Peptides and proteins are essential biopolymers that play critical roles in biological functions. Derivatives of these molecules in which functional moieties are chemically introduced have found widespread applications as molecular probes.
In the synthesis of these protein probes, total chemical synthesis or semi-synthesis methods are employed to allow for the precise introduction of functional groups at specific positions. Protein synthesis involves the preparation of peptide chains through solid-phase synthesis or expression, followed by analysis and purification using HPLC, chemoselective ligation of the peptides, and, if necessary, post-ligation steps.
While the unit operations involved in protein chemical synthesis are being optimized, there is a demand for further improvements in these processes. Here we have developed a set of tools based on hydrazide chemistry to accelerate protein chemical synthesis.
The water solubility of synthetic intermediates is a critical factor that impacts purification and reaction efficiency, mainly since many steps following solid-phase synthesis are performed in aqueous media. Peptide solubilization has been employed by introducing water-soluble tags to handle peptides with poor solubility. However, the available methods for tag introduction after solid-phase synthesis have been limited. To overcome this limitation, we have developed a method to solubilize peptides at a late stage by employing the reductive N-alkylation reaction of hydrazide with solubilizing tags having a formyl group.
Additionally, peptide hydrazides are commonly used as thioester precursors in protein synthesis. However, peptide hydrazides often exhibit peak tailing during HPLC analysis, making purification and analysis challenging. To address this issue, we have discovered that alkyl hydrazides improve peak shape and have developed a novel solid-phase linker that directly synthesizes alkyl hydrazides.
With the development of these tools, easier access to chemically synthesized proteins will contribute to the further advancement of protein science.