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Journal of Colloid and Interface Science 283 (2005) 555–564www.elsevier.com/locate/jcis

Aggregation behaviors of gemini nucleotide at the air–water interfaand in solutions induced by adenine–uracil interaction

Yujie Wanga, Bernard Desbatb, Sabine Maneta, Carole Aiméa, Thomas Labrota, Reiko Odaa,∗

a Institut Européen de Chimie et Biologie, 2 rue Robert Escarpit, 33607 Pessac Cedex, Franceb Laboratoire de Physico-Chimie Moléculaire, 351 cours de la libération, 33405 Talence Cedex, France

Received 27 May 2004; accepted 2 September 2004

Available online 9 December 2004

Abstract

Cationic gemini surfactants having nucleotides as counterions (called nucleo-gemini hereafter) were synthesized and their abehavior at air–water surfaces as well as in bulk solutions were studied. Fluid solutions of these nucleo-gemini surfactants show trahydrogels upon addition of complementary nucleoside bases or other nucleo-gemini surfactants having complementary bases asThe FTIR-ATR measurements show that the carboxylate groups of uridine form hydrogen bonds with the amine groups of adenoaggregation behavior was also confirmed at the air–water interface by Brewster angle microscopy as well as surface pressure methe monolayer of a gemini nucleotide was observed to undergo a transition to multilayers when nucleosides with complementary badded into the subphase. Isotherm curves of surface pressure monitored in parallel show a decrease in molecular area upon addnucleosides. 2004 Elsevier Inc. All rights reserved.

Keywords: Hydrogels; Molecular recognition at air–water interface; Gemini surfactants

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1. Introduction

Molecular recognition is an essential process for the cstruction of biological assemblies; the double helical strture of DNA provides the ultimate example. Such subtuned molecular recognition tools have attracted attenfor their potential use in materials science, for examplebiosensors. However, the elementary constituents ofbiological polymers, mononucleotides, do not show bpairing recognition in solution, since this particular reconition is driven by hydrogen bonds, and in aqueous soluspecific intermolecular interactions are in competition wnonspecific hydrogen bonds with water molecules[1]. For-mation of an isolated base pair is therefore energeticunfavorable. Cooperative processes where the pairingmation is influenced by neighboring constituents are

* Corresponding author. Fax: +33(0)540-00-30-66.E-mail address: r.oda@iecb.u-bordeaux.fr(R. Oda).

0021-9797/$ – see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.jcis.2004.09.003

portant. Pörschke has shown that nucleotides have toleast tetramers in order to exhibit recognition behaviosolution between complementary strands[2]. Extensive syn-thetic procedures are required to obtain very small amoof such materials for materials science applications. Altetive systems to covalently bonded oligonucleotides arecleotides which interact with each other via noncovalentteractions. The use of nucleo-amphiphiles has been propin such a context. The objective is to confine nucleo-basethe surfaces of amphiphilic aggregates to give effects simto those of covalent bonds between nucleotides[3].

To investigate molecular recognition between compmentary bases of nucleo-lipids, the most common conurations are at interfaces, such as spectroscopic studimonolayer isotherm measurements at the air–water inface[4,5], water–solid interface[6], or Langmuir–Blodgettfilms [7]. Complex formation between complementary bacan increase (or decrease) the molecular area at thewater interface, while the formation of hydrogen bondstween the nucleobases can be detected by FTIR. The su

556 Y. Wang et al. / Journal of Colloid and Interface Science 283 (2005) 555–564

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force apparatus[8], and mass spectrometry[6,9] have alsobeen used to measure the interaction between aggreof nucleo-lipids with complementary bases. The interacbetween supramolecular aggregates such as vesicles ocelles with nucleo-lipids have been investigated[10–13].These aggregates can provide the appropriate environfor internucleobase recognition, and the molecular organtion can be perturbed locally inducing variations in specscopic signals.

Almost all of these systems use nucleolipids wherenucleobases are directly connected to amphiphilic molecby covalent bonds. Herein we report a family of nucleamphiphiles formed by the complexation of cationic sfactants and anionic nucleotides, uracil 5′-monophosphat(U5′MP) or adenine 5′-monophosphate (A5′MP). Theirbehavior in bulk solution as well as at the air–waterterface was investigated with and without complemenbases. As cationic surfactants, dimeric (gemini) surfactcomposed of two conventional single-tail surfactants cnected covalently at their head groups were used. Tsurfactants show quite unusual phase behaviors andrecently motivated numerous physicochemical and biopical studies (for reviews see[14,15]). The most studiedclass of cationic gemini amphiphiles are bis-quaternammonium surfactants having the formula CsH2s-α,ω-((CH3)2N+CmH2m + 1Br−)((CH3)2N+CmH2m + 1Br−) de-noted withm-s-m notation[16]. For this group of surfactants, the effects of the length and the nature of the spchain on the critical micelle concentration (cmc) their mocular areas, their behavior at the air–water interface, andmorphology of their aggregates have been reported[17–21].The effects of various counterions[22–24], and the physicochemical properties of the equivalent trimers and tetramhave also been reported[25].

For the present study, we have chosen gemini surfacwith an ethylene spacer (n-2-m) [26,27]. Cationic surfac-tants with various chain lengths were complexed withcleic acids by ion exchange as shown inFig. 1. The condi-tions were chosen so that two nucleic acids form a comwith one gemini molecule.

The reasons for choosing such molecules were aslows. First of all, it is easier to obtain nucleo-amphiphilwith different bases by cation–anion complexation asposed to synthesizing new molecules each time. This is qimportant when one wants to compare the properties ofious base pairs. Furthermore, it allows us to easily oblarge amounts of sample, which is essential for performstructural studies on bulk samples since our objectivestudy the effect of recognition mechanisms on the aggation morphology in the bulk. Second, gemini molecuhaving an-2-m structure have a much higher packing pameter, as defined by Israelachvili[28], as compared to themonomeric counterparts, especially when both hydropbic chains have the same length (n = m) [27]. Therefore,these molecules form aggregates with low interfacial cvature (cylindrical micelles, bilayers, etc.) at very low co

s

i-

t

e

r

Fig. 1. Gemini nucleotide (for this figure, the counterions are U5′MP) n-2-n2U5′MP.

centrations (∼mM), which is interesting from the point oview of structural studies. Also, by forming a complextwo nucleotides per gemini molecule, one can improveplane internucleotide interactions. Finally, in order to mimbase pair and stacking interactions, it is favorable to hthe internucleotide distance close to that found in D(3.4 Å); the smallest distance between ammonium catto give a stable gemini molecule is∼4 Å (N–N distanceis ∼3.5 Å, but cationic charges are distributed among fmethyl groups) when an ethylene spacer is used. Inpresent paper, we report the transition of solution todrogels of nucleo-gemini surfactant induced by complemtary base pairing using electron microscopy and FTIR-ATheir interactions were followed in parallel at the air–wainterface by Brewster angle microscopy (BAM) and surfpressure measurements.

2. Materials and methods

2.1. Synthesis

Gemini molecules having various hydrocarbon chlengths were synthesized as previously reported[27]. Oncesynthesized, the bromide counterions were exchangednucleotides. We have observed that in order to obtainproducible results, it was very important to have goodichiometry between cations and anions, and the bestexchange method depends on the type of anion. For pphate ions, we found that the best method was to exchbromide ions with acetate, then couple these gemini acewith nucleic acids[29].

2.2. Gemini (n-2-n) acetates

n-2-n 2Br (2 g) was dissolved in a mixture of wat(200 ml) and MeOH (100 ml) at 60◦C. Ag acetate (ACROS

Y. Wang et al. / Journal of Colloid and Interface Science 283 (2005) 555–564 557

on.res

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Organics) (2.2 mol. equiv.) was added into the solutiThe flask is covered with aluminum foil and the mixtuis stirred (10 min 60◦C). After evaporation, MeOH waadded to separate then-2-n 2Ac (soluble in MeOH) andAgBr and excess AgAc (precipitate) (2 h 10◦C). After fil-tration, the solution is evaporated, and the same procewas repeated in water then lyophilized yielding then-2-n2Ac (90%).

2.3. Gemini (n-2-n) nucleotides

Dissolve 2 equivalents of 5′ nucleotides, e.g., SIGMAU5′MP 120.53 mg, andn-2-n 2Ac, e.g., 22-2-22,2Ac150 mg, in water (20 ml 45◦C). Consecutive lyophilizationand dissolution in water were repeated until total evaption of acetic acid took place (controlled by1H NMR).

2.4. Surface pressure measurements

The surface pressure measurements were performedcomputer-controlled Langmuir film balance (Nima Technogy, Coventry, UK). Two rectangular Teflon troughs (1and 350 cm2) were used. The troughs were filled with utrapure water (Milli-Q, Millipore, 18.2 M� cm) andT =25± 2◦C. The surface pressure (Π ) was measured by thWilhelmy method using a filter paper plate. The comprsion rate was 5 cm2 min−1.

Amphiphiles were solubilized in a mixture of chloroforand methanol at various ratios (8:2 to 10:0) dependingthe solubility of the molecules and spread at the air–winterface.

2.5. Brewster angle microscopy

The morphology of gemini-nucleotide layers at the awater interface was observed using a Brewster angle mscope (NFT BAM2plus, Göttingen) mounted on the TefloLangmuir trough. The microscope was equipped withfrequency-doubled Nd:Yag laser (532 nm, 20 mW), poizer, analyzer, and CCD camera. The spatial resolution oBAM was about 2 µm and the image size was 625×500 µm.The reflectivity calibration obtained from the gray level athe layer thickness estimate, using the refractive indnwater= 1.33 and the monolayernmonolayer= 1.49, were cal-culated with the BAM2plus software package (I-Elli2000(seeFig. 2).

2.6. FTIR-ATR spectroscopy measurements

The FTIR-ATR spectra were recorded on a Nicolet 5spectrometer equipped with a HgCdTe detector coole77 K. Generally, 200 or 300 scans were added at aolution of 4 cm−1. The spectra were recorded withGolden Gate single-reflection ATR cell, Specac, OrpingtUK.

s

a

Fig. 2. Layer thickness as a function of reflectivity.

Fig. 3. Freeze fracture TEM image of a gel of 20-2-20 2U5′MP at 50 mM.

2.7. Freeze-fracture electron microscopy

Freeze-fracture experiments were performed with a Bers BAF 300 vacuum chamber (Balzers, LiechtensteA small droplet of mixture was sandwiched between tcopper specimen holders. The sandwich was then frowith liquid propane cooled with liquid nitrogen. The frozesandwich was additionally fixed to a transport unit underuid nitrogen and transferred to the fracture replication stin a chamber that was then pumped down to 10−6 mbarat −120◦C. Immediately after fracturing, replication tooplace by first shadowing with platinum/carbon at 45◦ andthen with carbon deposition at 90◦. The sample was alloweto warm to room temperature. Replicas were retrievedcleaned in water and mounted on 200-mesh copper gObservations were made with a cryo-electron microscFEI EM120 (120 kV), and the images were recorded ondak SO163 films and developed using standard procedseeFig. 3for such an image.

558 Y. Wang et al. / Journal of Colloid and Interface Science 283 (2005) 555–564

Table 1Solution behaviors at 10 mM (unless specified) of gemini surfactants of various hydrophobic chain lengths with A5′MP or U5′MP as counterions at 22◦C

14-2-14 16-2-16 18-2-18 20-2-20 22-2-22

U5′MP Solution(TK < 5◦C)

Solution(TK < 5◦C)

Solution(TK = 7◦C)

Solutiona

gel at 70 mM(TK = 28◦C)

Solutiona

gel at 20 mM(TK = 45◦C)

A5′MP Solution/precipitate(TK = 27◦C)

Precipitate/gelb

(TK = 45◦C)Precipitate/gelb

(TK = 52◦C)Precipitate(TK = 57◦C)

Precipitate(TK = 63◦C)

a Once heated aboveTK, those are apparently fluid and transparent solutions; neither precipitation nor gel is observed.b 16-2-16 A5′MP forms a gel when cooled rapidly, whereas it precipitates when cooled slowly.

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3. Results and discussion

3.1. Gemini nucleotides

Table 1shows the Krafft temperature (TK), as well assolution behaviors at 10 mM of gemini nucleotide with hdrophobic chain lengths between C14 and C22. Withuracil 5′ monophosphate (U5′MP) counterion, all the molecules formed a fluid solution at 10 mM upon heating abTK. When the solutions were cooled down to room tempature (controlled at 22◦C), beyond a certain concentratiowhich varied with the chain length, they formed transparhydrogels[30] within a few days, 22-2-22 2U5′MP formeda gel at 20 mM and 20-2-20 2U5′MP at 70 mM. The freezefracture image of a gel of 20-2-20 2U5′MP at 50 mM showsan entangled network of fibrillar structures. The fibers doshow apparent internal structure, and are∼12 nm in diam-eter, which is about four times the molecular length inextended form suggesting that it is not simply a cylindrimicellar structure.

Here, it is interesting to note that since theTK of 20-2-202U5′MP as well as 22-2-22 2U5′MP are beyond the roomtemperature, if these solutions have not been heated aTK, they will not be solubilized, and will remain preciptated. On the other hand, once they are solubilized abTK, and as long as they are below the gelling concentratshown in the table (for example at 10 mM), neither precitation nor gel formation was observed for several moneven though they were underTK; i.e., these solutions wernot at equilibrium—their equilibrium state is to be precitated. And after a long time, as long as 3 months, somthe solutions became gels. Such an extremely slow kinof the nonequilibrium state in the solution state is rarelyserved. Detailed studies on the kinetics of these systemwell as structural studies on these fibers are being perfor

With the adenine 5′monophosphate (A5′MP) counterion,the 14-2-14 2A5′MP forms precipitates below and fluid mcellar solutions above 27◦C. 16-2-16 2A5′MP and 18-2-182A5′MP show history-dependent behavior; i.e., when twere cooled rapidly in a refrigerator, gel formation wasserved, but when the solution was cooled slowly at rotemperature, precipitation was observed. Here, it is imtant to note that physical gels (with a few exceptions sucworm-like micellar solutions) are in general a thermodynaically metastable state; that is, the gel is an arrested sta

e

s.

n

the way of precipitation. Most often they are observed bethe Krafft temperature, and such history-dependent behais commonly observed[31]. Gemini nucleotides with longechains, 20-2-20, and 22-2-22 2A5′MP also resulted in precipitate formation. The difference in aggregation behavbetween gemini-U5′MP and gemini-A5′MP stems from thehydrophobicity of the counterions.

In order to evaluate the effect of complementary batwo types of studies were performed: (1) mixture of nuclgemini n-2-n 2U5′MP with nucleoside adenosine orn-2-n2A5′MP with nucleoside uridine; (2) mixture of two nuclegemini is carrying U5′MP and A5′MP as counterions.

3.2. Gemini nucleotide + ribonucleoside

To avoid electrostatic interactions between cationicphiphiles and the added nucleotides, the ribose form onucleoside was chosen because of its charge neutrality.thermore, ribose is more soluble than the base alone.

We added adenosine (SIGMA) and uridine (SIGMA)10 mM solutions of 18-2-18, 20-2-20, and 22-2-22 2U5′MP.Separately, all three of these amphiphiles form fluid sotions at this concentration. Upon addition of adenosinconcentrations above 10 mM, solutions of 20-2-20 2U5′MPand 22-2-22 2U5′MP formed gels within A few hours tofew days, whereas addition of uridine up to 100 mM didinfluence the fluid aspect of the solution. SeeFig. 4.

For the system of 22-2-22 2U5′MP (10 mM)+adenosinemore detailed studies on the phase behavior as a funof the adenosine concentration were performed. Whenadenosine concentration was below 5 mM, the solutionfluid for weeks at room temperature (controlled at 22◦C)as well as in the refrigerator (5◦C). As the concentration oadenosine increased, at 5 mM, the solutions became tparent gels at 5◦C after 2 days, but remained fluid at rootemperature. When the concentration of adenosine watween 10 and 20 mM, the solutions formed transparentafter one or 2 days even at room temperature. Interestiin this range of concentrations, the melting temperaturthe gels remained constant as the adenosine concentwas increased (e.g., 45◦C). As the concentration of adensine was further increased to 30 mM, the gel became opat 5◦C, and at room temperature, it became a viscousagain. At higher adenosine concentrations (>40 mM), thesolution became fluid again with the presence of precipit

Y. Wang et al. / Journal of Colloid and Interface Science 283 (2005) 555–564 559

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Fig. 4. Solution of 20-2-20 2U5′MP at 10 mM. Upon addition of 60 mMuridine the solution is still fluid, whereas upon addition of 10 mM adesine, the solution gels.

Since both gemini-UMP and adenosine are soluble alonthe investigated range of concentration, such gel formabehavior can be understood as a result of variation in inmolecular interactions upon addition of adenosine.

Cytidine and guanosine were also added to the 10solution of 22-2-22 2U5′MP. For cytidine, similar gel for-mations were observed, although at different concentions, whereas with guanosine, only precipitation wasserved[29]. Such behavior of cytidine can be explainedthe tendency of monomeric nucleotides to form noncommentary base pairs by base stacking or hydrogen bondsto their higher conformational freedom[1,29].

3.3. FTIR-ATR measurements

This phase transition behavior may also be explaisimply by the higher hydrophobicity of adenine bases,therefore their preference for being at the aggregate in

e

Fig. 5. FTIR-ATR spectra of 22-2-22 2U5′MP solution, adenosine solutionas well as 22-2-22 2U5′MP+adenosine gel in the region of stretching banof C=O groups of uracil bases and CNC stretching bands of purine ba

face, or by their preference for forming stacks at thegregate interface. FTIR-ATR measurements on the soluas well as the gel were performed in order to monitorinteraction between uracil base and adenosine. Since thbration peaks of nucleobases, which are implicated ininteraction, are found at around 1600 cm−1, solutions inD2O were prepared in order to avoid the big absorptpeak of H2O after examination of whether the gelling bhavior in the two solvents are comparable.Fig. 5 shows thesymmetrical and antisymmetrical stretching bands resufrom the coupling between the two C=O of uracil basesat 1695 and 1655 cm−1, respectively, as well as the CNstretching bands of the purine base of adenosine at 16241577 cm−1. Table 2summarizes the shift in the positionof these peaks of 22-2-22 2U5′MP solution, adenosine solution, and 22-2-22 2U5′MP + adenosine gel. Among thtwo peaks corresponding to the stretching bands of theC=O of uracil bases, the one at 1655 cm−1 shifts towardhigher wavenumber, whereas the other at 1695 cm−1 shiftstoward lower wave number, for the gel. The stretching bof the purine base of adenosine at 1624 cm−1 shifts towardlower wavenumber, whereas the peak at 1577 cm−1 shifts to-ward higher wave number. The stretching band of NH (in D2O) of both bases is buried in the signal of H2O (D2O)and could not be observed. Interestingly, the bending banND2 of adenosine in D2O showed very a similar peak postion for the 22-2-22 2U5′MP+ adenosine solution when thgel was heated above melting temperature (45◦C), but wasobserved to shift toward higher wavenumber upon formaof gel (Fig. 6).

The ensemble of these shifts suggest that the NH2 (ND2)and C=O of adenine and uracil form hydrogen bonds ingel. The small shift in peak positions at around 1600 cm−1

indicates that these hydrogen bonds are quite weak, w

560 Y. Wang et al. / Journal of Colloid and Interface Science 283 (2005) 555–564

Table 2The shift in peak positions of C=O stretching (uracil) and purine stretching bands (adenine)

Peak position Stretching bands of purine base νantisym(C=O) νsym (C=O)

22-2-22 2U5′MP 1654.7 cm−1 1696.3 cm−1

22-2-22 2U5′MP+ adenosine (gel) 1622.8 cm−1 1579.0 cm−1 1655.7 cm−1 1693.6 cm−1

Adenosine 1624.6 cm−1 1577.5 cm−1

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Fig. 6. FTIR-ATR spectra of 22-2-22 2U5′MP solution, adenosine solution22-2-22 2U5′MP+adenosine melt solution at 45◦C, as well as gel at 20◦C,in D2O in the region of ND2 bending vibration.

can be easily broken upon increase of temperature resuthe melting of gel as shown inFig. 6.

3.4. Gemini 2U5′MP + gemini 2A5′MP

The effect of adding gemini (C14-C22) 2A5′MP to a10 mM solution of gemini (C18, 20, and 22) 2U5′MP at var-ious concentrations was subsequently investigated as sin Table 3.

The solution forms gels only for some cases: both chhave to be quite long (>C18). The longer the chains, widethe gel-forming concentration range. Also, when the dif

n

ence in hydrophobic chain length of two gemini is more thfour carbons, precipitate is observed without going throa gel phase. This observation can be compared to themeasurements of the fluid-to-gel transition of phospholmixtures by Mabrey and Stuntevant[32]. They had reportedthat when the difference in the chain lengths of two phphocholine lipids was less than a equal to four carbonsand 16 or 14 and 18), they observed complete miscibboth in gel and liquid crystalline states, while when the dference was six carbons (12 and 18), phase separationobserved in the gel phase. In the present case with gemnucleotides systems, the phase separation was observmixtures with the difference in carbon numbers greater t4 and gels were observed only for the cases where thegemini molecules had similar chain lengths. This suggethat the observed gels were formed with the two geminicleotides locally well miscible.

For mixtures where gel formation was observed, thesults were similar to those observed for the gemini 2U5′MP+adenosine. For example for the mixture 22-2-22 2U5′MPand 22-2-22 2A5′MP, at fixed concentration of 22-2-22U5′MP (10 mM) and at very low concentration of 22-2-2A5′MP (<1.7 mM), the solution was fluid. As the concetration of 22-2-22 2A5′MP was increased, a transparentwas formed, first only at 3◦C (at 1.7 mM), but then also aroom temperature (at 2.3 mM). The concentration rang22-2-22 2A5′MP for which gel was observed at room teperature was around 2.3–10 mM. With a further increof gemini 2A5′MP, the gel became opaque, and eventua precipitate formed and the mixture became fluid agA precipitate formed for all the systems when the concention of gemini 2A5′MP exceeded 10 mM.

For both cases, upon addition of adenosine or gem2A5′MP, the fluid solutions of gemini 2U5′MP became gelsThe gel with lower adenine concentration was transpar

)

arenthe-

Table 3Phase behavior (room temperature controlled at 22◦C) of the mixture of two nucleo-gemini surfactants

2A5′MP2U5′MP

14-2-14 16-2-16 18-2-18 20-2-20 22-2-22

18-2-18 Sol→pre Sol→pre Sol→gel→pre(3.8–4.5 mM)

20-2-20 Sol→pre Sol→pre Sol→gel→pre Sol→gel→pre Sol→gel→pre(1.7–2.0 mM) (3.0–4.0 mM) (2.0–10.0 mM

22-2-22 Sol→pre Sol→pre Sol→gel→pre Sol→gel→pre Sol→gel→pre(5.0–6.5 mM) (4.8–6.5 mM) (2.3–10 mM)

Note. The concentration of gemini A5′MP increases from left to right and the concentrations for which gel formation was observed are shown in the pses. Sol= solution, pre= precipitate.

Y. Wang et al. / Journal of Colloid and Interface Science 283 (2005) 555–564 561

amecip-inegelsngeltnts

-

hy,ith a

tureureace.o-

d asoro-

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,

oareem

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whereas with higher adenine concentration, the gel becopaque before the solution became fluid again with preitate formation. Interestingly, the ratio of uracil to adenbases for which the best (transparent and no precipitate)were observed was not always 1:1 but rather in the raof 3:1 to 1:1 [33]. It is interesting to compare this resuwith the results on the nucleolipid monolayer experimereported by Morisue et al.[34], which showed from UVabsorption data that the A·T·T base trimer with multiple hydrogen bonding is more stable than the A·T pair with doublehydrogen bonding. This trimer formation can explain win our case also, the most stable gels were obtained whigher ratio of uridine than adenosine.

3.5. Surface pressure and Brewster angle microscopestudies

The interaction between gemini-2U5′MP and ribonucle-oside as well as gemini-2U5′MP and gemini-2A5′MP werestudied at the air–water interface. In the case of the mixof two gemini surfactants, a solution of an equimolar mixtof chloroform/methanol was deposited at the water surfIn the case of gemini+ ribonucleoside, a uridine or adensine solution with 40–1000 times (5×10−6–1.25×10−4 M),the number of molecules in the monolayer was preparethe subphase solution; then the gemini-nucleotide chlform/methanol solution was deposited at the surface.

In Fig. 7, Π–A curves are shown for various gemini ncleotides, gemini nucleotide+ ribonucleoside.Fig. 7a shows20-2-20 2U5′MP (abbreviated as 20U), 20-2-20 2A5′MP(abbreviated as 20A), and a mixture of 20-2-20 2U5′MP and20-2-20 2A5′MP, as well as a mixture of 22-2-22 2U5′MP(22U) and 22-2-22 2A5′MP (22A).Fig. 7b shows 22U, 22Aand 22U+ adenosine in the subphase, 22A+ uridine in thesubphase, and 22U+ 22A. In the case of the mixture of twgemini surfactants, adjustments were made so that theper molecule represented the area of the sum of both gini molecules (measured surface ofx Å2 for y molecules of20U + y molecules of 20A givesx/2y Å2 per molecule).The BAM images were monitored simultaneously.

For both nucleotides, uracil and adenine, gemini nuctides alone show very similar compression curves if the gini is the same molecule; i.e., 22-2-22 2A5′MP and 22-2-222U5′MP or 20-2-20 2A5′MP and 20-2-20 2U5′MP showvery similar curves.

The C22 gemini showed a phase transition at apprmately 90 Å2, whereas with C20, no phase transition wobserved. BAM images inFig. 8show bright points as sooas the surface pressure starts to increase. The number aintensity of these bright points increase as the surface psure increases, however, they never form large aggregand remain distinct points. The reflectivity of the surfacecreases homogeneously with increasing surface pressuexpected for monolayer compression without aggregatemation. Using the graph inFig. 2, the thickness of film athe air–water interface was estimated. Interestingly, at s

a-

e-,

s

(a)

(b)

Fig. 7. Π–A curves of (a) 20U, 20A, 20U+ 20A, and 22U+ 22A, and(b) 22U, 22A, 22U+ A (concentration of adenosine in the subphase5 × 10−6 mM), 22A+ U (concentration of uridine is 5× 10−6 mM), and22A+ 22U.

ilar surface pressures, the monolayer thickness of 22-2U5′MP was much smaller than that of the 22-2-22 2A5′MPmonolayer; 8 Å for 22-2-22 2U5′MP (3 mN m−1) vs 13 Åfor 22-2-22 2A5′MP (6 mN m−1), 13 Å for 22-2-22 2U5′MP(22 mN m−1) vs 20 Å for 22-2-22 2A5′MP (18 mN m−1),18 Å for 22-2-22 2U5′MP (33 mN m−1) vs 31 Å for 22-2-22 2A5′MP (37 mN m−1). This difference cannot simply bexplained by the molecular size difference between adeand uracil. Rather, it is probably due to the difference inganization of the two bases.

Upon addition of complementary ribonucleoside to22-2-22 nucleotide monolayers, the spreading isotheshifted toward smaller areas. Both 22-2-22 2U5′MP +adenosine and 22-2-22 2A5′MP + uridine showed similaisotherms (Fig. 7b).

The monolayers formed from a mixture of gemini sfactants having uracil and adenine as counterions, 22-2A5′MP+22-2-22 2U5′MP and 20-2-20 2A5′MP+20-2-202U5′MP, showed a very similar shift toward smaller areexcept that the system with C20 did not have a transitwhile C22 did (seeFig. 6a).

562 Y. Wang et al. / Journal of Colloid and Interface Science 283 (2005) 555–564

r-

sionwere

s ofeac

dis-e

forere

pe-gategate

oundhout. Thaggr

rdAfteton-40t in

inshinge

(ar-11

mk-2-2-22

b-

llow

re.500 s

Fig. 8. BAM images of the 22-2-22 2U5′MP monolayer at various suface pressures: (a) 3 (R = 6.19 × 10−7); (b) 22 (R = 1.36 × 10−6);(c) 33 mN m−1 (R = 2.27× 10−6); and 22-2-22 2A5′MP monolayer atvarious surface pressures: (d) 6 (R = 1.41× 10−6); (e) 18 (R = 3.20×10−6); (f) 37 mN m−1 (R = 7.22× 10−6).

BAM images taken at various points of these comprescurves showed that very bright and large aggregatesformed at surface pressure as low as 5 mN m−1. SeeFig. 9.

From the intensity of these domains, the thicknessesuch aggregates were estimated at various regions ofimage. Roughly two or three different regions, could betinguished:!1 dark background region:!2 aggregated moror less large domains (presented by line arrows), andsome of the figures, very highly aggregated domains wpresent (highlighted with arrow heads). InFig. 10, the mono-layer thicknesses are shown as a function of the areamolecules for 22-2-22 2U5′MP (22U) as well as for 222-22 2U5′MP + adenosine background area and aggrearea. This clearly shows that with adenosine, the aggrearea is at least a few angstroms thicker and the backgrarea is a few angstroms thinner than the monolayer witadenosine. In parallel, the surface pressure decreasessuggests that adenosine induces segregation betweengated areas with higher packing of 22-2-22 2U5′MP, and thebackground areas with lower packing of 22-2-22 2U5′MP.

In order to confirm the shift of the isotherm curve towasmaller areas, another measurement was performed.the monolayer of 22-2-22 2U5′MP was compressed down51 Å2 (39 mN m−1), the adenosine solution (similar concetration (100 µl of 1 mM solution, which corresponds totimes the number of molecules on the monolayer) to tha

h

r

ise-

r

Fig. 9. BAM images of various gemini nucleotides with nucleosidesthe subphase. Layer thicknesses were estimated for two distinguiregions of the images:!1 background and!2 aggregated domains (linarrows), very highly aggregated domains were also often observedrow heads). 22-2-22 2U5′MP + adenosine at surface pressures of (a)mN m−1, !1 = 10 Å, !2 13 Å; (b) 29 mN m−1, !1 = 15 Å, !2 22 Å,adenine was added to the subphase before compression; (c) 24 mN−1,!1 = 21 Å, !2 > 120 Å (reflectivity was too high to estimate thicness), adenine was added to the subphase after compression. 22A5′MP+ uridine at various surface pressures: (d) 15 mN m−1, !1 = 14 Å,!2 17 Å; (e) 26 mN m−1, !1 = 17 Å; !2 30 Å, uridine was added to the suphase before compression; and (f) 26 mN m−1, !1 = 28 Å, !2 35 Å, uridinewas added to the subphase after compression. 22-2-22 2U5′MP + 22-2-222A5′MP: (g) 8 mN m−1, !1 = 13 Å, !2 20 Å; (h) 22 mN m−1, !1 = 16 Å,!2 20 Å; (i) 31 mN m−1, !1 = 19 Å, !2 23 Å.

the experiments above) was injected after an hour to athe surface pressure to equilibrate.

Fig. 11shows the time evolution of the surface pressuThe sudden decrease in pressure observed at around 3

Y. Wang et al. / Journal of Colloid and Interface Science 283 (2005) 555–564 563

. Th

ed

n

beatesnotupo

yrdsracimesno-

no-n didhen

thein-ly a

ses,re-theub-yas

dularob-

c-ayert theace.air–t theentsase

ityarecia

ag,

993)

ka,

ura,7.

ev.

m.

Fig. 10. Layer thickness and isotherm curves of the 22-2-22 2U5′MP mono-layer with and without adenosine in the subphase.

Fig. 11. Surface pressure of the 22-2-22 2U5′MP monolayer as a functionof time after the compression was stopped at 39 mN m−1. Adenosine wasinjected into the subphase at 3500 s.

corresponds to when the adenosine solution was addedrelaxation occurred in two steps.

BAM images after addition of uridine to a compress22-2-22 2A5′MP monolayer (at 26 mN m−1) as well asadenosine to a 22-2-22 2U5′MP monolayer (at 24 mN m−1)are shown inFigs. 9c and 9f. Again, aggregate formatiowas observed.

In both cases, when uridine or adenosine was addedfore or after the compression, formation of large aggregwere observed, while without them, aggregates wereobserved. The decrease in surface pressure observedaddition of adenosine inFig. 11, the images obtained bBAM, as well as the shift of the isotherm curves towalower surface pressures, indicate that the adenine–umixture leads to aggregation at the surface which consuthe amphiphiles that would otherwise make up the molayer.

e

-

n

l

4. Summary

The fluid solution of gemini-nucleotide 2U5′MP showedgelling behavior upon addition of complementary adesine bases, whereas uridine bases added to the solutionot induce gelation. The same behavior was observed wgemini-2U5′MP and gemini-2A5′MP were mixed. FT-IRmeasurements suggest that the NH2 and C=O of adenineand uracil form hydrogen bonds in the gel. However,peak shift is not very pronounced, indicating that theteraction is not very strong. The aggregation is probabcooperative process between the hydrogen bonds, theπ–π

stacking, and the hydrophobic character of adenine bapreferring to be at an amphiphilic interface. Such agggation behavior was also observed with monolayers atair–water interface. Upon addition of adenosine in the sphase, the molecular area of gemini-2U5′MP decreases bapproximately 20 Å2. A decrease in molecular area walso observed for the 1:1 mixture of gemini-2U5′MP andgemini-2A5′MP. Monolayer compressions were followeby BAM, and in the cases where a decrease in molecarea was observed, highly multilayered aggregates wereserved. The fact that the mixture of gemini-2U5′MP andgemini-2A5′MP formed a monolayer with smaller moleular area than either of them alone, and that the monolshows large aggregates observed by BAM, indicates thaadenine–uracil mixture leads to aggregation at the surfSuch aggregation behavior, both in the bulk and at thewater interface, is certainly partly due to base stacking ahydrophile–hydrophobe interface, but FTIR measuremclearly showed hydrogen bond formation, indicating bpair formation.

Acknowledgments

This work was supported by the CNRS, the Universof Bordeaux I, and the Aquitaine region. The authorsgrateful to I. Huc for many useful suggestions and L. Cucfor his help in improving the manuscript.

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