Click chemistry has been proven to be very useful in drug delivery. Due to the availability of a large number of click reactions with a various characteristics, selection of appropriate chemistry for a given application is often not a trivial task. This review is written for pharmaceutical researchers who are interested in click chemistry applications and yet may not be click chemistry experts. For this, the review gives an overview of available click reactions organized by application types. Further, the general understanding of click reactions being fast and high yielding sometimes overshadows the need to analyze reaction kinetics in assessing suitability of a given reaction for certain applications. For this, we highlight the need to analyze the relationship among reaction kinetics, concentration effects, and reaction time scales, knowing that lack of such analysis could easily lead to failures. Further, possible issues such as chemical stability with various click reagents are also discussed to aid experimental designs. Recent examples and extensive references are also provided to aid in-depth understanding of technical details. We hope this review will help those interested in using click chemistry in drug delivery to select the appropriate reactions/reagents and minimize the number of pitfalls.

Carbon monoxide (CO) is endogenously produced in mammals, with blood concentrations in the high micromolar range in the hemoglobin-bound form. Further, CO has shown therapeutic effects in various animal models. Despite its reputation as a poisonous gas at high concentrations, we show that CO should have a wide enough safety margin for therapeutic applications. The analysis considers a large number of factors including levels of endogenous CO, its safety margin in comparison to commonly encountered biomolecules or drugs, anticipated enhanced safety profiles when delivered via a noninhalation mode, and the large amount of safety data from human clinical trials. It should be emphasized that having a wide enough safety margin for therapeutic use does not mean that it is benign or safe to the general public, even at low doses. We defer the latter to public health experts. Importantly, this Perspective is written for drug discovery professionals and not the general public.

Thioether oxidation to sulfoxide by H₂O₂ has been widely reported as an ROS-sensitive trigger in drug delivery applications. Through a number of straightforward kinetic experiments with a series of aryl thioethers, we show that H₂O₂ oxidation under near-physiological conditions is expected to have half-lives on the scale of hundreds of hours at pathophysiologically relevant H₂O₂ concentrations. On the other hand, hypochlorite can oxidize thioethers at much faster rates with half-lives in the range of seconds to sulfoxide and minutes to sulfone under similar conditions. Such information means that hypochlorite likely plays a much more important role than H₂O₂ in activating thioether-based drug delivery systems.

Reactive oxygen species (ROS) play critical roles in normal physiological processes including cellular signaling and immune responses. Various pathological conditions including infections of various types, inflammation, cancer, and respiratory conditions are associated with elevated levels of ROS. Therefore, there is widespread interest in understanding ROS concentrations under various pathophysiological conditions for diagnostic and therapeutic applications including ROS-triggered drug delivery. However, in determining ROS concentration, there are major concerns of inappropriate use of various methods that lead to erroneous results; this has prompted the publication of a consensus paper in Nature Metabolism by a group of ROS experts stating “Unfortunately, the application and interpretation of these measurements are fraught with challenges and limitations. This can lead to misleading claims.” Along this line, we have identified an overlooked factor, which can significantly skew the results and results interpretation: the organic co-solvent. DMSO is one of the most widely used organic co-solvents to dissolve a reagent for bioassays. Herein, we describe the rapid oxidation of DMSO by hypochlorite and how this oxidation impacts results of ROS determination in buffer, cell culture media, cell culture, and cell lysates. We hope to use this one example to draw attention to the convoluted roles that DMSO and possibly other organic co-solvents can play and skew experimental results. We also hope to stimulate additional studies to bring more rigor to studying ROS concentration and biology.

Reactive oxygen species (ROS) play critical roles in pathophysiological processes. Therefore, there is widespread interest in learning ROS concentrations under various conditions. However, literature numbers in ROS concentration vary significantly, and most cannot be readily compared against each other, largely because of the lack of understanding of the effects of various factors that significantly impact the experimental outcome. In this study, we examine an overlooked factor: the chemical reactivity of commonly used organic buffer molecules toward ROS and how such reactivity affects the results interpretation. Specifically, we examined HEPES, Tris, MES, citrate, ammonium acetate, and phosphate-buffered saline (PBS) and found that most organic buffer components can rapidly consume NaOCl (the second most abundant ROS) and/or directly interact with certain ROS probes such as a boronate for H₂O₂ determination, leading to significant errors in experimental findings and interpretations of results. For example, 20 mM HEPES, MES, ammonium acetate, and Tris are found to consume 1 mM hypochlorite within 1 s, leading to false negative results. Additionally, these organic buffer components have been found to cause false negative results in the detection of ONOO^- when using a boronate-based probe. As such, these organic buffers should be avoided in the determination of ROS concentrations. We use these examples to draw attention to the profound effects of buffer components on ROS detection and examine chemistry issues in detail. We hope the findings described will lead to improved rigor in designing ROS experiments by considering factors that were previously considered as nothing but bystanders or benign.

Despite current vaccines and therapeutics targeting SARS-CoV-2, the causative agent of the COVID-19 pandemic, cases remain high causing a burden on health care systems. Spike-protein mediated membrane fusion of SARS-CoV-2 is a critical step in viral entry. Herein, we describe entry inhibitors identified by first screening a library of about 160 compounds and then analogue synthesis. Specifically, compound 261 was found to inhibit SARS-CoV-2 infection in a tissue model with IC₅₀ of 0.3 µM. Using NMR, we found that 261 interacts with key residues in the aromatic-rich region of the spike protein directly next to the transmembrane domain. Molecular dynamic simulations of the 261 binding pocket in the spike protein was also mapped to the transmembrane domain, consistent with NMR findings. The amino acids in the binding site are conserved among different coronaviruses known to infect humans; therefore, inhibitors targeting this conserved binding site could be a useful addition to current therapeutics and may have pan-coronavirus antiviral activities.