Peptide synthesis Merrifield method

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. 

In preparing these various libraries, extensive use is made of solid phase synthetic methods. These methods are all derived from the solid phase peptide synthesis (SPPS) method developed by Merrifield in 1963. When performing a large number of syntheses, it is preferable to perform the synthetic steps on a solid bead rather than completing the entire synthesis in the solution phase. The solid-phase technique makes byproduct removal and final compound purification easier. The organic chemistry literature contains a wealth of different types of solid-phase supports and novel linkers for attaching the synthetic substrate to the bead. 

First we shall consider reactions for traditional chemical synthesis of peptides and then we look at reactions used in automated solid-phase peptide synthesis. The method for solid-phase peptide synthesis was invented by R. B. Merrifield (Rockefeller University), for which he earned the 1984 Nobel Prize in Chemistry. Solid-phase p>eptide synthesis reactions are so reliable that they have been incorporated into machines called peptide synthesizers (Section 24.7D). 

We shall now exemplify the solid-phase peptide synthesis approach by c )c/o-[-L-Val-[)-Pro-D-Val-L-Pro-]], which was prepared by Merrifield himself, the inventor of the method (B.F. Gisin, 1972). 

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... 

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). 

Solid-Phase Peptide Synthesis The Merrifield Method... 

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

Merrifield method See solid-phase 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 ... 

Automated Peptide Synthesis The Merrifield Solid-Phase Method 1037... 

Peptide synthesis requires the use of selective protecting groups. An N-protected amino acid with a free carboxyl group is coupled to an O-protected amino acid with a free amino group in the presence of dicydohexvlcarbodi-imide (DCC). Amide formation occurs, the protecting groups are removed, and the sequence is repeated. Amines are usually protected as their teit-butoxy-carbonyl (Boc) derivatives, and acids are protected as esters. This synthetic sequence is often carried out by the Merrifield solid-phase method, in which the peptide is esterified to an insoluble polymeric support. 

The novel concept of synthesizing a molecule while attached to a swollen cross-linked resin bead was introduced and demonstrated by R. B. Merrifield with the solid-phase peptide synthesis method about 20 years ago (1,2). The procedure involves the covalent attachment of an amino-acid residue to the polymer bead followed by the addition of subsequent amino-acid units in a stepwise manner under conditions that do not disrupt the attachment to the support. At the completion of the assembly of the peptide, the product is cleaved from the resin and recovered. The macro-scopically insoluble support provides convenient containment of the desired product so that isolation and purification from soluble co-products in the synthesis can be achieved by simple... 

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],... 

RB Merrifield, AR Mitchell, JE Clarke. Detection and prevention of urethane acylation during solid-phase peptide synthesis by anhydride methods. J Org Chem 39, 660, 1974. 

JP Tam, RB Merrifield. Strong acid deprotection of synthetic peptides mechanisms and methods, in The Peptides Analysis, Synthesis, Biology, Vol. 9, pp 185-248, Academic Press, New York, 1987. 

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. 

A number of approaches may be adopted to achieve chemical synthesis of a peptide. The Merrifield solid phase synthesis method is perhaps the most widely used. This entails sequential addition of amino acids to a growing peptide chain anchored to the surface of modified polystyrene beads. The modified beads contain reactive chloromethyl (—CH2CI) groups. 

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. 

Solid-phase peptide synthesis does not solve all purification problems, however. Even if every coupling step in the ribonuclease synthesis proceeded in 99% yield, the product would be contaminated with many different peptides containing 123 amino acids, 122 amino acids, and so on. Thus, Merrifield and Gutte s six weeks of synthesis was followed by four months spent in purifying the final product. The technique has since been refined to the point that yields at the 99% level and greater are achieved with current instrumentation, and thousands of peptides and peptide analogs have been prepared by the solid-phase method. 

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 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. 

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). 

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