University of Birmingham Suzuki homo-coupling reaction based fluorescent sensors for monosaccharides

Palladium catalysed, aryl boronic acid homocoupling, is explored as a ﬂ uorescence sensing regime for saccharides. The catalytic formation rate of ﬂ uorescent bi-aryls, under control of a palladium catalyst, is modulated by the presence of saccharides. The nature of the aryl group, rate of biaryl formation and limits of detection are investigated.

The reversible covalent binding of boronic acids with saccharides has been extensively explored in the area of direct saccharide sensing. 5,[9][10][11] Colorimetric and uorometric boronic acid based saccharide sensors rely on a linear response generated by saccharides interacting with a dened stoichiometry of boronic acid sensor, to elicit the colour or uorescence responses they display. The synthesis of new sensor systems can oen be challenging yet the search for more sensitive sensors remains a highly active area of research. Indicator displacement assays partially relieve synthetic effort since the signal reporter moiety does not need to be directly linked to the binding unit (boronic acid moiety). 12-14 Using a similar concept a boronic acid-modied quencher was used by Singaram et al., 15 in this system binding between boronic acid (quencher) and saccharide released an anionic dye, resulting in signal recovery. While saccharide selectivity oen requires multiple boronic acid receptors, Jiang and co-workers have developed a series of glucose-selective monoboronic acid derivatives using the noncovalent "linking" of boronic acid moieties to afford optimal binding conformation for D-glucose. 16,17 However, the reversible interaction between a boronic acid and diols typically allow binding of sugar at mM or sub-mM levels. Thus a high concentration of chemosensor is required to generate a detectable signal.
New chemosensing strategies with enhanced sensitivity are in great demand. In previous work by some of the authors included in this report, the palladium catalysed formation of biphenyl via a homocoupling of phenylboronic acid was used as a probe to detect saccharide binding. 18 The approach relies on the catalytic formation of the reporter molecule, therefore the sensitivity is not limited to the detection of the added probe rather the formation of biphenyl giving enhanced opportunities for sensitivity in colorimetric and uorometric sensing, since the background and noise are minimised. In this system it is the rate of probe formation that gives information on the concentration of the saccharide analyte.
In a palladium catalysed Suzuki homo-coupling reaction the nature of the boron species affects the rate of reaction. Boronic esters react more slowly than boronic acids in these reactions; therefore the formation of a boronic ester with a saccharide slows bi-aryl formation. The boronic ester formed between an aryl boronic acid moiety and probe saccharides slows the rate of reaction towards Suzuki homo-coupling.
In the earlier work with phenylboronic acid (1a) as the substrate for catalytic saccharide sensing, the product biphenyl has excitation and emission wavelengths of 250 nm and 311 nm, respectively. These wavelengths are sub-optimal for practical sensing applications, since exogenous uorophores could interfere and impair the accuracy of this sensing system. As such alternative boronic acid substrates are required in order to deliver output molecules with longer wavelength excitation and emission proles to avoid interference from exogenous uorophores.
For naphthalene-1-boronic acid (1b) when the catalyst was added, a new emission at 380 nm was observed along with the disappearance of its intrinsic emission at 330 nm (S1 †), which was a very encouraging result since as well as extending the wavelength, the system is now a ratiometric uorescence sensing platform for saccharides. The reaction was monitored using TLC and the binaphthyl product was veried using an authentic sample of 1,1 0 -binaphthyl (S2 †). The optimum time for each measurement to obtain the maximum difference between the catalysed reaction with and without fructose was 25 minutes (S3 †). The titration of 1b (Fig. 3) with D-fructose revealed that 1 mM of D-fructose (4 mM in the case of the PBA system, shown in S4 †) was required to fully quench the reaction, demonstrating that the sensitivity of the catalytic sensing strategy can be modied by changing substrates. More importantly the ratiometric sensing observed could eliminate possible interference in the system and also lead to a calibration free system. Using the same protocol, saccharide titrations with Dgalactose and D-glucose were also investigated and the usual saccharide order of affinities towards the boronic acid moiety was observed (Table 1). 19 The limit of detection (LOD) indicates that an improved level of sensitivity was observed using the naphthalene-1-boronic acid, compared with our previous work. The improved sensitivity might be attributed to the establishment of the internal calibration of this sensing system. 1,4-Phenydiboronic acid (1c) was also utilised under the same conditions; in this system the uorescent intensity displayed an initial increase, but the intensity reduced over time. We attributed this to poor water solubility of the resulting polymer product, i.e. the uorescence decreases as the product desolvates. However, a good linear relationship between uorescent emission and the concentration of saccharide is obtained before the uorescence intensity decreases. The Dfructose titration was carried out by collecting uorescent spectra aer six minutes at different concentrations of saccharides. The 1,4-phenyldiboronic acid displayed a red-shied maximum emission and quicker response, which might be due to the extended conjugation of the generated products and also the reactivity of the substrate towards Suzuki homo-coupling reactions (Fig. 4).
It is worth noting that all the substrates were investigated under the same experimental conditions as the previous work without further optimisation toward each substrate for easier comparison. Given the popularity of the Suzuki coupling reaction and the multitude of boronic acid derivatives that are Scheme 1 Structures of phenylboronic acid (1a), naphthalene-1boronic acid (1b) and 1,4-phenyldiboronic acid (1c) and their corresponding homocoupling products (3a, 3b and 3c respectively).  readily available to use, saccharide recognition/sensing based on the Suzuki homo-coupling reaction could be easily tuned through the variation of substrates to afford practically applicable saccharide selective sensing platforms.

Conclusions
The magnitude of inhibition for all the boronic acids investigated 1a, 1b and 1c follow the same order as the affinities of saccharides toward the boronic acid moiety. 19 This demonstrates that the Suzuki homo-coupling reaction can be used as a general platform for the sensing of saccharides. More interestingly boronic acid 1b affords a ratiometric chemosensor with improved sensitivity and di-boronic acid 1c displays faster response and an improved linear relationship with increasing concentrations of saccharide. Given the multitude of boronic acids, our results indicate that it should be possible to extend this sensing platform and develop application specic chemosensors, with appropriate sensitivity and response time to match any desired saccharide sensing requirements.

Experimental section
Reagents and solvents were purchased from Sigma-Aldrich and Fisher, and were used directly without further purication. Fluorescence spectra were recorded on a Perkin Elmer Luminescence Spectrophotometer LHB50 uorescence spectrometer using a 1 cm quartz cell. Buffer solutions were 0.02 M NaHCO 3 -Na 2 CO 3 , calibrated by Hanna Instruments HI 9321 Microprocessor pH meter. NaHCO 3 -Na 2 CO 3 buffer solution was made up by dissolving 0.53 g Na 2 CO 3 and 1.68 g NaHCO 3 into 250 mL deionised water ($18.2 MU), with a pH value of 9.3. The stock solution of palladium catalyst was 1.0 Â 10 À3 M, made from PdCl 2 dissolved in MeOH. All the experiments were carried out at room temperature.