Automated Synthesis of Heavily Phosphorylated Peptides

Abstract

Multi phosphorylated peptides are key tools in understanding the biological roles of protein phosphorylation patterns. In this work, we focused on multi phosphorylated peptides with over four, clustered, phosphorylation sites that are termed herein heavily phosphorylated peptides (HPPs). The synthesis of heavily phosphorylated peptides is extremely difficult and requires the use of a wide temperature range. Standard peptide synthesizers are incapable of both cooling and heating, which impedes the automated synthesis of those peptides. Herein, we used the oligosaccharide synthesizer Glyconeer 2.1 to develop a protocol for the automated synthesis of heavily phosphorylated peptides. The Glyconeer 2.1 is able to both cool and heat, which enabled the development of highly controlled coupling and deprotection conditions that were used for the automated synthesis of four different heavily phosphorylated peptides with five or more, clustered, phosphorylation sites. Our approach paves the way for an easy automated synthesis of a variety of heavily phosphorylated peptides.

Dissecting the role of protein phosphorylation: a chemical biology toolbox

“…More efficient control of the temperature, in particular in the Fmoc-deprotection step, allowed the synthesis of peptides bearing up to nine clustered phosphorylations in reasonable yield and purity. 83 Works from Becker and Geyer demonstrated that global phosphorylation could provide access to multiphosphorylated peptides. In their synthesis of silaffin peptides, which play an important role in biomineralization, Lechner and Becker masked serine phosphorylation sites by means of Trt protection.…”

Accelerated Multiphosphorylated Peptide Synthesis

“… 13 − 15 This allowed the synthesis of MPPs with up to nine p-sites with minimal risk of β-elimination byproducts, but the process is very tedious and time-consuming. 18 An average synthesis time of an MPP via one of the above methods ranges from several hours to days depending on the sequence and the phosphorylation pattern and is much slower than the synthesis of nonphosphorylated peptides. 19 , 20 Accelerated SPPS processes are in high demand as peptide libraries are an essential tool for biological studies.…”

“…The automated approach provides MPPs with five p-sites in crude purity of above 50%, and the targeted approach can provide even higher purities. 15 , 18 Although crude purities obtained by those methods are higher than AMPS, a synthesis of 10 residues in the Glyconeer 2.1 takes ∼14 h, and via the targeted approach it takes days. 15 , 18 This makes the synthesis of each MPP about 40 times longer than via AMPS.…”

“… 15 , 18 Although crude purities obtained by those methods are higher than AMPS, a synthesis of 10 residues in the Glyconeer 2.1 takes ∼14 h, and via the targeted approach it takes days. 15 , 18 This makes the synthesis of each MPP about 40 times longer than via AMPS. Both these methods suffer from poor mixing of the solid support and the reaction reagents, whether by shaking or gas bubbling which translates to a longer reaction duration in addition to the cooling–heating cycles that are applied.…”

Translating solution to solid phase glycosylation conditions

“…18 We aimed to automate the optimization of glycosylation temperature using the Glyconeer TM (Scheme 1, bottom). [19][20][21][22] We set three challenges: develop an economically viable protocol to optimize the glycosylation temperature, transfer the optimized conditions from solution phase to solid phase, and decrease the excess of donors used in AGA (Scheme 1 bottom). The design of the strategy relied on these guidelines: (1) The optimization will require a tiny amount of donor and acceptor.…”