Methyleneation of Peptides by N,N,N,N-Tetramethylethylenediamine (TEMED) under Conditions Used for Free Radical Polymerization: A Mechanistic Study

Abstract

Free radical polymerization is often used to prepare protein and peptide-loaded hydrogels for the design of controlled release systems and molecular imprinting materials. Peroxodisulfates (ammonium peroxodisulfates (APS) or potassium peroxodisulfates (KPS)) with N,N,N,N-tetramethylethylenediamine (TEMED) are frequently used as initiator and catalyst. However, exposure to these free radical polymerization reagents may lead to modification of the protein and peptide. In this work, we show the modification of lysine residues by ammonium peroxodisulfate (APS)/TEMED of the immunostimulant thymopentin (TP5). Parallel studies on a decapeptide and a library of 15 dipeptides were performed to reveal the mechanism of modification. LC-MS of APS/TEMED-exposed TP5 revealed a major reaction product with an increased mass (+12 Da) with respect to TP5. LC-MS(2) and LC-MS(3) were performed to obtain structural information on the modified peptide and localize the actual modification site. Interpretation of the obtained data demonstrates the formation of a methylene bridge between the lysine and arginine residue in the presence of TEMED, while replacing TEMED with a sodium bisulfite catalyst did not show this modification. Studies with the other peptides showed that the TEMED radical can induce methyleneation on peptides when lysine is next to arginine, proline, cysteine, aspargine, glutamine, histidine, tyrosine, tryptophan, and aspartic acid residues. Stability of peptides and protein needs to be considered when using APS/TEMED in in situ polymerization systems. The use of an alternative catalyst such as sodium bisulfite may preserve the chemical integrity of peptides during in situ polymerization.

Synthesis and Evaluation of a Thermoresponsive Degradable Chitosan-Grafted PNIPAAm Hydrogel as a “Smart” Gene Delivery System

“…It was assumed that the grafting of PNIPAAm chains onto Cs occurred at the carbon C-2 position of the pyranose ring. The formation of amine radicals is highly favored over hydroxyl radicals [54,55], supporting work by Shirangi et al [56] that demonstrated that primary amines exhibit increased affinities for the α-TEMED radical species (Figure 9a). The aforementioned evidence points to the directed radical formation C-2-NH 2 species of chitosan [57].…”

“…Hydrogen atom abstraction could have occurred from NIPAAm homopolymer but was more likely from the radical initiators [58]. In addition, the higher concentrations of TEMED (0.2 mmol) compared to APS (0.131 mmol) supported the likelihood of increased formation of TEMED radical to facilitate the secondary initiation mechanism (Figure 9b) [56]. In contrast to gel permeation chromatography (GPC) and high-performance liquid chromatography (HPLC), for which retention times do not provide specific detail on the structure of the graft [59], or preparative liquid chromatography-mass spectrometry (LC-MS) methods where mass spectrometry (MS) fragmentation spectra would be the same irrespective of where the NIPAAm chain extension is grafted onto the Cs, the grafting position could be confirmed with the use of 1 H-NMR in dimethyl sulfoxide (d6-DMSO).…”

Nucleophile sensitivity of Drosophila TRPA1 underlies light-induced feeding deterrence

“…Therefore, the effect of the nucleophile scavenger NMM on free radical-induced TRPA1(A) activation was tested in heterologous frog oocytes. Addition of tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) initiates polymerization reactions, such as solidification of polyacrylamide gel, by generating free radicals ( Shirangi et al, 2015 ). To examine the responsiveness of TRPA1(A) to free radicals, frog oocytes expressing agTRPA1(A) were exposed to a mixture of 0.01 mM TEMED and 0.1 mM APS.…”

Nanogels for intracellular delivery of biotherapeutics

“…This can be solved by increasing the crosslink density of nanogels to stably entrap their payloads during gel formation. However, once the biotherapeutics are loaded in hydrogel particles during preparation, this might result in chemical modification of the loaded molecules [40][41][42][43]. In other alternative methods, strongly charged biotherapeutics, such as nucleic acids, can be post-loaded into oppositely charged nanogels and stably immobilized by strong electrostatic interaction under physiological conditions [44][45][46][47][48].…”