Development of ketalized unsaturated saccharides as multifunctional cysteine-targeting covalent warheads

Dong, S., Huang, H., Li, J. et al.

Commun Chem 7, 201 (2024). 

https://doi.org/10.1038/s42004-024-01279-z

Multi-functional cysteine-targeting covalent warheads possess significant therapeutic potential in medicinal chemistry and chemical biology. Herein, we present novel unsaturated and asymmetric ketone (oxazolinosene) scaffolds that selectively conjugate cysteine residues of peptides and bovine serum albumin under normal physiological conditions. This unsaturated saccharide depletes GSH in NCI-H1299 cells, leading to anti-tumor effects in vitro. The acetyl group of the ketal moiety on the saccharide ring can be converted to other carboxylic acids in a one-pot synthesis. In this way, the loaded acid can be click-released during cysteine conjugation, making the oxazolinosene a potential multifunctional therapeutic agent. The reaction kinetic model for oxazolinosene conjugation to GSH is well established and was used to evaluate oxazolinosene reactivity. The aforementioned oxazolinosenes were stereoselectively synthesized via a one-step reaction of nitriles with saccharides and conveniently converted into a series of α, β-unsaturated ketone N-glycosides as prevalent synthetic building blocks. The reaction mechanisms of oxazolinosene synthesis were investigated through calculations and validated with control experiments. Overall, these oxazolinosenes can be easily synthesized and developed as cysteine-targeted covalent warheads carrying useful click-releasing groups.

From Mechanism-Based Retaining Glycosidase Inhibitors to Activity-Based Glycosidase Profiling

 Marta Artola, Johannes M. F. G. Aerts, Gijsbert A. van der Marel, Carme Rovira, Jeroen D. C. Codée, Gideon J. Davies, and Herman S. Overkleeft

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c08840

Activity-based protein profiling (ABPP) is an effective technology for the identification and functional annotation of enzymes in complex biological samples. ABP designs are normally directed to an enzyme active site nucleophile, and within the field of Carbohydrate-Active Enzymes (CAZymes), ABPP has been most successful for those enzymes that feature such a residue: retaining glycosidases (GHs). Several mechanism-based covalent and irreversible retaining GH inhibitors have emerged over the past sixty years. ABP designs based on these inhibitor chemistries appeared since the turn of the millennium, and we contributed to the field by designing a suite of retaining GH ABPs modeled on the structure and mode of action of the natural product, cyclophellitol. These ABPs enable the study of both exo- and endo-acting retaining GHs in human health and disease, for instance in genetic metabolic disorders in which retaining GHs are deficient. They are also finding increasing use in the study of GHs in gut microbiota and environmental microorganisms, both in the context of drug (de)toxification in the gut and that of biomass polysaccharide processing for future sustainable energy and chemistries. This account comprises the authors’ view on the history of mechanism-based retaining GH inhibitor design and discovery, on how these inhibitors served as blueprints for retaining GH ABP design, and on some current and future developments on how cyclophellitol-based ABPs may drive the discovery of retaining GHs and their inhibitors.

SuFEx Chemistry Enables Covalent Assembly of a 280-kDa 18-Subunit Pore-Forming Complex

Lee Schnaider, Sophia Tan, Pratik R. Singh, Floriana Capuano, Alistair J. Scott, Richard Hambley, Lei Lu, Hyunjun Yang, E. Jayne Wallace, Hyunil Jo, and William F. DeGrado

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c07920

Proximity-enhanced chemical cross-linking is an invaluable tool for probing protein–protein interactions and enhancing the potency of potential peptide and protein drugs. Here, we extend this approach to covalently stabilize large macromolecular assemblies. We used SuFEx chemistry to covalently stabilize an 18-subunit pore-forming complex, CsgG:CsgF, consisting of nine CsgG membrane protein subunits that noncovalently associate with nine CsgF peptides. Derivatives of the CsgG:CsgF pore have been used for DNA sequencing, which places high demands on the structural stability and homogeneity of the complex. To increase the robustness of the pore, we designed and synthesized derivatives of CsgF-bearing sulfonyl fluorides, which react with CsgG in very high yield to form a covalently stabilized CsgG:CsgF complex. The resulting pores formed highly homogeneous channels when added to artificial membranes. The high yield and rapid reaction rate of the SuFEx reaction prompted molecular dynamics simulations, which revealed that the SO2F groups in the initially formed complex are poised for nucleophilic reaction with a targeted Tyr. These results demonstrate the utility of SuFEx chemistry to structurally stabilize very large (here, 280 kDa) assemblies.