Peptide synthesizer, automatic

At this time, a solid phase automatic peptide synthesizer was acquired, so we attempted total synthesis of Sh I. A solid phase synthesis of Ax I (antho-pleurin A) had previously been reported in an abstract (36). The synthetic Ax I possessed only 11% of the toxicity of the natural toxin. 

Automatic peptide synthesizer, Milligen 9050 PepSynthesizer (Milligen, Milford, MA). 

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... 

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. 

Ikota (20) reported the first solid-phase synthesis of motilin in 1980, and the overall yield was 1.8%. Coy et al. (21) improved the synthesis and got a 10% yield. Coy s synthesis was carried out on a Beckman model 990 automatic peptide synthesizer using A -Boc chemistry. Motilin was assembled stepwise on a 1% cross-linked BHA resin with Boc-a-benzyl-Glu as the first amino acid to be incorporated. In this manner, C-terminal Gin could be directly formed on HE cleavage of the final peptide from the resin. The detailed coupling schedule is shown in Table 2 (22). The symmetric anhydride method was used for... 

The fully protected peptides were synthesized either on a manual shaker or after optimization of coupling conditions [18] on an automatic peptide synthesizer (ACT 200) from Advanced Chemtech using the 2-chlorotrityl chloride resin and Fmoc/t-Butyl strategy [2]. The coupling with TBTU/DIEA [2] and the... 

This vessel was used in the first automatic peptide synthesizer designed by Stewart and Merrifield.P 1 The volume of solvent/reagent used should always fill the vessel over half full, so that all vessel walls are washed. An unfortunate consequence of this half-full requirement is that coupling reactions are carried out in more dilute conditions than are optimal. 

Polypeptoid synthesis reactions are carried out on automatic peptide synthesizers (ABI433A Applied Biosystems, Foster City, CA). Fmoc-rink amide resin (0.25 mmol Nova Biochem, San Diego, CA) is deprotected by treating with 20% (v/v) piperdine (ABI) in dimethylformamide (DMF Fisher Scientific, Itasca, IL) in two 15-min treatments. To assemble the polypeptoid chain, alternating cycles of bromoacetylation and amine displacement of the bromine from the A-terminal alkyl halide are performed (Figure 12.14). To bromoacetylate, the resin is vortexed for 45 min with 4.3 mL of... 

This method lends itself so well to automation that automatic peptide synthesizers are now available conunercially. One automatic peptide synthesizer was employed, for instance, in the synthesis of ribonuclease, an enzyme. In another instance an enzyme, ferredoxin, that consists of 55 amino acid residues, was synthesized. ... 

Syro II, fully automatic peptide synthesizer (MultiSynTech GmbH, Germany now from Biotage AB, Sweden). 

Robotic peptide synthesizers are now used to automatically repeat the coupling, washing, and deprotection steps with different amino acids. Each step occurs in high yield, and mechanical losses are minimized because the peptide intermediates are never removed from the insoluble polymer until the final step. Using this procedure, up to 25 to 30 mg of a peptide with 20 amino acids can be routinely prepared. 

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 high-throughput peptide synthesizer is shown in Fig. 9. Wash solutions and reagents common to all synthetic entities are automatically delivered through a multichannel distributor connected to a six-port selector valve. Building blocks and other specific reagents are individually delivered to their respective wells... 

Bartak, Z., Bolf, J., Kalousek, J., et al. (1994) Design and construction of the automatic peptide library synthesizer. Methods A Companion to Methods in Enzy-mology 6, 432-437. 

The Merrifield method has now been completely automated, so it is possible to purchase peptide synthesizers that automatically carry out all of the above operations and form polypeptides in high yield in a matter of hours, days, or weeks, depending on the length of the chain of the desired product. The instrument is pictured in Figure 28.8. For example, the protein ribonucle-ase, which contains 128 amino acids, has been prepared by this technique in an overall yield of 17%. This remarkable synthesis involved 369 separate reactions, and thus the yield of each individual reaction was > 99%. 

Peptide arrays on paper or cellulose membranes generated by SPOT synthesis generally are low density, even with the commercially available automatic SPOT synthesizer. Such a low-density peptide chip (several thousand peptides) now is available commercially, for example, PepChip microarray from Mimotopes. Foder et al. (4) first described the... 

The quantitative ninhydrin test has not been automated. The Applied Biosystems Models 430, 431, and 433 Peptide Synthesizers, however, have the capability to automatically remove 3-10 mg resin samples after each coupling for a manual ninhydrin test. 

Nevertheless, Merrifield and his co-workers introduced the picric acid test into a commercial peptide synthesizer to refine it into an almost fully automatic system controlled by a spectrophotometric unit, which determines the concentration of picrate released from the polymer support [137]. Though the machine performs the physical measurements with the highest accuracy available in this connection to date, the best analytical device cannot compensate inherent variable chemical deviations of a test reaction employed on polymer phase. 

As already indicated in the preceding paragraphs, an automatically operating peptide synthesizer needs complet monitoring of the execution of all orders programmed, and a feedback signal from the course and efficiency of the chemical reactions on polymer. 

The third method for automatic monitoring of the gel phase synthesis has long been known [ 196] - the perchloric acid titration on polymer — and recently was built into a computer-controlled peptide synthesizer [134]. Here, the results of the analytical determinations are fed back into the monitor system, which is programmed to optimize the course of the synthesis based on the titration data. This example of best realized automatization to date, however, is restricted to acid-tolerant synthesis strategies. The limiting chemical conditions of the analytical principle in use were already discussed on p. 43—44. 

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