Incorporation of 2,3‐Diaminopropionic Acid into Linear Cationic Amphipathic Peptides Produces pH‐Sensitive Vectors

Abstract

Non-viral vectors that harness the change in pH in endosomes are increasingly being used to deliver cargoes, including nucleic acids, to mammalian cells. Here we present evidence that the pKa of the β-NH 2 in 2,3-diaminopropionic acid (Dap) is sufficiently lowered, when incorporated in peptides, that its protonation state is sensitive to the pH changes that occur during endosomal acidification. The lowered pKa around 6.3 is stabilised by the increased electron withdrawing effect of the peptide bonds, by inter-molecular hydrogen bonding and from contributions arising from the peptide conformation, including mixed polar/apolar environments, Coulombic interactions and inter-molecular hydrogen bonding. Changes of the charged state are therefore expected between pH 5 and 7 and large scale conformational changes are observed in Dap rich peptides, in contrast with analogues containing lysine or ornithine, when the pH is altered through this range. These physical properties confer a robust gene delivery capability on designed cationic amphipathic peptides that incorporate Dap.

Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs

“…Because the p K a of the sidechain of peptide-incorporated amino-Ala (RNH 3 + to RNH 2 pKa is ~8 [36]) is dramatically different from that of the sidechain of Ser (ROH to RO- is ~13 [37]), we hypothesized that the GOAT-catalyzed acyl transfer reactions with the two substrates might show sharply different pH sensitivities if proton transfer of the nucleophile were rate-determining. We measured the rate of ghrelin octanoylation for Ser3-containing Ghrelin10 and Dap3-Ghrelin10 at 7.5 μM (sub-saturating), and 100 μM (saturating) as shown in Figure 6A–C.…”

“…The serine hydroxyl has a pKa of approximately 13 and will not change protonation over this range. In contrast, the p K a of the β-NH 2 group in Dap is approximately 8 [36, 37]. We note that sidechain pKa may be shifted by a number of pH units in the microenvironment of an enzyme active site [38] and may also be reduced in the hydrophobic context of microsomes.…”

Histidine‐rich designer peptides of the LAH4 family promote cell delivery of a multitude of cargo

“…Therefore, in some environments, this bulky residue may shift the conformational equilibria in a subtle manner, thereby also introducing changes in charge distribution and hydrophobic moment, at least during one of the multiple key steps during the transfection process. Whereas an amphipathic α-helical conformation may still be obtained in a membrane environment [53], other structures concomitant with changes in peptide self-association may be adopted in aqueous solution [14,32], a notion supported when the CD spectra of LAH4 and LAH4X2 are compared with each other (not shown), or when interacting with the cargo [23]. In this context, it is noteworthy that the transfection efficiency of LAH4 and LAH6 variants has been shown to be affected by their propensity to self-associate in solution [14].…”

Endowment of pH Responsivity to Anticancer Peptides by Introducing 2,3‐Diaminopropionic Acid Residues

“…Dap is a non‐proteinogenic amino acid found in several species of plants, in the biosynthesis of antibiotics, and in meteorites . The p K a value of the Dap side chain in a peptide is about 6.3, so it is more sensitive to acidification in cancer tissues than that of His. The template peptide was designed by introducing lysine residues into MG, and a series of peptides were generated by replacing several lysine residues with Dap.…”