Merrifield method

SOLID-PHASE PEPTIDE SYNTHESIS THE MERRIFIELD METHOD... 

Merrifield method See solid phase peptide synthesis Meso stereoisomer (Section 7 11) An achiral molecule that has chirality centers The most common kind of meso com pound IS a molecule with two chirality centers and a plane of symmetry... 

Solid-Phase Peptide Synthesis The Merrifield Method... 

What protected amino acid would you anchor to the solid support in the first step of a synthesis of oxytocin (see Figure 27.8) by the Merrifield method ... 

Merrifield method See solid-phase peptide synthesis. 

One of the cornerstones of combinatorial synthesis has been the development of solid-phase organic synthesis (SPOS) based on the original Merrifield method for peptide preparation [19]. Because transformations on insoluble polymer supports should enable chemical reactions to be driven to completion and enable simple product purification by filtration, combinatorial chemistry has been primarily performed by SPOS [19-23], Nonetheless, solid-phase synthesis has several shortcomings, because of the nature of heterogeneous reaction conditions. Nonlinear kinetic behavior, slow reaction, solvation problems, and degradation of the polymer support, because of the long reactions, are some of the problems typically experienced in SPOS. It is, therefore, not surprising that the first applications of microwave-assisted solid-phase synthesis were reported as early 1992 [24],... 

The Merrifield method has a number of attractive characteristics beyond the simple steps outlined here. For example, because the product molecule (such as the monomer, dimer, or trimer) is attached to a solid support, a chemist can apply other operations to the system without fear of losing that product. The reaction system can be washed at almost any point. This property is very useful, for example, because it allows the chemist to remove excess reactant or undesired by-products of the reaction. 

R. B. Merrifield, Methods. Enzymol. 289 3 (1997) K. B. Merrifield, in Peptides Synthesis, Structure, and Applications, B. Gutte, ed., Academic Press, San Diego, 1995, p. 93 Atherton and R. C. Sheppard, Solid Phase Peptide Synthesis, IRL Press, Oxford, U.K., 1989 P. Lloyd-Williams, F. Albericio, and E. Giralt, Chemical Synthesis of Peptides and Proteins, CRC Press, Boca Raton, Florida, 1997. 

Show by equations how the Merrifield method can be used to synthesize the tripeptide glycylvalylphenylalanine (Gly Val Phe)... 

The elongation step under a simulated environmental pressure is possible, as a never-born protein with 43 residues was obtained, although not by catalytic fragment condensation but by the Merrifield method (Chessari et al, unpublished data). 

Formation of an amide bond (peptide bond) will take place if an amine and not an alcohol attacks the acyl enzyme. If an amino acid (acid protected) is used, reactions can be continued to form oligo peptides. If an ester is used the process will be a kinetically controlled aminolysis. If an amino acid (amino protected) is used it will be reversed hydrolysis and if it is a protected amide or peptide it will be transpeptidation. Both of the latter methods are thermodynamically controlled. However, synthesis of peptides using biocatalytic methods (esterase, lipase or protease) is only of limited importance for two reasons. Synthesis by either of the above mentioned biocatalytic methods will take place in low water media and low solubility of peptides with more than 2-3 amino acids limits their value. Secondly, there are well developed non-biocatalytic methods for peptide synthesis. For small quantities the automated Merrifield method works well. 

Section 27.18 In the Merrifield method the carboxyl group of an amino acid is anchored to a solid support and the chain extended one amino acid at a time. When all the amino acid residues have been added, the polypeptide is removed from the solid support. 

In the synthesis of the enzyme ribonuclease by the Merrifield method, the 124 amino acids were arranged in the ribonuclease sequence through 369 reactions and some 12,000 individual operations of the automated peptide-synthesis machine without isolation of any intermediates. 

It is the C-terminal amino acid that is anchored to the solid support in the preparation of peptides by the Merrifield method. Refer to the structure of oxytocin in Figure 27.8 of the text and note that oxytocin, in fact, has no free carboxyl groups all the acyl groups of oxytocin appear as amide functions. Thus, the carboxyl terminus of oxytocin has been modified by conversion to an amide. 

The next stage is to link the carboxyl group of the second amino acid on to the amino group of the first. The Boc group (Chapter 24) is usually used for amino group protection in the Merrifield method and DCC (dicyclohcxylcarbodiimide) is used to activate the new amino acid. Here is a summary of this step, using symbols again for polymer and spacer. 

This process is routinely automated in commercially available machines. Solutions of all of the protected amino acids required are stored in separate containers and a programmed sequence of coupling and deprotection leads rapidly to the complete peptide in days rather than the years needed for solution chemistry. The most dramatic illustration of this came with the publication of a heroic traditional synthesis of bovine pancreatic ribonuclease A (an enzyme with 124 amino acids) by Hirschmann, side-by-side with one by Merrifield using functionalized polystyrene as we have described. The traditional method required 22 co-workers, while the Merrifield method needed only one. 

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. 

The physicochemical incompatibility of the polystyrene supports with the peptides has been observed to be one of the problems associated with the original Merrifield method. This has been approached successfully by Sheppard and coworkers by the introduction of the polar polyacrylamide supports. Poly(JV-acrylylpyrrolidine) resin is another polar polymeric support developed on similar grounds. The use of these polar resin supports facilitated the solid phase synthesis of a number of peptides and protein sequences in higher yield and purity than with the polystyrene resins. However, the inadequate availability of these polar supports appears to be a limiting factor for a wider utilization at present. 

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

In the Merrifield method an amino acid is attached to an insoluble polymer. Amino acids are sequentially added, one at a time, thereby forming successive peptide bonds. Because impurities and by-products are not attached to the polymer chain, they are removed simply by washing them away with a solvent at each stage of the synthesis. 

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%. 

Write out the steps for the synthesis of each peptide using the Merrifield method (a) Ala-Leu-Phe-Phe (b) Phe-Gly-Ala-Ile. 

Manning M. Synthesis by the Merrifield method of a protected nonapeptide amide with the amino acid sequence of oxytocin. J. 

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