The spontaneous esterification of boronic acids with polyols provides a promising opportunity to generate self-assembled bisubstrate-like inhibitors within the binding pocket of cAMP-dependent protein kinase (PKA). As a first substrate component, we designed amino acids, which have either a boronic acid or ribopyranose side chain and introduced them to the substrate-like peptide protein kinase inhibitor (PKI). The second component was derived from the active-site inhibitor Fasudil, which was functionalized with phenylboronic acid. NMR spectroscopy in dimethylsulfoxide proved spontaneous reversible condensation of both components. Reinforced by the protein environment, both separately bound substrates were expected to react via boronic-ester formation bridging the two binding sites of PKA. Multiple crystal structures of PKA with bound PKIs, positionally modified with residues such as a ribopyranosylated serine and threonine or a phenylboronic acid attached to lysine via amide bonds, were determined with the phenylboronic acid-linked Fasudil. Although PKA accepts both inhibitors simultaneously, the expected covalent attachment between both components was not observed. Instead, spontaneous reaction of the terminal boronic acid group of the modified Fasudil with the carboxylate of Glu127 was detected once the latter residue is set free from a strong salt bridge formed with arginine by the original peptide inhibitor PKI. Thus, the desired self-assembly reaction occurs spontaneously in the protein environment by an unexpected carboxylic acid boronate complex. To succeed with our planned self-assembly reaction between both substrate components, we have to redesign the required reaction partners more carefully to finally yield the desired bisubstrate-like inhibitors in the protein environment.

The incorporation of diamondoid amino acids (DAAs) into peptide‐like drugs is a general strategy to improve lipophilicity, membrane permeability, and metabolic stability of peptidomimetic pharmaceuticals. We designed and synthesized five novel peptidic DAA‐containing kinase inhibitors of protein kinase A using a sophisticated molecular dynamics protocol and solid‐phase peptide synthesis. By means of a thermophoresis binding assay, NMR, and crystal structure analysis, we determined the influence of the DAAs on the secondary structure and binding affinity in comparison to the native protein kinase inhibitor, which is purely composed of proteinogenic amino acids. Affinity and binding pose are largely conserved. One variant showed 6.5‐fold potency improvement, most likely related to its increased side chain lipophilicity. A second variant exhibited slightly decreased affinity presumably due to loss of hydrogen‐bond contacts to surrounding water molecules of the first solvation shell.

Six natural amino acids bearing hydroxy or thiol groups are transformed into N‐Fmoc and O‐acetyl protected ribopyranosylated amino acids (Raa) for further use in solid‐phase peptide synthesis (SPPS). Although the different O‐ and S‐glycosidic bonds influence the 1C4/4C1 equilibrium of the ribopyranosyl side chain significantly, the oligopeptides containing Raa are capable of spontaneous boronic ester formation. Ligation of model peptides with pyrene boronic acid and competition experiments between different peptides are monitored by NMR spectroscopy. In addition to boronic ester formation, we further propose ribopyranosylated peptides with adjustable orientation of the trihydroxy ax‐eq‐ax disposition for other applications.

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