Synergic “Click” Boronate/Thiosemicarbazone System for Fast and Irreversible Bioorthogonal Conjugation in Live Cells

Abstract

Fast, high-yielding, and selective bioorthogonal "click" reactions employing nontoxic reagents are in high demand for their great utility in the conjugation of biomolecules in live cells. Although a number of click reactions were developed for this purpose, many are associated with drawbacks and limitations that justify the development of alternative systems for both single- or dual-labeling applications. Recent reports have highlighted the potential of boronic ester formation as a bioorthogonal click reaction between abiotic boronic acids and diols. Boronic ester formation is a fast dehydrative process; however it is intrinsically reversible in aqueous medium. We designed and optimized a synergic system based on two bifunctional reagents, a thiosemicarbazide-functionalized nopoldiol and an ortho-acetyl arylboronic acid. Both reagents were shown to be chemically stable and nontoxic to HEK293T cells at concentrations as high as 50 μM. The resulting boronate/thiosemicarbazone adduct is a medium-sized ring that forms rapidly and irreversibly without any catalyst at low μM concentrations, in neutral buffer, with a rate constant of 9 M s as measured by NMR spectroscopy. Control experiments in the presence of competing boronic acids showed no crossover side-products and confirmed the stability and lack of reversibility of the boronate/thiosemicarbazone conjugates. Formation of the conjugates is not affected by the presence of biological diols such as fructose, glucose, and catechol, and the thiosemicarbazide-functionalized nopoldiol is inert to aldehyde electrophiles of the sort found on protein-bound glyoxylyl units. The suitability of this system in the cell-surface labeling of live cells was demonstrated using a SNAP-tag approach to install the boronic acid reagent onto the extracellular domain of the Beta-2 adrenergic receptor in HEK293T cells, followed by incubation with the optimal thiosemicarbazide-functionalized nopoldiol reagent labeled with fluorescein dye. Successful visualization by fluorescence microscopy was possible with a reagent concentration as low as 10 μM, thus confirming the potential of this system in biological applications.

Boronic acids as building blocks for the construction of therapeutically useful bioconjugates

“…14B). 126 This new system is similar to the previous one but, through the introduction of an additional hydrazone ligation, the reaction became irreversible. This click type reaction was successfully applied to site selective protein labelling on mammalian HEK293T cells.…”

Recent Progress in Chemical Modification of Proteins

“…31 They also showed that the 2-acetylphenylboronate unit forms more stable bonds with hydrazide, hydrazine and aminooxy functionalities, 32 indicating that 2-acetylphenylboronic acid would be potentially applicable as a novel reaction handle for bioorthogonal click chemistry. 33…”

Boronsäuren als bioorthogonale Sonden für zentrenselektives Protein‐Labeling

“…[63] Die initiale Boronatbildung ist nachweislich essenziell fürd ie intramolekulare Thiosemicarbazonbildung und liefert ein mittelgroßes Ringaddukt. Dies kçnnte ihre Anwendung bei der Abbildung lebender Zellen erschweren, da das Boronataddukt unerwünschte Spaltungsreaktionen eingehen kçnnte,w ährend überschüssiges Reagenz in einem Waschschritt entfernt wird.…”