Ortho-substituted aryl monoboronic acids have improved selectivity for d-glucose relative to d-fructose and l-lactate
Graphical abstract
Introduction
Aryl boronic acids are often used in carbohydrate recognition1, 2, 3, 4, 5, 6 because they are small and flexible, compared to other classes of carbohydrate binders, such as lectins7 and artificial macrocycles.8, 9 These features make aryl boronic acids easy to incorporate as recognition motifs in spectral probes or peptides, without dramatically changing the physical properties of the larger structures. The recognition event appears since boronic acids and boronates react with 1,2-cis-diols or 1,3-diols by reversible formation of the corresponding boronate esters. It is commonly believed that optimal binding affinity is achieved when pH is above pKa for a specific boronic acid. Earlier literature10, 11 reports that the binding strength depends on pKa of both the diol and the boronic acid. The event of carbohydrate binding is however complicated, since carbohydrates are polyols, which can exist in different anomeric configurations of six membered and five membered heterocyclic rings.
Glucose monitoring is of great importance in the treatment of diabetes mellitus. Boronic acids with displacement constants around 15–16 mM for d-glucose are desired,12 because blood glucose is fluctuating between 2 and 30 mM in diabetes patients. The maximum sensitivity is achieved when Kd is in the middle of the binding curve.
Aryl boronic acids with selective recognition of d-glucose over other polyol species are therefore of great interest, since d-glucose is the major carbohydrate present in human blood (≈5 mM)13 compared to d-fructose (<0.1 mM, even after a fructose-rich meal).14, 15 However d-fructose generally shows stronger binding affinity to most aryl monoboronates compared to d-glucose. An explanation might be, that under physiological conditions, d-fructose mainly exists in the furanose form, which can bind to boronates in tridentate configuration, while d-glucose mainly exists in the glucopyranose form, and binds in a bidentate configuration.16, 17 Notably l-lactate is also present in human blood as a metabolite after anaerobic biological processes, and α-hydroxy acids are also capable of forming boronate esters with aryl boronic acids.18, 19 Diboronates20, 21, 22 can show selectivity towards d-glucose, but they are synthetically challenging, compared with the synthesis of aryl monoboronates, and usually their solubility is low in aqueous media. Carbohydrate affinities of some boronates can be measured by exploiting their UV/vis or fluorescence properties, or in cases were such spectroscopic properties are missing, by competitive binding to spectroscopic probes.23, 24, 25, 26, 27, 28, 29 Another commonly used technique is the pH-depression method,30 where the drop in pH can be correlated with the binding constant between these two species. However this method suffers from the fact that it requires a high amount of boronic acid in solution. Furthermore this method assumes that the boronic acid diester is fully converted to the tetrahedral anionic form, as a consequence of the lowered pKa value.
In order to overcome the lack of spectroscopic properties of the given aryl boronic acids, we have adopted the method of UV/vis-titration experiments with the colored compound alizarin red sodium (ARS) in a three component competitive binding assay. This assay has successfully been employed by Wang and co-workers.11, 31, 32 As shown in Fig. 1, ARS binds reversibly to the aryl boronate, forming the corresponding aryl boronate ester, which displaces the absorption spectrum, and changes the color of the aqueous solution from clear red towards yellow.
Upon addition of the polyol to the ARS-boronate solution, ARS is released competitively, changing the color of the solution back towards red.
We used the ARS assay to screen a series of aryl boronic acids performing the measurements in a physiological environment at neutral pH. Comparing our data with data obtained using a non-physiological phosphate buffer, we report small changes in the respective binding affinities. Generally decreased selectivity towards d-fructose is achieved, due to the presence of ortho-substituents at the aryl monoboronic acids.
We have discovered that ortho-substituted aryl monoboronic acids bind d-fructose with a reduced strength compared to aryl monoboronic acids with no ortho-substituents. This is a valuable discovery, because a lot of synthetic effort can be avoided in the preparation of suitable boronic acid dyes, since aryl monoboronic acids generally are easier to access, and more soluble.
Section snippets
Results and discussion
Screening studies of a series of aryl boronic acids were performed in order to obtain selectivity towards binding of d-glucose. The measurements were performed in a physiological saline buffer, containing 10 mM phosphate, 2.7 mM KCl, and 137 mM NaCl in water, pH 7.4. The structures are shown in Fig. 2, and calculated Kd-values are shown in Table 1. Compound 1 and 2 are very similar in structure at the binding site, since they both contain a methyl group attached in the ortho-position to the
Conclusion
The experimental outcome shows that ortho-substituted aryl monoboronic acids are capable of binding d-fructose with a decreased selectivity relative to d-glucose in a physiological buffer, presumably due to a steric effect of the ortho-positioned methyl substituent, disfavoring the tridentate boronate-d-fructose complex. This is supported by computational calculations on B3LYP/6-31G(d) level with Gaussian.
Competitive intramolecular hydrogen bonding, in the case of ortho-positioned fluorine
General
The computational calculations were performed on B3LYP/6-31G(d) level with Gaussian.
The aryl boronic acids 1–6, 11, and 12 were purchased from Combi Blocks and used as received. Compounds 7 and 8 were purchased from Sigma–Aldrich and used as received. 9 and 10 were prepared by oxidation of the corresponding aldehydes with KMnO4.40 Alizarin red sodium, d-glucose, d-fructose, l-lactate, P4417 (phosphate buffered saline pellets), and P7994 (phosphate buffer pellets) were purchased from
Acknowledgements
Financial support from the University of Copenhagen, Department of Chemistry, and from Novo Nordisk A/S is gratefully acknowledged.
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