Synthesis and biological properties of dicationic arginine-diglycerides

Article abstract of DOI:10.1039/b203896j

A novel family of dicationic arginine-diglyceride surfactants, 1,2-diacyl-3-O-(L-arginyl)-rac-glycerol·2HCl (XXR) with alkyl chain lengths in the range of C₈-C₁₄ was prepared. These new surfactants can be regarded as analogues of lecithins. They have two hydrophobic tails of identical fatty acid chains attached to the glycerol through ester bonds and a dicationic polar head from arginine instead of the zwitterionic on the lecithins. These new compounds can be classified as multifunctional surfactants with self-aggregation behaviour comparable to that of short-chain lecithins. They have antimicrobial activity similar to that of the conventional cationic surfactants and are as harmless as amphoteric betaines.

Full text of DOI:10.1039/b203896j

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Synthesis and biological properties of dicationic arginine–diglycerides
a
L. P e´ rez, A. Pinazo, P. Vinardell, P. Clap e´ s, M. Angelet and M. R. Infante
a
c
b
a
a
a
Departamento de Tecnolog ı´ a de Tensioactivos, IIQAB, CID, CSIC, Jordi Girona 18-26, 08034,
Barcelona, Spain
Departamento de P e´ ptidos y Proteinas, IIQAB, CID, CSIC, Jordi Girona 18-26, 08034,
Barcelona, Spain
Departament de Fisiolog ı´ a-Divisi o´ IV, Facultat de farm a` cia, Av. Joan XXIII s/n, 08028,
Barcelona, Spain
b
c
Received (in London, UK) 22nd April 2002, Accepted 31st May 2002
First published as an Advance Article on the web 11th July 2002
A novel family of dicationic arginine–diglyceride surfactants, 1,2-diacyl-3-O-(L-arginyl)-rac-glycerolꢀ2HCl
(
8
XXR) with alkyl chain lengths in the range of C –C14 was prepared. These new surfactants can be regarded as
analogues of lecithins. They have two hydrophobic tails of identical fatty acid chains attached to the glycerol
through ester bonds and a dicationic polar head from arginine instead of the zwitterionic on the lecithins. These
new compounds can be classified as multifunctional surfactants with self-aggregation behaviour comparable to
that of short-chain lecithins. They have antimicrobial activity similar to that of the conventional cationic
surfactants and are as harmless as amphoteric betaines.
Introduction
Novel high-quality components of consumer friendly market
products require surfactants with multifunctional characteris-
tics such as: biodegradability, mildness, low potential toxicity,
water solubility, wide hydrophilic–lipophilic balance (HLB),
and ability to form lamellar phases and vesicles for potential
use in drug-delivery systems with antimicrobial activity.
Naturally occurring amino acids have been of particular
interest in the field of environmentally friendly surfactants.
Surfactant molecules from renewable raw materials that mimic
natural lipoamino acids are one of the preferred choices for
food and cosmetic applications. Given their natural and simple
structure, they show low toxicity and quick biodegradation. In
this sense, our group has gained wide experience of the synth-
esis of amino acid-based surfactants obtained from the combi-
nation of natural saturated fatty acids, alcohols and amines
with different amino acid head groups through ester, ether
and amide linkages. Thus, saturated single-chain surfactants
1
2
3
from arginine, tryptophan, double-chain ones from lysine,
4
glutamic acid and aspartic acid and gemini from arginine
5
Fig. 1 (a) Molecular structure of lecithins. (b) Molecular structure of
1,2-diacyl-3-O-(N-L-arginyl)glycerolꢀ2HCl (XXR), where X is the
number of carbon atoms in the alkyl chains.
with different ionic characters have proved to be in all cases
highly biodegradable, with low toxicity, ecotoxicity and irrita-
6
,7
tion effects. Water solubility and self-aggregation properties
have been associated directly with the chemical structure of the
molecule and only cationic lipoamino acids possessed antimi-
arginine amino acid instead of the phosphate derivative groups
of lecithins.
6
,8
crobial activity.
Polar glycerol-based lipids such as diacylglycerol derivatives
or short-chain lecithins are environmentally friendly com-
pounds with excellent dispersion properties. However, their
low water solubility and absence of antimicrobial activity limit
their uses as surfactants in many industrial applications.
In this paper, we describe a novel family of dicationic argi-
nine based surfactants, 1,2-diacyl-3-O-(L-arginyl)-rac-glycer-
olꢀ2HCl (XXR) (Fig. 1) with alkyl chain lengths in the range
The novel family of compounds proposed in this work
would combine in one molecule the physicochemical properties
of the glycero derivatives and those of the polar arginine-based
surfactants. It is also expected that they will be more hydrophi-
lic than diacylglycerol derivatives and therefore will have
higher water solubility properties. A dibasic amino acid such
as arginine was selected to introduce antimicrobial activity into
the molecule.
of C
8
–C14 . Structurally, they can be considered analogues of
Preliminary basic surface active properties of XXR com-
pounds including adsorption and self-aggregation in aqueous
9
lecithins (Fig. 1). These new surfactants have two hydrophobic
tails of identical fatty acid chains attached to the glycerol back-
bone through ester bonds and a cationic polar head from the
solution reveal that they show a critical micelle concentration,
ꢁ
CMC, in the range of 5–0.25 mM at 25 C. Comparing these
DOI: 10.1039/b203896j
New J. Chem., 2002, 26, 1221–1227
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This journal is # The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2002

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sodium bicarbonate was added. The remaining amount of
reactant N-Cbz-L-arginine (5.45 min) and the formation of
the 00RZ (5.23 min) in the reaction media was followed by
HPLC analysis. A conversion of 97% based on HPLC analysis
was obtained. The purification of 00RZ was carried out by pre-
parative HPLC and Ion Exchange Chromatography following
1
0
the procedure described by Mor a´ n et al. M 418.5; HPLC
¼ 5.23 min; Elemental Analysis calculated for C17
Clꢀ2H O in %: C, 44.9; H, 6.8; N, 12.3; Cl, 7.8. Found: C, 44.6;
H, 6.8; N, 12.2; Cl, 7.7. Spectral characteristics: MS: 382.9. H
NMR: Assignments corresponding to Scheme 1. d (ppm)
OD), 1.6–2.0 [m, 4H,(9)]; 3.2 [t, 2H,(10)]; 3.5 [d,
H,(1)]; 3.58 [m, 1H,(2)]; 4.0–4.6 [m, 3H,(3,5)]; 5.1 [s,
t
r
27 6 4
H O N -
2
1
H
(
CD
3
2
2
1
3
H,(8)]; 7.3 [m, 5H,(15,16,17,18,19)]. C NMR: Assignments
corresponding to Scheme 1. dC (ppm) (CD OD), 26.23 and
3
2
7
9.76 (9), 41.86 (10), 55.08 (5), 63.88 (1), 67.22 (8), 67.75 (3),
0.95 (2), 128.79 (19), 129.04 (17,18), 129.48 (15,16), 138.04
(
14), 158.58 (12,7), 173.60 (4).
The stability of the 00RZ compound as a function of the pH
Fig. 2 Polarised optical microscopy image of the liquid crystal for
ꢁ
2
the 88R/H O system at 35 C.
and the temperature was evaluated by HPLC analysis to estab-
lish the experimental conditions for the synthesis of XXRZ. 10
mg of 00RZ were dissolved in an aqueous solution (5 mL) at
the appropriate pH and temperature. HPLC analyses of these
solutions were carried out every 24 hours, monitoring the
decrease of the 00RZ peak and the formation and enlargement
of those corresponding to the N-Cbz-L-arginineꢀHCl com-
pound.
values with those of the lecithins with the same alkyl chain
length, the CMCs of the new compounds are one order of
magnitude higher due to the better solubility properties of
the hydrophilic head group for XXR. Furthermore, as in
lecithins, the new compounds formed lamellar and nematic
liquid crystal phases at room temperature (Fig. 2).
The chemical synthesis, the minimum inhibitory concentra-
tions against a series of microorganisms together with the
hemolytic activity and irritation effects are described.
Synthesis of 1,2-diacyl-3-O-(N-Cbz-L-arginyl)-rac-glycerolꢀHCl
(XXRZ)
A solution of 40 mL of pyridine containing 00RZ (0.015 mol, 5
g) of 96% purity by HPLC was placed in a round-bottomed
flask. To this solution, 0.05 mol of the corresponding long
chain acid chloride was added dropwise, stirring continually
at room temperature for 4 hours. After completion of the reac-
tion, pyridine was removed under reduced pressure. The result-
ing solid was dissolved in methanol and washed three times
with petroleum ether to remove the fatty acid excess. The sol-
vent was evaporated and the solid was dissolved in chloro-
form. This solution was then extracted with water to
eliminate water soluble impurities. After purification, transpar-
ent and viscous solids with 85% purity and yields exceeding
Experimental
All solvents were reagent grade and were used without further
purification. Trifluoracetic acid (TFA) was obtained from
Merck. N-Cbz-L-arginineꢀHCl (Cbz ¼ benzyloxycarbonyl)
was purchased from Bachem. Anhydrous glycerol and the long
chain acid chlorides with 8, 10, 12 and 14 carbon atoms were
supplied by Fluka. Boron trifluoroetherate (BF
3
2 5 2
ꢀO(C H ) )
was from Sigma. The progress of the reactions and purity of
the final products were monitored by analytical HPLC, model
Merck-Hitachi D-2500 with a Lichrospher 100 CN (propyl-
cyano) 5 mm, 250 ꢂ 4 mm column, using UV-VIS detector L-
9
0% were obtained. Two chromatographic purification techni-
ques were tested to obtain XXRZ compounds with purity
higher than 95%: preparative RP-HPLC and silica acid flash
chromatography.
4
250 at 215 nm. A gradient elution profile was employed from
the initial composition of A/B 75/25 (by volume), changing
over 24 min to a final composition of 5/95 where A is 0.1%
(
v/v) TFA in H
:4. The flow-rate through the column was 1.0 ml min
2
O and B is 0.085% of TFA in H
2
O/CH
3
CN
.
H
ꢃ1
1
1
The structures of the pure compounds were checked by
Preparative RP-HPLC purification. 1 g of the product
XXRZ was loaded into a preparative PrePack (Waters) col-
1
3
and C NMR analyses, which were recorded with a Varian
00 MHz spectrometer. Chemical shifts are reported in parts
umn (47 ꢂ 300 mm) filled with DeltaPack C , 300 mm, 15 mm
4
3
3
stationary phase. The product was eluted using a CH CN gra-
per million (d, in ppm) downfield of tetramethylsilane (TMS)
as internal reference. Mass spectroscopy (MS) spectra with fast
atom bombardment (FAB) or electrospray techniques were
also conducted with a VG-QUATTRO spectrometer from
Fisons Instruments. Elemental analyses of the final com-
pounds were recorded at the Microanalytical Service at the
IIQAB-CSIC. Preparative reversed-phase (RP)-HPLC runs
were performed on a Waters (Milford, MA, USA) Prep LC
dient (50 to 70% B in 30 minutes) in 0.1% aqueous TFA. The
ꢃ1
flow rate was 100 mL min and detection at 220 nm. Analysis
of the fractions was accomplished under isocratic conditions
using a Lichrosphere 100 CN (propylcyano), 5 mm, 250 ꢂ 4
2
mm column, eluted with 67% of B (0.085% TFA in H O/
3
CH CN 1:4) and 33% A (0.1% aqueous TFA), flow rate 1
ꢃ1
mL min and detection at 215 nm. The pure fractions were
pooled and lyophilised.
4
000 pumping system.
Silica acid flash chromatography purification. Silica (100 mL
Chromagel 60A CC, 70–230) was packed into a flash chroma-
tography column. Then XXRZ (3 g, purity of 85%) dissolved
in chloroform was loaded into the column and was eluted with
a gradient from chloroform to chloroform/methanol, 90/
10(v/v). The fractions containing the desired products were
pooled and evaporated to dryness.
Synthesis of 1-O-(N-Cbz-L-arginyl)-rac-glycerolꢀHCl (00RZ)
1
7.24 g of N-Cbz-L-arginineꢀHCl (50 mmol) were dissolved in
ꢁ
2
ml of BF
00 mL of glycerol. The solution was heated to 55 C and 25
3
2 5 2
ꢀO(C H ) was added to the stirred mixture drop-
wise. After 1 h the addition was completed and then the reac-
tion mixture was stirred for 5 h. Then, 400 mL of 0.6 M
1
222
New J. Chem., 2002, 26, 1221–1227

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Scheme 1 Synthetic pathway for the preparation of XXR compounds.
1
,2-Dioctyl-3-O-(N-Cbz-L-arginyl)-rac-glycerol, 88RZ (as
trifluoroacetate salt). Yield 40% (purified by RP-HPLC);
HPLC t ꢀ0.5H O.
¼ 19.23 min, M 748.79: C35
Anal. calc. (%) C, 55.4, H, 7.4, N, 7.5. Found: C, 55.4; H,
1.22 [s, 28H, (14CH
2
of the alkyl chain)], 1.42–1.86 [m, 8H,
(–CH –CH –COO–), (9)], 2.22–2.28 [2t, 6H (–CH –COO–)],
2
2
2
r
H
55
O
10
N
4
F
3
2
3.07 [m, (10)], 4.03–4.29 [m, 5H, (1)(3)(5)], 5.02 [s, 2H (8)],
5.12–5.22 [m, 1H (2)], 7.22–7.37 [m, 9H, (13)(15)(16)(17)-
1
13
7
.65; N, 7.5. MS: 635.1; H NMR: d
H
(CD
2CH of the alkyl chain)], 1.30 [s, 20H, (10CH of the alkyl
3
OD), 0.89 [t, 6H,
C 3
(18)(19)]. C NMR: d (DMSO), 13.72 [CH –, alkyl chain],
(
chain)], 1.40–2.00 [m, 8H, (–CH
21.93–33.34 [–CH – alkyl chain and (9), (10)], 53.47 [(5)],
3
2
2
2
–CH
2
–COO–), (9)], 2.29–
61.68 [(1)], 62.29 [(3)], 65.48 [(8)], 68.55 [(2)], 127.54 [(19)],
127.66 [(17)(18)], 128.19 [(15)(16)], 136.72 [(14)], 155.97 [(12)],
157.05 [(7)], 171.64 [(4)], 172.05 [(20)], 172.33 [(21)].
2
.35 [2t, (–CH –COO–)], 3.19 [t, (10)], 4.12–4.40 [m, 5H,
2
1)(3)(5)], 5.10 [s, 2H (8)], 5.23–5.32 [m, 1H, (2)], 7.29–7.35
(
[
1
3
m, 5H, (15)(16)(17)(18)(19)]. C NMR: d
CH –, alkyl chain], 23.68–41.85 [–CH – alkyl chain and (9),
10)], 55.02 [(5)], 63.26 [(1)], 64.04 [(3)], 67.79 [(8)], 70.45
(2)], 128.82 [(19)], 129.05 [(17)(18)], 129.48 [(15)(16)], 138.08
(14)], 158.63 [(12)], 173.17 [(4)], 174.47 [(20)], 174.83 [(21)].
H 3
(CD OD), 14.42
[
3
2
1,2-Dilauroyl-3-O-(N-Cbz-L-arginyl)-rac-glycerolꢀHCl,
(
[
[
1212RZ. Yield 70%; HPLC, t ¼ 21.71 min. M 782.5: Anal.
r
calc. (%) for C H O N Cl, C, 62.9; H, 9.1; N, 7.15; Cl, 4.5.
4
1
71
8
4
1
Found: C, 62.65; H, 9.2; N, 7.1; Cl, 4.55. M/S: 748.14; H
NMR: dH (CD OD), 0.88 [t, 6H, (2CH of the alkyl chain)],
3
3
1
,2-Didecyl-3-O-(N-Cbz-L-arginyl)-rac-glycerolꢀHCl,
1.29 [s, 32H, (16CH
(–CH –CH –COO–), (9)], 2.27–2.35 [2t, 6H (–CH –COO–)],
2 2 2
2
of the alkyl chain)], 1.57–1.86 [m, 8H,
1
010RZ. Yield 62%. HPLC, t ¼ 20.54 min. M 726.5: Anal.
r
calc. (%) for C37
H
63
O
8
N
14ClꢀH
2
O. C, 59.6; H,8.7; N, 7.52;
Cl, 4.8. Found: C, 59.54; H, 9.1; N, 7.52; Cl,4.7. MS: 692.09;
3.20 [t, (10)], 4.13–4.44 [m, 5H, (1)(3)(5)], 5.10 [s, 2H (8)],
5.22–5.26 [m, 1H (2)], 7.29–7.38 [m, 5H, (15)(16)(17)(18)(19)],
1
13
H NMR: d
H
(CD OD), 0.83 [t, 6H, (2CH
3
3
of the alkyl chain)],
7.73 [d, 1H, (6)]. C NMR: d
C
(CD
3
OD), 14.50 [CH
3
–, alkyl
New J. Chem., 2002, 26, 1221–1227
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chain], 23.76–41.88 [–CH – alkyl chain, (9), (10)], 55.03 [(5)],
2
Synthesis of 1,2-diacyl-3-O-(L-arginyl)-rac-glycerolꢀ2(HCl)
6
1
1
3.30 [(1)], 64.02 [(3)], 67.77 [(8)], 70.44 [(2)], 128.84 [(19)],
29.06 [(17)(18)], 129.50 [(15)(16)], 138.09 [(14)], 158.57 [(12)],
73.17 [(4)], 174.42 [(20)], 174.78 [(21)].
(XXR)
The corresponding pure XXRZ (0.0008 moles) was dissolved
in 35 mL of methanol and hydrogenated (25 minutes) in the
presence of Pd on activated charcoal (10% Pd) as catalyst.
To control the pH of the reaction between 5–8, a solution of
methanol with 0.0008 moles of HCl in water was added. The
catalyst was filtered off on celite and rinsed with methanol.
The filtrates were pooled and concentrated under reduced pres-
sure. Pure compounds were obtained by several crystalliza-
tions from methanol/acetone. See Tables 1 and 2 for
analytical and spectroscopic data.
1
414RZ. Yield 76%; HPLC, t
,2-Dimiristoyl-3-O-(N-Cbz-L-arginyl)-rac-glycerolꢀHCl,
1
calc. (%). for C H O N Cl, C, 64.6; H, 9.4; N, 6.6; Cl, 4.8.
r
¼ 22.79 min. M 835.5: Anal.
4
5
79
8
4
1
Found: C, 64.5; H, 9.2; N, 6.4; Cl, 4.4. M/S: 803.43;
NMR: d (CD OD), 0.90 [t, 6H, (CH of the alkyl chain)],
.28 [s, 40H, (20CH of the alkyl chain)], 1.57–1.96 [m, 8H,
–CH –CH –COO–), (9)], 2.29–2.34 [2t, (–CH –COO–)], 3.21
m, (10)], 4.12–4.43 [m, 5H, (1)(3)(5)], 5.11 [s, 2H (8)], 5.21–
H
H
3
3
1
2
(
2
2
2
Hemolytic activity
[
5
1
3
.34 [m, 1H (2)], 7.24–7.41 [m, 5H, (15)(16)(17)(18)(19)].
(CD OD), 14.52 [CH –, alkyl chain], 23.77–41.88
– alkyl chain and (9), (10)], 55.04 [(5)], 63.31 [(1)],
4.02 [(3)], 67.75 [(8)], 70.44 [(2)], 128.79 [(19)], 129.04
C
Erythrocytes preparation. Human blood was obtained from
the Blood Bank of the Hospital Clinic (Barcelona). Blood
was drain into heparinized tubs. The erythrocytes were washed
three times in phosphate buffer isotonic saline (PBS), contain-
NMR: d
–CH
6
C
3
3
[
2
ꢃ1
ꢃ1
ꢃ1
[
[
(17)(18)], 129.49 [(15)(16)], 138.07 [(14)], 158.56 [(12)], 173.13
(4)], 174.38 [(20)], 174.78 [(21)].
ing: 22.2 mmol L Na
2
HPO4; 5.6 mmol L KH
2
PO
4
, 123.3
mmol L NaCl, glucose 10.0 mmol L in distilled water
ꢃ1
Table 1 Analytical data of 1,2-diacyl-3-O-(N-L-arginyl)-rac-glycerolꢀ2(HCl) (XXR)
Elemental analysis calcd. (with 1 mol H
2
O)/found
Molecular formula;
molecular weight
HPLC retention
time (min.)
Compound
Yield (%)
%C
%H
%N
%Cl
a
b
c
a
b
c
a
a
8
1
1
1
8R
C
25
H
H
H
H
52
N
58
N
66
N
74
N
4
4
4
4
O
6
O
6
O
6
O
6
Cl
2
Cl
2
Cl
2
Cl
2
60
66
12.77
16.0
18.0
19.3
48.5
48.3
52.3
52.8
56.3
56.0
57.0
57.4
8.9
8.8
9.3
9.5
9.7
9.7
10.0
9.9
9.05
9.3
11.5
11.7
10.7
10.5
10.1
10.4
9.2
574
b
b
c
d
010R
212R
414R
C
29
8.4
8.7
629
c
C
33
71
78
8.0
8.3
685
d
d
d
C
37
7.2
6.8
741
9.2
a
b
O. Calculated with 2 mol H
c
O. Calculated with 1 mol H
d
O. Calculated with 2 mol H
Calculated with 2.5 mol H
2
2
2
2
O.
Table 2 Spectral assignments for 1,2-diacyl-3-O-(N-L-arginyl)-rac-glycerolꢀ2(HCl) (XXR) compounds
+
1
a
13
a
Compound m/z (M ) H NMR (DMSO), d
C NMR (DMSO), d
8
1
1
1
8R
501.2
557.4
613.7
669.7
0.90 [m, 6H, (2 CH
3
of the alkyl chain)],
14.43 [CH
3
2
–, alkyl chain], 23.68–41.75 [–CH – alkyl chain and (9),
1.31 [s, 16H, (8 CH
1.59–2.01 [m, 8H, (–CH
2.31–2.38 [2t, 2 (–CH –COO–)], 3.25–3.31
2
of the alkyl chain)],
(10)], 53.57 [(5)], 63.17 [(1)], 65.42 [(3)], 70.31 [(2)], 158.64 [(12)],
170.10 [(4)], 174.45 [(20)], 174.81 [(21)]
2
–CH –COO–), (9)],
2
2
[
m, (10)], 4.12–4.61 [m, 5H, (1)(3)(5)],
.32–5.36 [m, 1H (2)]
0.86 [m, 6H, (2 CH of the alkyl chain)],
.26 [s, 24H, (12 CH of the alkyl chain)],
.55–2.02 [m, 8H, (–CH –CH –COO–), (9)],
.27–2.33 [2t, (–CH –COO–)],
5
010R
2,12,R
4,14,R
3
3 2
14.47 [CH –, alkyl chain], 23.73–41.79 [–CH – alkyl chain and (9),
1
1
2
3
5
2
(10)], 53.68 [(5)], 63.197 [(1)], 65.25 [(3)], 70.34 [(2)], 158.64 [(12)],
170.63 [(4)], 174.46 [(20)], 174.81 [(21)]
2
2
2
.25 [m, (10)], 4.02–4.55 [m, 5H, (1)(3)(5)],
.28–5.37 [m, 1H (2)]
0.90 [m, 6H, (2 CH
3
of the alkyl chain)]
.29 [s, 32H, 16 CH of the alkyl chain)]
.57–2.03 [m, 8H, (–CH –CH –COO–), (9)]
.31–2.38 [2t, (–CH –COO–)] 3.27 [m, (10)],
3 2
14.48 [CH –, alkyl chain] 23.75–41.73 [–CH – alkyl chain and (9),
1
1
2
4
5
2
(10)] 53.56 [(5)], 63.19 [(1)], 65.30 [(3)] 70.29 [(2)], 158.62 [(12)],
170.01 [(4)], 174.36 [(20)], 174.76 [(21)]
2
2
2
.12–4.60 [m, 5H, (1)(3)(5)]
.31–5.39 [m, 1H (2)]
0.90 [m, 6H, (2 CH
3
of the alkyl chain)],
.29 [s, 40H, (20 CH of the alkyl chain)],
.57–2.31 [m, 8H, 2 (–CH –CH –COO–), (9)],
.31–2.37 [2t, (–CH –COO–)], 3.27 [m, (10)],
3 2
14.49 [CH –, alkyl chain], 23.76–41.74 [–CH – alkyl chain and (9),
1
1
2
4
5
2
(10)], 53.57 [(5)], 63.21 [(1)], 65.39 [(3)], 70.31 [(2)], 158.64 [(12)],
170.06 [(4)], 174.44 [(20)], 174.78 [(21)]
2
2
2
.12–4.60 [m, 5H, (1)(3)(5)],
.31–5.39 [m, 1H (2)]
a
Assignments corresponding to Scheme 1C.
1
224
New J. Chem., 2002, 26, 1221–1227

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(
pH ¼ 7.4). The cells were then suspended in the PBS at a cell
yielding a fast, clean and high (95%) conversion of N-Cbz-L-
arginineꢀHCl to the glycerol arginine ester. Purification of
the 00RZ product was carried out by two chromatographic
techniques, preparative HPLC (yield ¼ 45%) and ion
exchange chromatography (yield ¼ 84%).
9
ꢃ1
.
density of 8 ꢂ 10 cell mL
Hemolysis assay. Different volumes from 10 to 80 mL of sur-
factant solution (1 mg mL ) were pipetted into polystyrene
ꢃ1
tubs. Aliquots of 25 mL of erythrocyte suspension were added
to the tubs and incubated for 10 minutes, whilst shaking, at
room temperature. Following incubation, the tubes were cen-
trifuged (5 min at 5000 rpm) and the percentage of hemolysis
was determined by comparing the absorbance (540 nm) of
the supernatant with that of control samples totally hemolysed
with distilled water.
The next synthetic step 1B (Scheme 1) consists of the pre-
paration of 1,2-diacyl-3-O-(N-Cbz-L-arginyl)-rac-glycerolꢀHCl
(XXRZ) by acylation of the two remaining free hydroxyls
groups of 1-O-(N-Cbz-L-arginyl)-rac-glycerolꢀHCl (00RZ)
with the corresponding long chain acid chloride. Several meth-
ods of preparation described in the literature for the acylation
1
7
of the hydroxyl groups of glycerol use high temperatures and
basic or acid catalyst. Under these conditions hydrolysis of the
ester bond between the arginine and the glycerol may occur.
In order to assess the appropriate experimental conditions
for the acylation of the 00RZ compound without hydrolysis
of the ester bond of this compound, the stability of 00RZ as
a function of pH and temperature was studied (Figs. 3, 4).
Figs. 3(a) and (b) show that the compound 00RZ is stable in
aqueous solution over a wide range of pH (3.0–10.0) at room
temperature. At this pH range, percentages of decomposition
lower than 5% in 144 hours were observed. Alkaline aqueous
solution (pH > 10) or acidic aqueous solution (pH < 3) of
the 00RZ at room temperature decomposed in a few minutes
Protein denaturation. Aliquots of 100 mL of test sample were
added to 875 mL of PBS and 25 mL of erythrocyte suspension
were added to the tubs and incubated for 10 minutes. The
tubes were centrifuged and the absorbance was determined
at the supernatant at 575 nm and 540 nm in a dual-beam
UV/VIS spectrophotometer. The ratio of absorbance at 575
nm and 540 nm allowed the calculation of the hemoglobin
denaturation index (DI) which was compared with the SDS
as the internal standard.
L/D ratio. The relationship between hemolysis (HC50) and
the denaturation index (DI) is defined as the lysis/denatura-
tion quotient or L/D ratio used to predict the potential ocular
as a result of hydrolysis. Fig. 4 shows the percentage of the
ꢁ
00RZ hydrolysis versus temperature. At 25 C the compound
remains stable during the whole period of study. At higher
1
1,12
irritation of the surfactant.
The surfactants were classified
ꢁ
temperatures, 40 and 50 C, percentages of hydrolysis of 5%
are found.
in accordance with the following criteria: L/D < 0.1 very irri-
tant, L/D from 0.1 to 1 irritant, L/D from 1 to10 moderately
irritant, L/D from 10 to 100 slightly irritant and L/D > 100
non irritant.
Antimicrobial activity
The antimicrobial activities were determined ‘‘in vitro’’ on the
1
3
basis of the minimum inhibitory concentration (MIC) values,
defined as the lowest concentration of antimicrobial agent
which inhibits the development of visible growth after 24 h
ꢁ
of incubation at 37 C. The micro-organisms used (15 bacteria
and one yeast) were the following: gram-negative bacteria:
Alcaligenes faecalis ATCC 8750, Bordetella bronchiseptica
ATCC 4617, Citrobacter freundii ATCC11606, Enterobacter
aerogenes ATCC 10938, Salmonella typhimurium ATCC
1
4028, Streptococcus faecalis ATCC 1054, Escherichia coli
ATCC 27325, Klebsiella pneumoniae ATCC 9721, Pseudomo-
nas aeruginosa ATCC 9721, Arthrobacter oxydans ATCC
8
010; gram-positive bacteria: Bacillus cereus var. mycoides
ATCC 11778, Bacillus subtilis ATCC 6633, Staphylococcus
aereus ATCC 25178, Staphylococcus epidermidis ATCC
12228, Micrococcus luteus ATCC 9341; yeast: Candida albicans
ATCC 10231.
Results and discussion
The synthesis of the XXR compounds was achieved following
a three step procedure, starting from commercially available
N-Cbz-L-arginineꢀHCl. The synthetic pathway for the prepara-
tion of these compounds is outlined in Scheme 1.
The first step 1A (Scheme 1) consists of the preparation of 1-
O-(N-Cbz-L-arginyl)-rac-glycerolꢀHCl (00RZ) by chemical
esterification of the a-carboxyl group of N-Cbz-L-arginineꢀHCl
HCl with the primary hydroxyl function of glycerol using
boron trifluoroetherate as catalyst. Boron trifluoroetherate is
widely used in the esterification of various kinds of carboxylic
1
4
acids with alkyl alcohols and in the synthetic preparation of
amides from carboxylic acids. This reagent has also been
employed to synthesise glycerol esters of several amino acids.
In our case the use of boron trifluoroetherate as catalyst allows
us to develop the esterification reaction under mild conditions
1
5
Fig. 3 (a) Stability of the 00RZ compounds at room temperature and
pH 1 (L); pH 2 (S); pH 3 (G); pH 4 (;); pH 5 (˘); pH 6 (+). (b) Sta-
bility of the 00RZ compounds at room temperature and pH 12 (;);
pH 11 (G); pH 10 (+); pH 9 (S); pH 8 (˘); pH 7 (L).
1
6
New J. Chem., 2002, 26, 1221–1227
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View Article Online
1
3
The C NMR spectrum of the main compound provides
four signals, corresponding to the three carbons of the glycerol
skeleton [70.397 ppm, C(2); 63.994 ppm, C(3); 63.266 ppm,
C(1)], and C(8) [67.716 ppm] (see Scheme 1B). The same spec-
trum region for the 2-O-regioisomer only gives three signals.
The C(1) and C(3) of the glycerol have the same chemical shift
(
62.975 ppm), which means that these carbons are magneti-
cally and chemically equivalent. This is only possible if the
arginine is linked to C(2) of glycerol.
It has been described for monoglycerides that acyl migration
takes place easily giving an equilibrium mixture of the two
regioisomers: 2-monoglyceride (10%) and 1-monoglyceride
1
8
(
(
90%). In the case of 1-O-(N-Cbz-L-arginyl)glycerolꢀHCl
00RZ) this type of migration occurs probably when it is dis-
solved in pyridine to carry out the acylation of the hydroxyl
groups of this compound. Because of this, the 2-O-regioisomer
1,3-diacyl-2-O(N-Cbz-L-arginyl)glycerolꢀHCl (XRZX) in per-
centages of 5–10% was obtained.
The target compounds XXR were obtained by catalytic
hydrogenation of the Cbz group using Pd over charcoal
(Scheme 1C). The reaction was carried out controlling the
pH between 4–7 by the addition of a solution of HCl in metha-
nol. This allowed us to obtain the products as dihydrochloride
salts and avoid the hydrolysis of the ester linkages. Hydroge-
nation was controlled by HPLC by monitoring the disappear-
Fig. 4 Stability of the OORZ compounds with the temperature;
ꢁ
ꢁ
ꢁ
2
5 C (s); 40 C (L); 50 C (S).
Accordingly, the reaction for the esterification of the two
hydroxyl groups of 00RZ was carried out with long chain acid
chloride in pyridine at room temperature (Scheme 1B). Under
these conditions, the reaction mixture was kept in the pH
range 5–7 and the starting compound 00RZ remained stable.
The desired glycerol ester was obtained in an overall yield
exceeding 90%.
The main advantage of this reaction was the simple proce-
dure of the product purification owing to the clean reaction
mixtures obtained. The crude reaction mixture consisted basi-
cally of the product (XXRZ) and the excess of acid chloride.
By successive hexane and water extractions, products with a
purity of 85–90% (by HPLC) with a 80–90% yield were
achieved. Products with purity exceeding 98% were obtained
by both preparative RP-HPLC and silica gel chromatography.
By preparative RP-HPLC yields of 45% were obtained. Better
yields, 60–80%, were obtained by purification of the crude
reaction with silica gel column chromatography.
1
13
ance of the protected compounds. H NMR and C NMR of
all the final compounds showed the characteristic peaks corre-
sponding to the expected compounds (Table 2). The mass spec-
tra of XXR compounds showed a molecular ion peak
+
corresponding to [M + H] ꢃ 2Cl.
Antimicrobial activity
The dilution antimicrobial susceptibility test was carried out
and the minimum inhibitory concentration (MIC) values were
determined (Table 3). In contrast to short-chain lecithins,
XXR compounds exhibited antimicrobial activity. As
expected, the gram-negative bacteria were more resistant than
the gram-positive bacteria. This antimicrobial activity can
make them suitable for the subsequent biodegradation of
these surfactants. It is well known that some gram-negative
bacteria are relatively insensitive because their outer mem-
branes are impermeable to some hydrophobic–hydrophilic
For the four compounds synthesized, the HPLC chromato-
gram shows two peaks at the end of the reaction, a main peak
and a minor peak constituting 5–10% of the product. After
careful separation by silica gel column chromatography, by
1
9
compounds.
1
3
C NMR the main peak was identified as the desired products
XXRZ) and the minor peak was identified as the 2-O-regioi-
The maximum antimicrobial activity corresponds to the
compound with 8 carbon atoms in the alkyl chains, then, the
activity decreases with increasing alkyl chain length. This
(
somer 1,3-diacyl-2-O-(N-Cbz-L-arginyl)glycerolꢀHCl (XRZX).
ꢃ1
Table 3 Minimum inhibitory concentrations (mg L ) of the XXR compounds
Microorganism
88R
1010R
1212R
1414R
Gram-positives
Bacillus cereus var. mycoide ATCC 11778
Bacillus subtillis ATCC 6633
64
64
4
16
2
> 256
> 256
> 256
> 256
4
> 256
> 256
256
Staphylococcus aereus ATCC 6538
Staphylococcus epidermidis ATCC 12228
Micrococcus luteus ATCC 9341
Candida albicans ATCC 10231
16
8
> 256
1
32
16
1
> 256
16
16
64
Gram-negatives
Salmonella typhimurium ATCC 14028
Pseudomonas aeruginosa ATCC 9027
Escherichia coli ATCC 8793
16
64
8
32
> 256
64
> 256
> 256
> 256
> 256
2
> 256
> 256
> 256
> 256
Arthrobacter oxidans ATCC 8010
Streptoccocus faecalis ATCC 19434
Bortedella bronchiseptica ATCC 4617
Citrobacter freundii ATCC 22636
Alcaligenes faecalis ATCC 8750
32
8
16
4
0.25
0.5
0.25
256
0.25
0.25
32
8
> 256
4
> 256
> 256
> 256
> 256
256
16
4
Enterobacter aerogenes CECT 689
Klebsiella pneumoniae v. preumonial CIP 104216
32
8
> 256
16
1
226
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View Article Online
Table 4 Hemolytic activity of the XXR compounds, lauroyl arginine methyl ester (LAE), hexadecyltrimethylammonium bromide (HTAB) and
commercial betaine (Tego-Bet)
a
1
414R
1212R
1010R
88R
HTAB
LAE
Tego-Bet
ꢃ1
HC50/mg L
ID (%)
L/D
64.3
12.8
60.1
11.7
20.5
11.9
24.0
19.1
11.6
46.5
14.7
38.4
14.4
34.4
5.0
Moderate
5.6
Moderate
1.7
Moderate
1.3
Moderate
0.2
Irritant
2.6
Moderate
2.4
Moderate
Classification
a
From Goldschmidt (Essen, Germany).
maximum for 88R can be attributed to the combination of a
number of physicochemical parameters: hydrophobicity,
adsorption, CMC, and aqueous solubility, the solubility being
the limiting step for the transport. A loss of activity occurs for
the surfactants with longer alkyl chain lengths because they are
less water-soluble.
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1
2
3
4
5
6
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2
0
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1
285.
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1
surfactants synthesised in our lab and is also similar to some
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teric surfactants. The potential ocular irritation predicted by
the red blood cell method indicates that these surfactants are
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10
11
12
1
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2
1
benzalkonium chloride, cetyltrimethylammonium chloride).
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2
ternary group in the polar head. MonoQuats are capable of
1
inducing lysis of erythrocytes at low concentrations (0.05–0.1
ꢃ1 21
14 P. K. Kadada, Synthesis, 1971, 316–317.
mg L
at concentration ranges between 19–60 mg L
)
whereas these surfactants show hemolytic effects
.
1
1
5
6
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ꢃ1
In summary the novel synthesized compounds can be classi-
fied as multifunctional surfactants with a self aggregation
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7–112.
9
behaviour comparable to that of short-chain lecithins. More-
9
1
8
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activity similar to those of conventional cationic surfactants.
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incorporation of ester functionality in a cationic surfactant
considerably accelerates biodegradation. This has also been
demonstrated in the case of glucoside esters, which exhibited
1
9
Biochemistry of Antimicrobial Action, eds. T. S. Franklin and
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1
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2
3
very rapid biodegradation.
¨
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2
Acknowledgements
Financial support from the Spanish CICYT ref. PPQ2000-
687-C02-01 is gratefully acknowledged.
1
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1227