The use of high efficiency solid phase peptide synthesis (HE-SPPS) provides benefits for the synthesis of peptides due to its high purity, rapid speed, and low chemical usage characteristics [1]. This has been advanced further through the development of a one pot coupling and deprotection process, an evaporation enhanced post deprotection base removal at elevated temperature, and an improved carbodiimide coupling that enhances both O-acylisourea formation and the subsequent acylation [2]. Together these features maximized achievable synthesis purity, reduced the entire cycle time to < 3 minutes, and required only a single washing step per cycle. Therefore, using this process a difficult 10 mer peptide (0.1mmol scale) can be made in < 30 minutes with < 100mL total waste.
Realization of these benefits provides two practical benefits for peptide synthesis. First, enhanced synthesis purity compared to standard HE-SPPS conditions was achieved due to improvements in both the deprotection and coupling steps. Second, an automated sequential version of this process creates a powerful high throughput synthesis approach. For example, a batch of 24 different peptides (with lengths up to 75 amino acids) was synthesized automatically using this process within only a day while featuring an unprecedented reduction in chemical waste.
The application of this technology was then expanded through the use of the hyper-acid sensitive Cl-TCP linker [3] and a production scale microwave peptide synthesizer with reactor volumes up to 17L. The Cl-TCP linker provided a unique combination of stability and protection against C-terminal side reactions at elevated temperatures compared to the well-known 2-Cl-Trt and Trityl (TCP) linkers.
[1] J. M. Collins, K. A. Porter, S. K. Singh, G. S. Vanier, Org. Lett. 16, 940 (2014).
[2] US20160176918
[3] C. Heinlein, D. Silva, A. Trӧster, J. Schmidt, A. Gross, C. Unverzagt, Angew. Chem. 50, 6406 (2011).