Bioorthogonal Removal of 3‑Isocyanopropyl Groups Enables the Controlled Release of Fluorophores and Drugs in Vivo

Dissociative bioorthogonal reactions allow for chemically controlling the release of bioactive agents and reporter probes. Here we describe 3-isocyanopropyl substituents as masking groups that can be effectively removed in biological systems. 3-Isocyanopropyl derivatives react with tetrazines to afford 3-oxopropyl groups that eliminate diverse functionalities. The study shows that the reaction is rapid and can liberate phenols and amines near-quantitatively under physiological conditions. The reaction is compatible with living organisms as demonstrated by the release of a resorufin fluorophore and a mexiletine drug in zebrafish embryos implanted with tetrazine-modified beads. The combined benefits of synthetic ease, rapid kinetics, diversity of leaving groups, high release yields, and structural compactness, make 3-isocyanopropyl derivatives attractive chemical caging moieties for uses in chemical biology and drug delivery

Temporally Controlled Targeting of 4‑Hydroxynonenal to Specific Proteins in Living Cells

In-depth chemical understanding of complex biological processes hinges upon the ability to systematically perturb individual systems. However, current approaches to study impacts of biologically relevant reactive small molecules involve bathing of the entire cell or isolated organelle with excess amounts, leading to off-target effects. The resultant lack of biochemical specificity has plagued our understanding of how biological electrophiles mediate signal transduction or regulate responses that confer defense mechanisms to cellular electrophilic stress. Here we introduce a target-specific electrophile delivery platform that will ultimately pave the way to interrogate effects of reactive electrophiles on specific target proteins in cells. The new methodology is demonstrated by photoinducible targeted delivery of 4-hydroxynonenal (HNE) to the proteins Keap1 and PTEN. Covalent conjugation of the HNE-precursor to HaloTag fused to the target proteins enables directed HNE delivery upon photoactivation. The strategy provides proof of concept of selective delivery of reactive electrophiles to individual electrophile-responsive proteins in mammalian cells. It opens a new avenue enabling more precise determination of the pathophysiological consequences of HNE-induced chemical modifications on specific target proteins in cells

Enhancing Multistep DNA Processing by Solid-Phase Enzyme Catalysis on Polyethylene Glycol Coated Beads

Covalent immobilization of enzymes on solid supports provides an alternative approach to homogeneous biocatalysis by adding the benefits of simple enzyme removal, improved stability, and adaptability to automation and high-throughput applications. Nevertheless, immobilized (IM) enzymes generally suffer from reduced activity compared to their soluble counterparts. The nature and hydrophobicity of the supporting material surface can introduce enzyme conformational change, spatial confinement, and limited substrate accessibility, all of which will result in loss of the immobilized enzyme activity. In this work, we demonstrate through kinetic studies that flexible polyethylene glycol (PEG) moieties modifying the surface of magnetic beads improve the activity of covalently immobilized DNA replication enzymes. PEG-modified immobilized enzymes were utilized in library construction for Illumina next-generation sequencing (NGS) increasing the read coverage across AT-rich regions

Substoichiometric Hydroxynonenylation of a Single Protein Recapitulates Whole-Cell-Stimulated Antioxidant Response

Lipid-derived electrophiles (LDEs) that can directly modify proteins have emerged as important small-molecule cues in cellular decision-making. However, because these diffusible LDEs can modify many targets [e.g., >700 cysteines are modified by the well-known LDE 4-hydroxynonenal (HNE)], establishing the functional consequences of LDE modification on individual targets remains devilishly difficult. Whether LDE modifications on a single protein are biologically sufficient to activate discrete redox signaling response downstream also remains untested. Herein, using T-REX (targetable reactive electrophiles and oxidants), an approach aimed at selectively flipping a single redox switch in cells at a precise time, we show that a modest level (∼34%) of HNEylation on a single target is sufficient to elicit the pharmaceutically important antioxidant response element (ARE) activation, and the resultant strength of ARE induction recapitulates that observed from whole-cell electrophilic perturbation. These data provide the first evidence that single-target LDE modifications are important individual events in mammalian physiology