Automated Microwave-Enhanced Total Synthesis of Proteins

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. Recently, rapid flow-based methodology has shown the significant ability to assemble long sequences with extremely quick cycle times. 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. Optimized carbodiimide-based coupling conditions with microwave heating (CarboMAX^TM) allow for minimal epimerization and higher purity synthesis than more aggressive activation based methods using onium salts with strong bases. 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.

In 2022, a new series of microwave peptide synthesizers (Liberty PRIME 2.0 and Liberty Blue 2.0) were released that utilize a new headspace flushing technology for ensuring cleaner surfaces of the reaction vessel during peptide synthesis. This headspace flushing technology is useful for preventing volatile reagents from re-condensing and contaminating subsequent reactions. The small purity increases to each reaction become significant gains in purity over the course of a long protein synthesis. The successful synthesis of several proteins including ubiquitin (76mer), proinsulin (86mer), barstar (89mer), a collagen-like sequence (99mer), HIV protease (99mer), and MDM2 (127mer) was achieved on the Liberty PRIME 2.0.