Biochemical probes that exhibit turn on fluorescence in response to a defined target serve as enabling tools in research and technology development. While bioorthogonal conjugation chemistry has made it possible to fluorescently label a wide variety biomolecules via incorporation of probes via natural or engineered metabolic processes, the reported chemistries are limited by the need for excess reagents, which results in high background fluorescence. To circumvent this issue, we have synthesized weakly fluorescent coumarin analogs bearing an acetylphenyl boronic acid (APBA). The conjugation of APBA to a wide variety of reactive partners, including simple amines, alpha-nucleophiles and aminothiols, which is both accelerated and stabilized by the electron withdrawing ortho-boronic acid moiety, has been extensively documented. Importantly, upon conjugation to these defined reactive partners, the non-fluorescent APBA-presenting coumarin analogs become strongly fluorescent. Thus far, we have applied this fluorogenic conjugation chemistry towards conjugation of both natural (cysteine) and non-natural (semicarbazide) targets, both of which are known react quickly with APBA (~103 M-1s-1) with high affinity and high selectivity over the plethora of molecules in the biological milieu. To apply this stable fluorogenic conjugation chemistry to biological systems, we have designed a semicarbazide-presenting unnatural amino acid, which readily incorporates this unnatural chemical functionality into the cell wall of the pathogenic bacteria Staphalococcus aureus. The bioorthogonal conjugation of this installed semicarbazide with the designed courmarin derivatives enables facile labeling of the bacteria via turn on fluorescence, which displays low background interference. Thus, the development of such turn on fluorescent probes with wide-ranging conjugation partners enables targeted detection of a variety of different biomolecules in biological system, both through conjugation to naturally existing molecules, as well as metabollically incorporated chemical moieties.

Fluorogenic chemoselective biocompatible conjugation chemistries of acetylphenyl boronic acid for biological applications

256th National Meeting (2018)

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