title: Novel methods for identifying and targeting cellsurface carbohydrates associated with disease
Creators: name: Garget, Taylor A
Publisher: name: Griffith University
Year: 2022
Subjects: subject: Boronic acids
subject: cell-surface carbohydrates
subject: Neu5Ac

description: Boronic acids are a relatively new tool that could be of significant use in medicinal chemistry and medical diagnostics. Their most interesting feature is their ability to reversibly form boron ester groups with 1,2 and 1,3 diols in aqueous conditions under neutral pH. Much work has gone into designing boronic acid containing molecules that can be used as spectroscopic tools for separation, detection, and quantification of target diols. Boronic acids reactivity can be modulated by both steric and electronic factors to increase their selectivity. One of the primary uses of boronic acids is as artificial lectins (termed boronolectins) which target specific carbohydrates. This work aims to develop fluorescent boronic acid sensors that could be used in the detection and monitoring of the glycans in a clinical setting. The success of each sensor was judged on the following criteria, 1) their ability to bind and interact with the target glycans in simulated biologically relevant conditions, 2) their fluorescent properties including excitation, emission, and brightness and how these properties change. Chapter 1 is an introduction to the concept of boronic acid binding and the targets, several different Neu5Ac oligosaccharides. This covers the variety of sensing methods that have been developed, the pros and cons of each, and how each of these molecules are designed to specifically interact with Neu5Ac. Chapter 2 focuses on the synthesis and testing of boronic acid containing polymers. Benzoboroxoles were attached to 20-35% of the subunits of four different polymers: alginate, polysialic acid (PSA), hyaluronic acid and chitosan. These polymers had significant issues with solubility, limiting the methods that could be used for evaluating their binding. For this reason, surface plasmon resonance (SPR) was chosen, and small linker chains were attached to the polymers so they could be immobilized, thus avoiding problems with solubility. Through testing, only chitosan showed high immobilization due to its large number of primary amines, whereas alginate and PSA polymers showed low immobilization limiting the Rmax to &lt;1RU and hyaluronic acid had immobilization too low to allow binding data to be determined. Alginate and PSA showed binding to the control sugars fructose, galactose, glucose, and Neu5Ac with KD values between 16-80 μM but the results were not significant different (Students ttest, p&gt;0.4) from the control polymers. Chitosan was the only boronated polymer to show detectable interaction toward PSA with KD values between 0.27-0.40μM compared to 140-350 μM fructose, galactose and Neu5Ac with glucose being between 11-18 μM. The KD’s for short and long chain boronated chitosan were not significant different (Students t-test, p&gt;0.4) from the control polymers. While this research provides limited practical information on the use of free polymers for glycan binding ii it does show the potential of chitosan as a base to produce solid phase structures for producing, purifying, or enriching glycan samples. Chapter 3 analyses the utility of a novel amidination reaction; with the aim of utilizing this method to create boronic acid sensors. The electron withdrawing effect of the salicylic acid (SA) was found to increase the Lewis acidity of the boronic acid. This led to a B-N bond between 2-aminophenylboronic acid (2APB) and the reactant nitriles, this was then susceptible to attack by the primary amine group of 2APB rendering the amidination reaction intramolecular. Building from this, attempts were made to further enhance the reaction by selecting electron withdrawing halogenated alcohols and evaluating their utility as protecting/promoting groups. Trials using acetonitrile as the reactant nitriles showed increased conversion from 33% with SA, to 78% with 2,2,2-trifluoroethanol and finally quantitative yields with perfluorinated pinacol. This protecting group proved to be highly efficient catalysing the amidination of several different nitrile groups with only mild heating. DFT calculations confirmed the experimental data with perfluorinated pinacol being having lowest B electrostatic charge of 0.412 compared to 2,2,2- trifluoroethanol and SA which had charges of 0.564 and 0.634 respectively. However, this failed to yield a new sensor motif as the amidine version of 2APB was not fluorescently active. Attempts were made at synthesizing new amidine sensors using the traditional Pinner amidination reaction but attempts using 3-aminophenylboronic acid and 6-amionobenzoboroxole both failed to produce any products. One final attempt was performed to make a boronic acid sensor using a unique boroamidine fluorophore published within the last year. Of the three attempts only one of these boroamidines was produced with only a 6% yield. This fluorophore was found to have a constant fluorescence unaffected by the binding interaction of the boronic acid. While this section failed to discover a unique boronic acid fluorophore it has better described the synthetic chemistry behind a novel amidination reaction and more fully described its mechanism of action. Chapters 4 deals with the design, synthesis and testing of small molecule fluorescent sensors. Two lead molecules were investigated, 6-aminobenzylboroxole (6ABB) and 4-isoquinalineboronic acid (4IQBA); both were found to be fluorescent and possess a fluorescent switching mechanism triggered by binding to Neu5Ac. 6ABB was initially used as the basis for six different diboronates with varying binding locations to determine the optimum positioning of boronic acids to interact with the targets. Based on the positive binding of trends 4IQBA the aromatic nitrogen was chosen as a key design element and several diboronates were synthesized via the Metashulkin reaction. The fluorescent properties of the above molecules and several controls were tested, determining their fluorescent intensity, λex, λem, quantum yield and how these were affected by the addition of Neu5Ac, galactose, fructose, and glucose. SPR was then used to test the binding of these molecules to PSA, α2-3, and α2- 6 sialyllactose. This chapter contains a submitted publication that analyses 4-borono-2-(3- boronobenzyl)isoquinolin-2-ium (54) a diboronate based on 4IQBA that had the ability to differentiate between galactose and glucose based on their respective positive and negative fluorescent trends. Using iii various alternative sugars, the exact binding preferences of the different boronic acids were able to be determined showing the isoquinoline boronic acid of 54 favours the non-reducing end of the sugar 4,6 diols whereas the phenyl boronic acid favours the reducing end of the sugar 1,2-diol of glucose. Of the other sensors 2-aminopyrimidine-5-boronic acid had the best fluorescence with having up to a x10 increase in fluorescence when exposed to [Gal]*10-1; however, this molecule lacked specificity. 2- ((E/Z)-((3-borono)methylidene)amino)boronic acid (76) showed the best binding with KD’s of &lt;2μM for Glc, Lac and Neu5Ac. This molecule was the only one to show a three-shift fluorescent response with λem shifting from 380nm to 310nm before shifting to 345nm as the glycan concentration rose. This report has explored several different methods for the synthesis of boronic acid molecules targeting Neu5Ac oligosaccharides. We have shown that chitosan is a viable scaffold for post polymerisation modification through the addition of boroxole groups; with the resulting product having strong interactions with Neu5Ac. We showcased and enhanced amidination reaction that had increased synthetic efficiency using perfluorinated pinacol. Finally, we analysed the fluorescent ability of various small molecule sensors with special attention given to 54 which showed the ability to differentiate between glucose and galactose.