Merrifield polypeptide synthesis

T Wieland, C Birr, H Wissenbach. 3-Nitrophthalic anhydride as blocking agent in Merrifield polypeptide synthesis. Angew Chem 8, 764, 1969. 

The most widely nsed method to construct polypeptides is the use of a polymeric substrate to achieve multiple reactions. The Merrifield polypeptide synthesis on a polymeric substrate (crosslinked polystyrene) as the solid phase, revolutionized the synthesis of sequential polypeptides. The initial attachment of a protected amino acid to chloromethyl group containing polystyrene is achieved with triethylamine followed by hydrolysis to give 679 (Scheme 2.338, Step A). After removal of the protecting group, coupling of... 

Other authors concluded that the length of the spacer chain was irrelevant from the point of view of catalytic activity, but these statements were based on the comparison of reactions carried on under different conditions. Long-spacer chains exhibit a similar positive effect in the classical Merrifield polypeptide synthesis... 

Polypeptide Synthesis and Analysis. Sihca or controUed-pore glass supports treated with (chloromethyl)phenylethyltrimethoxysilane [68128-25-6] or its derivatives are replacing chloromethylated styrene—divinylbenzene (Merrifield resin) as supports in polypeptide synthesis. The sdylated support reacts with the triethyl ammonium salt of a protected amino acid. Once the initial amino acid residue has been coupled to the support, a variety of peptide synthesis methods can be used (34). At the completion of synthesis, the anchored peptide is separated from the support with hydrogen bromide in acetic acid (see Protein engineering Proteins). 

As is apparent from this example, the synthesis of a polypeptide requires numerous steps. At each step, the product must be isolated, and if you have worked in an organic chemistry laboratory, you are certainly aware of how much time and energy are required to isolate and purify a product. This makes a polypeptide synthesis quite tedious. In addition, the mechanical losses that occur in each isolation step contribute to lower yields for the overall process. Motivated by these problems, R. B. Merrifield developed a method, called solid phase synthesis, that makes the preparation of a polypeptide much easier. Merrifield was awarded the 1984 Nobel Prize in chemistry for this work. Let s see how it works. 

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... 

Many different support materials were developed [3, 4] since the original use by Merrifield of a polystyrene-based support material for polypeptide synthesis [5]. The work of Merrifield is described in Chap. 8 (see section on proteins). Beads of copolymers of styrene with divinyl benzene are available commercially and have been widely used as supports for many reactions. Many other polymeric materials are also used. These can be various other type of copolymers of styrene or with other polymers. The list includes cellulose, starch, polyalkanes, polyamides, poly(glycidyl methacrylate), polyisobutylene, polynorbomene, polyacrylamide, and others. In some instances, even glass was used. The more prominent support materials are presented below. 

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. 

Another example of the increased reactivity of carboxylate ions is the catalysis by 18-crown-6 (28) of the loading of A -t-butyloxycarbonyl amino acid salts onto chloromethylated polystyrene (Equation 12) for Merrifield-type polypeptide synthesis. However, a less dramatic acceleration than expected was observed, possibly because of steric effects of the polymer backbone. Naked fluoride ion is another unusually strong nucleophile, but its enhanced basic character, although useful in some cases, may induce competing eliminations. ... 

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. 

Polypeptide synthesis requires end-protected amino acids that are coupled by dicyclohexyl-carbodiimide. The product can be selectively deprotected at either end to allow for fvuther extension of the chain. The use of solid supports, as in the Merrifield synthesis, can be automated. 

In more recent years new developments in synthesis have advanced the synthetic technology to a highly efficient level. In particular, synthesis on polymer supports, a concept first developed by Merrifield in polypeptide synthesis, has resulted in enormous improvements and speed in synthesis of large size polynucleotides. 

In the synthesis of polypeptides with biological activity on a crosslinked polymer support as pioneered by Merrifield (1 2) a strict control of the amino acid sequence requires that each of the consecutive reactions should go virtually to completion. Thus, for the preparation of a polypeptide with 60 amino acid residues, even an average conversion of 99% would contaminate the product with an unacceptable amount of "defect chains". Yet, it has been observed (13) that with a large excess of an amino acid reagent —Tn the solution reacting with a polymer-bound polypeptide, the reaction kinetics deviate significantly from the expected exponential approach to quantitative conversion, indicating that the reactive sites on the polymer are not equally reactive. 

Prize in 1963 for inventing a new general method to synthesize important polymers, a method that uncovered much new basic science. A Nobel Prize in 1984 went to Robert Bruce Merrifield for his invention of a general approach to the synthesis of polypeptides and proteins, in a style directly reminiscent of the biological method used in such synthesis. 

Graft copolymers of nylon, protein, cellulose, starch, copolymers, or vinyl alcohol have been prepared by the reaction of ethylene oxide with these polymers. Graft copolymers are also produced when styrene is polymerized by Lewis acids in the presence of poly-p-methoxystyrene. The Merrifield synthesis of polypeptides is also based on graft copolymers formed from chloromethaylated PS. Thus, the variety of graft copolymers is great. 

The Merrifield synthesis is not without limitations since anything less than quantitative conversion in each chemical step with the complete absence of side reactions and the complete removal of reagents prior to the next chemical step would yield an impure polypeptide compared to the naturally occurring biological macromolecule. Thus, for the synthesis of a... 

Likewise, in the preparation of many ion-exchange resins, suitable functional groups are introduced by secondary reactions of macromolecular substances (that are generally crosslinked see Sect. 5.2). In this context the utilization of crosslinked polystyrene resins or poly(acrylamide) gel in the solid-phase synthesis of polypeptides (Merrifield technique) or even oligonucleotides should be mentioned. After complete preparation of the desired products they are cleaved from the crosslinked substrate and can be isolated. 

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). 

Tam has pioneered the use of dendrimers as templates for simultaneous growth of identical polypeptides [145]. Tam et al. were also the first to use the Merrifield approach to the synthesis of dendrimers [145-148]. Starting from phenylaceta-... 

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