Manual synthesis

A procedure for Boc-PNA manual synthesis is given below. If the coupling is incomplete (Kaiser test) double coupling is performed. The synthesis cycle is shown in Scheme 10 and the procedure is given in Table 1. 

Fmoc-(3-alanine/DMSO solution to all spot positions (see Note 4). For manual synthesis we recommend marking the spot positions with a pencil and to use a multistep pipettor (see Fig. 2). Repeat the delivery after 20 min. After that allow the reaction to occur once more for at least 20 min. 

The apparatus routinely used in this laboratory for the manual synthesis of peptides consists of a flask attached to a glass manifold that can support up to four sintered funnels, thereby permitting the simultaneous synthesis of up to four peptides. The side arm of the flask is attached to a vacuum pump to allow for solvents to be aspirated from each funnel. The manifold also contains valves... 

In manual synthesis, use at least 30 min coupling time for PNA monomers. 

The initial synthesis of the EGF-like peptide carried out on a synthesizer using an Fmoc approach failed. As identified by both HPLC and MS, the resulting peptide with 48 residues was found to be a mixture containing no detectable amount of the desired peptide. Manual synthesis was then attempted. Since the... 

A list of peptides to be synthesized was prepared and the number of peptides in the required array, the spot size, and the scale were defined. The spot positions were marked on the membrane with pencil dots for manual synthesis. The membrane sheet was placed in the reaction trough. An NMP solution, which was 0.3 M with respect to the second anchor compound [e.g., Fmoc-PAla or Boc-Lys(Fmoc)], 0.45 M with respect to HOBt, and 0.35 M with respect to DIC, was prepared and allowed to stand for 30 min. Aliquots of this solution were then spotted onto all positions according to the chosen array configuration. After 15 min, the spotting was repeated and reactions were allowed to proceed for 30 min. The sheet was washed with 2% AC2O/DMF (20 mL) for 30 s, once again for 2 min, and finally left in the solution overnight. The array was washed with DMF (3 x 20 mL), incubated for 10 min with 20% pi-... 

However, some other depsipeptides require additional tools. Many depsipep-tides of natural origin possess complex architectures such a cyclic or a bicyclic skeleton and may include unnatural or N-methylated amino acids in their structure. In these cases, a manual synthesis combined with careful optimization of each step is required. In this regard, syntheses of callipeltin B [171] and oxathio-coraline [172] have recently been described. 

M Ruvo, P Scardino, G Cassani, G Fassina. Facile manual synthesis of peptide libraries. Peptide Protein Lett 1(3) 187-192, 1994. 

PNA assembly with t-Boc synthesis chemistry may be employed by either manual or automated methods (Fig. 4). Manual synthesis based on the r-Boc chemistry yields PNA of very high quality but is labor intensive and requires a chemist trained in t-Boc SPPS. Manual synthesis has the advantage of being easily monitored at each monomer inclusion and also allows great flexibility of scale. 

Protocol 3. Manual Synthesis of PNA. Prepare the following solutions ... 

The t-Boc/Z chemistry provides oligomers of the highest quality and allows the preparation of PNA by manual synthesis. The shortcomings of this method, however, include the use of extremely harsh chemicals and long cycle times. The Fmoc/Bhoc protection method provides PNA with a shortened cycle time and has been adapted to a DNA synthesis platform. Custom PNA suppliers have adopted this method for more rapid oligomer production. 

Manual solid-phase synthesis requires intervention by a chemist during the assembly of a biopolymer. A manual synthesis can vary from manual addition of all reagents and solvents to manual intervention at a single step (such as addition of the monomer). 

Although automated solid-phase instruments are increasingly being used to construct biopolymers, manual synthesis is still used extensively with methods that are used to create molecular diversity (see Chapter 15). The libraries of molecules produced by these methods are often used for screening against biological targets. 

Automated synthesis can ran unattended and is less prone to human error than manual synthesis. As the biopolymer assembly process was automated, there was a tendency to produce longer sequences. This, in turn, required better synthetic methods, which has led to continual improvements in solid-phase chemistries and instrument design. 

At larger scales, the system volume may become insignificant, whereas at small scales the system volume often exceeds Kupport- In a manual synthesis, the system volume typically consists of the walls and cap of the reactor vessel in an automated synthesizer, the system volume comprises the tubing and valves. 

The reliability of the software in an automated synthesizer is as important as that of the instrument hardware. The software controls most of the tasks of the chemist in the manual synthesis. It runs the instrument, issuing the commands telling how much, when, and where to deliver reagents. The main features of instrument software are the user interface, system control, and system monitoring. 

Automated peptide synthesizers have been in use for several decades and have provided much needed assistance for manual synthesis of long peptides. In 2003, CEM Corporation introduced the first automated microwave peptide synthesizer, Odyssey. This system was updated shortly afterwards to the current version, Liberty (Fig. 20.6). 

The combination of automatic and manual synthesis allows a quick and broad exploration of the design space in real time. The response time of Amical is very short, making it a genuine interactive system. Several large examples have already been used for Amical evaluation with excellent results, including a telephone answering machine controller, which is used as an illustrating example in this chapter. 

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