Genetic Encoding and Enzymatic Deprotection of a Latent Thiol Side Chain to Enable New Protein Bioconjugation Applications

Abstract

The thiol group of the cysteine side chain is arguably the most versatile chemical handle in proteins. To expand the scope of established and commercially available thiol bioconjugation reagents, we genetically encoded a second such functional moiety in form of a latent thiol group that can be unmasked under mild physiological conditions. Phenylacetamidomethyl (Phacm) protected homocysteine (HcP) was incorporated and its latent thiol group unmasked on purified proteins using penicillin G acylase (PGA). The enzymatic deprotection depends on steric accessibility, but can occur efficiently within minutes on exposed positions in flexible sequences. The freshly liberated thiol group does not require treatment with reducing agents. We demonstrate the potential of this approach for protein modification with conceptually new schemes for regioselective dual labeling, thiol bioconjugation in presence of a preserved disulfide bond and formation of a novel intramolecular thioether crosslink.

Air‐Stable Bis‐Cyclometallated Iridium Catalysts for Ortho‐Directed C(sp2)−H Borylation

“…Previously Smith had reported an example on CÀ H borylation of enamines, [8d] but to the best of our knowledge, products 13 a, 13 b represent the first example of CÀ H borylation of acrylamides. Finally, ImIr-4 also promotes C(sp 3 )À H borylation, as exemplified on the substrate 13; [29] after oxidation of the crude mixture, product 14 was isolated.…”

Enzymatically Triggered Peptide–Lipid Conjugation of Designed Membrane Active Peptides for Controlled Liposomal Release

“…The Phacm-protection group is compatible with both Fmoc and Boc solid-phase peptide synthesis and can be selectively deprotected using the enzyme penicillin G acylase (PGA) under physiological conditions. 23 The deprotection process is cytocompatible 24 and rapid, 25 leaving free thiols 23 accessible for conjugation to thiol-reactive lipids. The release of carboxyfluorescein (CF) from the vesicles was used as an indicator of successful MAP conjugation.…”

Self-Sulfhydrated, Nitro-Fixed Albumin Nanoparticles as a Potent Therapeutic Agent for the Treatment of Acute Liver Injury

“…This process enables thiol-containing proteins, antioxidant enzymes, and cellular low-molecular-weight thiol (glutathione) to function as essential antioxidant buffering pool components in vivo and maintain redox homeostasis, protecting organisms from oxidative and xenobiotic stress. − Exogenous replenishment of thiol-containing agents, such as small molecular drugs (like N -acetyl- l -cysteine), sulfhydrated polymers, or bioconjugates, can restore the redox state of pathological microenvironments in diseases associated with oxidative stress, enabling rapid tissue damage reversal and organ function restoration. However, the low oxidative efficiency, sluggish ROS reaction rate, poor pharmacokinetics, and off-target effects of monothiol compounds result in low bioavailability, a narrow therapeutic window, a variety of allergic reactions, and so forth. − Besides, the chemical modification of polythiol groups into polymers is typically hampered by low thiol modification efficiency, susceptibility to oxidative deactivation and self-cross-linking of thiols, cumbersome and time-consuming reaction steps, the use of large amounts of toxic organic solvents, and so forth. − In this instance, it is conceivable that these factors hinder the engineering transformation and clinical application of current exogenous sulfhydryl supplementation strategies, and there is an urgent need for the development of formulations that have wide accessibility to raw material sources, high thiol modification efficiency, a simple and green preparation process, long-term storage stability, and good pharmacokinetic and pharmacodynamic efficiency in vivo to broaden their widespread cilincal uses.…”