Automated Microwave-Enhanced Total Synthesis of Proteins

Summary

Proteins and long peptides can be synthesized rapidly and efficiently by microwave-enhanced SPPS on the Liberty Blue™ 2.0 and Liberty PRIME™ 2.0. The use of optimized microwave SPPS incorporating a new headspace flushing technology allows for higher purity synthesis of protein sequences. This technology was demonstrated on a series of biologically relevant proteins (ubiquitin, barstar, proinsulin, collagen, HIV protease, and MDM2) from 76-127 amino acids which were obtained in good purity through stepwise assembly without any ligation steps. High purity samples were isolated from the crude material by elevated temperature chromatography at 60 °C on the Prodigy™ Preparative HPLC Peptide Purification system.

Introduction

Proteins and long peptides are critical components of biological systems and comprise many important therapeutics, but their research is slowed by time-intensive expression or native chemical ligation production methods. Total synthesis by SPPS provides a direct synthetic route to target specific sequences and allows for rapid generation of analogs. However, long peptides and proteins can be challenging targets for SPPS, due to the iterative accumulation of impurities and tendency for aggregation to occur. Historically, SPPS was limited to making fragments for native chemical ligation and longer sequences have been of limited accessibility.1 Recently, rapid flow-based methodology has shown the significant ability to assemble long sequences with extremely quick cycle times.2 However, this process requires large excesses of amino acid (≥ 100 equivalents) and produces large amounts of waste.

Microwave heating is now widely used and has demonstrated the ability to overcome aggregation and drive the completion of difficult reactions on long peptide chains.3, 4 Optimized carbodiimide-based coupling conditions with microwave heating (CarboMAX™) allow for minimal epimerization and higher purity synthesis than more aggressive activation based methods using onium salts with strong bases.5 Additionally, the use of a one-pot coupling and deprotection process without any draining between the steps results in a faster and more efficient process.