This is the accepted version of the journal article: Pi i Boleda, Bernat; Bouzas, Mireia; Gaztelumendi, Nerea; [et al.]. «Chiral pH-sensitive cyclobutane beta-amino acid-based cationic amphiphiles : Possible candidates for use in gene therapy». Journal of Molecular Liquids, Vol. 297 (January 2020), art. 111856. DOI 10.1016/j.molliq.2019.111856 This version is available at https://ddd.uab.cat/record/274089 under the terms of the license 1 Chiral pH-Sensitive Cyclobutane -Amino Acid-Based 2 Cationic Amphiphiles: 3 Possible Candidates for Use in Gene Therapy 4 5 Bernat Pi-Boleda,1 Mireia Bouzas,1§ Nerea Gaztelumendi,2 Ona Illa,1 6 Carme Nogués,2 Vicenç Branchadell,1 Ramon Pons,*3 Rosa M. 7 Ortuño*1 8 9 1: Departament de Química, Universitat Autònoma de Barcelona, 08193 Cerdanyola del 10 Vallès, Barcelona, Spain. 11 2: Departament de Biologia Cel·lular, de Fisiologia i d'Immunologia, Universitat 12 Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain. 13 3: Departament de Tecnologia Química i de Tensioactius, Institut de Química Avançada 14 de Catalunya, IQAC-CSIC, c/ Jordi Girona, 18-26, 08034 Barcelona, Spain. 15 16 17 1 18 ABSTRACT. 19 7.0 6.5 cis-8 C12 6.0 pKa = 8.5 5.5 pKa < 0 pH 5.0 cmc = 1.00 mM CMC = 1.0 mM 4.5 pKa = 0 strong acid 4.0 pKa = 6.25 weak acid 3.5 0.01 0.1 1 10 Concentration (mM) 7.0 6.5 trans-8 6.0 T12 pKa = 8.2 5.5 pH pKa < 0 5.0 cmc = 0.60 mM 4.5 pKa = 6.9 CMC = 0.60 mM 4.0 3.5 0.01 0.1 1 10 Concentration (mM) 20 21 Chiral cis/trans diastereomeric cationic amphiphiles have been synthesized and 22 studied. They are based on -amino acids and contain C12- and C16-alkyl chains, 23 respectively, as hydrophobic tails while the polar head consists of an ammonium cation 24 linked to a cyclobutane ring. Their physicochemical properties, such as the cmc (critical 25 micellar concentration), the pKa, the ratio of cationic versus non-ionic species, and the 26 surface tension are strongly dependent on the pH of the medium. At the same time the 27 aggregation state influences on the apparent pKa values of the aggregates, with release 28 of protons at the cmc whose values, as well as those of the adsorption effectiveness, 29 account for their efficient surfactant behavior. A tail-length effect is manifest because 30 surfactant cmc of compounds with a C16-alkyl chain are smaller than the C12-ones, 31 although not as small as expected. On the other hand, while for C12-surfactants the role 32 of the stereochemistry on the physicochemical parameters is patent, it is not very clear 33 for C16-surfactants. The stereochemistry also determines the predominant mode of self- 2 34 assembly since the trend for cis-isomers is to form micelles or vesicles while trans- 35 isomers preferably form fibers. CD spectroscopy confirmed the aggregation through the 36 formation of intermolecular hydrogen bonds between the amide groups of the 37 monomers. An alternative method to determine the cmc of these surfactants is provided 38 by considering the relationship of the CD max with concentration, although it is 39 restricted to those surfactants in which the chromophore is located in a chiral 40 environment. Furthermore, the non- toxicity of the surfactants has been verified by the 41 MTT assay. This characteristic, jointly with the efficiency and the good properties 42 shown, the fact that the cationic species are present in a high concentration at 43 physiological pH, as well as the weak acid behavior of the aggregates, confirm these 44 amphiphiles as promising candidates to be used as non-viral vectors for gene therapy 45 applications. 46 47 3 48 1. INTRODUCTION 49 Gene therapy has gained significant attention over the past two decades as a potential 50 method for treating genetic disorders [1-5] as well as an alternative way to traditional 51 chemotherapy used in treating cancer [6]. It mostly involves the use of DNA fragments 52 that encodes a therapeutic protein or gene in order to replace a mutated gene. In these 53 cases, DNA is packed within a vector forming DNA-vector complexes in order to 54 protect the information of the chain and to get inside specific cells within the body [7]. 55 Depending on the temperature, the pH, or the ionic strength, a single DNA chain in 56 solution could adopt different structures. 57 The role of chemists in the field of gene therapy is to design and prepare new non- 58 viral vectors thus avoiding the risks derived of the work with virus materials. Special 59 attention has been paid to cationic surfactants due to their properties [8]. Cationic polar 60 heads can interact with negatively charged phosphate groups of the DNA while 61 hydrophobic chains can stabilize the aggregates. In order to transfect into cells it is 62 important to control DNA compaction and the neutralization of negative charges to 63 avoid repulsive interactions with phospholipids in the cell membrane. One of the most 64 studied surfactants is commercial CTAB (hexadecyl trimethylammonium bromide) [9- 65 10]. It shows high efficiency compacting DNA compared with other shorter surfactants, 66 but most trimethylammonium derivatives show cytotoxicity [11]. 67 Amino acid-based surfactants are getting importance due to their good levels of 68 biodegradability and biocompatibility [12-16]. The combination of amino acids or 69 peptides with hydrocarbon chains of variable length has given rise to a variety of 70 compounds with an amphiphilic structure and with good surfactant properties. Although 71 most examples use natural -amino acids, the design of pH-sensitive amphiphiles can 72 be achieved from synthetic amino acids. The possibility to tune the properties of the 4 73 surfactants depending on the pH of the medium confers on these compounds a great 74 potential for biomedical applications [16-17]. Otherwise, the presence of amide groups 75 can help the self-assembly of surfactants through intermolecular hydrogen bonding [18]. 76 In our laboratories [19], different cyclobutane-containing anionic, 1 [20], or non- 77 ionic, 2 [21], -amino acid-based amphiphiles were synthesized and studied. Results 78 pointed out the relevance of the relative stereochemistry and of the stereochemical 79 constraints imposed by the cyclobutane ring on the aggregation properties of these 80 compounds (Figure 1). 81 82 83 Figure 1. Anionic (1), non-ionic (2), and cationic (3 and 4) amphiphiles previously investigated 84 in our laboratories. 85 86 Cationic bolaamphiphiles based on chiral cyclobutane scaffolds, 3 and 4, were also 87 prepared and investigated concluding that regiochemistry (N- versus C-centered 5 88 derivatives) plays a minor role on the cmc value but determines the morphology of the 89 supramolecular aggregates formed [22] (Figure 1). 90 In this paper, we describe the synthesis of four new cationic -amino acid-based 91 surfactants cis- and trans-8, and cis- and trans-9 (Scheme 1). All these compounds are 92 chiral and include two pairs of cis/trans diastereomers; in turn, 8 and 9 differ in the 93 alkyl-chain length (C12 and C16, respectively). Both the influence of the relative 94 stereochemistry and of the chain length have been considered in their physicochemical 95 characterization and in the analysis of the chiral supramolecular systems formed as 96 surfactants. Special attention has been paid to the influence of the pH on these 97 properties. Furthermore, bearing in mind the possibility of the future use of these 98 surfactants as non viral vectors for gene therapy, preliminary studies on their 99 cytotoxicity have been carried out. We present and discuss herein all the results 100 obtained. 101 102 103 Scheme 1. Synthesis of the cationic amphiphiles cis- and trans-8, and cis- and trans-9. 104 105 106 6 107 2. EXPERIMENTAL SECTION 108 2.1 Synthesis of the amphiphiles. 109 The detailed synthesis and characterization of surfactants cis- and trans-8, and cis- and trans-9 110 and their precursors (Scheme 1) are reported in the Supporting Information. 111 112 2.2 pKa Measurements. 113 The pKa values were determined from the potentiometric titration of 1 mL of 5 mM aqueous 114 surfactant solutions by using 5 mM NaOH aqueous solutions. The pH electrode was an ORION 115 8103SC semimicro and the potentiometer was a Thermo Orion model 720A+. All titrations 116 were conducted at 25 ± 0.1 °C and under nitrogen atmosphere. pKa was determined as the pH of 117 the corresponding semi equivalence point. Once the titration is finished, a new titration with 5 118 mM HCl is done to determine the reversibility. De-ionized water (milli-Q) was used to prepare 119 the solutions. 120 The pH values of different concentrations of surfactant water solutions under nitrogen were 121 measured using a pH electrode (model ORION 8103SC Ross Semimicro). Measurements were 122 made at increasing concentrations of surfactant to minimize errors from possible contamination 123 from the electrode. The acid−base equilibrium was modelled by Equation 1 for a weak base, 124 assuming dilute ideal behavior and complete salt dissociation. This equation was obtained 125 taking into account the equilibrium constant definition, mass and charge balances, and the ionic 126 product of water. 127 128 (1) [𝐻 + ] 𝐾𝑤 129 𝐶𝑎 = (1 + ) ([𝐻 + ] − + ) 𝐾𝑎 [𝐻 ] 130 131 132 The pKa values both below the cmc (monomeric surfactant) and above the cmc (apparent 133 pKa) were evaluated by using this equation. 134 135 2.3 Surface Tension Measurements. 136 The surface tension was measured using a home-made pendant drop tensiometer as in reference 137 22. Surface tension in acidic (0.01 M HCl) and basic conditions (0.01 M NaOH) have been 138 compared with unbuffered aqueous solutions. When using Gibbs isotherm the values of n to 139 estimating the area per molecule at cmc are 1 for both buffered conditions and undefined for the 140 unbuffered conditions. We are aware of the problems with estimating area per molecule from 7 141 surface tension, however, we think that these values have a strong comparative meaning when 142 compared to other in the literature, please refer to reference 22 and literature cited herein. 143 2.4 Cryogenic Transmission Electron Microscopy (cryo-TEM). 144 A drop of the surfactant solution with 2.5 mM concentration was placed on a carbon-coated 145 copper grid. Then, the sample-grid assembly was rapidly frozen using liquid ethane and kept at - 146 180 C during the imaging using liquid nitrogen. The images were acquired with Hitachi H- 147 7000 microscope operating at 200 kV. 148 149 2.5 Computational Calculations. 150 The structure of the surfactants in solution was optimized with DFT in water solution using 151 M06-2X density functional [23] and 6-31G(d) level of theory. These calculations have been 152 carried out using the Gaussian-09 program [24].Circular dichroism spectra were calculated by 153 taking the optimized structure of the monomer in water solution and calculating 30 excited 154 states with Gaussian-09 using the TDDFT method [25-27] at the M06-2X/6-311++G(2df,2pd) 155 level of theory. 156 157 2.6 Circular Dichroism (CD) Spectroscopy. 158 0.5-5.0 mM samples of surfactants were prepared in water and measured in a 1 cm width quartz 159 cuvette. CD spectra were recorded with JASCO-715 spectropolarimeter and were processed 160 using Spectra Manager Software. 161 162 2.7 Ultraviolet – visible (UV - vis) Spectroscopy. 163 The absorption was measured in a Hewlett Packard 8453 spectrophotometer in aqueous 164 solutions in a range between 180 and 500 nm. 165 166 2.8 Biological Experiments. 167 The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [28], was used 168 to analyze the cell viability and, proportionally, the surfactant toxicity. MTT (yellow color) is 169 reduced by the mitochondrial enzymes to formazan, an insoluble purple crystal which 170 absorbance can be read with a spectrophotometer after its solubilization by an organic solvent. 171 The higher the absorbance value, the higher the enzymatic activity, and therefore the more 172 number of living cells are present. In separate experiments, HeLa cells [29] were incubated in 173 the presence of the different surfactants at about 50 M concentration in water and MTT 174 (Sigma-Aldrich) in a 0.1 mg/mL concentration was added. After 3 h of incubation at 37 ºC in 175 darkness, the MTT was removed and formazan crystals were dissolved using DMSO. The 176 absorbance was recorded at 540 nm in an X3 Multilabel Plate Reader coupled to a PerkinElmer 8 177 2030 Manager control software. The experiments were replicated 4 times for each surfactant. 178 The control experiment was performed under the same conditions but in the absence of a 179 surfactant. 180 The normalization of the results was achieved considering the average of the absorbance 181 value of the control population as 100% of cellular proliferation (Equation 2). 182 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 183 % 𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 = 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑛𝑜𝑛−𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 𝑥 100 (2) 184 185 3. Results and Discussion 186 187 3.1 Synthesis of the amphiphiles. 188 cis- and trans-Diastereoisomers of compounds 8 and 9 were synthesized from the 189 appropriate and partially protected amino acids cis- and trans-5, respectively, by means 190 of analogous synthetic routes (Scheme 1). These amino acids were prepared, in turn, in 191 an enantioselective manner as previously described [30-31]. 192 Condensation of acid cis-5 [30] with dodecylamine, in the presence of 193 diisopropylamine (DIPEA) and (benzotriazol-1-yloxy)tripyrrolidinophosphonium 194 hexafluorophosphate (PyBOP) as a coupling agent, afforded cis-6 in 92% yield for the 195 two steps. Similarly, condensation with hexadecylamine led to cis-7 in 79% yield. 196 Subsequently, quantitative deprotection of the respective amines by acidolysis of the 197 Boc carbamate with 2 M HCl in Et2O provided cationic amphiphiles cis-8 and cis-9, 198 respectively. Following similar synthetic routes starting from trans-5 [31], the 199 diastereomers trans-8 and trans-9 were prepared in 82 and 78% yield, respectively. 200 201 3.2 Determination of the pKa of the surfactants and study of the pH dependence. 9 202 The pKa of the surfactants investigated was determined by using two different and 203 complementary techniques: (a) Measurement of the pH at various surfactant 204 concentrations; (b) acid-base titration with sodium hydroxide and retro-titration with 205 hydrochloric acid. Surfactants 8 and 9 present an acid-base equilibrium in aqueous 206 medium that is the average of several equilibria involving ionic or non-ionic monomers 207 and aggregates, which depend on the pH of the medium and the concentration of the 208 surfactant. 209 210 3.2.1 Measurement of the pH at diverse surfactant concentrations. 211 First, the dependence of the pH on the concentration of surfactant was investigated at 25 212 ºC. The obtained plots are shown in Figure 2 and from data therein, the relation of pKa 213 with respect to the concentration of surfactant was determined according to Equation 1 214 in the Experimental Section. 215 For pH-sensitive amphiphiles, at the extremes of concentration, the pH values 216 follow the slope of 0.5 expected for weak acids, with pKa corresponding to the 217 monomers at low concentrations and to the average of monomers and micelles above 218 the cmc (Figure 2). The onset of micellization is marked by the first change of slope, 219 which corresponds to the critical micellar concentration (cmc). Indeed, when the 220 formation of micelles occurs, a release of protons is produced due to the apparent pKa 221 change of the micelle with respect to the monomer [17] The dependence of the pKa 222 values on the concentration, for each surfactant, is detailed in the Supporting 223 Information (figure S1) . 224 Comparing diastereomeric surfactants cis- and trans-8, both with a C12-alkyl chain, 225 the pKa values before the cmc are very similar: 8.5 for cis-8 and 8.2 for trans-8 (Figure 226 2). Therefore, it seems that cis/trans stereochemistry does not exert a significant 10 227 influence on the behavior of the monomeric surfactants. Nevertheless, the slightly 228 weaker acidity of the cis isomer could be understood by the possibility of hydrogen 229 bonding between a proton of the ammonium cation and the oxygen of the carbonyl 230 group in the amide (see Figure 6 below). This possibility does not exist in the trans 231 stereoisomer. These values show that both surfactants are weak acids and their structure, 232 cationic or non-ionic, will depend on the pH of the solution. However, these surfactants 233 are more acid than alkylammonium chlorides (DAC), of use in gene therapy, which pKa 234 has been determined at several concentrations dodecylamine pKa=8.8 at 5 mM, or 235 pKa=9.16 at 2 mM, while tetradecylamine has a pKa=7.56 at 2 mM [32]. Taking into 236 account that the pH of the human blood is around 7.4, we can expect that, under 237 physiological conditions, cis- and trans-8 will be mostly in the protonated form. 238 (a) (b) 7.0 7.0 6.5 6.5 6.0 cis-8 6.0 trans-8 pKa = 8.2 pKa = 8.5 5.5 5.5 pKa < 0 pKa < 0 5.0 5.0 pH pH pKa = 0 pKa = 6.9 4.5 4.5 cmc = 1.00 mM cmc = 0.60 mM 4.0 pKa = 6.25 4.0 3.5 strong acid 3.5 3.0 3.0 weak acid 2.5 2.5 0.01 0.1 1 10 0.01 0.1 1 10 Concentration (mM) Concentration (mM) (c) (d) 7.0 pKa = 9.0 7.0 6.5 6.5 6.0 cis-9 6.0 trans-9 5.5 5.5 pKa = 5.4 5.0 5.0 pH pH 4.5 cmc = 0.32 mM 4.5 4.0 4.0 cmc = 0.16 mM 3.5 3.5 3.0 3.0 2.5 2.5 0.01 0.1 1 10 0.01 0.1 1 10 Concentration (mM) Concentration (mM) 239 240 Figure 2. Plots of pH versus surfactant concentration for: (a) cis-8, (b) trans-8, (c) cis-9, (d) 241 trans-9. Measurement at 25 ºC. 242 11 243 The situation is different for cis- and trans-9 bearing a C16-alkyl chain. Indeed, cis-9 244 presents a pKa 9.0 before the cmc while trans-9 shows a very low pKa, around 5.4. This 245 unexpected behavior could suggest that, in this case, stereochemistry plays a very 246 important role in the acidity of the proton, in contrast with surfactants cis-and trans-8 247 with a shorter carbon chain. Surfactant trans-9 was the less soluble and, indeed, some 248 turbidity was observed at high concentration (5 – 10 mM) while making the 249 measurements. This suggests the possible formation of oligomers involving both 250 cationic and non-ionic species. 251 Otherwise, a pKa shift depending on the surfactant concentration was observed in all 252 cases (Figure 2 and Figure S1 in the Supporting Information). After the cmc, the pKa 253 value decreases, cis-8 presenting a higher pKa shift than that shown for trans-8. Thus, 254 these compounds can behave as two different weak acids, as monomeric molecules 255 before the cmc and as micelles after the cmc. A similar behavior was observed for 256 diastereomers cis- and trans-9, although their lower solubility made difficult the study 257 at high concentration. 258 The pKa shift of molecules undergoing aggregation has been extensively studied [33- 259 34], and two main contributions to this fact were identified. One contribution comes 260 from the virtual charge effect due to a discontinuity in dielectric constant at the micelle 261 surface [35] and a second contribution comes from the polar heads interaction where 262 some protons are released to the solution in order to stabilize the aggregates. 263 Once molecules begin to self-assemble, the pH of the solution starts to decrease with 264 a higher slope (Figure 2). From the intersection of the two lines, the cmc value is 265 obtained: 1.0 mM and 0.60 mM for cis-8 and trans-8, respectively. Smaller values were 266 obtained for longer-chain surfactants 9 with cmc 0.32 mM and 0.16 mM for cis-9 and 267 trans-9, respectively. DAC, a cationic surfactant with similar chain length, presents a 12 268 cmc of 9 mM (pH 7) and 12 mM (pH 5) at 25 °C [36], which are higher values than 269 those for 8 and 9. Therefore, the presence of the cyclobutane unit and the amide bond 270 seems to have a significant role on those values. This finding seems to contradict what 271 was found in the case of anionic surfactants with polar heads also containing 272 cyclobutane and amide bond but which cmc value did not change appreciably with 273 respect to the a carboxylate of the same chain length (i.e. 12 mM for sodium laurate). 274 Moreover, the charge distribution in function of the concentration was determined 275 for each surfactant by plotting [NH3+]/[R-NH2]=[H+]/Ka versus concentration (see 276 Figure S2 in the Supporting Information). These graphics provide information about the 277 behavior of these surfactants as acids. All of them have a maximum of charge at the 278 cmc point. Before the cmc, each surfactant behaves as a normal weak acid but, once the 279 cmc is reached, the amount of cationic species divided by the amount of non-ionic 280 species (R-NH3+/R-NH2) starts to decrease in order to stabilize the aggregates and to 281 reduce electrostatic repulsion between the molecules. It is interesting to note that, for 282 trans-9, the charge ratio presents values between 3 and 5 showing that the concentration 283 of the cationic surfactant is only 3-5 times higher than the concentration of the non- 284 ionic surfactant. This ratio is much higher for the other surfactants studied. 285 The behavior of these amphiphiles could be interesting in medicinal chemistry, 286 because changes in the protonation state would lead to changes the in headgroup area 287 and, as a result, in their aggregation state. Surfactants with this type of properties were 288 shown to be efficient vectors for gene therapy because the release of DNA into the cells 289 is improved and, consequently, the level of gene transfection may be increased [37]. 290 291 3.2.2. Acid-base titrations. 13 292 As a second methodology to determine the pKa, acid-base titration of the surfactant 293 aggregates with aqueous NaOH and subsequent retro-titration with HCl were carried 294 out. The resultant curves for each surfactant are shown in Figure 3. 295 12 12 11 11 10 10 9 9 8 8 7 7 pH pH 6 6 5 5 4 4 3 3 2 2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 titrant volume/ml titrant volume/ml 296 297 Figure 3. Titration curves of 5 mM aqueous solutions of cis-8 (full squares), trans-8 (open 298 squares) and the corresponding back-titrations (full and open triangles respectively), cis-9 ( full 299 down triangles) and trans-9 (open down triangles) and the corresponding back-titrations (full 300 and open diamonds respectively), at 25 °C. 301 302 The pKa values can be obtained from the pH at the half-equivalence point, and refer 303 to the aggregate, which contains a 1:1 ratio of cationic and non-ionic species. At the end 304 of the first titration with NaOH, the obtained aggregate consists mostly of non-ionic 305 surfactants, while at the end of the retro-titration with HCl, the aggregate will be mainly 306 made by cationic surfactants. 307 We can observe that the pKa of these aggregates depends mainly on the length of the 308 chain. The pKa of the half-charged aggregates for the C12-alkyl chain surfactants 8 is 309 7.2±0.2 without significant influence of the stereochemistry while the pKa for the C16 - 310 surfactants is 6.6±0.1 for the cis stereoisomer and smaller (5.9 in the direct titration and 311 7.1 for the back-titration) for the trans- stereoisomer. 14 312 Looking at the profiles of the titration curves, we can conclude that for cis-8, trans-8 313 and cis-9 there is a clear equivalence point while for trans-9 the equivalence point is 314 less defined. Moreover, there is not a buffering effect with this molecule and the pH 315 changes at almost constant slope (with some unexpected buffering effect at pH around 316 3-3.5). This different behavior could be due to the presence of strongly different 317 aggregates than in the other cases. That means that the starting aggregate is different 318 from the others, as was observed in the previous pKa measurements. Furthermore, once 319 the non-ionic aggregate is obtained and the retro-titration is carried out, the profile of all 320 the surfactants is quite similar, showing perfectly the two equivalence points. As 321 observed in Figure 3, once the non-ionic aggregate has been formed, surfactant trans-9 322 behaves as the others remarking the strong influence of the starting aggregate on the pH 323 measurements. This difference seems a bit contradictory in the present case, since the 324 passage through an insoluble phase would allow to reaching a thermodynamic state. 325 326 3.3 Surface tension measurements. 327 As stated before, these pH-sensitive surfactants are not purely cationic, but they are a 328 mixture of cationic and non-ionic species depending on the pH. Therefore, in order to 329 study their surfactant behavior, different measurements of the surface tension () were 330 carried out at fixed pH values under buffered conditions or unbuffered (pH changing 331 with surfactant concentration) conditions (see the Experimental Section). Plots of the 332 surface tension versus surfactant concentration at three different pH values are shown in 333 Figure 4. From these graphics, the parameters characterizing the surface properties of 334 these surfactants, at 25 ºC and different pH values, could be determined (Table 1). 335 15 a) (a) b) (b) 70 cis-8 C12 70 T12 trans-8 60 60 pH==22 pH Surface Tension (mN/m) Surface Tension (mN/m) pH = 2 pH=2 pH==natural pH natural 50 pH=nat pH = natural 50 pH pH==12 12 pH=12 pH = 12 40 40 30 30 20 20 CMC CMC CMC CMC CMC CMC 10 10 pH = 12 pH nat pH = 2 pH = 12 pH = 2 pH nat 0.18 mM 0.87 mM 0.87 mM 0.06 mM 0.42 mM 1.08 mM 0 0 1E-3 0.01 0.1 1 10 100 1E-3 0.01 0.1 1 10 100 Concentration (mM) Concentration (mM) (c)c) d) (d) 70 C16 cis-9 70 T16 trans-9 60 pH=2 pH = 2 60 Surface Tension (mN/m) Surface Tension (mN/m) pH pHnat = natural pH = 2 pH=2 50 pH=12 pH = 12 50 pHnat pH = natural pH = 12 pH=12 40 40 30 30 20 20 CMC CMC CMC CMC CMC CMC 10 10 pH = 12 pH = 2 pH nat pH = 12 pH = 2 pH nat 0.06 mM 0.13 mM 0.14 mM 0.06 mM 0.15 mM 0.14 mM 0 0 1E-3 0.01 0.1 1 10 1E-3 0.01 0.1 1 10 Concentration (mM) Concentration (mM) 336 Figure 1. Surface tension versus surfactant concentration for a) C12, b) T12, C) C16 and d) 337 Figure 4. Plot of surface tension versus surface concentration for cis-8 (a), trans-8 (b), cis-9 T16 at three different pH values (2, natural and 12). 338 (c), and trans-9 (d) at pH 2 (blue), natural (black) and 12 (red). 339 340 The four compounds behave as good surfactants because they reduce the surface 341 tension from 70 to less than 40 mN/m, which has been taken as an indication of 342 saturation of the water-air interface with hydrocarbon tails [38]. In Figure 4, the cmc 343 values at each pH were determined, for each surfactant, as the intersection of the two 344 fitting straight lines in the plots of  versus concentration. From the slope of the 345 curve just before the cmc, the Gibbs surface excess (max) at the air-surface 346 corresponding to the cmc was obtained. From this value, the area per molecule (Am) 347 at the surface could be calculated at pH=2 and pH=12. (See the Experimental 348 Section). 349 350 16 351 Table 1. Surface Properties of Surfactants cis- and trans-8, and cis- and trans-9 in 352 Water at 25 ºC. surfactant pH surface excess area Am (Å2) Effectiveness Πcmc cmc (mM) Γmax (μmol/m2) (mN/m) 2 4.1±0.2 41±4 41.5±1 0.87±0.15 cis-8 natural 6.7-3.4 --- 46.8±2 0.87±0.15 12 7.3±0.3 23±2 48.0±1 0.18±0.03 2 4.2±0.2 40±4 40.8±1 1.08±0.10 trans-8 natural 4.6-2.3 --- 44.3±2 0.42±0.05 12 7.6±0.3 22±2 49.9±1.5 0.08±0.04 2 4.3±0.2 39±4 44.6±1 0.13±0.02 cis-9 natural 5.0-2.5 --- 44.6±2 0.14±0.02 12 7.5±0.3 22±2 37.4±1 0.06±0.01 2 4.2±0.2 39±4 43.6±1 0.14±0.02 trans-9 natural 6.2-3.1 --- 44.4±2 0.15±0.02 12 7.8±0.3 21±2 40.2±1 0.06±0.01 353 354 355 At natural pH, for each surfactant, a mixture of species adsorbed at the surface is 356 expected. Then, at this pH, Am has not been determined, because of the lack of physical 357 meaning. Looking at the obtained values at pH 2 and 12, we can observe that all the 358 surfactants in the cationic state occupy around 40 Å2 while, when non-ionic, the area is 359 around 22 Å2, which corresponds to the minimum area that a tail alkyl chain occupies at 360 the surface [39]. This result shows that in all cases they are able to saturate the surface 361 of the solution with the corresponding carbon chains. This implies a high density of 362 methyl terminal groups, which agree with the very low surface tension. In the case of 363 C16-alkyl chain surfactant, the area per molecule of the neutral compounds is also low. 364 However, it does not correspond to a so low surface tension and the reasons for this 365 behavior are probably related to the previously observed anomaly for these C16- 366 compounds, that is, premicellar aggregation. The area per molecule of the cationic 17 367 surfactants is bigger than that of the non-ionic surfactants due to the repulsion between 368 the charges. We can compare the values obtained here with the values obtained for 369 trimethyl ammonium bromides and chlorides. For instance, the area per molecule 370 determined for tetradecyl trimethyl ammonium bromide is 61 with a small reduction to 371 59 in the presence of NaBr at 0.1 M. Chloride salts produce smaller area per molecule 372 as deduced for dodecyl trimethyl ammonium chloride in 0.1M NaCl (38 Å2) or the 373 longer chain hexadecyl trimethyl ammonium chloride in 0.1 M NaCl (46 Å2) [38]. 374 Surprisingly, the present surfactants show an area per molecule at full protonation 375 comparable to the dodecyl trimethyl ammonium chloride with small or nil increment 376 due to the bulky cyclobutane scaffold. 377 Special attention has to be paid to the obtained cmc values because they will mark 378 the working concentrations for a possible further study of the interaction with DNA. At 379 pH 2, both cis and trans diastereomers have a very similar cmc value: around 1 mM for 380 cis- and trans-8 and around 0.15 mM for cis- and trans-9. The behavior of these 381 cationic surfactants can be compared with commercial dodecyltrimethylammonium 382 bromide (DTAB), with cmc 16 mM at 25 °C [40], and CTAB, with cmc 0.98 mM, at 25 383 °C [41]. For surfactants 8 and 9, the difference in the cmc value while increasing the 384 alkyl chain length from 12 two 16 carbon atoms is smaller than in the case of DTAB 385 and CTAB. This is probably due to the presence of the cyclobutane moiety and points 386 out that surfactants 8 and 9 are more efficient than the commercial ones. Therefore, at 387 pH 2, having only cationic surfactants, stereochemistry seems not to play any 388 significant role. Nevertheless, at pH 12, the cmc values of the compounds give the 389 impression of being more dependent on the stereochemistry and on the chain length. For 390 the non-ionic surfactants, the cmc is 0.18 mM for cis-8, while cmc is 0.06 mM for 391 trans-8. Then, the trans isomer seems to be more efficient than the cis one. However, 18 392 for both cis- and trans-9 the cmc is around 0.06 mM. So, in this case, the length of the 393 chain has more influence on the solubility and, consequently, on the cmc value than the 394 stereochemistry. Therefore, both diastereomers behave similarly as non-ionic 395 surfactants at this pH. Finally, at natural pH, where there is a mixture of cationic and 396 non-ionic surfactant, the cmc values appear to have the same behavior as at pH 2, as 397 observed in Table 1, and can be compared with the obtained ones when measured in the 398 bulk of the solution. It is noteworthy the different behavior of trans-9 with respect to pH 399 and surface tension. While in surfactants 8 former there are strong differences between 400 both stereoisomers, for surfactants 9 there are not very significant differences. 401 Anomalous trends in the dependence of cmc with chain length have been previously 402 observed, particularly in the case of gemini surfactants [42]. In those cases, the decrease 403 of cmc with chain length is smaller than for classical surfactants and, even increases of 404 cmc have been observed for C16-alkyl chain surfactants compared with C12-ones. This 405 anomalous behavior has been attributed to the formation of premicellar aggregates. 406 Although the present structures are by no means gemini, it is not unconceivable that a 407 dimer could behave in similar way as to a gemini surfactants. 408 3.4 Cryo-TEM Studies. 409 The morphology of the aggregates formed by these four surfactants above the cmc, at 410 three different pH values (2, natural and 12), was studied by using cryo-TEM at –180 411 °C. Selected cryo-TEM images are shown in Figure 5. (For a more complete set of cryo- 412 TEM micrographs, see the Supporting Information, figures S3-S6). The images 413 obtained showed irregular aggregates along with some vesicles and/or fibers with 414 different size. Nevertheless, some trends were perceived allowing us to conclude that, 415 independently of the pH, stereochemistry has some influence on the morphology of the 416 aggregates and that cis surfactants seem to self-assemble preferably forming vesicles, 19 417 sometimes as multilayer aggregates, while trans surfactants, in addition to vesicles, 418 form fibers at basic pH. 419 (a) (b) (c) (d) 420 421 Figure 5. Selected cryo-TEM micrographs of a) cis-8 at natural pH, b) trans-8 at pH 12, c) cis-9 422 at pH 2, d) trans-9 at pH 12. 423 424 3.5 Computational calculations. 425 In order to gain insight in the structural features of monomeric surfactants and their 426 ability to form intramolecular hydrogen bonds, computational calculations were carried 427 out. The predicted structures are shown for cis- and trans-8 in Figure 6. The same 428 geometry of the polar head was found for cis- and trans-9, respectively. Distances of 429 hydrogen bonds between the carbonyl oxygen of the amide and hydrogen from the free 430 amine or the ammonium cation are listed in Table 3. Cationic cis-isomers form stronger 431 intramolecular hydrogen bonds than non-ionic surfactants cis-8 and cis-9. As expected, 432 the trans diastereomers present very weak intramolecular hydrogen-bonding both in the 433 cationic and non-ionic species The length of the alkyl chain also plays a role in 434 agreement with the lower solubility in water of surfactants 9 with respect to that of 8. 20 435 cationic cis-8 non-ionic cis-8 cationic trans-8 non-ionic trans-8 436 437 Figure 6. Calculated structures of cationic and non-ionic species for surfactants cis- and trans- 438 8. Non-polar hydrogen atoms are omitted for clarity. 439 440 441 Table 3. Hydrogen-bond Distances in Monomeric Surfactants cis- and trans-8, 442 and cis-and trans-9. Surfactant C=O····H-N (cationic) (Å) C=O····H-N (non-ionic) (Å) cis-8 1.979 2.469 trans-8 2.853 2.863 cis-9 2.042 2.639 trans-9 2.858 2.869 443 444 445 3.6. Circular Dichroism (CD) studies. 446 Aggregation of chiral surfactants into chiral aggregates can be followed using CD 447 spectroscopy [20,43-44]. Therefore, the analysis of the corresponding spectra between 448 200 and 250 nm would afford information about the chirality of the system [45] and one 21 449 could expect different CD spectra before and after the cmc. This assumption lies on the 450 fact that Cotton effects are extremely sensitive to changes in the chiral environment 451 close to the chromophore and to the interchromophoric interactions in supramolecular 452 aggregates [46]. The resultant CD spectra are shown in Figure 7. The shape and sign of 453 the CD signal was the same than that predicted by the theoretical CD spectrum of each 454 monomer of cis- and trans-8 in solution (See the figure S7 in Supporting Information). 455 (a) 20 (b) C12 20 T12 [θ] mdeg mM-1 cm-1 [θ] mdeg mM-1 cm-1 0 0 5.00 mM 3.76 mM 3.01 mM 5.00 mM 2.50 mM 3.76 mM -20 3.01 mM 1.25 mM 0.94 mM -20 2.50 mM 0.75 mM 1.25 mM 0.63 mM 0.94 mM 0.75 mM 0.63 mM -40 -40 190 200 210 220 230 240 250 260 190 200 210 220 230 240 250 260 Wavelength (nm) Wavelength (nm) 456 457 Figure 7. Experimental CD spectra at different concentrations of surfactants cis-8 (a) and trans- 458 8 (b). 459 460 When increasing the concentration of surfactant, the signal of the monomer is shifted 461 to longer wavelengths. The red shift could be attributed to the formation of aggregates 462 through the creation of hydrogen bonds involving the chromophore group, i.e. the 463 amide, that provokes a change in the transition energy [18,46]. 464 Measurements of the UV-vis absorption of each surfactant in aqueous solution were 465 carried out (see figure S8 in the Supporting Information) and, in fact, a slight red shift 466 can be observed from low to higher concentrations, in agreement with CD results. 467 However, max in UV-vis is not the same as max in CD because there is a change from 468 single molecules to aggregates at the cmc, as can been appreciated in the representation 469 of CD max values in function of the surfactant concentration (Figure 8). 22 470 a) b) 220 220 215 215 Wavelength (nm) Wavelength (nm) 210 210 T12 C12 205 205 200 CMC = 1.1 mM 200 1.1 mM 1.2 mM CMC = 1.2 mM 195 0 1 2 3 4 5 6 0 1 2 3 4 5 Concentration (mM) Concentration (mM) 471 472 Figure 8. Plots of CD max versus concentration of surfactant cis-8 (a) and trans-8 (b). 473 474 Both graphics show a sharp change in the slope at values closely related to those 475 determined for the cmc in solution (see previous sections in this manuscript). Therefore, 476 in these cases, CD spectroscopy can be used for the determination of the cmc because it 477 is known that this parameter contains a range of concentrations and its true value can 478 vary up to one order of magnitude depending on the method used and on the measured 479 physical property [17,20]. The determination of the cmc of a surfactant by the change in 480 UV-vis absorption related to the formation of aggregates had been already reported [47- 481 49]. However, these techniques are based only on the intensity of the absorption and not 482 on the max value and a clear peak in the spectra should be observed in order to 483 determine the maximum. Even so, this technique is restricted to surfactants that self- 484 assemble through a chromophore located in a chiral environment. 485 Surfactants cis- and trans-9 were also investigated (see figure S9 in the Supporting 486 Information). Nevertheless, due to their low solubility and, consequently, their low cmc 487 value (around 0.1-0.2 mM) the method became limited to the study of a narrow range of 488 concentrations and reliable conclusions could not be obtained. 489 3.7. Cytotoxicity of the surfactants. 23 490 Encouraged by the interesting properties found for surfactants 8 and 9 and focusing on 491 the possibility to use them as non viral vectors for gene therapy, a preliminary 492 investigation of their cytotoxicity was carried out by means of the MTT assay. Standard 493 HeLa cells were used for this study. The 100% living cells was considered as the result 494 of a control experiment performed in absence of the surfactants and under the same 495 conditions as explained in the Experimental Section. The average toxicity results of four 496 replicates for each surfactant are shown in Figure 9. We can observe that surfactants 8 497 and 9 are non-toxic in contrast to DTAB and CTAB, which are commonly used in 498 biological experiments. 499 120 100 % Living cells 80 60 40 20 0 DTAB C12 T12 CTAB C16 T16 DTAB cis-8 trans-8 CTAB cis-9 trans-9 Surfactant Surfactant 500 501 Figure 9. Toxicity results of the MTT assay with surfactants DTAB, cis- and trans-8, CTAB, 502 cis- and trans-9 using HeLa cells. Error bars represent standard deviations (SD) of four 503 independent experiments. 504 505 4. Conclusions 506 Two pairs of cis/trans diastereomeric -amino acid-based cationic amphiphiles have 507 been synthesized and studied. They contain C12- and C16-alkyl chains, respectively, as 508 hydrophobic tails and the polar head consists of an ammonium cation linked to a 24 509 cyclobutane ring. The pH of the medium modulates their physicochemical properties, 510 such as the cmc, the ratio of cationic versus non-ionic species and the surface tension 511 magnitude. At the same time the aggregation state influences on the apparent pKa values 512 of the aggregates, with release of protons at the cmc. The cmc for all of them is lower 513 than that for commercial DTAB and CTAB, which is probably due to the steric 514 restrictions imposed by the cyclobutane unit. Moreover, the adsorption effectiveness is 515 around 40 mN/m in all cases, which is in accordance with an efficient surfactant 516 behavior. The tail-length influences the surfactant properties and those with a C16-alkyl 517 chain are better than those with a shorter tail. In terms of surface tension there is some 518 effect of stereochemistry regarding the position of cmc for the C12- derivatives, while 519 there is no effect for C16- or on the area per molecule. Aggregation influences on the 520 apparent pKa of these molecules with trans-9 behaving as a much more acidic molecule 521 than expected, but this particular behavior does not translate to other properties. The 522 stereochemistry also determines the predominant mode of self-assembling since the 523 trend for cis-isomers is to form micelles or vesicles while trans-isomers preferably form 524 fibers. CD spectroscopy supports the formation of aggregates through intermolecular 525 hydrogen-bonding between the amide groups of the monomers. Moreover, by 526 considering the relationship of the CD max with the concentration, an alternative 527 method to determine the cmc of these surfactants is provided. This methodology could 528 be general but is restricted to those surfactants in which the chromophore is located in a 529 chiral environment. Finally, the non- toxicity of the surfactants has been verified by the 530 MTT assay. This characteristic, jointly with the efficiency and the good properties 531 shown, the fact that the cationic species are present in a high concentration at 532 physiological pH, as well as the weak acid behavior of the aggregates, confirm these 25 533 amphiphiles as promising candidates to be used as non-viral vectors for gene therapy 534 applications. Further investigation in this field is being carried out in our laboratories. 535 536 ASSOCIATED CONTENT 537 Supporting Information 538 Synthetic procedures, characterization, and 1H and 13 C NMR spectra of the new 539 compounds; charge distribution at different concentration of the surfactants; cryo-TEM 540 micrographs of the surfactants at different pH values; calculated CD spectra for cis- and 541 trans-8; CD and UV-vis absorption spectra of cis- and trans-9. 542 AUTHOR INFORMATION 543 Corresponding Authors 544 *E-mail: ramon.pons@iqac.csic.es (R.P.). 545 *E-mail: rosa.ortuno@uab.es (R.M.O.). 546 ORCID 547 Bernat Pi-Boleda: 0000-0003-1050-6788 548 Ona Illa : 0000-0001-7390-4893 549 Vicenç Branchadell : 0000-0003-3480-1669 550 Ramon Pons : 0000-0003-4273-9084 551 Rosa M. Ortuño: 0000-0001-7635-7354 552 Present Address 553  Kao Corporation, S.A.; Puig dels Tudons, 10, 08210 Barberà del Vallès, Barcelona, 554 Spain 555 § Kern Pharma, S.L. ; Venus, 72, Pol. Ind. Colón II, 08228 Terrassa, Barcelona, Spain 556 26 557 Notes 558 The authors declare no competing financial interest. 559 ACKNOWLEDGMENTS 560 Financial support from MINECO (CTQ2016-77978-R, AEI/FEDER, UE, and 561 CTQ2017-88948-P) is gratefully acknowledged. Ms. Imma Carrera is gratefully 562 acknowledged for help with the surface tension measurements. 563 564 REFERENCES [ 1 ] Cavazzana-Calvo, M. Gene Therapy of Human Severe Combined Immunodeficiency (SCID)-X1 Disease. Science 2000, 288 (5466), 669–672. [2] Manno, C. S.; Pierce, G. F.; Arruda, V. R.; Glader, B.; Ragni, M.; Rasko, J. J.; Rasko, J.; Ozelo, M. C.; Hoots, K.; Blatt, P.; Konkle, B.; Dake, M.; Kaye, R.; Razavi, M.; Zajko, A.; Zehnder, J.; Rustagi, P. K.; Nakai, H.; Chew, A.; Leonard, D.; Wright, J. F.; Lessard, R. R.; Sommer, J. M.; Tigges, M.; Sabatino, D.; Luk, A.; Jiang, H.; Mingozzi, F.; Couto, L.; Ertl, H.C.; High, K. A.; Kay, M. A. Successful Transduction of Liver in Hemophilia by AAV-Factor IX and Limitations Imposed by the Host Immune Response. Nat. Med. 2006, 12, 342–347. [3] Gene and Stem Cell Therapy; Boyd, A. C., Ed.; Karger: Basel, New York, 2006. [4] Maguire, A. M.; Simonelli, F.; Pierce, E. A.; Pugh, E. N.; Mingozzi, F.; Bennicelli, J.; Banfi, S.; Marshall, K. A.; Testa, F.; Surace, E. M.; et al. Safety and Efficacy of Gene Transfer for Leber’s Congenital Amaurosis. N. Engl. J. Med. 2008, 358, 2240–2248. [5] Kaplitt, M. G.; Feigin, A.; Tang, C.; Fitzsimons, H. L.; Mattis, P.; Lawlor, P. A.; Bland, R. J.; Young, D.; Strybing, K.; Eidelberg, D.; et al. Safety and Tolerability of Gene Therapy with an Adeno-Associated Virus (AAV) Borne GAD Gene for Parkinson’s Disease: An Open Label, Phase I Trial. Lancet 2007, 369, 2097–2105. [6] Yang, Z. R.; Wang, H. F.; Zhao, J.; Peng, Y. Y.; Wang, J.; Guinn, B.-A.; Huang, L. Q. Recent Developments in the Use of Adenoviruses and Immunotoxins in Cancer Gene Therapy. Cancer Gene Ther. 2007, 14, 599–615. [7] Advances in Genetics, Volume 89; Friedmann, T., Dunlap, J. C., Goodwin, S. F., Eds.; Elsevier Inc.: Oxford, 2015. [8] DNA Interaction with Polymers and Surfactants:; Dias, R. S., Lindman, B., Eds.; John Wiley & Sons, Inc: New Jersey, 2008. 27 [9] Carlstedt, J.; Lundberg, D.; Dias, R. S.; Lindman, B. Condensation and Decondensation of DNA by Cationic Surfactant, Spermine, or Cationic Surfactant-Cyclodextrin Mixtures: Macroscopic Phase Behavior, Aggregate Properties, and Dissolution Mechanisms. Langmuir 2012, 28, 7976–7989. [10] Grueso, E.; Cerrillos, C.; Hidalgo, J.; Lopez-Cornejo, P. Compaction and Decompaction of DNA Induced by the Cationic Surfactant CTAB. Langmuir 2012, 28, 10968–10979. [11] Pinnaduwage, P.; Schmitt, L.; Huang, L. Use of a Quaternary Ammonium Detergent in Liposome Mediated DNA Transfection of Mouse L-Cells. Biochim. Biophys. Acta 1989, 985, 33–37. [12] Pérez, N.; Pérez, L.; Infante, M. R.; García, M. T. Biological Properties of Arginine-Based Glycerolipidic Cationic Surfactants. Green Chem. 2005, 7, 540–546. [13] Infante, M. R.; Pérez, L.; Morán, M. C.; Pons, R.; Mitjans, M.; Vinardell, M. P.; Garcia, M. T.; Pinazo, A. Biocompatible Surfactants from Renewable Hydrophiles. Eur. J. Lipid Sci. Technol. 2010, 112, 110–121. [ 14 ] Pinazo, A.; Pons, R.; Pérez, L.; Infante, M. R. Amino Acids as Raw Material for Biocompatible Surfactants. Ind. Eng. Chem. Res. 2011, 50, 4805–4817. [15] Chandra, N.; Tyagi, V. K. Synthesis, Properties, and Applications of Amino Acids Based Surfactants: A Review. J. Dispers. Sci. Technol. 2013, 34, 800–808. [16] Foley, P.; Kermanshahi pour, A.; Beach, E. S.; Zimmerman, J. B. Derivation and Synthesis of Renewable Surfactants. Chem. Soc. Rev. 2012, 41, 1499–1518. [17] Mezei, A.; Pérez, L.; Pinazo, A.; Comelles, F.; Infante, M. R.; Pons, R. Self Assembly of pH-Sensitive Cationic Lysine Based Surfactants. Langmuir 2012, 28, 16761–16771. [18] Bordes, R.; Tropsch, J.; Holmberg, K. Role of an Amide Bond for Self-Assembly of Surfactants. Langmuir 2010, 26, 3077–3083. [19] Sorrenti, A.; Illa, O.; Ortuño, R. M. Amphiphiles in Aqueous Solution: Well Beyond a Soap Bubble. Chem. Soc. Rev. 2013, 42, 8200-8219. [20] Sorrenti, A.; Illa, O.; Pons, R.; Ortuño, R. M. Chiral Cyclobutane -Amino Acid-Based Amphiphiles: Influence of cis/trans Stereochemistry on Solution Self-Aggregation and Recognition. Langmuir 2015, 31, 9608-0618. [21] Sorrenti, A.; Illa, O.; Ortuño, R. M.; Pons, R. Chiral Cyclobutane -Amino-Acid Based Amphiphiles: Influence of cis/trans Stereochemistry on Condensed Phase and Monolayer Structure. Langmuir 2016, 32, 6977-6984. [22] Pi-Boleda, B.; Sorrenti, A.; Sans, M.; Illa, O.; Pons, R.; Branchadell, V.; Ortuño, R. M. Cyclobutane Scaffold in Bolaamphiphiles: Effect of Diastereoisomerism and Regiochemistry on Their Surface Activity Aggregate Structure. Langmuir, 2018, 34, 11424-11432. 28 [ 23 ] Zhao, Y.; Truhlar, D. G. The M06 Suite of Density Functionals for Main Group Thermochemistry, Thermochemical Kinetics, Noncovalent Interactions, Excited States, and Transition Elements: Two New Functionals and Systematic Testing of Four M06-Class Functionals and 12 Other Functionals. Theor. Chem. Acc. 2007, 120, 215–241. [24] Gaussian 09, Revision E.01, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.et al. Gaussian, Inc., Wallingford CT, 2009. [25] Bauernshmitt, R.; Ahlrichs, R. Treatment of Electronic Excitations within the Adiabatic Approximation of Time Dependent Density Functional Theory. Chem. Phys. Lett. 1996, 256, 454–464. [26] Autschbach, J.; Ziegler, T.; van Gisbergen, S. J. A.; Baerends, E. J. Chiroptical Properties from Time-Dependent Density Functional Theory. I. Circular Dichroism Spectra of Organic Molecules. J. Chem. Phys. 2002, 116, 6930. [27] Stratmann, R. E.; Scuseria, G. E.; Frisch, M. J. An Efficient Implementation of Time- Dependent Density-Functional Theory for the Calculation of Excitation Energies of Large Molecules. J. Chem. Phys. 1998, 109, 8218-8224. [28] Mosnann, T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays J. Immunol. Methods 1983, 65, 55-63. [29] Rahbari, R.; Sheahan, T.; Modes, V.; Collier, P.; Macfarlane, C.; Badge, R.M. A Novel L1 Retrotransposon Marker for HeLa Cell Line Identification. BioTechniques. 2009, 46, 277-284. [30] Izquierdo, S.; Rúa, F.; Sbai, F.; Parella, T.; Álvarez-Larena, A.; Branchadell, V.; Ortuño, R. M. J. Org. Chem. 2005, 70, 7963-7971. [31] Fernandes, C.; Pereira, E.; Faure, S.; Aitken, D. J. J. Org. Chem. 2009, 74, 3217-3220. [32] Matulis, D.; Bloomfield, V.A. Thermodynamics of the hydrophobic effect. I. Coupling of aggregation and pKa shifts in solutions of aliphatic amines. Biophysical Chemistry 2001, 93, 37- 51. [33] Goldsipe, A.; Blankschtein, D. Molecular-Thermodynamic Theory of Micellization of pH- Sensitive Surfactants. Langmuir 2006, 22, 3547–3559. [34] Goldsipe, A.; Blankschtein, D. Titration of Mixed Micelles Containing a pH-Sensitive Surfactant and Conventional (pH-Insensitive) Surfactants: A Regular Solution Theory Modeling Approach. Langmuir 2006, 22, 9894–9904. [35] Söderman, O.; Jönsson, B.; Olofsson, G. Titration of Fatty Acids Solubilized in Cationic and Anionic Micelles. Calorimetry and Thermodynamic Modeling. J. Phys. Chem. B 2006, 110, 3288–3293. [36] Dai, Q.; Laskowski, J.S. The Krafft point of dodecylammonium chloride: pH effect. Langmuir 1991, 7, 1361-1364 29 [37] Fielden, M. L.; Perrin, C.; Kremer, A.; Bergsma, M.; Stuart, M. C.; Camilleri, P.; Engberts, J. B. F. N. Sugar-Based Tertiary Amino Gemini Surfactants with a Vesicle-to-Micelle Transition in the Endosomal pH Range Mediate Efficient Transfection in Vitro. Eur. J. Biochem. 2001, 268, 1269–1279. [38] Rosen, M. J.; Kunjappu, J. T. Surfactants and Interfacial Phenomena, 4th ed.; John Wiley & Sons, Inc.: New York, 2012. [39] Nagarajan, R. Self Assembly of Bola Amphiphiles. Chem. Eng. Commun. 1987, 55, 251– 273. [40] Causi,S.; DeLisi, R.; Milioto, S.; Tirone, N. Dodecyltrlmethylammonium Bromide in Water-Urea Mixtures. Volumes, Heat Capacities, and Conductivities. J. Phys. Chem., 1991, 95, 5664-5673. [41] Okuda, H.; Imae, T.; Ikeda, S. The Adsorption of Cetyltrimethylammonium Bromide on Aqueous Surfaces of Sodium Bromide Solutions. Colloids and Surfaces 1987, 27, 187–200. [ 42 ] Menger, F. M.; Littau, C. A. Gemini Surfactants: A New Class of Self-Assembling Molecules. J. Am. Chem. Soc. 1993, 115, 10083-10090. [43] Mohanty, A.; Dey, J. Effect of the Headgroup Structure on the Aggregation Behavior and Stability of Self-Assemblies of Sodium N-[4-(n-Dodecyloxy)benzoyl]-L-Aminoacidates in Water. Langmuir 2007, 23, 1033–1040. [44] El-Hachemi, Z.; Mancini, G.; Ribó, J. M.; Sorrenti, A. Role of the Hydrophobic Effect in the Transfer of Chirality from Molecules to Complex Systems: From Chiral Surfactants to Porphyrin/surfactant Aggregates. J. Am. Chem. Soc. 2008, 130, 15176–15184. [45] Circular Dichroism: Principles and Applications, 2nd ed.; Berova, N., Nakanishi, K., Woody, R. W., Eds.; Wiley-VCH: Weinheim, 1994. [46] Pescitelli, G.; Di Bari, L.; Berova, N. Application of Electronic Circular Dichroism in the Study of Supramolecular Systems. Chem. Soc. Rev. 2014, 43, 5211–5233. [47] Yu, D.; Huang, F.; Xu, H. Determination of Critical Concentrations by Synchronous Fluorescence Spectrometry. Anal. Methods 2012, 4, 47–49. [48 ] Tanhaei, B.; Saghatoleslami, N.; Chenar, M. P.; Ayati, A.; Hesampour, M.; Mänttäri, M. Experimental Study of CMC Evaluation in Single and Mixed Surfactant Systems, Using the UV–Vis Spectroscopic Method. Journal of Surfactants and Detergents, 2013, 16, 357-362. [49] Polavarapu, P. L.; Vijay, R. Chiroptical Spectroscopy of Surfactants. J. Phys. Chem. A 2012, 116, 5112–5118. 30