Merrifield solid phase synthesis, synthetic 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. 

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 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). 

The first synthesis reported by Merrifield produced the desired tetrapeptide (Leu-Ala-Gly-Val).f l Amino acids, dipeptides, and tripeptides were all detected in the crude product released from the resin. Through continued improvements of the method, the high speed of the amino acid incorporation and automation, the solid-phase peptide synthetic methodology has become the method of choice for most laboratories synthesizing peptides. Shortly after the introduction of the solid-phase procedure it was used to synthesize insulin. This impressive achievement awakened the biochemical community to the promise of synthetic chemistry and initiated a period in which large numbers of peptide analogues were prepared and analyzed. 

An important feature of Merrifield s method of sequential synthesis on the polymer support is that the synthesis goes unchecked. Unless the coupling reaction proceeds to completion in every step, the final product obtained after cleavage is bound to be contaminated with peptides differing from the desire sequence by one or more amino acid residues. Thus, because of the multiple uncertainties associated with solid-phase synthesis, it is highly desirable to have rapid analytical control of the two major synthetic operations, i.e., coupling and deprotection, in order to achieve unambiguous synthesis of the desired peptide. 

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. 

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 1963, R. B. Merrifield reported an unconventional method of peptide synthesis for which the name solid-phase peptide synthesis was coined. This method differed from general organic synthetic methods in that one of the reactants was reversibly and covalently bound to an insoluble, solid polymer support which was then reacted with the reagent to give a resin-bound product. After filtering the latter from the reaction mixture and after repetition of as many steps as necessary to achieve the synthesis, the product was obtained by a suitable cleavage reaction (Merrifield, 1963 Marshall and Merrifield, 1965). It may be recalled that, in... 

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