Design, Synthesis, and Biological Studies of New Fluorescent Arylboronic Acid Chemosensor Dyes for the Detection of Mycolactone Toward the Diagnosis of Buruli Ulcer

Abstract

Background: Buruli ulcer (BU) is a devastating neglected tropical skin disease caused by Mycobacterium ulcerans (MU). The disease is prevalent in rural areas, accounting for approximately 30% of all ulcer cases that are reported in highly endemic health facilities in Africa. In 2023, 1952 new cases were reported from 12 countries, with 1573 (81%) of the reported cases from the African Region alone and 379 (19%) from the Western Pacific Region. Early and accurate diagnosis of BU is required for appropriate antibiotic therapy. However, the current diagnostic methods including microscopy, culture, histopathology, and PCR are associated with limitations such as not being readily available in endemic areas, low sensitivities and specificities, long turn-around time for results to be obtained, expensive, requiring sophisticated instrumentation, and highly skilled technical expertise. The fluorescent thin layer chromatography (f-TLC) method which involves the chemical derivatization of mycolactone with a 2-naphthylboronic acid (BA) for its direct detection has been evaluated. The method is rapid and promising; however, it suffers interferences from other co-extracted and co-eluted lipids. In addition, the read-out has not been automated and leads to subjective interpretations of results. This research seeks to provide an improved f-TLC diagnostic method that largely satisfies the ASSURED criteria (Affordable and cost-effective, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and Delivered to those who need it) for the diagnosis of BU. Methods: Firstly, the f-TLC method was used to screen a series of 27 commercially available arylboronic acids as potential replacements for the 2-naphthylboronic acid (BA) for mycolactone detection. Secondly, High-Performance Thin Layer Chromatography (HPTLC) was employed for the quantitative detection of mycolactone. Thirdly, considering that fluorescent chemosensors are increasingly becoming relevant in recognition chemistry due totheir great sensitivity, selectivity, fast response time, real-time detection capability, and low cost, a library of fluorescent chemosensors with various signalling moieties (i.e. fluorophores and chromophores with certain beneficial photophysical characteristics) and a recognition moiety (i.e. boronic acid unit) were (i) rationally designed; (ii) synthesized using combinatorial approaches; and (iii) fully characterized using a set of complementary spectrometric and spectroscopic techniques such as NMR, LC-MS, FT-IR, and X-ray crystallography. In addition, a complete set of basic photophysical quantities such as absorption maxima (absmax), emission maxima (emmax), Stokes shift (∆λ), molar extinction coefficient (ε), fluorescence quantum yield (ΦF), and brightness were determined using UV-vis absorption and fluorescence emission spectroscopy techniques. Results: Three commercially available boronic acids i.e. BA15, BA18, and BA21 yielded fluorescent bands on TLC with intensities that were superior to BA when coupled to mycolactone. Among the three, BA18 produced the most outstanding TLC profiles, and thus, was validated using clinical samples from patients including five (5) positive and four (4) negative samples that were confirmed by gold standard PCR. These produced bands on TLC plates that were easier to interpret even in the presence of background interference. The automated HPTLC method achieved satisfactory quantification of mycolactone with an RF value of 0.32. The linear range was 4 – 45 ng for mycolactone with an R2 = 0.98. The limit of detection (LOD) and limit of quantification (LOQ) were estimated to be 6.6 and 22 ng/spot respectively. Conclusion: BA18 was found to be a potential alternative to BA from the commercially available aryl boronic acids. A total of 12 fluorescent arylboronic acids were synthesized including aminoacridine, aminoquinoline, azo, BODIPY, coumarin, fluorescein, and rhodamine variants. After assessing the photophysical properties together with the binding ability of the dyes to mycolactone on TLC, AAG113 emerged as the best (absmax = 456 nm, (emmax = 590 nm, ∆λ = 134 nm, ε = 52816.1 M-1cm-1, ΦF = 0.78, and brightness = 41196.6 M 1cm-1). Preliminary results show that HPTLC offers advantages over conventional f-TLC because a number of the steps are automated giving rise to increased resolution, and more accurate quantitative measurements of mycolactone.