New alquil amide type cationic surfactants from arginine

Article abstract of DOI:10.1039/a705565j

The synthesis, stability, surface activity and antimicrobial properties of a new family of cationic surfactants (the long chain arginylalkylamide dihydrochloride salts) derived from the condensation of the amino acid arginine and a long chain alkylamine are described. The surface active parameters reported are c.m.c. (critical micellar concentration), pC₂₀ (negative log of the surfactant molar concentration required to reduce the surface tension of the solvent by 20 mN m^-1), γ_c.m.c (the surface tension at the c.m.c.), Γ_max (the maximum surface excess concentration) and A_min (the minimum area per surfactant molecule at the interface). These data and those obtained from the evaluation of the antimicrobial properties are compared with the data corresponding to another family of cationic surfactants reported earlier by our group: the long chain N^α-acylarginine methyl ester salts. Moreover, the synthesis of analogues possessing a reactive group capable of bonding to wool or cotton fibres is described: the long chain N^α-dichlorotriazinylarginylalkylamide monohydrochloride salts. We expect these compounds to bond to the textile substrate by the formation of a covalent bond. Confirmation of this is, however, necessary.

Full text of DOI:10.1039/a705565j

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New alquil amide type cationic surfactants from arginine
a
,
Eulàlia Piera, Francisco Comelles, Pilar Erra and M Rosa Infante* †
Department of Surfactant Technology, CID, (CSIC), Jordi Girona 18-26, 08034 Barcelona,
Spain
The synthesis, stability, surface activity and antimicrobial properties of a new family of cationic
surfactants (the long chain arginylalkylamide dihydrochloride salts) derived from the condensation of the
amino acid arginine and a long chain alkylamine are described. The surface active parameters reported are
c.m.c. (critical micellar concentration), pC20 (negative log of the surfactant molar concentration required
Ϫ1
to reduce the surface tension of the solvent by 20 mN m ), ã
(the surface tension at the c.m.c.),
c.m.c.
Ãmax (the maximum surface excess concentration) and Amin (the minimum area per surfactant molecule
at the interface). These data and those obtained from the evaluation of the antimicrobial properties are
compared with the data corresponding to another family of cationic surfactants reported earlier by our
á
group: the long chain N -acylarginine methyl ester salts.
Moreover, the synthesis of analogues possessing a reactive group capable of bonding to wool or cotton
á
fibres is described: the long chain N -dichlorotriazinylarginylalkylamide monohydrochloride salts.
We expect these compounds to bond to the textile substrate by the formation of a covalent bond.
Confirmation of this is, however, necessary.
α
Introduction
data will be compared with those of the long chain N -
7
acylarginine methyl ester salts reported earlier. The surface
Natural proteinaceous fibres such as wool are susceptible to
biological degradation by bacteria and to attack by insects such
as moths. In order to increase the lifetime of woollen items, the
wool needs to be protected from such agencies by antimicrobial
and mothproofing finishing treatments. However, traditional
antimicrobial and mothproofing finishing systems like quater-
active parameters studied include c.m.c. (critical micellar con-
centration), pC20 (negative log of the surfactant molar concen-
tration required to reduce the surface tension of the solvent
Ϫ1
by 20 mN m ), γ
(the surface tension at the c.m.c.), Γmax
c.m.c.
(
the maximum surface excess concentration at the air/aqueous
solution interface) and Amin (the minimum area per surfactant
molecule at the air/aqueous solution interface). Moreover,
the synthesis of their analogues, possessing a reactive group
capable of bonding to wool or cotton fibres and thus endow-
ing them with permanent properties is also described: the
1
nary ammonium surfactants have recently become less desir-
able from an environmental viewpoint and the use of systems
based on, e.g. chlorinated phenols, regardless of their effective-
ness, is severely limited by environmental legislation. Given
the significance of this field, the search for new biocompat-
ible alternatives constitutes an important challenge to wool
α
long chain N -dichlorotriazinyl arginylalkylamide monohy-
drochloride salts, [DCT᎐ArgNH᎐C ]ؒHCl with n = 12 and 14.
2,3
n
researchers.
It is well known that amino acid derivatives with alkyl chains
4
5,6
Cl
from fatty acids or fatty amines of varying chain length
C –C ) produce biocompatible lipoaminoacids with an inter-
N
(
12
16
CH3 (CH2)n NH CO CH NH
N
esting surface active behaviour. Our group has, since 1985,
undertaken research and development of cationic surfactants
N
(CH2)3
NH
α
Cl
of the long chain N -acyl arginine alkyl ester type with excellent
antimicrobial and non-toxic properties. It has been demon-
strated that the antimicrobial activity is directly associated with
the presence of a critical alkyl chain length of C12 carbon atoms
and the cationic charge of the protonated guanidino group of
the arginine available for interaction with the cell membrane in
C
H2N
+
NH2
–
Cl
n = 11 and 13
7–10
the molecule.
It is expected that if a reactive electrophilic grouping such as the
dichlorotriazine DCT is incorporated into the arginylalkyl-
amide cationic surfactant, the resulting compound will bond to
a textile substrate such as wool or cotton by way of a covalent
bond in a manner analogous to that of a reactive dye.
analogue N -dichlorotriazinylarginine methyl ester mono-
hydrochloride salt [H᎐DCT᎐ArgOMe]ؒHCl (without alkylic
In this paper, the synthesis, surface activity and antimicrobial
properties of a new family of cationic surfactants of the type
long chain arginylalkylamide dihydrochloride salts, [H᎐Arg-
11–14
NH᎐C ]ؒ2HCl with n = 10, 12, 14 and 16 are described. These
n
The
α
+
–
CH3 (CH2)n NH CO CH NH3Cl
(
CH2)3
Cl
NH
C
N
CH3O CO CH NH
CH2)3
N
H2N
+
–
NH2
Cl
N
(
Cl
n = 9, 11, 13, 15
NH
C
H2N
+
NH2
–
†
Fax: (34-3) 2045904; E-Mail: rimste@cid.csic.es
Cl
J. Chem. Soc., Perkin Trans. 2, 1998
335

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chain) was also prepared in order to use it as a model to
study the stability of the triazine reactive group. Data on this
parameter are also reported.
The UV spectra were recorded on a UV–VIS recording spec-
trophotometer 265FW Shimadzu.
The new class of compounds, whose synthesis is described in
this work, contains a fatty amine of C –C chain length con-
General procedure for the preparation of the long chain arginyl-
alkylamide dihydrochloride salts [H᎐ArgNH᎐C ]ؒ2HCl
10
16
n
á
densed to the α-carboxyl group of the arginine dihydrochloride
and optionally the 4,6-dichlorotriazin-2-yl grouping (–DCT)
attached to the α-amino group of the arginylalkylamide
homologue. Arginine was combined with fatty amines in order
to leave the α-amino group free to react with the triazinyl
reactive compound. All these compounds were designed as new
antimicrobial alternative surfactants of a presumably low tox-
icity for application as possible antibacterial agents to materials
such as wool or cotton. Treatments of these amphiphiles with
wool fibres are in progress and the results will be reported in the
near future.
Synthesis of long chain N -tert-butoxycarbonylarginyl-
alkylamide monohydrochloride salts: [Boc᎐ArgNH᎐C ]ؒHCl.
n
Boc᎐Arg (10.0 g, 30 mmol) was dissolved in DMF (130 ml) at
25 ЊC. To this solution triethylamine (8.4 ml, 30 mmol) was
added. After 30 min, the corresponding 1-alkylamine (30
mmol) was added, and finally BOP (13.28 g; 30 mmol). The
reaction mixture was left stirring overnight at 25 ЊC, after which
time TLC analysis was performed. The presence of a new
arginyl derivative product in the reaction mixture was
demonstrated.
The reaction mixture containing a precipitate was filtered
under vacuum and TLC analysis showed that the solid collected
was triethylamine hydrochloride. The DMF was evaporated
from the filtrate to yield an oil. Diethyl ether was added and left
in a freezer overnight, which resulted in the formation of a
precipitate that was isolated by filtration. The solid was washed
successively with diethyl ether, acetone, ethyl acetate and diethyl
ether. The residue was dissolved in methanol–hexane (1:9) and
Experimental
Synthesis
α
N -tert-Butoxycarbonyl arginine monohydrochloride [Boc᎐
Arg]ؒHCl (extra pure grade) was supplied by Novabiochem;
triethylamine (TEA), dimethylformamide (DMF) and tri-
fluoroacetic acid (TFA) (all of synthesis grade) were supplied by
Merck; [benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate] reagent (BOP) was supplied by Neo-
system Laboratoire; 1-decylamine, 1-dodecylamine, 1-tetra-
decylamine and 1-hexadecylamine (99%) were supplied by
Fluka; 2,4,6-trichloro-1,3,5-triazine grouping [TCT] was sup-
plied by Aldrich. All the other solvents, acids, alkalis and
electrolytes used were general purpose grade.
washed with H O. The organic layer was evaporated to dryness
2
under vacuum, dissolved in ethyl acetate and evaporated again
to yield a white solid. Although routine HPLC analysis of these
compounds was not possible because of their strong hydro-
phobic adsorption to the solid phase, [Boc᎐ArgNH᎐C ]ؒHCl
n
α
yielded one peak by CE.‡ N -tert-Butoxycarbonylarginyl-
decylamide monohydrochloride, [Boc᎐ArgNH᎐C ]ؒHCl, M
1
0
450.064, yield 40%, Rf (CHCl –MeOH 50:50) 0.64; mp
3
TLC analysis: the following TLC systems were used
throughout the experiments performed in this work to monitor
the progress of the reaction. Solvent: chloroform–methanol
133.6 ЊC (Found: C, 56.03; H, 9.96; N, 15.51. Calc. for
D
C H O N Clؒ¹H O: C, 56.04; H, 9.85; N, 15.56%); [α] Ϫ6.60
2
1
44
3
5
2
¯
²
α
(2% MeOH); CE t = 47.11 min. N -tert-Butoxycarbonyl-
M
(
1:1). Substrate: aluminium backed silica gel sheets (Merck).
arginyldodecylamide
monohydrochloride,
[Boc᎐ArgNH᎐
The plates were developed using the following techniques. (a)
C ]ؒHCl, M 478.118, yield 76%, R (CHCl –MeOH 50:50)
12
f
3
Ninhydrin: this detects the presence of primary amino groups;
0.67; mp 140.7 ЊC (Found: C, 56.81; H, 10.03; N, 14.48. Calc.
D
(
b) chlorine–toluidine: this detects any compounds containing
nitrogen after chlorine bleaching and spraying with toluidine;
c) sakaguchi: (α-naphthol-bromine): this detects the presence
for C H O N Clؒ¹H O: C, 56.66; H, 10.36; N, 14.31%); [α]
2
3
48
3
5
2
¯
²
α
Ϫ5.48 (2% MeOH); CE t = 48.81 min. N -tert-Butoxy-
M
(
carbonylarginyltetradecylamide monohydrochloride, [Boc᎐
ArgNH᎐C ]ؒHCl, M 506.172, yield 52%, R (CHCl –MeOH
of the arginyl guanidino group.
14
f
3
The purity of intermediate and final compounds was checked
by HPLC, capillary electrophoresis (CE) and elemental
analysis.
50:50) 0.69, mp 167.2 ЊC (Found: C, 58.90; H, 10.43; N, 13.71.
Calc. for C H O N Clؒ¹H O: C, 59.32; H, 10.35; N, 13.84%);
25
52
3
5
2
¯
²
D
α
[α] Ϫ10.75 (2% MeOH); CE t = 50.34 min. N -tert-
M
HPLC conditions: a Merck–Hitachi Model L-6200 liquid
chromatograph was used with an injection valve fitted with a 20
µl loop. The detection system consisted of a UV–VIS detector,
Butoxycarbonylarginylhexadecylamide
monohydrochloride,
[Boc᎐ArgNH᎐C ]ؒHCl, M 534.226, yield 65.5%, R (CHCl –
16
f
3
MeOH 50:50) 0.60, mp 159.4 ЊC (Found: C, 62.26; H, 11.23;
N, 11.27. Calc. for C H O N Clؒ¹H O: C, 60.70; H, 10.57; N,
2
10 nm wavelength Merck–Hitachi Model (L-4250). Chroma-
tography was accomplished by using a Lichrospher 100 RP-18
5 µm) in LichroCart 125-4 column (Merck). Flow rate: 1 ml
2
7
56
3
5
2
¯
²
D
13.11%); [α] Ϫ6.1 (2% MeOH); CE t = 52.17 min.
M
α
(
Spectral characteristics of long chain N -tert-butoxycarbon-
Ϫ1
min . The mobile phase was a solvent gradient 50–100% B in
5 min, A = 0.1% trifluoroacetic acid (TFA) in water, B = 0.21%
ylarginylalkylamide monohydrochloride salts, [Boc᎐ArgNH᎐
Ϫ1
2
C ]ؒHCl.—ν(KBr)/cm —3280 (NH), 3159 (NH amide), 2927
n
TFA in acetonitrile (ACN).
(CH ), 1686 (CO᎐N amide), 1510 (CO᎐N amide Boc-group);
2
CE conditions: the separations were performed with an
Applied Biosystems Model 270-A (USA) apparatus. A single
column was used with 72 cm × 50 µm id, uncoated fused silica
from Composite Metal Services (Works, UK). The separation
unit was equipped with a UV–VIS detector operating at a
δ (300 MHz, C D O ) 0.80 (t, 3H, CH ), 1.18 (m, 18H, CH ),
H
2
6
5
3
2
1.33 (s, 9H, CH Boc), 1.4–1.7 (m, 3H, CH ), 2.99 (m, 5H,
3
2
CH ᎐NH), 3.80 (m, 1H, CH), 6.69 (m, 1H, NH Boc), 7.10
2
(m, 4H, NH guanidine group), 7.73 (m, 2H, NH arginine);
2
δ (300 MHz, C D O ) 12.94 (CH ), 21.11–30.30 (CH ),
C
2
6
5
3
2
2
10 nm wavelength. Separation was performed at 5 kV applied
27.79–27.99 (CH , Boc), 52.88 (CH), 76.98 (C Boc group),
3
voltage; aqueous buffer 0.05  sodium dihydrogenphosphate
dihydrate, pH = 4.5, 50% ACN as organic modifier, 0.5 s injec-
tion time.
154.22 (CO Boc group), 156.04 (C᎐N guanidine group), 170.62
(CO amide).
Synthesis of long chain arginylalkylamide dihydrochloride
15
Melting points were determined with a FP81 measuring
cell of the FP90 Mettler System. Optical rotations were meas-
ured with a 141 Perkin-Elmer spectropolarimeter (Norwalk,
CT).
salts [H᎐ArgNH᎐C ]ؒ2HCl. Boc᎐ArgNH᎐C ؒHCl (4.9 mmol)
n
n
was dissolved in trifluoroacetic acid (TFA) (12.2 ml), at 4–5 ЊC.
The mixture was left stirring for 30 min at 4–5 ЊC, after which
time diethyl ether was added. The remaining TFA was elimin-
The structures of the intermediate and final products were
1
13
checked by H and C NMR analyses, which were recorded
with a Gemini 300 MHz spectrometer. The IR spectra were
recorded on an IRFT Nicolet 510 spectrophotometer.
‡
CE specific conditions: aqueous buffer 0.05  sodium dihydrogen-
phosphate dihydrate, pH = 4.5, 75% THF, 1 s injection time, 15 kV
applied voltage.
3
36
J. Chem. Soc., Perkin Trans. 2, 1998

View Article Online
Ϫ1
ated by passing N through the mixture. Then, methanol–HCl
HCl.—ν(KBr)/cm 3413 (NH), 3294 (NH amide), 2923 (CH ),
2
2
(
0.5 ) was added and stirred for five minutes. The remaining
1655–1700 (CO amide) and (C᎐N DCT group); δ (300 MHz,
᎐
H
2
HCl and the methanol were eliminated by evaporation and the
resulting oil-paste was dissolved in water and dried in the freeze
dryer yielding a pure solid by analytical HPLC and CE. Argin-
[ H ]methanol) 0.894 (t, 3H, CH ), 1.28 (m, 18H, CH ), 1.4–
4 3 2
2.0 (m, 6H, CH ), 3.23 (m, 4H, CH ᎐NH), 4.45 (m, 1H, CH);
2
2
2
δ (300 MHz, [ H ]acetone) 0.86 (t, 3H, CH ), 1.26 (m, 18H,
H
6
3
yldecylamide dihydrochloride [H᎐ArgNH᎐C ]ؒ2HCl,
M
CH ), 1.4–1.9 (m, 6H, CH ), 3.2–3.4 (m, 4H, CH ᎐NH),
10
2
2
2
3
9
86.408, R (CHCl –MeOH 50:50) 0.33 (Found: C, 47.43; H,
4.60 (m, 1H, CH), 7.08 (m, 4H, NH2 guanidine group),
f
3
2
.79; N, 17.28. Calc. for C H ON Cl ؒH O: C, 47.52; H, 9.72;
8.14 (m, 2H, NH); δ (300 MHz, [ H ]acetone) 14.32 (CH ),
16
37
5
2
2
C
6
3
D
N, 17.32%); [α] 22.3 (2% MeOH); HPLC t = 1.75 min, CE
23.27–46.81 (CH ), 55.78 (CH), 158.40 (C᎐N guanidine
R
2
tM = 35.79 min. Arginyldodecylamide dihydrochloride [H᎐
ArgNH᎐C ]ؒ2HCl, M 414.456, R (CHCl –MeOH 50:50) 0.36,
group), 166.83 (CO amide), 170.32, 170.99, 171.65 (C᎐N DCT
group).
᎐
12
f
3
mp 110–115 ЊC (Found: C, 48.13; H, 10.14; N, 15.50. Calc. for
D
á
C H ON Cl ؒH O: C, 47.99; H, 10.06; N, 15.54%); [α] 15.3
Procedure for the preparation of N -dichlorotriazinylarginine
18
41
5
2
2
(
2% MeOH), HPLC t = 21.28 min,§ CE tM = 36.66 min. Argin-
methyl ester monohydrochloride salt [DCT᎐ArgOMe]ؒHCl
Arginine methyl ester dihydrochloride (H᎐ArgOMe) (1 g, 3.83
mmol) was dispersed in DMF (3.2 ml) at 25 ЊC. Then, triethyl-
amine (0.53 ml, 3.83 mmol) was added and the reaction mixture
was left stirring overnight at 25 ЊC.
R
yltetradecylamide dihydrochloride [H᎐ArgNH᎐C ]ؒ2HCl, M
4
C, 52.22; H, 10.70; N, 15.16. Calc. for C H ON Cl ؒH O: C,
14
42.510, R (CHCl –MeOH 50:50) 0.37, mp 132 ЊC (Found:
f 3
20
45
5
2
2
D
5
2.16; H, 10.29; N, 15.21%); [α] 16.68 (2% MeOH), HPLC
tR = 5.80 min, CE tM = 37.53 min. Arginylhexadecylamide
dihydrochloride [H᎐ArgNH᎐C ]ؒ2HCl, 470.564, Rf
CHCl –MeOH 50:50) 0.47, mp 155–160 ЊC (Found: C, 57.78;
The reaction mixture was then filtered and TLC analysis
showed that the solid collected was triethylamine hydro-
chloride. Then, the DMF was evaporated from the filtrate
which contains the arginine methyl ester monohydrochloride
salt to yield an oil. This oil was dissolved in water–acetone (1:1)
and was added dropwise to a solution containing TCT (0.73 g,
3.96 mmol) in acetone. The pH was held at about 7–8 with
sodium carbonate throughout the reaction. The reaction mix-
ture was left stirring at 4–5 ЊC for one hour. Some more acetone
was added to the reaction mixture which was left in the freezer
overnight. This resulted in the precipitation of sodium carbon-
ate and sodium chloride. The reaction mixture was filtered and
the acetone was evaporated to obtain an aqueous medium in
which the residual TCT precipitated. This precipitate was isol-
ated by filtration. Finally, the filtrate was dried in the freeze
dryer yielding a solid. M 371.04, yield 59.3%, R (CHCl –MeOH
M
16
(
3
H, 11.51; N, 11.93. Calc. for C H ON Cl : C, 56.15; H, 10.50;
N, 14.88%); [α] 10.47 (2% MeOH), HPLC t = 11.66 min, CE
tM = 38.55 min.
22
49
5
2
R
Spectral characteristics of long chain arginylalkylamide
Ϫ1
dihydrochloride salts [H᎐ArgNH᎐C ]ؒ2HCl.—ν(KBr)/cm 3355
(
δ (300 MHz, C D O ) 0.83 (t, 3H, CH ), 1.22 (m, 14H, CH ),
1
CH), 7.34 (m, 4H, NH guanidine group), 8.00 (m, 1H, NH
arginine), 8.35 (m, 2H, NH ), 8.75 (m, 1H, NH amide); δ (300
MHz, C D O ) 13.97 (CH ), 22.11–31.32 (CH ), 51.56 (CH),
1
n
NH), 3178 (NH amide), 2917 (CH ), 1663 (CO᎐N amide);
2
H
2
6
5
3
2
.3–1.9 (m, 6H, CH ), 3–3.2 (m, 4H, CH ᎐NH), 3.8 (m, 1H,
2
2
2
2
C
2
6
5
3
2
57.12 (C᎐N guanidine group), 168.07 (CO amide).
᎐
f
3
á
General procedure for the preparation of N -dichlorotriazinyl-
50:50) 0.52 (Found: C, 32.66; H, 4.64; N, 25.13; Cl, 27.07. Calc.
arginylalkylamide monohydrochloride salts [DCT᎐ArgNH᎐ C ]ؒ
HCl
n
10 16
2
7
3
D
[
H᎐ArgNH᎐C ]ؒ2HCl (n = 10, 12) (2.6 mmol) was dispersed in
n
DMF (7 ml). Then, triethylamine was added (equimolar) and
the mixture was left stirring overnight at 25 ЊC. The reaction
mixture was filtered to remove the triethylamine hydrochloride
and then DMF was evaporated from the filtrate containing the
arginylalkylamide monohydrochloride [H᎐ArgNH᎐C ]ؒHCl.
2H, CH ᎐NH), 4.42 (m, 1H, CH), 7.24 (m, 4H, NH guanidine
n
2
2
[
H᎐ArgNH᎐C ]ؒHCl (2.6 mmol) was dissolved in water–
group), 7.87 (m, 1H, NH arginine), 9.54 (d, 1H, CH᎐NH᎐
DCT); δ (300 MHz, D O) 25.941, 28.36, 35.99 [(CH ) ᎐CH],
n
acetone (1:1) (20 ml), and was added dropwise to a solution
C
2
2 3
containing TCT in 21 ml of acetone (equimolar ϩ 10%) at 4–
52.55 (O᎐CH ), 54.92 (CH), 158.35 (C᎐N guanidine group),
3
5
ЊC. Sodium carbonate (0.5 equimolar) was added in order to
166.70 (C᎐O methyl ester), 170.19, 170.61, 171.84 (C᎐N DCT
hold the pH at about 7 throughout the reaction. Then, the reac-
tion was left stirring at 4–5 ЊC for one hour, after which time
the presence of the new product was demonstrated by HPLC
analysis.
group).
Stability
The stability of [DCT᎐ArgOMe]ؒHCl and [DCT᎐ArgNH᎐ C ]ؒ
n
The acetone was evaporated from the reaction mixture,
which was dried in the freeze dryer yielding a white solid. The
pure product was obtained from this solid by successive extrac-
HCl as a function of pH and temperature was evaluated by UV
spectrometry at 232 nm and C NMR spectroscopy. Given that
13
[DCT᎐ArgNH᎐C ]ؒHCl compounds are not soluble in water
n
α
tion with acetone. N -Dichlorotriazinylarginyldodecylamide
even at very low concentrations, these compounds were sus-
pended in an aqueous solution at the appropriate pH and
temperature.
monohydrochloride [DCT᎐ArgNH᎐C ]ؒHCl, M 525.953, R
12
f
(
CHCl –MeOH 50:50) 0.59, mp 137 ЊC (Found: C, 45.14; H,
3
7
.46; N, 20.81; Cl, 17.48. Calc. for C H ON Cl ؒ2H O: C,
21 39 8 3 2
4
4.84; H, 7.65; N, 19.93; Cl, 18.93%); HPLC t = 12.97 min.
Surface active properties
R
α
N -Dichlorotriazinylarginyltetradecylamide monohydrochlor-
ide [DCT᎐ArgNH᎐C ]ؒHCl, M 554.006, R (CHCl –MeOH
The surface tension measurements at equilibrium (γ) were
determined with a tensiometer (Krüss K-12) with a Wilhelmy
plate. All solutions, at different surfactant concentrations, were
prepared with deionized water and allowed to equilibrate for
2 h at 25 ЊC in appropriate cells. Effectiveness of adsorption
(pC ), critical micellar concentration (c.m.c.), maximum
14
f
3
5
0:50) 0.59, mp 145 ЊC (Found: C, 44.68; H, 7.33; N, 17.62;
Cl, 16.23. Calc. for C H ON Cl ؒ3H O: C, 45.39; H, 8.06;
N, 18.42; Cl, 17.49%); HPLC t = 15.84 min.
23
43
8
3
2
R
α
Spectral characteristics of long chain N -dichlorotriazinyl-
20
arginylalkylamide monohydrochloride salts [DCT᎐ArgNH᎐C ]ؒ
n
¶
The mobile phase was a solvent gradient 5–70% B in 32 min, A = 0.1%
§
The mobile phase was a solvent gradient 20–100% B in 40 min,
TFA in water, B = 0.085% TFA, in ACN–H O 80:20.
|| Aqueous buffer 0.05  sodium dihydrogenphosphate dihydrate,
pH = 4.5, 25 kV applied voltage, 5 s injection time.
2
Ϫ1
Ϫ1
A = 0.1% TFA, 70 µl l TFA in water, B = 0.085% TEA, 70 µl l TEA
in ACN.
J. Chem. Soc., Perkin Trans. 2, 1998
337

View Article Online
surface excess concentration (Γmax) and area per molecule (Amin)
were calculated from the γ/log C curves at equilibrium.
In this strategy the heart of the synthesis involves the use of
16
(benzotriazol-1-yloxytris(dimethylamino)phosphonium hexa-
fluoroposphate) BOP reagent in the presence of an activating
base to condense the α-carboxyl group of the Boc–Arg to
the primary amino group of the 1-alkylamine (n = 10, 12, 14
Antimicrobial properties
The minimum inhibitory concentration values (m.i.c.) were
determined by using a ‘microtriters’ system (disposable
multiple-well plates. Reg: 25850-96, Corning Lab. Sci. Co.,
USA) consisting of a plate containing a series of cups classified
in files and columns. Each cup is filled with the same concen-
tration of microorganism in Müller Hilton broth and a variable
concentration of surfactant. The growth of each micro-
organism is visually controlled at 24 h of incubation at 37 ЊC.
α
and 16) to yield a long chain N -tert-butoxycarbonylarginyl-
alkylamide monohydrochloride [Boc᎐ArgNH᎐C ]ؒHCl. The
n
employment of BOP to condense a primary alkylamine to the
carboxylic group of the arginine obviates the need for convert-
ing the essential non-electrophilic carboxylic group into the
highly electrophilic acyl chloride using reagents such as thionyl
chloride and phosphorus() chloride. The coupling reaction
was achieved rapidly by using Et N as a base without the pro-
3
tection of the guanidino group of arginine.
The subsequent deprotection procedure yielded quantitat-
Results and discussion
Synthesis
ively the required H᎐ArgNH᎐C compounds which were not
n
The synthetic pathways for the preparation of [H᎐ArgNH᎐C ]ؒ
further purified. White hygroscopic solids, such as dihydro-
chloride salts with optical activity were thus prepared as final
products. The effectiveness of the reaction was demonstrated
by HPLC and CE analysis of the final compounds. HPLC
n
2
HCl (n = 10, 12, 14 and 16) as dihydrochloride salts and
[
DCT᎐ArgNH᎐C ]ؒHCl (n = 12, 14) as monohydrochloride
n
salts are outlined in Scheme 1(a) and (b), respectively.
analysis yielded one peak for each of the H᎐ArgNH᎐C ؒ2HCl
n
compounds, whose tR decreased when the chain length
decreased. CE analyses also yielded one peak for each of the
compounds, whose tM decreased when the chain length
decreased. The purity of these compounds was about 99.5% on
the basis of elemental analysis, HPLC and CE.
(
a)
O
C
(
CH3)3C
O
NH CH CO2H
CH2)3
(
NH
Reactive DCT᎐ArgNH᎐C derivatives were prepared using
the arginylalkylamide salts as starting materials. The syn-
C
n
H2N
+
–
NH2
BOP
Cl
thetic mechanism for DCT᎐ArgNH᎐C ؒHCl (n = 12 and 14)
n
1
-alkylamine
compounds consisted of nucleophilic attack of the α-amino
group of the H᎐ArgNH᎐C ؒHCl on one of the C-atoms in the
TCT molecule. The purity of these compounds was about 93%
on the basis of elemental analysis, HPLC and CE.
n
O
C
(CH3)3C
O
NH CH CO NH (CH2)n CH3
The preparation of the H᎐DCT᎐ArgOMeؒHCl model
(
CH2)3
resembled that of DCT᎐ArgNH᎐C ؒHCl. The synthetic con-
n
NH
C
ditions yielded the required product with a purity of 99.7%,
showing a single peak by HPLC and CE analyses. Satisfactory
elemental analyses were obtained from these materials giving
FABMS and NMR spectra which were consistent with the
desired compounds.
H2N
+
–
NH2
Cl
(
(
i) TFA
ii) HCL
+
–
Cl H3N
CH CO NH (CH2)n CH3
Stability
(CH2)3
Whereas the H᎐ArgNH᎐C ؒ2HCl compounds were very stable
n
NH
C
in solid state or in aqueous solution over a wide range of pH
(3.0–9.0) even up to a temperature of 50 ЊC, the compounds
containing a ᎐DCT group showed a short lifetime. H᎐DCT᎐
ArgOMeؒHCl, stored dessicated at 0 ЊC showed purity for only
H2N
+
–
NH2
Cl
(
b)
3
–4 months; in alkaline aqueous solution at pH > 7 and at
+
–
room temperature it decomposed as a result of hydrolysis in a
few hours. The reaction with hydroxide ions in the aqueous
medium results in the displacement of one of the chloride
atoms, producing a less reactive N -monochloromonohydroxy
compound or a non-reactive N -dihydroxy compound. This
Cl H3N CH CO NH (CH2)n CH3
(
CH2)3
NH
α
DMF
C
α
H2N
+
–
NH2
TEA
Cl
H2N CH CO NH (CH2)n CH3
CH2)3
(a)
(b)
(
OH
N
OH
N
NH
N
N
N
N
C
CH3O CO CH NH
CH3O CO CH NH
Cl
N
H2N
+
–
NH2
(CH )
2 3
(CH )
2 3
N
N
Cl
Cl
OH
Cl
NH
NH
Cl
C
C
H2N
+
NH2
H2N
+
NH2
Cl
N
–
–
Cl
Cl
N
N
CH3 (CH2)n NH CO CH NH
CH2)3
reaction occurs despite the fact that the DCT᎐ArgNH᎐C ؒHCl
n
compounds are not soluble in water. They are present in
aqueous solution as a solid suspension. Even in this form, the
greater electronegativity of the chlorine atoms in the molecule
makes them labile and readily susceptible to stepwise nucleo-
philic displacement by an hydroxide ion. Dye-stuffs incorpor-
(
Cl
NH
C
H2N
+
–
NH2
Cl
17,18
Scheme 1
ating TCT groups show a similar stability behaviour.
3
38
J. Chem. Soc., Perkin Trans. 2, 1998

View Article Online
ties are influenced by the alkyl chain length, in particular, for
(a)
the homologue of 16 carbon atoms. The solubility properties
of arginylalkylamide salts are higher than those of the long
α
chain N -acylarginine alkyl esters reported earlier as a con-
sequence of possessing two ionic groups per molecule (δ-
guanidine and α-amino groups); for example, the water solubil-
α
ity was 25% (w/v) for N -dodecylarginine methyl ester (LAM)
α
19
and 10% (w/v) for N -tetradecylarginine methyl ester (TAM).
The introduction of a group such as dichlorotriazine into the
H᎐ArgNH᎐C molecule implies a dramatic loss of water solu-
n
bility. These reactive compounds were not soluble in water even
at very low concentrations. To ascertain whether or not these
compounds could be applied in a water solution, the water
solubility of mixtures DCT᎐ArgNH᎐C12 and H᎐ArgNH᎐C12
up to 50% (mol/mol) were determined in the range of temper-
atures 25–40 ЊC. All the solutions were water soluble at 25 ЊC
except the most concentrated mixture (50/50), which was sol-
uble at 40 ЊC. This solution maintained its solubility when the
temperature decreased up to 25 ЊC. Given the solubility proper-
ties of these compounds, the surface active properties were only
1
90 200
225
250
275
300
(b)
evaluated for the H᎐ArgNH᎐C dihydrochloride salts.
n
One of the main characteristics of surfactants is their ten-
dency to adsorb at interfaces in an oriented fashion as a con-
sequence of their amphipathic structure, consequently reducing
the surface tension of the solvent. This adsorption is important
in determining the amount of surfactant adsorbed per unit area
of the saturated interface or saturation absorption Γmax, which
is a measure of the change undergone by the interface because
of the surfactant. This depends on the structural groupings in
the surfactant molecule and its orientation at the interfaces. The
effectiveness of adsorption is related to the interfacial area
occupied by the surfactant molecule Amin; the smaller the effect-
ive cross-sectional area of the surfactant at interface the greater
190 200
225
250
λ /nm
275
300
16
its effectiveness of adsorption. Micelle formation is an alter-
native mechanism to adsorption for removing hydrophobic
groups from contact with water in which a number of inter-
facial phenomena such as solubilization, surface or interfacial
tension reduction are involved. For surfactants in aqueous solu-
tion the adsorption and micellization processes are both related
tothehydrophobic–hydrophilicbalanceofthemoleculeandthey
intensify with the increase in the hydrophobic character of the
surfactant, since this distorts the structure of the water and
Fig. 1 UV spectra for (a) the model H᎐DCT᎐ArgOMeؒHCl and (b) its
hydrolysed form
Table 1 Solubility of H᎐ArgNH᎐C ؒ2HCl compounds and DCT᎐
n
ArgNH᎐C ؒHCl compounds in 100 ml of water at 25 ЊC. The results
n
are indicated in % (w/v).
Compound
% (w/v)
H᎐ArgNH᎐C ؒ2HCl
34.37
32.22
29.82
1.53
1
0
16
H᎐ArgNH᎐C ؒ2HCl
therefore increases the free energy of the system. Fig. 2 shows
the surface tension (γ) vs. log surfactant concentration plots
for the four homologues in water solutions. c.m.c. values were
determined from the surface tension γ/log C curves at 25 ЊC.
These were taken as the concentrations at the point of intersec-
tion of the two linear portions of γ/log C plots. Maximum sur-
face excess concentration values or saturated adsorption (Γmax),
1
2
H᎐ArgNH᎐C ؒ2HCl
1
4
H᎐ArgNH᎐C ؒ2HCl
1
6
DCT᎐ArgNH᎐C ؒHCl
<0.1
<0.1
1
2
DCT᎐ArgNH᎐C ؒHCl
1
4
α
The presence of these compounds in a satisfactory N -
dihydrochloride form could be demonstrated by their UV
spectra, which show a characteristic maximum peak at 232 nm
changing the maximum toward the short wavelength region of
UV light, about 210 nm. The UV spectrum for the model ana-
logue H᎐DCT᎐ArgOMeؒHCl and the one for its hydrolysed
form are shown in Fig. 1.
Ϫ2
in mol cm and the minimum area per molecule (Amin) in the
air/water interface were calculated using the Gibbs adsorption
equation (1), where (∂γ/∂log C) is the slope of the γ/log C plot
T
Ϫ1
∂γ
Γmax
=
ͩ ͪ
(1)
2
.303nRT ∂ log C
T
13
α
The C NMR analysis of the N -dichlorotriazine com-
pounds exhibits characteristic signals of the three carbon atoms
in triazine bonding to chlorine atoms (C᎐Cl) at δ 170–174,
whereas, when hydrolysed, the resulting compound containing
C᎐OH groups shows a characteristic signal at about δ 150. It is
noticeable that the alkylamide compounds H᎐ArgNH᎐Cnؒ
Ϫ1
16
Amin = (N Γ) × 10
(2)
A
Ϫ1
Ϫ1
at a constant absolute temperature, T, R = 8.314 J mol K , n
is the number of species whose concentration at interface alters
with changes in the surfactant concentration in the solution.
For our cationic surfactants n = 3 (the dicationic amphiphile
residue and two chlorides) and N is the number of Avogadro.
Table 2 summarizes all these parameters together with the
γc.m.c. and pC20 which measure the effectiveness and the effi-
ciency of adsorption of the surfactants respectively.
2
HCl (in which no ester bonds exist) are more stable at alkaline
α
pH than the earlier described long chain N -acylarginine alkyl
ester surfactants.
Surface active properties
The solubilities in 100 ml of water at 25 ЊC were determined for
all H᎐ArgNH᎐C and DCT᎐ArgNH᎐C hydrochloride salts.
The results are indicated in Table 1. An isotropic liquid phase
was observed for the arginylalkylamide salts. Their solubili-
The c.m.c. values of H᎐ArgNH᎐C compounds show a fall
n
with the rise in the number of methylene groups in the alkyl
chain, as would be expected from the increase in the hydro-
phobic character of the molecule. In agreement with the rel-
n
n
J. Chem. Soc., Perkin Trans. 2, 1998
339

View Article Online
Fig. 2 Surface tension vs. log concentration at 25 ЊC for (a) H᎐ArgNH᎐C , (b) H᎐ArgNH᎐C , (c) H᎐ArgNH᎐C and (d) H᎐ArgNH᎐C16
1
0
12
14
Table 2 Surface active parameters for H᎐ArgNH᎐C ؒ2HCl compounds at 25 ЊC
n
Ϫ3
Ϫ1
Ϫ3
Ϫ3
14
Ϫ2
a
2
2
Ϫ1
Compound
γc.m.c./10 N m
pC20
c.m.c./10 mol dm
Γmax/10 mol m
Amin /10 nm molecule
H᎐ArgNH᎐C10
H᎐ArgNH᎐C12
H᎐ArgNH᎐C14
H᎐ArgNH᎐C16
34
35
35
43
2.60
4.00
4.80
5.09
20
1.0
0.16
0.038
1.17
0.99
0.98
0.92
142 (95)
167 (111)
169 (113)
180 (120)
a
Values in parentheses correspond to n = 2.
α
ationship observed for the long chain N -acylarginine methyl
of the new molecules contains two cationic groups per molecule
(instead of one in the case of N -acylarginine methyl ester
20
α
ester salts, the variation of log c.m.c. of H᎐ArgNH᎐C with
n
the number of carbon atoms (10, 12, 14 and 16) of the alkyl
chain is linear, with a slope significantly higher than that for the
N -acylarginine derivatives, thereby suggesting that the hydro-
compounds) which tend to spread out on the interface and
increase the repulsion inter–intramolecular forces yielding
higher Amin values. However, since Amin values of H᎐ArgNH᎐Cn
compounds when n = 3 are higher than expected, we are not
sure whether n = 3 or 2 for these surfactants (values in paren-
theses for n = 2 are more reasonable). The constant n depends
on the number of species constituting the surfactant which are
adsorbed at the interface. It has been argued that for other
surfactants of the type bisQuats with two quaternary ammo-
nium groups per molecule, one counterion is associated with
one ionic head group and, therefore, the value n = 2 should be
α
phobic contribution to micellation of the present surfactants
is higher (Fig. 3). As in a conventional series of homologues
with a different alkyl chain length, the efficiency of adsorption,
pC , increases as the number of carbon atoms grows. The
20
larger the pC20 value, the more efficiently the surfactant is
adsorbed at the interface and the more efficiently it reduces
surface tension.
For surfactants with a single hydrophilic group, the area
occupied by a surfactant molecule at the surface appears to be
determined by the area occupied by the hydrated hydrophilic
group, rather than by the hydrophobic group. For the H᎐Arg-
21
used. Amin values of H᎐ArgNH᎐C salts are consistent with
n
the values of γc.m.c.. The higher the value of Amin, the higher the
α
γc.m.c. values. They exceed those of the N -acylarginine methyl
NH᎐C compounds, the minimum area per molecule at the
ester derivatives because of the number of molecules adsorbed
at the surface and do not change significantly with the alkyl
n
aqueous solution/air interface, Amin (obtained from the values
of the saturated adsorption Γmax), assuming n = 3 in the Gibbs
chain length, except for the H᎐ArgNH᎐C derivative whose
16
2
2
2
2
Ϫ1
equation, is very large (A in the range 142–180 × 10 nm
Amin value is 180 × 10 mm molecule . For this homologue the
value of γc.m.c. is 43 mN m . It has been postulated that when
min
Ϫ1
α
Ϫ1
molecule ) if this is compared with the Amin values of the N -
2
acylarginine methyl ester derivatives: A (LAM) = 70.1 × 10
the number of carbon atoms in a straight-chain hydrophobic
group exceeds 16 there is a significant decrease in the effective-
ness of adsorption (the maximum change in γ), which has been
attributed to the coiling of the long chain with a subsequent
increase in the cross-sectional area of the molecule at the
min
2
Ϫ1
2
2
Ϫ1
nm molecule ; A (TAM) = 67.2 × 10 nm molecule . This
min
fact seems to indicate that given the size and nature of their
hydrophilic head groups, the molecules of arginylalkylamide
α
surfactants are less packed at the interface than those of N -
22
acylarginine methyl ester derivatives. The hydrophilic portion
interface.
3
40
J. Chem. Soc., Perkin Trans. 2, 1998

View Article Online
Ϫ1
Table 3 Minimum inhibitory concentration (m.i.c.) (µg ml ) of H᎐ArgNH᎐C ؒ2H O, H᎐ArgNH᎐C ؒ2H O, LAM and TAM
1
2
2
14
2
Ϫ1
m.i.c./µg ml
H᎐ArgNH᎐C ؒ2HCl
Microorganism
Gram negative
H᎐ArgNH᎐C ؒ2HCl
LAM
TAM
1
2
14
1
2
3
4
5
6
7
8
9
Alcalígenes faecalis ATCC 8750
Bordetella bronchiseptica ATCC 4617
Citrobacter freundii ATCC 11606
Enterobacter aerogenes ATCC 10938
Salmonella typhimurium ATCC 14028
Streptococcus faecalis ATCC 1054
Escherichia coli ATCC 27325
32
>128
>128
128
128
32
>128
32
>128
128
4
128
128
128
64
64
>128
32
128
64
128
16
>128
128
32
8
128
32
128
8
—
—
209
—
—
—
104
209
—
Klebsiella pneumoniae ATCC 13882
Pseudomonas aeruginosa ATCC 9721
1
0 Arthrobacter oxydans ATCC 8010
—
Gram positive
1
1
1
1
1
1
1 Bacillus cereus var. mycoides ATCC 1178
2 Bacillus subtilis ATCC 6633
3 Staphylococcus aureus ATCC 25178
4 Staphylococcus epidermis ATCC 155-1
5 Micrococcus luteus ATCC 9341
6 Candida albicans 10231
>128
128
>128
>128
128
64
32
1
128
64
64
64
26
4
6
8
—
52
52
13
52
—
32
32
α
long chain N -acylarginine methyl ester derivatives (owing to
the differences in the hydrophilic head groups) but with a
higher stability at alkaline pH values. They exhibit antimicro-
bial activity which depends on the alkyl chain length of the
hydrophobic part. These arginylalkylamide antimicrobial
surfactants can be regarded as a possible alternative to con-
ventional quaternary ammonium surfactants and as a good
starting material for preparing DCT᎐ArgNH᎐Cn reactive
compounds for covalent bonding to keratine or cotton
substrates.
These compounds were prepared with a view to developing a
new system of cationic surfactants with antimicrobial activity
for textile applications. Treatments of DCT᎐ArgNH᎐C com-
n
pounds solubilized in H᎐ArgNH᎐C solutions with wool fibres
n
and the testing of microbial resistance of fibres are currently in
progress. Synergistic and toxic studies of these mixtures are also
being carried out.
Fig. 3 Effect of length of the hydrophobic group on the c.m.c., (᭿)
arginylalkylamide salts, (᭡) N-acylarginine methyl ester salts
Acknowledgements
We are grateful to Professor D. Lewis and Dr A. N. Jarvis for
their useful contribution to the study on dichlorotriazine
derivatives. We also acknowledge financial support from
DGICYT (Grant PB93-0026), from Ministerio de Educación
y Ciencia and from the British Council (Acción Integrada
HB94-129B).
Antimicrobial properties
The evaluation of antimicrobial activity was carried out by
determination of the m.i.c. parameter (Table 3) (the lowest
concentration of surfactant at which the microorganisms tested
do not show visible growth). Given the water insolubility of
DCT᎐ArgNH᎐Cn compounds, the antimicrobial study was
only restricted to the H᎐ArgNH᎐C surfactants. Comparing
n
data for 16 selected microorganisms demonstrated that
References
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n
1
2
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a
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5
6
α
TAM in the series of N -acylarginine methyl esters salts, in the
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a
a
12
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9, 7.
a
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n
From these results we conclude that H᎐ArgNH᎐C ؒ2HCl
a
n
1
salts are a new family of amino acid based surfactants with a
surface active performance which is slightly lower than that of
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a
1
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1
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a
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3
42
J. Chem. Soc., Perkin Trans. 2, 1998