Efficient flow synthesis of human antimicrobial peptides

Abstract

Organisms from all kingdoms of life have evolved a vast array of peptidic natural products to defend against microbes. These are known collectively as antimicrobial peptides (AMPs) or host defense peptides, reflecting their abilities to not only directly kill microbes, but also to modulate host immune responses. Despite decades of investigation, AMPs have yet to live up to their promise as lead therapeutics, a reality that reflects, in part, our incomplete understanding of these diverse agents in their various physiological contexts. Toward improving our understanding of AMP biology and the ways in which this can be best leveraged for therapeutic development, we are interested in large-scale comparisons of the antimicrobial and immunological activities of human AMPs, an undertaking that requires an efficient workflow for AMP synthesis and subsequent characterization. We describe here the application of flow chemistry and reverse phase flash chromatography to the generation of 43 AMPs, approaches that, when combined, significantly expedite synthesis and purification, potentially facilitating more systematic approaches to downstream testing and engineering.

Figure 1:

A. Schematic of the flow synthesizers used in this study. Three pumps on the right dedicated to amino acids (blue), base (green), and activators or deprotection reagents (yellow - depending on cycle step) control flow of selected reagents (amino acids in blue / purple, base in green, activators in yellow, deprotection solution in red) and solvent (brown) on the left through the selector valves in the center and into to the heating loops and heated reactor on the right, eventually passing through an ultraviolet (UV) detector and on to the central waste bin. All functions are computer-controlled. See also Experimental. B. Workflow for peptide synthesis and characterization. In brief, flow-synthesized peptides are treated with strong acid to effect sidechain deprotection and release of the linear polypeptide from resin followed by trituration, lyophilization, and characterization by liquid chromatography-mass spectrometry (LCMS) and high performance liquid chromatography (HPLC). Crude peptides are purified by reverse phase flash chromatography (RPFC) or preparative HPLC and folded if required. Quality control LCMS and HPLC are then used to characterize purified peptides ahead of assays.

Figure 2:

Automated flow synthesis of antimicrobial peptide LL-37 (reproduced here from Supplementary Figure 7). A. LL-37 sequence (red = cationic, blue = anionic, orange = polar, green = nonpolar, purple = aromatic). B. Synthesizer UV trace showing resolved Fmoc deprotection peaks alternating with saturated amino acid coupling peaks. Synthesizer settings are specified for each peptide; here, 3^rd Generation synthesizer - optimized for Length. Spaces in the x-axis represent optional, user-initiated pauses. C. Fmoc deprotection integrals and peak width and height expressed as percentages relative to the first cycle. D. Left panel: total ion chromatogram (TIC) of crude AMP overlaid on Blank run with the predicted average and monoisotopic masses as well as the observed mass as calculated from the most abundant ion. Right panel: extracted ion chromatogram (EIC) of crude AMP for the specified m/z range. The LCMS method is specified for each AMP; here, LCMS Method 5. E. TIC and EIC of purified AMP, LCMS Method 3. F-G. Mass spectra associated with the dominant peaks of D and E, respectively. The charge states of the labeled ions are indicated in parentheses. H-I. Analytical high performance liquid chromatography (HPLC) traces of crude and purified peptide, respectively, with the integrated percentage of the dominant peak (retention time in parentheses). The HPLC method is specified for each AMP; here, HPLC Method 1.