A simple supramolecular complex of boronic acid-appended β-cyclodextrin and a fluorescent boronic acid-based probe with excellent selectivity for d-glucose in water

Various diboronic acid-based chemosensors for d-glucose have been developed for use in diabetes diagnostic systems. However, most of these chemosensors have limitations, such as poor water solubility, difficulties in synthesis, and inability to selectively detect d-glucose from among other saccharides. We report a simple chemosensor based on a supramolecular complex of fluorophenylboronic acid-appended β-cyclodextrin (FPB-βCyD) and an anthracene-based probe having a boronic acid moiety (1). Hydrophobic 1 is encapsulated in the cyclodextrin cavity of FPB-βCyD, making the supramolecular complex (1/FPB-βCyD) applicable in a water-rich solvent mixture (98% water). Interestingly, 1/FPB-βCyD showed a strong turn-on response to d-glucose with a 9.6-fold enhancement in fluorescence intensity, and no response to other saccharides. This study uncovers an innovative approach based on the supramolecular assembly of simple components for the development of a water-soluble d-glucose chemosensor with excellent selectivity.


Introduction
The modern diet based on high-calorie foods has increased the number of diabetic patients globally, and the total number is projected to exceed 300 million by 2030. 1 There is a strong demand for an accurate blood glucose sensing system for the early diagnosis of diabetes because conventional enzyme-based D-glucose biosensors have low heat and pH stability and poor reproducibility.
Recently, heat-and pH-stable diboronic acid sensors, which contain two boronic acid moieties that are precisely positioned to simultaneously react with the two 1,2-diol moieties of Dglucose, have emerged for D-glucose sensing. 2,3 Nevertheless, the development of a truly D-glucose selective diboronic acidbased chemosensor has been continuously pursued as diboronic acids can also react with other saccharides possessing two 1,2-diol moieties (e.g., D-galactose). In addition, many diboronic acid compounds require complicated organic synthesis, and the presence of a highly hydrophobic uorophore decreases their solubility in water. The aim of our research is to develop an ideal chemosensor with (1) high selectivity for D-glucose, (2) a simple structure, and (3) high water solubility to achieve D-glucose sensing.
We present a supramolecular complex consisting of a m-uorophenylboronic acid-based probe (1) and m-uorophenylboronic acid-appended b-cyclodextrin (FPB-bCyD) as a uorescent chemosensor for D-glucose that can be used in a water-rich solvent mixture of dimethyl sulfoxide (DMSO)/ water (2/98 in v/v, Scheme 1). The structures of these Scheme 1 Molecular structures of the supramolecular complexes evaluated in this study.
components are so simple that the synthesis can be completed effortlessly. Cyclodextrin (CyD) is water-soluble and has a hydrophobic cavity that can encapsulate the hydrophobic probe; this allows the supramolecular complex to function as a sensor in an aqueous environment. [4][5][6][7] To enhance the affinity of the boronic acid moiety for D-glucose, we introduced an electron-withdrawing uorine group into the phenylboronic acid moiety to increase the Lewis acidity of the boron centre. 5 We also elucidated the sensing mechanism of supramolecular complex 1/FPB-bCyD through comparison with a complex of 1 and phenylboronic acid-appended b-cyclodextrin (PB-bCyD).

Instrumentation
The solution pH was measured by a HORIBA pH meter F-52 (Horiba, Japan). Fluorescence spectra were measured using a Hitachi F-7000 uorescence spectrophotometer (Hitachi, Japan) equipped with a temperature controller (Hitachi, Japan) to keep the temperature constant at 25 C. Induced circular dichroism (ICD) spectra were measured using a JASCO J-820 spectrophotometer (JASCO, Japan) equipped with a Peltier temperature controller (JASCO, Japan) and an EYELA Cool Ace CA-1111 (EYELA, Japan) to keep the temperature constant at 25 C under a nitrogen atmosphere.

Preparation of sample solutions
The ionic strength of sample solutions was adjusted to 0.10 M with sodium chloride. The pH of sample solutions was adjusted by adding hydrochloric acid aq. and 50% sodium hydroxide solution.

Measurement of uorescence spectra under various pH conditions
Sample solutions that contain DMSO, 0.2 mM of a cyclodextrin compound, 0.10 M of NaCl, 10 mM of phosphate buffer, a saccharide (0 or 30 mM), and 10 mM of 1 in a mixed solvent of DMSO/water (2/98 in v/v) were prepared. The solutions were acidied with a diluted hydrochloric acid aqueous solution until ca. pH 3. Fluorescence spectra for each of the sample solutions were measured under various pH conditions by the titration of NaOH aq. into the acidic sample solution. The solution pH was increased until ca. pH 11. Before every measurement, the sample solution was stirred for 25 minutes aer each titration.

Determination of acid dissociation constants
pH dependence of the uorescence intensity at a specic wavelength was analysed using the KaleidaGraph program according to a theoretical sigmoidal curve derived from the acid dissociation model of a monobasic acid.

Measurement of uorescence spectra at various saccharide concentrations
A series of solutions that contains DMSO, NaCl, a cyclodextrin compound, carbonate buffer, and a saccharide with various concentrations (0-30 mM) was prepared individually. The pH of all the solutions was adjusted to 10. Before the measurement of uorescence and ICD spectra, the solutions were stirred for 25 minutes aer the addition of an appropriate amount of 1 DMSO solution to prepare solutions containing 10 mM of 1, 0.2 mM of a cyclodextrin compound, 0.10 M of NaCl, 10 mM of carbonate buffer, and a saccharide (0-30 mM) in a mixed solvent of DMSO/ water (2/98 in v/v).
Supramolecular complex 1/FPB-bCyD was prepared by dissolving FPB-bCyD (0.2 mM) and 1 (10 mM) in DMSO/water (2/98 in v/v). According to our previous studies, CyD compounds can encapsulate hydrophobic probes in their CyD cavities. [4][5][6][7] Induced circular dichroism (ICD) spectra were measured to clarify the structure of 1/FPB-bCyD (Fig. S1 †). In the absence and presence of saccharides, a weak positive Cotton effect was observed in the 270-290 nm region. In the presence of D-fructose, this Cotton effect was not observed because of the strong positive Cotton effect of D-fructose itself at 280 nm. 8 We previously reported that bisignate Cotton effects are observed in the ICD spectra when two molecules of 1 are simultaneously held in the native g-CyD cavity, and only one molecule of 1 is held in the native b-CyD. 5 Therefore, 1 and FPB-bCyD form a supramolecular complex of 1 : 1 stoichiometry. The uorescence spectrum of 1/FPB-bCyD showed a broad vibrational structure with peaks at 413 nm and 431 nm, with no band corresponding to anthracene dimer emission observable at around 520 nm (Fig. S2 †). 9 These features were identical to those of the 1 : 1 stoichiometric complex of 1 and native b-CyD, suggesting that 1/FPB-bCyD is a supramolecular complex of 1 : 1 stoichiometry. 5 Anthracene compounds form photodimers that are non-emissive upon irradiation; 10 however, the uorescence spectra of 1/FPB-bCyD showed no change over time with excitation at 323 nm (Fig. S3 †), suggesting that 1 molecules in solution are not positioned sufficiently close to each other to form photodimers, as these molecules are trapped by excess FPB-bCyD to form 1 : 1 stoichiometric complexes.
The uorescence and UV-vis absorption spectra of 1/FPB-bCyD were measured at pH 10 in the absence and presence of Dglucose, D-fructose, and D-galactose, i.e., common saccharides in blood ( Fig. 1 and S4 †). Interestingly, whereas no spectral change was observed in the UV-vis absorption spectra, a 9.6-fold enhancement in the uorescence intensity of 1/FPB-bCyD was observed at 431 nm upon the addition of 30 mM D-glucose. In contrast, the spectra of 1/FPB-bCyD showed little change when D-fructose and D-galactose were added. Whereas 1/FPB-bCyD solutions exhibited weak uorescence that was not visible to the naked eye in the absence of saccharides as well as in the presence of D-fructose and D-galactose, strong blue uorescence was observed in the presence of D-glucose under 365 nm UV irradiation using a commercial UV lamp (Fig. 2). The uorescence spectra of 1/FPB-bCyD were measured under various pH conditions in the absence and presence of the saccharides (Fig. S5 †). Fluorescence intensities at 413 nm and 431 nm increased with increasing pH. Given that FPB-bCyD is nonemissive in the entire pH region investigated, this pH dependence is likely attributable to the acid dissociation of 1, i.e., structural change in the boronic acid moiety from trigonal boronic acid (non-uorescent) to tetrahedral boronate ion (weakly uorescent) (Scheme S1a †). The uorescence spectrum of 1/FPB-bCyD measured in the absence of saccharides at pH > 10 was almost identical to that measured in the presence of Dfructose or D-galactose. Interestingly, in the presence of Dglucose, 1/FPB-bCyD exhibited much stronger uorescence than that observed in the absence of saccharides at pH > 10 ( Fig. S5-5 †). By applying non-linear least squares tting, the apparent acid dissociation constants (pK app a1 ) were 9.14 AE 0.03 in the absence of saccharides, 8.54 AE 0.05 in the presence of Dglucose, 7.52 AE 0.04 in the presence of D-fructose, and 8.44 AE 0.03 in the presence of D-galactose (Fig. S6 †). The decrease in pK app a1 values in the presence of saccharides is due to the formation of boronate ester ions through the reaction of a boronic acid and a saccharide (Scheme S1b †), suggesting that 1 reacts with saccharides to give boronate ester ions. 11 It should be emphasised that D-fructose and D-galactose inherently react with 1 at pH 10, but do not form a uorescent product with 1/FPB-bCyD. Therefore, 1/FPB-bCyD enables the highly selective detection of D-glucose in a water-rich solvent at pH > 10 with a turn-on response, emitting blue uorescence visible to the naked eye.
To evaluate the sensitivity of 1/FPB-bCyD for the saccharides, the uorescence spectra of 1/FPB-bCyD were measured at various concentrations of common saccharides (D-glucose, D-fructose, and D-galactose) and less common saccharides (D-mannose, D-ribose, and D-xylose) at pH 10 ( Fig. S7 †). Spectral measurements   were performed 25 minutes aer mixing 1/FPB-bCyD with each saccharide (Fig. S8 †) because the reaction took about 20 minutes to complete. Surprisingly, 1/FPB-bCyD showed extremely high selectivity for D-glucose, exhibiting a strong turn-on response with uorescence enhancement at 431 nm (Fig. 3). On the other hand, 1/FPB-bCyD showed almost no uorescence response to the other saccharides. The uorescence intensity increased linearly in the D-glucose concentration range of 0-5 mM and reached a plateau at ca. 20 mM. Because D-glucose in blood is present at the mM level, 1/FPB-bCyD could potentially be used for monitoring glucose in actual blood samples. 12 The conditional equilibrium constant K 0 at pH 10 for the reaction of 1/FPB-bCyD with D-glucose was determined to be 139 AE 9 M À1 according to a theoretical equation derived from the 1 : 1 binding model (eqn (S1) and Fig. S9 †).
The K 0 values for the other reaction systems could not be determined because none of the other saccharides evaluated caused sufficient uorescence enhancement. Using a 3s approach and the data of reproducibility (n ¼ 3) at the lower measurement points in Fig. 3, the limit of detection (LOD) was estimated to be 90 mM for D-glucose with the calibration curve of I ¼ 562.66C glu. + 575.57 (R 2 ¼ 0.983).
Competitive experiments indicated that the origin of uorescence, i.e., the reaction product of 1/FPB-bCyD and D-glucose (1/FPB-bCyD-glc), was robust in the presence of other saccharides except D-fructose, which decreased the uorescence intensity by only 6% (Fig. S10 †). These results demonstrate that 1/FPB-bCyD selectively recognises D-glucose in the presence of other saccharides. When 0.1 mM of D-fructose was also present, the uorescence intensity of 1/FPB-bCyD-glc decreased to 83%. This is due to the much higher affinity of D-fructose for monoboronic acids compared with D-glucose, thereby inhibiting the formation of 1/FPB-bCyD-glc. 13 Nevertheless, as blood D-fructose concentrations are in the mM range, the presence of D-fructose is unlikely to interfere with D-glucose sensing by systems based on 1/FPB-bCyD in practical situations. 14 To elucidate the D-glucose sensing mechanism of 1/FPB-bCyD, we examined the saccharide recognition characteristics of 1/PB-bCyD, a control supramolecular complex of probe 1 and PB-bCyD with 1 : 1 stoichiometry (Scheme 1), for comparison. The phenylboronic acid moiety of PB-bCyD has a lower affinity for saccharides than the m-uorophenylboronic acid moiety of FPB-bCyD as the Lewis acidity of phenylboronic acid is lower than that of m-uorophenylboronic acid. 15 The uorescence spectra of 1/PB-bCyD were measured under various pH conditions in the absence and presence of Dglucose, D-fructose, and D-galactose (Fig. S11 †). When the pH of the solution was increased from 7 to around 10, an increase in uorescence intensity was observed in the absence and presence of saccharides, as in the case of 1/FPB-bCyD; this was considered to be due to the deprotonation of the boronic acid moiety of 1 in 1/PB-bCyD. At pH # 6, an emission band at 447 nm appeared, which possibly originated from exciplex formation between 1 and the phenylboronic acid moiety of PB-bCyD. The analysis of 1/PB-bCyD uorescence intensity at 413 nm under various pH conditions revealed pK app a1 values of 9.25 AE 0.05 in the absence of saccharides, 8.62 AE 0.02 in the presence of D-glucose, 7.84 AE 0.06 in the presence of D-fructose, and 8.95 AE 0.05 in the presence of D-galactose (Fig. S12 †). These pK app a1 values were larger than those of 1/FPB-bCyD under the corresponding conditions, suggesting that the acidity of 1 is slightly inuenced by the structure of the boronic acid moiety appended to the b-CyD. 1/PB-bCyD exhibited a marked increase in uorescence intensity in response to D-glucose at pH > 10, similar to 1/FPB-bCyD (Fig. S11-2 †). It is notable that 1/FPB-bCyD displayed a much stronger turn-on uorescence response to D-glucose than 1/PB-bCyD (9.6-fold vs. 2.8-fold enhancement in uorescence intensity at 431 nm). This indicates that the m-uorophenylboronic acid moiety of FPB-bCyD, which has a higher affinity for saccharides than the phenylboronic acid moiety of PB-bCyD, plays a key role in the uorescence detection of D-glucose. On the basis of these ndings, we propose the following mechanism for the sensing of saccharides by 1/FPB-bCyD (Scheme 2). The reaction of 1/FPB-bCyD with D-glucose produces uorescent product 1/FPB-bCyD-glc through the simultaneous binding of the two boronic acid moieties of 1/FPB-bCyD to the two 1,2-diol moieties of Dglucose. This rigidies the anthracene moiety of 1 in the cavity of FPB-bCyD, giving rise to enhanced uorescence. As the affinity of FPB-bCyD for saccharides is higher than that of PB-bCyD, 1/FPB-bCyD forms a more stable product with Dglucose, showing a stronger (3.4-fold) turn-on uorescence response to D-glucose than 1/PB-bCyD. In contrast, no such recognition through the two boronic acid moieties of 1/FPB-bCyD occurs with other saccharides; each boronic acid moiety reacts with a saccharide in 1 : 1 stoichiometric ratio, and thus, the structure of 1/FPB-bCyD is inherently unchanged, and the uorescence spectrum is also unchanged. Remarkably, 1/FPB-bCyD exhibited no uorescence enhancement in response to Dgalactose, which also has two 1,2-diol moieties. The excellent selectivity of 1/FPB-bCyD for D-glucose originates from its two boronic acid moieties that are precisely positioned to match the distance between the two 1,2-diol moieties of D-glucose, which is shorter than that of D-galactose. 16 Our results demonstrate that 1/FPB-bCyD selectively detects D-glucose via Scheme 2 Plausible saccharide sensing mechanism of 1/FPB-bCyD: D-glucose and other saccharides. the formation of a stable supramolecular complex, 1/FPB-bCyD-glc, through the hydrophobic interaction of the anthracene moiety of 1 with the hydrophobic cavity of FPB-bCyD and the dynamic covalent binding of the two boronic acid moieties to the two 1,2-diol moieties of D-glucose.

Conclusions
We have demonstrated that 1/FPB-bCyD, a water-soluble supramolecular complex composed of two simple components, recognises D-glucose with extremely high selectivity in a water-rich solvent (98% water). The two boronic acid moieties of 1/FPB-bCyD are precisely positioned to simultaneously react with the two 1,2-diol moieties of D-glucose. The reaction of 1/ FPB-bCyD with D-glucose gives 1/FPB-bCyD-glc, which results in a 9.6-fold enhancement in uorescence intensity that is visible to the naked eye, by the rigidication of 1 in the hydrophobic cavity of FPB-bCyD. In contrast, other saccharides including Dgalactose are hardly recognised by the two reaction sites of 1/ FPB-bCyD, and hence, no rigidication of 1 occurs. Consequently, the excellent selectivity of 1/FPB-bCyD for D-glucose is achieved. The uorescence intensity of 1/FPB-bCyD linearly increased with increasing D-glucose concentration in the mM range, suggesting that 1/FPB-bCyD is a potential candidate for the basic structure of glucose sensing systems for diabetes diagnosis. Moreover, we have demonstrated that the novel approach that exploits the supramolecular assembly of boronic acid-appended CyD and boronic acid-based hydrophobic probe enables extremely selective detection of D-glucose in water. Furthermore, it requires neither difficult multi-step synthesis nor substantial amounts of organic solvents to dissolve the hydrophobic probe, unlike most of the conventional boronic acid-based chemosensors. The present approach represents a completely novel strategy and has the potential to become a breakthrough in the development of practical boronic acidbased chemosensors that are highly selective for D-glucose in water.

Conflicts of interest
There are no conicts to declare.