Automated polypeptide synthesizer

Commercially available computer-automated peptide synthesizers allow synthesis of a polypeptide in a time of about one hour or less per amino acid unit. The ribonuclease synthesis takes about one week or less. 

In addition to the above-mentioned, rather painstaking, techniques of polypeptide syntheses, a very elegant technique was developed by Merrifield. This solid-phase peptide synthesis automates the reaction sequences. The method makes use of an insoluble crosslinked polymer substrate with pendant reactive groups for attachment of peptide chains. Chloromethylated polystyrene microgels are often used (see Chapter 8 for more discussions on the use of chloromethylated polystyrene for reactions of polymers). The chloromethyl moieties serve as the initiating sites for formation of the polypeptides ... 

One widely used method of formation of protected compounds involves polymer-supported reagents, with the advantage of simple workup by filtration and automated syntheses, especially of polypeptides. Polymer-supported reagents are used to protect a terminal — COOH group as a polymer-bound ester (RCOOR —( ) during peptide syntheses, to protect primary alcohols as... 

One widely used method involving protected compounds is solid-phase synthesis (polymer-supponed reagents). This method has the advantage of requiring only a simple workup by filtration such as in automated syntheses, especially of polypeptides, oligonucleotides, and oligosaccharides. 

The Merrifield method has now been completely automated, so it is possible to purchase peptide synthesizers that automatically carry out all of the above operations and form polypeptides in high yield in a matter of hours, days, or weeks, depending on the length of the chain of the desired product. The instrument is pictured in Figure 28.8. For example, the protein ribonucle-ase, which contains 128 amino acids, has been prepared by this technique in an overall yield of 17%. This remarkable synthesis involved 369 separate reactions, and thus the yield of each individual reaction was > 99%. 

Combinatorial chemistry is a sophisticated set of techniques used to synthesize, pnrify, analyze, and screen large numbers of chemical compounds, far faster and cheaper than was previously possible. The direct precursor of combinatorial chemistry was the solid-phase synthesis of polypeptides developed by American biochemist Robert Bruce Merrifield in the 1960s, followed by the advances in laboratory automation since then. Initial development of the field has been led by the pharmaceutical industry in the search for new drugs, but its applications are spreading into other fields of chemistry. Other terms associated with this field are parallel array synthesis and high-throughput chemistry. 

Merrifield used this procedure to prepare a number of peptides. For example, he synthesized the nonapeptide bradykinin in 68% yield in only eight days, a remarkable feat at the time.The biological activity of the synthetic peptide was identical with that of the natural peptide. Merrifield was ultimately able to automate all the steps in his technique for solid-phase peptide synthesis and demonstrated its power by using a homemade machine to prepare bovine pancreatic ribonuclease, an enzyme that contains 124 amino acids. This synthesis proceeded in 17% overall yield and required 369 chemical reactions and 11,931 individual operations The synthetic ribonuclease had a specific activity that was 13-24% that of the native enzyme. The lower activity of the synthetic enzyme can probably be attributed to the fact that each coupling step did not proceed with 100% efficiency, so some polypeptides lacking one or more individual amino acids in the sequence were also produced. Because of their close similarity to ribonuclease, it was not possible to separate these polypeptides from the synthetic enzyme. 

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