Polypeptides Merrifield solid-phase peptide

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 major disadvantage of solid-phase peptide synthesis is the fact that all the by-products attached to the resin can only be removed at the final stages of synthesis. Another problem is the relatively low local concentration of peptide which can be obtained on the polymer, and this limits the turnover of all other educts. Preparation of large quantities (> 1 g) is therefore difficult. Thirdly, the racemization-safe methods for acid activation, e.g. with azides, are too mild (= slow) for solid-phase synthesis. For these reasons the convenient Merrifield procedures are quite generally used for syntheses of small peptides, whereas for larger polypeptides many research groups adhere to classic solution methods and purification after each condensation step (F.M. Finn, 1976). 

Peptide synthesis has once again stepped into the limelight in the last 10 years with the discovery of neuropeptides, peptides of the renin-angiotensin system, and immuno-active peptides. Above all, the solid-phase peptide synthesis of R. B. Merrifield, besides conventional peptide syntheses, has made an inestimable contribution to the production of biological and medically important polypeptides. 

Without doubt, the solid-phase peptide synthesis (Merrifield method) remains a preferred method for controlling all five critical molecular design parameters (size, shape, topology, flexibility, and surface chemistry) by precisely producing amino-acid sequences in a stepwise fashion. The scope and limitations of this approach have been reviewed [34] and widely recognized [35]. These solid-phase syntheses with protection/deprotection procedures are used routinely to produce numerous, previously unattainable [36], polypeptides and polynucleotides. One of the ultimate synthetic efforts in the control of CMDPs was the total synthesis by Khorana et al. [37] of a DNA molecule in the 1960s. 

Despite the achievements of polypeptide synthesis in solution, currently at the research level, most polypeptides are synthesized by solid phase peptide synthesis (SPPS) [5, 6]. This approach, pioneered by Bruce Merrifield revolutionized the synthesis of peptides and the principles have been applied... 

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. 

The method described in Section 28.6 works well for the synthesis of small peptides. It is extremely time-consuming to synthesize larger proteins by this strategy, however, because each step requires isolation and purification of the product. The synthesis of larger polypeptides is usually accomplished by using the solid phase technique originally developed by R. Bruce Merrifield of Roekefeller University. 

Berg, R.H., Almdal, K., Watsberg Pedersen, W., Holm, A., Tam, J.P., and Merrifield, R.B. In Innovation and Perspectives in Solid Phase Synthesis Peptides, Polypeptides and Oligonucleotides, Macro-Organic Reagents and Catalysts-1990. Epton, R. (ed.) SPCC Birmingham, pp. 453-459 (1990). 

In spite of these murky aspects, which concern the adventures in the field of syntheses of polypeptides and proteins on solid phase by incremental chain elongation, the method of Merrifield is invaluable in the quick and convenient preparation of smaller peptides... 

This situation changes considerably if one conducts even an extended stepwise peptide synthesis on solid phase with small peptide units for building blocks instead of amino acid derivatives. In this way, the number of stages to synthesize a polypeptide is diminished to a fraction of those necessary in the original Merrifield procedure, with all the advantageous consequences of a limited statistical expansion of failures along the synthesis run. 

It was left to the ingenuity of R. Bruce Merrifield, an organic chemist at the Rockefeller University, to develop an innovative solution to this problem that totally revolutionized the field of peptide synthesis. His plan was to assemble the peptide chain in a stepwise manner by adding new amino acids at the A terminus while the Oterminal end was attached to a solid polymeric support of chloromethylated polystyrene, which is now referred to as the Merrifield. In this fashion, all of the excess reagents, impurities, and by-products could be easily removed by washing the resin after each operation, and the pure polypeptide could be cleaved from the solid support as the last step in the synthesis. Merrifield was awarded the Nobel Prize in Chemistry in 1984 in recognition of his contributions to the invention and development of the solid-phase method for the synthesis of peptides. 

Merrifield s revolutionary concept of solid-phase synthesis was not limited to peptides, and similar techniques have been developed for the synthesis of nucleic acids and carbohydrates on solid supports. For each application, specialized instrumentation that is computer-controlled is commercially available. Access to such equipment has enabled researchers in areas of biology, medicine, material science, and biomedical engineering to prepare thousands of peptides and polypeptides for study. In the pharmaceutical industry, for example, solid-phase synthesis has been used to prepare relatively large numbers of related molecules, so-called compound libraries, that... 

A major advance in polypeptide synthesis came in 1962 when R. Bruce Merrifield of the Rockefeller University described a solid-phase synthesis (alternatively called polymer-supported synthesis) of the tetrapeptide, Leu-Ala-Gly-Ala, by a technique that now bears his name. Merrifield was awarded the 1984 Nobel Prize in Chemistry for his work in developing the solid-phase method for peptide synthesis. 

In the Merrifield method, the C-terminal amino acid is joined as a benzyl ester to the solid polymer support and then the polypeptide chain is extended one amino acid at a time from the N-terminal end. The advantage of polypeptide synthesis on a solid support is that the polymer beads with the peptide chains anchored on them are completely insoluble in the solvents used in the synthesis. Furthermore, excess reagents (e.g., DCC) and by-products (e.g., DCU) are removed after each step simply by washing the polymer beads. When synthesis is completed, the polypeptide is released from the polymer beads by cleavage of the benzyl ester. The steps in solid-phase synthesis of a polypeptide are summarized in Figure 27.11. 

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