Flow‐based SPPS for protein synthesis: A perspective

Abstract

Flow‐based approaches to solid phase peptide synthesis (SPPS) have been pursued since the method's early days, with anticipated gains in speed, reaction monitoring, and ease of automation. Here, we discuss how these advantages are being realized by synthesis at elevated temperature, facilitated by a 'preheat/activation' loop. This important modification both accelerates peptide synthesis—providing a wealth of new data from in‐line monitoring—and in conjunction with an optimized protocol, extends the length of peptides routinely accessible by stepwise synthesis. Streamlined synthesis of longer peptides will address a major challenge in chemical protein synthesis: preparation of peptide segments for use in chemical ligation. The context of recent results in flow‐based SPPS is discussed, highlighting remaining challenges and future opportunities.

Circular Aqueous Fmoc/t‐Bu Solid‐Phase Peptide Synthesis

“…Furthermore, significant excess of AAs and process aids are needed, and in some cases up to 100 equiv. of AAs in couplings are employed [24] . Although minimized use of AAs [25] and solvents [26] has been reported, these methods were hampered by the use of the hazardous DMF.…”

Flow-based Methods in Chemical Peptide and Protein Synthesis

“…[6] In contrast to traditional synthesis methods, flow-based SPPS offers high control over reaction conditions, and in-line analysis gives valuable information on individual coupling and deprotection steps. We have recently reviewed the general principle of flowbased SPPS and set the method into a historical context, [4] thus this review will focus on recent developments.…”

“…[2] Continuous flow offers four key advantages for peptide chemistry: (1) Reactive or transient intermediates can be generated and immediately consumed under highly controlled reaction conditions (flash chemistry), [3] (2) continuous flow ensures minimal exposure of reactor contents to reactive by-products, (3) energy transfer is improved, and (4) time-resolved reaction monitoring is achieved by in-line analysis. [4]…”

Posttranslational Chemical Mutagenesis Methods to Insert Posttranslational Modifications into Recombinant Proteins

“…The NCL approach is the most utilized ligation method, owing to the accessibility of the building blocks and the mild reaction conditions. Recent advances in protecting group chemistry [17,18], functional tags [19,20], continuous flow synthesis [21,22], and post-synthetic modifications [7,9] allow for the preparation of large and complex protein targets with a broad range of PTMs for various applications. Moreover, the development of the protein semi-synthesis technique has expanded the scope of modified protein production by combining synthetic peptides and large recombinant protein fragments in Chemical protein synthesis provides a powerful means to prepare native and modified proteins in an effective and controlled manner [7,8].…”