Automated synthesis reaction cycle

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. 

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. 

The breakthrough in peptide chemistry, which opened up applications in biochemistry and molecular biology, was the development of solid phase synthesis by Merrifield in 1963. This formed the basis of automated synthetic procedures in which the nascent peptide chain was covalently linked to a solid support such as a styrene-divinylbenzene copolymer the complex isolation and purification procedures needed to separate reactants and products at the end of each reaction cycle, which characterised previous solution methods of peptide synthesis, were replaced by a simple washing step. With modern automated methods of peptide synthesis, the time for an Fmoc reaction cycle has been reduced to 20 min, so that a 50-residue peptide can be synthesised in a day (Chan and White 2000). 

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. 

The technology for chemical peptide synthesis is now automated. As in the sequencing reactions already considered, the most important limitation of the process is the efficiency of each chemical cycle, as can be seen by calculating the overall yields of peptides of various... 

Incorporation of the F-Tag Cap into the Automated Solid-Phase Synthesis Cycle. The resin (50 /imol) was swelled in a 0.1 M solution of 2,6-lutidine in CH2C12 (4 ml, 8.0 equiv.). After vortexing for 5 s, a 0.1 M solution of the F-Tag triflate in CH2C12 (2.5 ml, 5.0 equiv., loaded into cartridges) was delivered to the reaction vessel. Mixing of the suspension was performed (10 s vortex, 50 s rest) for 15 min. 

The automation of laboratory operations to enhance the process development chemist s armory has also grown as an activity. Equipment is available (e.g., Buchi Syncore8) to aid in the evaluation and optimization of such as time-temperature cycles in a given reaction, or in combinatorial and parallel synthesis endeavors. 

Solid-phase parallel synthesis mimics the previously described solution phase strategy. This approach easily lends itself to both semi- and full automation. In contrast to the solution phase method, purification is easily achieved by simply washing the resin beads, and the reactions can be driven to completion by excess reagents, multiple cycles, and microwave techniques. The initial building block or scaffold is attached to the resin bead by a detachable linker. At the end of the synthesis, the final construct is released under the appropriate cleavage conditions for automated purification, usually by high-pressure liquid chromatography (HPLC). This allows bioanalysis of the final product in aqueous solution under standard assay conditions. 

The hardware of an automated solid-phase synthesizer consists of all the electromechanical components of the system (reaction vessel, tubing, valves, pumps, detectors, power supplies, circuit boards, sensors, etc.). It is imperative that all these components are reliable and can operate without failure for many synthesis cycles before repair or replacement. Before using any component in an automated system, it should undergo compatibility and lifetime testing with the reagents that would be in contact during the synthesis. 

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