Electrostatic control of peptide side-chain reactivity using amphiphilic homopolymer-based supramolecular assemblies

Abstract

Supramolecular assemblies formed by amphiphilic homopolymers with negatively charged groups in the hydrophilic segment have been designed to enable high labeling selectivity toward reactive side chain functional groups in peptides. The negatively charged interiors of the supramolecular assemblies are found to block the reactivity of protonated amines that would otherwise be reactive in aqueous solution, while maintaining the reactivity of nonprotonated amines. Simple changes to the pH of the assemblies' interiors allow control over the reactivity of different functional groups in a manner that is dependent on the pKa of a given peptide functional group. The labeling studies carried out in positively charged supramolecular assemblies and free buffer solution show that, even when the amine is protonated, labeling selectivity exists only when complementary electrostatic interactions are present, thereby demonstrating the electrostatically controlled nature of these reactions.

Figure 1

Mass spectra of malantide reacted with sulfo-NHSA (a) inside the negatively charged polymer reverse micelle at pH 7.0 and (b) in free aqueous solution at pH 7.0.

Figure 2

Mass spectrum of PSTAIR reacted with sulfo-NHSA (a) inside the positively charged polymer reverse micelle at pH 7.0 and (b) in free aqueous solution at pH 7.0.

Figure 3

Mass spectra of β-Amyloid 10–20 reacted with sulfo-NHSA (a) inside the negatively charged polymer reverse micelle at pH 7.0 and (b) inside the positively charged polymer reverse micelle at pH 7.0.

Figure 4

Mass spectra of malantide reacted with DEPC (a) in free aqueous solution at pH 7.0, (b) inside the negatively charged reverse micelles at pH 7.0, (c) in free solution at pH 11.2, and (d) inside the negatively charged reverse micelles at pH 11.2.

Figure 5

Mass spectra of kinetensin reacted with DEPC (a) in free aqueous solution at pH 5.5, (b) inside the negatively charged reverse micelles at pH 5.5, (c) in free solution at pH 7.0, and (d) inside the negatively charged reverse micelles at pH 7.0.

Figure 6

Mass spectra of Apelin 13 reacted with DEPC (a) in free aqueous solution at pH 5.5, inside negatively charged reverse micelles at b) pH 5.5, (c) pH 7.0, and (d) pH 10.0. Note: The unlabeled peaks in each spectrum correspond to oxidative modifications to the side chain of methionine. These oxidative modifications are also observed in control experiments, indicating that the peptide is oxidized in the pure sample.

Figure 7

Mass spectra of malantide and PSTAIR reacted with sulfo-NHSA in negatively charged assemblies at pH 7.0 (a) malantide and (b) PSTAIR.

Scheme 1

Schematic illustrating the labeling of extracted peptides inside negatively charged reverse micelles and their analysis by MALDI-MS.

Scheme 2

Schematic illustrating the electrostatic interaction driven selectivity and the detection event

Scheme 3

Reaction of sulfo-NHSA with lysine residue.

Scheme 4

DEPC labeling of histidine residue increases m/z by 72 units.

Chart 1

Structure of the amphiphilic homopolymer based on carboxylic acid moiety (negatively charged, 1) and quaternary ammonium moiety (positively charge, 2)