Oligopeptides 423

It is now well established that the 13C chemical shifts of amino acid residues removed more than two units from the chain ends in unstructured polypeptides are not influenced, except by proline moieties, by the peptide sequence (Table 5.27, [96, 791, 792, 802-810]). Chemical shift changes of carbons of the same amino acid at different positions within a peptide or protein are therefore due to effects caused by secondary or tertiary structure. 

Straightforward assignments of the 13C signals of synthetic oligopeptides were therefore achieved by proton off-resonance decoupling and by comparison with the spectra of the 

In future, the methods of two-dimensional NMR spectroscopy (Section 2.10) will be the ones used for the unequivocal signal identifications of longer chain peptides. Complete 13C shift assignments are achieved from carbon-proton shift correlations via two- and three-bond couplings, as is demonstrated for the orange-red chromopeptide antibiotic actinomycin D (Fig. 5.13, Table 5.28, [603, 812]). 

The oxidation state of cysteine in peptides such as glutathione may easily be recognized by the downfield shift of the signal of Cp in the cysteine residue upon oxidation from — CH2SH to -CH2-S-S- [89,790], The signal of the CH2 S-S-CH2- moiety at 41.6 ppm is also recognized in the spectra of oxytocin, vasopressin and insulin [790], 

A large number of 13C NMR studies on proline derivatives and proline peptides have appeared in the literature [815-830]. As the electron charge density of cis-proline carbons is different from that of franx-prolinc carbons, these isomers can be differentiated by nCNMR spectroscopy [826, 830]. On the basis of calculations Tonelli [831] predicted four conformations for the dipeptide Boc-Pro-Pro-OBzl, three of which could be detected by 13C NMR spectroscopy [826, 830], In proline-containing peptides the stereochemistry of the proline residue plays an important role for the conformation of these oligomers. The 13C chemical shift data of cis and trans proline derivatives, collected in Table 5.29, are useful to determine the stereochemistry of the amino acid-proline bond, e.g. in cyclo-(Pro-Gly)3, melanocyte-stimulating hormone release-inhibiting factor or thyrotropin-releasing hormone. 

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J. Kostrowicki and H.A. Scheraga, Application of the diffusion equation method for global optimization to oligopeptides, J. Phys. Chem. 96 (1992), 7442-7449. M. Levitt, A simplified representation of protein confomations for rapid simulation of protein folding, J. Mol. Biol. 104 (1976), 59-107. 

The following short descriptions of the steps involved in the synthesis of a tripeptide will demonstrate the complexity of the problem amino acid units. In the later parts of this section we shall describe actual syntheses of well defined oligopeptides by linear elongation reactions and of less well defined polypeptides by fragment condensation. 

In more recent publications the analytical standards have been raised considerably (HPLC, H-NMR, CD R.F. Nutt, 1980), and one may predict that in the near future it will be possible to characterize fully synthetic oligopeptides of moderate size. 

First the protected oligopeptide is coupled with polymer-bound nitrophenol by DCC. N"-Deblocking leads then to simultaneous cycliiation and detachment of the product from the polymer (M. Fridkin, 1965). Recent work indicates that high dilution in liquid-phase cycli-zation is only necessary, if the cyclization reaction is sterically hindered. Working at low temperatures and moderate dilution with moderately activated acid derivatives is the method of choice for the formation of macrocyclic lactams (R.F. Nutt, 1980). 

Short chains of amino acid residues are known as di-, tri-, tetrapeptide, and so on, but as the number of residues increases the general names oligopeptide and polypeptide are used. When the number of chains grow to hundreds, the name protein is used. There is no definite point at which the name polypeptide is dropped for protein. Twenty common amino acids appear regularly in peptides and proteins of all species. Each has a distinctive side chain (R in Figure 45.3) varying in size, charge, and chemical reactivity. 

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