Merrifield procedure, peptide synthesis

Although the steps just shown can be repeated to add one amino acid at a time to a growing chain, the synthesis of a large peptide by this sequential addition is long and arduous. An immense simplification is possible, however, using the Merrifield solid-phase method. In the Merrifield method, peptide synthesis is carried out with the growing amino acid chain covalently bonded to small beads of a polymer resin rather than in solution. In the original Merrifield procedure, polystyrene resin was used, prepared so that 1 of... 

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... 

The polymer-bound p-nitrobenzophenone oxime (71d) has been found to be a suitable support for stepwise peptide synthesis. Protected peptides can be assembled on 70d by coupling and deprotection steps similar to those employed in the usual Merrifield solid-phase procedures (Scheme 39). Cleavage of peptides from 71d can be accomplished with hydrazine and amino acid esters under mild conditions, which do not affect benzyl ester side-chain protecting groups. 

Figure 3-15 Procedure for solid-phase peptide synthesis devised by Merrifield.299...

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). 

Now we consider an example to illustrate how these procedures are combined in the Merrifield solid-phase peptide synthesis. 

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 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. 

Peptides of 33 amino acid residues in length were synthesized on a Milligen Biosearch 9600 automated peptide synthesizer. The procedures used were based on Merrifield solid phase synthesis utilizing Fmoc-BOP chemistry [38] as described by Mayo et al. [36]. 

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. 

II. Merrifield s "Solid Phase" Method (SPPS) - The isolation and purificaton of each intermediate during the assembly of a peptide chain is of importance in order to limit the presence of by-products in the final product. However, these procedures are not ideally suited to the s thesis of long chain polypeptides in solution, because of the technical difficulties mentioned above. In 1963, Merrifield reported his method of "solid phase peptide synthesis"(SPPS) (4), which simplified the preparation of long peptides and was amenable to fiirtiier acceleration by automation. 

The design and application of new polymer supports for peptide synthesis remains a rapidly developing field. The use of glass beads coated with the polystyrene chloromethylated resin in a packed bed has been studied in detail. With a model tetrap>eptide the procedure was developed to a point where it became ten times faster than the usual Merrifield method but the average yield of each amino acid attachment was about 98.8% and the tetrapeptide... 

Each step from the chemical and practical view has its own problems and therefore will be discussed in detail later on in separate sections. The succession of cycles during a solid phase synthesis characterizes the various stages of the peptide elongation. Since all operations in the Merrifield method are often repeated, the procedure is predestined for automatic peptide synthesis. Consequently the first apparatus for this purpose had already been constructed in the incipience of the methodical development by R. B. Merrifield and J. M. Stuart [39] (see p. 72). 

For that reason, the applicability of the other strategy introduced by R. L. Letsinger and M. J. Kornet in 1963 [42] is in contrast to the Merrifield procedure limited. In the second method the stepwise peptide synthesis starts from the opposite direction, beginning from the N-terminal amino acid bound by its amino function to an insoluble support. The peptide is elongated by activations of the solid phase carboxyl component on each... 

This situation changes considerably if one conducts even an extended stepwise peptide synthesis on solid phase with small peptide units for building blocks instead of amino acid derivatives. In this way, the number of stages to synthesize a polypeptide is diminished to a fraction of those necessary in the original Merrifield procedure, with all the advantageous consequences of a limited statistical expansion of failures along the synthesis run. 

Besides this fundamental reason one has to realize that the result of even a most accurate multistep synthesis on polymer can be questioned by the chemical reaction necessary to release the final product from its support. Parts of the target sequence can be destroyed and desired protecting groups on side functions may be cleaved. These facts furthermore multiply the possibility for contaminations. Therefore, the overall success of any Merrifield peptide synthesis is inherently related also to the quality of isolation and purification procedures (see for example [168]). 

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