Merrifield solid phase synthesis, synthetic

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). 

Solid phase synthesis lends itself easily to automation. This faet was readily apparent to Merrifield, who built the first automated synthesizer (S,5S,5P). Merri-field s pioneering effort was soon followed by other laboratories (60-87) and several synthesizers appeared on the market. The development of the Fmoe synthetic strategy (88,89) allowed for the substantial simplification of automatic synthesizers, since handling of the very unfriendly reagent, hydrofluoric acid, was no longer necessary. Cambridge Research Biochemicals was the first to introduce a Fmoc-based synthesizer, PEPSYNthesizer, to the market. This machine was capable of only one step of the synthesis, but it applied the... 

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. 

Synthesis on solid supports was first developed by Merrifield [1] for the assembly of peptides. It has expanded to include many different applications including oligonucleotide, carbohydrate, and small-molecule assembly (see Chapters 11 and 14). The repetitive cycle of steps involved in the solid-phase synthesis of biopolymers can be performed manually using simple laboratory equipment or fully automated with sophisticated instrumentation. This chapter examines typical solid-phase reaction kinetics to identify factors that can improve the efficiency of both manual and automated synthesis. The hardware and software features of automated solid-phase instruments are also discussed. The focus of this discussion is not on particular commercial model synthesizers but on the basic principles of instrument operation. These considerations can assist in the design, purchase, or use of automated equipment for solid-phase synthesis. Most contrasting features have advantages and disadvantages and the proper choice of instrumentation depends on the synthetic needs of the user. 

Polymers as solids are ubiquitous in our modern society. They are some of the most common synthetic materials. Biologically derived macromolecules are also abundant. Whether it is a piece of wood, a natural fiber, or a lobster shell, nature uses solid organic macromolecular materials as key architectural material. This abundance of examples of synthetic and natural solid polymeric materials is mirrored in the prevalence with which insoluble cross-linked polymer supports are used in synthesis and catalysis [23-25]. However, while solid-phase synthesis and related catalysis chemistry most commonly employ cross-linked supports that resemble those originally used by Merrifield [26], the polymers found in nature are neither always insoluble nor always cross-linked. Indeed, soluble polymers are as common materials as their insoluble cross-linked analogs. Moreover, nature quite commonly uses soluble polymers as reagents and catalysts. Thus, it is a bit surprising that synthetic soluble polymers are so little used in chemistry as supports for reagents, substrates, and catalysts. 

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. 

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