Structures and Surface Properties of âCyclicâ Polyoxyethylene Alkyl

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Structures and Surface Properties of "Cyclic" Polyoxyethylene Alkyl Ethers: Unusual Behavior of Cyclic Surfactants in Water Yuki Hirose, Toshiaki Taira, Kenichi Sakai, Hideki Sakai, Akira Endo, and Tomohiro Imura Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b01553 • Publication Date (Web): 27 Jul 2016 Downloaded from http://pubs.acs.org on August 6, 2016

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Structures and Surface Properties of "Cyclic" Polyoxyethylene Alkyl Ethers: Unusual Behavior of Cyclic Surfactants in Water

Yuki Hirose,a Toshiaki Taira,b Kenichi Sakai,a Hideki Sakai,a Akira Endo,b and Tomohiro Imura,b,* a Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan b Research Institute for Chemical Process Technology, National Institute of Advanced Industrial Science and Technology (AIST), Central 5-2, 1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan.

KEYWORDS Cyclic surfactant, polyoxyethylene alkyl ether, self-assembly, cloud point, detergent enzyme

ACS Paragon Plus Environment

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ABSTRACT

The cyclization of amphiphiles has emerged as an attractive strategy for inducing remarkable properties in these materials without changing their chemical composition. In this study, we successfully synthesized three cyclic polyoxyethylene dodecyl ethers (c-POEC12's) with different ring sizes and explored the effects of their topology on their surface and self-assembling properties related to their function, comparing them with those of their linear counterparts (lPOEC12's). The surface activity of the c-POEC12's remained almost constant despite the change in their hydrophobic and hydrophilic balance (HLB) value, while that of the l-POEC12's decreased with an increase in the HLB value as general surfactants. In contrast to the normal micelles seen in the case of the l-POEC12's (3.4–9.7 nm), the cyclization of the POEC12's resulted in the formation of large spherical structures 72.8–256.8 nm in size. It also led to a dramatic decrease of 28 °C in the cloud point temperature. Furthermore, the cyclization of the POEC12's markedly suppressed the rate of protease hydrolysis caused by the surfactants. The initial rate of reduction of a detergent enzyme from Bacillus licheniformis was increased by more than 40% in the case of c-POE600C12 and c-POE1000C12, even though they exhibited surface activities almost equal to or higher than those of their linear counterparts. These results suggest that cyclization induces unusual aqueous behaviors in POEC12, making the surfactant milder with respect to detergent enzymes while ensuring it exhibits increased surface activity.


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1. INTRODUCTION Cyclic molecules are often observed in nature (e.g., DNA,1 peptides,2 and membrane lipids in archaea3). They play critical roles in various biological systems and exhibit unique properties, which arise from their cyclic topology. It is well known that several archaea living in harsh conditions such as high-temperature, acidic and/or salt-rich environments have unique membranes of ether lipids with a cyclic topology.3 The cyclic structure of single-walled bilayer membrane lipids is believed to be responsible for their thermal and salt resistances. Inspired by this, Tezuka et al. recently studied the thermal resistance of micelles formed from cyclic amphiphilic diblock copolymers.4 They synthesized a cyclized poly(butyl acrylate)-bpoly(ethylene oxide) by the ring-closing "metathesis" of the terminal allyl telechelic precursors under highly dilute conditions and found that the cloud point of the micelles was more than 40 °C higher than that of their linear counterpart, namely, (poly(butyl acrylate)-b-poly(ethylene oxide)-b-poly(butyl acrylate)). Another end-to-end ring closure strategy using linear α-alkyne-ωazido precursors was reported by Grayson and coworkers.5 Poly(N-isopropylacrylamide)s (PNIPAM)s, which are thermoresponsive polymers, were cyclized by the click-ring closure of their linear α-alkyne-ω-azido PNIPAM precursors. The cyclic form of the PNIPAM derivative formed flower-like micelles6 and exhibited a lower phase-separation temperature7 in aqueous solution, owing to the stringent restrictions with respect to the backbone conformations. Thus, the glass-transition temperature of the cyclic PNIPAMs in bulk was higher than that of their linear counterparts.8 The effects of the cyclic topology on the dynamic or crystallization behavior of polymers have also been studied. It was found that cyclic poly(ε-caprolactones) exhibit higher segmental mobilities and crystallinities.9


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More recently, it was reported that cyclic poly(ethylene imine) (PEI) architectures exhibit potential for use as gene delivery systems, as cyclic PEIs show a higher transfection efficiency than do linear PEIs, as well as reduced cytotoxicity in the case of in-vitro transfection assays.10 Although remarkable progress has been made in the synthesis and purification of cyclic molecules in recent years,11 the synthesis is still generally performed under highly diluted conditions (< 10-5 M), in order to avoid their intermolecular oligomerization. The lack of efficient synthesis protocols that do not involve the addition of undesirable functional groups (e.g., triazole or olefin) has limited the widespread use of cyclic molecules, in spite of their remarkable polymeric properties. In addition to showing promise as polymers, cyclic molecules also have potential as surfactants. For instance, one of the most powerful microbial biosurfactants, the so-called "surfactin," has a unique cyclic structure. It consists of a heptapeptide ring and dramatically decreases the surface tension of water to 27 mN/m at the critical micelle concentration (CMC, 2.7 ×10-5 M).12 The restriction in the conformational freedom caused by cyclization increases its area per molecule at the interface; this allows the molecules to self-assemble into large structures even at extremely low concentrations.13 In addition to exhibiting these unusual properties, surfactin is less toxic than linear synthetic surfactants.14 However, only a few studies15, 16 have been performed on the synthesis and properties of the cyclic forms of surfactants. Although the formulation of surfactants in aqueous liquid detergents without a loss in the protease activity remains an issue,17 the cyclization of normal surfactants is likely to induce unusual aqueous behaviors, along with changes in their interactions with biological molecules such detergent enzymes.


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In this study, the "cyclic" forms of a polyoxyethylene dodecyl ether (c-POEC12) with different ring sizes and their exact linear counterparts (l-POEC12) were systematically synthesized without using the highly dilution method. The effect of the cyclic topology on the surface and self-assembly properties were examined by measuring the surface tension and cloud point temperature, as well as through dynamic light scattering (DLS) measurements and freezefracture transmission electron microscopy (FF-TEM). Moreover, it was found that the cyclization of POEC12 significantly retards the further suppression of the detergent protease hydrolysis rate attributable to the surfactant.

2. EXPERIMENTAL SECTION 2.1. Materials. Commercial reagents were purchased from Wako Pure Chemical Industries, Ltd., unless otherwise stated. PEG400 and PEG600 were freeze-dried, while PEG1000 was dried under reduced pressure before use. DMF (99.5%) and hexane (96.0%) were obtained by dehydration

with

molecular

sieves

overnight.

1,12-dibromododecane

(97.0

%),

1-

bromododecane (98.0 %, Tokyo Chemical Industry Co., LTD.), sodium hydride (50~72%), dodecyl 3-mercaptopropionate (98.0 %), and 2,2’-azodiisobutylronitrile (AIBN) (98.0 %) were used as received. 3N-HCl was prepared by the dilution of concentrated HCl with ultrapure water. Chloroform (99.0%) and acetonitrile (99.7%) were used as received, and so was sodium chloride. N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA) (98.0%, Sigma-Aldrich) and the protease from B. licheniformis were used as received. Tris-HCl was prepared by dissolving Tris-HCl powder in ultrapure water.

2.2. Synthesis and purification 2.2.1. Cyclic POE400C12. A 0.96 g (0.024 mol) sample of sodium hydride and 500 mL of dry DMF were placed in a flame-dried, argon-purged 1 L three-neck flask equipped with a


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magnetic stir bar. A 60 mL portion of 1.60 g (0.004 mol) of PEG400 in DMF and 240 mL of dry hexane were added to the flask under stirring. After being added to the flask, the solution was stirred at 55 °C on a mantle heater for 1 h. Next, 1.31 g (0.004 mol) of 1,12dibromododecane was added; this was followed by continuous stirring at 55 °C for 24 h. The reaction was then stopped by adding 3N-HCl for neutralization. The solvent was evaporated under reduced pressure and chloroform was added subsequently. After filtration to remove the salts, the chloroform was vaporized to obtain a crude sample. Preparative RP-HPLC was performed to obtain cyclic POE400C12 and the linear byproduct (an HPLC apparatus consisting of a GL-7410 system (GL Sciences Inc.) and a YRD-880 midget refractive index detector (Shimamura Tech Co, Ltd.) with a Tosoh model CO-8011 column (C18) was used at 40 °C). Acetonitrile/water (=65/35) was used as the eluent at a flow rate of 9.0 mL/min. A mixture of the two compounds (0.75 g) was obtained after the evaporation of the eluent and drying in vacuo. The ratio of cPOE400C12 and the linear byproduct was 45/55 as estimated by the peak ratio of the 1HNMR spectrum. To remove the linear byproduct, which included olefin, a thiol-ene reaction was performed. The mixture, dodecyl 3-mercaptopropionate, and AIBN (molar ratio [C=C]/[SH]/[AIBN] = 1 : 2 : 1) were placed in a test tube and then stirred at 80 °C in an oil bath for 1 h in an argon atmosphere. After the confirmation of the completion of the reaction, cyclic POE400-C12 was purified by preparative RP-HPLC (acetonitrile/water = 55/45). This was followed by evaporation and drying under reduced pressure. cPOE400C12 was obtained as a clear oil. Yield: 0.24 g (9%). MALDI-TOF MS: [M+Na]+=647.64(n=10, calcd=647.84), 1H-NMR: (400 MHz, D2O): δ (ppm) = 3.80-3.62 (OCH2CH2O-), 3.56 (t, J = 6.5 Hz, 4H, -CH2O-), 1.59 (m, 4H, -CH2CH2O-), 1.42-1.27 (16H, CH2-), 13C-NMR: 71.5, 70.7, 70.3, 29.4, 26.1. 2.2.2. Cyclic POE600C12. A 0.96 g (0.024 mol) sample of sodium hydride and 500 mL of dry DMF were placed in a flame-dried, argon-purged 1 L three-neck flask equipped with a magnetic stir bar. A 60 mL portion of 2.40 g (0.004 mol) of PEG600 in DMF and 240 mL of dry hexane were added under stirring. The solution was then stirred at 55 ºC on a mantle heater for 1 h. Next, 1.31 g (0.004 mol) of 1,12-dibromododecane was added; this


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was followed by continuous stirring at 55 °C for 24 h. The reaction was stopped by adding 3N-HCl for neutralization. The solvent was evaporated under reduced pressure and chloroform was added subsequently. After filtration to remove the salts, the chloroform was vaporized to obtain a crude sample. Preparative RP-HPLC (acetonitrile/water = 65/35) was performed to obtain cyclic POE400C12 and a linear derivative possessing an olefin bond on an end group. The mixture of the two compounds was obtained (1.20 g) after the evaporation of the eluent and drying in vacuo. The ratio of c-POE600C12 and the linear byproduct was 42/58 as calculated by the peak ratio of the 1H-NMR spectrum. The mixture, dodecyl 3-mercaptopropionate, AIBN (molar ratio [C=C]/[SH]/[AIBN] = 1 : 2 : 1) were placed in a test tube and then stirred at 80 °C in an oil bath for 1 h in an argon atmosphere. After the confirmation of the completion of the reaction, c-POE600C12 was purified by preparative RP-HPLC (acetonitrile/water = 50/50) followed by evaporation and drying under reduced pressure. c-POE600C12 was obtained as a clear oil. Yield: 0.24 g (9%). MALDI-TOF MS：[M+Na]+=735.68(n=12, calcd=735.95), 1H-NMR: (400 MHz, D2O): δ (ppm) = 3.80-3.64 (-OCH2CH2O-), 3.56 (t, J = 6.6 Hz, 4H, -CH2O-), 1.60 (m, 4H, CH2CH2O-), 1.43-1.26 (16H, -CH2-), 13C-NMR: 71.5, 70.7, 70.2, 29.7, 26.1. 2.2.3. Cyclic POE1000C12. A 0.96 g (0.024 mol) sample of sodium hydride and 500 mL of dry DMF were placed in a flame-dried, argon-purged 1 L three-neck flask equipped with a magnetic stir bar. A 60 mL portion of 4.00 g (0.004 mol) of PEG1000 in DMF and 240 mL of dry hexane were added under stirring to the flask. The solution was then stirred at 55 °C on a mantle heater for 1 h. Next, 1.31 g (0.004 mol) of 1,12-dibromododecane was added; this was followed by continuous stirring at 55 °C for 24 h. The reaction was stopped by adding 3N-HCl for neutralization. The solvent was evaporated under reduced pressure and chloroform was added subsequently. After filtration to remove the salts, the chloroform

was

vaporized

to

obtain

a

crude

sample.

Preparative

RP-HPLC

(acetonitrile/water = 60/40) was performed to obtain c-POE600C12 and the linear derivative, which contained an olefin bond on an end group. A mixture of the two compounds (1.23 g) was obtained after the evaporation of the eluent and drying in vacuo. The ratio of c-POE600C12 and the linear byproduct was 43/57 as calculated by the peak


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ratio of the 1H-NMR spectrum. The mixture, dodecyl 3-mercaptopropionate, AIBN (molar ratio [C=C]/[SH]/[AIBN]=1:2:1) were placed in a test tube and then stirred at 80 °C in an oil bath for 1 h in an argon atmosphere. After the confirmation of the completion of the reaction, c-POE1000C12 was purified by preparative RP-HPLC (acetonitrile/water = 50/50) followed by evaporation and drying under reduced pressure. c-POE1000C12 was obtained as a white solid. Yield: 0.33 g (7%). MALDI-TOF MS: [M+Na]+=1219.80(n=23, calcd=1220.53), 1H-NMR: (400 MHz, D2O): δ (ppm) = 3.78-3.64 (-OCH2CH2O-), 3.58 (t, J = 6.6 Hz, 4H, -CH2O-), 1.60 (m, 4H, -CH2CH2O-), 1.42-1.25 (16H, -CH2-), 13C-NMR: 71.4, 70.5, 70.1, 29.6, 26.1. 2.3. Nuclear Magnetic Resonance (NMR). The 1H-NMR spectra were recorded on a NMR spectrometer (Avance 400M, Bruker Biospin Co., Inc.) operated at 400 MHz. D2O was used as the deuterated solvent. The 13C-NMR spectra were recorded on the same instrument at 100 MHz. CDCl3 was used as the deuterated solvent. Both types of spectra were calibrated using the residual undeuterated solvent as the internal reference (D2O: 4.79 ppm, CHCl3: 77.16 ppm). 2.4.

Matrix-Assisted

Laser

Desorption/Ionization

Time-of-Flight

Mass

Spectrometry (MALDI-TOF MS). The MALDI-TOF spectra were recorded on a mass spectrometer (AutoflexTM Series, Bruker Daltonics Co., Inc.) in the reflector mode. 2,5Dihydroxybenzoic acid was used as the matrix, and trifluoroacetic acid was added to improve the degree of ionization. The absolute molecular weights and PDI values were determined as per previously reported procedures.22 2.5. Gel Permeation Chromatography (GPC). The GPC curves were obtained using a HPLC apparatus consisting of a Tosoh model DP-8020 system and a RI-8020 refractive index detector with tandem-linked Shodex LF-804 columns at 40 °C. Chloroform was used as the eluent at a flow rate of 1.0 mL/min. The concentration of the samples was 1 mg/mL. The relative average molecular weights and PDI values were determined from the calibration curve obtained using polystyrene standards. The relative average molecular weights as determined by GPC were as follows: c-POE400C12; Mn = 683 g/mol, Mw/Mn =


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1.06, c -POE600C12; Mn = 868 g/mol, Mw/Mn = 1.03, c-POE1000C12; Mn = 1594 g/mol, Mw/Mn = 1.05, l-POE400C12; Mn = 705 g/mol, Mw/Mn = 1.05, l-POE600C12; Mn= 1146 g/mol, Mw/Mn = 1.05, l-POE1000-C12; Mn = 1980 g/mol, Mw/Mn = 1.05. 2.6. Reverse-Phase High Performance Liquid Chromatography (RP-HPLC). The RPHPLC profiles were obtained using a HPLC apparatus consisting of a Tosoh model DP8020 system and a RI-8020 refractive index detector with a Lichrospher 100 RP-18 column (C18) (Agilent Technology Co., Inc.) at 40 °C. A mixed solvent of HPLC-grade acetonitrile and ultrapure water (65/35) was sufficiently degassed and used as the eluent at a flow rate of 1.0 mL/min. For each sample, the concentration was set at 1 mg/mL. 2.7. Surface Tension Measurements. The surface tensions of the POEC12 aqueous solutions were determined by the pendant drop method at 25 °C, which was performed using an apparatus consisting of an automatic interfacial tensiometer (DM500, Kyowa Interface Science) and the Drop Shape Analysis software FAMAS (version 2.01). A drop was formed at the tip of a syringe by pressing the solution out by means of a setscrew. The drop shape analysis was performed as follows: a drop profile was extracted from the image of the drop. Then, a curve fitting program compared the experimental drop profile with a theoretical one (Young-Laplace method) and gave the corresponding surface tension value. The samples were prepared by diluting the stock solutions with ultrapure water, and ageing them overnight. For each concentration, the evolution of the drop surface tension was followed over 15 min. 2.8. Dynamic Light Scattering (DLS) Measurements. The size distributions of the assemblies of the synthesized POEC12's were measured with a DLS instrument (DLS7000, Otsuka Electronics Co.) using an Ar laser with a wavelength of 488 nm as the light source at 75 mW at 25 °C. The time-dependent correlation function of the scattered light intensity was measured at a scattering angle of 60° or 90°. The DLS intensity data were processed using the instrumental software to obtain the hydrodynamic diameter, PDI, and mass-diffusion coefficient values of the samples. The mass-diffusion coefficient (D) was derived from the decay time (τc) of the intensity autocorrelation function using


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D=(2kL2τc)-1, in which kL is the scattering wave vector. The hydrodynamic mass-diffusion coefficient, D0, was obtained as the limit of D as kL tended to zero. In this study, the autocorrelation function was analyzed using the histogram method.27 2.9. Freeze-Fracture Transmission Electron Microscopy (FF-TEM). FF-TEM was used to determine the structure of the assembly formed by c-POE600C12 at 20 mM. The sample was frozen with liquid nitrogen at -189 ºC. The fracturing process was performed with a freeze-fracture specimen preparation system (JFD-V, JEOL). Subsequently, sublimation was peformed in order to be able to visualize the structure clearly. Then, the fractured surface was replicated by first evaporating platinum-carbon and then carbon to strengthen the replica. After the replica had been washed, it was examined and photographed using a transmission electron microscope (JEM-1010, JEOL) at 110 kV. 2.10. Cloud Point Measurements. The optical transmittances of the aqueous POEC12 solutions (10 mg/ml) at a wavelength of 650 nm were acquired on a UV/Vis spectrometer (V-560, JASCO) equipped with a ETC-505T temperature controller at the heating rate of 1 °C/min. The cloud point was defined as the temperature at which the transmittance of the sample solution became less than 50%. 2.11. Protease Activity Assay. The protease activity against Suc-AAPF-pNA was measured using a UV/Vis spectrometer. A 6.5 × 10-2 mM solution of Suc-AAPF-pNA in 50 mM Tris-HCl (pH=7.5) was hydrolyzed by adding 4.2×10-2 mM of protease in the presence or absence of the POEC12's (10 mM). The initial proteolysis rate (t = 0–20 s) was calculated based on the absorbance at 410 nm.14a

3. RESULTS AND DISCUSSION 3.1. Synthesis of cyclic and linear POEC12's. A number of cyclic polyoxyethylene alkyl ethers (c-POEAEs), whose one side of the ring is hydrophilic and the other is hydrophobic, were designed and synthesized by varying the ring size. Scheme 1

shows the structures of the


10

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compounds and their hydrophilic-lipophilic balance (HLB) values as calculated using Griffin’s method,18 where the dodecyl group (C12) was chosen as the most popular hydrophobic group in the surfactants. The synthesis was performed using the Williamson ester synthesis via the bimolecular cyclization of 5 mM polyoxyethylene glycols (PEG400; Mw/Mn = 1.04, PEG600; Mw/Mn = 1.04, PEG1000; Mw/Mn = 1.06) and equimolar 1,12-dibromododecane (5 mM) with NaH (6 eq.) in dry N, N-dimethylformamide (DMF)/hexane (7/3) at 55 °C overnight. The use of an alkane (alkanes are poor cosolvents of PEG) is known to facilitate the ring closure reaction by reducing the end-to-end distance,19 which allows for cyclization even at concentrations (10-3 M) two orders of magnitude higher than that corresponding to highly diluted conditions (< 10-5 M), at which intermolecular oligomerization is strictly restricted. While the pure DMF system without hexane did not result in the cyclic forms of the compounds, the addition of hexane yielded cyclic polyoxyethylene dodecyl ethers (c-POEC12's) with diminishing linear oligomers or terminal olefin byproducts caused by E2 elimination. The DMF/hexane ratio and alkane species (hexane, heptane, and octane) were optimized, as shown in Supporting Information (Figure S1 and Table S1). After purification by reverse-phase high-performance liquid chromatography (HPLC), the purified c-POEC12 samples were subjected to 1H-,

13

C-nuclear magnetic resonance (NMR)

analyses and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The NMR spectra did not contain any signals attributable to the terminal methyl groups. Further, the spectra could be assigned to c-POE400C12 (Figure S2), c-POE600C12 (Figure S3), and c-POE1000 (Figure S4) and reflected the molecular symmetry of the compounds. The molecular ion peaks [M + Na]+ of the peak tops in the MALDI spectra (Figure S5-S7) were seen at 647.64 (calcd. 647.84) for POE400-C12, 735.68 (calcd. 735.95) for


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POE600-C12, and 1219.80 (calcd. 1220.53) for POE1000-C12. The isolated yields were 240 mg (9%) for c-POE400C12, 290 mg (10%) for c-POE600C12, and 330 mg (7%) for cPOE1000C12; these amounts were adequate for further exploring the relationship between the cyclic molecules and their properties. Normal polyoxyethylene dodecyl ethers (l-POEC12's), which are exact the “linear” counterparts of the above-mentioned cyclic compounds, were also synthesized by the Williamson ester synthesis without the addition of hexane. The detailed procedure was shown in supporting information. The l-POEC12's were also investigated using 1H-, 13C-NMR (Figure S8S10) analyses and MALDI-TOF MS spectroscopy (Figure S11-S13). The molecular ion peaks [M+Na]+ of the peak tops were seen at 605.20 (calcd. 605.81) for l-POE400-C12, 737.48 (calcd. 737.96) for l-POE600-C12, and 1177.31 (calcd. 1178.49) for l-POE1000-C12. Thus, three cyclic POEC12 compound as well as their linear counterparts were successfully synthesized without loading any undesirable functional groups.

3.2. GPC traces of cyclic and linear POEC12's. The gel permeation chromatography (GPC) elution curves for the samples of the cyclic and linear POEC12's in chloroform are shown in Figure 1. It can be seen that the retention times for both the c-POEC12's and l-POEC12's gradually shifted to lower values with an increase in the polyoxyethylene chain length. On comparing the c-POEC12 compounds with their linear counterparts, it was seen that the retention times of the c-POEC12's (red line) were higher than those of l-POE400C12 (blue dashed line) as well as the POE400C12 system (Figure 1(a)). The difference in the retention times of the cPOEC12's and l-POEAC12's was because of the compact and shrunken form of the c-POEC12's,


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which resulted from the conformational restriction caused by cyclization, as has been frequently reported for other cyclic polymers as well.20,21 This topologic effect was small when the polyoxyethylene chains were short. The values of the polydispersity index (Mw/Mn), as estimated by GPC, were 1.06 for cPOE400C12, 1.03 for c-POE600C12, and 1.05 for c-POE1000C12; these agreed well with those of the PEGs used as the substrates. Since the relative molecular weights determined using GPC were found to be influenced by the topology, the absolute average molecular weight22 of each compound was estimated from its MALDI-TOF MS spectrum (Figures S5-S7) and used to further quantitatively studying the c- and l-POEC12's.

3.3. Surface activities of cyclic and linear POEC12's. Surface-tension measurements were performed to explore the effects of the cyclic topology on the surface activity of the POEC12's. Figure 2 shows the relationship between the surface tension of water and the POEAE concentration, as measured by the pendant drop method at 25 °C. Although the synthesized cPOEC12 compounds are unique cyclic amphiphiles whose one side of the ring is hydrophilic and the other side is hydrophobic, they gradually reduced the surface tension by adsorption at the air/water interface in a manner similar to that of the l- POEC12's, with the surface tension becoming constant above the CMC. The CMC and γCMC values were then estimated from the cross-over point of the two fitted lines in Figure 2 and are listed in Table 1. The CMC values of the c-POEC12 compounds were found to be one order of magnitude higher than those of the lPOEC12 compounds, indicating that the c-POEC12's were less hydrophobic, in keeping with the conclusion drawn earlier from their RP-HPLC peak positions (Figure S14). The CMC values of


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c-POE600C12 and c-POE1000C12 were the same in spite of the difference in the polyoxyethylene chain length, this trend was also observed for l-POE600C12 and lPOE1000C12. While the γCMC of c-POE400C12 was higher than that of l-POE400C12, it was almost similar to that of c-POE400C12. Further, the γCMC of c-POE1000C12 was lower than that of l-POE1000C12, indicating clearly that the cyclization of POEC12 increased the surface activity when the polyoxyethylene chains became longer. The other surface parameters such as excess amount of adsorption (Γ) and area per molecule (A) were calculated using the Gibbs adsorption equation (Eq. (1)) and Eq. (2)23, 24 (Table 1). = − =

1 (1) 2.303 log

1 (2)

where γ is the surface tension, R is the ideal gas constant, T is the absolute temperature, (⁄ ) is the slope of the linear fit of the surface tension plot before the CMC has been reached. Further, N is the number of species (n = 1), and NA is Avogadro's number. Resent papers by Menger et al25 have questioned the use of the Gibbs adsorption equation for analyzing surface tension data to obtain the surface parameters. Neutron reflection (NR) was then used to answer the issue and the limitations were found to apply the Gibbs adsorption equation to surface tension data, especially for anionic26 and cationic27 surfactants. However, Li and Penfold et al proved28 that the Gibbs adsorption equation is reliable and applicable for nonionic surfactants to obtain surface parameters. In this study, the Gibbs adsorption equation


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was then applied to estimate the surface parameter such as Γ and A, even the values in table 1 were not directly confirmed by the other techniques such as neutron reflection.

The gradual decrease in the Γ value of the l-POEC12's with an increase in their HLB value agreed with the lower surface activity of the l-POEAEs for longer polyoxyethylene chains. On the other hand, the Γ value of the c-POEAEs remained almost constant even as the polyoxyethylene chains became longer; this is what resulted in the surface activity of the cPOEC12's being higher than those of the corresponding l-POEC12's with the longer polyoxyethylene chains. In contrast, cyclization is like to disturb the adsorption of POEC12's with the shorter polyoxyethylene chains at the air/water interface. The lower Γ value of the cPOE400C12 results in the lower γCMC than that of l-POE400C12. These results indicate that the restructuring of the surfactants by cyclization allowed them to exhibit unusual behaviors, which could not be predicted from the general classification of the surfactants based on their HLB values.18 More interestingly, the topologically cyclic POEC12 compounds exhibited significantly larger areas per molecule at the air/water interface than those of the linear molecules (Table 1), resulting in the former exhibiting distinctive molecular assemblies in aqueous solutions.

3.4. Self-assembly of cyclic and linear POEC12's. DLS measurements were performed to examine the effects of the cyclic topology on micelle formation in aqueous c-POEC12 and lPOEC12 solutions. This was because the size or shape of the micelles has a critical effect not only on the basic properties such as the degree of solubilization.29 First, the autocorrelation functions of the solutions of the cyclic and linear POEC12 compounds were measured at 25 °C (Figure S15). In contrast to the case for the l-POEC12's, a dramatic increase in the relaxation


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times was observed for the c-POEC12's; this suggested that the c-POEC12's exhibited much lower diffusion coefficients in aqueous solutions. The histogram analysis30 of the DLS correlation functions is shown in Figure 3. The hydrodynamic diameters (Dh) of the l-POEC12 (blue) micelles were 3.4 ± 1.1 nm for lPOE400C12, 4.9 ± 1.6 nm for l-POE600C12, and 9.7 ± 3.4 nm for l-POE1000C12. It is generally accepted that micellar size increases with an increase in the molecular length, since the micellar size corresponds to twice the length of the surfactants for normal spherical micelles. Surprisingly, the c-POEC12's (red) were found to form “giant” assemblies, which were more than 20 times larger than those of their linear counterparts. The Dh values of the c-POEC12 micelles were 72.8 ± 22.8 nm for c-POE400C12, 256.8 ± 29.8 nm for c-POE600C12, and 231.3 ± 72.2 nm for c-POE1000C12. Although the DLS analysis confirmed giant assemblies in the case of the c-POEC12's, the opposite trend has often been observed in previous studies: Grayson et al. determined31 the Dh value of aqueous micelles of amphiphilic poly(ethylene glycol)-b-poly(ε-caprolactone) (PEGx-bPCLy), reporting that it decreased from 27 nm to 15 nm on cyclization. Booth and coworkers32 reported that the Dh value of cyclic poly(ethylene oxide)-b-poly(butylene oxide) (PEOx-b-PBOy) was 8.8 nm, which is lower than that of its linear counterpart (14.2 nm) in water. Unlike these amphiphilic diblock copolymers having flexible polymer chains, the cyclic amphiphiles synthesized in this study had a rigid and inflexible hydrocarbon as the hydrophobic site. The fact that the areas per molecule (A) of the c- POEC12's (Table 1) were large allowed them to align in a bulky topology at the interface, resulting in the formation of the giant assemblies observed in water. In fact, giant micelles or vesicle-like structures ranging from 150


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nm to 200 nm in size have been reported for the cyclic form of surfactin13,33 and lactone-ringtype biosurfactants34 having large areas per molecule (A) at the air/water interface. The large spherical objects whose sizes were smaller than those from DLS were observed in the case of cPOE600C12 using freeze-fracture TEM (FF-TEM) (Figure S16). The particle size obtained from FF-TEM generally depends on how to fracture the particles. Although further studies such as small angle X-ray scattering (SAXS) or small angle neutron scattering (SANS) are required to determine detailed morphology, the solutions were optically isotropic and stable for at least six months. We preliminary checked the binary c-POE400C12/water phase behavior by polarized light microscopy. Although normal l-POE400C12 formed lyotropic liquid crystals at relatively high concentrations, c-POE400C12 did not form any optically anisotropic phases. This partially supports that cyclization causes the difference in the self-assembly, which generally dominated by critical packing parameter (CPP).

3.5. Clouding point temperatures of cyclic and linear POEC12's. The turbidities of aqueous solutions of the c-POEC12's and l-POEC12's were measured by varying the temperature from 25 °C to 95 °C in a stepwise fashion. It generally known that cloud point temperature depends on its concentration. Here we compare the clouding point temperatures at the same concentration (10gm/ml). We found that the cloud point of the linear-form POEC12's dropped by more than 28 °C after cyclization. Figure 4 shows the temperature dependence of the transmittance (%) at 650 nm for the aqueous solutions of both the c-POEC12's (red circles) and the l-POEC12's (blue triangles) at concentrations higher than the CMC. The cloud points at which the transparent


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solutions quickly became turbid (blue triangles) were 76 °C for l-POE400C12, 88 °C for lPOE600C12, and higher than 95 °C for l-POE1000C12; these corresponded well with the values reported previously for l-POEC12's having almost similar polyoxyethylene chain lengths.35 Meanwhile, the cyclization process caused a dramatic decrease in the cloud point temperatures, with the cloud point temperatures of c-POE400C12, c-POE600C12, and c-POE1000C12 being 46 °C, 60 °C, and 82 °C, respectively. The clouding behavior of POEAE is known to be related to the dehydration of the oxyethylene units and their subsequent gauche-to-trance conformational change. Around the oxyethylene units, the hydrated water molecules are less structured and more mobile owing to a lowering of the conformational entropy because of the cyclization of the chain. This is probably what results in the decrease in the cloud point temperature to a value lower that of the linear counterpart. A similar trend, that is, a decrease in the cloud point after cyclization, was reported not only for polyoxyethylene without an alkyl chain but also for poly(N-isopropylacrylamide)s (PNIPAM)s7 and poly(N-vinylcaprolactam).36 On the other hand, Tezuka et al. recently reported37 that the thermal and salt stabilities of amphiphilic copolymers exhibit the opposite trend. The cloud point of a micellar solution of cyclic poly(methyl acrylate)-b-poly(oxyethylene)(PMA-b-POE, diblock copolymer) was 40 °C higher than that of the linear counterpart of poly(methyl acrylate)-b-poly(oxyethylene-bpoly(methyl acrylate) (PMA-b-POE-b-PMA, triblock copolymer). They concluded that the triblock linear counterpart (PMA-b-POE-b-PMA) with hydrophobic ends causes intermicellar bridging by entangling the hydrophobic segments between micelles, resulting in a decrease in the cloud point to a level lower than that of cyclic PMA-b-POE, which has the same molecular weight.38


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To further explore this phenomenon, a triblock linear counterpart, C6-POE1000-C6, with hydrophobic segments at both ends was synthesized; its cloud point was found to be 55 °C (Figure S17), which is 25 °C lower than that of c-POE1000C12, as shown in Figure 4 (b)(blue triangle). This result was in good agreement with the trend reported by Tezuka et al. Further, the salt stability of c-POE1000C12 improved, since the slope of the curve of the cloud point after the addition of NaCl increased after cyclization, as shown in Figure S18. These results demonstrate clearly that not only a cyclic topology but also the design of the hydrophobic segments allows one to tune the cloud point temperature of the above-mentioned compounds without having to change their chemical composition.

3.6. Proteolysis kinetics in cyclic and linear POEC12 solutions. The effect of the cyclization of the POEC12's on the activity of detergent enzymes was examined using Suc-AAPF-pNA as a model peptide substrate. To be able to formulate surfactants in aqueous solutions of liquid detergents containing POEAEs without inducing a loss of enzyme activity still remains a challenge. This is because POEAEs are known to cause the denaturation of enzymes such as subtilisin Carlsberg from Bacillus licheniformis, even though the degree of denaturation is not as severe as that of anionic sodium dodecyl sulfate or cationic dodecyl trimethylammonium bromide.17 Figure 5 shows the ratios of the initial rate of hydrolysis (t = 0–20 s) of Suc-AAPF-pNA by the protease from Bacillus licheniformis using the c-POEC12's or l-POEC12's as well as those without them (v/vo). All the measurements were performed at a POEAE concentration of 10 mM, since little change was observed in the peptide hydrolysis rate at concentrations higher that the


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CMC values of the surfactants.17 The initial hydrolysis rate (v/vo) with l-POE400C12 (blue) was approximately 40% lower compared to that without the surfactant (v/vo = 1) and decreased further when the polyoxyethylene chain was lengthened. The cyclization of the POEAEs was found to prevent a marked decrease in the protease activity: c-POE400C12 (red) suppressed the decrease in the initial rate to 10% 10% of the initial rate reduction. More interestingly, the initial rates in the case of c-POE600C12 and c-POE1000C12 were more than 40% higher than those of the linear counterparts, in spite of exhibiting surface activities equal to or higher than those of lPOE600C12 and l-POE1000C12. After the reaction, the surface tensions of all the aqueous solutions were determined; the values were almost similar to their γCMC values listed in Table 1. It has been reported by Kinbara et al. and co-workers that the enzymatic activity of lysozymes increases when cyclized PEG is used as an additive.39 To the best of our knowledge, this is the first report suggesting that the cyclization of POEAEs significantly suppresses the decrease in the protease hydrolysis rate, while also ensuring that the compounds exhibit higher surface activities than those of their linear counterparts. Although the HLB values of c-POE400C12 and l-POE400C12 are the same, the difference in CPP as predicted in Section 3.4 could cause the difference in affinity for the detergent enzyme. The cyclization of POEAEs not only induces unusual behavior in aqueous solutions but also allows for the formulation of POEAEs with proteases in liquid detergents merely by changing the topology.

4. CONCLUSIONS


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The cyclic forms of polyoxyethylene dodecyl ethers (c-POEC12's) with different ring sizes were systematically synthesized by the addition of hexane as a poor cosolvent under relatively high conditions (10-3 M), together with their exact linear counterparts (l-POEC12's). The surface activity of the c-POEC12's remained almost constant even as their HLB value was changed; on the other hand, the l-POEC12's behaved as conventional surfactants. In contrast to the normal spherical micelles seen in the case of the l-POEC12's (3.4–9.7 nm), the cyclization of the POEC12's led to the formation of large spherical structures 72.8–256.8 nm in size and resulted in a marked decrease in their cloud point temperature by more than 28 °C. The cloud point temperature could be tuned by changing the topology and subsequent segment design without having to change the chemical composition of the compounds. Furthermore, the cyclization of the POEC12's significantly mitigated the marked suppression of the rate of protease hydrolysis caused by the surfactants, regardless of the fact that the surface activity of the surfactants was almost similar or higher. This should allow the issue of the damage caused to detergent enzymes by surfactants during formulation in liquid detergents to be addressed.

■ Associated Content Supporting Information, Table S1, and Figure S1-S20.

■ Author Information


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Corresponding Author E-mail: [email protected] The authors declare no completing financial interests.

■ Acknowledgments We are grateful to Dr. Luke Lemmon of the Scripps Research Institute (TSRI) and Dr. Sho Kataoka and Dr. Hiroyuki Minamikawa of the National Institute of Advanced Science and Technology (AIST) for valuable discussions. This work was financially supported by the Strategic Research Fund of AIST, Japan.

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Figure captions

Scheme 1. Structures of cyclic and linear polyoxyethylene dodecyl ethers. Figure 1. GPC traces of cyclic and linear a) POE400C12, b) POE600C12, and c) POE1000C12 at a concentration of 1 mg/mL in CHCl3, The flow rate was 1 mL/min. Figure 2. Surface tension versus concentration plots of cyclic and linear a) POE400C12, b) POE600C12, and c) POE1000C12 at 25 ºC. Figure 3. Size distributions of the assemblies prepared using a) POE400C12, b) POE600C12, and c) POE1000C12 at a concentration of 20 mM (25 ºC), as determined by DLS measurements. Figure 4. Results of the temperature-dependent turbidity measurements of a) POE400C12, b) POE600C12, and c) POE1000C12 at a concentration of 10 mg/mL. The scan rate was 1 ºC/min.


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Figure 5. Effects of the cyclic and linear POEC12’s on the hydrolysis rate of suc-PPAF-pNA hydrolysis at 40 ºC. v/v0 represents the ratio between the rate with POEC12 and that without POEC12. The concentration was 10 mM.


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Insert Table of Contents Graphic and Synopsis Here


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Y. Hirose et al. Scheme 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

“Cyclic”

c-POE400C12 c-POE600C12 c-POE1000C12 (HLB = 14.2) (HLB = 15.5) (HLB = 17.1)

“Linear”

l-POE400C12 (HLB = 14.2)

l-POE600C12 (HLB = 15.5)


l-POE1000C12 (HLB = 17.1)

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a) POE400C12

Refractive index (Arb.Unit)

18 18

19 19

20 21 20 21 Retention time (min)

Cyclic Linear

22 22

b) POE600C12

18 18


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58


Y. Hirose et al. Fig 1

19 19


Cyclic Linear

22 22

c) POE1000C12

18 18

19 19


23 23

23 23

Cyclic Linear

22 22


23 23

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Surface tension (mN/m) Surface tension (mN/m)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

Surface tension (mN/m)


70 70

a) POE400C12

60 60 50 50 40 40

Cyclic Linear

30 30 10 -6-6

70 70

10 -5-5

10 10 -4-4 -3-3 Concentration (M)

10 -2-2

10 -1-1

10 -2-2

10 -1-1

-2-2 10

-1-1 10

b) POE600C12

60 60 50 50

Cyclic Linear

40 40

30 30 10 -6-6

10 -5-5

10 10 -4-4 -3-3 Concentration (M)

70 70

c) POE1000C12 60 60 50 50

40 40 30 30 -6-6 10

Cyclic Linear -5-5 10

-4-4 -3-3 10 10 Concentration (M)


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80 80

Number (rel. %)

a) POE400C12 60 60

Cyclic Linear

40 40 20 20

0 11 80 80

10 100 10 100 diameter (nm)

Number (rel. %)

b) POE600C12 60 60

1000 1000 Cyclic Linear

40 40 20 20

0 11

10 100 10 100 diameter (nm)

1000 1000

80 80

C) POE1000C12 Number (rel. %)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

60 60

Cyclic Linear

40 40

20 20

00 11

10 100 10 100 diameter (nm)


1000 1000

Langmuir

Page 32 of 34


Transmittance (%)

140 140

a) POE400C12

120 120

100 100 80 80 60 60 40 40

Cyclic Linear

20 20 00 25 25

35 35

45 45

55 65 75 55 65 75 Temperature (ºC)

85 85

95 95

85 85

95 95

85 85

95 95

Transmittance (%)

140 140

b) POE600C12

120 120 100 100

80 80 60 60 40 40

Cyclic Linear

20 20 00 25 25

35 35

140 140

Transmittance (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

45 45

55 65 75 55 65 75 Temperature (ºC)

c) POE1000C12

120 120 100 100 80 80 60 60

40 40

Cyclic Linear

20 20 00 25 25

35 35

45 55 65 75 45 55 65 75 Temperature (ºC)


Page 33 of 34

Langmuir


1.4 1.4

Cyclic Linear

1.2 1.2

1.01

v/v0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2

0 POE400C12

POE600C12

POE1000C12


Langmuir

Page 34 of 34

Y. Hirose et al. Table 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

Table 1. Surfactant parameters for the cyclic and linear POEC12’s

Cyclic

Linear

CMC (mM)

γCMC (mN/m)

POE400C12

4.3

37.3

1.9

88.1

POE600C12

7.0

37.1

1.1

150.7

POE1000C12

7.6

38.8

1.1

153.1

POE400C12

0.1

33.5

4.4

38.1

POE600C12

0.3

37.3

2.7

60.7

POE1000C12

0.3

45.4

1.6

101.9


(10-6

Γ A 2 2 mol/m ) (Å /molecule)

Structures and Surface Properties of âCyclicâ Polyoxyethylene Alkyl

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