Peptide solid-phase

One step further, namely to monomeric sequence-defined polymeric vehicles, leads to the synthetic strategy based on standard peptide solid phase chemistry. 

Deprotected target peptide Solid phase peptide synthesis... 

To avoid the possibility that a small peptide from a phage-displayed library will not bind adequately when immobilized on a solid support, Baumbach and Hammond suggested that combinatorial peptide libraries for protein purification be synthesized directly on resins that could be used as chromatographic supports on a large scaled, in this way, any ligand that is identified is already on a platform or format that would facilitate implementation in downstream processing. The one-bead-one-peptide solid phase library format is ideally suited for this purpose, if the library is built on chromatographic resins that can withstand the harsh solvent conditions used for peptide synthesis. 

Kumler and Boyer (1976) used esr spectroscopy to study the glass transition of several spin-labeled polymers and copolymers, and spin-labeled polymeric matrices have been used to study solvent effects on chain flexibility in polystyrenes usually used in peptide solid-phase syntheses (see Section IV). 

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). 

The major disadvantage of solid-phase peptide synthesis is the fact that ail 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 Menifield 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). 

Sections 27 15 through 27 17 describe the chemistry associated with the protection and deprotection of ammo and carboxyl functions along with methods for peptide bond formation The focus m those sections is on solution phase peptide synthesis Section 27 18 shows how these methods are adapted to solid phase synthesis... 

Solid Phase Peptide Synthesis The Mernfield Method... 

SOLID-PHASE PEPTIDE SYNTHESIS THE MERRIFIELD METHOD... 

The actual process of solid phase peptide synthesis outlined m Figure 27 15 begins with the attachment of the C terminal ammo acid to the chloromethylated polymer m step 1 Nucleophilic substitution by the carboxylate anion of an N Boc protected C terminal... 

FIGURE 27 14 A section of polystyrene showing one of the benzene rings modified by chloromethylation Indi vidual polystyrene chains in the resin used in solid phase peptide synthesis are con nected to one another at various points (cross linked) by adding a small amount of p divinylbenzene to the styrene monomer The chloromethylation step is carried out under conditions such that only about 10% of the benzene rings bear —CH2CI groups... 

Memfield successfully automated all the steps m solid phase peptide synthesis and computer controlled equipment is now commercially available to perform this synthesis Using an early version of his peptide synthesizer m collaboration with coworker Bemd Gutte Memfield reported the synthesis of the enzyme ribonuclease m 1969 It took them only SIX weeks to perform the 369 reactions and 11 391 steps necessary to assemble the sequence of 124 ammo acids of ribonuclease... 

Solid phase peptide synthesis does not solve all purification problems however Even if every coupling step m the ribonuclease synthesis proceeded in 99% yield the product would be contaminated with many different peptides containing 123 ammo acids 122 ammo acids and so on Thus Memfield and Gutte s six weeks of synthesis was fol lowed by four months spent m 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... 

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... 

FIGURE 27 15 Peptide synthesis by the solid phase method Ammo acid residues are attached sequentially beginning at the C terminus... 

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... 

Solid phase peptide synthesis (Section 27 18) Method for peptide synthesis m which the C terminal ammo acid is co valently attached to an inert solid support and successive ammo acids are attached via peptide bond formation At the completion of the synthesis the polypeptide is removed from the support... 

J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd ed.. Pierce Chemical Company, Rockford, IL, 1984. 

E. Atherton and R. C. Sheppard, Solid Phase Peptide Synthesis. A Practical Approach, Oxford-IRL Press, New York, 1989. 

H2/Pd-C, EtOH, 20°. < -Bromobenzyl carbonates have been developed for use in solid-phase peptide synthesis. An aryl o-bromobenzyl carbonate is stable to acidic cleavage (CF3CO2H) of a /-butyl carbamate a benzyl carbonate is cleaved. 

Polymer-supported esters are widely used in solid-phase peptide synthesis, and extensive information for this specialized protection is reported annually. Some activated esters that have been used as macrolide precursors and some that have been used in peptide synthesis are also described in this chapter the many activated esters that are used in peptide synthesis are discussed elsewhere. A useful... 

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