CHAPTER 1: Supramolecular Chemistry of Boronic Acids

Various organic solvents are gelatinized by low molecular weight gelators. These phenomena are interesting in that the fibrous aggregates formed by non-covalent interactions between gelators are responsible for the gelation. In particular, cholesterol-based gelators show excellent gelation ability towards various organic solvents at sufficiently low concentrations. The resulting gels display chirally oriented structures that are imparted from the cholesterol skeleton having chiral centers.

James et al. synthesized a new gelator by combining a boronic acid moiety with the cholesterol skeleton (cholesterylphenylboronic acid 1).^1,2  It was found that saccharide complexes of 1 efficiently gelatinize several organic solvents. The gelation properties such as the sol–gel phase transition temperature, xerogel fiber structure, gel stability difference between the d- versus l-complexes, etc. are changeable by a slight difference in the saccharide structure (Figure 1.1).

Inoue et al. utilized the xerogel fibers prepared from 1 to host matrixes exhibiting binding ability towards saccharides.³  The process consists of three stages. In the initial stage, benzene is gelatinized by the 1 : 2 complex between xylose and 1, and then the gel is freeze-dried. In the next stage, the resulting xerogel is washed with aqueous acetic acid solution and a water/methanol mixture to remove xylose from the xerogel. In the final stage, the xylose-removed xerogel is dispersed in aqueous xylose solutions, and the amount of re-bound xylose is determined after stirring for 40 h. Interestingly, the xerogel prepared with l-xylose as a template exhibits four-times higher re-binding ability for l-xylose than for d-xylose. This chiral discrimination ability indicates that the “memory” for the originally imprinted saccharide is retained in the xerogel.

Kimura et al. prepared another type of boronic acid-appended gelator 2 consisting of long alkyl chains and l-glutamate segment.⁴  Aqueous solutions containing 2 were gelatinized in the presence of various saccharides, and the aggregation structures of the gelator were observed by TEM measurements. It was revealed that various types of higher-order structures are developed depending on the saccharide used.

A gel-based fluorocolorimetric sensor for polyols was reported by Ikeda et al.⁵  A boronic acid-appended receptor bearing 7-nitrobenzoxal[1,2,5]diazole (NBD) (3) is incorporated into self-assembled nanofibers consisting of gelator 4 and hydrophobic coumarin dye 5. In the absence of polyols, FRET (fluorescence resonance energy transfer) from the NBD moiety of 3 to the coumarin unit in 5 is observed. With increasing polyol concentration, the spectral change appeared due to cancellation of FRET. This is attributed to the migration of 3 from the hydrophobic nanofiber phase to the hydrophilic aqueous phase upon binding of polyols (Scheme 1.1). The authors demonstrated that the gel-based sensor is capable of detecting polyols such as catechol, dopamine, and catechin under dry conditions by integrating the gel-based sensor into a filter paper.

Zhou et al. developed a new boronic acid-based gelator 6 that can gelate several organic solvents by self-assembling to form a nanofiber network.⁶  The driving force for the aggregation is attributed to the hydrogen bonding and the π–π stacking between the gelators. It was found that the addition of glucose induces a gel–sol transition, due to the formation of a gelator–glucose complex. This gel exhibits excellent sensitivity towards glucose among six saccharides (mannitol, galactose, lactose, maltose, sucrose, and fructose). The gelator is reusable by dissociating the complex with an acidic solution and then extracting with an organic solvent.

Stimuli-responsive polymer gels have attracted much attention due to their potential application for the design of self-regulated materials and systems. So far, many attempts have been made to design of glucose-regulated insulin delivery systems using stimuli-responsive hydrogels. Usually, two different types of approaches have been utilized for endowing hydrogels with glucose-responsiveness: (1) enzymatic reactions between glucose oxidase and glucose and (2) complementary binding of lectin (concanavalin A) to glucose. The boronic acid-based system is a third candidate.

Matsumoto et al. developed boronic acid-based hydrogels showing glucose responsiveness.⁷  They were synthesized by copolymerizing boronic acid monomer 7, N-isopropylmethacrylamide, and 2-carboxyisopropylacrylamide with a crosslinker (N,N′-methylene-bis-acrylamide). The hydrogels tend to shrink with increasing temperature due to the thermo-responsive nature of the main chain [poly(N-isopropylmethacrylamide)]. The gel prepared under the optimal monomer composition is shrunken in the absence of glucose, whereas the gel volume increases with increasing glucose concentration. The observed glucose responsiveness is derived from the formation of anionic boronate esters that make the polymer chain more hydrophilic. This totally-synthetic material is potentially applicable to insulin-delivery diabetes-devices that can tolerate long-term use and storage.

It is known that polycations and polyanions form charge-neutralized polyion complexes in aqueous solutions. By using polyion complex formation reactions, Kanekiyo et al. invented a novel molecular imprinting method for nucleotides as templates.⁸  Firstly, a polycation (8) was mixed with a boronic acid-containing polyanion (9) in the presence of AMP (adenosine monophosphate). Then, the obtained polyion complex containing AMP was washed with an acidic solution to remove the template AMP. Finally, the resultant “cleft” polyion complex was tested for the re-binding ability towards nucleotides. It was proven that the “cleft” polyion complex shows high affinity and selectivity towards AMP. This means that the memory for AMP is retained in the polyion complex matrix. Interestingly, the removal and re-binding processes for AMP coincide with the swelling and shrinking of the polyion complex (Scheme 1.2): without AMP, it is swollen due to existence of excess cationic charges, which create electrostatic repulsion within the polyion complex matrix, whereas the re-binding of AMP neutralized the excess cationic charges resulting in the shrinkage of the polyion complex. This stimuli-responsive polyion complex was subsequently applied as a sensing element in a QCM (quartz crystal microbalance) system.⁹  For this purpose, the polyions were alternatingly adsorbed onto a QCM resonator surface in the presence of AMP (Scheme 1.3). After removal of AMP from the surface polyion complex, a swollen gel layer with excess cationic charges resulted. It was confirmed that this QCM system selectively responds to AMP among various adenosine derivatives. The responsiveness is derived from the mass decrease induced by the shrinkage of the surface.

Kanekiyo et al. also developed nucleotide-responsive hydrogels by copolymerizing boronic acid monomer 10 and cation monomer 11 with a crosslinker (N,N′-methylene-bis-acrylamide).¹⁰  The hydrogels efficiently bind nucleotides such as AMP and ATP (adenosine triphosphate) by a cooperative action of the boronate ester formation and the electrostatic interaction between the cationic units and the phosphate group. The binding process coincides with the swelling and shrinking behavior of these hydrogels. For the hydrogel with the specific monomer composition, a unique “charge inversion” is observable: with increasing nucleotide concentrations, the cation-rich hydrogel is gradually shrunken due to charge neutralization, then it is swelled again because of the introduction of excess anionic charges (Scheme 1.4). These nucleotide-induced swelling and shrinking phenomena are applicable to nucleotide sensors by reproducing the gels on the surface of a QCM resonator.

Porphyrin is a useful scaffold for developing molecular recognition elements, since it shows highly sensitive UV-vis absorption and fluorescence emission. By combining porphyrin and boronic acid, one can construct supramolecular systems that exhibit unique guest-induced spectroscopic changes.

Imada et al. synthesized a porphyrin derivative bearing four boronic acid moieties (12).¹¹  It was confirmed that 12 forms a one-dimensionally stacked aggregate in water/DMSO mixture at pH 6.9. After adding saccharides to the solution, CD (circular dichroism) spectra were measured. In the presence of saccharides (except fructose), the solutions of 12 become CD-active and the sign of the exciton-coupling band (ECB) changes depending on the added saccharides. These results demonstrate that the absolute configuration of saccharides is predictable by CD measurements. Subsequently, a further sophisticated procedure was reported by Takeuchi et al.¹²  A porphyrin derivative bearing only one boronic acid moiety (13) was synthesized, and 1 : 2 sugar–boronic acid complexes were prepared. Then, the photochemical properties of the 1 : 2 complexes were studied by UV, fluorescence, and CD spectroscopy. It was confirmed that the extinction coefficients and fluorescence intensities are linearly correlated with the theoretically calculated dihedral angles between the two porphyrin moieties in the 1 : 2 complexes. In addition, the CD signs are explained by the absolute configurations of saccharides. These results establish that the dihedral angle between the two porphyrins plays a decisive role in electronic properties of the 1 : 2 complexes, and the saccharide structure can be conveniently determined by CD measurements.

The sugar sensing utilizing aggregation properties of boronic acid-appended porphyrin 14 were investigated by Murakami et al.^13,14  In the absence of saccharides, 14 forms aggregates that are non-fluorescent. The aggregates are dissociated by the addition of saccharides, resulting in strong fluorescence. Among four monosaccharides tested, the spectral change occurs in the order d-fructose > d-arabinose > d-mannose > d-glucose. Sugar-controlled aggregate formation of 15 was studied by Arimori et al.¹⁵  It was demonstrated that the morphology of oriented aggregates in aqueous media can be controlled by adding saccharides. Well-developed fibrous aggregates were obtained in the presence of d-fructose and d-glucose, whereas less-developed coagulated fibrous aggregates were obtained in the presence of d-ribose and d-fucose.

Arimori et al. succeeded in controlling photo-induced electron transfer process of porphyrins by saccharides.¹⁶  For this purpose, positively-charged porphyrins bearing boronic acids (16) were synthesized. When anionic fluorophores such as naphthalenedisulfonate and anthraquinonedisulfonate are mixed with 16 in aqueous solutions, fluorescence emission from these fluorophores is largely quenched. This change is attributed to the formation of electrostatically associated complexes between cationic 16 and the anionic fluorophores in which photo-induced electron transfer between the two components can efficiently take place. Addition of fructose dissociates the complexes because the positive charges on 16 are neutralized by the anionic charges on the boronate groups. As a result, the fluorescence intensity increases with fructose concentration since the quenching efficiency is sufficiently lowered by the dissociation of the complexes. An interaction between 16 and DNA was investigated by Suenaga et al.¹⁷  At pH 8.01, 16 is strongly bound to DNA. Comparison of the absorption spectra and the CD spectra established that poly(dGdC)·poly(dGdC) double strand intercalates 16, whereas poly(dAdT)·poly(dAdT) double strand binds 16 to the outside of the main chain. When d-fructose is added, 16 is dissociated from DNA through complexation with d-fructose. These results show that one can conveniently control the association–dissociation equilibrium between 16 and DNA by saccharides.

The cooperative action of two boronic acids is indispensable to the selective binding of saccharides in aqueous solution. However, it is not so easy to synthesize porphyrin derivatives bearing two appropriately arranged boronic acid groups within a molecule. To overcome this difficulty, Takeuchi et al. designed a boronic acid-based porphyrin receptor utilizing the metal coordination property in a metalloporphyrin with an axial ligand.¹⁸  For example, a boronic acid-appended Zn(ii) porphyrin (17) was synthesized and mixed with 3-pyridylboronic acid to create a self-organized diboronic acid system. When saccharides are added to this system, characteristic CD patterns inherent to the absolute configurations of saccharides are observed. Imada et al. utilized 17 for selective binding of glucose-6-phosphate and 3,4-dihydroxyphenylalanine (DOPA).^19,20  It was shown that 17 can bind these guest molecules in a two-point interaction manner: one between the diol and the boronic acid and the other between the phosphate or amino group and Zn(ii) in the metalloporphyrin moiety.

Hirata et al. designed porphyrin derivatives bearing a pair of boronic acid groups (18, 18-Zn, and 18-Cu).²¹  These compounds have a diethynyl porphyrin axis, which act as a saccharide-binding modulator. Saccharide binding studies were conducted in water–methanol (1 : 1, v/v) mixed solvent by UV-vis, fluorescence, and CD spectroscopies. It was found that 18-Zn can bind mono- and oligo-saccharides to produce 1 : 1 host–saccharide complexes with association constants (log K) of 2–3. The CD spectra indicate that the two boronic acid groups of 18-Zn are cooperatively used to bind one saccharide. The porphyrin unit efficiently works as a read-out functional moiety for the saccharide-binding information to give sharp spectral changes (Figure 1.2). The binding signal can be finely turned by metalation of the porphyrin unit. Following this study, Hirata et al. designed porphyrin derivatives bearing two pairs of boronic acid groups 19 to construct an allosteric saccharide-sensing system.²²  The conditions utilized for saccharide-binding studies are identical to those used for 18. The stepwise binding constants (log K₁ and log K₂) were, respectively, evaluated to be 3.58 and 3.48 for l-fucose and 3.95 and 3.69 for d-xylose. These K₂ values are significantly larger than those that are statistically expected (K₁ = 4K₂). Therefore, the obtained data imply that once a pair of boronic acids in 19 binds the first guest saccharide, another pair of boronic acids enhances its affinity toward the second guest saccharide. Binding of the first guest saccharide is entropically disfavored since the host molecule has to lose its rotational freedom, whereas the second guest binding is entropically favored due to preorganization and alignment of the second binding site (Scheme 1.5). Thus, 19 can behave as a saccharide receptor exhibiting a positive allosteric effect.

Hargrove et al. synthesized another porphyrin derivative (20) bearing a pair of boronic acid groups.²³  This receptor was used for sensing ginsenoside derivatives such as 21 through fluorescence spectroscopy. The fluorescence intensity is decreased with increasing ginsenosides concentrations, and the obtained quenching curves are used to estimate the 1 : 1 binding constants. The results support a view that the sugar units in the ginsenosides are bound to the boronic acid groups, while the steroid core and porphyrin ring participate in hydrophobic interactions.

To achieve successful two-point binding of a specific saccharide, it is important to manipulate two boronic acid moieties in an appropriate special position. In the previous section, porphyrins bearing two or more boronic acid groups display specific saccharide selectivity and the resultant complexes become CD-active only when two boronic acid groups are intramolecularly bridged by a saccharide molecule. To arrange two molecules of boronic acid-appended porphyrins in an appropriate special position, Takeuchi et al. utilized a µ-oxo dimer of a metalloporphyrin (22) to manufacture “sugar tweezers’”.^24,25  The µ-oxo dimer is stably formed in basic aqueous solutions where complexes between boronic acids and saccharides are also formed. The saccharide binding process of 22 can be conveniently monitored by CD spectroscopy, and the association constants (log K) for glucose and galactose were determined to be 5.18 and 4.39, respectively. In contrast, other monosaccharides are CD-silent. The origin of the CD activity is attributed to the formation of 1 : 1 µ-oxo dimer/saccharide complexes, in which two porphyrin rings are chirally bridged by one saccharide molecule.

The first example of positive allosterism in an aqueous saccharide-binding system was achieved by Sugasaki et al. using a Ce(iv) bis(porphyrinate) double decker scaffold bearing two pairs of boronic acid groups (23).²⁶  In this system, the binding of the first guest saccharide suppresses the rotational freedom of the two porphyrin planes, which facilitates the binding of the second guest saccharide. As a result, two pairs of boronic acid groups in 23 can auto-acceleratively bind two saccharide molecules and yield CD-active species. The 1 : 2 association constants of 23 for saccharides were determined by analysis of the CD intensity–guest concentration plots: log K = 4.57 for d-fructose, 5.98 for d-glucose. Compound 23 was also used for oligosaccharide binding.^27,28  Oligosaccharides such as malto-oligosaccharides, laminari-oligosaccharides, and Lewis oligosaccharides are bound by 23 in aqueous media through the boronic acid–diol interaction with association constants (log K) of 5–6. Characteristic sigmoidal binding isotherms are observed (Figure 1.3), indicating that the binding of two equivalents of oligosaccharides to 23 occurs cooperatively.

A meso–meso-linked porphyrin dimer bearing four boronic acid groups (24) was reported by Ikeda et al.²⁹  A strong CD band is observed when maltotetraose is added to the solution containing 24, while virtually no CD band is observed when glucose is added. The results indicate that maltotetraose bridges two boronic acid groups in 24, whereas glucose is too small to bridge the two boronic acid groups. The CD intensity measured as a function of maltotetraose concentration provides a sigmoidal curve indicating that the 1 : 2 complex is formed in a cooperative manner. The obtained association constant (log K) for maltotetraose is 5.78. A computational study predicts that the distance between the two boronic acid groups is comparable with that between 1,2-diol and 4,6-diol in the two terminal glucose units of maltotetraose.

Arimori et al. utilized the electrostatic interaction to generate dimeric boronic acid-appended porphyrins.³⁰  Anionic porphyrin 25 and cationic porphyrins 16 form 1 : 1 complexes, which give the specific exciton-coupling bands in CD spectroscopy only in the presence of glucose and xylose. The CD sign is characteristic for the absolute configuration of the saccharides. Structural examination established that only these monosaccharides can bridge two porphyrins by ester formation with boronic acid and twist them asymmetrically.

The air–water interface has interesting features as a medium for molecular recognition. For example, (1) a molecularly flat environment is formed at the interface, (2) a boundary region is facing the two phases with different dielectric constants, (3) macroscopically dynamic changes can be taken place within the plane of the interface, and (4) an access point between hydrophilic and hydrophobic compounds is provided. By utilizing these features, one can construct fascinating supramolecular systems exhibiting unique molecular recognition behaviors.

Shinkai et al. investigated the molecular recognition ability of amphiphilic boronic acids 26 towards mono- and di-saccharides at the air–water interface.³¹  Compound 26 forms a stable monolayer at the interface, and the surface pressure–molecular area (π–A) isotherms are affected by the addition of saccharides in the water subphase. Ludwig et al. conducted more detailed examinations and found that the detection of saccharides by the monolayers of 26 at the air–water interface becomes more sensitive by adding a polycation in the subphase.³²  Figure 1.4 depicts typical π–A isotherms of 26 at pH 10.0 when the subphase contains d-fructose and a polycation [quaternized poly(4-vinylpyridine)]. The molecular area and compressibility increase with saccharide concentration, and 0.05 mM of d-fructose or 0.1 mM of d-glucose are unequivocally detected. The effect of polycation is explained by the fact that the polycation facilitates hydrolysis of boronic acid group to form anionic boronate [–B(OH)₃⁻], which makes the formation of boronate ester with saccharide thermodynamically favorable. Amphiphilic diboronic acids 27–31 were also synthesized and used for sugar recognition at the air–water interface. Mono- and di-saccharides are selectively detected because of the fixed distance between the boronic acid moieties in the amphiphilic molecule and the organized structure of the monolayer.^33,34 

Ludwig et al. demonstrated that cholesterol-substituted phenylboronic acid 1 can be utilized for chiral discrimination of monosaccharide at the air–water interface.³⁵  Langmuir–Blodgett (LB) films were prepared with 1, and π–A isotherms were recorded in the presence of monosaccharide in the water subphase. It was found that the phase transition pressures of the monolayers are correlated with the Ph–Ph dihedral angle of the 1 : 2 saccharide–1 complexes (Figure 1.5). The monolayer exhibits chiral discrimination towards optical isomers of monosaccharides.

Polymeric amphiphile 32 carrying boronic acid groups in its polar head regions was prepared by Kitano et al.³⁶  The amphiphile forms a stable monolayer and the π–A profile is changeable by the addition of sugars in the subphase. The limiting molecular area of 32 at pH 11 is in the following order: lactose > mannose > no sugar ≈ fructose ≈ galactose > glucose. Notably, glucose decreases the molecular area, which is attributed to shrinkage of the boronic acid-containing head group by the formation of inter- and intramolecular crosslinks consisting of 1 : 2 glucose–boronate complexes. The polymeric amphiphile 32 was also used for the recognition of sugar proteins. By the addition of ovalbumin, which has a sugar chain consisting of two N-acetylglucosamine residues and seven mannose residues, the surface area of monolayer of 32 is greatly increased.

Recently, a mechanically controlled molecular recognition at the air–water interface was applied to the indicator displacement assay (IDA) by Sakakibara et al.³⁷  For that purpose, the amphiphilic molecule 33 consisting of phenylboronic acid, cholesterol, and fluorescein was synthesized. The cholesterol unit provides a hydrophobic functionality, and the carboxyfluorescein was chosen as a fluorescent probe because it can serve as an acceptor of fluorescence resonance energy transfer (FRET) for coumarin-based indicators such as 4-methylesculetin (ML). Firstly, a monolayer of 33 was formed at the interface in the presence of ML, then fluorescence spectra of the monolayer were measured at different surface pressures (π). At a π of 10 mN m⁻¹, excitation at 373 nm produces a blue emission at around 450 nm. As the surface pressure increases to 20 mN m⁻¹, a new green emission appears at around 530 nm. These phenomena indicate that compression of the monolayer can switch the energy transfer between excited ML and ground state fluorescein. Next, the effect of glucose on the fluorescence behavior was evaluated. With increasing glucose concentration in the water subphase, the energy transfer gradually turned off in a ratiometric fashion (Figure 1.6). This result indicates the displacement of indicator (ML) from 33 by glucose.

Metal nanoparticles (NPs) have been widely investigated for nanoscale optical devices because of their unique properties. Organic monolayer-protected metal NPs are particularly attractive for their photostability, size-controlled fluorescence properties, quantitative control of multiple functionalization, etc.

Gold nanoparticle/polyaniline nanocomposites bearing boronic acid moieties were synthesized by reducing HAuCl₄ with 3-aminophenylboronic acid in the presence of PVA [poly(vinyl alcohol)] by Ma et al.³⁸  The process is shown in Scheme 1.6. The Au nanocomposite shows good dispersiveness in aqueous solutions since PVA acts as a stabilizer. With increasing glucose concentration, the surface plasmon resonance (SPR) intensity decreases and the absorbance maximum shifts towards longer wavelength. This change is attributed to the replacement of PVA from the nanocomposite with boronic acid groups by competitive binding of glucose, resulting in the aggregation of the nanocomposite.

Liu et al. established a colorimetric sensing strategy for dihydronicotinamide adenine dinucleotide (NADH, 34).³⁹  When 4-mercaptophenylboronic acid (MPBA) is introduced on the surface of AuNPs through Au–S interaction, it begins to form aggregates. The authors insist that the aggregation is caused by the dehydration condensation of boronic acid groups. In the presence of NADH, aggregation of AuNPs is suppressed due to the formation of a boronate ester between MPBA and NADH. As a result, an obvious color change from blue to red is observed with increasing NADH concentration (Figure 1.7). The calibration curve has a linear range from 8 nM to 8 µM with a detection limit of 2 nM. The MPBA-functionalized AuNP was also used for glucose detection by Wang et al.⁴⁰  In this case, the AuNP is water-soluble in the absence of saccharides. In the presence of glucose, formation of 1 : 2 glucose–boronate complexes results in aggregation of the AuNP. Glucose concentration can be determined by monitoring the average particle size change of the assay solution using dynamic light scattering (DLS) analysis.

The synthesis of molecularly imprinted Au nanoparticle composite (Scheme 1.7) was developed by Frasconi et al.⁴¹  AuNPs were functionalized with thioaniline and mercaptophenylboronic acid, and electropolymerization was carried out in the presence of antibiotics such as streptomycin (35). After removal of the template antibiotics, sensing ability towards the antibiotics were evaluated by means of SPR. It was found that the detection limit for analyzing antibiotic 35 is as low as 200 fM. This strategy was also applied for imprinting and sensing mono- and di-saccharides by Ben-Amram et al.⁴² 

Zhang et al. synthesized boronic acid-capped silver nanoparticles using thiolated boronic acid 36.⁴³  When a polysaccharide (dextran) is added to an aqueous dispersion of the NPs, aggregation takes place and the aggregated NPs display a decrease of absorbance at 397 nm and an increase at 640 nm (Figure 1.8). The luminescence intensity shows an upward deviation with increasing concentration of dextran (Figure 1.9). The luminescence spectral change is ascribed to surface-enhanced fluorescence by the enhanced field from the aggregated silver NPs. In contrast, glucose induces minor spectral changes compared with the case of dextran.

Tang et al. developed a metal-enhanced quantum dot (QD) fluorescence system by conjugating CdSe QDs with Ag nanoparticles.⁴⁴  The boronic acid-functionalized CdSe QDs and the mercaptoglycerol-modified AgNPs are assembled into AgNP–CdSe QD complexes through the formation of a boronate ester bond (Scheme 1.8). As compared to that of bare CdSe QDs, up to a nine-fold enhancement and a clear blue-shift in the fluorescence emission peak for AgNP–CdSe QD complexes are observed. These effects are interpreted as due to the surface plasmon resonance of AgNPs inducing metal-enhanced fluorescence (MEF). In the presence of glucose, the AgNP–CdSe QD complexes are gradually disassembled by competitive binding of glucose with the boronic acid groups (Scheme 1.8), resulting in weakening of the fluorescence enhancement (Figure 1.10A). A linear decrease in fluorescence intensity in the range 2–52 mM with a detection limit of 1.86 mM was achieved (Figure 1.10B). A similar strategy was used for the fluorescence imaging of fluoride anion (F⁻) in living cells by Xue et al.⁴⁵  In this case, CdTe QDs are disassembled from AuNPs by the addition of fluoride, because F⁻ breaks the boronate eater linkages by the formation of trifluoroboronates.

Wu et al. demonstrated that metal nanoparticles can be immobilized in stimuli-responsive microgels, and this system offers the possibilities of external switching and manipulation of sensor devices.⁴⁶  The CdS hybrid microgels were synthesized through the in situ formation of CdS QDs in the interior of the copolymer microgel of poly(N-isopropylacrylamide–acrylamide–acrylamidophenylboronic acid) [p(NIPAM–AAm–PBA)]. It was found that the saccharide-induced volume phase transition of the hybrid microgels significantly affects the photoluminescence (PL) properties of the CdS QDs embedded in the interior of the microgels (Scheme 1.9). In the absence of saccharides, the hybrid microgels are in the shrunken state and exhibit PL emission centered at 638 nm. With increasing saccharide concentrations, the microgels are gradually swelled with an accompanying gradual quenching of the PL emission. At pH 8.8, the PL quenching almost linearly increases with glucose concentration from 0 to 13.5 mM.

Self-assembly is a viable method for constructing highly-ordered molecular architectures. Since boronic acids form covalent yet reversible bonds with diols, they can be regarded as useful building blocks to obtain stably self-assembled macromolecules exhibiting dynamic self-repairing capabilities. Since one boronic acid reacts with two OH groups, one can expect that the polycondensation reaction of diboronic acids and monosaccharides yields a sugar-containing linear polymer. To realize this idea, Mikami et al. synthesized diboronic acid 37 and reacted it with an equimolar amount of saccharides in the presence of molecular sieves (Scheme 1.10).⁴⁷  When 37 is polycondensed with l-fucose, the CD spectra shows an exciton-coupling band and the intensity increases with increasing molecular mass of the resulting polymers. By using d-fucose instead of l-fucose, a symmetrical CD spectrum is obtained. The highest molecular weight estimated by light-scattering measurements was 1.06 × 10⁵ for l-fucose. Diboronic acid 37 was also used for polycondensation with tetraols 38 and 39.⁴⁸  The average molecular weights are 8.6 × 10³ for d-38, 1.06 × 10⁴ for l-39, and 1.4 × 10⁴ for d-39. Another type of boronate ester-based polymer was reported by Niu et al.⁴⁹  A diboronic acid 40 was synthesized and condensed with 1,2,4,5-tetrahydroxybenze (Scheme 1.11). The resulting polymer had a molecular weight of ∼2.5 × 10⁴. Since the polymer has an extended π-conjugation system, absorption and emission spectra are significantly redshifted compared to the non-conjugated polymer.

Sarson et al. combined two kinds of reversible interactions, namely, boronic acid–diol and metal–pyridine interactions, to generate self-assembled polymers.⁵⁰  A 1 : 2 mixture of magnesium dicatechol porphyrin 41 and pyridine-3-boronic acid self-assembles into a polymer. The molecular weight was estimated to be ∼10⁹ by light scattering measurements.

Boronic acid-appended polymers are a unique class of stimuli-responsive polymers with potential applications as self-regulated drug delivery systems, therapeutic agents, self-healing materials, and sensors for saccharides and their derivatives. As an initial and basic study for saccharide-responsive polymers based on the boronic acid–diol interactions, Nagasaki, Kimura, and co-workers synthesized a boronic acid-modified poly(l-lysine) (42).^51–53  It is known that polypeptides show the helix–coil transition in aqueous solution that can be monitored by CD spectroscopy. When monosaccharides are added to the solution of 42, the helix content increases and the pH giving the maximum helix content shifts to lower pH region. In the absence of saccharides, the maximum helix content is 78% at pH 9, whereas in the presence of 32 mM d-fructose the maximum helix content reaches nearly 100% at pH 7. The increase in helix content is attributable to the hydrogen bonding interactions among OH groups in bound fructose by which the helical structure is stabilized. The chirality of 42 was also utilized to control the two-dimensional orientation of cyanine dye 43. In the presence of saccharides, 43 is associated onto 42 through the electrostatic interaction between cationic 43 and anionic boronate groups in 42, resulting in a significant color change. The chiral orientation can be evaluated by CD spectroscopy. Kobayashi et al. incorporated a fluorophore into the boronic acid-modified poly(l-lysine). Using the poly(l-lysine) derivative 44, the saccharide-induced conformational changes can readily be monitored by fluorescence spectroscopy.⁵⁴ 

Friggeri et al. presented a new molecular imprinting method based on a poly(l-lysine) derivative 45 bearing –SH groups in addition to boronic acid groups (Scheme 1.12).⁵⁵  The interaction between boronic acid groups and a template saccharide induces a conformational change in the polymer. Subsequent adsorption of the polymer onto a gold surface through the Au–S interaction fixes the polymer conformation. By removing the template saccharide, a molecularly imprinted interface is created. The binding ability of the interface was examined by QCM measurements, which revealed that the glucose-imprinted interface could selectively detect glucose over fructose.

Another type of boronic acid-appended polymer exhibiting saccharide-induced conformational changes was developed by Kanekiyo et al.⁵⁶  The synthesized copolymer 46 consists of boronic acid and pyrene units. In aqueous solution, binding with saccharide turns the polymer conformation from a contracted to an expanded form due to electrostatic repulsion between ionized boronate groups. This conformational change is conveniently detected by monitoring the excimer (480 nm) to monomer (377 nm) emission intensity ratio in the fluorescence spectra (Figure 1.11).

Sugar-responsive block copolymers 47 were synthesized by Roy et al. through RAFT (reversible addition–fragmentation chain transfer) polymerization of 3-acrylamidophenylboronic acid (APBA) and N,N-dimethylacrylamide.⁵⁷  The behaviors of the block copolymer in aqueous solution were investigated by dynamic light scattering measurements (Figure 1.12). In the absence of saccharides, 47 exists as unimers at pH 10.7, whereas it forms aggregates with an average hydrodynamic diameter of 35 nm at pH 8.7. This pH responsiveness arises from the ionization equilibrium of the boronic acid groups. The aggregates observed at pH 8.7 are supposed to be micelles composed of hydrophilic poly(N,N-dimethylacrylamide) coronas and a hydrophobic polyAPBA core. Upon addition of glucose, the average hydrodynamic diameter decreases to 9 nm, indicative of disassembly of the aggregates due to hydrophilization of the sugar-bound polyAPBA. To introduce a thermo-responsive nature, N-isopropylacrylamide (NIPAM) was copolymerized with APBA.⁵⁸  The resulting block copolymer 48 possesses sugar- and pH-responsiveness like 47. When a solution of 48 is heated from 25 to 50 °C at pH 11, aggregates 78 nm in size are observed. PolyNIPAM is known to exhibit a phase transition at around 32 °C, above which the polymer is dehydrated. Therefore, it is proposed that heating of the solution leads to the formation of micelles with a polyAPBA corona and a polyNIPAM core.

A label-free detection of saccharides was proposed by Chung et al.⁵⁹  A boronic acid-containing copolymer was prepared from APBA and acrylamide. The polymer was mixed with a cationic platinum complex 49 in aqueous solution buffered at pH 9.0, and the fluorescence spectra were recorded by excitation at 448 nm. In the absence of saccharides, an emission band at 577 nm is observed. With increasing glucose concentration, this band decreases while a new emission band at 800 nm grows. These spectral changes are caused by the saccharide-induced aggregation of cationic 49 onto the anionic boronate polymer.

Tsuchiya et al. utilized well-known “iodo-starch reaction” for sensing polyhydroxy compounds.⁶⁰  To realize that, amylose was modified with boronic acid moieties (Scheme 1.13). An aqueous solution containing the boronic acid-modified amylose and iodine (mixture of I₂ and KI) is colored bluish purple due to encapsulation of iodine within the helical cavity of amylose. With increasing concentration of alditols such as sorbitol, the solutions gradually lose their color (Figure 1.13). This color change is attributable to the dissociation of iodine from the amylose cavity induced by alditols. It was concluded that the origin of the responsiveness is derived from an electrostatic repulsion between anionic boronate groups and iodine that exists as anionic polyiodide form (I₃⁻ or I₅⁻).

Schizophyllan (SPG, 50) is a natural polysaccharide produced by the fungus Schizophyllum commune, and its repeating unit consists of three β-1,3-glucoses and one β-1,6-glucose side chain linked at every third main-chain glucose. In the side-chain glucose unit, the 4-OH and the 6-OH groups remain unsubstituted, which can form complexes with boric and boronic acids. Tamesue et al. utilized this interaction for regularly aligned single-walled carbon nanotubes (SWNTs).⁶¹  An aqueous solution containing sodium borate was added to an aqueous dispersion of SWNT-SPG composites. When the sample was observed by TEM, it was clearly seen that the SWNT-SPG composite forms sheet-like structures with periodical stripes (Figure 1.14a–d). This kind of ordered structure could not be observed in the absence of borate ion. It is supposed, therefore, that borate ions assemble and align the SWNT-SPG composites as illustrated in Figure 1.14e. Tamesue et al. also used the interaction between SPG and boronic acids in designing pH- and sugar-responsive hydrogels.⁶²  By mixing boronic acid-modified poly(acrylic acid) (51) and SPG in water at pH 9.9, a hydrogel is readily formed due to the formation of crosslinks created by the interaction between the boronate groups in 51 and the side-chain glucose units in SPG. This hydrogel shows reversible transformation from gel to sol by (1) changing medium pH from 9.9 to 8.4 and (2) adding fructose.

Kanekiyo et al. developed a fluorescent sensing system for ATP (adenosine 5′-triphosphate) utilizing the ATP-mediated aggregation process of pyrene-appended boronic acid 52 on a polycation (8).⁶³  In the absence of ATP, 52 shows monomer emission in the range 360–430 nm. With increasing ATP concentration, excimer emission centered at 482 nm is intensified (Figure 1.15). Two kinds of interactions should induce supramolecular aggregate formation (Scheme 1.14): (1) electrostatic interaction between ATP and polycation 8 and (2) boronate–diol interaction between 52 and ATP. It was found that this system selectively detects ATP among related compounds such as ADP, AMP, and deoxy-ATP. In addition, when glucose is added to the aqueous mixture of 52 and 8, excimer emission is observed.⁶⁴  This is attributable to the formation of 1 : 2 complex between glucose and 52, in which two pyrene groups are intramolecularly stacked. Other saccharides such as fructose, galactose, and ribose scarcely induce such excimer emission.

As a practical saccharide sensor for personal use, it would be convenient if the sensor shows easily observable distinct color changes. However, boronic acid-based sensors sometimes lack distinct color changes since the phenylboronic acid moiety itself has no visible color. Therefore, it seems to be necessary to introduce novel methodology for the development of practically applicable saccharide sensors. Kanekiyo et al. reported a “saccharide-responsive polymer”, from which anionic dyes are sequentially released.⁶⁵  A copolymer containing boronic acid and amine units was synthesized from monomers 10 and 53, and then anionic dyes having different color were adsorbed on it. By immersing the dye-adsorbed copolymer in aqueous saccharide solution, blue dye 54 and yellow dye 55 are sequentially released from the copolymer with increasing saccharide concentrations. As a result, the aqueous solution changes color from colorless to blue, and then to green.

The saccharide-responsive polymer was produced as a thin film on a glass plate by Iwami et al.^66,67  By copolymerizing 10 and 53 on a glass plate, a boronic acid-containing thin film was obtained. After adsorbing anionic dyes (54 and 55), the thin film was immersed in aqueous saccharide solutions containing the cationic red dye 56. As the saccharide concentration increases, the thin film shows color changes from green to red via yellow (Figure 1.16). The origin of the distinct color changes is attributed to a stepwise release and binding of dyes as illustrated in Scheme 1.15. The saccharide-responsive thin film was further extended to a multicolor sensor array.⁶⁸  Copolymerization was conducted on a pattern-printed microscope slide that is covered with hydrophobic coating (20 µm thick) having circular holes (8 mm in diameter). Each spot in the slide was then stained with various anionic dyes. The sensor array thus obtained distinctly changes its color as shown in Figure 1.17. This methodology enables us to measure saccharide concentration by pattern-based sensing utilizing multiple color changes of the sensor array.

The above-mentioned sensing chips take a relatively long time (∼1 h) for the appearance of sufficient color changes. To shorten the response time, the film thickness was greatly reduced by applying the layer-by-layer adsorption technique by Takayoshi et al.⁶⁹  The saccharide-responsive thin films were obtained on a pattern-printed microscope slide via alternating adsorption of a boronic acid-containing polycation (57) and sodium polyacrylate. The film thickness was determined by SEM analysis to be about 1 µm, which is ten-times thinner than the previously reported film. After adsorbing anionic dyes, the sensing chip was immersed in aqueous saccharide solution. As the saccharide concentration increases, the thin film shows a multi-patterned color change within 10 min (Figure 1.18).