2 research outputs found

    Methyleneation of Peptides by <i>N</i>,<i>N</i>,<i>N</i>,<i>N</i>‑Tetramethylethylenediamine (TEMED) under Conditions Used for Free Radical Polymerization: A Mechanistic Study

    No full text
    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 <i>N</i>,<i>N</i>,<i>N</i>,<i>N</i>-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<sup>2</sup> and LC–MS<sup>3</sup> 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 <i>in situ</i> polymerization systems. The use of an alternative catalyst such as sodium bisulfite may preserve the chemical integrity of peptides during in situ polymerization

    Chemoenzymatic Approach for the Preparation of Asymmetric Bi‑, Tri‑, and Tetra-Antennary <i>N</i>‑Glycans from a Common Precursor

    No full text
    Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards that are needed to fabricate the next generation of microarrays, to develop analytical protocols to determine exact structures of isolated glycans, and to elucidate pathways of glycan biosynthesis. We describe here a chemoenzymatic methodology that makes it possible, for the first time, to prepare any bi-, tri-, and tetra-antennary asymmetric <i>N</i>-glycan from a single precursor. It is based on the chemical synthesis of a tetra-antennary glycan that has <i>N</i>-acetylglucosamine (GlcNAc), <i>N</i>-acetyllactosamine (LacNAc), and unnatural Galα­(1,4)-GlcNAc and Manβ­(1,4)-GlcNAc appendages. Mammalian glycosyltransferases recognize only the terminal LacNAc moiety as a substrate, and thus this structure can be uniquely extended. Next, the β-GlcNAc terminating antenna can be converted into LacNAc by galactosylation and can then be enzymatically modified into a complex structure. The unnatural α-Gal and β-Man terminating antennae can sequentially be decaged by an appropriate glycosidase to liberate a terminal β-GlcNAc moiety, which can be converted into LacNAc and then elaborated by a panel of glycosyltransferases. Asymmetric bi- and triantennary glycans could be obtained by removal of a terminal β-GlcNAc moiety by treatment with β-<i>N</i>-acetylglucosaminidase and selective extension of the other arms. The power of the methodology is demonstrated by the preparation of an asymmetric tetra-antennary <i>N</i>-glycan found in human breast carcinoma tissue, which represents the most complex <i>N</i>-glycan ever synthesized. Multistage mass spectrometry of the two isomeric triantennary glycans uncovered unique fragment ions that will facilitate identification of exact structures of glycans in biological samples
    corecore