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Peptide Synthesis
 

OVERVIEW

 



The Symphony/Multiplex TM automated peptide synthesizer is an instrument with 12 reaction vessels, which operate independently, i.e. 12 peptides at a time, and is dedicated to Fmoc synthetic chemistry. The coupling time for one amino acid addition is 30 minutes, although this may be extended where troublesome couplings are anticipated. The "average" cycle time for one amino acid addition can be assumed to be approximately sixty minutes. Futhermore, the synthetic protocol allows the option of an additional "capping step" (~ 10 minutes) at any cycle. This can be included to terminate failure sequences and produces truncated peptides for easier purification. In addition, we have two Perkin Elmer/ABI 433A automated peptide synthesizers that are dedicated to Fmoc synthetic chemistry. The standard cycle time for an addition of an amino acid is one hour. The instrument has the capabiltiy of monitoring the Fmoc deprotection step and will extend the deprotection step automatically, where it is needed.

Fmoc chemistry as it applies to solid phase peptide synthesis, is as simple in theory as in its application. All protecting groups that are used to protect the side chain functional groups of individual amino acids are acid labile, while the N-terminal amino function of the amino acid is protected by the Fmoc group which is base labile. Therefore, incorporation of new amino acids is simply a process of treating an Fmoc amino acid that is already attached to a resin with base (i.e. 20% piperidine/DMF) and adding a new Fmoc amino acid activated ester along with the appropriate activator (i.e. N-hydroxy benzotriazole (HOBT). Within the Fmoc synthetic chemistry scheme there is some flexibility at the coupling stage. Typically, HBTU/HOBT is the most widely used coupling reagent used by these automated peptide synthesizers, but other coupling chemistries may be used such as HATU/HOAT, PyBOP/HOBT, or OPFP preactivated amino acids/HOBT. Peptides can be synthesized as the free carboxyl or as the C-terminal amide. The N- terminus can be free or acetylated. In addition, the bromoacetylation of the N-terminus and inclusion of norleucine for quantitating the peptide loading onto the carrier protein are routine procedures. Incorporation of unusual amino acid derivatives are only restricted by the availability of the Fmoc activated esters. Both the Symphony and the Perkin Elmer/ABI instruments incorporates the use of HBTU/HOBT for its coupling chemistry. Consequently, each protected amino acid is coupled as the free acid. The first residue may be attached to the HMP (Wang) resin using DMAP following by a capping step using benzoic anhydride or one can use a variety of preloaded resins.

 

The standard scale for the Symphony/ Multiplex instrument is 25 micromoles which will normally be expected to yield 20-30 mg for a crude 20-mer. Purification of the peptide is not offered for this scale. The standard scale for the Perkin Elmer/ABI 433A instrument is 0.25 millimoles which will normally yield 300-400 mg for a crude 20-mer. One can usually expect a 50% recovery, if high purity is required.

While cleavage is performed on instrument for the Symphony/Multiplex synthesizer, the cleavage must be performed off instrument for the Perkin Elmer/ABI 433A. The resin is treated with trifluoroacetic acid (TFA) in the presence of appropriate scavengers. These scavengers include phenol, water, and triisopropylsilane. The slowest deprotection is with the methoxytrimethylbenzene sulfonyl (Mtr) group of Arg, therefore peptides with multilple Mtr groups have to be carefully monitored. Trp residues can be oxidized under these TFA conditions and require shorter treatments. Peptides that contain both Arg and Trp residues pose particular problems and frequently require extensive purification.

 

All peptides synthesized are routinely analyzed by reverse phase HPLC and Mass Spectrometry. HPLC conditions are as follows: * 0.4 x 25 cm Vydac C18 analytical column * 10-50% Acetonitrile (0.1% TFA)/water * 40 minute linear gradient * Flow rate 1.0 ml/min * UV detector--220 nm * Beckman Diode Array Detector Model 168 * chart speed 0.5 cm/min PAN is currently capable of performing mass spectrometric analysis which is an excellent technique for confirming mass numbers. Our instrument is a Voyager-DE RP Biospectrometry Workstation. This spectrometer is a MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) instrument. It is equipped with delayed extration (DE) for improved mass accuracy and a reflector (RP) for fragmentation analysis. If the N-terminal amino function of the peptide is free, then N-terminal sequencing of the peptide may be used, if the situation calls for it. For peptides that contain one or more cysteine residues, it is not uncommon to obtain multiple peaks on an HPLC chromatogram due to oxidation of the sulfhydryls. Likewise, methionine can be oxidized to its corresponding sulfoxide which will exhibit itself as an additional peak on the HPLC chromatogram. These products can be much more difficult to reduce in peptides than in proteins. Other peaks can produced by incomplete deprotection of the Mtr group of Arg, deletion peptides (no capping) or truncated peptides (with capping). Unpurified preparations normally contain 60-95% of the expected product. However, this tends to be peptide specific.

 

Under normal synthetic conditions, where the product of interest constitutes the majority of the preparation, yields are of the order of 50%. However, this is highly dependent upon the complexity of the amount of impurities found in the crude peptide as well as of the purification conditions themselves. A typical preparative run can handle 100-150 mg of crude peptide. The preparative column consist of a Millipore 25 mm x 10 cm C18 column RCM. Conditions vary and are dependent on the analytical HPLC chromatogram profile. Both isocratic and gradient conditions can be used. Again whether one method is used over the other, is dependent on the analytical HPLC chromatogram profile.