Covalent Chemical Tools for Profiling Post-Translational Modifications.

Nature increases the functional diversity of the proteome through posttranslational modifications (PTMs); a process that involves the proteolytic processing or catalytic attachment of diverse functional groups onto proteins. These modifications modulate a host of biological activities and responses. Consequently, anomalous PTMs often correlate to a host of diseases, hence there is a need to detect these transformations, both qualitatively and quantitatively. One technique that has gained traction is the use of robust chemical strategies to label different PTMs. By utilizing the intrinsic chemical reactivity of the different chemical groups on the target amino acid residues, this strategy can facilitate the delineation of the overarching and inclusionary roles of these different modifications. Herein, we will discuss the current state of the art in post-translational modification analysis, with a direct focus on covalent chemical methods used for detecting them.

Rapid Arene Triazene Chemistry for Macrocyclization.

Here, we report a novel rapid arene triazene strategy for the macrocyclization of peptides that generates an inbuilt chromophoric triazene moiety at the site of cyclization within a minute. The rapid arene triazene chemistry is chemoselective for secondary amines and p-amino phenylalanine. Importantly, the resulting triazene cyclic peptide is highly stable at neutral pH and under harsh conditions but rapidly responds to various external stimuli such as UV radiations and acidic conditions, resulting in the ring opening to generate the linear peptides in an unchanged form, which further cyclizes under neutral pH conditions. This method works with completely unprotected peptides and has been applied for the synthesis of 18- to 66-membered monocycles and bicycles with various amino acid compositions in one pot under neutral pH conditions. Due to the high stability of triazene cyclic peptides, the postcyclization modification was carried out with various functional groups. This rapid, macrocyclization strategy featuring a triazene scaffold, amenable to late-stage diversification and responsive to external stimuli, should find application in various fields of chemical biology, selective drug delivery, and identification of cyclic peptide hits after library screening.

Selective Triazenation Reaction (STaR) of Secondary Amines for Tagging Monomethyl Lysine Post-Translational Modifications.

Lysine monomethylation (Kme) is an impactful post-translational modification (PTM) responsible for regulating biological processes and implicated in diseases, thus there is great interest in identifying these methylation marks globally. However, the progress in this area has been challenging because the addition of a small methyl group on lysine leads to negligible change in the bulk, charge, and hydrophobicity. Herein, we report an empowering chemical technology selective triazenation reaction, which we term "STaR", of secondary amines using arene diazonium salts to achieve highly selective, rapid, and robust tagging of Kme peptides from a complex mixture under biocompatible conditions. Although the resulting triazene-linkage with Kme is stable, we highlight the efficient decoupling of the triazene-conjugate to afford unmodified starting components under mild conditions when desired. Our work establishes a unique chemoselective, traceless bioconjugation strategy for the selective enrichment of Kme PTMs.

Secondary amine selective Petasis (SASP) bioconjugation.

Selective modification of proteins enables synthesis of antibody-drug conjugates, cellular drug delivery and construction of new materials. Many groups have developed methods for selective N-terminal modification without affecting the side chain of lysine by judicious pH control. This is due to lower basicity of the N-terminus relative to lysine side chains. But none of the methods are capable of selective modification of secondary amines or N-terminal proline, which has similar basicity as lysine. Here, we report a secondary amine selective Petasis (SASP) reaction for selective bioconjugation at N-terminal proline. We exploited the ability of secondary amines to form highly electrophilic iminium ions with aldehydes, which rapidly reacted with nucleophilic organoboronates, resulting in robust labeling of N-terminal proline under biocompatible conditions. This is the first time the Petasis reaction has been utilized for selective modification of secondary amines on completely unprotected peptides and proteins under physiological conditions. Peptide screening results showed that the reaction is highly selective for N-terminal proline. There are no other chemical methods reported in literature that are selective for N-terminal proline in both peptides and proteins. This is a multicomponent reaction leading to the synthesis of doubly functionalized bioconjugates in one step that can be difficult to achieve using other methods. The key advantage of the SASP reaction includes its high chemoselective and stereoselective (>99% de) nature, and it affords dual labeled proteins in one pot. The broad utility of this bioconjugation is highlighted for a variety of peptides and proteins, including aldolase and creatine kinase.

Amino-Acid-Catalyzed Direct Aldol Bioconjugation.

A site-specific bioconjugation was developed based on direct aldol coupling using amino-acid-derived organocatalysts. The functionalization exhibits fast kinetics and occurs under mild, biocompatible conditions (viz., aqueous media, moderate temperature, and neutral pH). The resulting bioconjugates were found to be stable toward abundant aldolase enzymes, as well as acidic and basic pH. The methodology was demonstrated through conjugation of a variety of small molecules, dyes, and peptides to proteins, including a single-domain antibody, which was then used for cellular imaging.