Rapid flow-based synthesis of post-translationally modified peptides and proteins: a case study on MYC's transactivation domain.

Protein-protein interactions of c-Myc (MYC) are often regulated by post-translational modifications (PTMs), such as phosphorylation, and crosstalk thereof. Studying these interactions requires proteins with unique PTM patterns, which are challenging to obtain by recombinant methods. Standard peptide synthesis and native chemical ligation can produce such modified proteins, but are time-consuming and therefore typically limited to the study of individual PTMs. Herein, we report the development of flow-based methods for the rapid synthesis of phosphorylated MYC sequences (up to 84 AA), and demonstrate the versatility of this approach for the incorporation of other PTMs (N ε-methylation, sulfation, acetylation, glycosylation) and combinations thereof. Peptides containing up to seven PTMs and phosphorylation at up to five sites were successfully prepared and isolated in high yield and purity. We further produced ten PTM-decorated analogues of the MYC Transactivation Domain (TAD) to screen for binding to the tumor suppressor protein, Bin1, using heteronuclear NMR and native mass spectrometry. We determined the effects of phosphorylation and glycosylation on the strength of the MYC:Bin1 interaction, and reveal an influence of MYC sequence length on binding. Our platform for the rapid synthesis of MYC sequences up to 84 AA with distinct PTM patterns thus enables the systematic study of PTM function at a molecular level, and offers a convenient way for expedited screening of constructs.

Merging Flow Synthesis and Enzymatic Maturation to Expand the Chemical Space of Lasso Peptides.

Many peptidic natural products, such as lasso peptides, cyclic peptides, and cyclotides, are conformationally constrained and show biological stability, making them attractive scaffolds for drug development. Although many peptides can be synthesized and modified through chemical methods, knot-like lasso peptides such as microcin J25 (MccJ25) and their analogues remain elusive. As the chemical space of MccJ25 analogues accessible through purely biological methods is also limited, we proposed a hybrid approach: flow-based chemical synthesis of non-natural precursor peptides, followed by in vitro transformation with recombinant maturation enzymes, to yield a more diverse array of lasso peptides. Herein, we established the rapid, flow-based synthesis of chemically modified MccJ25 precursor peptides (57 amino acids). Heterologous expression of enzymes McjB and McjC was extensively optimized to improve yields and facilitate the synthesis of multiple analogues of MccJ25, including the incorporation of non-canonical tyrosine and histidine derivatives into the lasso scaffold. Finally, using our chemoenzymatic strategy, we produced a biologically active analogue containing three d-amino acids in the loop region and incorporated backbone N-methylations. Our method provides rapid access to chemically modified lasso peptides that could be used to investigate structure-activity relationships, epitope grafting, and the improvement of therapeutic properties.

Flow-based Methods in Chemical Peptide and Protein Synthesis.

Flow chemistry has emerged as a powerful method for on-demand chemical synthesis and modification of peptides and proteins. Herein, we discuss the characteristics of flow chemistry and how they are applied to various aspects of peptide chemistry. We highlight recent advances in automated flow-based peptide synthesis, which extend the length of peptides routinely accessible to single-domain proteins and allow for the collection of time-resolved synthesis data. Applications of this data for the prediction of synthesis outcome and the potential for the development of more sustainable synthesis methods are also discussed. Finally, we will review solutionphase approaches, including flow-based ligation strategies and peptide cyclization. Throughout this review, the current challenges and potential future developments are highlighted.

Automated access to well-defined ionic oligosaccharides.

Ionic polysaccharides are part of many biological events, but lack structural characterisation due to challenging purifications and complex synthesis. Four monosaccharides bearing modifications not found in nature are used for the automated synthesis of a collection of ionic oligosaccharides. Structural analysis reveals how the charge pattern affects glycan conformation.