Polypeptides automated peptide synthesis

The preparation of polypeptides and proteins using automated peptide synthesis—the Merrifield method (Section 28,7) The structure of spider silk (Section 28.8B)... 

A variety of chromophores can be introduced as nonnatural amino acids into polypeptide chains, in any order, using conventional or automated peptide synthesis techniques. 

Many different insoluble polymers, called resins, are currently available for automated peptide synthesis. For example, the Wang resin contains benzene rings substituted with -CH2OH groups that serve as sites of attachment for amino acids. Propose reaction conditions that would bind an Fmoc-protected amino acid to a Wang resin. What reaction conditions could be used to remove the polypeptide from the resin after the synthesis is complete ... 

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. 

II. Merrifield s "Solid Phase" Method (SPPS) - The isolation and purificaton of each intermediate during the assembly of a peptide chain is of importance in order to limit the presence of by-products in the final product. However, these procedures are not ideally suited to the s thesis of long chain polypeptides in solution, because of the technical difficulties mentioned above. In 1963, Merrifield reported his method of "solid phase peptide synthesis"(SPPS) (4), which simplified the preparation of long peptides and was amenable to fiirtiier acceleration by automation. 

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 ... 

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. 

Thanks to automation, the synthesis of polypeptides is now a routine procedure in chemical research. It is common for researchers to order several peptides at a time for use in fields as diverse as medicine, biology, material science, and biomedical engineering. 

Memfield first introduced the concept of solid-phase peptide synthesis nearly half a century ago, and since then the use of heterogeneous materials to facilitate synthesis has evolved and become widespread in many contexts. For example, the automated solid-phase synthesis of oligomeric biomolecules, such as polypeptides and polynucleotides, has become the standard methodology for the production of such compounds. 

Polypeptide synthesis has been automated. This ingenious method, known as the Merrifield solid-phase peptide synthesis, uses a solid support of polystyrene to anchor a peptide chain. 

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%. 

Chemistry for the Automated C-Terminal Sequence Analysis of Proline Containing Polypeptides. Application of the acetic anhydride/TMS-ITC/TFA procedure, used for the synthesis of TH-proline, to the tripeptide, N-acetyl-Ala-Phe-Pro, in our laboratory, found that thiohydantoin proline was formed in low yield (approx. 1-2% of theoretical). Recovery of the peptide products after the reaction revealed that approximately half of the starting peptide was unchanged and the remaining hdf had been decarboxylated at the C-terminus, thereby blocking it to C-terminal sequence analysis. This was most likely caused by the high concentration of trifluoroacetic acid, the excess of acetic anhydride present, and the high temperature (80°C) at which the reaction was performed. 

One common example of repetitive synthetic strategy comes from the field of natural products. The preparation of oligo- and polypeptides is a repeating sequence of protection and activation of the reactants, followed by the addition of a new amino acid. In 1963, one of the highlights in repetitive synthesis was the automation of peptide production by Merrifield8 (Figure 1). In the literature, authors often speak of a protein machine since it is now possible to build up peptides mechanically without the preparative help of a chemist. 

Polypeptide synthesis on insoluble polymer supports was pioneered by R. B. Merrijield [108]. This method could be automated and has facilitated protein synthesis enormously [109]. Chemical ligation of even unprotected peptide segments has recently been reported [110],... 

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