ABSTRACT
We present a process for solid phase peptide synthesis (SPPS) that completely eliminates all solvent intensive washing steps during each amino acid addition cycle. A key breakthrough is the removal of a volatile Fmoc deprotection base through bulk evaporation at elevated temperature while preventing condensation on the vessel surfaces with a directed headspace gas flushing. This process was demonstrated at both research and production scales without any impact on product quality and when applied to a variety of challenging sequences (up to 89 amino acids in length). The overall result is an extremely fast, high purity, scalable process with a massive waste reduction (up to 95%) while only requiring 10-15% of the standard amount of base used. This transformation of SPPS represents a step-change in peptide manufacturing process efficiency, and should encourage expanded access to peptide-based therapeutics.
Subject(s)
Peptides , Solid-Phase Synthesis Techniques , Peptides/chemistry , Amino Acids/chemistryABSTRACT
The unique combination of microwave heating with optimized carbodiimide activation has proven to be an indispensable technique for high-throughput peptide production. Here, we describe new methods in microwave-assisted solid phase peptide synthesis and optimized post-synthesis modifications that have been recently developed. These methods have drastically reduced synthesis time and solvent requirement while delivering peptides in high crude purities.
Subject(s)
Microwaves , Peptides/chemical synthesis , Solid-Phase Synthesis Techniques/methods , Amino Acid Sequence , Antifreeze Proteins/chemistry , Automation , Chemistry Techniques, Synthetic , Chromatography, High Pressure Liquid , Disulfides , High-Throughput Screening Assays , Mass Spectrometry , Peptides/analysis , Peptides/isolation & purification , Phosphopeptides/chemical synthesis , Phosphopeptides/chemistry , Solid-Phase Synthesis Techniques/instrumentation , SolventsABSTRACT
A series of improvements to the standard solid phase peptide synthesis (SPPS) process allowing for significant gains in product purity along with only a 4 min standard cycle time and a 90% reduction in total waste produced is reported. For example, syntheses of the well-known (65-74)acyl carrier protein (ACP) and (1-42)ß-amyloid peptides were accomplished with 93 and 72% purity (UPLC-MS) in only 44 and 229 min, respectively.
Subject(s)
Acyl Carrier Protein/chemical synthesis , Amyloid beta-Peptides/chemical synthesis , Oligopeptides/chemical synthesis , Peptide Fragments/chemical synthesis , Acyl Carrier Protein/chemistry , Amyloid beta-Peptides/chemistry , Molecular Structure , Oligopeptides/chemistry , Peptide Fragments/chemistry , Solid-Phase Synthesis TechniquesABSTRACT
As the range of techniques for microwave heating has expanded, so have the areas in which it can have a profound impact. Two emerging areas are the application of microwave heating for the synthesis of peptides, peptoids, oligopeptides and carbohydrates and in the field of proteomics.
Subject(s)
Carbohydrates/radiation effects , Microwaves , Peptides/radiation effects , Carbohydrate Conformation/radiation effects , Carbohydrates/chemical synthesis , Carbohydrates/chemistry , Peptides/chemical synthesis , Peptides/chemistry , Protein Conformation/radiation effects , ProteomicsABSTRACT
Microwave energy represents an efficient manner to accelerate both the deprotection and coupling reactions in 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). Typical SPPS side reactions including racemization and aspartimide formation can occur with microwave energy but can easily be controlled by routine use of optimized methods. Cysteine, histidine, and aspartic acid were susceptible to racemization during microwave SPPS of a model 20mer peptide containing all 20 natural amino acids. Lowering the microwave coupling temperature from 80 degrees C to 50 degrees C limited racemization of histidine and cysteine. Additionally, coupling of both histidine and cysteine can be performed conventionally while the rest of the peptide is synthesized using microwave without any deleterious effect, as racemization during the coupling reaction was limited to the activated ester state of the amino acids up to 80 degrees C. Use of the hindered amine, collidine, in the coupling reaction also minimized formation of D-cysteine. Aspartimide formation and subsequent racemization of aspartic acid was reduced by the addition of HOBt to the deprotection solution and/or use of piperazine in place of piperidine.
