Reductive amination of ω-conotoxin MVIIA: synthesis, determination of modification sites, and self-assembly

Abstract

Peptide drugs have disadvantages such as low stability, short half-life and side effects, which limit their widespread use in clinical practice. Therefore, peptide drugs can be modified to improve these disadvantages. Numerous studies have shown that alkyl-modified peptide drugs can self-assemble to prolong the duration of efficacy and/or reduce side effects. However, the commonly used solid-phase synthesis method for alkyl-modified peptides is time-consuming. To overcome this, a simple reductive amination reaction was employed, which can directly graft the alkyl chain to the peptide sequence and effectively avoid stepwise synthesis from C- to N-terminal with amino acids. In this study, ω-conotoxin MVIIA was used as the peptide drug, while myristic aldehyde was used as the alkylating agent. To obtain the maximum productivity of modified peptides, the molar ratio of peptide MVIIA to myristic aldehyde in the reductive amination reaction was optimized. Furthermore, the peptide modification sites in this reaction were confirmed by secondary mass spectrometry analysis. Besides, alkyl-modified peptide MVIIA was able to form micelles by self-assembly and improved stability in serum, which was related to our previous work where myristoylated peptide MVIIA micelles can improve the drug stability. Finally, this study was intended to provide a methodological basis for modifying the alkyl chain of peptide drugs.

Keywords: Conotoxin; MVIIA; Modification; Reductive amination; Self-assembly.

Fig. 1

Structure and reductive amination route of MVIIA. (A) The structure of MVIIA with disulfide bonds. (B) Reductive amination of MVIIA

Fig. 2

Liquid chromatograms optimized for reactions with molar ratios of (A) 1:0.5 (B) 1:1 (C) 1:3 (D) 1:10 for peptide and myristic aldehyde, respectively

Fig. 3

Modification site identification of single-modified peptides. (A) MS spectrum corresponding to the peak at 40 min (B) Schematic of theoretical peptide chain cleavage. When peptide chain was broken to generate ions, if the charge remained on the fragment at the N-terminus, we named a, b and c ions. If the charge remained on the fragment at the C-terminus, we named x, y and z ions. (C) MS/MS spectra of single-modified MVIIA. (D) Schematic of key fragment ions by program-assisted analysis

Fig. 4

Isolation and characterization of the two-site modified peptide MVIIA. (A) The high-performance liquid chromatography (HPLC) of peptide MVIIA and myristic aldehyde with molar ratios of 1:2. (B) The MS spectrum of the two-site modified MVIIA 1. (C) The MS spectrum of the two-site modified MVIIA 2. (D) The MS spectrum of the two-site modified MVIIA 3

Fig. 5

Identification by secondary mass spectrometry of the modification sites of the two-site modified peptide MVIIA 1 (A), MVIIA 2 (B), and MVIIA 3 (C)

Fig. 6

Characterization of aggregates formed by MVIIA, K2-MVIIA, K2-K4-MVIIA. The anionic 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence traces for MVIIA (A), K2-MVIIA (B) and K2-K4-MVIIA (C). Transmission electron microscopy (TEM) images of MVIIA (D), K2-MVIIA (E), and K2-K4-MVIIA (F) aggregates at 37 °C for 24 h

Fig. 7

Degradation kinetics of peptides MVIIA, K2-MVIIA and K2-K4-MVIIA in human serum. Data are presented as mean ± SEM, n = 4. *p ≤ 0.05 and ***p ≤ 0.001 by one-way ANOVA with Dunnett’s T3 multiple comparison test