Longer Peptides

Because longer peptides enable more coverage of a protein and better ability to study its interactions, the ability to synthesize longer chains is highly desirable. Conventional Fmoc SPPS has enabled routine assembly of peptide sequences of up to 30 to 40 amino acids when aggregation is not an important issue. 

Microwave synthesis of the 30mer peptide Val-Tyr-Trp-Thr-Ser-Pro-Phe-Met-Lys-Leu-Ile-His-Glu-Gln-Cys-Asn-Arg-Ala-Asp-Gly-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly with crude purity of 80.0% has also been reported. 

Gly-NH2 1 = product 2 = del[Arg], Synthetic conditions 20% piperidine in DMF = deprotection PyBOP-HOBt-DlEA 0.9 1 2, x6 excess = coupling, reagent K = cleavage. 

The synthesis was performed at the 0.1 mmol level using 5% piperazine in DMF as the deprotection reagent and HBTU activation with a fivefold excess of reagents and was complete in 12.5 h [36]. 

Erythropoietin (EPO) is a naturally occurring protein responsible for red blood cell production. A 20mer peptide mimic of this natural protein has been synthesized in 8 h by use of microwave SPPS. Purity was higher than that obtained in 5 days by conventionally synthesis [33] and the biological activity was reported to be equal to that of the conventionally synthesized material, as shown in Fig. 20.16. 

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Dipeptides and longer peptides are typically synthesized by solid-phase chemistry at polymer beads, a route discovered by and named after Merrifield [5, 88]. Disadvantages of this approach are that the polymer support is expensive and additional steps for linkage to and cleavage from the polymer are required. Hence solution chemistries are an alternative to the Merrifield approach. 

If longer peptides are to be synthesized, the dipeptides as well as the monomers must be activated. This leads to a side reaction which can endanger the required chain-forming reaction, the formation of cyclic diketopiperazines ... 

Peptide length, as longer peptides have more groups where additional protons can be attached (basic residues). 

Recently, a SAMDI-MS assay was described by means of which endogenous caspase protease activities in cell lysates can be determined [26], Similar to the assay used to determine anthrax lethal factor inhibitors, peptide substrate SAMs for either caspase-3 or -8 were treated with cell lysates. In contrast to fluorescence assays, also longer peptide substrates could be used, thus enabling a better resolution of the two caspase activities. 

The barrel-shaped architecture of the 20S proteasome allows substrate proteins to be degraded in a processive manner, i.e. without the release of degradation intermediates (Akopian et al. 1997). It is noteworthy, that the cleavage sites found when longer peptides or proteins are used as substrates, do not reflect the specificities defined by means of short fluorogenic... 

Similiar to albomycin and ferrichromes, the linear tripeptide of A -hydroxy-A -acetyl-ornithine can be used as the simplest chiral template for artificial siderophores. It can be used alone as a tripeptide like for 80, can be conjugated to a hydrophobic fatty acid 81132 pj. p j.j longer peptide 82, 83, and 85. ... 

The peptide (melittin) was foimd associated to RMs in a single state as opposed to involvement of at least two forms of melittin with Upid in phospholipid vesicles. Folding and dynamics of this peptide in RMs were also investigated In RMs, activity of a-chymotrypsin was imaffected by pressurization while lipase lost its activity at low pressures and regained on depressurization. The use of pressure as a switch for lipase catalysis is discussed Partition coefficients for 11 amino acids, 17 dipeptides and 5 longer peptides in RMs were determined. 

Both Met-enkephalin and Leu-enkephalin have their own pro- and prepro forms.29 Bovine preproenkephalin A is a 268-residue protein containing a 20-residue signal sequence and four sequences of Met-enkephalin and one of Leu-enkephalin, each flanked by pairs of basic residues. There are also Met-enkephalin-Arg-Gly-Leu (YGGFMRGL) and Met-enkephalin-Arg-Phe sequences. Not all of these are cut out cleanly, and other peptides such as Met-enkephalin-Arg-Arg-Val-NH2 are also found in brain. Proenkephalin B contains three copies of Leu-enkephalin contained within longer peptides. One of these, P-dynorphin (Table 30-4), is also a potent opioid compound. The enkephalins are thought to act as neurotransmitters, which are rapidly degraded after their release by two or three membrane-bound... 

Fig. 26. The preparation of well-defined peptidic surfaces [65,66]. A Using a thiol spacer, homopolypeptides (poly-A in this case) that adopt an a-helical conformation are synthesized on a gold surface. B Using an aminopeptidase, the longer peptidic chains are hydrolyzed to yield a more homogeneous surface. (Reproduced with the permission of Ref. 65,66)...

Longer peptides having these arginine-related phosphonates at the C-terminal are inhibitors of some trypsin-like enzymes, e.g. Boc-D-Phe-Pro-(4-AmPhg)p(OPh)2 is an inhibitor of thrombinJ44 This peptide was synthesized from Boc-D-Phe-Pro-OH and the corresponding... 

To avoid the problems with kinetic control of a reaction during peptide bond formation, it is recommended that longer peptide phosphonates be synthesized stepwise by extension in the N-terminal direction, starting with diphenyl l-(aminoalkyl)phosphonates. The longer coupling time will provide peptide products that are usually an equivalent (or close to equivalent) mixture of diastereomers. The synthesis of longer peptides is demonstrated by the synthesis of Boc-Val-Pro-Valp(OPh)2, which is an excellent inhibitor of human neutrophil elastase (kobs/[I] = 27,000 M 1s 1)J38 ... 

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