Surface Monolayers of Polymeric Amphiphiles Carrying a Copolymer

monolayer, the sp3 hybridization of boronic acid moiety was presumed to be facilitated by both incorporation of amino group in the copolymer segment a...
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Langmuir 1998, 14, 3916-3920

Surface Monolayers of Polymeric Amphiphiles Carrying a Copolymer Segment Composed of Phenylboronic Acid and Amine. Interaction with Saccharides at the Air-Water Interface Masazo Niwa,* Takashi Sawada, and Nobuyuki Higashi* Department of Molecular Science & Technology, Faculty of Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan Received November 17, 1997. In Final Form: May 4, 1998 A novel polymer amphiphile (1), having the copolymer segment containing phenylboronic acid and tertiary amino groups, formed a stable monolayer on water. From the result of pH dependence of the monolayer, the sp3 hybridization of boronic acid moiety was presumed to be facilitated by both incorporation of amino group in the copolymer segment and its alignment at the air-water interface. The sugar recognition by this polymer monolayer was achieved even at a pH around 6 and became extremely sensitive compared with that of a monolayer consisting of a monomeric boronic acid amphiphile. The 1 monolayer was found to have an ability to discriminate between linkage isomers of disaccharides, which were estimated as molecular area changes of the monolayer upon addition of saccharides in the subphase.

Introduction Higher-ordered structures of biopolymers such as proteins and nucleic acids are basically macromolecular assemblies resulting from specific interactions among characteristic polyelectrolyte elements. It should be of significance to model such structural features of biopolymers and to define the interaction mechanisms among the component chains as developing molecularly controlled polymeric materials mimic biofunctions, such as molecular recognition. Langmuir monolayers are a useful tool for assembling molecules two-dimensionally. We have devised a strategy in which purely synthetic polyelectrolytes are aligned at the air-water interface. The polyacids such as poly(methacrylic acid) (PMAA) and poly(L-glutamic acid) (PLGA) have been selected as polyelectrolytes because of their ease of preparation and well-defined conformational characteristics in bulk water phase.1 To form monolayers on water, these polymers were modified with hydrophobic long alkyl chains at the polymer chain end. These polymer assemblies have shown unique properties different from those in bulk water: for instance, the PMAA-based amphiphiles have the ability to read out the chain length of the corresponding guest polymers such as polyethylene glycol,2 and the secondary structure (Rhelix, β-sheet and so on) of the PLGA monolayer can readily be controlled by varying the monolayer phase.3 In the present study, we have prepared a novel sugarresponsive polymeric amphiphile (1) that contains phenylboronic acid units and tertiary amino groups in the polymer segment. Boronic acid has been demonstrated to make stable complexes with diol compounds including poly(vinyl alcohol), glucose, and sorbitol.4-7 However, the (1) For a recent review, see: Higashi, N.; Niwa, M. Colloids Surf. A 1997, 123/124, 433-442. (2) (a) Higashi, N.; Shiba, H.; Niwa, M. Macromolecules 1989, 22, 4650-4651. (b) Higashi, N.; Matsumoto, T.; Niwa, M. Langmuir 1994, 10, 4651-4656. (3) Higashi, N.; Shimoguchi, M.; Niwa, M. Langmuir 1992, 8, 15091511. (4) Wulff, G. Pure Appl. Chem. 1982, 54, 2093-2102. (5) Schott, H.; Rudloff, E.; Schmidt, P.; Roychoudhury, R.; Ko¨ssel, H. Biochemistry 1973, 12, 932-938.

efficient complexation with these diol compounds requires the boron atom to be sp3 hybridized (tetrahedral anion, -B(OH)3-), which is achieved at high pH. To facilitate sp3 hybridization, amino groups have been incorporated in the vicinity of the boronate moiety. Such an incorporation stabilizes the complex formation through the charge 7.4.7,8 We report herein the monolayer properties of 1 on water and its interaction with saccharides in the subphase.

Results and Discussion Preparation and Characterization of 1. The preparation of 1 was carried out by photopolymerization of 3-methacrylamide phenylboronic acid and N,N-dimethylaminoethyl methacrylate in the presence of a xanthate derivative (2) having two long alkyl chains. Xanthate derivatives are useful for preparing a “diblock” type copolymer because they have been established to serve as a living radical photoinitiator in polymerizations of vinyl monomers.9 The resulting polymer amphiphile, 1, was confirmed by 1H NMR spectroscopy to consist of two long alkyl chains and a copolymer segment composed of a phenylboronic acid moiety and a tertiary amino group with a composition of almost 1:1 and a mean degree of polymerization (n) of 43. (6) Weith, H. L.; Weibers, J. L.; Gilham, P. T. Biochemistry 1970, 9, 4396-4401. (7) Westmark, P. R.; Valencia, L. S.; Smith, B. D. J. Chromatogr. 1994, A664, 123-128. (8) Kitano, S.; Hisamitsu, I.; Koyama, Y.; Kataoka, K.; Okano, T.; Sakurai, Y. Polym. Adv. Technol. 1991, 2, 261-264. (9) Niwa, M.; Higashi, N.; Shimizu, M.; Matsumoto, T. Makromol. Chem. 1988, 189, 2187-2199.

S0743-7463(97)01261-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 06/18/1998

Surface Monolayers of Polymeric Amphiphiles

Figure 1. pH dependence of the absorption maximum (λmax) of 1 in methanol-water (6:4 v/v).

Figure 2. 11B NMR spectra obtained for 1 in the presence of various amounts of D-fructose at 30 °C: solvent, CD3OD/H2O (8:2 v/v); external standard, B(OCH3)3; [boronic acid unit in 1] ) 9.3 × 10-2 M; [D-fructose]/[boronic acid unit in 1] ) 0 (a), 0.4 (b), and 1.0 (c).

To elucidate effect of incorporation of amino group in the polymer segment, the pKa value of phenylboronic acid moiety was evaluated by phototitration. Figure 1 shows the shift of absorption maximum (λmax) of 1 in methanolwater (6:4 v/v) by varying pH. The λmax 241 and 243 nm are assigned to neutral and anionic species, respectively. From the titration curve, the pKa was estimated to be 7.3. The result indicates that the pKa value is effectively lowered in the presence of tertiary amino groups, since the pKa of monomeric phenylboronic acid was 8.8. The interaction of 1 with diol compounds in solutions was subsequently examined by means of 11B NMR spectroscopy. D-Fructose and adenosine were employed as the diol compound. Figure 2 shows 11B NMR spectra of 1 in the presence of various amounts of D-fructose in CD3OD/H2O (8:2 v/v) at 30 °C. Spectrum a obtained in the absence of D-fructose gives a peak at around 10 ppm, which can be assigned to the free (sp2 hybridized) boronic acid.10 By addition of D-fructose with a ratio of [D-fructose]/ [boronic acid in 1] ) 0.4, a new peak appears at -12 ppm, and simultaneously the peak intensity at around 10 ppm decreases. Such a drastic upfield shift has been described to be due to formation of boronate ester with D-fructose.11 (10) Mikami, M.; Shinkai, S. Chem. Lett. 1995, 603-604. (11) Suenaga, H.; Nakashima, K.; Shinkai, S. J. Chem. Soc., Chem. Commun. 1995, 29-30.

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Figure 3. Surface pressure (π)-area (A) isotherm of 1 spread on pure water at pH 5.8, 20 °C.

Figure 4. pH Dependence of the limiting molecular area (A0) evaluated on the basis of π-A isotherms depicted in the inset. π-A isotherms were measured at 20 °C.

Further addition of D-fructose (spectrum c) causes the complete disappearance of the peak at 10 ppm and the 1:1 ester formation. A similar spectral feature was observed for the combination of 1 and adenosine; i.e., the boronic acid groups of 1 can also interact with the cis-diol moiety of adenosive and form ester. Monolayer Properties of 1 on Water and on Aqueous D-Fructose. Figure 3 displays a surface pressure-area (π-A) isotherm of 1 on pure water at pH 5.8, 20 °C. It can be seen from the figure that the polymeric amphiphile provides a well-condensed, stable monolayer with a collapse pressure of about 40 mN m-1. The limiting molecular area, estimated by extrapolating the solidlike region to zero surface pressure, 0.42 nm2, is very close to the cross section of the vertically aligned two hydrocarbon chains (0.40 nm2). This means that the polymer segment containing boronic acid and amino groups of 1 prefers to exist in aqueous phase without affecting the monolayer phase at that pH. Figure 4 (inset) depicts typical π-A isotherms of 1 measured at different pH values. π-A isotherms are found to vary dramatically by both lowering and elevating pH. Accordingly, the pH dependence of the limiting molecular area, on the basis of these π-A isotherms, was examined and is shown in the same figure. The monolayer is most expanded at pH 3.0, probably due to expansion of the copolymer segment caused by electrostatic repulsion among the completely protonated ammonium cations. With increasing pH, the limiting molecular area is found to decrease steeply and give a minimum at around pH 6.

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Niwa et al.

Figure 5. π-A isotherms of 1 spread on pure water (a) and on aqueous D-fructose (2.0 × 10-3 M) (b) at pH 5.8, 20 °C.

Such a steep decrease in the limiting area can be supposed to stem from weakening the electrostatic repulsive force working among ammonium cations by ionization of the boron atoms. Beyond pH 6, the limiting area has a tendency to increase gradually, probably because the anionic phenylboronate groups contribute predominantly to the electrostatic repulsion among polymer segments. The minimum of the limiting area at around pH 6 can be assigned to an apparent pKa of the boronic acid unit of the copolymer segment in the monolayer state. In a bulk solution, as described above, the pKa value of the boronic acid unit was evaluated to be 7.3, which is considerably higher than that estimated in the monolayer state. This result suggests that the interaction among copolymer segments of 1 would be appreciably strong within the monolayer. Subsequently, the effect of addition of D-fructose on the π-A isotherm of 1 was examined at pH 6, at which information about interaction of 1 monolayer with Dfructose would be most sensitively read out because the monolayer was the most compact at that pH region. D-Fructose was chosen as a diol compound since it is wellknown to have a large binding constant in complexation with phenylboronic acid derivatives in solution.12 Figure 5 shows π-A isotherms of 1 on pure water and on aqueous D-fructose (2 × 10-3 M) at 20 °C. The monolayer on aqueous D-fructose is found to expand markedly compared with that on pure water, suggesting that the phenylboronic acid moiety in the monolayer interacts with D-fructose. If so, there should be a remarkable pH dependence of the π-A isotherm since the binding ability of phenylboronic acid to diol compounds depends strongly on the surrounding pH as mentioned above. The relative change in the limiting area, ∆A0 (∆A0 ) A0′ - A0) in the absence (A0) and presence (A0′) of D-fructose at different pH values is displayed as a function of D-fructose concentration in Figure 6. The A0 increases with increasing D-fructose concentration and is leveled off beyond a concentration of 1 × 10-4 M. The changes of A0 are considerably larger at higher pH values such as 5.8 and 10.9. At pH 3.0, as was predicted, the area change is relatively small, implying that the interaction of boronic acid moiety in the monolayer with D-fructose might be weak, probably because the boronic acid took the free form at that pH. At pH 10.9, surprisingly, the ∆A0 value at a D-fructose concentration of 2 × 10-4 M goes up to over 1 nm2/molecule, resulting from the effective interaction of the sp3-hybridized boronate anion of 1 with D-fructose, as shown in Scheme 1. This extremely large expansion of the monolayer upon (12) Lorand, J. P.; Edwards, J. O. J. Org. Chem. 1959, 24, 769-774.

Figure 6. Relative change in the limiting molecular area (∆A0) at various pH values as a function of D-fructose concentration. Scheme 1

addition of D-fructose must be due to the monolayer component containing a polymer segment, in which the boronic acid moiety is incorporated; i.e., the monolayer expansion would be caused not only by the interaction of boronate anion and D-fructose but also by its following conformational change of the copolymer segment. In fact, a monolayer, composed of a small amphiphilic molecule containing phenylboronic acid as the hydrophilic group, has been reported to show only a small area change (0.2 nm2/molecule) upon addition of D-fructose.13 At pH 6.0, interestingly, the concentration dependence of ∆A0 is almost fitted with that at pH 10.9, meaning that even at pH 5.8 the boron atoms of the copolymer segment were sp3 hybridized (-B(OH)3-) and could readily interact with D-fructose. Effect of Various Diol Compounds on π-A isotherms of 1. The interaction of the 1 monolayer with various diol compounds such as mono- and disaccharides and nucleosides have been examined by using the area change in π-A isotherms, which was established for D-fructose. Figure 7 summarizes the concentration dependence of ∆A0 for D-fructose, D-galactose, and D-glucose on the basis of π-A isotherms of 1 on their aqueous solutions. There is a significant difference in the concentration dependence of ∆A0 among monosaccharides. The most sensitive concentration dependence is observed for D-fructose and reduced in the order D-glactose > D-glucose. This order is consistent with that in the binding constant for the boronic ester formation between phenylboronic acid and monosaccharides evaluated in water.12 Figure 8 shows the result obtained for disaccharides of cellobiose, lactose, and maltose, all of which are 1,4-linked dimers and have a glucose residue as the reducing terminus. As is well-known for arylboronic acids,12,14 the cis vicinal OH groups on the anomeric carbon and the adjacent one of a reducing sugar provide the primary site of boronic ester formation. In fact, Nagai et al. reported that there was no significant different in the binding (13) Ludwig, R.; Ariga, K.; Shinkai, S. Chem. Lett. 1993, 1413-1416. (14) Tsukagoshi, K.; Shinkai, S. J. Org. Chem. 1991, 56, 4089-4090.

Surface Monolayers of Polymeric Amphiphiles

Figure 7. Relative change in the limiting molecular area (∆A0) at pH 5.8 as a function of monosaccharide concentration: D-fructose (a); D-galactose (b); D-glucose (c).

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Figure 9. Relative change in the limiting molecular area (∆A0) at pH 5.8 as a function of nucleoside concentration: adenosine (a); uridine (b); cytidine (c).

cytidine, which have cis-diol moiety, was examined in the same manner described above. The concentration dependence of ∆A0 is shown in Figure 9. There is a definite difference among nucleosides, which is not so large compared with that for saccharides. The relative affinities increase in the order cytidine < uridine < adenosine. The structural distinction among these nucleosides exists only in nucleic acid bases, implying that the 1 monolayer has the ability to discriminate between nucleic acid bases. Conclusions

Figure 8. Relative change in the limiting molecular area (∆A0) at pH 5.8 as a function of disaccharide concentration: cellobiose (a); lactose (b); maltose (c).

constant of an arylboronic acid among three disaccharides.15 In our polymer monolayer (Figure 8), however, there appears a remarkable difference in the concentration dependence among such disaccharides; i.e., the extent of A0 change with the saccharide concentration increases in the order maltose < lactose < cellobiose. We do not have any conclusive evidence so far to explain this phenomenon, but one possible explanation, which will require further exploration, is as follows. In bulk solution, the interaction of boronic acid with saccharide is governed mainly by the binding constant. However, in the case of our polymeric monolayers, the guest saccharides must at first permeate the aligned polymer segments at the interface and then interact with phenylboronic acid groups. Thus, in this case a steric factor of the guest molecule would also play an important role in complexation. Cellobiose and lactose are β-linked dimers, and in contrast maltose is R-linked. Therefore, it is supposed that the permeation of the extended, β-linked cellobiose and lactose could be enhanced compared with that of the bent, R-linked maltose, although the difference between cellobiose and lactose cannot be reasonably explained at the present time. Finally, the interaction with nucleosides of adenosine, uridine, and (15) Nagai, Y.; Kobayashi, K.; Toi, H.; Aoyama, Y. Bull. Chem. Soc. Jpn. 1993, 66, 2965-2971.

The present study demonstrates that (i) a novel polymer amphiphile (1), carrying the copolymer segment containing phenylboronic acid and tertiary amino groups, gives a stable monolayer on water, (ii) the sp3 hybridization of boronic acid is facilitated by both incorporation of amino group in the copolymer segment and its alignment at the interface, (iii) the sugar recognition by the polymer monolayer (1) is achieved even at around pH 6 and becomes extremely sensitive compared with that by a monolayer consists of a monomeric boronic acid amphiphile, and (iv) the polymer monolayer is likely to have abilities to discriminate linkage isomers of disaccharides and nucleosides. These findings emphasize potential contribution of the copolymer segment and its assembled structure at the air-water interface to the sugar-binding processes. Experimental Section Materials. The polymer amphiphile 1 was prepared according to Scheme 2. Preparation of the photoinitiator, 2, was carried out by reaction of 3-(N,N-dioctadecylamino)propylamine and chloroacetyl chloride and then by reaction of the resultant chloride with sodium xanthate according to the manner described elsewhere.16 The purity and structure of the final product were confirmed by thin layer chromatography (TLC) and 1H NMR spectroscopy, respectively.

Scheme 2

The vinyl monomer of phenylboronic acid (3-methacrylamide phenylboronic acid, 3) was prepared as follows. Into a THF solution (30 mL) of m-aminophenylboronic acid monohydrate (0.39 g, 2.5 mmol) and triethylamine (0.42 g, 3.0 mmol) was added (16) Niwa, M.; Date, M.; Higashi, N. Macromolecules 1996, 29, 36813685.

3920 Langmuir, Vol. 14, No. 14, 1998 dropwise an excess of methacryloyl chloride (1.51 g, 14.5 mmol) in THF under ice cooling. The mixture was stirred at room temperature for 24 h. The precipitate was removed by filtration. After removal of the solvent, the residue was recrystallized with CHCl3 giving a pale yellow crystal (1.00 g, 89%): TLC (CHCl3: MeOH ) 9:1) Rf ) 0.6 single spot; IR 3300 cm-1(N-H), 1655 (CdC), 1620 (CdO, amide); 1H NMR(CDCl3) δ 1.95 (s, 3H), 5.48 (s, H), 5.82 (s, H), 7.27 (t, H), 7.51 (d, H), 7.74 (d, H), 7.95 (s, H), 8.00 (s, 2H), 9.70 (s, H). The another vinyl monomer of N,N-dimethylaminoethyl methacrylate (4) was purchased from Wako Chemical Co. (Japan). Photopolymerization of 3 and 4 (total monomer concentration, [3]0 + [4]0 ) 2.0 M; [3]0 /([3]0 + [4]0) ) 0.50) was carried out in the presence of 2 ([3]0 + [4]0)/[2]0 ) 40) in acetone under nitrogen atmosphere at 30 °C. After 40 h of UV lamp irradiation (a lowpressure Hg lamp), the precipitate was separated by centrifuge and then washed with THF repeatedly and dried in vacuo. The structure of the polymer thus obtained was determined by 1H NMR spectroscopy.

Niwa et al. All sugars and nucleosides were used as commercial products without further purification. Instruments and Measurements. UV spectra were determined using a UV-2100 (Shimadzu Co. Ltd., Japan). 11B NMR spectra were taken at 30 °C for CD3OD/H2O (8:2 in vol.) solutions using a JEOL JNM GX-400 spectrometer. External B(OCH3)3 was used as a reference. The monolayers are obtained by spreading a MeOH/benzene (2:8 in vol.) solution (about 1 mg mL-1) of 1 on water that was purified (F > 18 MΩ cm) using a Milli-Q system (Millipore Ltd.). Twenty minutes after spreading, the monolayer was compressed continuously with a rate of 1.20 cm2 s-1. Wilhelmy’s plate method and a Teflon-coated trough with a microprocessor-controlled film balance, FSD-50 (USI system Ltd., Japan) with a precision of 0.01 mN m-1, were used for surface pressure measurements. pH in the subphase was adjusted with aqueous HCl and KOH.

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