Studies and applications of aldehyde chemistry for peptide modification and formaldehyde detection

Abstract

Aldehyde is a versatile functional group and synthetic building block in organic synthesis. Significant advancement has been made to demonstrate the key role of aldehydes in synthetic chemistry through diverse organic transformation reactions including C-C, C-O, and C-N bond formations. In this work, we would like to expand the impact of aldehyde chemistry to the interdisciplinary research areas on chemical biology and food safety. Selective modification of peptides and proteins has attracted great attention in chemical biology, as it allows the generation of novel bioconjugates used in the study of complex biological systems. The N-terminal α-amino group of peptides/proteins is utilized for site-specific modification as most proteins have only one N-terminus. In my work, site-specific N-terminal α-amino group modification of peptides by an oxime formation/exchange sequence was developed. We found that oxone (2KHSO₅.KHSO₄.K₂SO₄) is a convenient and chemoselective reagent for oxidation of N-terminal α-amino group to oximes. The substrate scope of the present oxime formation reaction was studied by using a series of unprotected peptides XSKFR (X = 20 natural amino acids). The N-terminal modifications were found in the peptides in a range of conversions (21-99%) as confirmed by ESI-MS/MS analyses. With the above promising results, subsequent functionalization of the oxime-modified peptides via an oxime exchange with O-substituted hydroxylamines was studied. Moreover, incorporation of biophysical probes such as fluorescent dansyl and PEG-linked hydroxylamine by the oxime exchange reaction was performed, leading to the corresponding functionalized peptides in 99% conversion. These findings indicated that N-terminal α-amino group modification by using an oxime formation/exchange reaction sequence could be performed under mild reaction conditions and the present method demonstrates the potential of oxone towards peptide/protein modification. Formaldehyde is known to be a natural metabolite in various living organisms, and is thus present in many fresh and preserved foods as a normal component. Formaldehyde is not permitted for food use in Hong Kong and China. However, formaldehyde was recently found to be deliberately used in food processing in China for bleaching, protein coagulation and preservation purposes, causing a serious hazard to public health. In my work, a gold(III) complex-catalyzed three component coupling reaction of formaldehyde, an amine and an alkyne for propargylamine synthesis was established to allow formaldehyde detection within 1 hour at 50 ℃. The high selectivity of the coupling reaction to detect formaldehyde is achieved by the steric bulkiness around the amino group of the amine, as formaldehyde is the smallest aldehyde in nature. Different parameters such as catalysts, reagent ratios and solvents were optimized using a HPLC-based assay, with up to 56% yield achieved in 1 hour. Different fluorophores such as coumarin and dansyl were employed for fluorescent detection. The reaction system could also be immobilized on solid supports such as 2-chlorotrityl chloride resin for washing of excess reagents. Measurement of fluorescent intensity on the resin showed excellent linearity (R² = 0.99). The reaction was applicable to detect formaldehyde in dried shiitake mushroom samples.