A new and versatile class of cyclic diazodicarboxamides that reacts efficiently and selectively with phenols and the phenolic side chain of tyrosine through an ene-like reaction is reported. This mild aqueous tyrosine ligation reaction works over a broad pH range and expands the repertoire of aqueous chemistries available for small molecule, peptide, and protein modification. The tyrosine ligation reactions are shown to be compatible with the labeling of native enzymes and antibodies in a buffered aqueous solution. This reaction provides a novel synthetic approach to bispecific antibodies. We believe this reaction will find broad utility in peptide and protein chemistry and in the chemistry of phenol-containing compounds.

The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivatives in peptide and protein chemistry, we synthesized PTAD derivatives possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivatives and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective tri-functionalization of proteins is readily achieved. In particular cases, we noted PTAD decomposition resulted in formation of a putative isocyanate by-product that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addition of a small amount of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene) glycol chains onto proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated products. Finally, we applied the tyrosine click reaction to create a novel antibody drug conjugate. For this purpose, we synthesized a PTAD derivative linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the potential of this reaction in creating protein conjugates with small molecules. The tyrosine click linkage demonstrated stability to extremes of pH, temperature and exposure to human blood plasma indicating that this linkage is significantly more robust than maleimide-type linkages that are commonly employed in bioconjugations. These studies support the broad utility of this reaction in the chemoselective modification of small molecules, peptides, and proteins under mild aqueous conditions over a broad pH range using a wide variety of biologically acceptable buffers such as phosphate buffered saline (PBS) and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) buffers as well as others and mixed buffered compositions.

4-Formylbenzene diazonium hexafluorophosphate (FBDP) is a novel bench-stable crystalline diazonium salt that reacts selectively with tyrosine to install a bioorthogonal aldehyde functionality. Model studies with N-acyl-tyrosine methylamide allowed us to identify conditions optimal for tyrosine ligation reactions with small peptides and proteins. FBDP-based conjugation was used for the facile introduction of small molecule tags, poly(ethylene) glycol chains (PEGylation), and functional small molecules onto model proteins and to label the surface of living cells.

The three major members of non-coding RNAs (ncRNAs), named microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), play an important role in hepatocellular carcinoma (HCC) development. Recently, the competing endogenous RNA (ceRNA) regulation model described lncRNA/circRNA as a sponge for miRNAs to indirectly regulate miRNA downstream target genes. Accumulating evidence has indicated that ceRNA regulatory networks are associated with biological processes in HCC, including cancer cell growth, epithelial to mesenchymal transition (EMT), metastasis, and chemoresistance. In this review, we summarize recent discoveries, which are specific ceRNA regulatory networks (lncRNA/circRNA-miRNA-mRNA) in HCC and discuss their clinical significance.

Epigenetic alterations, such as histone modification, DNA methylation, and miRNA-mediated processes, are critically associated with various mechanisms of proliferation and metastasis in several types of cancer. To overcome the side effects and limited effectiveness of drugs for cancer treatment, there is a continuous need for the identification of more effective drug targets and the execution of mechanism of action (MOA) studies. Recently, epigenetic modifiers have been recognized as important therapeutic targets for hepatocellular carcinoma (HCC) based on their reported abilities to suppress HCC metastasis and proliferation in both in vivo and in vitro studies. Therefore, here, we introduce epigenetic modifiers and alterations related to HCC metastasis and proliferation, and their molecular mechanisms in HCC metastasis. The existing data suggest that the study of epigenetic modifiers is important for the development of specific inhibitors and diagnostic targets for HCC treatment.