Straightforward method for the preparation of lysine-based double-chained anionic surfactants

Article abstract of DOI:10.1080/00397910801997827

Double-chained surfactants with potential biocompatibility have been prepared in high yields by lysine acylation with four natural saturated fatty acids (C6 to C12) and with cis-undec-5-enoic acid. The surfactants were found to assemble into nanotubules in aqueous medium and, when mixed with a commercial cationic surfactant, to spontaneously form liposomes. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/00397910801997827

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Straightforward Method for
the Preparation of Lysine-
Based Double-Chained Anionic
Surfactants
Paula Gomes a , Maria João Araújo a , Eduardo F.
Marques a , Soraia Falcão a & Rodrigo O. Brito a
a CIQUP, Department of Chemistry, Faculty of
Sciences , University of Porto , Porto, Portugal
Published online: 12 Jun 2008.
To cite this article: Paula Gomes , Maria João Araújo , Eduardo F. Marques , Soraia
Falcão & Rodrigo O. Brito (2008) Straightforward Method for the Preparation of
Lysine-Based Double-Chained Anionic Surfactants, Synthetic Communications: An
International Journal for Rapid Communication of Synthetic Organic Chemistry, 38:12,
2025-2036, DOI: 10.1080/00397910801997827
To link to this article: http://dx.doi.org/10.1080/00397910801997827
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Synthetic Communications1, 38: 2025–2036, 2008
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910801997827
Straightforward Method for the Preparation
of Lysine-Based Double-Chained
Anionic Surfactants
˜
´
Paula Gomes, Maria Joao Araujo, Eduardo F. Marques,
˜
Soraia Falcao, and Rodrigo O. Brito
CIQUP, Department of Chemistry, Faculty of Sciences, University of Porto,
Porto, Portugal
Abstract: Double-chained surfactants with potential biocompatibility have been
prepared in high yields by lysine acylation with four natural saturated fatty acids
(C6 to C12) and with cis-undec-5-enoic acid. The surfactants were found to
assemble into nanotubules in aqueous medium and, when mixed with a com-
mercial cationic surfactant, to spontaneously form liposomes.
Keywords: Carbodiimide; Coupling; Fatty acid; Liposome; Lysine; Peptide;
Surfactant
INTRODUCTION
Fatty-acid acylation is a widespread posttranslational modification of
natural peptides and proteins aimed at different functional and structural
biological purposes.[1–3] Thus, the increase of protein’s lipophilicity con-
ferred by fatty-acid acylation can, among other effects, (i) increase the
protein’s stability against proteolytic degradation, (ii) target the protein
Received in the U.K., August 22, 2007.
Address correspondence to Paula Gomes, Centro de Investigac¸a˜o em Quımica
´
´
ˆ
da Universidade do Porto, Departamento de Quımica da Faculdade de Ciencias
do Porto, R. Campo Alegre 687, P-4169-007, Porto, Portugal. E-mail: pgomes@
fc.up.pt
2025

2026
P. Gomes et al.
for certain intracellular compartments, and (iii) promote the protein’s
interaction and association with phospholipid bilayers in cell mem-
branes.[1–6] This latter feature of lipoproteins has been the driving
force for N-fatty-acid acylation of antimicrobial peptide’s N-terminal
and=or lysine residues as a strategy to improve their antibiotic
activity.[7–16] However, the potential interest of N-acylation of protein-
ogenic amino acids with natural fatty acids to produce surface-active
agents of gemini type has been little explored, despite the urgent need
for new double-chain surfactants that are simultaneously efficient
detergents with anti bacterial properties, biodegradable, and biocom-
patible.[17] A few authors have been devoted to amino acid–based
surfactants since the 1980s, and their work has been a valuable contri-
bution to the unveiling of the enormous potential of amino acid– and
peptide-based surfactants. Some pioneer works are, for instance, those
from Hidaka et al., who reported the interesting self-assembling
properties of chiral N-(2-hydroxydodecyl) amino acids,[18] and from
Imae et al., who have studied N-acyl derivatives of L-aspartic acid,
L-glutamic acid, and b-Alanine using capric, lauric, miristic, palmitic,
or stearic acid as the acyl component.[19] In the latter case, the authors
have found interesting solution properties and morphologies for their
amino acid amphiphiles, from gels to cylindrical or helical fibers and
even vesicles, which opened new perspectives for biomedical applica-
tions of biocompatible surfactants. Over the past decade, Infante
and coworkers have been devoted to the synthesis of amino acid–
based single-chain, double-chain, and gemini surfactants through
employment of several chemical and chemoenzymatic methodologies
and characterized their self-aggregation, biodegradability, and toxicity
properties.[20–30] Among the different types of lipoamino acids created
and studied by these authors, those derived from basic amino acids
such as arginine presented interesting properties because of their low
toxicity, high biodegradability, and antimicrobial activity.[26] The same
research group has also explored some lysine-derived surfactants,
including Na,Ne-dioctanoyl-lysine, which performed significantly
better than classical anionic surfactants in terms of ocular and skin
irritancy, haemolytic activity, antimicrobial properties, and aquatic
toxicity.[31–36]
The wide potentialities of lysine-based double-chain anionic amphi-
philes as bio- and ecofriendly surfactants with prospective industrial
interest prompted us to explore alternative methods for their preparation.
Synthetic methodologies were developed to give high yields at a multi-
gram scale and involve inexpensive, safe, simple, and high-yield experi-
mental procedures, as these are key factors for a feasible industrial
development of novel surfactants.

Lysine-Based Double-Chained Surfactants
MATERIALS AND METHODS
2027
L-lysine methyl ester di-hydrochloride and N-[(1H-benzotriazole-1-yl)
(dimethylamino)methylene]-N-methylmethanaminium N-oxide tetrafluoro-
borate (TBTU) were from Bachem (Switzerland). The solvents were of pro
analisi (p.a.) quality from Merck (Germany). Fatty acids and other chemicals
were all from Sigma-Aldrich (Germany). The reaction progress was moni-
tored by thin-layer chromatography (TLC) on aluminium plates covered
by a 2.5-mm-thick layer of silica gel 60 F254 (Merck).
Fatty acid coupling to L-lysine methyl ester was achieved by stan-
dard peptide chemistry methods, using TBTU as coupling agent to yield
the desired di-acylation products 1a–e (Scheme 1i) that were easily iso-
lated by acid–base liquid extraction. Compounds 1a–e were then submit-
ted to saponification at room temperature with an aqueous methanolic
solution of sodium hydroxide, from which the target anionic amphiphiles
Scheme 1. Synthetic path to anionic amphiphiles 2a–e: (i) TBTU=DIEA, dichloro-
methane=toluene, 0 ꢀC!rt; (ii) NaOH in methanol–water, 0 ꢀC.

2028
P. Gomes et al.
2a–e (Scheme 1ii) were easily isolated. The structure and purity of all
1
compounds were checked by H NMR, 13C NMR, and ESI-MS.
Compounds 1a–e
Lysine methyl ester hydrochloride (5.0 mmol) was added to a solution
of the fatty acid (10.0 mmol) in dichloromethane (20 mL) and the
mixture was stirred in an ice-water bath. After addition of N-ethyl-
N,N0-diisopropylamine (DIEA, 20.0 mmol) and TBTU (10.0 mmol), the
mixture was kept at 0 ꢀC for 2 h. The reaction was allowed to proceed
at room temperature for 72 h, after which the solvent was evaporated
and the oily residue dissolved in toluene (40 mL). This solution was
washed three times with an aqueous solution of 5% HCl (20 mL of
10% HCl þ 20 mL of boiling water) and as many times with an aqueous
solution of 15% Na2CO3 (20 mL of 30% Na2CO3 þ 20 mL of boiling
water). The organic layer was then washed with boiling water
(2 ꢁ 15 mL), dried over anhydrous Na2SO4, and evaporated to dryness.
The products were isolated as white solids and presented correct spectral
data, as exemplified for 1d.
N,N 0-Di-dodecanoyl-lysine Methyl Ester (1d)
g ¼ 82%; mp ¼ 84–85 ꢀC. dH (CDCl3, 300 MHz): 6.21 (d, 1H, J ¼ 7.8 Hz,
CO-NH-CH); 5.73 (br s, 1H, CH2-NH-CO); 4.56 (dt, 1H, J ¼ 8.1 and
7.8 Hz, NH-CH-COOCH3), 3.72 (s, 3H, CH3-O); 3.22 (m, 2H, CH2-
NH); 2.22 (t, 2H, J ¼ 7.7 Hz, CH2-CO-NH); 2.14 (t, 2H, J ¼ 7.7 Hz,
NH-CO-CH2); 1.68 (m, 8H, CH-CH2, CH2-CH2-NH and CO-CH2-
CH2); 1.24 (m, 34H, CH2-(CH2)8-CH3 and CH2-(CH2)2-NH); 0.864
(t, 6H, J ¼ 6.8 Hz, CH2CH3).
dC (CDCl3, 75 MHz): 173.5 and 173.3 (CO-NH); 173.1 (COOCH3);
52.35 (CH); 51.60 (CH3-O); 38.69 (CH2-NH); 36.81 and 36.53 (CH2-CO);
32.01, 31.89, 29.61, 29.50, 29.34, 29.32, 29.27, 28.82, 25.82, 25.62, 22.66,
and 22.28 (CH2); 14.09 (CH3).
Calculated for C31H61N2O4 (MHþ ) 525.83, found 525.42.
Compounds 2a–e
To a stirred solution of 1a–e (6 mmol) in methanol (12 mL), the equiva-
lent volume of 1M aqueous NaHO (6 mmol) was added, and the reaction
allowed to proceed for 3 h at 0 ꢀC. In the case of compounds 2c and 2d,
a white solid extensively precipitated and was collected by suction

Lysine-Based Double-Chained Surfactants
2029
filtration, after which was rinsed with ice-cold water. In the remaining
three cases, no extensive precipitation was observed, and the reaction
mixtures were freeze-dried to yield the desired products as white solids
that presented correct spectral data, as exemplified for 2d.
Sodium N,N0-Di-dodecanoyl Lysinate (2d)
g ¼ 87%; mp ¼ 205–206 ꢀC. dH (CD3OD, 300 MHz): 4.23 (m, 1H, NH-
CH-COO ꢂ Naþ ); 3.14 (t, 2H, J ¼ 6.8Hz, CH2-NH); 2.22 (t, 2H,
J ¼ 7.5Hz, CH2-CO); 2.15 (t, 2H, J ¼ 7.6 Hz, CH2-CO); 1.83 (m, 2H, CH-
CH2); 1.58 (m, 6H, CH2-CH2-CH2-NH and CO-CH2-CH2); 1.28 (m, 34H,
CH2-CH2-CH2-NH and (CH2)8-CH3); 0.886 (t, 6H, J ¼ 6.5Hz, CH2CH3).
dC (CD3OD, 75 MHz): 179.4 (COOꢂ ); 176.2 and 175.4 (CO-NH);
56.07 (CH); 40.29 (CH2-NH-CO); 37.44 and 37.25 (CH2-CO); 33.73
(CH2-CH); 33.12 (CH2-CH2-CH3); 30.82, 30.80, 30.73, 30.69, 30.57,
30.47, 30.42, 30.16, 27.17, 27.09, 24.28 and 23.78 (CH2); 14.51 (CH3).
Calculated for C30H57N2O4Na (MNaþ ) 555.76; found 555.44.
RESULTS AND DISCUSSION
Establishment of the Synthetic Method for Compounds 1a–e
The first synthetic approach to prepare lysine Na,Ne-diacyl derivatives
1a–e was based on the employment of N,N0-dicyclohexylcarbodiimide
(DCCI) as coupling agent, as this is an inexpensive, mild, and safe acyla-
tion method.[37] The acylation reaction was tested using lauric (dodeca-
noic) acid as the acyl component, and a product was formed and
isolated by column chromatography on silica. 1H NMR and electrospray
ionization mass spectrometry (ESI-MS) spectra of the product identified
it as N,N0-dicyclohexyl-N-dodecylurea instead of the target compound
(Scheme 2). N-Acylurea formation can become a serious problem in
DCCI-mediated coupling when the attack of the nucleophile (lysine
amino groups in our case) to the reactive O-acylisourea intermediate is
sluggish. This favors the ON acyl migration that leads to the stable
(i.e., unreactive) N-acylurea, decreasing or even zeroing the probability
of obtaining the desired condensation product.[38]
Attempts to Prepare 1d by DCCI-Mediated Coupling
Lysine methyl ester hydrochloride (5.0 mmol) was added to a solution of
the fatty acid (10.0 mmol) in dichloromethane (20 mL), and the mixture
was stirred in an ice-water bath. After addition of DCCI (11.0 mmol),

2030
P. Gomes et al.
Scheme 2. Formation of N,N0-dicyclohexyl-N-dodecanoylurea in the place of
the desired condensation product when DCCI was used as coupling reagent.
the mixture was kept at 0 ꢀC for 2 h. The reaction was allowed to proceed
at room temperature for 24 h, after which a second portion of DCCI
(5 mmol) was added. After 48 h, the solid fraction was removed by suc-
tion filtration and identified (TLC) as unreacted DCCI. The filtrate
was evaporated to dryness and observed (TLC) to be composed by one
major product and unreacted starting materials. This mixture was sepa-
rated by column chromatography on silica using dichloromethane=
acetone 10:1 (v=v) as eluant. The pure white waxy product was isolated
1
and identified by H NMR and ESI-MS.
N,N0-Dicyclohexyl-N-dodecanoylurea
g ¼ 32%. dH (CDCl3, 300 MHz): 7.16 (s, 1H, CO-NH-CH); 3.79 (m, 2H,
cyclohexyl CH); 2.40 (t, 2H, COCH2), 1.26 (m, 38H, dodecanoyl and
cyclohexyl CH2); 0.88 (t, 3H, CH3).
Calculated for C25H46N2O2 (MHþ ) 407.36; found 407.43.
We then used TBTU,[39] a more efficient, yet more expensive, coup-
ling agent than DCCI. TBTU-mediated coupling not only led to the

Lysine-Based Double-Chained Surfactants
2031
exclusive formation of target compounds 1a–e (Scheme 1i) in high yields
but also enabled product isolation with high purity through acid–base
extractions that are faster, easier, and cheaper than chromatographic
techniques. Thus, unreacted starting materials and both the tetramethy-
lurea and 1-hydroxybenzotriazole (HOBt) by-products were completely
removed by washing the organic layer with 5% aqueous HCl followed
by 15% aqueous Na2CO3, so that products 1a–e were easily isolated in
high yield and purity by simply evaporating to dryness the resulting
organic layer. In the course of the extractions, compounds 1a–e displayed
surface-active properties that were easily dealt with. Therefore, toluene
was used as the organic solvent, as others such as dichloromethane, ethyl
acetate, hexane, or ethyl ether promoted the formation of intractable
emulsions or the preferred localization of the compounds at the liquid–
liquid interface. Further, use of boiling aqueous solutions in the extrac-
tions was seen to avoid emulsification. These simple procedures led to
excellent results up to a 5-gram synthesis of 1a–e.
Infante and coworkers have previously synthesised similar Na,Ne-
diacyl-lysine derivatives by addition of lysine sodium salt to the
corresponding acyl chloride, followed by product precipitation with
hydrochloric acid and a final recrystallization step to yield the final
product with adequate purity.[32] This is indeed an efficient method,
but our findings clearly show that TBTU-mediated synthesis of 1a–e is
advantageous from all points of view, as it involves safer starting materi-
als and milder reaction conditions and avoids recrystallizations.
As compared to cheaper coupling agents, even if carbodiimide-based
coupling could be made to work (for instance, by addition of HOBt as
auxiliary nucleophile),[40] it would most certainly require chromato-
graphic purification steps to fully eliminate the remains of unreacted
carbodiimide and its urea by-product. It could be argued that this
problem would be solved by employment of the water-soluble carbodi-
imide (WSC) N-ethyl-N0-(3-dimethylaminopropyl)carbodiimide[41] that,
together with its urea, can be removed by liquid–liquid extraction. How-
ever, the price of WSC is ca. four times higher than that of TBTU, which
makes the latter a more attractive choice.
Preparation of Compounds 2a–e as Potentially Bio- and
Ecofriendly Soaps
The lysine Na,Ne-diacyl derivatives 1a–e were saponified with sodium
hydroxide in aqueous-methanolic medium (Scheme 1ii). The saponi-
fications were carried out at 0 ꢀC with no excess alkali to avoid
amino acid racemisation[42] and reached completion within 3 h. The

2032
P. Gomes et al.
higher-molecular-weight saturated products 2c and 2d readily precipi-
tated from the reaction mixture and were simply isolated by suction fil-
tration followed by extensive rinsing of the solid with ice-cold water.
As compounds 2a, 2b, and 2e did not precipitate under the conditions
employed, their reaction mixtures were freeze dried to give the pure pro-
ducts. Once more, experimental procedures to obtain the target anionic
amphiphiles 2a–e from their ester precursors 1a–e were fast, clean, inex-
pensive, and straightforward, leading to excellent results.
Self-assembly Properties of Lysine-Based Surfactants
Preliminary studies on the aqueous self-assembly of the newly synthesized
anionic surfactants have revealed some very interesting features. The
shortest chain 2a and the unsaturated 2e amphiphiles are soluble at room
temperature, whereas 2b–d are insoluble. The surfactants alone in water
do not form micellar solutions nor liposome dispersions, as is the case
with conventional single- and double-chained compounds. Instead, they
have a tendency to self-aggregate into large tubular structures or fibers,
for which electrostatic interactions and hydrogen bonding play a key
structural role.[43] In Fig. 1, one can observe the type of micron-sized,
flexible tubules formed by the unsaturated amphiphile, in a 0.5 wt% dis-
persion in water, as observed under a light microscope with differential
interference contrast. Another technically very relevant property dis-
played by these amphiphiles is exemplified by compound 2d and can be
seen in Fig. 2. Upon mixing with a conventional cationic single-chained
surfactant such as dodecyltrimethyl ammonium bromide, spontaneouly
formed liposomes with high long-term stability can be obtained.[43] Such
observations open up a possible application of these amphiphiles in the
design of efficient encapsulation-based drug delivery systems.
Figure 1. Light micrograph for a 0.5 wt% aqueous dispersion of compound 2e,
displaying self-assembled long tubular aggregates (400ꢁ , h ¼ 25 ꢀC).

Lysine-Based Double-Chained Surfactants
2033
Figure 2. Cryotransmission electron micrograph of a 0.5 wt% aqueous dispersion
of compound 2d with the cationic surfactant DTAB (molar ratio 1:3), where self-
assembled unilamellar liposomes can be seen (h ¼ 25 ꢀC).
CONCLUSION
Ten lysine-based double-chained anionic surfactants have been prepared
by a clean and straightforward two-step synthesis. The synthetic pro-
cedures were successfully carried out at up to a 5-g scale, leading to
excellent yields and involving only simple and inexpensive compound
isolation techniques, such as liquid–liquid extraction, freeze drying, or
product precipitation.
Physicochemical studies carried out on some of the anionic amphi-
philes produced have shown that the neat compounds self-assemble in
water into tubular aggregates but can also form stable unilamellar lipo-
somes in binary mixtures. These properties are good indicators for the
potential pharmaceutical interest of these compounds.
In conclusion, the double-chain anionic amphiphiles herein
described, based on a proteinogenic amino acid and natural fatty acids,
are promising candidates for the industrial development of novel surfac-
tants with prospective biocompatibility, biodegradability, and versatility.
ACKNOWLEDGMENTS
ˆ
The authors thank Fundac¸a˜o para a Ciencia e Tecnologia (FCT, Lisbon-
Portugal) for financial support through project POCTI=QUI=
44296=2002 and through pluriannual funding to research unit Centro

2034
P. Gomes et al.
´
de Investigac¸a˜o em Quımica da Universidade do Porto (CIQUP, Porto-
Portugal). Thanks are due to Claudia Correia for laboratory assistance.
ꢀ
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