A Simple and Versatile Pathway for the Synthesis of Visible Light Photoreactive Nanoparticles

This work pioneers the design of visible (415 nm) and UV‐B light (300 nm) reactive nanoparticles via radical polymerization in aqueous heterogeneous media based on methyl methacrylate (MMA) and unique acrylates bearing tetrazole functionalities in a simple and straightforward two step reaction. Stable colloidal nanoparticles with an average diameter of 150 nm and inherent tetrazole functionality (varying from 2.5 to 10 wt% relative to MMA) are prepared via one‐pot miniemulsion polymerization. In a subsequent step, fluorescent pyrazoline moieties serving as linkage points are generated on the nanoparticles by either photoinduced nitrile imine‐mediated tetrazole‐ene cycloaddition (NITEC) or nitrile imine carboxylic acid ligation (NICAL) in water, thus enabling the particles as fluorescent tracers. Through in‐depth molecular surface analysis, it is demonstrated that the photoreactive nanoparticles undergo ligation to a variety of substrates bearing functionalities including maleimides, acrylates, or carboxylic acids, illustrating the versatility of the particle modification process. Critically, the unique ability of the photoreactive nanoparticles to be activated with visible light allows for their decoration with UV light–sensitive molecules. Herein, the ligation of folic acid—a vitamin prone to degradation under UV light—to the photoreactive nanoparticles using visible light is exemplified, demonstrating the synthetic power of our photoreactive fluorescent nanoparticle platform technology.     

Modular Enzyme- and Light-Based Activation of Cyclopropene–Tetrazine Ligation

We describe a modular activation strategy for cyclopropene–tetrazine ligation. This activation strategy uses chemically diverse enzyme- or photolabile protecting groups as cyclopropene reactivity cages. The linkages between the caging groups and cyclopropene are through carbamates, thus permitting the application of diverse cages to allow bioorthogonal reactivity by administering enzymes or light.     

Cycloadditions of Trans-Cyclooctenes and Nitrones as Tools for Bioorthogonal Labelling

Trans-cyclooctenes (TCOs) represent interesting and highly reactive dipolarophiles for organic transformations including bioorthogonal chemistry. Herein we show that TCOs react rapidly with nitrones and that these reactions are bioorthogonal. Kinetic analysis of acyclic and cyclic nitrones with strained-trans-cyclooctene (s-TCO) shows fast reactivity and demonstrates the utility of this cycloaddition reaction for bioorthogonal labelling. Labelling of the bacterial peptidoglycan layer with unnatural d -amino acids tagged with nitrones and s-TCO-Alexa488 is demonstrated. These new findings expand the bioorthogonal toolbox, and allow TCO reagents to be used in bioorthogonal applications beyond tetrazine ligations for the first time and open up new avenues for bioorthogonal ligations with diverse nitrone reactants.     

The Anti-Inflammatory Role of Bilirubin on “Two-Hit” Sepsis Animal Model

Introduction: Bilirubin is a product of the heme catabolism pathway, and it is excreted in bile and removed from the body through the urine. Bilirubin has potent antioxidant properties but also plays a role in anti-inflammation by protecting the body against endotoxin-induced lung inflammation, down-regulating the expression of adhesion molecules, and inhibiting the infiltration of inflammatory cells. Thus, bilirubin is a promising agent that could use in inflammation disease treatment. The application of bilirubin on the “two-hit” sepsis animal model has been, to date, unknown. Methods: we used lipopolysaccharide to induce initial insults in C57BL/6 mice. After 24 h, mice underwent cecal ligation and puncture to induce the “two-hit” sepsis model. Next, mice were administered 30 mg/kg bilirubin and we observed an improvement. Results: We observed that bilirubin inhibited the expression of pro-inflammatory cytokines, while the levels of anti-inflammatory cytokines were significantly augmented in the lung. Bilirubin improved the survival rate in the sepsis model. Furthermore, we suggest that bilirubin can modulate the accumulation of T-regulatory cells and myeloid-derived suppressor cells. Notably, bilirubin suppressed the activation and functions of T-cells. Conclusions: These results clarified that bilirubin might improve tissue injury in sepsis through anti-inflammatory mechanisms.     

Tetrazine-Triggered Release of Carboxylic-Acid-Containing Molecules for Activation of an Anti-inflammatory Drug

In addition to its use for the study of biomolecules in living systems, bioorthogonal chemistry has emerged as a promising strategy to enable protein or drug activation in a spatially and temporally controlled manner. This study demonstrates the application of a bioorthogonal inverse electron-demand Diels–Alder (iEDDA) reaction to cleave trans-cyclooctene (TCO) and vinyl protecting groups from carboxylic acid-containing molecules. The tetrazine-mediated decaging reaction proceeded under biocompatible conditions with fast reaction kinetics (<2 min). The anti-inflammatory activity of ketoprofen was successfully reinstated after decaging of the nontoxic TCOprodrug in live macrophages. Overall, this work expands the scope of functional groups and the application of decaging reactions to a new class of drugs.     

Bacterial Cell-Surface Display of Semisynthetic Cyclic Peptides

Semisynthetic cyclic peptides containing both non-proteinogenic building blocks, as the synthetic part, and a genetically encoded sequence amenable to DNA-based randomization hold great potential to expand the chemical space in the quest for novel bioactive peptides. Key to an efficient selection of novel binders to biomacromolecules is a robust method to link their genotype and phenotype. A novel bacterial cell surface display technology has been developed to present cyclic peptides composed of synthetic and genetically encoded fragments in their backbones. The fragments were combined by protein trans-splicing and intramolecular oxime ligation. To this end, a split intein half and an unnatural amino acid were displayed with the genetically encoded part on the surface of Escherichia coli. Addition of the synthetic fragment equipped with the split intein partner and an aminooxy moiety, as well as the application of a pH-shift protocol, resulted in the onsurface formation of the semisynthetic cyclic peptide. This approach will serve for the generation of cyclic peptide libraries suitable for selection by fluorescence-activated cell sorting, and more generally enables chemical modification of proteins on the bacterial surface.     

Triple Orthogonal Labeling of Glycans by Applying Photoclick Chemistry

Bioorthogonal labeling of multiple biomolecules is of current interest in chemical biology. Metabolic glycoengineering (MGE) has been shown to be an appropriate approach to visualizing carbohydrates. Here, we report that the nitrile imine–alkene cycloaddition (photoclick reaction) is a suitable ligation reaction in MGE. Using a mannosamine derivative with an acrylamide reporter group that is efficiently metabolized by cells and that quickly reacts in the photoclick reaction, we labeled sialic acids on the surface of living cells. Screening of several alkenes showed that a previously reported carbamate-linked methylcyclopropene reporter that is well suited for the inverse-electron-demand Diels–Alder (DA_inv) reaction has a surprisingly low reactivity in the photoclick reaction. Thus, for the first time, we were able to triply label glycans by a combination of DA_inv, photoclick, and copper-free click chemistry.     

Engineering the Mycomembrane of Live Mycobacteria with an Expanded Set of Trehalose Monomycolate Analogues

Mycobacteria and related organisms in the Corynebacterineae suborder are characterized by a distinctive outer membrane referred to as the mycomembrane. Biosynthesis of the mycomembrane occurs through an essential process called mycoloylation, which involves antigen 85 (Ag85)-catalyzed transfer of mycolic acids from the mycoloyl donor trehalose monomycolate (TMM) to acceptor carbohydrates and, in some organisms, proteins. We recently described an alkyne-modified TMM analogue (O-AlkTMM-C7) which, in conjunction with click chemistry, acted as a chemical reporter for mycoloylation in intact cells and allowed metabolic labeling of mycoloylated components of the mycomembrane. Here, we describe the synthesis and evaluation of a toolbox of TMM-based reporters bearing alkyne, azide, trans-cyclooctene, and fluorescent tags. These compounds gave further insight into the substrate tolerance of mycoloyltransferases (e.g., Ag85s) in a cellular context and they provide significantly expanded experimental versatility by allowing one- or two-step cell labeling, live cell labeling, and rapid cell labeling via tetrazine ligation. Such capabilities will facilitate research on mycomembrane composition, biosynthesis, and dynamics. Moreover, because TMM is exclusively metabolized by Corynebacterineae, the described probes may be valuable for the specific detection and cell-surface engineering of Mycobacterium tuberculosis and related pathogens. We also performed experiments to establish the dependence of probe incorporation on mycoloyltransferase activity, results from which suggested that cellular labeling is a function not only of metabolic incorporation (and likely removal) pathway(s), but also accessibility across the envelope. Thus, whole-cell labeling experiments with TMM reporters should be carefully designed and interpreted when envelope permeability may be compromised. On the other hand, this property of TMM reporters can potentially be exploited as a convenient way to probe changes in envelope integrity and permeability, facilitating drug development studies.     

An Extended Approach for the Development of Fluorogenic trans-Cyclooctene–Tetrazine Cycloadditions

Inverse-electron-demand Diels–Alder (iEDDA) cycloaddition between 1,2,4,5-tetrazines and strained dienophiles belongs among the most popular bioconjugation reactions. In addition to its fast kinetics, this cycloaddition can be tailored to produce fluorescent products from non-fluorescent starting materials. Here we show that even the reaction intermediates formed in iEDDA cycloaddition can lead to the formation of new types of fluorophores. The influence of various substituents on their photophysical properties and the generality of the approach with use of various trans-cyclooctene derivatives were studied. Model bioimaging experiments demonstrate the application potential of fluorogenic iEDDA cycloaddition.     

Accurate Determination of Human CPR Conformational Equilibrium by smFRET Using Dual Orthogonal Noncanonical Amino Acid Labeling

Conjugation of fluorescent dyes to proteins—a prerequisite for the study of conformational dynamics by single-molecule (sm) FRET—can lead to substantial changes in a dye's photophysical properties, ultimately biasing the determination of inter-dye distances. In particular, cyanine dyes and their derivatives, the most commonly used dyes in smFRET experiments, exhibit such behavior. To overcome this, we developed a general strategy to equip proteins site-specifically with FRET pairs through chemoselective reactions with two distinct noncanonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli. Application of this technique to human NADPH-cytochrome P450 reductase (CPR) demonstrated the importance of homogenously labeled samples for accurate determination of FRET efficiencies and unveiled the effect of NADP⁺ on the ionic-strength-dependent modulation of the conformational equilibrium of CPR. Thanks to its generality and accuracy, the presented methodology establishes a new benchmark for deciphering of complex molecular dynamics in single molecules.