In Vivo Targeting Using Arylboronate/Nopoldiol Click Conjugation

Abstract

Bioorthogonal click reactions yielding stable and irreversible adducts are in high demand for in vivo applications, including in biomolecular labeling, diagnostic imaging, and drug delivery. Previously, we reported a novel bioorthogonal “click” reaction based on the coupling of ortho-acetyl arylboronates and thiosemicarbazide-functionalized nopoldiol. We now report that a detailed structural analysis of the arylboronate/nopoldiol adduct by X-ray crystallography and 11B NMR reveals that the bioorthogonal reactants form, unexpectedly, a tetracyclic adduct through the cyclization of the distal nitrogen into the semithiocarbazone leading to a strong B–N dative bond and two new 5-membered rings. The cyclization adduct, which protects the boronate unit against hydrolytic breakdown, sheds light on the irreversible nature of this polycondensation. The potential of this reaction to work in a live animal setting was studied through in vivo capture of fluorescently labeled molecules in vivo. Arylboronates were introduced into tissues through intradermal injection of their activated NHS esters, which react with amines in the extracellular matrix. Fluorescently labeled nopoldiol molecules were administered systemically and were efficiently captured by the arylboronic acids in a location-specific manner. Taken together, these in vivo proof-of-concept studies establish arylboronate/nopoldiol bioorthogonal chemistry as a candidate for wide array of applications in chemical biology and drug delivery.

Supramolecular “Click Chemistry” for Targeting in the Body

“…In the field of bioconjugate chemistry, a molecular-scale pretargeting approach has been demonstrated using different bioorthogonal ligations to capture circulating agents at specific sites in the body through spontaneous formation of covalent bonds. − Spatial localization can be achieved within the body by covalent bond formation in situ using a two-step application of a pretargeted entity bearing one component of a bioorthogonal motif followed by application of the second motif attached to a drug or imaging agent (Figure B,C). − The attachment of a drug to a group for click chemistry offers certain prodrug benefits of attenuated systemic activity; such agents also incorporate labile linkages for subsequent release of the active therapeutic via linker hydrolysis following local accumulation. Others have demonstrated so-called “click-to-release” and “catch and release” chemistries wherein an active agent releases from its bioorthogonal motif-bearing prodrug precursor by spontaneous ring isomerization simultaneous to in situ formation of a covalent bond. , …”

Boronic acid based dynamic click chemistry: recent advances and emergent applications

“…Interestingly, the reaction was shown to work in a live animal setting. 61 Following their previous report on the stable conjugates between a-amino hydrazides and 2-FPBA/2-APBA, the Bane group reported the formation of highly stable products from the reaction of 2-FPBA with b-hydroxy hydrazides. 62 An apparent second-order rate constant for the hydrazone formation step was calculated to be $955 M À1 s À1 , and the combined ring closure event was $0.014 s À1 .…”

Engineered Polymer-Supported Biorthogonal Nanocatalysts Using Flash Nanoprecipitation

“…Bioorthogonal chemistry is a versatile strategy for modulating bioprocesses using abiotic reactions. − Transition-metal catalysts (TMCs) present excellent candidates for bioorthogonal reactions, catalyzing transformations that mimic the catalytic behavior displayed by natural enzymes. − Unmasking reactions mediated by TMCs are rapidly being developed for biomedical applications, using the ability of these catalysts to generate therapeutic agents in situ and minimizing off-target effects. − However, the direct application of TMCs in living systems is challenging due to insolubility, instability, and hence low catalytic efficiency of “naked” TMCs. , The encapsulation of TMCs into polymeric nanoparticles generates bioorthogonal polyzymes that provide enhanced solubility and stability while preserving high catalytic activity. , …”