Multiwell Assay for the Analysis of Sugar Gut Permeability Markers: Discrimination of Sugar Alcohols with a Fluorescent Probe Array Based on Boronic Acid Appended Viologens

Abstract

With the aim of discerning between different sugar and sugar alcohols of biomedical relevance, such as gut permeability, arrays of 2-component probes were assembled with up to six boronic acid-appended viologens (BBVs): 4,4′-o-BBV, 3,3′-o-BBV, 3,4′-o-BBV, 4,4′-o,m-BBV, 4,7′-o-PBBV, and pBoB, each coupled to the fluorophore 8-hydroxypyrene, 1,3,6-trisulfonic acid trisodium salt (HPTS). These probes were screened for their ability to discriminate between lactulose, l-rhamnose, 3-O-methyl-d-glucose, and xylose. Binding studies of sugar alcohols mannitol, sorbitol, erythritol, adonitol, arabitol, galactitol, and xylitol revealed that diols containing threo-1,2-diol units have higher affinity for BBVs relative diols containing erythro-1,2 units. Those containing both threo-1,2- and 1,3-syn diol motifs showed high affinity for boronic acid binding. Fluorescence from the arrays were examined by principle component analysis (PCA) and linear discriminant analysis (LDA). Arrays with only three BBVs sufficed to discriminate between sugars (e.g., lactulose) and sugar alcohols (e.g., mannitol), establishing a differential probe. Compared with 4,4′-o-BBV, 2-fold reductions in lower limits of detection (LOD) and quantification (LOQ) were achieved for lactulose with 4,7-o-PBBV (LOD 41 μM, LOQ 72 μM). Using a combination of 4,4′-o-BBV, 4,7-o-PBBV, and pBoB, LDA statistically segregated lactulose/mannitol (L/M) ratios from 0.1 to 0.5, consistent with values encountered in small intestinal permeability tests. Another triad containing 3,3′-o-BBV, 4,4′-o-BBV, and 4,7-o-PBBV also discerned similar L/M ratios. This proof-of-concept demonstrates the potential for BBV arrays as an attractive alternate to HPLC to analyze mixtures of sugars and sugar alcohols in biomedical applications and sheds light on structural motifs that make this possible.

Boronic acid recognition of non-interacting carbohydrates for biomedical applications: increasing fluorescence signals of minimally interacting aldoses and sucralose

“…It is also clear that the glycitol product can be readily quantified using boronic acid-based detection systems such as our two-component fluorescent probe (4,4′- o -BBV–HPTS). 22 …”

“…Based on earlier findings, certain sugar alcohols have higher binding affinities for the boronic acid viologen receptor (4,4′- o -BBV) compared to the corresponding aldose forms. 22,29 Further, in situ modification of sucralose using reactive oxygen and hydroxide intermediates generated by the Fenton reaction or by photooxidation of H 2 O 2 by ultraviolet light is demonstrated. Compared to native sucralose, significantly increased fluorescence recovery was obtained by the dechlorination reaction.…”

“…This system has been extensively studied by our group for the recognition of various saccharides and sugar alcohols (glycitols). 20–22 …”

“…It is known that acyclic sugar alcohols can have higher binding affinity toward boronic acids compared to the corresponding aldose or ketose forms. 22,29 We explored pre-treatment reactions for saccharides with low or no binding affinity ( i.e. , fucose, rhamnose, or xylose) by a simple reductive conversion to corresponding alditols.…”

“…31,32 Our recent work has involved developing a non-enzymatic, multiwell fluorescent assay to quantify these sugar markers which are routinely used to monitor gut permeability. 22,33 We were interested in quantifying xylose and l -rhamnose, as these aldoses have also been used to study intestinal permeability. 34 Sucralose, a non-reducing trichlorinated sucrose, is used as a marker for colon permeability as it is resistant to fermentation by colonic bacteria.…”