Peptide solid phase technique

Memfield s concept of a solid phase method for peptide synthesis and his devel opment of methods for carrying it out set the stage for an entirely new way to do chem ical 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 hun dreds of related compounds (called libraries) at a time It is one of the most active areas of organic synthesis especially m the pharmaceutical industry... 

In the case of (Ala-Gly-Pro)n with n = 5-15, the tripeptide chains were synthesized by the liquid-solid phase technique. As mentioned above, the coupling of longer preformed peptide chains was difficult and the yield of the trimer was low. Therefore, a liquid-solid phase technique was applied in which a trimer was grown in a stepwise manner, beginning from a trifunctional crosslinked base A. 

Although the solid-phase technique was first developed for the synthesis of peptide chains and has seen considerable use for this purpose, it has also been used to synthesize chains of polysaccharides and polynucleotides in the latter case, solid-phase synthesis has almost completely replaced synthesis in solution. The technique has been applied less often to reactions in which only two molecules are brought together (nonrepetitive syntheses), but many examples have been reported. 

Peptide synthesis was amenable to solid-phase techniques since the process was repetitive. The C-terminal amino acid is attached to polymeric surface and the peptide chain is assembled via a two-step process coupling of the incoming amino acid that has the alpha-amino group protected... 

In general, solid-phase synthesis, rather than solution-phase synthesis, can be the preferred method for the generation of combinatorial libraries because of the greater abihty to automate a solid-phase protocol, primarily due to the use of excess reagents in solution to effect cleaner reactions and to the ease of workup by simple filtration. The solid-phase method of peptide synthesis has had many notable successes. However, the preparation of peptides containing more than 20 amino acids in length using the solid-phase technique often causes major problems in that very extensive purification of the final product is needed. 

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. 

Kato and Anfinsen 245) have reported on the use of agarose-bound S-protein in the form of a column for the purification of synthetic S-peptide derivatives. This procedure has shown the presence of closely related products in material from the Merrifield solid phase techniques. Some of these products produced enzymically inactive complexes with S-protein. 

Different solid-phase techniques for the synthesis of C-terminal peptide aldehydes have gained much attention and allowed greater accessibility to such compounds. Solid-phase techniques have been used to synthesize peptide aldehydes from semicarbazones, Weinreb amides, phenyl esters, acetals, and a, 3-unsaturated y-amino acids)47-50,60 63 The examples presented below use unique linkers to enhance the automated efficiency of C-terminal peptide aldehyde synthesis)47 For instance, the reduction of phenyl esters led to the aldehyde as the major product, but also a small amount of alcohol)50 The cleavage of u,p-unsaturated y-amino acids via ozonolysis yielded enantiomeric pure C-terminal peptides)49,61 The semicarbazone from reduction of peptide esters technique laid the initial foundation for solid-phase synthesis. Overall, Weinreb reduction is an ideal choice due to its high yields, optical purity, and its adaptability to a solid-phase platform)47 ... 

A new solid-phase technique used a relatively nontoxic cleaving agent for peptide aldehydes, which would prevent lithium aluminum hydride reduction byproduct contamination 51 A support-bound threonine or serine was treated with the substrate aldehyde to give an imine intermediate that immediately cyclized to give the stable oxazolidine linker, which was stable to Fmoc conditions including treatment with TFA. The peptide was cleaved under mild aqueous conditions at 60 °C 51 ... 

A solid-phase technique that uses an N-protected u,p-unsaturated y-amino acid proved to be effective for ozonolysis due to its ethylenic functionality. The linker was prepared from the Wittig reaction between (ethoxycarbonylethylene)triphenylphosphorane and either the Boc or Fmoc N-protected a-amino aldehyde. The Boc or Fmoc protecting group was removed and the chain was elongated by standard BOP coupling 49,52 53 61 The peptide-resin was then cleaved by ozonolysis, which afforded the desired aldehyde 49 ... 

The method described in Section 28.6 works well for the synthesis of small peptides. It is extremely time-consuming to synthesize larger proteins by this strategy, however, because each step requires isolation and purification of the product. The synthesis of larger polypeptides is usually accomplished by using the solid phase technique originally developed by R. Bruce Merrifield of Roekefeller University. 

How To Synthesize a Peptide Using the Merrifield Solid Phase Technique 1097... 

This peptide was synthesized by Fisher et al. (44) in 1974 employing the solid-phase technique using A -Boc chemistry. BHA-type resin was used for the synthesis on a Beckman Model 990 solid-phase peptide synthesizer. Dried BHA-resin hydrochloride was neutralized with triethylamine and stirred overnight with an excess of A -Boc-Met and DCI in DCM. The remaining unreacted amino groups of the resin were acetylated with a mixture of AC2O and triethylamine in DMF overnight. Amino-acid analysis of the resin, after hydrolysis in 6 A... 

These peptides have only been reported and prepared by traditional solution-phase syntheses (144-146). However, because their structures are not complicated, they can be readily prepared by modem solid-phase techniques. 

Since the introduction of sohd-phase peptide synthesis (SPPS) on lightly crosslinked polystyrene (PSty) by Merrifield in 1962,this technique has been optimized in all its aspects and has subsequently found widespread application in many other fields of organic chemistry, e.g. for the synthesis of oligonucleotides, for catalysis, for the immobilization of enzymes in biotechnology, and for the preparation of polymeric reagents. A new but very rapidly growing field of application of this revolutionary method is the use of solid-phase techniques in combinatorial chemistry for the synthesis of hbraries of compounds whose constituents differ widely in their chemical nature. ... 

This combination together with protecting groups such as Aloc,f l aUyl,f l and the Ddef groups, has widened the synthetic options considerably for sohd-phase peptide synthesis. This is now the method of choice for making most side-chain-modified peptides, for example phospho- and glycopeptides by solid-phase techniques. 

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