A novel method for repetitive peptide synthesis in solution without isolation of intermediates

Abstract

A novel method was developed for the large-scale manufacture of peptides in solution, called DioRaSSP-Diosynth Rapid Solution Synthesis of Peptides. This method combines the advantages of the homogeneous character of classical solution-phase synthesis with the universal character and the amenability to automation inherent to the solid-phase approach. The process consists of repetitive cycles of coupling and deprotection in a permanent organic phase and is further characterized by the fact that intermediates are not isolated. Couplings are mediated by water-soluble carbodiimide. Several types of function may be applied for temporary amino protection depending on the sequence of the actual peptide, including Z, Fmoc, Msc and Nsc. Formate is the preferred hydrogen donor during hydrogenolysis of the Z function, while 1,8-diazabicyclo[5.4.0]undec-7-ene is used to deprotect Fmoc, Msc and Nsc. Morpholine is added during the deprotection of Msc and Nsc to scavenge the arising alkenes. Processes according to this highly efficient synthesis method are easy to scale up and yield products of reproducible high purity, which is guaranteed by a new quenching method for residual activated compounds, applying an anion-forming amine such as a beta-alanine ester. This ester should display a lability similar to that of the temporary amino-protecting function, allowing simultaneous deprotection of the growing peptide and the quenched compound. The DioRaSSP approach assures the completely quantitative removal of deprotected quenched compounds before the coupling step of the next cycle of the synthesis by basic aqueous (that is active) extraction, while the growing peptide remains anchored in the organic phase due to the presence of hydrophobic protecting functions.

Subtle Recognition of 14-Base Pair DNA Sequences via Threading Polyintercalation

“…38 To minimize coupling steps and maximize yield, Fmoc-(β-Ala) 3 -OH and Fmoc-(Gly) 3 -OH were synthesized prior to SPPS 39,40 (Supporting Information). While 2 was left with a terminal free amine, the N-terminus of 3 was capped as the acetamide (using acetic anhydride) to produce species with +5 and +4 charges, respectively, at neutral pH.…”

Sustainability in peptide chemistry: current synthesis and purification technologies and future challenges

“…Essentially, in the DioRaSSP® approach, the growing peptide is kept in an organic phase, typically EtOAc, by using a simple hydrophobic C-terminal ester, generally a tert -butyl group. 164 As shown in Fig. 17, a DioRaSSP® cycle comprises three key steps: the coupling, generally performed using EDC/HOBt and a benzyloxycarbonyl-protected amino acid (30–60 min); the quenching of residual activated carboxylic compound using β-alanine benzyl ester; and the deprotection of the N-terminus of growing peptide and by-products via palladium-catalysed hydrogenolysis, using 3–4 equivalents of formate (30–60 min).…”

Implication of Side Reactions in Iterative Biopolymer Synthesis: The Case of Membrane Enhanced Peptide Synthesis

“…A considerable number of biological and chemical approaches have been developed for preventing or minimizing the formation of error sequences. [28][29][30][31][32][33][34] From the process operation perspective, two key decision variables in MEPS are the reaction extent for each coupling and the extent of removal of amino acid and piperidine before a new coupling. These variables have significant implications for the formation of general error sequences (i.e.…”