Fluorine-thiol displacement probes for acetaminophen's hepatotoxicity.

Chemicals possessing reactive electrophiles can denature innate proteins leading to undesired toxicity, and the overdose-induced liver injury by drugs containing electrophiles has been one of the major causes of non-approval and withdraw by the US Food and Drug Administration (FDA). Elucidating the associated proteins could guide the future development of therapeutics to circumvent these drugs' toxicities, but was largely limited by the current probing tools due to the steric hindrance of chemical tags including the common "click chemistry" labels. Taking the widely used non-steroidal anti-inflammatory drug acetaminophen (APAP) as an example, we hereby designed and synthesized an APAP analogue using fluorine as a steric-free label.

Unprotected peptide macrocyclization and stapling via a fluorine-thiol displacement reaction.

We report the discovery of a facile peptide macrocyclization and stapling strategy based on a fluorine thiol displacement reaction (FTDR), which renders a class of peptide analogues with enhanced stability, affinity, cellular uptake, and inhibition of cancer cells. This approach enabled selective modification of the orthogonal fluoroacetamide side chains in unprotected peptides in the presence of intrinsic cysteines. The identified benzenedimethanethiol linker greatly promoted the alpha helicity of a variety of peptide substrates, as corroborated by molecular dynamics simulations.

Steric-Free Bioorthogonal Labeling of Acetylation Substrates Based on a Fluorine-Thiol Displacement Reaction.

We have developed a novel bioorthogonal reaction that can selectively displace fluorine substitutions alpha to amide bonds. This fluorine-thiol displacement reaction (FTDR) allows for fluorinated cofactors or precursors to be utilized as chemical reporters, hijacking acetyltransferase-mediated acetylation both in vitro and in live cells, which cannot be achieved with azide- or alkyne-based chemical reporters. The fluoroacetamide labels can be further converted to biotin or fluorophore tags using FTDR, enabling the general detection and imaging of acetyl substrates.

Site-specific antibody fragment conjugates for targeted imaging.

This work details the use of amber suppression-mediated genetic incorporation of unnatural amino acids (UAAs), specifically p-azido-l-phenylalanine (pAzF) and p-acetyl-l-phenylalanine (pAcF), to develop site-specifically labeled antibody Fab fragments. These antibody fragment conjugates represent a novel class of imaging agents with optimal stability, efficacy, and pharmacological properties, which have demonstrated promising potential for probing and understanding the in vivo bio-distributions of protein targets of interest. This chapter provides general guidelines for preparing these Fab conjugates, and details of follow-up bioassays such as single-agent based positron emission tomography (PET) imaging of immune-checkpoint protein PD-L1, and the use of GCN4-mediated switchable antibody conjugates for near-infrared fluorescent imaging of cancer-related biomarkers.

Site-Specific Immuno-PET Tracer to Image PD-L1.

The rapid ascension of immune checkpoint blockade treatments has placed an emphasis on the need for viable, robust, and noninvasive imaging methods for immune checkpoint proteins, which could be of diagnostic value. Immunoconjugate-based positron emission tomography (immuno-PET) allows for sensitive and quantitative imaging of target levels and has promising potential for the noninvasive evaluation of immune checkpoint proteins. However, the advancement of immuno-PET is currently limited by available imaging tools, which heavily rely on full-length IgGs with Fc-mediated effects and are heterogeneous mixtures upon random conjugation with chelators for imaging.

A Switchable Site-Specific Antibody Conjugate.

Genetic incorporation of unnatural amino acids (UAAs) provides a unique approach to the synthesis of site-specific antibody conjugates that are homogeneous and better defined constructs than random conjugates. Yet, the yield varies for every antibody, and the process is costly and time-consuming. We have developed a switchable αGCN4-Fab conjugate that incorporates UAA p-acetylphenylalanine.

Interrogating the Roles of Post-Translational Modifications of Non-Histone Proteins.

Post-translational modifications (PTMs) allot versatility to the biological functions of highly conserved proteins. Recently, modifications to non-histone proteins such as methylation, acetylation, phosphorylation, glycosylation, ubiquitination, and many more have been linked to the regulation of pivotal pathways related to cellular response and stability. Due to the roles these dynamic modifications assume, their dysregulation has been associated with cancer and many other important diseases such as inflammatory disorders and neurodegenerative diseases.