Pyrocinchonimides Conjugate to Amine Groups on Proteins via Imide Transfer.

Advances in bioconjugation, the ability to link biomolecules to each other, small molecules, surfaces, and more, can spur the development of advanced materials and therapeutics. We have discovered that pyrocinchonimide, the dimethylated analogue of maleimide, undergoes a surprising transformation with biomolecules. The reaction targets amines and involves an imide transfer, which has not been previously reported for bioconjugation purposes. Despite their similarity to maleimides, pyrocinchonimides do not react with free thiols. Though both lysine residues and the N-termini of proteins can receive the transferred imide, the reaction also exhibits a marked preference for certain amines that cannot solely be ascribed to solvent accessibility. This property is peculiar among amine-targeting reactions and can reduce combinatorial diversity when many available reactive amines are available, such as in the formation of antibody-drug conjugates. Unlike amides, the modification undergoes very slow reversion under high pH conditions. The reaction offers a thermodynamically controlled route to single or multiple modifications of proteins for a wide range of applications.

Macrocyclic Peptides Derived from Familial Alzheimer's Disease Mutants Show Charge-Dependent Oligomeric Assembly and Toxicity.

This work probes the role of charge in the oligomeric assembly, toxicity, and membrane destabilization of a series of peptides derived from Aß and the E22Q and E22K familial mutants. In the mutant Aß peptides, an acidic residue (E) is replaced with either a neutral or basic residue (Q or K), thus altering the net charge of the peptide. Acetylation at peripheral positions permits modulation of charge of the peptides and allows investigation of the role of charge in their oligomeric assembly, cytotoxicity, and membrane disruption. Peptides with the same net charge generally behave similarly even if the amino acid residue at position 22 differs. As the net charge of the peptide decreases, so does the extent of assembly, cytotoxicity, and membrane destabilization, which were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lactate dehydrogenase (LDH)-release assays with SH-SY5Y cells, and dye leakage assays using liposomes. These findings suggest that the charge of the amino acid side chain, rather than its size or hydrophobicity, accounts for the differences in the oligomeric assembly and toxicity of the E22 familial mutants of Aß.