Optimizing for Speed
Automated peptide synthesizers can enable high-throughput, scalable production of high purity peptides for research or clinical applications.
“The speed of automated peptide synthesis will depend both on the optimization of reaction parameters and the throughput of the instrument used,” says James Cain, Ph.D., applications manager at Protein Technologies.
Variation of factors such as coupling and deprotection reagents, reagent excesses and concentrations, solvent selection, resin types, and resin loading make it possible to obtain high purity products with relatively short total reaction times, Dr. Cain explains.
Highly active coupling reagents—such as HATU and HCTU, for example—may not be well-suited for use on some robotic multiple peptide synthesizers due to long reagent dispensing times.
Dr. Cain points to human b-amyloid (1-42) as an example of a peptide that is difficult to synthesize and is of research and commercial interest. Its synthesis is difficult due to the high hydrophobicity of the C-terminal segment and tendency for on-resin aggregation.
Traditional approaches for making this peptide might require 50 to 60 hours of synthesis time, according to Dr. Cain. By optimizing a number of variables, it is possible to synthesize 24 human b-amyloid (1-42) peptides on the company’s Symphony X™ automated synthesizer in less than 14 hours using short reaction times and the instrument’s fluid delivery system.
In this example, the variables modified included use of low-loaded resin, a modified deprotection mixture containing 2% DBU added to the standard 20% piperidine in DMF, and HCTU as coupling reagent. Further efficiency is achieved through simultaneous addition of reagents to multiple vessels and washing of valve blocks and other components at the same time.
The Symphony’s IntelliSynth UV monitoring system, which captures UV readings every 10 seconds during the deprotection reaction, is particularly helpful for difficult sequences, such as the poly-alanine (Ala)10K, which commonly exhibits severe aggregation after the addition of the fifth residue, according to Dr. Cain.
Under standard deprotection conditions, longer deprotection times and more repetitions were required for full removal of the Fmoc protecting group after the onset of aggregation. More efficient removal could be achieved by adding 2% DBU to the deprotection mixture.
Infrared heating can also be incorporated into the design of the Symphony X. It has been shown to speed the synthesis of certain difficult sequences, such as an analog of ACP(65-74), in which the alanines have been replaced with sterically hindered aminoisobutyric acid (Aib) residues.
According to Dr. Cain, “While very pure product can be obtained using long coupling times at room temperature, the addition of IR heating allows for a ninefold reduction in the coupling time for the most difficult steps.”