Solid phase peptide synthesis equipment

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. 

This chapter provides a manual for a laboratory-hased short course to introduce the common techniques of solid-phase peptide synthesis (SPPS). The course provides students the opportunity to design and manually synthesize analogs of glutathione using relatively simple equipment available in any unsophisticated laboratory. The manual provides compact protocols for both the different steps of SPPS and the final cleavage of peptides from resin supports. We also introduce a simple method for the synthesis of combinatorial libraries of glutathione analogs that is suitable for those relatively unfamiliar with the field of peptide chemistry. 

In batch SPPS (solid-phase peptide synthesis), the resin is contained in a vessel, usually equipped for bottom filtration. After a washing solvent or a reagent is added, the resin is mixed and the solvent or reagent is removed by filtration. Batch synthesizers use a variety of methods to mix the resin the wrist-action shaker, vortex mixing, nitrogen bubbling, or overhead stirring. 

Since the original work of Merrifidd, the field of solid phase peptide synthesis has evolved enormously to sophisticated automated equipment. Today s modern instrument has been engineered to a level at which a researcher only has to input a sequence into a computer and allow the machine to produce the desired target peptide. Contrary to the belief of researchers who have just entered the field of solid phase peptide synthesis and claim that the field is a mature science, there is unfortunately no guarantee that an individual instrument will prepare a desired sequence effectively, due to chemical and sequence constraints. Although synthesizers have removed the tediousness of repetitive synthetic operations, chemists still must decide the appropriate chemical pathway, select an instrument that will satisfy their demands, and interpret the data generated. 

An alternative to microarrays, especially in the area of (cyclic) peptides, are one-bead-one-compound (OBOC) libraries. The technology for solid-phase peptide synthesis is well developed. Split-mix synthesis can be assisted by, for example, sorting equipment using radiolabels. All beads can then be screened at once, at least in theory, and active compounds can be detected by, for example, fluorescence [167]. There are three major challenges [168] ... 

C-terminal peptide tiiioesters are used extensively in synthetic protein chemistry, especially for native chemical ligation (NCL) and other chemoselective reactions, which has inspired a search for robust synthetic strategies. Initially, peptide thioesters were mainly prepared using solid-phase peptide synthesis with amino acids N -protected with Boc (Boc-SPPS) [1-3], see Chapter 4. However, this technique requires specialized equipment for handling of hydrofluoric acid (HP) for release of the peptide from the resin, and it is therefore currently not used in many laboratories. Furthermore, the HP treatment is incompatible with many post-translational modiflcations such as glycosylations or phosphorylations [4]. Boc-SPPS is described thoroughly in Chapter 4. 

Previously, the synthesis of peptidic materials was seen as a cumbersome and expensive method that lacked the precision of other polymeric synthesis methods [3, 10, 13, 16, 18, 33, 36-38, 63]. Synthesis of peptides has quickly advanced with cheaper methods, larger yield quantities, greater precision, and more adaptive equipment and methodology. There are two main pathways of creating a large number of customized peptide sequences, (a) engineered/recombinant DNA synthesis or (b) synthetic solid phase peptide synthesis (SPPS) [1,13, 18, 20, 33, 36, 39 5, 64, 65]. These methods are preferred to traditional methods because of the yield, automation, and time saved when compared to manual organic synthesis. 

The automated solid-phase synthesis of oligosaccharides was first reported in 2001 [38], A peptide synthesizer was modified to enable solid-phase oligosaccharide synthesis. The synthesizer was equipped with a cooling system for low-temperature glycosylations. As in the case with other automated platforms, a combination of new technologies and synthetic methods were developed, and distinctive breakthroughs were achieved. Many state-of-the-art synthetic and analytical methods were adopted and adjusted over the years. The first decade of automated solid-phase oligosaccharide synthesis relied on the development of new methods to access complex structures (Fig. 7.3). 

Solid phase methods have been adapted so that many different peptides can be synthesised in parallel on filter paper (Frank 1989). For example, by using a 10 x 10 grid, 100 different peptides can be synthesised simultaneously by incorporating different amino acid at different positions on the filter. This is a simple way of preparing many different peptides very economically for applications such as epitope mapping. Equipment also exists for carrying out automated peptide synthesis in parallel. 

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. 

Merrifield s revolutionary concept of solid-phase synthesis was not limited to peptides, and similar techniques have been developed for the synthesis of nucleic acids and carbohydrates on solid supports. For each application, specialized instrumentation that is computer-controlled is commercially available. Access to such equipment has enabled researchers in areas of biology, medicine, material science, and biomedical engineering to prepare thousands of peptides and polypeptides for study. In the pharmaceutical industry, for example, solid-phase synthesis has been used to prepare relatively large numbers of related molecules, so-called compound libraries, that... 

---