Accelerated Multiphosphorylated Peptide Synthesis

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.


Reagents for coupling
Amino acid solutions were prepared by adding a solution of 3 equiv. Fmoc-protected amino acids in 1 mL DMF. An activator solution was prepared by dissolving 2.9 equiv.
of HATU in 1 mL of DMF per coupling. A base solution was prepared by dissolving 8 equiv. of DIEA in 1 mL DMF per coupling.

Bases for Fmoc deprotection
Solutions of 0.5 and 5% and 20% (V/V) piperidine in DMF were prepared.

Computational details: Modeling Fmoc deprotection and β-elimination processes using piperidine base
Fmoc deprotection and β-elimination reactions were also modeled using piperidine as the base. As done with DBU, the Fmoc deprotection reaction was studied from a model system consisting of one Fmoc-Ser/(HPO3Bzl)-OH and one piperidine molecule. We found similar mechanisms with piperidine to those observed with DBU. The Fmoc deprotection is a two-step process, shown in Figure S11(i) and S12 (i). The first step is  Figure S11(ii-a). The second step consists of a proton being S14 transferred from the C of the [Ser/(PO3Bzl)-OH]to the piperidine resulting in the dephosphorylation of the Ser (ii-c). The energy barrier for this step is 16.6 kcal/mol. Figure S11. PES representing Fmoc deprotection (i) and β-elimination (ii) processes with piperidine. Above each bar, the associated chemical structure is presented. Black bars represent minima on the PES, and purple/red bars represent transition states. ∆ values are calculated with respect to the initial structures (i-a/ii-a). In the case of i-d), the ∆ value is omitted as, when going from i-c) to i-d), we have removed from the model system Fmoc-Ser/(HPO3Bzl)-OH the molecular fragment that has dissociated. The ∆ for i-e) and i-f) are relative to the energy of i-d).
Figure S12. PES representing Fmoc deprotection (i) and β-elimination (ii) processes with DBU. Above each bar, the associated chemical structure is presented. Black bars represent minima on the PES, and purple/red bars represent transition states. ∆ values are calculated with respect to the initial structures (i-a/ii-a). In the case of i-d), the ∆ value is omitted as, when going from i-c) to i-d), we have removed from the model system Fmoc-Ser/(HPO3Bzl)-OH the molecular fragment that has dissociated. The ∆ for i-e) and i-f) are relative to the energy of i-d).