Selective N-Terminal Acylation of Peptides and Proteins with Tunable Phenol Esters.

Selective modification of peptides and proteins is of foremost importance for the development of biopharmaceuticals and exploring biochemical pathways, as well as other applications. Here, we present a study on the development of a general and easily applicable selective method for N-terminal acylation of biomolecules, applying a new type of phenol esters. Key to the success was the development of highly tunable phenol activators bearing in the ortho-position, sulfonic acid or sulfonamide, acting as a steric shield for hydrolysis, and electron-withdrawing groups in the other ortho- and para-position for controlling the reactivity of the activated phenol esters. A library of heptapeptides, testing all 20 natural amino acids positioned at the N-terminal, were acylated in a selective manner at the N-terminus. The majority showed high conversion and excellent Nα-selectivity. Several biologically relevant biomolecules, including DesB30 insulin and human growth hormone, could also be modified at the N-terminal in a highly selective way, exemplified by either a fluorophore or a fatty acid sidechain. Finally, taking advantage of the possibility to accurately adjust the reactivity of the phenol esters, we present a potential strategy for the construction of dual active biopharmaceuticals through the employment of a bifunctional acylation linker and demonstrate its use in the creation of a GLP-1 insulin analogue, coupled through the lysine residue of GLP-1 and the N-terminal PheB1 amine of DesB30 insulin.

New Phenol Esters for Efficient pH-Controlled Amine Acylation of Peptides, Proteins, and Sepharose Beads in Aqueous Media.

This paper describes the discovery, synthesis, and use of novel water-soluble acylation reagents for efficient and selective modification, cross-linking, and labeling of proteins and peptides, as well as for their use in the effective modification of sepharose beads under pH control in aqueous media. The reagents are based on a 2,4-dichloro-6-sulfonic acid phenol ester core combined with a variety of linker structures. The combination of these motifs leads to an ideal balance between hydrolytic stability and reactivity. At high pH, good to excellent conversions (up to 95%) and regioselectivity (up to 99:1 Nε/Nα amine ratio) in the acylation were realized, exemplified by the chemical modification of incretin peptides and insulin. At neutral pH, an unusually high preference toward the N-terminal phenylalanine in an insulin derivative was observed (>99:1 Nα/Nε), which is up until now unprecedented in the literature for more elaborate reagents. In addition, the unusually high hydrolytic stability of these reagents and their ability to efficiently react at low concentrations (28 µM or 0.1 mg/mL) are exemplified with a hydroxy linker-based reagent and are a unique feature of this work.

A Nickel(II)-Mediated Thiocarbonylation Strategy for Carbon Isotope Labeling of Aliphatic Carboxamides.

A series of pharmaceutically relevant small molecules and biopharmaceuticals bearing aliphatic carboxamides have been successfully labeled with carbon-13. Key to the success of this novel carbon isotope labeling technique is the observation that 13 C-labeled NiII -acyl complexes, formed from a 13 CO insertion step with NiII -alkyl intermediates, rapidly react in less than one minute with 2,2'-dipyridyl disulfide to quantitatively form the corresponding 2-pyridyl thioesters. Either the use of 13 C-SilaCOgen or 13 C-COgen allows for the stoichiometric addition of isotopically labeled carbon monoxide. Subsequent one-pot acylation of a series of structurally diverse amines provides the desired 13 C-labeled carboxamides in good yields. A single electron transfer pathway is proposed between the NiII -acyl complexes and the disulfide providing a reactive NiIII -acyl sulfide intermediate, which rapidly undergoes reductive elimination to the desired thioester. By further optimization of the reaction parameters, reaction times down to only 11 min were identified, opening up the possibility of exploring this chemistry for carbon-11 isotope labeling. Finally, this isotope labeling strategy could be adapted to the synthesis of 13 C-labeled liraglutide and insulin degludec, representing two antidiabetic drugs.