Automated peptide synthesizers

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... 

One widely used method of formation of protected compounds involves polymer-supported reagents, with the advantage of simple workup by filtration and automated syntheses, especially of polypeptides. Polymer-supported reagents are used to protect a terminal — COOH group as a polymer-bound ester (RCOOR —( ) during peptide syntheses, to protect primary alcohols as... 

Fig. 10 Liberty (CEM Corporation), the first automated microwave solid-phase peptide synthesizer, is shown on the left-, the available reaction vessel setup for solid-phase synthesis is depicted on the right...

Such biosyntheses were models for the Merrifield-synthesis [8] (Fig. 3), which culminated in the development of fully automated peptide synthesizers [9]. In a repeated reaction cycle a N-terminal protected amino acid, which is attached with its C-terminal end to an insoluble solid support, is deprotected, activated and lengthened by a second protected amino acid unit. The deprotect -ing and coupling steps can be repeated until the entire peptide is assembled. 

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. 

The synthesis can be carried out manually or automated using a Symphony/Multiplex multiple peptide synthesizer or an Argonaut Nautilus. 

Commercially available computer-automated peptide synthesizers allow synthesis of a polypeptide in a time of about one hour or less per amino acid unit. The ribonuclease synthesis takes about one week or less. 

To expedite peptide syntheses of up to 124 amino acid units, beads of solid polystyrene— with chloromethyl groups, —CH CI, para to about every hundredth phenyl group—are used in an automated process. The sequence of steps is (1) attach C-terminal Boc-protected amino acid by... 

The basic advantage of the polymer support techniques is that the polymer (including all chains attached to it) is easily separated from all other reagents, because it is insoluble in the solvents used. Excess reagents, other reaction products (such as DHU), side products, and the solvents themselves are quickly washed away. Purification of the polymeric species (such as 104, 105, and 106) is rapid and complete. The process can even be automated,886 to the extent that six or more amino acids can be added to a peptide chain in one day. Commercial automated peptide synthesizers are now available.887... 

Caged peptides (NPY and angiotensin II) containing Tyr[Bzl(2-N02) were synthesized on an automated solid-phase peptide synthesizer using Fmoc chemistry and cleaved from the resin by treatment with TFA. Crude peptides were purified by semipreparative HPLC with a gradient phase of 0.1% TFA and MeCN. For fractions containing peptides, the eluant was not passed through the UV monitor to avoid potential photolysis. The collected peptide was examined by analytical HPLC and MS. 

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. 

The instrument chosen for the evaluation of carbohydrate synthesis was an ABI-433 peptide synthesizer (Fig. 2). The instrument was adapted for carbohydrate synthesis and customized coupling cycles were developed. A specially designed low-temperature reaction vessel was installed and interfaced with a commercially available cooling device.13 The necessary reagents were loaded onto the instrument ports and reaction conditions were programmed on the computer, in a fashion similar to the automated synthesis of peptides. 

Amino acids were added to the N -Boc-D-Aia-0-Resin (1.0 mmol of resin was replaced in the reaction vessel of 5.0 L Vega 296 automated solid phase peptide synthesizer in the following sequence ... 

PS3 automated peptide synthesizer (Protein Technologies) (see Note 1). 

Advances in solid-phase oligosaccharide synthesis led to the development of an automated platform that was first demonstrated by Peter Seeberger and coworkers in 2001 using a modified peptide synthesizer [38], Automated washing, deprotection, and glycosylation steps dramatically reduced the time and labor usually attributed to solution- and solid-phase synthesis of oligosaccharides. 

First, a peptide synthesizer was modified to allow solid-phase oligosaccharide synthesis. This platform had the basic function of an automated synthesizer and allowed repeating cycles of glycosylation and deprotection to be mostly software controlled (Fig. 7.2a) [38], This robust pressure-driven system relies on an established... 

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). 

For almost lOyears, automated solid-phase synthesis of oligosaccharides was performed on a modified peptide synthesizer. The first prototype of a specially made oligosaccharide synthesizer [39] features fully automated tailor-made syringe pump systems for accurate delivery of reagents. 

Fig. 15 The newly introduced principle of single amino acid chelators (42, SAAC) which can directly be subjected to automated solid phase peptide syntheses...

Another advantage of the solid phase method is that the steps are repetitive and can be automated. An automatic peptide synthesizer is commercially available. It consists of a flask with a fritted-glass filter at the bottom along with bottles containing solutions of BOC-protected amino acids and the other necessary reagents and wash solutions. The polymer with the first amino acid attached is placed in the flask, and the order of... 

While chemical synthesis is mostly an art, with specialized reactions for both inorganic and inorganic synthesis, the complexities of biochemistry have nurtured specialized instruments that can split or assemble biomolecules. An automated solid-phase peptide synthesizer was introduced by Merrifield15 in 1963 this allows for the facile synthesis of oligopeptides (up to 100 amino acid units) [2], The enzyme DNA polymerase I was discovered by Komberg16 in 1957 this allowed the assembly of DNA from fragments [3]. 

The progress in the development of improved monitoring techniques for the heterogeneous synthetic reactions involved in the solid phase method provided the feedback control which was necessary for the full automation of the peptide synthesizers. A monitoring system, which is based on the titration of the unreacted polymer-peptide chains with picric acid 34,35), on coupling with a peptide synthesizer provided an automatic feedback 36). This feedback is to implement the next step in the synthesis if the level of the unreacted peptide chains is below the acceptable preset value, or otherwise to repeat the last step. 

The monitoring system based on the potentiometric titration of resin-bound unreacted amino groups 29,30) has helped the development of much advanced fully automated peptide synthesizers 37,38). This computarized system evaluates automatically the synthetic data and decides whether to proceed to the next step, to repeat the last step, or to stop the synthesis. Very recently Edelstein et al.391 designed and im-... 

A number of automatic peptide synthesizers are commercially available now 40). However, in spite of all these mechanical and electronical advances in the automation of solid phase synthesis, as Barany and Merrifield state in their review on the solid phase peptide synthesis11), the limiting factor continues to be the chemistry of the process and automation can reach its full potential only when the assorted chemical difficulties are under control. 

Assay of Truncated Peptides. The truncated forms of allatostatin 4 were synthesized on a Pharmacia-LKB Biolynx model 4170 automated solid-phase peptide synthesizer by The Biotechnology Service Centre, Department of Clinical Biochemistry, Banting and Best Institute, Toronto, Canada. The assays comparing the activity of allatostatin 4 with the two analogues were carried out as for the HPLC eluates. 

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