Calixarenes in a Membrane Environment: A Monolayer Study on the Miscibility of Threep-tert-Butylcalix[4]arene â-Lactam Derivatives with1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine

Beata Korchowiec,†,‡ Adel Ben Salem,§,⊥ Yohann Corvis,† Jean-Bernard Regnouf de Vains,§Jacek Korchowiec,| and Ewa Rogalska*,†

Groupe d’Etude des Vecteurs Supramole´culaires du Me´dicament UMR 7565 CNRS/UniVersiteHenri PoincareNancy 1, Faculte´ des Sciences, BP 239, 54506 VandoeuVre-les-Nancy Cedex, France and Faculte´ dePharmacie, 5, rue Albert Lebrun, BP 80403, 54001 Nancy Cedex, France, Department of Physical Chemistryand Electrochemistry, Faculty of Chemistry, Jagiellonian UniVersity, and Department of Theoretical Chemistry,Faculty of Chemistry, Jagiellonian UniVersity, ul. R. Ingardena 3, 30-060 Krakow, Poland

ReceiVed: February 4, 2007; In Final Form: September 1, 2007

Literature data indicate that some calixarene derivatives with antimicrobial activities may be useful as drugs;one of the aspects of the biological activity of different classes of antibiotics concerns interactions with lipidmembranes. Here, the possibility of incorporation and/or translocation of three amphiphilicp-tert-butylcalix-[4]arene derivatives across membranes was studied using lipid monolayers. The derivatives used have6-aminopenicillanic acid or benzylpenicillin moieties grafted in alternate positions at the calixarene lowerrim; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), a model bacterial membrane lipid, was usedto prepare the monolayers. The miscibility of calixarene-antibiotic conjugates with lipid films was studiedusing surface pressure and surface potential measurements, as well as Brewster angle microscopy. The resultsobtained show that the miscibility is significantly different for the 6-aminopenicillanic acid and the twobenzylpenicillin derivatives. Molecular modeling allowed the assessment of the lowest energy conformationsof the calixarene derivatives and gave more insight into the interactions with the DMPE films.

1. Introduction

Staying one step ahead of resistance with new antibiotics andtreatments for infections is a huge challenge because bacteriaevolve quickly to counter them.1 Indeed, use or misuse ofantibiotics may result in the development of antibiotic resistanceby the infecting organisms. For example, nearly all strains ofStaphylococcus aureus, which could be treated successfully withpenicillin in the 1940s and 1950s, are at present resistant to it.2

Therefore, there is an urgent need for new antibiotics and newstrategies to combat bacteria, particularly those showing mul-tidrug resistance.

Calixarenes, a class of polyphenolic macrocycles, are receiv-ing increased attention due to their great fundamental andpractical importance.3-6 The calix[n]arenes (Scheme 1) can betailored synthetically by altering the R moieties. Literature dataindicate that some calixarene derivatives with antimicrobialactivities7 may be useful as drugs in the treatment of tuberculosisand mycobacterioses.8 It was shown as well that calixarene-based vancomycin antibiotic mimics were active against Gram-positive bacteria.9 Other calixarene derivatives, of whichsynthesis and applications are covered by patents, showedactivities against bacteria,10 fungi, cancerous cells, viruses,11,12

enveloped viruses,13 but also against thrombosis14 or fibrosic

diseases. Our group demonstrated recently that some ioniccalixarene derivatives were active against Gram-positive, as wellas Gram-negative bacteria.15,16

In our group, different synthetic methods have been developedto prepare calixarene derivatives.17-22 The derivatives bearingantibiotic moieties were conceived as possible drug carriers,releasing the antibiotic upon hydrolysis. Indeed, the release ofthe antibiotics from this kind of derivative occurs uponhydrolysis in vitro (results not published), and the same processcould be expected to take place in physiological conditions.Along this line, 6-aminopenicillanic acid was grafted via amidebonds on the 1,3-bis(O-acetyl)-p-tert-butylcalix[4]arene coneconformer in alternate positions (Calix I ).21 On the other hand,different penicillins (Calix II andIII ), as well as an antibioticfrom the quinolone group, the nalidixic acid, were grafted onthe p-tert-butylcalix[4]arene via ester bonds.22

One of the aspects of the biological activity of differentclasses of antibiotics are interactions with lipid membranes.23-29

The derivatives used in this study are liposoluble amphiphiles;their structures might allow incorporation into the bilayer ortranslocation across the lipid membrane. The length ofCalix I ,Calix II , and Calix III in a fully extended conformation is,respectively, 20.0, 18.9, and 21.9 Å. These values are signifi-cantly lower compared to the thickness of a phosphoglyceridebilayer and the thickness of the bilayer hydrocarbon core, whichare around 35-4030 and 32 Å,23,31 respectively. As a result ofthis difference in thickness, the orientation and structure of thesecalixarene-based molecules within the bilayer is currently underinvestigation using molecular modeling techniques. On the other

* Corresponding author. E-mail: rogalska@lesoc.uhp-nancy.fr.† Facultedes Sciences, Universite´ Henri Poincare´ Nancy 1.‡ Department of Physical Chemistry and Electrochemistry, Jagiellonian

University.§ Facultede Pharmacie, Universite´ Henri Poincare´ Nancy 1.| Department of Theoretical Chemistry, Jagiellonian University.⊥ Current address: Institut Supe´rieur des Sciences Applique´es et de

Technologie de Gabe`s, Rue Omar, El Khattab 6072, Gabes, Tunisie.

13231J. Phys. Chem. B2007,111,13231-13242

10.1021/jp070970+ CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 11/01/2007

hand, an impact of calixarene-based antibiotics on the activityof surface-active enzymes, lipases and phospholipases wasobserved in vivo.8 The latter effect might be linked to themodification of the structure and physicochemical propertiesof lipid aggregates by the incorporated drug molecules. Indeed,we showed recently that the presence of an antifungal agent,griseofulvin, in phospholipid monolayers stimulates phospho-lipase A2 (PLA2) activity.32 Other literature data indicate thatPLA2 is sensitive to the lipid aggregate morphology.33-36

Taking the above considerations into account, we decided toproceed with a study on the calixarene-antibiotic conjugate-lipid systems. This study aimed to evaluate the capacity of thethree new derivatives to interact with biological membranes;to this end, the Langmuir technique was used. Indeed, weshowed recently that different amphiphilic calixarene derivativesformed stable monomolecular films at the air-water interface.37

First, the interfacial behavior of the pure calixarene derivativeswas examined in the monolayers formed at the air-waterinterface. Second, mixed 1,2-dimyristoyl-sn-glycero-3-phos-phoethanolamine (DMPE)/calixarene derivative films werestudied. Surface pressure-molecular area (Π-A) isotherms,surface potential-molecular area (∆V-A) isotherms, Brewsterangle microscopy (BAM), and molecular modeling were usedto characterize the monolayers. The results obtained gave usan insight into the inter- and intramolecular interactions of thesemolecules and showed that the calixarenes studied differsignificantly in their interfacial behavior and in the interactionswith DMPE. In the case ofCalix II andCalix III , disorganizingof the lipid monolayer was observed. The disorganizing effectwas more pronounced in the case ofCalix II compared toCalixIII , albeit the only structural difference between these twoderivatives is the one methylene-different length of the linkers

by which the benzylpenicillin is attached to the aromatic crown.The results obtained in this study prepare the ground for furtherresearch on the biological activity of calixarene-antibioticconjugates.

2. Experimental Section

Characterization. Melting points (°C, uncorrected) weredetermined on an Electrothermal 9100 in capillary apparatus.1H and13C NMR spectra were recorded on a Bruker DRX 400(400.130 and 100.612 MHz, respectively; CDCl3, TMS asinternal standard, chemical shifts in ppm). Mass spectra (elec-trospray, ES) were recorded on a Platform Micromass apparatusat the Service Commun de Spectrome´trie de Masse, Nancy.Infrared spectroscopy was performed on a Bruker Vector 22apparatus (KBr,ν in cm-1), and UV spectra were recorded ona SAFAS UV mc2 apparatus,λmax in nm, ε in L mol-1 cm-1.Elemental analyses were performed on a ThermofinniganFlashEA 1112 apparatus, at the Service Commun de Microanal-yse, Nancy. All chemicals were used without further purificationunless otherwise specified.

Synthesis. Calix I, bearing an amide-linked 6-pivAPAderivative was synthesized as described previously.21 Calix IIandCalix III were obtained by attaching PG to the calixarenemoiety via a bifunctional ester-ether alkyl linkage. 1,3-Bis-(bromoalkyl)-calixarene calixarene derivatives were esterifiedusing PG sodium salt (PGNa). To this end, bromoethyl andbromopropyl derivatives3 and4 (Scheme 2) were prepared asdescribed in the literature.38 Subsequently,3 and4 were reactedwith 2 equiv of PGNa in DMF to giveCalix II (47%) andCalixIII (32%), respectively.

An alternative synthetic route, with theâ-lactam used as analkylating agent, was explored as well; this approach was aimed

SCHEME 1: Calixarene Structure and Derivatives Used in This Studya

a (A) General calixarene structure. In the molecules used here R) N-acetyl-pivaloyloxymethyl-6-aminopenicillanic acid (6-pivAPA, inCalix I ),benzylpenicillin (penicillin G or PG) ethyl ester (inCalix II ), or propyl ester (inCalix III ); (B) numbering of the atoms in the antibiotic substituents;(C) derivativeCalix I; (D) derivativeCalix II ; (E) derivativeCalix III .

13232 J. Phys. Chem. B, Vol. 111, No. 46, 2007 Korchowiec et al.

at a regioselective functionalization of the calixarene.20,39-42 Thereaction of PGNa with 1,2-dibromoethane or 1,3-dibromopro-pane in DMF gave the bromoester1 (70% yield) or2 (50%yield), respectively. Unfortunately, in the next step, compounds1 and2 underwent degradation.

The new derivativesCalix II andCalix III were characterizedusing infrared, UV-vis, mass spectrometry, elemental analyses,and 1D and 2D NMR methods. IR analyses showed in bothcases the presence of amide, ester, and lactam CdO bonds inthe 1685-1709, 1745-1752, and 1786-1792 cm-1 regions,respectively. The UV-vis spectrometry performed in CHCl3

showed a transition at around 284 nm. The electrospray massspectrometry performed in the positive mode using MeOH withtrace amounts of H2O as solvent yielded base peaks attributedto the monocharged, monosodio derivatives and to the dicharged,disodio derivatives. Elemental analyses were in accordance withthe molecular formulas; some amount of CH2Cl2 detected inCalix III with NMR was probably due to its complexationwithin the calixarene cavity, as already observed for othercalixarene species.17

Structural and conformational analysis ofCalix II andCalixIII in CDCl3 solution was done using1H and13C NMR; COSY,HSQC, and HMBC experiments (results not shown). The Ar-CH2-Ar resonance signals observed with13C NMR between31 and 32 ppm showed that both derivatives were in a coneconformation.43,44It is worth noticing thatCalix II bearing ethyllinkers exhibits two very close resonance signals for the Ar-CH2-Ar protons, suggesting that the proximity of the penicillinchiral centers induces distortion of the calixarene crown.

The 1H NMR spectra ofCalix II andCalix III display allcharacteristic signals of both penicillin and calixarene moieties.The influence of the chiral penicillin moiety on the calixareneconformation was evidenced by the presence of two or moreAr-CH2-Ar AB systems and, in the ethyl derivativeCalix II

(Figure 1), of two strong AX systems for the aromatic protons.In the intermediate1, the ester methylene group appears as adecuplet at around 4.50 ppm, whereas in2 it gives a well-definedABm resonance signal at around 4.32 ppm. This differencesuggests that, in the absence of steric or other constraint due tothe calixarene moiety, the influence of the pename chirality onthe ester methylene group through the carboxylic function ismore pronounced in the propyl intermediate2.

The study of the ethyl and propyl linkers in the calixarenederivatives showed that the ether methylene group appears asan ABm system at around 4.20 ppm inCalix II (ethyl) and asa triplet (J ) 6 Hz) at around 4.10 ppm inCalix III (propyl),while the ester methylene groups appear as a broad multiplet

SCHEME 2: Synthetic Pathway Used to Prepare the Calixarene Derivatives

Figure 1. Partial 1H NMR spectra of the (A) propyl linker analogCalix III and (B) PG ethyl linkerCalix II (CDCl3, 400 MHz, roomtemperature).

Calixarenes in a Membrane Environment J. Phys. Chem. B, Vol. 111, No. 46, 200713233

at around 4.65 ppm inCalix II and as a well-defined ABmsystem at around 4.72 ppm inCalix III (Figure 1).

It can be concluded that increasing the length of the linkerdecreases the influence of the calixarene moiety on the penicillinsubstituent. This influence is absent inCalix III , while it isclearly seen in the ethyl derivativeCalix II , as shown in thediastereotopic differentiation of the ether methylene protons.At low temperatures, the ester multiplet ofCalix II can be seento be a spread out and poorly resolved ABm system; it is alsothe case with the ether ABm signal (results not shown).

A similar diastereotopic differentiation observed in tensedhelicoidal calixarene-based bipyridyl Cu(I) complexes18 suggeststhat the ethyl penicillin substituent inCalix II is blocked in aspecific conformation relative to the calixarene main axis. TheAX resonance signals of the calixarene aromatic protonsobserved inCalix II , which result from a twisting of themacrocycle around its main axis, support the suggestion of theconformational hindrance. Moreover, the penicillin H(6) andH(5) protons in the ethyl derivativeCalix II are shifted upfieldaround 0.35 and 0.1 ppm, respectively, compared to thepenicillin bromoethyl ester1 and, as shown in Figure 1, toCalixIII . This phenomenon can be interpreted in terms of shieldingdue to a short distance between these protons and the calixarenearomatic cycle and the consequent steric constraints.

A ROESY (rotational frame nuclear Overhauser effectspectroscopy) experiment performed withCalix II (Figure 2)showed that H(5) and H(6) were correlated to the aromatic andhydroxyl protons of the unsubstituted and, to a lesser extent, tothe substituted phenolic units. This observation suggests thatin CDCl3 solution the penicillin substituents are oriented parallelrelative to the calixarene core axis. This result is in accordancewith the results of molecular modeling (Figure 3). Finally, theresidual phenolic-OH groups appear at around 7.00 and 7.50ppm in Calix II and Calix III , respectively. This effect maybe due to H bonding,45 as already observed in other carboxylatedcalixarenes.19

Compound 1.A solution of PGNa (0.2 g, 5.6 10-4 mol) and1,3-dibromoethane (2 mL, 23.1 10-3 mol) in dry DMF (10 mL)was stirred at 50°C during 18 h under Ar. The solvent wasevaporated to dryness, and the residue was dissolved in CH2-Cl2, washed with water, then dried over Na2SO4. The solutionwas concentrated and chromatographed (SiO2, CH2Cl2/Et20 10:1) to give1 (0.17 g, 70%) as an oily solid.1H NMR (CDCl3):1.498 (s, 3 H, CH3), 1.502 (s, 3 H, CH3), 3.546 (t,J ) 5.7 Hz,

2 H, OCH2CH2Br), 3.649 (s, 2 H, CH2C6H5), 4.418 (s, 1 H,H(2)), 4.476 (dec, 2 H, OCH2CH2Br), 5.330 (d,J ) 4.2 Hz, 1H, H(5)), 5.677 (dd,J1 ) 4.2 Hz,J2 ) 9 Hz, 1H,H(6)), 6.151(d, J ) 9 Hz, 1 H, NH), 7.250, 7.420 (m, 5 H,HC6H5). 13CNMR (CDCl3): 27.35 (CH3), 28.47 (OCH2CH2Br), 32.05 (CH3),43.78 (CH2C6H5), 59.16 (C(6)), 64.92 (C(3)), 65.40 (OCH2-CH2Br), 68.49 (C(5)), 70.70 (C(2)), 128.06, 129.54, 129.95(Cp,m,o of C6H5), 134.24 (Ci of C6H5), 167.60 (COester), 170.73(COamide), 173.94 (COlactam).

Compound 2.A solution of PGNa (0.2 g, 5.6 10-4 mol) and1,3-dibromopropane (2 mL, 19.6 10-3 mol) in dry DMF (20mL) was stirred at 35°C during 30 h under Ar. The solventwas evaporated to dryness, and the residue was dissolved inCH2Cl2, washed with water, then dried over Na2SO4. Thesolution was concentrated and chromatographed (SiO2, CH2-Cl2) to give2 (0.13 g, 50%) as an oily solid. UV (CHCl3): 326.0(675.4).1H NMR (CDCl3): 1.438 (s, 3 H, CH3), 1.453 (s, 3 H,CH3), 2.204 (quint,J ) 6.3 Hz, 2 H, CH2CH2CH2), 3.465 (t,J) 6.3 Hz, 2 H, OCH2CH2CH2Br), 3.639 (s, 2 H, CH2C6H5),4.306 (ABm, 2 H, OCH2CH2CH2Br), 4.386 (s, 1 H,H(2)), 5.503(d, J ) 4.2 Hz, 1 H,H(5)), 5.646 (dd,J1 ) 4.2 Hz,J2 ) 9 Hz,1 H, H(6)), 6.214 (d,J ) 9 Hz, 1 H, NH), 7.240, 7.407 (m, 5H, HC6H5). 13C NMR (CDCl3): 27.14 (CH3), 29.39 (OCH2-CH2CH2Br), 31.53 (OCH2CH2CH2Br), 32.54 (CH3), 43.73(CH2C6H5), 59.35 (C(6)), 63.69 (OCH2CH2CH2Br), 64.79(C(3)), 68.59 (C(5)), 70.69 (C(2)), 128.04, 129.52, 129.96 (Cp,m,o

of C6H5), 134.28 (Ci of C6H5), 167.77 (COester), 170.77(COamide), 173.78 (COlactam).

Calix II. A solution of PGNa (0.455 g, 1.27 10-3 mol) and1,3-bis(bromoethyl)calixarene3 (0.5 g, 5.8 10-4 mol) in dryDMF (20 mL) was stirred under argon at 50°C during 27 h.The solvent was evaporated to dryness, and the residue dissolvedin CH2Cl2, then washed with H2O. The organic phase was driedover Na2SO4, concentrated, then chromatographed (SiO2, CH2-Cl2/Et2O 10:1) to giveCalix II (0.37 g, 46%) as a white powder.Mp: 123 °C. IR (KBr): 1686.46 cm-1 (CO amide), 1751.61cm-1, 1787.22 cm-1 (CO lactam, CO ester). UV-vis (CHCl3):284 (7360).1H NMR (CDCl3): 0.959 (s, 18 H, 2Me3C A),1.307 (s, 18 H, 2Me3C B), 1.451 (s, 6 H, 2 CH3), 1.549 (s, 6H, 2 CH3), 3.308, 4.250 (AB,JAB ) 13.2 Hz, 4 H, Ar-CH2--Ar), 3.331, 4.327 (AB,JAB ) 13.2 Hz, 4 H, Ar-CH2-Ar), 3.614(s, 4 H, C6H5CH2), 4.200 (ABm, 4 H, OCH2CH2OCO), 4.499(s, 2 H, H(2)), 4.590 (m, 4 H, OCH2CH2OCO), 5.263 (dd,J1

) 9 Hz, J2 ) 4.2 Hz, 2 H, H(6)), 5.442 (d,J ) 4.2 Hz, 2 H,H(5)), 6.04 (d,J ) 9 Hz, 2 H, NH), 6.780, 6.808 (AX,JAX )2.4 Hz, 4 H, ArH A), 7.031 (s, 2 H, OH), 7.050, 7.097 (AX,JAX ) 2.3 Hz, 4 H, ArH B), 7.24-7.40 (m, 10 H, Hm,p,o ofC6H5).13C NMR (CDCl3): 27.27 (CH3), 31.37 (Me3C A), 31.73(CH3), 32.06 (Me3C B), 31.73, 31.87 (Ar-CH2-Ar), 34.25(Me3C B), 34.34 (Me3C A), 43.81 (CH2C6H5), 58.86 (C(6)),64.85 (C(3)), 64.85 (OCH2CH2OCO), 68.26 (C(5)), 70.87(C(2)), 73.51 (OCH2CH2OCO), 125.61, 125.77 (C(H) of Ar B),125.94, 126.22 (C(H) of Ar A), 127.71, 128.18 (Co of Ar B),127.98 (Cp,(C6H5)), 129.49, 129.92 (Co,m (C6H5)), 132.62,132.94 (Co of Ar A), 134.34 (Ci,(C6H5)), 142.22 (Cp of Ar B),147.75 (Cp of Ar A), 149.63 (Ci of Ar A), 150.69 (Ci of Ar B),168.03 (COO), 170.45 (CONH), 173.96 (CONH). Anal. Calcdfor C80H96N4O12S2 (1369.77): C, 70.15; H, 7.06; N, 4.09.Found: C, 70.06; H, 7.05; N, 4.01. ES-MS (positive mode):1391.33 [Calix II + Na]+; 707.25 [Calix II + 2Na]2+/2.

Calix III. A solution of 1, 3-bis(bromopropyl)calix[4]arene4 (1 g, 1.12 10-3 mol) and PGNa (0.9 g, 0.25 10-3 mol) in dryDMF (100 mL) was heated at 40°C under argon. The reactionwas monitored by chromatography (SiO2; CH2Cl2/Et2O 5:1).

Figure 2. ROESY correlations betweenâ-lactam and aromatic protonsin Calix II (CDCl3; 400 MHz, room temperature).

13234 J. Phys. Chem. B, Vol. 111, No. 46, 2007 Korchowiec et al.

After 72 h, the solvent was evaporated to dryness at roomtemperature; the residue was dissolved in CH2Cl2, washed withH2O, and then dried over Na2SO4. The solvent was evaporated,and the final solid residue was chromatographed (SiO2; CH2-Cl2/Et2O 5:1) to giveCalix III (0.5 g, 32%) as a white solid.Mp: 142 °C. IR (KBr): 1685.81 cm-1 (CO amide), 1747.54cm-1 (CO lactam), 1786.62 cm-1 (CO ester). UV-vis(CHCl3): 285 (6950).1H NMR (CDCl3): 1.020 (s, 18 H,Me3CA), 1.297 (s, 18 H,Me3C B), 1.431 (s, 6 H, CH3), 1.465 (s, 6H, CH3), 2.359 (quint,J ) 6.0 Hz, 4 H, OCH2CH2CH2OCO),3.331, 4.210 (AB,JAB ) 13.0 Hz, 4 H, ArCH2Ar), 3.342, 4.192(AB, JAB ) 13.0 Hz, 4 H, ArCH2Ar), 3.658 (s, 4 H, CH2C6H5),4.074 (t,J ) 6 Hz, 4 H, OCH2CH2CH2OCO), 4.436 (s, 2 H,H(2)), 4.703 (ABm, 4 H, OCH2CH2CH2OCO), 5.507 (d,J )4.2 Hz, 2 H,H(5)), 5.621 (dd,J1 ) 9.0 Hz,J2 ) 4.2 Hz, 2 H,H(6)), 6.145 (d,J ) 9 Hz, 2 H, NH), 6.870 (s, 4 H, ArH A),7.062 (s, 4 H, ArH B), 7.26-7.42 (m, 10 H, C6H5), 7.549 (s, 2H, OH). 13C NMR (CDCl3): 27.05 (CH3), 29.76 (OCH2CH2-

CH2OCO), 31.43 (Me3C A), 32.08 (Me3C B), 32.18 (ArCH2-Ar), 32.48 (CH3), 34.24 (Me3C B), 34.42 (Me3C A), 43.83(CH2C6H5), 59.24 (C(6)), 62.95 (OCH2CH2CH2OCO), 64.79(C(3)), 68.52 (C(5)), 70.74 (C(2)), 72.49 (OCH2CH2CH2OCO),125.61 (Cm of Ar B), 126.11 (Cm of Ar A), 128.10 (Cp of C6H5),129.54 (Cm of C6H5), 129.99 (Co of C6H5), 127.81, 127.83 (Co

of Ar B), 132.94, 132.94 (Co of Ar A), 134.28 (Ci of C6H5),142.16 (Cp of Ar B), 147.73 (Cp of Ar A), 149.64 (Ci of Ar A),150.87 (Ci of Ar B), 167.88 (COO), 170.68 (CONH), 173.80(COlactam). Anal. Calcd for C82H100N4O12S2‚0.5CH2Cl2(1440.29): C, 68.80; H, 7.07; N, 3.89. Found: C, 69.12; H,7.05; N, 3.84. ES-MS (positive mode): 1420.22 [Calix III +Na]+; 721.37 [Calix III + 2Na]2+/2.

Compression Isotherms and Brewster Angle Microscopy.Monolayer experiments were carried out with a KSV 5000Langmuir balance (KSV, Helsinki). A Teflon trough (15 cm×58 cm× 1 cm) with two hydrophilic Delrin barriers (symmetriccompression) was used in all experiments. The system was

Figure 3. Optimized structures ofCalix I (A), Calix II (B), and Calix III (C). Left and middle column, side view; right column, top view.Hydrogen bonds are not shown.

Calixarenes in a Membrane Environment J. Phys. Chem. B, Vol. 111, No. 46, 200713235

equipped with an electrobalance and a platinum Wilhelmy plate(perimeter 39.24 mm) as a surface pressure (Π) sensor. Surfacepotential was measured using KSV Spot 1 with a vibrating plateand a stainless steel counter electrode. The apparatus was closedin a Plexiglas box, and temperature was kept constant at 20°C.Before each utilization, the trough and the barriers were cleanedusing cotton soaked in chloroform, gently brushed with ethanol,then with tap water, and finally rinsed with water purified byreverse osmosis (Millipore, France). All solvents used forcleaning the trough and the barriers were of analytical grade.Pure water (Millipore Milli-Q system, 18 MΩ‚cm), surfacetension (γ) of 72.75 mN m-1 at 20°C, was used as a subphasein all experiments. Any residual surface-active impurities wereremoved before each experiment by sweeping and suction ofthe surface. Monolayers were spread on pure water usingcalibrated solutions (concentration∼0.5 mg mL-1) of purecalixarenes, pure 1,2-dimyristoyl-sn-glycero-3-phosphoethano-lamine (DMPE; 99%, Sigma) and DMPE/calixarene mixtures.Calixarene solutions were prepared with spectrophotometricgrade chloroform (Aldrich, ACS). DMPE and DMPE/calixarenemixtures were dissolved in chloroform/methanol 4:1 v/v. Afterthe equilibration time of 20 min, the films were compressed atthe rate of 2.5 mm min-1 barrier-1 (750 mm2 min-1). A PCwas used with KSV software to control the surface pressure-molecular area (Π-A) compression isotherm experiments. Thestandard deviation obtained from compression isotherms re-peated systematically at least three times for each system studiedwas(0.5 Å2 on molecular area,(0.01 V on surface potential,and(0.2 mN m-1 on surface pressure.

The morphology of the films was imaged with a computer-interfaced KSV 2000 Langmuir balance combined with aBrewster angle microscope (KSV Optrel BAM 300, Helsinki).The light source was a standard HeNe laser, 10 mW, 633 nm;the spatial resolution of the device is 2µm. The Teflon troughdimensions were 6.5 cm× 58 cm× 1 cm; other experimentalconditions were as described above.

Computational Details.The geometrical structure of inves-tigated calixarenes was initially optimized at the semiempiricallevel of theory (AM1). The conelike shape of the calixarenepart was taken into account, according to NMR experiments.The other possible structures, namely, partial cone, 1,2-alternate,and 1,3-alternate, were excluded from the optimization process.The conelike calixarene crown and the penicillin moieties wereoptimized independently. The entireCalix I , II , andIII systemswere assembled from these smaller fragments, with the termi-nating hydrogen atoms removed. Since all three calixarenederivatives have several single bonds in both substituents, a largenumber of local minima exist on the hyperenergy surfaces; thefull search on the energy surfaces is practically impossible. Wehave performed a limited search on the energy hypersurfacewith restricted (C2) and unrestricted (C1) symmetries, respec-tively. In the former case, the surface was scanned bysimultaneously changing two selected dihedral angles usingC2

symmetry. In the latter case, both rotation angles were inde-pendent. It was found that for the reduced,C1 symmetry of themolecules, the located conformers were less stable comparedto those forC2. For this reason, theC2 symmetry was assumedin more extended ab initio calculations at the Hartree-Fock(HF) level of theory with 6-31G basis set.46,47The Gamess48,49

and Gaussian50 program packages were used in the calculations.

3. Results and Discussion

Calixarene Modeling.The located structures have a column-like shape. The substituents are brought closely together and

stretch along the long molecule axis below the aromatic crown.The lowest energy conformers are depicted in Figure 3.Hydrogen bonds form between the free hydroxyl groups andthe oxygen atoms of the substituted hydroxyl groups on thelower calixarene rim. InCalix I , the two amide linkers arehydrogen bonded via the nitrogen and oxygen atoms of theamide groups. These hydrogen bonds rigidify the moleculeskeleton and favor the column-like shape. An alternative,propeller-like structure ofCalix I (structure not shown) is around20.5 kcal mol-1 less stable. The column-like conformation isfavored as well in the case ofCalix II and Calix III ; theseconformations are stabilized by the intersubstituent hydrogenbonds, namely, the twoâ-lactam carbonyl oxygens hydrogenbond to the amide linker nitrogens.

In Table 1 the total energies, dipole moments, and calculatedmolecular areas of the three derivatives are listed. The molecularareas were computed by integrating the density plot in the planeperpendicular to the long molecule symmetry axis. The sameplane, cutting through the most rigid part of the molecule atthe level of thet-butyl moieties of the calixarene upper rim,was taken into account with the three derivatives.

Calixarene One-Component Monolayers.The propertiesof the monolayers formed with the calixarene derivatives werecharacterized using surface pressure-area (Π-A) isotherms,surface potential-area (∆V-A) isotherms, and BAM. Surfacepressure-area isotherms recorded on pure water subphase at20 °C are shown in Figure 4.

All derivatives form stable monolayers at the air-waterinterface. The monolayers have limiting areas,A0, of 175, 180,and 181 Å2 with Calix I , Calix II , andCalix III , respectively.The experimental limiting area values were determined by alinear fitting of the upper part of the isotherm corresponding tothe most condensed film and extrapolating to zero surfacepressure. The comparison of the experimental,A0

exp, and

TABLE 1: Total Energies, Dipole Moments, and SurfaceAreas Computed at Hartree-Fock Level of Theory with6.31G Basis Set for the Most Stable Conformers ofCalixarene Derivatives

E (au) µ (D) S(Å2)a

Calix I -5144.98313 9.11 149Calix II -4994.79176 14.04 158Calix III -5072.83142 12.24 160

a Surface areas were integrated to 0.0001 contour.

Figure 4. Surface pressure isotherms ofCalix I (solid line),Calix II(dotted line), andCalix III (dashed line) spread on pure water at20 °C.

13236 J. Phys. Chem. B, Vol. 111, No. 46, 2007 Korchowiec et al.

theoretical,A0theor, values shows that the latter are lower (Tables

1 and 2); note that the computed surface areas were obtainedby integrating the electron density contour plot.

The similar values ofA0exp/A0

theor (1.174, 1.139, and 1.131,for Calix I , Calix II , andCalix III , respectively) indicate thata quantitative agreement between the experimental and theoreti-cal results can be obtained by choosing an adequate moleculecontour for the calculations.

The maximum values of the compressibility modulus,Cs-1,

corresponding to the highest packing of molecules in themonolayer are given in Table 2. It can be seen that themonolayer formed withCalix II is more condensed comparedto those formed withCalix I andCalix III . Indeed, theCs

-1

values with theCalix I andCalix III monolayers are situatedbetween the liquid expanded and condensed phase, whereas thatof Calix II can be considered as condensed phase.51 TheΠ-A

isotherms ofCalix II andCalix III bearing similar substituentsexhibit similar phase characteristics. However, the slope of theisotherm can be seen to decrease upon introduction of anadditional methylene group between the phenolic unit andbenzylopenicillin moiety. Indeed, with an increased length ofthe linker inCalix III , the monolayer becomes more compress-ible and less stable. The lower stability of the monolayer, whichshows in the lower collapse pressure values (Table 2), may resultfrom a higher conformational mobility and lower ordering oftheCalix III polar head groups in the monolayer. These resultsare in accordance with the NMR results on the conformationalmobility of these two derivatives. However, the highest com-pressibility and the lowest stability of the monolayer areobserved in the case ofCalix I . This effect may be due to thepresence of alkyl moieties in the penicillin substituents comparedto Calix II andCalix III , bearing benzyl moieties. Indeed, in

Figure 5. Surface potential-area (A) and surface dipole moment-area (B) isotherms ofCalix I (solid line),Calix II (dotted line), andCalix III(dashed line) spread on water at 20°C.

TABLE 2: Characteristic Parameters of Pure Calixarene Isotherms

Πcoll

(mN m-1)Acoll

(Å2 molecule-1)Cs

-1

(mN m-1)A0

(Å2 molecule-1)∆Vmax

(V)µ⊥(D)

Calix I 36.1 113 82.7 175 0.795 2.39Calix II 39.1 133 129 180 0.442 1.56Calix III 37.4 116 87.9 181 0.554 1.71

Figure 6. Π-A and∆V-A isotherms of binary mixtures DMPE/CalixI spread on water at 20°C. Solid lines correspond to pure components;dashed lines,xDMPE ) 0.75; dotted lines,xDMPE ) 0.50; dashed-dottedlines,xDMPE ) 0.25.

Figure 7. Π-A and∆V-A isotherms of binary mixtures DMPE/CalixII spread on water at 20°C. Solid lines correspond to pure components;dashed lines,xDMPE ) 0.75; dotted lines,xDMPE ) 0.50; dashed-dottedlines,xDMPE ) 0.25.

Calixarenes in a Membrane Environment J. Phys. Chem. B, Vol. 111, No. 46, 200713237

the latter case theπ-π stacking could contribute to themonolayer stabilization. It is worth noticing that the obtainedvalues of the molecular areas at the collapse of the monolayers(Acoll) compare well with those reported for other amphiphiliccalixarene derivatives.37

The surface potential of the investigated calixarenes plottedas a function of the molecular area is shown in Figure 5A. Thedifference between the∆V-A isotherm ofCalix I andCalixII or Calix III is more pronounced compared to the differencebetweenCalix II andCalix III . As indicated by the∆V values,Calix I is oriented more vertically relative to the water surfacecompared toCalix II andCalix III . In the case ofCalix I thesurface potential increases smoothly with the decreasing mono-layer area. The∆V-A isotherm shows a liquid expanded phaseeven for the highest measured molecular area of 468 Å2

molecule-1 (data not shown). In the case ofCalix II , ∆V isclose to zero for the areas larger than 240 Å2 molecule-1

showing that the monolayer is in a gas phase. Upon compres-sion, ∆V sharply increases at 240 Å2 molecule-1 indicating areorientation of the molecules and an LE-G phase transition.The highest value of∆V corresponds to the maximum packingof molecules in the monolayer (∆Vmax values are reported inTable 2).Calix III , having one additional methylene unit inthe linker, is in a liquid expanded phase at the molecular areasat which gas phase is observed withCalix II , and its orientationrelative to the water surface is more vertical.

On the basis of the∆V values, the effective dipole momentof molecules at the interface,µ⊥, was calculated. As followsfrom the Helmholtz equation

whereA is the area per molecule,ε0 is the vacuum permittivity,and ε is the relative permittivity of the monolayer, which isassumed to be 1.52

whereµR represents the change in the effective dipole momentin the hydrated polar group region, including the effective dipolemoment of the hydrophilic group and changes in the dipolemoment due to the reorientation of the water dipoles, whereasµω corresponds to the hydrophobic region of the monolayer.53,54

The plotsµ⊥-A are presented in Figure 5B.The calixarenes studied here have an identical hydrophobic

calixarene crown. The contribution to the total effective dipolemoment of the hydrophobic part being identical for all threemolecules, the differences between the effective dipole momentsof Calix I , Calix II , andCalix III reflect different contributionsof the polar head groups and the associated water molecules.The maximum dipole moments (Table 2) indicate reducing ofthe tilt angle of the head group in the orderCalix II , Calix III ,Calix I . It can be noticed that in the case ofCalix II andCalixIII the slopes of the surface potential-area (Figure 5A) andsurface dipole moment-area (Figure 5B) isotherms, as well asthe∆Vmax andµ⊥ values (Table 2) are similar. We propose thatthe hydrogen bonding to the penicillin heteroatoms may beresponsible for structuring a water layer around the polar sidechains; the water layer contribution would have an equalizingeffect on the surface potential results, masking the smallstructural difference betweenCalix II andCalix III .

Phospholipid/Calixarene Binary Mixtures. The surfacepressure and surface potential-area isotherms of mixed DMPE/calixarene monolayers at the air-water interface are shown inFigures 6-8 for DMPE/Calix I , DMPE/Calix II , and DMPE/Calix III , respectively. The characteristic parameters of theisotherms are given in Table 3.

The DMPE monolayer shows a typical first-order transitionfrom the liquid expanded to liquid condensed phase (LE-LC)at around 5 mN m-1 and 0.330 V.55 The isotherms of the mixedfilms are situated between those corresponding to the puremonolayers. The decrease of the calixarene molar fraction resultsin a shift of the isotherm toward smaller molecular areas. Theonly exception is theΠ-A isotherm corresponding to the highestcalixarene content in DMPE/Calix I system, which is shiftedto higher molecular areas compared to that of the pure calixarenemonolayer. This effect may be due to the disrupting of theCalixI network by DMPE.

The profiles of the isotherms corresponding to the mixed films(Figures 6-8) as well as compressibility modulus (Cs

-1) values(Table 3) indicate that the properties of the mixed systems aredominated by the calixarene derivatives (for pure DMPE

Figure 8. Π-A and∆V-A isotherms of binary mixtures DMPE/CalixIII spread on water at 20°C. Solid lines correspond to pure components;dashed lines,xDMPE ) 0.75; dotted lines,xDMPE ) 0.50; dashed-dottedlines,xDMPE ) 0.25.

TABLE 3: Characteristic Parameters of DMPE/Calixarene Isotherms

xDMPE

Πcoll

(mN m-1)Acoll

(Å2 molecule-1)Cs

-1

(mN m-1)A0

(Å2 molecule-1)∆Vmax

(V)µeff

(D)

Calix I 0.25 35.9 118 88.3 179 0.764 2.400.50 35.7 101 86.4 153 0.741 1.990.75 35.5 80 226 106 0.677 1.44

Calix II 0.25 38.6 114 127 156 0.455 1.370.50 39.0 88 147 118 0.462 1.080.75 38.9 62 194 80 0.492 0.81

Calix III 0.25 37.2 102 78.3 169 0.540 1.470.50 37.0 80 82.0 129 0.539 1.150.75 36.8 60 107 91 0.536 0.86

∆V ) µ⊥/ε0εA

µ⊥ ) µR + µω

13238 J. Phys. Chem. B, Vol. 111, No. 46, 2007 Korchowiec et al.

Cs-1 ) 873.3 mN m-1). The fact that the collapse pressure for

all the mixed monolayers is close to that of the correspondingcalixarene might suggest phase separation in the two-componentfilms.56-58 However, the surface potential at the collapse (∆Vmax)gradually evolves from the value corresponding to the purecalixarene film to that of the pure DMPE (Table 3). It has to benoted that surface potential is usually considered as a moresensitive tool for providing information about the orientationand organization of the molecules in monolayers compared tosurface pressure. Consequently, on the basis of the surfacepotential measurements, it may be supposed that the calixarenesstudied are miscible with DMPE. Moreover, all mixed mono-layers undergo a phase transition, which occurs at the same

molecular areas with both types of isotherms. The increase ofthe phase transition surface pressure and surface potential withthe increasing calixarene molar fraction could be considered asa supplementary indication of the surface miscibility of the twocomponents.

To clarify the miscibility properties of the three calixarenederivatives, mean molecular area (MMA) and∆V dependenciesversus molar fraction of DMPE (xDMPE) were plotted forarbitrarily chosen surface pressures (10, 15, and 20 mN m-1)(Figure 9). Positive deviations from linearity in the MMA ofmixed films are observed for all three derivatives and surfacepressures investigated (Figure 9, parts A, C, and E). Thesedeviations, which imply polar or steric interactions in the mixed

Figure 9. Miscibility analysis of DMPE/Calix I (A and B), DMPE/Calix II (C and D), and DMPE/Calix III (E and F) monolayers at 10 (dottedlines), 15 (dashed lines), and 20 mN m-1 (solid lines). The straight lines represent the additive mixing.

Calixarenes in a Membrane Environment J. Phys. Chem. B, Vol. 111, No. 46, 200713239

films, are low in the case ofCalix II andCalix III , and increasein the orderCalix II , Calix III , andCalix I . This observationis in accordance with the results of modeling, showing that theantibiotic substituents inCalix I are hydrogen bonded via theamide linkers beneath the calixarene crown, compared tohydrogen bonding viaâ-lactam rings inCalix II andCalix III .A higher conformational liberty of the substituent extremities

in Calix I would make theCalix I and DMPE packing loosercompared to the derivativesCalix II andCalix III .

∆V-xDMPE dependencies show as well that the miscibilitybehavior ofCalix I is different compared to that ofCalix IIandCalix III . The negative excess∆V (∆V ex) observed withCalix I for xDMPE ) 0.00-0.25 indicates that the ordering ofthe Calix I molecules decreases in the presence of DMPE;inversely, the positive∆V ex observed at 15 and 20 mN m-1

for xDMPE ) 0.75-1.00 indicates thatCalix I has an orderingeffect on DMPE at these surface pressures; at 10 mN m-1 theordering effect is observed only forxDMPE ) 0.50. In the caseof Calix II /DMPE andCalix III /DMPE, the negative∆V ex

indicates a decreased ordering of the molecules forming the filmin the whole range of compositions, compared to pure sub-stances.

From another point of view,Calix I , contrary toCalix IIandCalix III , contributes to a more vertical average orientationof the dipoles in the mixed films; along this line, dipoles areoriented more vertically in the mixed films containingCalixIII , compared toCalix II ; obviously, the longer linker inCalixIII allows for an easier ordering of molecules in the mixed filmcompared toCalix II . These observations may be relevant forfurther studies on the biological activity of the three calixarenederivatives.

The miscibility of the three calixarenes with phospholipidmonolayers can be analyzed59 in terms of Gibbs energy of

Figure 10. Gibbs energy of mixing as function ofxDMPE, calculated atΠ ) 20 mN m-1. Solid line, DMPE/Calix I ; dotted line, DMPE/CalixII ; dashed line, DMPE/Calix III .

Figure 11. BAM images of calixarene, DMPE, and DMPE/calixarene monolayers. Line A-C: pureCalix II ; line D-F: pure DMPE; line G-I:mixed films. (A) Π ∼ 0 mN m-1; A ) 240 Å2 molecule-1; (B) Π ) 6 mN m-1, A ) 185.6 Å2 molecule-1; (C) collapse point; (D)Π ∼ 0 mN m-1,A ) 90 Å2 molecule-1; (E) Π ) 6 mN m-1; A ) 57.8 Å2 molecule-1; (F) collapse point; (G) DMPE/Calix I ; (H) DMPE/Calix II ; (I) DMPE/CalixIII . The G, H, and I images were taken at 12, 13, and 14 mN m-1 and 110.9, 82.5, and 87.5 Å2 molecule-1, respectively. The composition of allthe mixed films wasxDMPE ) 0.75. Scale: the width of the snapshots corresponds to 400µm.

13240 J. Phys. Chem. B, Vol. 111, No. 46, 2007 Korchowiec et al.

mixing, ∆Gex, expressed for the two-component system asfollows:

whereA12 is the mean molecular area in the mixture,A1 andA2

are the mean molecular areas of the pure components 1 and 2,andx1 andx2 are the molar fractions of the components in themixed film. The Gibbs energy of mixing for all investigatedsystems, obtained from the integration of theΠ-A isothermsfrom 0 to 20 mN m-1, is shown in Figure 10.

It can be seen that the energy of the formation of the mixedfilms, ∆G ex, is higher compared to pure films. Assuming thatthe enthalpy of film formation is comparable forCalix II andCalix III due to their structural similarities, the differences inthe∆G ex values can be attributed to entropic effects. Therefore,the DMPE/Calix III films, having a higher∆G ex, would formmore ordered systems than those formed with DMPE/Calix II .These results are in accordance with those obtained with∆V-Aisotherms (Figures 6-9). This reasoning cannot be used in thecase of structurally differentCalix I .

A BAM study was carried out to obtain complementaryinformation about the monolayer properties (Figure 11). Theimages of theCalix I and Calix III monolayers are notpresented, as these monolayers are isotropic all along compres-sions. In the pureCalix II monolayer formation of a foam isobserved at the molecular area of 240 Å2 molecule-1 and surfacepressures close to zero (Figure 11A);37 at this point LE-G phasetransition is observed in the corresponding∆V-A isotherms.At higher surface pressures this monolayer is isotropic (Figure11, parts B and C), which is in accordance with theΠ-A and∆V-A isotherm characteristics. BAM images of the pure DMPEmonolayer show formation of a foam in the LE-G phasetransition (Figure 11D) and large condensed domains in the LE-LC phase transition (Figure 11E). Upon compression the DMPEmonolayer becomes isotropic and breaks down at the collapsepoint (Figure 11F).

All mixed films go, at different surface pressures, throughan LE-LC phase transition manifested in BAM images bydomain structure. The BAM images corresponding to the LE-LC region in the mixedxDMPE ) 0.75 monolayers are presentedin Figure 11G-I. The shape of the domains resembles that ofthe pure lipid only in the case of the DMPE/Calix I film (Figure11G). The domains in the DMPE/Calix III and DMPE/CalixII mixtures (Figure 11, parts H and I, respectively) aresignificantly smaller compared to the pure DMPE. These resultssuggest that the mechanism of the LE-LC phase transition isdifferent in the DMPE/Calix I compared to the DMPE/CalixII and DMPE/Calix III films. The formation of small, numerousdomains in the latter two mixtures may be attributed to thedisorganizing effect of these calixarene derivatives observedwith the ∆V-A isotherms.

4. Conclusions

The results obtained show that the miscibility properties ofthe 6-pivAPA derivative,Calix I , are different compared to thetwo benzylpenicillin derivatives,Calix II andCalix III . Thiseffect is obviously linked to the aliphatic pivaloyl terminalmoieties present inCalix I substituents, which decrease themolecule packing in the mixed films compared to the benzylmoieties present inCalix II and Calix III; the decreasedmolecule packing in the case ofCalix I is accompanied by anincreased ordering of the DMPE molecules in the lipid-richphase. On the other hand, the higher∆G ex obtained withCalix

III compared toCalix II indicates that a higher conformationalflexibility of the former facilitates the interactions with DMPE.The results obtained suggest that the incorporation ofCalix IIand Calix III into biological membranes can be expected,whereasCalix I could be more easily translocated across themembrane compared to the benzylpenicillin derivatives. A studyof the biological effects of the calixarene derivatives in lipidmembranes is underway in our laboratory.

Acknowledgment. The Ministere de la Recherche et del’Enseignement Supe´rieur and the Centre National de laRecherche Scientifique are acknowledged for their financialsupport. A.B.S. acknowledges the Ministe`re de l’EnseignementSuperieur de Tunisie for a 6 month Ph.D. fellowship. Part ofthe calculation was performed at CYFRONET (MNiSW/SGI3700/UJ/161/2006 and MNiSW/SGI4700/UJ/061/2007). Wethank Dr. J.-M. Ziegler for mass spectroscopy, Ms. E. Eppigerfor NMR measurements, and Jean-Louis Vaucher and AlexisMartin from Service Technique de l’UHP for technical as-sistance. We thank Ms. N. Marshall for proof-reading themanuscript.

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