Conformational studies of hybrid aminoxy peptides & exploration of protein posttranslational modification by medium-chain fatty acid

Abstract

Conformations of aminoxy peptides with a wide range of backbones and substituents have been characterized over the past two decades. It has been discovered that aminoxy peptides composed of α-, β-, and γ-aminoxy acids comprise a novel class of foldamers that adopt unique and stable secondary structures, such as turn, helix and sheet, in the solid state and in non-polar solutions. However, hybrid aminoxy peptides assembled by different types of aminoxy acid residues were largely unexplored. In Chapter 1, α- and cyclic β2,3-aminoxy acid residues were coupled to form hybrid aminoxy peptides and their secondary structures were investigated to find the structural relationship between aminoxy acid monomers and the resulting hybrid peptides. Compounds 1–12 were synthesized and their conformations were characterized by X-ray crystallography, 1D and 2D NMR spectroscopy and circular dichroism. Consistent results obtained from different methods establish that consecutive turns are conformationally stable and conserved in such hybrid peptides. In addition, novel helical and turn-like structures have been observed in the crystal structures of 5 and 8. The NOE patterns and CD spectra of other peptides were similar to those of 5 and 8, indicating that either helical or reverse turn structure is adopted by this series of peptides, regardless of the solvent, ring size and stereochemistry of cyclic β2,3-aminoxy acid residues. Protein posttranslational modification (PTM) has attracted much attention in current epigenetic research, especially on target profiling, mechanism and physiological functions. Several methods of profiling potential protein targets have been discovered with the rapid development of highly sensitive mass spectrometry detection and bioconjugation chemistry. The successful application of ‘chemical reporters’ in detecting protein lipidation by long-chain fatty acids has inspired us to explore any unknown but plausible PTM, such as medium-chain fatty acid lipidation, which is physiologically relevant yet rarely reported. In Chapter 2, several ‘chemical reporters’ were synthesized to explore potential protein lipidation by medium-chain fatty acids. Compounds alk-6, 7 and 8 were prepared and subjected to metabolic labeling in RAW264.7 cells. The resulting cell lysates were conjugated with rhodamine-N3, and fluorescent protein bands were visualized after SDS-PAGE. Alk-7 was found to uniquely give a novel fluorescent protein band at around 50 kDa, in dose- and time-dependent manners. This protein band showed resistance towards basic NH2OH nucleophilic replacement, which hinted that alk-7 might covalently modify proteins through amide bond, that is, either lysine or N-terminal amino acid residue was modified. After conjugating cell lysates with biotin-N3, streptavidin beads enrichment, trypsin digestion and mass spectrometry analysis, the newly alk-7 labeled protein band was identified as mitochondrial aldehyde dehydrogenase. Furthermore, it was validated through immunoblotting ALDH2 that this alk-7 labeling abundantly existed in RAW264.7 and HCT116 cells, but scarcely in HepG2 cells. Alkyne-free endogenous medium-chain fatty acids were used to compete with alk-7 labeling; however, none of them showed obvious competition effect. Therefore, we conclude that mitochondrial ALDH2 can be labeled by chemical reporter alk-7, possibly through amide bond formation, in some cell lines. The functional significance of this labeling remains to be established.