Merrifield method, of peptide synthesis

A huge advantage of the Merrifield method of peptide synthesis is that the growing peptide can be purified by washing the column with an appropriate solvent after each step of the procedure. The impurities are washed out of the column because they are not attached to the solid support. Since the peptide is covalently attached to the resin, none of it is lost in the purification step, leading to high yields of purified product. 

Books and Review Articles - A number of book and review articles have appeared during the last year. Advances in the chemical synthesis of peptides were reviewed by Hardy, Katsoyannis and YoungJ. The description of the experimental details for the Merrifield method of peptide synthesis has been published by Stewart. It is also noted that the tentative rules for the abbreviated nomenclature of synthetic peptides have also been published. 

Merrifield was awarded the 1984 Nobel Prize in chemistry for developing the solid-phase method of peptide synthesis. 

Describe the synthesis of the dipeptide Lys-Ala by Merrifield s solid phase chemical method of peptide synthesis. What pitfalls might be encountered if yon attempted to add a leucine residue to Lys-Ala to make a tripeptide ... 

The breakthrough in peptide chemistry, which opened up applications in biochemistry and molecular biology, was the development of solid phase synthesis by Merrifield in 1963. This formed the basis of automated synthetic procedures in which the nascent peptide chain was covalently linked to a solid support such as a styrene-divinylbenzene copolymer the complex isolation and purification procedures needed to separate reactants and products at the end of each reaction cycle, which characterised previous solution methods of peptide synthesis, were replaced by a simple washing step. With modern automated methods of peptide synthesis, the time for an Fmoc reaction cycle has been reduced to 20 min, so that a 50-residue peptide can be synthesised in a day (Chan and White 2000). 

Merrifield introduced his solid-phase peptide synthesis (SPPS) methodology in 1963 t l this has since become the preferred method of peptide synthesis. Two decades later, Fur-kaP5,56] extended Merrifield s techniques to the synthesis of libraries of peptides used for the screening of new, desirable pharmaceutical products. Because peptides do not necessarily represent the ideal candidates for therapeutics, more recent attention has focused on other libraries of compounds that can be prepared by solid-phase multistep procedures. Among the approaches used for such purposes, the HO-3CR and the U-4CR, and their combination with other reactions, are finding increasing applications. 

Modern methods of peptide synthesis began with the solid-phase method introduced by Merrifield in 1962 (Fig. 3-15). To begin the synthesis a suitably protected amino acid is covalently linked to a polystyrene bead. The blocking f-butoxycarbonyl (Boc) group is removed as isobutene by an elimination reaction to give a bound amino acid with a free amino group. 

In addition to producing low overall yields, the method of peptide synthesis described in Section 23.9 is extremely time-consuming because the product must be purified at each step of the synthesis. In 1969, Bruce Merrifield described a method that revolutionized the synthesis of peptides because it provided a much faster way to produce peptides in much higher yields. Furthermore, because it is automated, the synthesis requires fewer hours of direct attention. Using this technique, bradykinin was synthesized with an 85% yield in 27 hours. Subsequent refinements in the technique now allow a reasonable yield of a peptide containing 100 amino acids to be synthesized in four days. 

M. Bodansky, Peptide Synthesis, 2nd ed., John Wiley Sons, Inc., New York, 1976 J. Meinhofet in Ref. 1, Chapt. 9, p. 297 G. R Pettit, Synthetic Peptides, Vols. 1—4, Van Nostrand Reinhold, New York, 1980, Vols. 5, 6, Elsevier New York, 1982 E. Shtoedet and K. Luehke, The Peptide, Vol. 1, Methods of Peptide Synthesis, Academic Press, New York, 1965 N. Izumiya and co-workers. Fundamentals andTLxpenments of Peptide Synthesis (in Japanese), Mamzen, Tokyo, Japan, 1987 R. B. Merrifield,/. Am. Chem. Soc. 85, 2149 (1963) G. Batany and R B. Merrifield in E. Gross and J. Meinenhofer, eds.. The Peptides Analysis, Synthesis, Biology, Vol. 2, Academic Press, New York, 1980, pp. 1—284 G. R Marshall, Peptides Chemistry and Biology, Escom, Leiden, The Nethedands, 1988. 

One of the most developed methods used in combinatorial chemistry libraries preparation is solid-phase organic synthesis (SPOS) based on the Merrifield method for peptide synthesis [128]. A great number of such libraries have been prepared on a solid support, generally a functionalized polystyrene resin cross-linked with a small amount of divinylbenzene. Recently, it was demonstrated that micro-wave irradiation can be applied to solid-phase immobilized reagents to reduce significantly the reaction time. Those readers who are interested in such processes we would like to refer to more extensive reviews published by Chamberlin et al. [129] and Kappe [130], while in this chapter we are giving most common examples. 

In 1963, R. B. Merrifield reported an unconventional method of peptide synthesis for which the name solid-phase peptide synthesis was coined. This method differed from general organic synthetic methods in that one of the reactants was reversibly and covalently bound to an insoluble, solid polymer support which was then reacted with the reagent to give a resin-bound product. After filtering the latter from the reaction mixture and after repetition of as many steps as necessary to achieve the synthesis, the product was obtained by a suitable cleavage reaction (Merrifield, 1963 Marshall and Merrifield, 1965). It may be recalled that, in... 

The choice of chloromethylated polystyrene as the resin support to bind the amino acid substrate by benzyl ester is amply justified on the basis of the frequent use of benzyl esters of amino acids in solution and methods of peptide synthesis (Greenstein and Winitz, 1961 Schroder and Lubke, 1965 Bodanszky and Ondetti, 1966). Such a bond is stable during the various reactions of peptide synthesis but is readily cleaved by anhydrous hydrogen bromide at the end of synthesis. Details for the improved synthesis of chloromethylated co(polystyrene-DVB) have been reported (Feinberg and Merrifield, 1974 see also Chapter 2). Both microporous (swellable) and macroreticular or macroporous polymers have been examined as support material (Tilak and Hollinden, 1971 Sano et aL, 1971), but in most synthetic reactions the swellable resins have an edge over the nonswellable resins. 

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). 

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],... 

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. 

Merrifield s concept of a solid-phase method for peptide synthesis and his development of methods for carrying it out set the stage for an entirely new way to do chemical reactions. Solid-phase synthesis has been extended to include numerous other classes of compounds and has helped spawn a whole new field called combinatorial chemistry. Combinatorial synthesis allows a chemist, using solid-phase techniques, to prepare hundreds of related compounds (called libraries) at a time. It is one of the most active areas of organic synthesis, especially in the pharmaceutical industry. 

In the Merrifield method, the C-terminal amino acid is covalently attached to a solid support contained in a column. Each N-terminal blocked amino acid is added one at a time, along with other needed reagents, so the protein is synthesized from the C-terminal end to the N-terminal end. Notice that this is opposite to the way proteins are synthesized in nature (from the N-terminal end to the C-terminal end Section 27.13). Because it uses a solid support and is automated, Merrifield s method of protein synthesis is called automated solid-phase peptide synthesis. 

The concept of peptide synthesis on a solid support known as solid phase peptide synthesis was developed in 1963 (Merrifield, 1963) and the concept has been extended and generalized to organic synthesis on polymeric supports. The method is particularly attractive to the synthesis of biomacromolecules (Bayer, 1991 Fields, 1997 Kates and Albericio, 2000 Merrifield 1985). 

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