Lysine−Bisglycidol Conjugates as Novel Lysine Cationic Surfactants

pubs.acs.org/Langmuir © 2009 American Chemical Society

Lysine-Bisglycidol Conjugates as Novel Lysine Cationic Surfactants Aurora Pinazo, Marta Angelet, Ramon Pons, Marina Lozano, Ma Rosa Infante, and Lourdes P
erez* Departament de Tecnologia Quı´mica i de Tensioactius, Institut de Quı´mica Avanc-ada de Catalunya, CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain Received October 15, 2008. Revised Manuscript Received May 28, 2009 The synthesis of a novel class of lysine-based cationic amphiphilic derivatives of the type Nε,Nε0 -bis(n-acyloxypropyl)L-lysine methyl ester salts combining several hydroxyl functions and aliphatic chains of 12 or 14 carbon atoms is described. The compounds have one, two, three, and four alkyl chains. Aggregation in water was studied by four different methods: surface tension, conductivity, chloride ion activity, and nuclear magnetic resonance (1H NMR). The critical aggregation concentration value of the new surfactants depends on both the number of alkyl chains and the alkyl chain length. The formation of vesicles at low concentrations was confirmed by H1 NMR and optical microscopy. Antimicrobial activity was determined on the basis of the minimum inhibitory concentration (MIC) values. These novel lysine-based surfactants showed moderate antimicrobial activity which may be an important advantage for many biomedical applications.

Introduction Surfactants are one of the most representative chemical products and are consumed in large quantities every day on a worldwide scale. Many traditional surfactants exhibit an insufficient rate of biodegradation and high aquatic toxicity. These undesirable effects on the environment are leading society to focus on the development of multifunctional “green” surfactants such as the biocompatible amino-acid-based surfactants developed in refs 1-4. This sphere of the knowledge, which has formed the bulk of our work since the 1980s, is dedicated to the synthesis, study of properties, and development of new, nontoxic, and readily biodegradable surfactants obtained from renewable natural products such as amino acids and vegetable oil derivatives.5-8 Our research has contributed to better understand the molecular requirements to increase biodegradability, reduce toxicity, and simultaneously show antimicrobial activity for the rational design of novel environmentally friendly surfactants for basic research as well as specific industrial applications.9,10 Acyl-glycerol amino acid conjugates constitute a class of specific biocompatible surfactants which can be considered analogues to partial glycerides and phospholipids. They consist *To whom correspondence should be addressed. E-mail: [email protected]. Fax: 34 93 2045904. Telephone: 34 93 4006164. (1) Yokohama, S.; Kouchi, J.; Tabohashi, T.; Harusawa, F.; Yamaguchi, A.; Sakai, H.; Abe, M. Chem. Pharm. Bull. 2001, 49, 1331–1335. (2) Varka, E. M.; Heli, M. G.; Coutouli-Argyropoulou, E.; Pegiadou, S. A. Chem.;Eur. J. 2006, 12, 8305–8311. (3) Ohta, A.; Toda, K.; Morimoto, K. Y.; Asakawa, T.; Miyagishi, S. Colloids Surf., A 2008, 317, 316–322. (4) Capone, S.; Walde, P.; Seebach, D.; Ishikawa, T.; Caputo, R. Chem. Biodiversity 2008, 5, 16–30. (5) Kunieda, H.; Nakamura, K.; Infante, Ma R.; Solans, C. Adv. Mater. 1992, 4, 291–293. (6) P
erez, L.; Torres, J. L.; Manresa, A.; Solans, C.; Infante, M. R. Langmuir 1996, 12, 5296–5301. (7) Infante, M. R.; Pinazo, A; Seguer, J Colloids Surf., A 1997, 123-124, 49–70. (8) Clap
es, P.; Infante, M. R. Biocatal. Biotransform. 2002, 20, 215–233. (9) Mor
an, M. C.; Pinazo, A.; P
erez, L.; Clap
es, P.; Angelet, M.; Garcı´ a, M. T.; Vinardell, M. P.; Infante, M. R. Green Chem. 2004, 6, 233–240. (10) Moran, M. C.; Clap
es, P; Comelles, F; Garcı´ a, M T.; Perez, L; Vinardell, M. P.; Infante, M. R. Langmuir 2001, 17, 5071–5075.

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of one or two aliphatic chains and one amino acid, as the polar head, linked together through ester bonds in the glycerol backbone11,12 (Figure 1a). The physicochemical properties of several anionic (aspartic acid, glutamic acid), cationic (arginine), and nonionic (asparagines, glutamine, tyrosine) amino-acid-derived glycerolipid structures have been reported (Figure 1a).13,14 These compounds form thermotropic liquid crystals and in aqueous media exhibit a broad polymorphism leading to micelles, bilayers, vesicles, or lyotropic liquid crystals depending on the concentration and temperature. Furthermore, they show some of the self-aggregation properties of lecithins, along with significant water solubility as well as valuable antimicrobial activity.15-17 They can be considered “soft preservatives” compared to traditional “hard” cationic surfactants, which are less biodegradable compounds. They are very interesting biocompatible antimicrobial products due to their self-aggregation and biological properties for application in cosmetic, pharmaceutical, and food formulations. However, an exhaustive examination of their chemical stability shows that the ester bond between the glycerol skeleton and the amino acid undergoes hydrolysis in a short time in aqueous solution as a function of pH and temperature.15 In this work, we report for the first time on the chemical synthesis, aggregation properties, and antimicrobial activity of a novel class of lysine-based cationic amphiphilic molecule derivatives of the type Nε,Nε0 -bis(acyloxypropyl)-L-lysine methyl ester salts, combining several hydroxyl functions and aliphatic chains (11) Mor
an, M. C.; Infante, M. R.; Clap
es, P J. Chem. Soc., Perkin Trans. 1 2001, 2063–2070. (12) Mor
an, M. C.; Infante, M. R.; Clap
es, P. J. Chem. Soc., Perkin Trans. 1 2002, 1124–1134. (13) Mor
an, M. C.; Pinazo, A.; Clap
es, P.; Infante, M. R.; Pons, R J. Phys. Chem. B 2004, 108, 11080–11088. (14) Mor
an, M. C.; Pinazo, A.; Clap
es, P.; Infante, M. R.; Pons, R J Phys. Chem. B 2005, 109, 22899–22908. (15) P
erez, L.; Infante, M. R.; Pons, R.; Mor
an, M. C.; Vinardell, M. P.; Mitjans, M.; Pinazo, A. Colloids Surf., B 2004, 35, 235–242. (16) P
erez, L.; Clap
es, P; Pinazo, A.; Angelet, M.; Vinardell, M. P.; Infante, M. R. New J. Chem. 2002, 26, 1221–1227. (17) P
erez, L.; Pinazo, A.; Garcı´ a, M. T.; Mor
an, M. C.; Infante, M. R. New J. Chem. 2004, 28, 1326–1334.

Published on Web 06/19/2009

DOI: 10.1021/la901675p

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Figure 1. (a) Schematic structure of acyl-glycerol amino acid conjugates. The sphere represents the amino acid polar head. (b) Schematic structure of the Nε,Nε0 -bis(n-acyloxypropyl)-L-lysine methyl ester salts.

with 12 (C12) or 14 (C14) carbon atoms. The chemical structure is shown in Figure 1b. The new compounds have been named LGG12, LGGdi12, LGGtri12, and LGGtetra12 for the homologues with alkyl chain of 12 carbon atoms and LGG14, LGGdi14, LGGtri14, and LGGtetra14 for the homologues with alkyl chains of 14 carbon atoms. These new cationic amphiphilic molecules show two main new features: the polar group is a lysine methyl ester residue, that confers the cationic character to the molecule and is linked to a bisglycidol chain, and a residue of bis(2,3-dihydroxypropil), that can carry from one to four aliphatic chains as a part of the hydrophobic moiety. In addition, the lysine group is bonded to the polyol skeleton through an N alkyl amine linkage which provides increased chemical stability compared to the mono- and diacyl-glycerol arginine derivatives.11,12 One important drawback of the classical cationic surfactants is their low biodegradability in aerobic conditions due to the N-C linkage between the alkyl chain and the cationic group. In the new compounds reported in this work, the presence of an ester type linkage between the hydrophilic moiety (polar headgroup) and the hydrophobic backbone (alkyl chain) will ensure high biodegradability. The probable rupture of the ester linkages will produce fatty acids that are easily biodegradable and a lysine linked to the polyol which will not have a surfactant character and will probably biodegrade easily. In fact, the mono- and diglycerides from arginine, with similar structure, are readily biodegradable compounds. These aforementioned features make these novel 7804 DOI: 10.1021/la901675p

multifunctional biocompatible amphiphiles promising molecules with probably novel nanoscale morphologies, novel solubilization and gelification properties, and suitable for preparation of controlled delivery vehicles in water or in organic systems. Moreover, the polyhydroxyl functionality of the precursor bisglycidolized lysine (III in Scheme 1) opens new routes of selective chemical modifications, leading to new materials of desirable physicochemical properties.

Experimental Section Materials. Unless stated otherwise, all solvents were reagent grade and used without further purification. Trifluoroacetic acid (TFA) was obtained from Merck. N-Benzyloxycarbonyl-L-lysine hydrochloride (N-Cbz-L-lysine 3 HCl) was obtained from Calbiochem-Novabiochem AG (Switzerland). Glycidol and the acyl chlorides with 12 and 14 carbon atoms were purchased from Sigma-Aldrich (USA). Strong cation exchanger Macro-Prep High S (50 μm) was from Bio-Rad (Hercules, CA). Silice 60 A C.C. 70-200 μm Chromatogel was from SDS. Thionyl chloride (SOCl2) and palladium on activated charcoal (Pd/C) (10%) were supplied by Merck (Darmstadt, Germany). Deuterated solvents were purchased from Eurotop, and Mueller-Hinton Broth was purchased from Difco Laboratories (USA). Water from a Milli-Q Millipore system was used to prepare aqueous solutions. Chemical Synthesis Methodology. Compounds of the type Nε,Nε0 -bis(acyl-2,3-dihydroxypropyl)-lysine methyl ester hydrochloride salt derivatives with one, two, three, and four C12 and C14 alkyl chains were prepared by chemical methodologies. The synthetic pathway is outlined in Scheme 1. Langmuir 2009, 25(14), 7803–7814

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Article Scheme 1. Synthesis Scheme for the Nε,Nε0 -Bis(n-acyloxypropyl) Lysine Methyl Ester Saltsa

a I: N-Cbz-L-lysine; II: N-Cbz-L-lysine methyl ester; III: Nε,Nε0 -bis(2,3-dihydroxyropyl) N-Cbz-L-lysine methyl ester; IVa: Nε-(2,3-dihydroxyropyl) Nε0 -(2-hidroxy-3-acyloxypropyl) N-Cbz-L-lysine methyl ester; IVb: Nε,Nε0 -bis(2-hidroxy-3- acyloxypropyl) N-Cbz-L-lysine methyl ester; IVc: Nε(2-hidroxy-3-acyloxypropyl) Nε0 -(2,3-diacyloxypropyl) N-Cbz-L-lysine methyl ester; IVd: Nε,Nε0 -bis(2,3-diacyloxypropyl) N-Cbz-L-lysine methyl ester; Va: Nε-(2,3-dihydroxyropyl) Nε0 -(2-hidroxy-3-acyloxypropyl) lysine methyl ester, LGG12 for n=10, LGG14 for n=12; Vb: Nε,Nε0 -bis(2-hydroxy3-acyloxypropyl) lysine methyl ester LGGdi12 for n=10, LGGdi14 for n=12; Vc: Nε-(2-hydroxy-3- acyloxypropyl) Nε0 -(2,3-diacyloxypropyl) lysine methyl ester, LGGtri12 for n=10, LGGtri14 for n=12; Vd: Nε,Nε0 -bis(2,3-diacyloxypropyl) lysine methyl ester, LGGtetra12 for n=10, LGGtetra14 for n=12.

1. Nε-2,3-Dihydroxyropyl Nε0 -2-Hydroxy-3-acyloxypropyl Lysine Methyl Ester Hydrochloride Salts (Va: LGG12 and LGG14). Synthesis of NR-Cbz-L-lysine Methyl Ester HCl (II). N-Cbz-L-lysine 3 HCl (25 g, 0.089 mol) was dissolved

in methanol (400 mL). The solution was cooled with dried ice to -80 °C, and SOCl2 (19.5 mL, 0.27 mol) was added dropwise to the stirred dissolution. The mixture of the reaction was stirred at room temperature for 20 h. Methanol, excess of SOCl2, and hydrochloride acid formed during the reaction were eliminated by successive vacuum evaporations, and then the residue was dissolved in water. After freeze-drying, a white solid was obtained with 99% purity by HPLC. Yield: 97%. HPLC: tr = 5.88 min. Langmuir 2009, 25(14), 7803–7814

MW: 294.33. Elemental analysis (%) found: C, 51.13; H, 6.89; N, 8.11. Calculated for C15H22O4N2 3 0.5 H20: C, 51.76; H, 7.19; N, 8.05. Molecular ion peak [MþHþ] m/z: 295.2. 1H NMR: δH (ppm) (CD3OD), 1.42-1.89 [m, 6H, 3CH2 side chain of lysine group)], 2.85 [t, 2H (-CH2-NH2)], 3.74 [s, 3H (-COO-CH3)], 4.21 [m, 1H (-NH-CH-COO-)], 5.17 [s, 2H (Cbz-CH2-)], 7.25-7.31 [m, 5H of the Cbz-group].13C NMR: δC 7 (CD3OD), 23.83-32.03 [(-CH2-) side chain of lysine group], 40.49 [(-CH2-NH2)], 52.75 (-COO-CH3)], 55.12 [(NH-CHCOO-)], 67.67 [(Cbz-CH2-)], 128.81-129.48 [(6C) of the Z group (Cbz)], 158.69 [(COO-NH) of group (Cbz)], 174.38 [(-COO-CH3)]. DOI: 10.1021/la901675p

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Synthesis of Nε,Nε -Bis(2,3-dihydroxypropyl)-N-Cbz-Llysine Methyl Ester (III). N-Cbz-L-lysine methyl ester (0.02 mol) was dissolved in dry methanol (150 mL) under nitrogen atmosphere. Glycidol (3.4 mL, 0. 05 mol) was added dropwise. The mixture was stirred at 70 °C during 48 h. The reaction progress was monitored by HPLC. After vacuum methanol evaporation, orange oil was obtained. The oil was dissolved in water, and the purification of the product was carried out by cationic exchange chromatography. The fractions containing the desired product were pooled, desalted, and freeze-dried. A white hygroscopic solid was obtained with 98% purity by HPLC. Yield: 89%. HPLC: tr=5.43 min. MW: 442.50. Elemental analysis (%) found: C, 59.92; H, 10.16; N, 9.97. Calculated for C21H35O8N2: C, 61.90; H, 10.12; N ,10.30. Molecular ion peak [M þ Hþ] m/z: 443.5. 1H NMR: δH (ppm) (CD3OD), 1.42-1.9 [m, 6H, 3CH2 side chain of lysine group], 2.4-2.7 [m, 6H (-CH2-NH-), (-CH2NH-CH2-)], 3.4-3.7 [m, 6H 2(CH2 OH-CHOH-CH2)], 3.74 [s, 3H (-COO-CH3)], 4.21 [m, 1H (-NH-CH-COO-)], 5.17 [s, 2H (Cbz-CH2-)], 7.25-7.31 [m, 5H of the Cbz-group (Z)]. 13 C NMR δC (ppm) (CD3OD): 24.3, 26.0, 32.3, 46.8 [CH2 lysine chain], 55.2 [(-CH3) methyl ester], 56.5 [(CH an)], 59.0 [(CH2N-CH2)], 65.7 [2(CH2OH-CHOH)], 67.6 [(Cbz-CH2)], 69 [2(N-CH2-CH-OH)], 128.8, 129.0, 129.5 [(Cbz CH)], 138.2 [(Cbz C)], 158.7 [(Cbz CdO)], 174.6 [(-COOCH3)].

Synthesis of Nε-(2,3-Dihydroxypropyl) Nε0 -(2-Hydroxy3-acyloxypropyl)-N-Cbz-L-lysine Methyl Ester (IVa). This

section describes the general procedure for the synthesis of compound IVa for fatty chains of 12 and 14 carbon atoms. Fatty acid chloride (0.07 mol) was added dropwise to a stirred mixture of III (0.07 mol) in dry pyridine (50 mL) at 0 °C. The reaction was left to warm at room temperature and stirred during 1 h. The mixture was then cooled again at 0 °C, acid chloride (0.018 mol) was added, and the mixture was stirred for 1 h at room temperature. Pyridine was then eliminated by evaporation under reduced pressure. The residue was subjected to chromatography on silica gel eluting with hexane/chloroform (50:50 to 0:100; v:v). The desired product was obtained in a yield of 50% as a white solid with a purity of 96.6% by HPLC. 1H NMR δH (ppm) (CD3OD): 0.86 [t, (-CH3) alkyl chain)], 1.27 [m, (-CH2) alkyl chain], 1.582.35 [m, (-CH2-) alkyl chain and (-CH2-) side chain of lysine group), 2.3 [t, (-CH2-) alkyl chain], 3.3-3.9 [m, 11 H, (-OCH3), (CH2OH-CHOH-CH2-N(CH2)-CH2-CHOH-CH2OCO)], 5.0 [s, 2H (Cbz-CH2-)], 4.3 [m, 4H, (CH2OH-CHOH-CH2N(CH2)-CH2-CHOH-CH2OCO), (NH2-CH-COO-)], 7.27.4 [m, 5H of the Cbz-group]. 13C NMR δC (ppm) (CD3OD): 14.4 [(-CH3 alkyl chain], 23.7-34.7 [(-CH2-) alkyl chain and (-CH2-) lysine chain], 54.9 [(-CH- an)], 52.7 [(-CH3) methyl ester], 57.4 [(-CH2-N-CH2-)], 65.1 [(-CHOH-CH2OH-), 66.7 [alkyl chain (-CH2-OCO-)], 66.8 [(CHOH-CH2OH)], 67.1 [Cbz-CH2], 67.6 [(-COO-CH2-CHOH)], 128.7-129.0129.4 [(Cbz, CH)], 138.1 [(Cbz, C)], 158.6 [(Cbz CdO)], 174.9 [(-COO-CH2-)], 174.3 [(-COOCH3)].

(-CH2-) side chain of lysine group], 2.3 [t, (-CH2-) alkyl chain], 3.2-3.8 [m, 11 H, (-OCH3), (-CH2OH-CHOHCH2-N(CH2)-CH2-CHOH-CH2OCO)], 4.1-4.3 [m, 4H, (-CH2OH-CHOH-CH2-N(CH2)-CH2-CHOH-CH2OCO)], (NH2-CH-COO-)]. 13C NMR δC (ppm) (CD3OD): 14.1 [(-CH3) alkyl chain], 23.1-34.7 [(-CH2-) alkyl chain and (-CH2-) lysine chain], 53.5 [CH an], 53.7 [(-CH3) methyl ester], 57.4 [(-CH2-NH-CH2-)], 65.1 [(-CHOH-CH2OH)], 66.7 [alkyl chain (-CH2-OCO-)], 66.8 [(-CHOH-CH2OH)], 67.6 [(-COO-CH2-CHOH)], 170.8 [(-COO-CH2-)], 175.0 [(-COOCH3)].

Synthesis of Nε-(2,3-dihydroxypropyl) Nε0 -(2-Hydroxy-3miristoyloxypropyl)-lysine Methyl Ester HCl (Va: LGG14).

The same procedure as that for LGG12 was used except that myristoyl chloride was used instead of the lauroyl chloride. Purity of 98% by HPLC. HPLC: tr = 12.5 min. Elemental analysis (%) found: C, 54.79; H, 9.37; N, 4.71, Calculated for C27H54N2O7 3 HCl 3 3H2O: C, 54.66; H, 10.36; N, 4.72. Molecular ion peak [M þ Hþ] m/z: 519.84. 1H NMR δH (ppm) (CD3OD): 0.86 [t, (-CH3-) alkyl chain], 1.27 [m, (-CH2-) fatty chain], 1.58-2.35 [m, (-CH2-) alkyl chain and (-CH2-) side chain of lysine group], 2.3 [t, (-CH2-) fatty chain], 3.2-3.8 [m, 11 H, (-OCH3), (-CH2OH-CHOH-CH2-N(CH2)-CH2-CHOH-CH2OCO-), 4.1-4.3 ppm [m, 4H, (-CH2OH-CHOH-CH2-N(CH2)CH2-CHOH-CH2OCO-), (NH2-CH-COO-)]. 13C NMR δC (ppm) (CD3OD): 14.1 [(-CH3) alkyl chain], 23.1-34.7 [(-CH2-) alkyl chain and (-CH2-) lysine chain], 53.5 [(-CH-) an], 53.7 [(-CH3)methyl ester], 57.4 [(-CH2-NHCH2-)], 65.1 [(-CHOH-CH2OH-)], 66.7 [alkyl chain (-CH2OCO-)], 66.8 [(CHOH-CH2OH)], 67.6 [(COO-CH2 CHOH)], 170.8 [(-COO-CH2-)], 175.0 [(-COOCH3)].

2. Nε,Nε0 -Bis(2-hydroxy-3-acyloyloxypropyl)-L-lysine Methyl Ester Hydrochloride Salts (Vb: LGGdi12 and LGGdi14). Synthesis of Nε,Nε0 -Bis(2-hydroxy-3-lauroyloxypropyl)-NCbz-lysine Methyl Ester Hydrochloride Salt (IVb). This

Synthesis of Nε-(2,3-Dihydroxypropyl) Nε0 -(2-Hydroxy3-lauroyloxypropyl)-lysine Methyl Ester HCl (Va: LGG12).

section describes the general procedure for the synthesis of compound IVa for 12 and 14 carbon atoms in the fatty chain. To obtain the compounds with two fatty chains, the same synthetic procedure as that used to obtain IVa was used, with the only difference being that in this case the molar ratio fatty acid chloride/compound III was 2.5/1. HPLC purity 98.73%. 1H NMR δH (ppm) (CD3OD): 0.9 [t, 6H, (-CH3) alkyl chain], 1.29 [m, (-CH2-) alkyl chain], 1.50-2.55 [m, (-CH2-) alkyl chain and CH2 side chain of lysine group], 2.4 [t, (-CH2-) alkyl chain], 3.7 [s, 3H, (-OCH3)], 3.9 [m, 2H, -CH2OH)], 4.0-4.1 [m, 2H, (-CH2COO-)], 4.4 [m, 1H, (-NH-CH-COOCH3)], 5.1 [s, 2H, (-CH2-Cbz)], 7.2-7.4 [ m, 5H of the Cbz-group]. 13C NMR δC (ppm) (CD3OD): 14.0 [(-CH3) alkyl chain], 22.6-34.0 [(-CH2-) alkyl chain and (-CH2-) lysine chain], 53.6 [(-CH-) an], 52.4 [(-CH3) methyl ester], 58.1 [(-CH2-N-CH2)], 66.2 [alkyl chain (-CH2-OCO-)], 67.7 [(CHOH-CH2OH)], 66.9 [Cbz-CH2], 67.6 [(-COO-CH2 CHOH-)], 128.1-128.2-128.5 [Cbz, CH], 136.1 [Cbz, C ], 155.9 [Cbz CdO], 172.8 [-COO-CH2-], 173.9 [-COOCH3].

A solution of the lauroyl derivative IVa (2 g) in methanol (100 mL) was kept under nitrogen atmosphere for 15 min and hydrogenated in the presence of the catalyst Pd/C (0.5 g). The pH of the mixture was checked periodically, and HCl in methanol was added slowly to maintain the pH between 4 and 6 to avoid methyl ester hydrolysis. The catalyst was removed by filtration using Celite; the filtrate was evaporated to dryness, dissolved in water, and finally freeze-dried. The solid was recrystallized in acetonitrile/ ethyl acetate. Overall yield was 35%. The desired product was obtained as a white solid with a purity of 98.55% by HPLC. HPLC: tr = 8.6 min. Elemental analysis (%) found: C, 54.49; H, 9.98; N, 4.83. Calculated for C25H50N2O7 3 HCl 3 3H2O: C, 54.68; H, 10.46; N, 5.10. Molecular ion peak [M þ Hþ] m/z: 491.5. 1H NMR δH (ppm) (CD3OD): 0.86 [t, (-CH3) alkyl chain], 1.27 [m, (-CH2-) alkyl chain], 1.58-2.35 [m, (-CH2-) alkyl chain and

The corresponding lauroyl derivative IVb was hydrogenated as described above. HPLC purity 98.55%. Overall yield: 35%. HPLC: tr =16.15 min. Elemental analysis (%) found: C, 56.77; H, 9.88; N, 3.53. Calculated for C37H72N2O8 3 HCl 3 6H2O: C, 57.75; H, 11.13; N, 3.64. Molecular ion peak [M þ Hþ] m/z: 674.40. 1H NMR δH (ppm) (CD3OD): 0.9 [t, 6H, (-CH3) alkyl chain], 1.27 [m, (-CH2-) alkyl chain], 1.50-2.64 [m, (-CH2-) alkyl chain and (-CH2-) side chain of lysine group], 2.3 [t, (-CH2-) alkyl chain], 3.8 [s, 3H, (-OCH3)], 3.7 [m, 2H, (-CHOH-)], 4.1-4.3 [m, 4H, (-CH2COO-)], 4.3 [m, 1H, (-NH-CH-COOCH3)]. 13C NMR δC (ppm) (CD3OD): 14.6 [(-CH3) alkyl chain], 22.8-34.9 [(-CH2-) alkyl chain and (-CH2-) lysine chain], 53.9 [(-CH-) an], 53.8 [(-CH3) methyl

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Synthesis of Nε,Nε0 -Bis(2-hydroxy-3-lauroyloxypropyl)N-L-lysine Methyl Ester Hydrochloride Salt (Vb:LGGdi12).

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Pinazo et al. ester], 57.6 [(-CH2-N-CH2-)], 66.9 [alkyl chain (-CH2OCO-)], 65.4 [(-CHOH-CH2OH)], 170.9 [(-COO-CH2-)], 174.9 [(-COOCH3)].

Synthesis of Nε,Nε0 -Bis(2-hydroxy-3-miristoyloxypropyl)N-L-lysine Methyl Ester Hydrochloride Salt (Vb:LGGdi14).

The corresponding myristoyl derivative IVb was hydrogenated as described above. HPLC purity 98.55%. HPLC: tr = 18.3 min. Elemental analysis (%) found: C, 58.99; H, 10.86; N, 3.25. Calculated for C41H80N2O8 3 HCl 3 6H2O: C, 59.64; H, 11.35; N, 3.39. Molecular ion peak [M þ Hþ] m/z: 730.01. 1H NMR δH (ppm) (CD3OD): 0.9 [t, 6H, (-CH3) alkyl chain], 1.27 [m, (-CH2-) alkyl chain], 1.50-2.64 [m, (-CH2-) alkyl chain and (-CH2-) side chain of lysine group], 2.3 [t, (-CH2-) alkyl chain], 3.8 [s, 3H, (-OCH3)], 3.7 [m, 2H, (-CHOH-)], 4.1-4.3 [m, 4H, (-CH2COO-)], 4.3 [m, 1H, (-NH-CH-COOCH3)]. 13C NMR δC (ppm): 14.6 (-CH3) alkyl chain], 22.8-34.9 [(-CH2-) alkyl chain and (-CH2-) lysine chain], 53.9 [(-CH-) an], 53.8 [(-CH3) methyl ester], 57.6 [(-CH2-N-CH2-)], 66.9 [alkyl chain (-CH2-OCO-)], 65.4 [(-CHOH-CH2OH)], 170.9 [(-COO-CH2-)], 174.9 [(-COOCH3)].

3. Nε-(2-Hydroxy-3-lauroyloxypropyl) Nε0 -(2,3-Dilauryloxypropyl)-lysine Methyl Ester Hydrochloride Salt (Vc: LGGtri12) and Nε,Nε0 -Bis-(2,3-dilauroyloxypropyl)-lysine Methyl Ester Hydrochloride Salt (Vc: LGGtetra12). These

two compounds were prepared under similar conditions to those described for Va. Depending on the lauroyl chloride/compound III molar ratio, LGGtri12 or LGGtetra12 was preferably obtained. LGGtri12 was purified by chromatography on silica gel eluting with hexane/chloroform (50:50 to 0:100 v/v). However, LGGtetra12 was almost always contaminated with a certain amount of LGGtri12. Isolation and identification of this compound was carried out by analytical HPLC. LGGtri12: overall yield, 35%; HPLC, tr= 23.22 min; mass spectroscopy, molecular ion peak [M þ Hþ] m/z, 855.25. LGGtetra12: HPLC, tr = 25.80 min; mass spectroscopy, molecular ion peak [Mþ Hþ] m/z, 1037.17. Compounds LGGtri14 and LGGtetra14 were also obtained, but they could not be isolated. Methods. HPLC Analysis. The progress of the reactions was monitored by HPLC, with a Merck-Hitachi D-2500 model using a UV-vis detector L-4250 at 215 nm. A Lichrospher 100 CN (propylcyano) 5 μm, 250  4 mm column was used at room temperature. A gradient elution profile was employed from the initial solvent composition of A/B 50/50 (by volume), changing during 24 min to a final composition of 100% B, where A is 0.1% (v/v) TFA in H2O and B is 0.085% of TFA in H2O/CH3CN 1:4. The flow-rate through in the column was 1.0 mL/min. An isocratic elution was employed with 33% B for monitoring the formation of N-Cbz-L-lysine methyl ester and Nε,Nε0 -(2,3-dihydroxypropyl)N-Cbz-L-lysine methyl ester. Column Chromatography. The purification of the products was performed by preparative ion exchange chromatography. The crude product (5 g) was loaded onto a preparative column filled with Macro-Prep High S Support strong cationic exchange (27  4 cm) stationary phase equilibrated with phosphate buffer solution 0.020 M, pH 6.4. Impurities were eluted first using water. The product was eluted with NaCl 0.5 M aqueous solution. Analysis of the fractions was carried out by HPLC using similar conditions as those used for the reaction monitoring. The pure fractions were pooled, desalted, and freeze-dried. Structure Characterization. The structures of the pure compounds were verified 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 decoupling to eliminate 13C-1H coupling. Langmuir 2009, 25(14), 7803–7814

Article The distortionless enhancement by polarization transfer (DEPT) spectra were run in a standard way to separate the CH/CH3 and CH2 lines phased up and down, respectively. Mass spectroscopy (MS) spectra with fast atom bombardment (FAB) or electrospray techniques were also conducted with a VGQUATTRO spectrometer from Fisons Instruments. Elemental analyses of the final products were recorded at the Microanalysis Service at the IIQAB-CSIC. Aqueous Sample Preparation. Water from a Milli-Q system was used. Before sample preparation, the water was filtered through a 0.2 μm ultrafiltration membrane apparatus from Osmonics Inc. Stock aqueous dispersions were prepared by weight, shaken first by hand for ca. 1 min, and then sonicated for 15 min at 25 °C in a sonicator bath (Ultrasons-H, Selecta S.A.) to confirm that the product dissolved completely. The samples were kept at 25 °C during at least 2 h for equilibration. Lower concentrations were prepared by dilution from stock aqueous dispersions. Surface Tension Measurements. The surface tension was measured using a homemade pendant drop surfactometer. In this technique, a surfactant solution drop is created at the end of a straight cut Teflon tube of 0.8 mm internal diameter and 1.58 mm external diameter. The image of the drop is taken using a web camera. The image is corrected for spherical aberration. The droplet contour is taken at the point of maximum intensity slope. This contour is fitted to the Laplace-Young equation18 using a golden section search algorithm that has as parameters the center coordinates, an angular correction for the vertical alignment, the radius of the droplet, and the interfacial tension. Evaporation was prevented by working in a saturated humidity atmosphere. The saturation adsorption values, Γmax, at the air/water interface were calculated by fitting the experimental points before the break to the Gibbs adsorption equation (eq 1) Γ ¼ -1=nRT dγ=d ln C

ð1Þ

where R is the gas constant, T is the absolute temperature, γ is the surface tension, C is the concentration, and n is the number of free species per original surfactant molecule (here n = 2). Conductimetry. Conductivity was measured using an Orion Cond. Cell 011010A with platinized platinum electrodes in conjunction with a Thermo Orion 550A instrument with a cell constant of 0.998 cm-1. The cell constant was calibrated with NaCl solutions of known conductivities and was used for calculating the conductivity of the surfactant solution. The conductivity of water was subtracted from the measured conductivity of each sample. Measurements were made at increasing concentrations to minimize errors from possible contamination from the electrode. Chloride Ion Activity Measurements. The chloride ion activities were measured with a chloride-specific combination electrode Orion 94-17B. For aqueous NaCl solutions, the plot of the emf versus log(C) was a straight line from 0.1 to 40 mM. The calculated electrode slope was 58 mV, which was close to the Nerst factor of 2.303RT/F=59.16 mV at 25 °C. For surfactant solutions, due to the micellization and counterion binding above the critical micelle concentration (cmc), a break can be observed in the emf versus log(C) plot. Therefore, this method can be used for detecting the cmc. Proton Nuclear Magnetic Resonance. The spectra were acquired with a simple one-pulse experiment using a spectral width of 10 000 Hz and a preacquisition delay of 2-8 s. Samples were prepared in 1 mL of D2O (2.5 and 5 mM) of at least 99.8% atom D%. The water impurity in the D2O was used as an internal reference. The sample was heated at 50 °C during 10 min. The NMR temperature measurements were carried out maintaining (18) Enhorning, G. J. Appl. Physiol. 1997, 43, 198–203.

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the sample at the established temperature during 10 min before the start of the measurements. Small-Angle X-ray Scattering (SAXS). X-ray scattering measurements were carried out using a Kratky compact camera of small angle (Hecus X-ray Systems, Graz) coupled to a Siemens KF 760 (3 KW) generator. Nickel-filtered radiation with a wavelength corresponding to the Cu KR line (1.542 A˚) was used. The collimation system produces a line beam. The linear detector was a PSD-OED 50 M-Braun, and the temperature controller was a Peltier KPR (Anton Paar, Graz). Samples were inserted between two Mylar sheets with a 1 mm separation. The SAXS scattering curves are shown as a function of the scattered vector modulus q according to eq 2 q ¼ 4π=λ sinðθ=2Þ

ð2Þ

where λ is the wavelength of the X-ray used (1.542 A˚) and θ is the scattering angle. The scattering curves shown are smeared due to the experimental slit length and width collimations. This induces both a line width increase of the scattering peaks and a small movement of the peak position to smaller q values. The difference is about 4% for a peak appearing at q=1.2 nm-1 and 2% for a peak with q=2.4 nm-1 representative of our results. Conversely, this produces an overestimation in the lattice parameters by the same amounts. The crystalline structures have been assigned from the correspondence of plots of peak positions as a function of the allowed Miller indices (h2 þ k2 þ l2) for the different structures. Using Tanford19 values for hydrophobic volumes and the volume of the polar head obtained from partial group contributions, we can calculate the structural parameters from the lattice parameter and geometric relationships for the different structures.20-23 The group volume contributions are the same as those in Pinazo et al.,24 and we have also used25 the values of 0.018 nm3 for the hydroxyl group (-OH), 0.029 nm3 for the chloride anion (Cl-), 0 nm3 for the trisubstituted nitrogen (N), and 0.021 nm3 for the -COO- groups. If Vt is the total molecular volume and d is the Bragg distance (d = 2π/q.) of the first maximum, we can figure out the area per molecule in lamellar structures by eq 3 A ¼

2Vt d

ð3Þ

The hydrophobic (lc) and hydrophilic lengths (lh) are defined as the ratio of the respective hydrophobic or hydrophilic volumes to the area per molecule, lc = Vc /A and lh= Vh /A. Qualitative Phase Behavior. Optical microscopy was employed to study the phase behavior of binary water/surfactant systems as a function of temperature. Optical observations were performed according to the “flooding” (penetration) method of Lawrence.26 A Reichert Polyvar 2 Leica polarizing microscope equipped with a hot stage was employed. A camcorder and a PC with Leica IM 500 software were used to capture images. In a flooding experiment, water was allowed to diffuse into an anhydrous surfactant placed between a slide and a coverslip. After a short time, gradients in composition were produced and different separated mesophases developed around the crystalline surfactant. Antimicrobial Activity. Antimicrobial activities were determined “in vitro” on the basis of minimum inhibitory concentration (19) Tanford, C. The Hydrophobic Effect; J. Wiley & Sons: New York, 1980; p 52 (20) Larsson, K. J. Phys. Chem. 1989, 93, 7304–7314. (21) Ropers, M. H.; St
eb
e, M. J. Phys. Chem. Chem. Phys. 2001, 3, 4029–4036. (22) Fontell, K. Adv. Colloid Interface Sci. 1992, 41, 127–147. (23) Alexandridis, P.; Olsson, U.; Lindman, B. Langmuir 1997, 13, 23–34. (24) Pinazo, A.; P
erez, L.; Lozano, M.; Angelet, M.; Infante, M. R.; Vinardell, M. P.; Pons, R. J. Phys. Chem. B 2008, 112, 8578–8585. (25) Hayter, J. B.; Penfold, J. Colloid Polym. Sci. 1983, 261, 1022–1030. (26) Lawrence, A. C. S. Discuss. Faraday Soc. 1958, 25, 51–58.

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(MIC) values,27 defined as the lowest concentration of antimicrobial agent which inhibits the development of visible growth after 24 h of incubation at 37 °C. The compounds tested were dissolved in Mueller-Hinton broth (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 manufacturers’ instructions. Then 10 μL of a nutrient broth starter culture of each bacterial strain was added to achieve final inoculums of ca. 5  10-4-5  10-5 colony forming units per milliliter. 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 concentration of antimicrobial agent at which no visible turbidity was observed was taken as the minimum inhibitory concentration. The microorganisms used (15 bacteria and 1 fungus) were the following. Gram-negative bacteria: Alcaligenes faecalis ATCC 8750, Bordetella bronchiseptica ATCC 4617, Citrobacter freundii ATCC11606, Enterobacter aerogenes ATCC 10938, Salmonella typhimurium ATCC 14028, Streptococcus faecalis ATCC 1054, Escherichia coli ATCC 27325, Klebsiella pneumonia ATCC 9721, Pseudomonas aeruginosa ATCC 9721, Arthrobacter oxidans ATCC 8010. Gram-positive bacteria: Bacillus cereus var. mycoides ATCC 11778, Bacillus subtilis ATCC 6633, Staphylococcus aereus ATCC 25178, Staphylococcus epidermidis ATCC 155-1, Micrococcus luteus ATCC 9341. Fungus: Candida albicans ATCC 10231.

Results and Discussion Synthesis and Characterization. New cationic amphiphile compounds of the type Nε,Nε0 -bis (n-acyloxypropyl)-lysine methyl ester salts with one, two, three, or four fatty chains of 12 or 14 (see Figure 1B) have been prepared for the first time. The synthesis, illustrated in Scheme 1, includes four steps: (1) esterification of Cbz-lysine derivative to give compound II with the carboxylic group of the amino acid protected, (2) aminoxylation of compound II (as free base) with excess of glycidol to give compound III, (3) esterification of the hydroxyl functions of compound III with fatty acyl chlorides, and (4) deprotection of the Cbz group by catalytic hydrogenation to obtain the final compounds. The key step in our strategy is the reaction between the protected lysine methyl ester and glycidol to obtain compound III. This reaction is easy to perform and guaranties good yields. Modification of the lysine Nε amine residue with a diglycidol occurs through the nucleophilic attack of the less hindered electrophilic epoxide carbon by the nitrogen group of the amines. Because lysine has two amino groups of similar reactivity against glycidol, protection of the NR amino group is an essential requirement for the synthesis of these compounds. A first approach based on the copper salt of lysine was designed to simultaneously protect the COOH group and NR function of the amino acid. Although the reaction between the lysine Cu complex and glycidol was excellent, we had serious difficulties in affording good results during the acylation and deprotection steps, and consequently, the method was abandoned. As we had previously observed,28 NR-Carbobenzoxylation of lysine was an improved (27) Jones, R. N.; Barry, A. L.; Gavan, T. L.; Washington, J. A. In II Manual of Clinical Microbiology; Lennette, E. H., Ballows, A, Hauser, W. J., Shadomy, H. J., Eds.; American Society for Microbiology: Washington, DC, 1980. (28) Pinazo, A.; Pons, R.; Angelet, M.; Lozano, M.; Infante, M. R.; P
erez, L. Book of Proceedings II Reuni
on Ib
erica de Coloides e Interfaces, RICI II; Department of Chemistry, University of Coimbra: Coimbra, 2007; pp 341-349.

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Figure 2. (a) Regioisomer mixture obtained for the LGG12 and LGG14, percentage compound (a1) > 90%. (b) Regioisomer mixture obtained for the LGGdi12 and LGGdi14, percentage compound (b1) > 90%.

choice to provide protection during the reaction (Scheme 1) which was easily hydrogenated at the end of the process. Accordingly, Nε,Nε0 -(2,3-dihydroxypropyl)-N-Cbz-L-lysine methyl ester (III) was obtained with 98% purity and in 80% yield by reacting II with glycidol at 70 °C. Preparative cationic exchange chromatography offers an efficient method to purify the crude reaction. Impurities were eluted first using water. Finally, compound III was recovered with NaCl in water and desalted in absolute ethanol. The product thus purified was suitable for acylation. The preparation of compounds IV (a-d) were acceptably carried out by acylation of the hydroxyl free groups of compounds III with the corresponding fatty acyl chlorides in pyridine at room temperature, as described by P
erez and co-workers to prepare mono- and diacyl-rac-glycero-3-O-(L-arginine) derivatives.17,29 Mono-, di-, and triacyl derivatives were separated over silica chromatography in a simple and clean manner. The resulting products IV (a, b) with 10 and 12 carbon atoms were obtained as white solids with a high purity. Compounds IV (c, d) with 10 and 12 carbon atoms coexist at 50% in the final reaction mixture independently of the III/acyl chloride ratio. The triacylated IVc with 10 carbon atoms was successfully obtained as colorless oil with a high purity by preparative silica gel chromatography. However, the tetraacylated surfactant (IVd) with 10 carbon atoms was always contaminated with a certain amount of IVc, even after purification by silica gel. Isolation and identification of this compound were carried out by analytical HPLC. For further studies, purification of this compound will be achieved by preparative HPLC as described in ref 12. Due to their high hydrophobicity, the compounds IV (c, d) with 12 carbon atoms were not isolated. The last synthetic step to obtain the target compounds V (a-d) consists of a catalytic hydrogenation of the Cbz group using Pd over charcoal. To prevent the hydrolysis of the ester linkages, the reaction was carried out keeping the pH in the range 5-7. Pure compounds were obtained after several crystallizations in acetonitrile/ethyl acetate. The overall yields of V (a-c) were low because of the purification processes. The structure of the prepared lysine-based amphiphilic molecules was confirmed by NMR and MS spectra. It has been reported for monoglycerides that acyl migration takes place, yielding a balance mixture of the two regioisomers: 2-monoglyceride (10%) and 1-monoglyceride (90%).30 Migration of the alkyl chains has also been observed for (29) P
erez, L.; Clap
es, P; Pinazo, A; Angelet, M.; Vinardell, M. P.; Infante, M. R. New J. Chem. 2002, 26, 1221–1227. (30) Born
e, J. Ph.D. Thesis, Lund University, Lund, Sweden, 2002.

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monoglycerides from arginine; in this case, the final compound also consists of a mixture of regioisomers with a 90% of the target compound 1-acyl-3-argynil-glycerol and 10% of the two possible regioisomers (1-acyl-2-arginine-glycerol and 1-arginine-2-acylglycerol).17 The 13C NMR and DEPT spectra of the LGGdi14 and LGGdi12 present the signal corresponding to the compound b1 (Figure 2) with very good intensity. Two small additional signals with low intensity (lower than 10%) in the region corresponding to the carbons of the glycidol group are also present. One corresponds to a CH group (73.0 ppm) that suggests the presence of regioisomers where the alkyl chain is linked to the CH group of the glycidol. The other signal corresponds to a CH2 group (62.5 ppm); therefore, the terminal CH2(OH) group of the molecule does not include any alkyl chain (Figure 2b). Similar behavior has been observed for the monoalkyl derivatives. Here, the final product also consists of a mixture of the possible regioisomers (Figure 2a). The DEPT spectra also show the resonance corresponding to the acylated CHO-CO-R group. The percentage of the target compound (1a in Figure 2) is also greater than 90%. Aggregation Properties. To check the performance of the synthesized compounds as surfactants in aqueous solution, their aggregation properties were studied. The critical micellar concentration and critical aggregation concentration (cac) have been determined by surface tension (pendant drop method), conductivity, and chloride ion activity. Table 1 summarizes the values of breaks in conductivity, ion chloride, and surface tension. Breaks are indicative of cmc or cac concentrations. The most striking feature of these values is the great disparity between the surface tension and the other two techniques. In view of these results, the obvious explanation is that for these surfactants the breaks in these three physical parameters do not correspond to the same phenomena. The discrepancy between surface tension and bulk techniques in the determination of the cmc/cac is present in the literature.31-37 Similar low surface tension cmc values compared to the bulk (31) Rosen, M. J.; Mathias, J. H.; Davenport, L. Langmuir 1999, 15, 7340–7346. (32) Pinazo, A.; Wen, X.; P
erz, L.; Infante, M. R.; Franses, E. I. Langmuir 1999, 15, 3134–3142. (33) Rosen, M. J.; Mathias, J. H.; Davenport, L. Langmuir 2001, 17, 6148–6154. (34) Zana, R. J. Colloid Interface Sci. 2002, 246, 182–190. (35) Kanicky, J. R.; Shah, D. O. Langmuir 2003, 19, 2034–2038. (36) Alvarez Alcalde, M.; Jover, A.; Meijide, F.; Galantini, L.; Viorel Pavel, N.; Antelo, A.; Vazquez Tato, J. Langmuir 2008, 24, 6060–6066. (37) Cui, X.; Mao, S.; Liu, M.; Yuan, H.; Du, Y. Langmuir 2008, 24, 10771– 10775.

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Pinazo et al. Table 1. Surface and Aggregation Properties of Aqueous Dispersions at 25°C

compound

cac surface tension (mM)

γmin (mN/m)

Γmax (106 mol/m2)

Amin (nm2)

cac conductivity (mM)

cac ion chloride (mM)

LGG12 LGG14 LGGdi12 LGGdi14

0.20 ( 0.05 0.07 ( 0.02 0.03 ( 0.01 0.06 ( 0.02

33 ( 1 31 ( 1 24.5 ( 1 25 ( 1

2.8 ( 0.4 2.5 ( 0.4 5.0 ( 3.0 8.8 ( 8

0.58 ( 0.10 0.66 ( 0.10 0.33 ( 0.2 0.19 ( 0.2

8 ( 0.01 0.7 ( 0.01 0.5 ( 0.01 0.4 ( 0.01

6 ( 0.01 1 ( 0.01 0.6 ( 0.01 0.3 ( 0.01

techniques have been found previously for dialquil glycerol surfactants,38 and this has been attributed to the different sensitivities of the techniques to different types of aggregates. A first examination of the surface tension-log concentration plots for the single chain surfactants (Figure 3A) shows that, as expected, the break for LGG14 is found at lower concentrations than that for LGG12.39 Moreover, the area per molecule found from the slope of these plots falls within what can be considered normal values. Therefore, the surface tension behavior of these two products is similar to other cationic surfactants of the same hydrophobic chain length but with the stabilization of the surface tension occurring at a concentration well below the one expected (2 orders of magnitude below that of trimethyl ammonium homologues). The surface tension as a function of log(C) curves of the diacyl products shows (Figure 3B) that the surface tension of LGGdi14 stabilizes at a higher concentration than LGGdi12. This is contrary to what would be expected for a cmc. Also, it is apparent that plots of the surface tension versus log(C) for both LGGdi14 and LGGCdi12 show a slope much higher than that of the corresponding single chain products. This strong slope implies that the area per molecule of these compounds cannot be established accurately. We have to remark that the surface tension stabilization as a function of time is very slow for these diacyl compounds, a matter of hours. Experiments on the same solution performed consecutively show large differences in their time evolution, 2 h compared to 6 h (see Figures S1 and S2 in the Supporting Information). In some cases, no change in surface tension has been recorded for up to 1 h (which could have been taken as the equilibrium surface tension) and then the surface tension starts a slow but remarkable decrease of several tens of mN/m. This surfactant adsorption kinetics is close to that observed for ionic surfactants in the absence of salt.40 The observations described above suggest anomalies in the surface tension behavior of the new compounds. Breaks in the surface tension at such low concentrations (for the LGG12 and LGG14) could be attributed to the adsorption of a significant amount of nonprotonated molecules. At these low concentrations, formation of nonprotonated basic form is favored.41 Two different factors can have an effect on the surface tension of the LGGdi12 and LGGdi14: the presence of significant amounts of nonprotonated species and formation of lamellar species. In these conditions, the surface tension break would not correspond to a cmc but to a cac. The slow and poor reproducibility of the LGGdi14 and LGGdi12 kinetics could also be attributed to this fact. For arginine-based diacyl surfactants, it was shown (using a combination of surface tension, pH, and LS measurements) that at very low concentration the adsorption of nonprotonated species dominated, while at higher concentrations the protonated species dominated the formation of micelles. (38) P
erez, L.; Infate, M. R.; Angelet, M.; Cl
apes, P.; Pinazo, A. Prog. Colloid Polym. Sci. 2004, 123, 210–216. (39) Rosen, M. J. Surfactants and Interfacial Phenomena, 3rd ed.; John Wiley & Sons, Inc: New Jersey, 2004. (40) Diamant, H.; Andelman, D. J. Phys. Chem. 1996, 100, 13732–13742. (41) Pinazo, A.; P
erez, L.; Infante, M. R.; Pons, R. Phys. Chem. Chem. Phys. 2004, 6, 1475–1481.

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Figure 3. Surface tension as a function of concentration curves at 25 °C. (A) (0) LGG12 and (4) LGG14; (B) (0) LGGdi12 and (4) LGGdi14.

The aqueous solution behavior of the new compounds was also studied by 1H NMR. Figure 4 shows the NMR spectrum of the 2.5 mM solution of LGGdi12 at 25 and 50 °C. Spectra of LGGdi14 at 25 and at 50 °C were similar. At 25 °C, the 1H NMR spectrum indicates the presence of vesicles and not micelles. The slow motion of the alkyl chains in the vesicles results in extremely broad NMR signals with low intensity.42 Surfactants LGGL12 and LGG14, with only one alkyl chain, yield 1H NMR typical high-resolution spectra with a Lorentzian band shape corresponding to the presence of classical micelles. The spectrum has also been determined at 50 °C. Using the water impurity in the D2O as an internal reference, the integrated 1 H NMR signal intensities increase with the temperature due to fast local anisotropic motions in the alkyl chain. We also observed a narrowing of the NMR signal of the alkyl chains, from 39.7 Hz for the spectra at 25 °C to 26.7 Hz at 50 °C. The observations under microscopy of the LGGdi12 solutions at 25 °C (2.5 and 5 mM) also showed that aggregation in large vesicles occurred (Figure 5). The microscopic images demonstrate (42) Wennerstr€om, H.; Ulmius, J. J. Magn. Reson. 1976, 23, 431–435.

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Figure 4. 1H NMR spectra of the LGGdi12 compound (2.5 mM aqueous solution) at 25 and 50 °C.

Figure 5. Light microscopic images of the LGGdi12 solutions at 25 °C: (a) 2.5 mM and (b) 5 mM.

the presence of a disperse population of vesicles with diameters larger than 500 nm. The two concentrations studied showed vesicles with similar size. Apparently, at 25 °C, we have large vesicles in the sample (not detected by NMR) and small vesicles, some of them in the gel state (not detected by NMR) and others in the fluid state (detected by NMR). At 50 °C, the fraction of alkyl chains in the fluid state increases, giving narrower NMR bandwidths due to fast anisotropic motions.43 Similar results have been obtained for the LGGdi14 compound. The NMR and microscopic observations suggest that for the dialkylated compounds we have vesicles of very different sizes. By microscopy, we can observe the large vesicles, while by NMR we only observe the smaller ones. From these studies, it can be concluded that in the diluted region of the system H2O/surfactant, lysine bisglycidol derivatives with one alkyl chain give rise to classical micelles while the compounds with two alkyl chains form vesicles. This different behavior can be attributed to the different geometrical shape of the molecules. The aggregate types formed by the surfactants depend on the dimensionless surfactant packing parameter44 P = V/Al, where V is the hydrophobic volume, l is the maximum hydrophobic chain length, and A is the surface area occupied by the headgroup which has been measured from surface tension. V and l have been calculated according to Tanford’s values.19 Surfactants with P < 1/3 give rise to spherical micelles changing to more elongated structures as P increases. For P=1/2, the preferred packing is cylindrical and packing tends to form bilayers (43) Saveyn, P.; Meeren, P. V.; Cocquyt, J.; Drakenberg, T.; Olofsson, G.; Olsson, U. Langmuir 2007, 23, 10445–10462. (44) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 1976, 2(72), 1525–1567.

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Table 2. Structural Parameters Derived from Geometrical Relationships Using the SAXS Pattern Assignation and Lattice Parameters for the Compounds in the Dry State at 25 °C compound

a (nm)a

lc (nm)b

lcmax (nm)c

Vc (nm3)d

A (nm2)e

Lh (nm) f

Pg

LGG12 4.78 1.00 1.42 0.325 0.33 1.39 0.39 LGG14 5.46 1.25 1.67 0.379 0.30 1.49 0.34 LGGdi12 5.14 1.52 1.42 0.650 0.43 1.06 LGGdi14 5.51 1.73 1.67 0.758 0.44 1.04 a Lattice parameter. b Calculated hydrophobic length. c Maximum hydrophobic length calculated from Tanford’s values. d Hydrophobic volume. e Area per molecule at the separation plane. f Headgroup length; the headgroup volume is nearly constant and equal to 0.454 nm3. g Packing parameter.

or vesicles for P values around unity. For LGG12 and LGG14, the packing parameters are 0.39 and 0.34, respectively (Table 2), which suggests that in solution they form ellipsoidal micelles. The large error in the area per molecule obtained for the diacyl compounds precludes their use for calculating the packing parameter. However, the minimum area per molecule for a two parallel chain compound would give rise to packing parameters around unity. The thermotropic behavior of LGG12, LGG14, LGGdi12, and LGGdi14 surfactants was investigated by SAXS measurements. The thermotropic liquid crystals form upon heating the dry solid, and they arise from anisotropic interaction between rigid or semirigid structures. The studies indicate that the four compounds present lamellar structures, which means that the surfactant molecules are arranged in layers with defined periodicity. At 25 °C, they all present a gel phase structure with characteristic equidistant peaks. At the chain melting temperature, DOI: 10.1021/la901675p

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Figure 6. Scattering intensity of the four products as a function of scattering vector at 25 °C.

a noticeable change in the peak positions and sharpness is detected (data not shown). In Figure 6, the scattering intensity of the four products is shown as a function of scattering vector at 25 °C. The scattering curves are smeared by the use of a line collimated beam. LGG12 and LGG14, single chain compounds, show a weak lamellar arrangement with characteristic positions in 1:2:3 patterns. The sharper peaks obtained for the LGG12 compound can be attributed to a more ordered structure. The main peak of the LGG14 compound appears at a somewhat smaller q vector, implying longer distances between layers. From the hydrocarbon chain and polar head volumes coupled with the lattice distance, we can calculate the molecular packing parameters (Table 2). The structure of the diacyl compounds, LGGdi12 and LGGdi14, is more ordered than that of the single chain surfactants, and this is reflected in the peak sharpness and the number of reflections found. The LGGdi12 main peak appears at a bigger q vector (smaller distance) than that for the LGGdi14 compound. The structural parameters are also shown in Table 2. Compared 7812 DOI: 10.1021/la901675p

with the single chain surfactants, the number of reflecting planes is quite impressive, up to five orders, displayed in the diacyl compound SAXS curves. As a result, diacyl compounds show both a stiffer bilayer arrangement and a crystalline domain size. This can be related to the intrinsic form of the molecules; while the area per headgroup of the single chain compounds is around 1.5 times the section of the hydrocarbon chain (0.21 nm2),19 the area per headgroup of the double chain compounds is very close to the section of the two hydrocarbon chains attached. This more cylindrical natural form of the double chain surfactant fits better in the lamellar arrangement than the more cone-shaped single chain surfactants. For the single chain surfactants, the hydrophobic chain length is significantly shorter than the maximum length according to Tanford0 s values,19 and this probably implies interdigitation of the hydrophobic chains. We can compare the area per molecule of the dry compounds with the area per molecule obtained from the surface tension isotherm. Both single chain surfactants present a minimum area per molecule of about 0.6 nm2 in solution, twice the dry state area per molecule. For short chain diacyl compounds with lysine as headgroup, we found Langmuir 2009, 25(14), 7803–7814

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Figure 7. Typical texture observed for binary water/LGGdi12 at 40 °C under polarized light.

an area per molecule of 1.4 nm2 in aqueous solution and 0.5 nm2 in the dry state.24 The behavior of the double chain surfactants is different; while the area per molecule in the dry state corresponds to 0.4 nm3, the area per molecule obtained from the adsorption isotherm is close to the same value. This observation may imply that the hydration of the polar head for these molecules is low and that the aggregates formed by LGGdi12 and LGGdi14 in solution have a lamellar structure, forming vesicles in solution. Indeed, vesicle structures have also been observed for the diacyl compounds under optical microscopic observations (see Figure 5). When the temperature is increased, the LGG12 compound undergoes a phase transition that, judging from the relative position of the peaks in the SAXS curves, could have a cubic Ia3d structure (data not shown). This structure seems to develop gradually with time, and no more profound characterization of this possible cubic phase will be attempted at this time. The LGG14 compound did not show this transition and seemed to directly melt. In the case of the double chain surfactants, an increase in temperature promotes changes in the SAXS patterns compatible with the transition from lamellar gel to lamellar liquid crystal. The reduction in interlamellar distance is comparable for both compounds (0.45 nm shorter at 65 °C than at 25 °C) and roughly corresponds to what would be expected for the melting of the hydrocarbon chains.14 In Figure 6B, a shoulder is observed for the LGGdi12 compound at 25 °C. This shoulder disappears at a higher temperature (data not shown). The origin of this shoulder is unclear, but it could be related to the presence of two or more phases coexisting because of regioisomer mixture.14 Finally, the lyotropism of the lysine derivatives synthesized was qualitatively evaluated using the Lawrence method.26 At room temperature, the four compounds exhibited birefringent texture under the crossed polarized microscopy, indicating the presence of lyotropic liquid crystals. On heating (temperatures around 30 °C), the initial phase transforms to a classical lamellar phase (Figure 7) which melts to a liquid isotropic phase at higher temperatures. Compounds that usually form vesicles in diluted solutions also tend to form lamellar phases at higher concentrations. This type of arrangement has also been described for the diacylglycerides from arginine as well as for lecithins and diacylglycerol compounds.16 Indeed, one of the most important properties of the phospholipids is their great capability to give rise to vesicles or liposomes. Antimicrobial Activity. The antimicrobial action of cationic surfactants is based on their ability to disrupt the integral bacterial Langmuir 2009, 25(14), 7803–7814

Article

membrane by a combined hydrophobic and electrostatic adsorption phenomenon at the membrane/water interface followed by membrane disorganization. The antimicrobial activity of the amino-acid-based surfactants depends on their structure and size, with the chain length being a critical structure parameter for their effectiveness. Minimum inhibitory concentration (MIC) values for the lysine-based surfactants LGG12 and LGGdi12 are summarized in Table 3. While LGG12 is not active at the maximum concentration tested (256 mg/L), LGGdi12 shows antimicrobial activity against a wide range of Gram-positive and Gram-negative bacteria with MIC values ranging from 32 to 256 mg/L. The antimicrobial activity of amphiphiles is usually modulated by the hydrophilic/lypophilic balance of the molecules. The LGG12 has only one hydrophobic alkyl chain of 12 carbon atoms and a big polar group that consists of the lysine with a positive charge in the R-amino group and the two glycidol moieties. It seems that the hydrophilic group is too big and it is necessary to introduce the second alkyl chain to increase the hydrophobicity of the molecule with the consequent enhancement of the antimicrobial power. Because of the different composition of the cellular membrane, LGGdi12 is more active against Gram-positive bacteria than against Gram-negative bacteria.45 The external layer of the outer membrane of the Gram-negative bacteria is almost entirely composed of lipopolysaccarides and proteins and restricts the entrance of biocides and amphiphilic compounds. In all cases, the MIC values are lower than the critical aggregation concentration. Thus, the monomers and not the vesicles are the ones that interact with the cellular wall of the bacteria. The activity of the LGGdi12 is similar to that reported for the cationic Nε-acyl lysine methyl ester surfactants and lower than that shown by the cationic NRacyl lysine and NR-acyl arginine derivatives.46 (NB In all cases, the MIC determinations were carried out using the same broth and also the same experimental procedures.) The modulation of the antimicrobial activity of these types of surfactants could be attributed to the different pKa values of the molecules. The headgroup charge of these surfactants is modulated 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 8.28 At pH equal to the pKa, the amino acid will be 50% protonated. As the pH is decreased more than 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 Nεlysine derivatives is around 8, the average charge of the compound could be lower than 1 at the pH of the test medium (pH 7), and hence, the compounds have more difficulties in disrupting the bacterial membrane. As for the effect of the compound’s net charge on its bactericidal activity, numerous studies show that the electrostatic interactions play a key role in the action of cationic systems, and that a decrease in the charge density of the cationic compound results in a reduction in adsorption and bacterial effects.47 Accordingly, the antibacterial activity varies with the pH in systems in which the net charge depends on the pH. For example, decreasing the pH (45) Oros, G.; Cserhati, T.; Forgacs, E. Chemosphere 2003, 52, 185–193. Rosen, M. J.; Fei, L.; Zhu, Y. P.; Morral, S. W. J. Surfactants Deterg. 1999, 2, 343–347. (46) Infante, M. R.; Molinero, J.; Erra, P.; Julia, R.; Garcı´ a, J. J. Fette, Seifen, Anstrichm. 1983, 87(8), 309–313. (47) (a) Ringstad, L.; Schmidtchen, A.; Malmsten, M. Langmuir 2006, 22, 5042– 5050. (b) Ringstad, L.; Kacprzyk, L.; Schmidtchen, A.; Malmsten, M. Biochim. Biophys. Acta, Biomembr. 2007, 1768, 715–727.

DOI: 10.1021/la901675p

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Table 3. MIC Values for LGG12 and LGGdi12 (pH of the Culture Medium = 7) microorganisms Gram positives

Gram negatives

Bacillus cereus var. mycoides CECT 193 Enterococcus hirae ATCC 10541 Micrococcus luteus ATCC 9341 Staphylococcus aureus ATCC 6538 Bacillus subtilis ATCC 6633 Staphylococcus epidermis ATCC 12228 Mycobacterium phlei ATCC 41423 Klebsiella pneumoniae CIP 143 Eschericia coli ATCC 8739 Salmonella typhimurium ATCC 14028 Pseudomonas aeuriginosa ATCC 9027 Bordetella bronchiseptica ATCC 4617 Serratia marcescens ATCC 274 Enterobacter aerogenes ATCC 13048 Candida albicans ATCC 10231

LGGdi12 (mg/L)

LGG12 (mg/L)

128

>256

128

>256

128

>256

64

>256

32

>256

128

>256

64

>256

128

>256

128

>256

256

>256

256

>256

32

>256

>256

>256

>256

>256

128

>256

Conclusions

increases the antimicrobial activity of chitosan and some peptides which take protons to become more cationic.48 Antimicrobial lipopeptides composed of palmitoyl mono-, di-, and tricationic lysines have been recently reported by Makovitzki et al.49 Interesting results have been obtained by these authors. On the one hand, the number of cationic amino acids in the molecule significantly affects the antimicrobial activity, and on the other hand, for the tricationic compounds, replacing only one of the three amino acids is sufficient to obtain compounds with very (48) Makovitzki, A.; Shai, Y Biochemistry 2005, 44, 9775–9784. (49) Makovitzki, A.; Baram, J.; Shai, Y. Biochemistry 2008, 47, 10630–10639.

7814 DOI: 10.1021/la901675p

different antimicrobial specificity. A single lysine attached to palmitic acid is inactive, two lysines show partial activity, and three lysines linked to the palmitic acid show strong activity, higher than that shown by the bisglycidol derivatives. Considering the findings obtained by Makovitzki et al., the moderate activity shown by LGG12, LGG14, LGGdi12, and LGGdi14 surfactants could be attributed to the fact than only one cationic amino acid is present in the structure. In these surfactants, a single lysine is enough to obtain partial activity, and probably with two lysine groups the antimicrobial activity would be enhanced. The moderate activity of LGGdi12 can be considered an advantage for many biomedical applications.50 The decrease in the antimicrobial activity in cationic surfactants usually indicates lower cytotoxicity and better biodegradation properties. Preliminary studies on the acute toxicity and biodegradation of these compounds agree with these assumptions. Novel lysine-bisglycidol conjugates of the type Nε,Nε0 -bis (acyl-2,3-dihydroxypropyl)-L-lysine methyl ester salts, combining several hydroxyl functions and aliphatic chains of 12 (C12) and 14 (C14) carbon atoms, have been synthesized at the laboratory scale. As a function of the number of alkyl chains, the compounds aggregate as micelles or vesicles at very low concentrations. In the dry state, all of the products present a gel phase structure at 25 °C with characteristic equidistant peaks; in the chain melting, a noticeable change in the peak positions and sharpness is detected. The antimicrobial activity is also modulated by the number of alkyl chains. While the monoacyl derivative LGG12 is not active, the homologue LGGdi12 shows a moderate activity against Gram-positive and Gram-negative bacteria. Acknowledgment. This work has been partially supported by the Spanish CICYT under Project CTQ2006-01582. J. Caelles from the SAXS-WAXS service at IQAC is kindly acknowledged for his help with SAXS measurements. I. Carrera is acknowledged for surface tension measurements. Dra. Ma Angels Manresa is also acknowledged for her aid in the antimicrobial activity determinations. Supporting Information Available: Surface tension versus time curves. This material is available free of charge via the Internet at http://pubs.acs.org. (50) Rosa, M.; Penacho, N.; Simoes, S.; Lima, M. C. P.; Lindman, B.; Miguel, M. G. Mol. Membr. Biol. 2008, 25, 23–34.

Langmuir 2009, 25(14), 7803–7814