Merrifield solid phase synthesis method

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. 

Of the three above-mentioned major classes of natural biopolymers, chemical synthesis of nucleic adds (in particular DNA) and proteins had already been achieved by the Merrifield solid-phase synthesis method a computer-controlled automated synthesizer became commerdally available more than 20 years ago. However, the chemical synthesis of polysaccharides like cellulose was far more difficult than that of the former two classes. Polysaccharide synthesis is the repetition of glycosylation, the most fimdamental reaction in carbohydrate chemistry (Scheme 2). 

Probtom 21.30 Describe the Merrifield solid-phase synthesis when carried out in the reverse order of the classical Sanger and Edman methods. <... 

Rapid Synthetic Procedures. The most interesting development of the year v/as the skillful use of a-amino acid N-carboxyanhydrides in a rapid synthesis in aqueous medi mi32. This procedure is much faster than the Merrifield "solid phase" synthesis, vmdoubtedly more econoraical, and probably easier to adapt to larger scale syntheses. It appears that more byproducts are formed, but both methods require chroniatographic or more elaborate purification of the final products. 

The method bears many similarities to the Merrifield solid-phase synthesis of peptides. A starter unit is attached to a solid support, nucleosides are attached one-by-one until the sequence is complete, whereupon the target oligonucleotide is removed from the support and purified. 

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. 

Preparation of Merrifield resin-bound nitro acetates, which is a suitable building block for the development of combinatorial solid phase synthesis, is reported.4 The anion of ethyl nitro acetate is generated in DMF by an electrochemical method using Pt cathode, magnesium rod anode, and tetrabutylammonium bromide as an electrolyte. Alkylaton of this anion with alkyl halides gives mono-alkylated products in 80% yield.5... 

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],... 

A major breakthrough in solid-phase synthesis (SPPS) was provided by Merrifield in 1962. The method was based on a stepwise strategy and involved the following ... 

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. 

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 a more recent study, Westman and Lundin described the solid-phase synthesis of aminopropenones and aminopropenoates, respectively30 as intermediates for heterocyclic synthesis. Two different three-step methods for the preparation of heterocycles have been developed. The first method involved formation of a polymer-bound ester from a IV-protected glycine derivative and Merrifield resin (Scheme 7.10a), while the second method employed an interesting approach utilising simple aqueous methy-lamine solution for functionalisation of the solid support (Scheme 7.10b). In this latter approach, a variety of hetero cycles were readily synthesised from the generated polymer-bound benzylamine using a two-step protocol (see Section 5.3.3). 

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). 

The split-mix method was developed for preparing peptide libraries. The method is based on Merrifield s solid phase procedure, published in 1963 [8], Each coupling cycle of the solid phase synthesis is replaced by the following simple operations ... 

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. 

Show a synthesis of Ala-Phe-Gly-Gly using the Merrifield solid phase method. 

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 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). 

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