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Sep 6, 2013 - Linyi Zhu , Yongqiang Tang , Yilin Wang. Journal of ... Meina Wang , Chunxian Wu , Yongqiang Tang , Yaxun Fan , Yuchun Han , Yilin Wang...
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Complex Formation and Aggregate Transitions of Sodium Dodecyl Sulfate with an Oligomeric Connecting Molecule in Aqueous Solution Linyi Zhu, Yuchun Han, Maozhang Tian, and Yilin Wang* Key Laboratory of Colloid and Interface Science, Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China S Supporting Information *

ABSTRACT: Anionic single-tail surfactant sodium dodecyl sulfate (SDS) and a molecule with multiple amido and amine groups (Lys-12-Lys) were used as building blocks to fabricate oligomeric surfactants through intermolecular interactions. Their interactions and the resultant complex and aggregate structures were investigated by turbidity titration, isothermal titration microcalorimetry, dynamic light scattering, cryogenic transmission electron microscopy, freeze-fracture transmission electron microscopy, 1H NMR, and 1D NOE techniques. At pH 11.0, the interaction between SDS and Lys-12-Lys is exothermic and mainly resulted from hydrogen bonding among the amido and amine groups of Lys-12-Lys and the sulfate group of SDS and hydrophobic interaction between the hydrocarbon chains of SDS and Lys-12-Lys. At pH 3.0, each Lys-12-Lys carries four positive charges and two hydrogen bonding sites. Then SDS and Lys-12-Lys form complexes Lys12-Lys(SDS)6 and Lys-12-Lys(SDS)4 through the head groups by electrostatic attraction and hydrogen bonds assisted by hydrophobic interaction. Moreover, the complexes pack more tightly in their aggregates with the increase of the molar ratio. Especially the Lys-12-Lys(SDS)4 and Lys-12-Lys(SDS)6 complexes behave like oligomeric surfactants taking Lys-12-Lys as a spacer group, exhibiting a series of aggregates transitions with the increase of concentration, i.e., larger vesicles, smaller spherical micelles, and long threadlike micelles. Therefore, oligomeric surfactants Lys-12-Lys(SDS)4 and Lys-12-Lys(SDS)6 have been successfully fabricated by using a single chain surfactant and an oligomeric connecting molecule through noncovalent association.

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INTRODUCTION Surfactants have been widely applied in many fields from industries to daily life. To find more efficient surfactants is one of the main tasks in surfactant studies. Over the past 20 years, gemini surfactants have been extensively studied.1−4 They exhibit superior physicochemical properties and special performances than traditional single-chain surfactants, such as higher surface activity, lower critical micelle concentration, and especially much more affluent aggregate structures. Subsequently, higher oligomeric analogues were synthesized and investigated,5−16 and the early investigations have been well reviewed by Laschewsky17 and Zana.18 Their unique properties and aggregation behaviors have made them an attractive new member of surfactant field. Oligomeric surfactants are made of three or more identical or nearly identical amphiphilic moieties chemically connected by spacer groups.5 They can be classified as linear, ring type, and star shape according to the features of their spacer groups. Zana’s group6,9 synthesized linear trimeric and tetrameric quaternary ammonium surfactants and found that these surfactants formed branched threadlike micelles and closed-looped micelles, respectively. Yoshimura and Esumi19 reported that a series of ring-type trimeric surfactants from cyanuric chloride formed large aggregates in aqueous solution. Our group synthesized a series of star-shaped oligomeric © 2013 American Chemical Society

quaternary ammonium surfactants with amide-type spacer groups.20−22 They all incline to form large size aggregates before micelles. The star-shaped trimeric surfactants20 generate vesicles at lower concentration and change to spherical micelles at higher concentration. Both tetrameric21 and hexameric starshaped22 surfactants form network-like premicellar aggregates below their critical micelle concentration and then transfer into spherical micelles with the increase of the surfactant concentration through different processes. Although the oligomeric surfactants above present unique properties and self-assembly behaviors, the synthesis and purification are so complicated and difficult that their applications are limited. Therefore, it is necessary to think how to obtain the properties of the oligomeric surfactants without the restriction of synthesis. Applying noncovalent interactions is a possible convenient approach to construct desired oligomeric surfactants, which will undoubtedly promote the development and application of novel surfactants. Zhang and co-workers have made great advancement in the construction of supramolecular amphiReceived: April 27, 2013 Revised: August 28, 2013 Published: September 6, 2013 12084

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philes using noncovalent interactions and have contributed comprehensive reviews23−25 in this area. Utilizing noncovalent interactions including hydrogen bonding, metal−ligand coordination, host−guest recognition, and electrostatic attraction, amphiphiles with special architectures and functions can be more easily constructed. Because of the reversibility of noncovalent interactions, the self-assembly of the amphiphiles constructed by this approach can be conveniently controlled by adjusting the ratio of building blocks and environment conditions, such as pH, salt, light, and so on. In recent several years, novel surfactants have been built by using surfactants as building blocks through noncovalent interactions.26−28 Kabanov et al.26 have successfully achieved stable vesicles through the interaction of single-tail cationic surfactants with diblock copolymer of poly(ethylene oxide) or poly(sodium methacrylate). Zhang’s group27 constructed stimulus-responsive vesicles using an azobenzene-containing surfactant and α-CD as the building blocks through photocontrolled inclusion and exclusion. Our group28 constructed a gemini-type surfactant system by using a “gemini-type” organic salt 1,2-bis(2-benzylammoniumethoxy)ethane dichloride (BEO) and sodium dodecyl sulfate (SDS). The existence of (SDS)2−BEO gemini-type structure was confirmed by DSC and 1H NMR, and it exhibited the characteristics of gemini surfactants. However, so far any attempt to construct oligomeric surfactants by noncovalent interactions has not yet been reported. Some mixtures may form surfactant structures similar to oligomeric surfactants, but this was normally discussed from the aspect of intermolecular interactions rather than the formation of new surfactant structures. The purpose of the present work is to fabricate oligomeric surfactants with intermolecular interaction. We synthesized a oligomeric connecting molecule Lys-12-Lys (Figure 1) which

Article

EXPERIMENTAL SECTION

Materials. Sodium dodecyl sulfate (SDS) (>99%) was purchased from Aldrich. (2R,2′R)-N,N′-(Dodecane-1,12-diyl)bis(2,6-diaminohexanamide) (Lys-12-Lys) was designed by us and synthesized by GL Biochem (Shanghai) Ltd. The structure of Lys-12-Lys was confirmed by 1H NMR and mass spectra (Figure S1 in Supporting Information), and the purity is better than 99.5%, checked by highperformance liquid chromatography. The inorganic reagents (>99.5%) were purchased from Beijing Chemical Co. Milli-Q water (18.2 MΩ· cm) was used throughout. Potentiometric pH Titration. Lys-12-Lys was first dissolved in pure water at a concentration of 1.0 mM, and then the solution pH was adjusted to 3.5 with a small volume of 1.0 M HCl standard solution. Then 1.0 M NaOH standard solution was gradually added into this solution in small portions. The titration process was monitored with a pHS-2C acidity meter, and the temperature was kept at 34.0 ± 0.1 °C. The titration was repeated three times, and the mean pKa values were calculated. Isothermal Titration Microcalorimetry (ITC). A TAM 2277-201 isothermal titration microcalorimeter (Thermometric AB, Järfälla, Sweden) with a 1 mL stainless steel sample cell was used to measure the enthalpy change. The reference cell was loaded with pure water at pH 3.0 or 11.0. The sample cell was initially loaded with 600−800 μL of water or 10.0 mM SDS solution at pH 3.0 or 11.0, and then the concentrated Lys-12-Lys solution of 25.0 mM or the mixture of Lys12-Lys and SDS at different molar ratios and the corresponding pH was injected into the stirred sample cell in portions of 5−8 μL using a 500 μL Hamilton syringe controlled by a Thermometric 612 Lund pump. The system was stirred at 60 rpm with a gold propeller continuously until the desired concentration range had been covered. The interval between two injections was 7−9 min, which depends on the time for the signal to return to the baseline. The observed enthalpies (ΔHobs) were obtained by integrating the areas of the peaks in the plot of thermal power against time. The reproducibility of experiments was within ±4%. All the measurements were conducted at 34.00 ± 0.01 °C. Turbidity Measurements. The turbidity change of injecting concentrated Lys-12-Lys solution into the SDS solution, reported as 100 − %T, was measured at 450 nm using a Brinkman PC920 probe colorimeter. During the whole titration process, a thermostated water circulating bath was used to keep the experiment temperature at 34.0 ± 0.1 °C. The concentration of the SDS solution was the same as that used for ITC. After the values became stable (about 3−5 min), the final turbidity titration curves were recorded, and they were corrected by subtracting the turbidity curve from a Lys-12-Lys free titration. Because of the broad concentration range, parts of the turbidity measurements for the mixed solution of Lys-12-Lys/SDS at different molar ratios were carried out with a Shimadau 1601 PC UV/vis spectrometer. It was monitored by UV absorbance at 400 nm. A cuvette with 1 cm pathway was used. All the measurements were performed at 34.0 ± 0.5 °C. Dynamic Light Scattering (DLS). A Nano ZS (Malvern) equipped with a solid-state He−Ne laser (λ = 633 nm) was used to measure the hydrodynamic size of aggregates in the mixed solution of Lys-12-Lys/SDS. All of the measurements were performed at θ = 173°. The aggregate size was derived from a Cumulants analysis of the measured correlation curve while the aggregates were assumed as spheres. The measurements for each sample were repeated five times, and mean diameter values were calculated. The experiments were performed at 34.0 ± 0.1 °C. NMR. 1H NMR measurements were carried out on a Bruker AV400 FT-NMR spectrometer operating at 400.1 MHz. The stock solutions of the Lys-12-Lys/SDS mixture were prepared with deuterium oxide (99.9%) which was purchased from CIL Cambridge Isotope Laboratories. About 500 μL of each solution was transferred into a 5 mm NMR tube for the measurement. The center of the HDO signal (4.79 ppm) was used as the reference in the D2O solutions. In all NMR experiments, the number of scans was adjusted sufficiently enough to achieve good signal-to-noise ratios, and it was recorded with

Figure 1. Chemical structure of Lys-12-Lys.

has two lysine groups connected by a dodecyl spacer through amido bonds. At basic condition, it is uncharged. While in acidic solution, the amino groups are protonated; thus, each Lys-12-Lys molecule carries four positive charges. Besides, the presence of N−H dipoles allows the amides of Lys-12-Lys to work as H-bond donors to form H-bonds with H-bond acceptors. Therefore, at acidic condition Lys-12-Lys may have four or six binding sites with an anionic single-tail surfactant, which may create tetrameric or hexameric surfactants. Herein, sodium dodecyl sulfate (SDS) was chosen as the anionic singletail surfactant. Through investigating the interaction between Lys-12-Lys and SDS at various pHs, molar ratios, and concentrations, it has been proved that “oligomeric surfactants” Lys-12-Lys(SDS)4 and Lys-12-Lys(SDS)6 have been fabricated. The mixed solution of Lys-12-Lys and SDS forms vesicles, spherical micelles, and threadlike micelles with the increase of concentration at pH 3.0. It is believable that this approach enriches the varieties of oligomeric surfactants without complicated synthesis. 12085

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a digital resolution of 0.04 Hz/data point. 1D selective nuclear Overhauser effect (NOE) experiments were carried on a Bruker Avance 600 spectrometer. They were obtained for the Lys-12-Lys/ SDS mixtures at 600.1 MHz with a mixing time of 800 ms and 11.8 μs 1 H 90° pulse width. The relaxation delay and acquisition time were set at 2 s and 911 ms, respectively. All the NMR experiments were carried out at 25 ± 2 °C, which was different from other experiments because of the limitation of NMR spectrometers. Cryogenic Transmission Electron Microscopy (Cryo-TEM). The Lys-12-Lys/SDS mixed solutions were embedded in a thin layer of vitreous ice on freshly carbon-coated holey TEM grids by blotting the grids with filter paper and then plunging them into liquid ethane cooled by liquid nitrogen. Frozen hydrated specimens were imaged by using an FEI Tecnai 20 electron microscope (LaB6) operated at 200 kV with the low-dose mode (about 2000 e/nm2) and the nominal magnification of 50 000. For each specimen area, the defocus was set to 1−2 μm. Images were recorded on Kodak SO163 films and then digitized by a Nikon 9000 with a scanning step 2000 dpi corresponding to 2.54 Å/pixel.29 Freeze-Fracture Transmission Electron Microscopy (FFTEM). A small amount of a Lys-12-Lys/SDS sample was placed on a 0.1 mm thick copper disk and then covered with a second copper disk. The sample-loaded copper sandwich was frozen by plunging this sandwich into liquid propane which was cooled by liquid nitrogen. Fracturing and replication were carried out in a freeze-fracture apparatus (BalzersBAF400, Germany) at −140 °C. Pt/C was deposited at an angle of 45° to shadow the replicas, and C was deposited at an angle of 90° to consolidate the replicas. The samples were imaged under an electron microscope (FEI Tecnai 20).

reflect the effect of protonation degree of Lys-12-Lys on its interaction with SDS. Phase Behavior at pH 3.0 and 11.0. The ITC and turbidity titration curves for titrating a small volume of Lys-12Lys into 10 mM SDS solution at pH 3.0 and 11.0 are shown in Figure 3. The observed enthalpy values ΔHobs are calculated by per mole Lys-12-Lys. The initial SDS concentration is beyond its critical micellar concentration (CMC), and the SDS concentration is 8.6 mM after the titration, which is still above the CMC, so the SDS solution is only slightly diluted. The ITC curve for titrating Lys-12-Lys into water is also shown in the figure for comparison. The phase separation domain is indicated by shading. The horizontal axis of the curves is the Lys-12-Lys/SDS molar ratio represented by R. The results at the two pHs show significant differences. At pH 11.0, both the turbidity and ITC curves gradually and slowly change. The turbidity is lower and the solution is transparent throughout the titration. Meanwhile, the ΔHobs is strongly exothermic all along with gradually reducing to the value close to the dilution curve of Lys-12-Lys to water. Although the turbidity is very low (less than 20), it is much higher than that of SDS solution without Lys-12-Lys, suggesting that the mixture of Lys-12-Lys and SDS forms larger aggregates than SDS micelles. Given that the Lys-12-Lys molecule is uncharged at pH 11.0, hydrogen bonding among its amido and amine groups, and sulfate group of SDS and hydrophobic interaction between the hydrocarbon chains of SDS and Lys-12-Lys are the most likely driving forces for the interaction and aggregation. The strong interaction leads to precipitation when temperature is lowered. It was found when the temperature drops to 25.3 °C at pH 11.0, the turbidity begins to increase sharply (as illustrated in Figure S2 of the Supporting Information), and white precipitates appear in the solution. Since the phase separation happens around room temperature at pH 11.0, considering limited practical application, the following investigations are focused on the Lys-12-Lys/SDS mixed solution at pH 3.0. At pH 3.0, both the ITC and turbidity curves indicates that the interaction mode between SDS and Lys-12-Lys changes with the Lys-12-Lys/SDS molar ratio. Because pH does not affect hydrophobic interaction between SDS and Lys-12-Lys, the differences of the ITC and turbidity curves at pH 3.0 from at pH 11.0 are mainly caused by the variations of electrostatic interaction and hydrogen bonding. As shown in the figure, before R1, the ITC curve shows an exothermic process with a large negative observed enthalpy ΔHobs, about −35 kJ/mol. At this pH, all the amino groups of Lys-12-Lys are positively charged, so the dodecyl sulfate ions of SDS tend to bind with these charge groups through electrostatic attraction assisted by hydrophobic interaction between the hydrocarbon chains of SDS and Lys-12-Lys. Thus, the exothermic enthalpies at the early stage of the titrations are mainly caused by the electrostatic binding between these oppositely charged ions as observed in other systems.30 Correspondingly, the turbidity is very low and the solution is optically transparent, which means that the aggregates are small at this stage although the Lys-12Lys molecules have bound with the SDS micelles. Above R1 (∼0.22), the ΔHobs value and turbidity suddenly start to vary, and the turbidity reaches to a very high value at R2 (∼0.26) while ΔHobs reaches a maximum. The sharp rise of the turbidity between R1 and R2 suggests the formation of large aggregates. So R1 is the initial point for the formation of large aggregates. Both the electrostatic binding of Lys-12-Lys with SDS and the

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RESULTS AND DISCUSSION Since Lys-12-Lys is a pH-sensitive compound, the interaction with SDS will be highly dependent on pH. Therefore, potentiometric pH titration was carried out to determine the protonation constants of Lys-12-Lys, and the result is shown in Figure 2. The pKb values of the amino groups in Lysine are

Figure 2. pH titration curve of 1.0 mM Lys-12-Lys at 34.0 ± 0.1 °C.

10.53 and 8.95, but the present pH titration curve shows only one pKa at 7.0 ± 0.1. This indicates that all the amino groups in the Lys-12-Lys molecule should be protonated almost at the same pH. Because the pKa determined from the pH titration curve cannot distinguish different ionizable groups in the molecule, it can only be called as an apparent pKa. According to the pH titration curve, pH 3.0 and 11.0 are chosen in the following studies. At pH 3.0, all the amino groups of Lys-12-Lys molecule are protonated. Thus, Lys-12-Lys carries four positive charges at this acidic condition. At pH 11.0, all of the amine groups are deprotonated, and then the Lys-12-Lys molecule is uncharged. Therefore, the investigations at these two pHs can 12086

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region). Because each Lys-12-Lys carries four negative charges as we described, R1 and R2 are around the equal charge point between Lys-12-Lys and SDS. That is to say, the charge neutralization promotes the growth and association of the Lys12-Lys/SDS aggregates and finally leads to the phase separation. Similar phenomena have been reported in many oppositely charged polymer/surfactant systems.31,32 The ΔHobs becomes progressively less exothermic after R2, and eventually it comes back to almost zero at R3, where the titration curve merges to the dilution curve of concentrated Lys-12-Lys into water, indicating that the added Lys-12-Lys does not interact with SDS anymore. The thermodynamic study in the later text will prove the formation of complexes between Lys-12-Lys and SDS before the precipitation. Before R2, the Lys-12-Lys molecules added in SDS are not enough to neutralize all the negative charges of SDS molecules in the solution, so the complexes of Lys-12-Lys with SDS are distributed in the mixed aggregates and the aggregates carry negative charges from the sulfate groups of SDS. The electrostatic repulsion among the charges keeps the aggregates soluble. After R2, all the SDS molecules have been electrostatically bound by Lys-12-Lys; therefore, the aggregates become uncharged and insoluble in water. Because Lys-12-Lys has no self-assembling ability under the concentration used in the titration, more Lys-12-Lys molecules cannot join in the aggregates. Thus, the precipitates cannot be redissolved by adding more Lys-12-Lys. According to the work on the redissolution of water-insoluble polyion−surfactant complexes reported by Piculell and co-workers,33 the present result indicates that the hydrophobic interaction of Lys-12-Lys with the formed Lys-12-Lys/SDS aggregates is very weak and is not enough to redissolve the precipitates. Obviously, the amount of unneutralized SDS molecules and the self-assembly ability of SDS plays a decisive role in the solubilization of the Lys-12Lys(SDS)n complexes. Interaction Model between Lys-12-Lys and SDS at pH 3.0. Since the Lys-12-Lys/SDS solution is homogeneous at pH 3.0 before R2, the following works are concentrated on this molar ratio range to investigate the interaction model and aggregate transitions in the system. Lys-12-Lys carries four positive charges at pH 3.0; thus, it has at least four binding sites for SDS molecules. Besides, the H-bond donors amides groups of Lys-12-Lys may provide another two binding sites. Therefore, the number of binding sites of Lys-12-Lys with SDS might be 4 or 6. In order to prove this, the ITC curve before R1 in Figure 3d was analyzed to obtain the binding constant (Kb) of Lys-12-Lys with SDS, the number of binding sites (n) on each Lys-12-Lys where SDS can choose to bind with, the binding enthalpy change (ΔHb), and the binding entropy change (ΔSb) by the standard Marquardt method with an ITC package (supplied by Microcal Inc.). The ITC analysis process for the binding of the single set of identical sites was given in the Supporting Information, and the thermodynamic fitting curve to the ITC experimental data is shown in Figure 4. The n value obtained from the ITC analysis is 5.8. Because n represents the number of the sites on each Lys-12-Lys where SDS can choose to bind with and each SDS molecule can only bind with one site, n is also the possible maximum number of SDS molecules bound with one Lys-12-Lys molecule. When the amount of the SDS molecules is enough, the binding sites of Lys-12-Lys may be saturated, and then the n value will be the number of SDS bound with one Lys-12-Lys molecule. Therefore, the n value suggests that about 6 SDS molecules

Figure 3. (a, c) Turbidity curves for titrating the Lys-12-Lys solution into 10 mM SDS solution at pH 11.0 and 3.0 and at 34.0 ± 0.1 °C. (b, d) ITC curves for titrating the Lys-12-Lys solution into water and 10 mM SDS solution at pH 11.0 and 3.0 and at 34.00 ± 0.01 °C. (e, f) Enlarged plots of the turbidity and ITC curves in (c, d) for the Lys-12Lys/SDS molar ratio R lower than 0.25.

aggregate growth cause the ΔHobs to become much more exothermic. Beyond R2, the solution becomes semitransparent and opalescent; i.e., precipitation occurs. Thus, R2 is the starting point for the precipitation. The precipitates are never redissolved even at the end of the titration (see the shadow 12087

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SDS anymore. These aggregates tend to associate with each other and cause precipitation. To further understand the complex formation between Lys12-Lys and SDS, the 1H NMR technique was used and the spectra are shown in Figure 5. After adding Lys-12-Lys into the

Figure 4. Thermodynamic fitting for the ITC experimental data of titrating Lys-12-Lys to SDS before R1 at pH 3.0. The binding parameters of SDS with Lys-12-Lys are n = 5.8 ± 0.1, Kb = (1.16 ± 0.72) × 104 (mol/L)−1, ΔHb = −1.527 ± 0.007 kJ/mol, and ΔSb = 0.073 kJ/(mol K).

are needed to saturate all the binding sites of each Lys-12-Lys molecule. Considering the four positive charges from the protonated amine groups of Lys-12-Lys, the number of binding sites with anionic SDS should be around 4. The extra two binding sites should be resulted from the following reason. The presence of N−H dipoles allows the amides to work as H-bond donors to form H-bonds with H-bond acceptors, i.e., the oxygen atoms from the sulfate group of SDS. So Lys-12-Lys may offer another two sites to bind with SDS assisted by the synergistic effect of the hydrophobic attraction among the tails of SDS and the spacer group of Lys-12-Lys. On the basis of the above results, it is reasonable to conclude that the complexes of Lys-12-Lys and SDS change with their mixing ratio. As shown in the enlarged plots of the turbidity and ITC curves in Figure 3e,f, both the turbidity and the observed enthalpy already start to change at R0, i.e., ∼0.14. The turbidity starts to increase slightly, and the observed enthalpy becomes less exothermic. This means that the aggregates begin to grow and the electrostatic interaction of Lys-12-Lys with SDS becomes weak beyond R0. Because one Lys-12-Lys molecule can bind with six SDS molecules at most, SDS is always in excess before R0, where the ratio of Lys-12-Lys to SDS is 1:7. So Lys-12-Lys and SDS prefer to form the Lys-12-Lys(SDS)6 complex with the maximum available binding sites before R0. The complex is like a hexameric surfactant, and it coexists with free SDS and gradually becomes the main component in the mixed aggregates. The unbound SDS molecules in the aggregates keep the mixed aggregates soluble. Beyond R0, almost all the SDS molecules have been bound by Lys-12-Lys. However, parts of the SDS molecules bind with Lys-12-Lys through hydrogen bonds, which makes the mixed aggregates still carry net negative charges from SDS and in turn keeps the aggregates soluble. With the further addition of the Lys-12-Lys molecules, the number of SDS molecules becomes less than 6 times of Lys-12-Lys. Because the electrostatic binding between Lys-12-Lys and SDS is stronger than the hydrogen bonding between them, SDS molecules prefer to bind on the four charged sites of Lys-12-Lys. Hence, more Lys-12-Lys molecules may generate the Lys-12-Lys(SDS)4 complexes in a dynamic equilibrium with Lys-12-Lys(SDS)6. The change of the components in the mixture reduces the charge density of the mixed aggregates and in turn induces the growth of the mixed aggregates. When the Lys-12-Lys/SDS ratio reaches R1, i.e., ∼0.23, which is very close to the equal charge point of 1:4, the Lys-12-Lys/SDS aggregates are mainly composed of the charge neutralized Lys-12-Lys(SDS)4 complexes without unneutralized

Figure 5. 1H NMR spectra and proton assignments of Lys-12-Lys and SDS in D2O at pH 3.0. The SDS concentration for all of the solutions is 10.0 mM.

SDS micellar solution, the chemical shifts of SDS almost do not change, and only the shape of peaks becomes smooth and broad, which indicates that these protons are spin-restricted. On the contrary, the signals of the Lys-12-Lys protons change dramatically. The triple peaks at 3.90 ppm (Ha) ascribed to −NHCOCHNH3+− and the multiple peaks at 3.20 (Hb) ascribed to −CH2NHCO− disappear. The triple peaks at 2.97 (Hc) adjacent to the amine group of the lysine residue, the quadruple peaks at 1.87 (Hd), and the multiple peaks at 1.67(He) around amine groups shift downfield. Normally, the changes of chemical shifts reflect the conformation and solvation of surfactants in aggregates.34,35 After adding Lys12-Lys to the SDS micellar solution, due to the long-range electrostatic attraction, the positively charged groups of Lys-12Lys get close to the negatively charged headgroup of SDS. Then, the hydrophobic part of Lys-12-Lys inclines to escape from the bulk water and associate into the hydrophobic core of the SDS micelles. The signals shift to downfield, indicating that the partial changeover of the molecular conformation of Lys12-Lys. Meanwhile, another reason to the downshift of the Lys12-Lys signals is that the hydrophilic groups of Lys-12-Lys transform from bulk water into the surface water pseudophase whose polarity is lower. The interaction between Lys-12-Lys and SDS can also be understood from the spatial relationship of Lys-12-Lys with SDS upon the binding. The 1D selective nuclear Overhauser effect (1D-NOE) was studied, and the NOE difference spectra are shown in Figure 6. Three protons whose chemical shifts do not overlap with the others were chosen to carry out the experiments. Obviously, irradiation the methyl protons Hd′ at the end of the hydrocarbon chain of SDS only produces NOE effect on the other protons of SDS itself. On the contrary, both the methylene protons (Hc and Hd) adjacent to the amine groups of Lys-12-Lys produce NOE effects on the protons (Ha′ and Hb′) adjacent to the headgroup of SDS. This indicates that Hc and Hd are spatial correlated with Ha′ and Hb′ within a distance of 5 Å. It reveals that the sulfate headgroup of SDS is indeed near the amine groups of Lys-12-Lys, confirming SDS 12088

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not induce obvious change in the mixed aggregates before R0. Beyond R0, the mixed aggregates grow as reflected by the turbidity (Figure 3). The cryo-TEM image shown in Figure 7F indicates that the mixed aggregates beyond R0 are threadlike micelles. That is to say, upon binding with Lys-12-Lys, the complex formation causes the SDS spherical micelles to transfer into long threadlike micelles. Zana’s group6,9 have found that linear trimeric and tetrameric quaternary ammonium surfactants formed threadlike micelles. The present results indicate that using an anionic single chain surfactant SDS and a positively charged oligomeric connecting molecule Lys-12-Lys has constructed the Lys-12-Lys(SDS)4 and Lys-12-Lys(SDS)6 complexes with similar properties of linear oligomeric surfactants. As observed above, the Lys-12-Lys(SDS)4 and Lys-12Lys(SDS)6 complexes have induced the aggregate transition in the mixed systems. Normally the structures of surfactant aggregates are strongly dependent on concentration. Therefore, the following investigations were carried out in order to understand the concentration dependence of the aggregate transitions in the Lys-12-Lys/SDS mixture containing the Lys12-Lys(SDS)4 and Lys-12-Lys(SDS)6 complexes. According to the turbidity and ITC results in Figure 3c,d, the Lys-12-Lys/ SDS molar ratios were chosen between R0 and R1, where the aggregates are obviously enlarged and stable enough without precipitation. Thus, the Lys-12-Lys/SDS molar ratio was fixed at 0.15 and 0.20, and the aggregate transitions with the increase of the total Lys-12-Lys and SDS concentration were studied by ITC, DLS, and turbidity as shown in Figure 8. For the Lys-12-Lys/SDS molar ratios of 0.15 and 0.20, the variations of the observed enthalpy, the aggregate size, and the solution turbidity display the same changing tendencies. With the increase of concentration, the turbidity expressed by the absorbance increases until a maximum and then reduces progressively. Before the maximum, precipitation takes place in the area shown by the dashed line. Meanwhile, the DLS results show that the average diameters of the aggregates gradually decrease from several hundred nanometers to less than 10 nm. However, the ITC curves are so complicated that we cannot properly explain them, although the variations of the curves do correspond to the phase separation and aggregate transitions in the systems. Herein, the ITC curve for R = 0.15 is used as a representative to illustrate the variations. To avoid misleading, the ITC curves will only be described but not be explained. Around Ca, the exothermic ΔHobs corresponds to the region of precipitation. Above Ca, precipitation begins to dissolve, and the ΔHobs becomes less exothermic. From Cb up to Cc, ΔHobs nearly remains constant. Considering the progressively decreased turbidity and size, the constant ΔHobs may be related to the continuous aggregate transition with the increase of concentration. After Cc, the ITC curve shows a “Z-type extension”, showing a maximum exothermic value at Cd and returning to baseline at Ce. The mixtures in different concentration ranges except for the precipitate were observed by using cryo-TEM and FF-TEM. The representative images are illustrated in Figure 7. Vesicles with a diameter of ∼50 nm are observed for the sample at Cb (CSDS = 1.0 mM, Figure 7A). The sample between Cb and Cc presents lots of vesicles with diameters ranging from 30 to 80 nm (CSDS = 2.0 mM, Figure 7B). The mixtures between Cc and Cd contain not only larger vesicles but also smaller micelles of ∼10 nm (CSDS = 3.0 and 4.0 mM, Figure 7C,D). When the concentration is between Cd and Ce, only small spherical

Figure 6. 1D NOE difference spectra upon irradiating the Hd′ at 0.87 ppm, Hd at 1.91 ppm, and Hc at 3.03 ppm at different Lys-12-Lys/SDS molar ratios indicated above the spectra.

interacts with Lys-12-Lys through head groups. In addition, all of the NOE effects are enhanced with the increase of the Lys12-Lys/SDS molar ratio, indicating that the molecules are packed more tightly in the aggregates when more Lys-12-Lys molecules are added in the solution. Since the Lys-12Lys(SDS)4 complexes are generated at higher Lys-12-Lys/ SDS ratio than Lys-12-Lys(SDS)6 complexes, the NOE results suggest that the aggregates of the Lys-12-Lys(SDS)4 complexes are packed more tightly than those of the Lys-12-Lys(SDS)6 complexes. Aggregate Transitions at pH 3.0. As revealed above, Lys12-Lys and SDS form the Lys-12-Lys(SDS)4 and Lys-12Lys(SDS)6 complexes, and the resultant mixed aggregates vary in size and in phase behavior. In order to know the aggregate transitions induced by the complex formation, cryo-TEM and FF-TEM were applied. As reported previously,36,37 SDS should form spherical micelles of ∼2 nm in diameter in the solution just above its cmc (∼8.7 mM). 10 mM SDS is used in the studies above, so the SDS molecules without Lys-12-Lys exists as such a kind of spherical micelles. After adding Lys-12-Lys, Lys-12-Lys does 12089

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Figure 7. Cryo-TEM and FF-TEM micrographs of the aggregates in the Lys-12-Lys/SDS mixed solutions at the molar ratio of 0.15 and at the concentration of SDS: (A) 1.0, (B) 2.0, (C) 3.0, (D) 4.0, (E) 5.0, and (F) 10.0 mM. (B) and (C) are FF-TEM micrographs, and the others are cryoTEM micrographs. All the samples are at pH 3.0.

micelles of ∼5 nm are observed (CSDS = 5.0 mM, Figure 7E), and the DLS result also shows that the mean diameter of the aggregates is less than ∼10 nm. The sample far beyond Ce, i.e., at 10 mM, forms long threadlike micelles with a diameter of ∼5 nm. Meanwhile, the DLS result shows that the average size distribution of the aggregates is ∼8 nm above 6 mM, and it no longer changes with the increase of concentration. The DLS experiment performed here was finite-angle dynamic light scattering, and spherical aggregates were assumed to fit the data. Therefore, it cannot represent the real size of threadlike micelles. In brief, the total concentration strongly affects the selfassembling ability and structure of the mixture. With the increase of the total Lys-12-Lys and SDS concentration while fixing their ratio, the mixture experiences the aggregate transitions of vesicles to spherical micelles and then to long threadlike micelles. But before the vesicles, the very low concentration only leads to precipitation rather than ordered self-assemblies. That is to say, below Ca, the concentration is too low, so SDS and Lys-12-Lys cannot self-assemble and the electrostatic binding of Lys-12-Lys with SDS only leads to precipitation. When the total concentration is above Cb, the formation of the Lys-12-Lys(SDS)4 and Lys-12-Lys(SDS)6 complexes significantly enhances the self-assembling ability of the mixture so that the concentration has reached the critical aggregation concentration. The structure of the Lys-12Lys(SDS)4 and Lys-12-Lys(SDS)6 complexes makes them tend to form low curvature aggregates, so vesicles are formed in the mixture solution. As the concentration further increases, the hydrophobic interaction among the SDS alkyl tails becomes stronger and makes the alkyl tails pack more tightly, while Lys12-Lys acts like a spacer group of oligomeric surfactant and thus keeps the surfactant with a larger headgroup. These factors decrease the packing parameter and result in the aggregate transition from large vesicles to small spherical micelles. This process is very similar to our previous observations in trimeric,

Figure 8. (a) ITC curves of titrating the Lys-12-Lys/SDS mixed solutions into water. (b) Turbidity of the Lys-12-Lys/SDS mixed solutions monitored by UV−vis absorbance at 400 nm. (c) The average diameter of the Lys-12-Lys/SDS aggregates by DLS as a function of the SDS concentration at various molar ratios. All the experiments are performed at pH 3.0 and 34.0 °C.

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aggregation behaviors of “tetrameric surfactant” Lys-12-Lys(SDS)4 and “hexameric surfactant” Lys-12-Lys(SDS)6 display the features of both the linear and star-shaped oligomeric surfactants. Consequently, oligomeric surfactants have been fabricated through noncovalent interactions. This approach presents a new way to develop oligomeric surfactants without complicated synthesis and should be able to be extended to construct other novel surfactants by choosing proper building blocks.

tetrameric, and hexameric surfactants.19−21 When the total concentration continuously increases to a high value, the very strong hydrophobic interaction of the SDS chains with the alkyl chain in the middle of the Lys-12-Lys molecules may cause the alkyl chain of Lys-12-Lys to bend into the hydrophobic core of micelles, just like gemini surfactants with a long spacer group. This effect can reduce the area of the surfactant headgroup and increase the packing parameter. Therefore, the spherical micelles transfer to long threadlike micelles. In addition, the turbidity, ITC and size distribution curves tell the same story that the phase boundary moves to high concentration with the increase of the Lys-12-Lys/SDS molar ratio (red → blue, Figure 8). This will be easily understood with a simple mathematical calculation on the basis of the ITC curves and the binding constants obtained. As estimated in Table S1, the total concentration of SDS (bound and unbound) required for each aggregate transition increases as the Lys-12Lys/SDS molar ratio increase from 0.15 to 0.20, but the concentration of unbound free SDS is on the contrary. This indicates that the main composition of the mixed solution changes from SDS to the complexes of Lys-12-Lys(SDS)6 and Lys-12-Lys(SDS)4 at higher R. At higher R, the self-assembly of the mixed solution mainly reflect the feature of “oligomeric surfactants” Lys-12-Lys(SDS)6 and Lys-12-Lys(SDS)4 themselves. Thus, this result confirms again that we have achieved oligomeric type surfactants by intermolecular interaction.

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ASSOCIATED CONTENT

S Supporting Information *

Characterization data of Lys-12-Lys by 1H NMR, mass spectrum, and high-performance liquid chromatography, temperature dependence of Lys-12-Lys/SDS, replotted Figure 3 with the Lys-12-Lys concentration as x-axis, the estimated concentrations of total and unbound SDS at different Lys-12Lys/SDS molar ratios, and analysis process of ITC curves. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (Y.W.).

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Notes

The authors declare no competing financial interest.

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CONCLUSIONS In this work, anionic single-tail surfactant sodium dodecyl sulfate (SDS) and an oligomeric connecting molecule Lys-12Lys were used as building blocks to fabricate the oligomeric surfactants through intermolecular interactions. The formation of complexes between Lys-12-Lys and SDS was verified by ITC, turbidity titration, and NMR, and the results revealed that the complexes change with their mixing ratio. At pH 3.0, Lys-12Lys carries four charged binding sites and two hydrogen binding sites, so SDS interacts with Lys-12-Lys through head groups by electrostatic attraction and hydrogen bonds beside the hydrophobic interaction between their hydrocarbon chains. They form Lys-12-Lys(SDS)6 complexes at lower Lys-12-Lys/ SDS molar ratio and Lys-12-Lys(SDS)4 complexes at higher Lys-12-Lys/SDS molar ratio. The latter is packed more tightly in the aggregates than the former. These two complexes can induce affluent aggregate structures in the mixed systems as oligomeric surfactants do. At fixed molar ratios, with the increase of the total Lys-12-Lys and SDS concentration, the mixture experiences the aggregate transitions from vesicles to spherical micelles and then to long threadlike micelles. This is attributed to the spacer function of Lys-12-Lys in the oligomeric surfactants constructed as well as the alterations of the hydrophobic interaction among the SDS alkyl tails and the alkyl chain in the middle of Lys-12-Lys. Concluding from the recent studies about the synthesized linear trimeric and tetrameric surfactants6,9 and the star-shaped trimeric, tetrameric, and hexameric surfactants,19−21 both the degree of oligomerization and the nature of the spacer groups play decisive roles in the aggregation behaviors of oligomeric surfactants. Linear oligomeric surfactants tend to form threadlike micelles, while star-shaped oligomeric surfactants incline to form large size aggregates before micelles. The positive charges and the hydrogen binding sites of Lys-12-Lys with which the SDS head groups bind are neither typical linear nor typical starshaped, but like a transition state between them. Therefore, the

ACKNOWLEDGMENTS We are grateful for financial support from the National Natural Science Foundation of China (21025313 and 21021003).

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REFERENCES

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