Diverted Natural Lossen-type Rearrangement for Bioconjugation through in Situ Myrosinase-Triggered Isothiocyanate Synthesis.

Fluorescein isothiocyanate (FITC) is one of the most extensively used fluorescent probes for the labeling of biomolecules. The isothiocyanate function reacts with lysine residues of proteins to provide a chemically stable thiourea linkage without releasing any byproduct. However, diversification of isothiocyanate-based reagents is still hampered by the lack of mild conditions to generate isothiocyanate chemical functions, as well as by their poor stability and limited solutions available to increase water solubility, restricting the use of isothiocyanate labeling to highly water-soluble fluorophores. Inspired by plant biological processes, we report a safe and biocompatible myrosinase-assisted in situ formation of isothiocyanate conjugates from a highly water-soluble and stable glucosinolate precursor. This method was applied for the fluorescence labeling of a plasmatic protein and fluorescence imaging of living cells.

Covalent Modification of Biomolecules through Maleimide-Based Labeling Strategies.

Since their first use in bioconjugation more than 50 years ago, maleimides have become privileged chemical partners for the site-selective modification of proteins via thio-Michael addition of biothiols and, to a lesser extent, via Diels-Alder (DA) reactions with biocompatible dienes. Prominent examples include immunotoxins and marketed maleimide-based antibody-drug conjugates (ADCs) such as Adcetris, which are used in cancer therapies. Among the key factors in the success of these groups is the availability of several maleimides that can be N-functionalized by fluorophores, affinity tags, spin labels, and pharmacophores, as well as their unique reactivities in terms of selectivity and kinetics. However, maleimide conjugate reactions have long been considered irreversible, and only recently have systematic studies regarding their reversibility and stability toward hydrolysis been reported. This review provides an overview of the diverse applications for maleimides in bioconjugation, highlighting their strengths and weaknesses, which are being overcome by recent strategies. Finally, the fluorescence quenching ability of maleimides was leveraged for the preparation of fluorogenic probes, which are mainly used for the specific detection of thiol analytes. A summary of the reported structures, their photophysical features, and their relative efficiencies is discussed in the last part of the review.

Photoprogramming Allostery in Human Serum Albumin.

Developing strategies to interfere with allosteric interactions in proteins not only promises to deepen our understanding of vital cellular processes but also allows their regulation using external triggers. Light is particularly attractive as a trigger being spatiotemporally selective and compatible with the physiological environment. Here, we engineered a hybrid protein in which irradiation with light opens a new allosteric communication route that is not inherent to the natural system. We select human serum albumin, a promiscuous protein responsible for transporting a variety of ligands in plasma, and show that by covalently incorporating a synthetic photoswitch to subdomain IA we achieve optical control of the ligand binding in subdomain IB. Molecular dynamics simulations confirm the allosteric nature of the interactions between IA and IB in the engineered protein. Specifically, upon illumination, photoconversion of the switch is found to correlate with a less-coordinated motion of the two subdomains and an increased flexibility of the binding pocket in subdomain IB, whose fluctuations are cooperatively enhanced by the presence of ligands, ultimately facilitating their release. Our combined experimental and computational work demonstrates how harnessing artificial molecular switches enables photoprogramming the allosteric regulation of binding activities in such a prominent protein.

Labelling Bacterial Nanocages with Photo-switchable Fluorophores.

The robustness and biocompatibility of bacterial nanocages holds promise for bio-nanotechnologies. The propensity of these nano-carriers to penetrate cells has been demonstrated, which calls for the development of tracking strategies, both in vitro and in vivo. Here, we label bacterial nanocages with photo-switchable fluorophores, to facilitate their imaging by super-resolution microscopy. We demonstrate the functionalization of the encapsulin from Brevibacterium linens with a spiropyran, which is not fluorescent, by covalent attachment to the amine residues at the outer encapsulin shell. Upon alternating irradiation with ultraviolet and visible light, the spiropyran switches forth and back to its fluorescent merocyanine photo-isomer and thus the fluorescence can be switched on and off, reversibly. We also show that the bacterial compartments preserve their structural integrity upon covalent modification and over at least five irradiation cycles.

Cyclodextrin polyrotaxanes as a highly modular platform for the development of imaging agents.

Selectively functionalized cyclodextrins with a bodipy fluorescent tag or Gd(3+) complex were synthetized and threaded onto a polyammonium chain to form polyrotaxanes. This modular supramolecular assembly makes an ideal platform for bimodal (fluorescent and MRI) imaging applications.

Mechanostereoselective One-Pot Synthesis of Functionalized Head-to-Head Cyclodextrin [3]Rotaxanes and Their Application as Magnetic Resonance Imaging Contrast Agents.

A versatile, five-component, one-pot synthesis of cyclodextrin (CD) [3]rotaxanes using copper-catalyzed azide-alkyne cycloaddition has been developed. Head-to-head [3]rotaxanes of α-CD selectively functionalized by one or two gadolinium 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid monoamide complexes were obtained mechanostereoselectively. The magnetic resonance imaging efficiency, expressed by the longitudinal proton relaxivity of the rotaxanes, was significantly improved as compared to the functionalized CD. In vitro and in vivo preclinical studies showed a higher contrast and retention in the kidney than gadolinium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complex, demonstrating the potential of these rotaxanes as MRI contrast agent.