure for the Synthesis of Nr,Nω-Bis(Nr-
lauroyl-Nε-carbobenzoxylysine) r,ω-Hexylendiamide (9a), Nε,
Nω-Bis(Nε-lauroyl-Nr-carbobenzoxylysine) r,ω-Hexylendiamide
(8a), and Nε,Nω-Bis(Nε-lauroyl-Nr-carbobenzoxylysine) r,ω-Sper-
midindiamide (7a). A solution of LZK or LZL (20 mmol) in 50 mL
of CH2Cl2 was treated with 50 mmol of DABCO. Then, 20 mmol
of hexanediamine or spermidine was added under stirring until
the total solubilization of the compounds. Finally, 40 mmol of
BOP was added, and the mixture was stirred for 3 h. After
complete conversion of the starting materials, the mixture was
cooled to 0 °C, and the crude precipitate was isolated by
filtration. The solid was washed with hexane and water and
dried over vacuum. Pure compound was obtained by several
crystallizations from methanol.
Analytical Data and Spectral Assignments for 8a. Yield, 70%;
HPLC, tr = 21.4 min; MW, 1005.4 g/mol; ESI-MS, m/z 1028
1
(M þ Na); H NMR, δH (DMSO-d6) 0.8 [t, 6H, -CH3, alkyl
chain], 1.3-1.8 [m, 56H, -CH2-, alkyl chain, lysine lateral chain,
and spacer chain], 2.1 [t, 4H, 2 -CH2-CONH-], 3.1 [m, 8H, CO-
NH-CH2-(CH2)4-CH2-NHCO, 2 -CONH-CH2], 3.9 [m,
2H, -CH-],5.0[m,4H,2-CH2-, Cbz group], 7.3 [m, 10H, -CH-,
aromatic ring]; 13C NMR, δC (DMSO-d6) 13.6 [-CH3, alkyl
chain], 21.8, 22.6, 25.0, 25.7, 28.4, 28.7, 31.0, 31.6 [-CH2-, alkyl
chain, lysine lateral chain, and spacer chain], 35.3 [-CH2-
CONH-], 38.0 [-CONH-CH2], 38.2 [-CONH-CH2], 54.5
[-CH-],65.1[-CH2-, Cbz group], 127.4, 128.0 [-CH-,aromatic
ring], 136.9 [-C-, aromatic ring], 155.6 [-CO-, Cbz group],
171.3 [-CONH-], 171.7 [-CONH-].
(c) General Procedure for the Synthesis of Nr,Nω-Bis(Nr-
lauroyl-lysine) r,ω-Hexylendiamide (C6(LL)2), Nε,Nω-Bis(Nε-lauroyl-
lysine) r,ω-Hexylendiamide (C6(LK)2), and Nε,Nω-Bis(Nε-laur-
oyl-lysine) r,ω-Spermidindiamide (C7NH(LK)2). Compounds
C6(LL)2, C6(LK)2, and C7NH(LK)2 were obtained by hydrogena-
tion of the corresponding pure 7a, 8a, and 9a (0.003 mmol) in
30 mL of MeOH/HCl (HCl/lysine derivative mol=2.3) using Pd
in activated charcoal (10% Pd) as catalyst. The reaction was
carried out at room temperature and atmospheric pressure.
Given that HCl is present in the reaction medium, the final
surfactant was obtained as HCl salt. At the end of the reaction,
the catalyst was filtered off on Celite. The solvent was evapo-
rated under reduced pressure. Pure compound was obtained by
crystallizations from MeOH/ACN.
Analytical Data and Spectral Assignments for C6(LL)2. Yield,
96%; HPLC, tr=17.6 min; MW, 810.0 g/mol; ESI-MS, m/z 738
(Mþ without 2Cl-); elem anal. found, C, 60.8; H, 10.6; N, 10.0;
Cl, 8.3; calcd for C42H86N6O4Cl2 1.5H2O, C, 60.2; H, 10.6; N,
3
10.1; Cl, 8.4; 1H NMR, δH (CD3OD) 0.9 [t, 6H, 2 -CH3, alkyl
chain], 1.2-1.8 [m, 56H, -CH2-, alkyl chain, lysine lateral
chain, and spacer chain], 2.2 [t, 4H, -CH2-CONH-], 2.9 [t,
4H, 2 -CONH-CH2-], 3.1 [m, 4H, -CH2-NH3þ], 4.2 [m, 2H,
2 -CH-]; 13C NMR, δC(CD3OD), 14.4 [-CH3, alkyl chain],
23.7, 23.9, 26.9, 27.3, 28.0, 30.2, 30.3, 30.4, 30.5, 30.6, 30.7, 32.5,

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1001
33.0 [-CH2-, alkyl chain, lysine lateral chain, and spacer chain],
36.8 [-CH2-CONH-], 40.1 [CO-NH-CH2-], 40.4 [-CH2-
NH3þ], 54.5 [-CH-], 174.1 [-CONH-], 176.3 [-CONH-].
Analytical Data and Spectral Assignments for C6(LK)2. Yield,
96%; HPLC, tr=17.8 min; MW, 810.0 g/mol; ESI-MS, m/z 738
(Mþ without 2Cl-); elem anal. found, C, 56.6; H, 10.4; N, 9.6;
compounds against Gram-positive and Gram-negative bac-
teria with very low cytotoxicity. Monolysine derivatives with
CMC values around 3-7 mM are active against a wide range
of bacteria, and they still have low hemolytic character.
Finally, gemini surfactants will be adequate for applications
that need nontoxic cationic active surfactants that aggregate
at very low concentrations with soft antimicrobial properties.
Cl, 9.0; calcd for C42H86N6O4Cl2 4H2O, C, 57.1; H, 10.6; N, 9.5;
3
Cl, 8.0; 1H NMR, δH (CD3OD) 0.8 [t, 6H, 2 -CH3, alkyl chain],
1.3-1.8 [m, 56H, -CH2-, alkyl chain, lysine lateral chain, and
spacer chain], 2.1 [t, 4H, 2 -CH2-CONH-], 3.1 [t, 8H, -CO-
NH-CH2-(CH2)4-CH2-NHCO-, 2 -CONH-CH2-], 3.8
[m, 2H, 2 -CH-]; 13C NMR, δC (CD3OD) 14.4 [-CH3, alkyl
chain], 23.2, 23.7, 27.1, 27.5, 30.0, 30.1, 30.3, 30.4, 30.6, 30.7,
32.3, 33.0 [-CH2-, alkyl chain, lysine lateral chain, and spacer
chain], 37.8 [-CH2-CONH-], 39.8 [-CONH-CH2-], 40.4
[-CONH-CH2-], 54.4 [-CH-], 169.9 [-CO-NH-], 176.3
[-CO-NH-].
Acknowledgment. Financial support from Spanish Plan
National IþDþI CTQ2009-14151-C02-01, CTQ2009-14151-
C02-02, and AGAUR 2009 SRG 246.
Supporting Information Available: Tables listing the MIC at
different pH values as well as conductivity curves against
concentration. This material is available free of charge via the
Internet at http://pubs.acs.org.
Analytical Data and Spectral Assignments for C7NH(LK)2.
Yield, 92%; HPLC, tr=18.7 min; MW, 875.5 g/mol; ESI-MS,
m/z 676 (Mþ without 3Cl-); elem anal. found, C, 55.5; H, 10.3;
References
N, 10.3; Cl, 12.0; calcd for C42H86N6O4Cl2 2.5H2O, C, 56.0; H,
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and Professional: London, U.K., 1994; pp 248-257.
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(3) Llies, M. A.; Seitz, W. A.; Johnson, B. H.; Ezell, E. L.; Miller, A. L.;
Thompson, E. B.; Balaban, A. T. Lipophilic pyrylium salts in the
synthesis of efficient pyridinium-based cationic lipids, gemini
surfactants, and lipophilic oligomers for gene delivery. J. Med.
Chem. 2006, 49, 3872–3887.
3
10.3; N, 10.6; Cl, 11.5; 1H NMR, δH (CD3OD) 0.8 [t, 6H, -CH3,
alkyl chain], 1.2-2.0 [m, 54H, -CH2-, alkyl chain, lysine
lateral chain, and spacer chain], 2.2 [t, 4H, -CH2-CO-NH-
Lys], 3.1 [m, 4H, CONH-CH2-(Spe)-CH2-NHCO-], 3.2 [t,
4H, 2 -CONH-CH2-], 3.3 [m, 4H, -CH2-NHþ2-CH2-, 3.9
[m, 2H, 2 -CH-]; 13C NMR, δC (CD3OD) 14.4 [-CH3 ], 23.3,
23.7, 24.5, 27.1, 27.3, 29.8, 30.3, 30.4, 30.6, 30.7, 32.1, 32.2, 33.0
[-CH2, alkyl chain, lysine lateral chain, and spermidine], 36.9
[-CH2-CONH-], 37.3 [-CONH-CH2-], 39.6, 39.9 [CONH-
CH2-(Spe)-CH2-NHCO-], 46.5, 48.3 [-CH2-NHþ2-CH2],
54.4 [-CH-], 170.80 and 170.22 [-CO-NH-], 176.62 and
176.61 [-CO-NH-].
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€
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(4) Vyas, S. M.; Turanek, J.; Knotigova, P.; Kasna, A.; Kvardova, V.;
Rankin, E.; Knutson, L.; Lehmler, H. J. Synthesis and biocompat-
ibility evaluation of partially fluorinated pyridinium bromides.
New J. Chem. 2006, 30, 944–951.
(d) General Procedure for the Synthesis of Nr,Nω-Bis(Nr-
lauroyl-Nε-trimethyl-lysine) r,ω-Hexylendiamide (C6(LK)2 TM).
To a solution of gemini C6(LK)2 (0.003 mmol) in MeOH was
added K2CO3 until pH 10. Then dimethyl sulfate (0.014 mmol)
was added dropwise, and the mixture was stirred at room tem-
perature until the reactants were consumed. The solvent was
evaporated to give the ammonium salts and was crystallized in
MeOH/ACN. The sulfate salts thus obtained were treated with
HCl in MeOH to obtain the corresponding chloride surfactants.
Analytical Data and Spectral Assignments for (C6(LK)2 TM).
Yield, 70%; HPLC, tr=20.5 min; MW, 894.2 g/mol; ESI-MS,
m/z 411.3; elem anal. found, C, 58.2; H, 10.9; N, 8.3; Cl, 7.2;
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calcd for C48H98N6O4Cl2 5H2O, C, 58.5; H, 10.9; N, 8.5; Cl, 7.2;
3
1H NMR, δH (CD3OD) 0.9 [t, 6H -CH3, alkyl chain], 1.2-1.8
[m, 56H, -CH2-, alkyl chain, lysine lateral chain, and spacer
chain], 2.2 [t, 4H, -CH2-CO-NH-], 3.1 [s, 18H, N-CH3], 3.2
[m, 4H, CONH-CH2-(CH2)4-CH2-NHCO], 3.4 [t, 4H CH2-
Nþ], 4,3 [m, 2H, -CO-CH-NHCO]; 13C NMR, δC(CD3OD)
14.4 [-CH3, alkyl chain], 23.4, 23.7, 26.9, 27.3, 30.2, 30.4, 30.5,
30.6, 30.7, 32.6, 33.0 [-CH2-, alkyl chain, lysine lateral chain,
and spacer chain], 36.9 [-CH2-CONH-] 40.2 [-CONH-
CH2-], 53.5 [N(CH3)3], 54.4 [-CH-], 67.4 [-CH2-Nþ-],
174.1 [-CO-NH-], 176.3 [-CO-NH-].
Conclusions
Cationic surfactants from lysine, monoalkyl derivatives
with one lysine on the polar head, monoalkyl derivatives with
two amino acids on the polar head, and gemini surfactants
have been prepared using simple and fast chemical methodolo-
gies. The CMC of these surfactants mainly depended on the
hydrophobicity of the molecule. Nevertheless, the antimicro-
bial activity as well as the hemolytic character depended also
on the charge density, the type of amino acid present on the
polar head, and the position of the cationic charge. Diamino
acid surfactants have CMC values around 20 mM, and they
can be suitable for applications that require antimicrobial
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