Automated SPPS

The biberty (Fig. 10), a monomode microwave reactor for automated SPPS, was recently introduced by the CEM Corporation [153]. Although this instrument was originally developed for SPPS, it also allows for a broader scale of solid-phase applications. The solid-phase vial is equipped with a polypropylene frit and cap at one end (the entire assembly fitting into the standard 10 mb CEM reaction vessel) to allow the processing of 0.1 to 1.0 mmol quantities of resin attached substrates. An integrated fiber optic probe provides... 

It has been reported by Patel and Gordon " that SPPS is limited by poor yields with hindered amines, deactivated aromatic aldehydes and slight over alkylation with aliphatic aldehydes. Johnson et al argued that because of the speed and convenience of automated SPPS, support-bound cyclization protocols are ideal for the preparation of numerous cyclo peptide analogues. 

Peptide synthesizers or Solid-phase synthesizers — To find automated SPPS machines... 

Figure 3 summarizes the steps involved in cyclic peptide synthesis and these are described in more detail in Subheadings 3.1 and 3.2 for Boc and Fmoc methods, respectively. Carry out all procedures at room temperature and in a well-ventilated fume hood unless otherwise specified. The amounts of reagents are given based on a 0.5 mmol scale synthesis, which typically yields >10 mg of final product after purification. These procedures have been used in our laboratory to synthesize several classes of disulfide-rich cyclic peptides, including SFTI-1 (14 amino acids, one disulfide bond), cyclic Vcl.l (22 amino acid, two disulfide bonds), kalata B1 (29 amino acids, three disulfide bonds), MCoTI-II (34 amino acids, three disulfide bonds), and cyclic chlorotoxin (43 amino acids, four disulfide bonds) (Fig. 4). The procedures are described for manual synthesis but are also applicable to automated SPPS.

In the general procedure manual peptide synthesis is described. Kaiser test see Note 5) should be performed after each amino acid coupling ensuring completeness of the reaction. In our lab, we use a Syro II robot (MultiSynTech) for automated SPPS. If you are equipped with a robot, use your established standard protocols for peptide preparation, because the sequence is not known to be difficult. 

These methodologies have been reviewed (22). In both methods, synthesis involves assembly of protected peptide chains, deprotection, purification, and characterization. However, the soHd-phase method, pioneered by Merrifield, dominates the field of peptide chemistry (23). In SPPS, the C-terminal amino acid of the desired peptide is attached to a polymeric soHd support. The addition of amino acids (qv) requires a number of relatively simple steps that are easily automated. Therefore, SPPS contains a number of advantages compared to the solution approach, including fewer solubiUty problems, use of less specialized chemistry, potential for automation, and requirement of relatively less skilled operators (22). Additionally, intermediates are not isolated and purified, and therefore the steps can be carried out more rapidly. Moreover, the SPPS method has been shown to proceed without racemization, whereas in fragment synthesis there is always a potential for racemization. Solution synthesis provides peptides of relatively higher purity however, the addition of hplc methodologies allows for pure peptide products from SPPS as well. 

The techniques for automated solid phase synthesis were first highly developed for polypeptides and the method is abbreviated as SPPS. Polypeptide synthesis requires the sequential coupling of the individual amino acids. After each unit is added, it must be deprotected for use in the next coupling step. 

A variety of commercial kits and automated systems are available to test the abilities of bacteria to assimilate, ferment, decarboxylate, or cleave selected organic compounds.46 Their reliability for species identification is usually greater with cultures from clinical samples, where a limited number of bacteria are commonly encountered, and less with environmental soil and water samples, where a great many uncommon or previously unidentified species not in the database are likely to be present.29,45 Additional tests beyond those found in the commercial kits may be necessary for example, the hydrolysis of various nitriles and amides is useful for identifying Rhodococcus spp.47 Some commercial kits for clinical use feature antimicrobial susceptibility testing.21... 

Fahr, A. M. Eigner, U. Armbrust, M. Caganic, A. Dettori, G. Chezzi, C. Bertoncini, L. Benecchi, M. Menozzi, M. G. Two-center collaborative evaluation of the performance of the BD Phoenix automated microbiology system for identification and antimicrobial susceptibility testing of Enterococcus spp. and Staphylococcus spp. J. Clin. Microbiol. 2003, 41,1135-1142. 

Duncanson, P., Wareing, D. R. A., and Jones, O. (2003). Application of an automated immunomagnetic separation-enzyme immunoassay for the detection of Salmonella spp. during an outbreak associated with a retail premises. Lett. Appl. Microbiol. 37,144-148. 

Shelef, L. A. and Tan, W. (1998). Automated detection of hydrogen sulfide release from thiosulfate by Salmonella spp. J. Food Prot. 61, 620-622. 

SPPS. In contrast to OPcp esters, these latter OPfp esters derived from 27 and those derived from HODhbt are the most commonly used active esters in SPPS and have been applied to automated continuous flow Fmoc/tBu synthesis. Their use notably simplifies the course of the solid-phase synthesis by avoiding individual preactivation procedures. 

From these experiments the following conclusions can be drawn With few modifications to usual protocols (such as those presented here), carrying out SPPS at elevated temperature (preferably 60 °C) will increase efficiency by reducing cycle times to significantly less than 30 minutes under automated and non-automated conditions. Toluene/DMSO mixtures are also compatible with SPPS at room temperature and should solve some aspects (cost and environmental) of disposal associated with chlorinated solvents but have not been shown to have advantages over DMF or NMP... 

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