Accelerated Multiphosphorylated Peptide Synthesis

Abstract

Preparing phosphorylated peptides with multiple adjacent phosphorylations is synthetically difficult, leads to β-elimination, results in low yields, and is extremely slow. We combined synthetic chemical methodologies with computational studies and engineering approaches to develop a strategy that takes advantage of fast stirring, high temperature, and a very low concentration of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to produce multiphosphorylated peptides at an extremely rapid time and high purity.

Figure 1

Fmoc deprotection (blue) and β-elimination (red) of protected phosphorylated Ser/Thr are competing reactions that take place under basic conditions. The common deprotection protocol, using 20% piperidine solution, results in complete deprotection at RT while promoting β-elimination at high temperature (right). New conditions are required for minimizing β-elimination during Fmoc deprotection at high temperatures (left).

Figure 2

(A) Potential energy surface (PES) representing Fmoc deprotection (i) and β-elimination (ii) processes with DBU. (B) PES representing Fmoc deprotection (i) and β-elimination (ii) processes using piperidine. The chemical structure associated with the initial, final, and intermediate compounds, annotated i-a to i-c and ii-a to ii-c, are described in the SI. ΔE values are calculated with respect to the initial structures (i-a/ii-a).

Figure 3

B2R-5p synthesis by either the MW-assisted (A) or AMPS (B) approaches. In both syntheses, three residues (marked with *) were coupled twice pSMGpT*LR*pT*pSIpS since the coupling involved an introduction of a bulky pThr or Arg to clustered regions_..