Alternative boronic acids in the detection of Mycolactone A/B using the thin layer chromatography (f-TLC) method for diagnosis of Buruli ulcer
Authors: Akolgo Gideon A. , Partridge Benjamin M. , D. Craggs Timothy , Amewu Richard K. 
Published: July 2023

Abstract
Background
Mycobacterium ulcerans is the causative agent of Buruli ulcer. The pathology of M. ulcerans disease has been attributed to the secretion of a potent macrolide cytotoxin known as mycolactone which plays an important role in the virulence of the disease. Mycolactone is a biomarker for the diagnosis of BU that can be detected using the fluorescent-thin layer chromatography (f-TLC) technique. The technique relies on the chemical derivatization of mycolactone A/B with 2-naphthylboronic acid (BA) which acts as a fluorogenic chemosensor. However, background interferences due to co-extracted human tissue lipids, especially with clinical samples coupled with the subjectivity of the method call for an investigation to find an alternative to BA.

Methods
Twenty-six commercially available arylboronic acids were initially screened as alternatives to BA using the f-TLC experiment. UV–vis measurements were also conducted to determine the absorption maximum spectra of mycolactone A/B and myco-boronic acid adducts followed by an investigation of the fluorescence-enhancing ability of the boronate ester formation between mycolactone A/B and our three most promising boronic acids (BA15, BA18, and BA21). LC–MS technique was employed to confirm the adduct formation between mycolactone and boronic acids. Furthermore, a comparative study was conducted between BA18 and BA using 6 Polymerase Chain Reaction (PCR) confirmed BU patient samples.

Results
Three of the boronic acids (BA15, BA18, and BA21) produced fluorescent band intensities superior to BA. Complexation studies conducted on thin layer chromatography (TLC) using 0.1 M solution of the three boronic acids and various volumes of 10 ng/µL of synthetic mycolactone ranging from 1 µL – 9 µL corresponding to 10 ng – 90 ng gave similar results with myco-BA18 adduct emerging with the most visibly intense fluorescence bands. UV–vis absorption maxima (λmax) for the free mycolactone A/B was observed at 362 nm, and the values for the adducts myco-BA15, myco-BA18, and myco-BA21 were at 272 nm, 270 nm, and 286 nm respectively. The comparable experimental λmax of 362 nm for mycolactone A/B to the calculated Woodward-Fieser value of 367 nm for the fatty acid side chain of mycolactone A/B demonstrate that even though 2 cyclic boronates were formed, only the boronate of the southern side chain with the chromophore was excited by irradiation at 365 nm. Fluorescence experiments have demonstrated that coupling BA18 to mycolactone A/B along the 1,3-diols remarkably enhanced the fluorescence intensity at 537 nm. High-Resolution Mass Spectrometer (HR-MS) was used to confirm the formation of the myco-BA15 adduct. Finally, f-TLC analysis of patient samples with BA18 gave improved BA18-adduct intensities compared to the original BA-adduct.

Conclusion
Twenty-six commercially available boronic acids were investigated as alternatives to BA, used in the f-TLC analysis for the diagnosis of BU. Three (3) of them BA15, BA18, and BA21 gave superior fluorescence band intensity profiles. They gave profiles that were easier to interpret after the myco-boronic acid adduct formation and in experiments with clinical samples from patients with BA18 the best. BA18, therefore, has been identified as a potential alternative to BA and could provide a solution to the challenge of background interference of co-extracted human tissue lipids from clinical samples currently associated with the use of BA.