New applications of the interaction between diols and boronic acids

Florine Duval - New applications of the interaction between diols and boronic acids – Summary

Chapter 1 introduces the theory and known applications of the interaction between boronic acids and diols, and explains the context of this thesis. Diagnosis of depression was the initial goal of this multidisciplinary project. The focus of the PhD project was the development of a strategy to immobilize antibodies on the surface of a chip in such a way that very low concentrations (~ 1 pM) of biomarkers for depression could be detected in urine. To achieve this, the immobilization of antibodies using boronic acids seemed promising.

However, preliminary experiments and further insights revealed the many challenges that this immobilization strategy faces, giving rise to Chapter 2. This chapter discusses several important points that need to be taken into account when one plans to immobilize antibodies via boronic acids: choice of the boronic acid structure and spacer to attach it to the surface, use of an antifouling polymer, choice of an antibody with suitable glycosylation, optimization of the conditions for antibody immobilization...

One big issue for antibody immobilization using boronic acids is the reversibility of the reaction between boronic acids and diols, hence the possible release of the antibody from the surface.

Chapter 3 describes the design and synthesis of boronic acid-containing linkers that would enable the oriented and irreversible immobilization of antibodies. Two linkers were designed with an amine for surface attachment, a boronic acid for capturing antibodies via the N-glycans in their Fc chain, and a diazirine for irreversible immobilization upon UV irradiation while maintaining antibody orientation. From a diazirine building-block that was obtained in three steps, the first linker was synthesized in four steps and the second linker was synthesized in three steps. Diol-functionalized silica was used for the chromatography of two boronic acid-containing intermediates, this method being novel (to the best of our knowledge) and likely based on boronic acid-diol interactions. High-resolution mass spectrometry, through matching exact masses, matching isotope patterns and observation of species corresponding to the esterification of boronic acids with MeOH, confirmed that both linkers were synthesized successfully.

During the synthesis of boronic acid-containing linkers, it was difficult to see which spots on TLC plates corresponded to boronic acids. To solve this problem, a new TLC staining method based on the reaction between boronic acids and alizarin was developed.

Chapter 4 presents this work in detail. After optimization experiments, 1 mM alizarin in acetone was shown to be the preferred staining solution. When the TLC plate was briefly dipped in this solution, allowed to dry in ambient air and observed under 365 nm light, bright yellow fluorescent spots were observed where boronic acids were present. Phenylboronic acid was detected at a concentration as low as 0.1 mM. A range of boronic acids and derivatives was successfully detected, and boron-free compounds resulted in no or very weak fluorescence. The staining method was further tested in the monitoring of three reactions involving boronic acids, and provided clear information about the consumption or formation of boronic acid-containing compounds.

Although TLC is useful to synthetic chemists, analysis of reaction mixtures by HPLC is sometimes necessary for obtaining more accurate information or for optimization of preparative HPLC conditions.

Chapter 5 presents the development and applicability of a method for the on-line HPLC detection of boronic acids using alizarin. After optimization experiments at an HPLC flow rate of 0.40 mL/min, the HPLC-separated analytes were mixed post-column with a solution of 75 μM alizarin and 0.1% triethylamine in ACN, which was delivered at a flow rate of 0.60 mL/min. The reaction between alizarin and boronic acids occurred in a reaction coil of dimensions of 3.5 m × 0.25 mm at a temperature of 50 °C, resulting in fluorescent complexes that were detected as positive peaks by a fluorescence detector (lexc 469 nm and lem 610 nm). The method enabled the selective detection of various boronic acids and derivatives, with a limit of detection of phenylboronic acid of 1.2 ng or 1 μM. It could successfully monitor the progress of two organic reactions involving boronic acid-containing compounds, and provided useful insights into the course of the reactions.

Chapter 6 provides a reflexion about the work presented in this thesis, suggestions for future research, and a general conclusion.