Solid-phase peptide synthesis automation

In addition to the above-mentioned, rather painstaking, techniques of polypeptide syntheses, a very elegant technique was developed by Merrifield. This solid-phase peptide synthesis automates the reaction sequences. The method makes use of an insoluble crosslinked polymer substrate with pendant reactive groups for attachment of peptide chains. Chloromethylated polystyrene microgels are often used (see Chapter 8 for more discussions on the use of chloromethylated polystyrene for reactions of polymers). The chloromethyl moieties serve as the initiating sites for formation of the polypeptides ... 

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... 

A recent development in this context is the Liberty system introduced by CEM in 2004 (see Fig. 3.25). This instrument is an automated microwave peptide synthesizer, equipped with special vessels, applicable for the unattended synthesis of up to 12 peptides employing 25 different amino acids. This tool offers the first commercially available dedicated reaction vessels for carrying out microwave-assisted solid-phase peptide synthesis. At the time of writing, no published work accomplished with this instrument was available. 

GE Reid, RJ Simpson. Automated solid-phase peptide synthesis use of 2-(17f-ben-zotriazol-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate for coupling of tert-butyloxycarbonyl amino acids. Anal Biochem 200, 301, 1992. 

W van den Nest, S Yuval, F Albericio. Cu(OBt)2 and Cu(OAt)2, copper(II)-based racemization suppressors ready for use in fully automated solid-phase peptide synthesis. J Pept Sci 7, 115, 2001. 

Solid-phase peptide synthesis has become widely used for the preparation of peptides built from a-amino acids of varying sizes and complexity, and also in the recent synthetic approaches to peptide libraries. It has been recognized that the use of solid-phase protocols for the synthesis of (3-peptides is likely to make them more attractive lead compounds in drug discovery. Although still at an early stage, work has begun to develop suitable protocols for automated (3-peptide synthesis. 

Less reactive than acyl halides, but still suitable for difficult couplings, are symmetric or mixed anhydrides (e.g. with pivalic or 2,6-dichlorobenzoic acid) and HOAt-derived active esters. HOBt esters smoothly acylate primary or secondary aliphatic amines, including amino acid esters or amides, without concomitant esterification of alcohols or phenols [34], HOBt esters are the most commonly used type of activated esters in automated solid-phase peptide synthesis. For reasons not yet fully understood, acylations with HOBt esters or halophenyl esters can be effectively catalyzed by HOBt and HOAt [3], and mixtures of BOP (in situ formation of HOBt esters) and HOBt are among the most efficient coupling agents for solid-phase peptide synthesis [2]. In acylations with activated amino acid derivatives, the addition of HOBt or HOAt also retards racemization [4,12,35]. 

A wide choice of peptide synthesizers is currently available, ranging from manual to fully automated. They are all based on solid-phase peptide synthesis methodologies in which either f-butoxy carbonyl (t-boc) (11), or 9-fluor-enylmethoxycarbonyl (Fmoc) (12) is the major protecting group during synthesis. A detailed description of peptide synthesis is clearly beyond the scope of this chapter, and further information on practical and theoretical approaches to this chemistry may be found elsewhere (13-15). However, a brief outline of solid-phase synthesis may prove useful. 

Automated Stepwise Solid-Phase Peptide Synthesis General Procedure 177-791... 

Like the solid phase peptide synthesis, this process has also been automated. Commercial automated synthesizers are available that can prepare polynucleotides containing more than 150 bases with a cycle time of about 10 minutes per base. 

Brunfeldt, K. (1973) Automation in solid phase peptide synthesis. In Peptides 1972, Proc.l2.EPS (Hanson, H. and Jakubke, H. D., eds.), North-Holland Publishing Company, Amsterdam, pp. 141-151. 

Geiser, T., Beilan, H., Bergot, B. J., and Otteson, K. M. (1988) Automation of solid-phase peptide synthesis. In Macromolecular Sequencing and Synthesis Selected Methods and Applications (Schlesinger, D. H., ed.), Alan R. Liss, New York, pp. 199-218. 

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