Oligopeptide Fragments

In collaboration with Eckard Wimmer (State University of New York, Stony Brook, New York) a breakthrough in our understanding of the replication mechanism of the poliovirus was achieved. Key to this study proved to be the accessibility of homogeneous uridylylated oligopeptide fragments. In itself, the chemical synthesis of peptide nucleic acid fragments of the complexity required for these studies, with the many synthetic hurdles that are caused by the incompatibility of standard peptide synthesis and nucleic acid synthesis protocols, represents a milestone in the oeuvre of Jacques van Boom and a hallmark in bioorganic chemistry. 

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. 

The use of SPPS allows synthesis of both native oligopeptides and important synthetic analogues. Many of these fragments or substituted analogues have been found to be equal to, or more potent than, the parent molecule and to exhibit long-acting and antagonistic activities. 

Another solid phase fragment condensation with CDI and 1-hydroxybenzotriazole in the synthesis of the human insulin B-chain afforded the oligopeptide in 75% yield. The reaction time with the coupling pair CDI/HOBt was shorter than in the case of the DCC/ HOBt system.136 The CDI/HOBt activation method was also applied to the synthesis of a... 

The third current approach is synthesis of peptide chains as models for the helical peptaibol (Section 8.2) and gramicidin (Section 8.3) ion channels. Considerable work has been carried out in the former area, involving synthesis of Aib-containing small peptides, in order to obtain conformational data applicable to the more complex oligopeptide antibiotics. By working with such fragments it has been possible to obtain valuable X-ray crystal structure information on the helical conformation of ala-methin 246), emerimicin 247), suzukacillin 248), and other members of the peptaibol series. 

Steps a and b of this synthetic procedure were performed under standard activation and oxidation conditions. It is advantageous that in the final deprotection step c the trimethylsilylethyl group is cleaved by / -fragmentation by the action of TBAF simultaneously with tert-hutyl protective groups present in the oligopeptide chain. 

Some characteristic properties of iron-sulfur clusters in proteins result from interactions with invariant amino acid fragments around the clusters. In particular, clusters in peptide environments exhibit positive shifts in redox potentials relative to those in nonpeptide environments. Such shifts are observed for a variety of oligopeptide model complexes of IFe, 2Fe-2S, and 4Fe-4S proteins. 

Once we have this library, fragment-condensation procedures might link the oligopeptides to one another so as to build longer chains. Prebiotic methods for fragment condensation are not really known. Kent s synthesis comes close to the target (Kent, 1999) but is restricted to particular chemical structures and cannot be seen as a generalized prebiotic method. 

Let us see where this scheme may bring us, and for this purpose refer to Figure 4.7. This shows a small library of oligopeptides, where one (or more) of these products, indicated in the figure with an asterisk, is endowed with peptidase activity. These peptides then, under the reaction conditions, can catalyze fragment condensation in a specific way - for example of those peptides with a terminal aromatic residue. Following the first condensation step, the catalytic center is inserted into a longer polypeptide, with retention of the catalytic activity. 

Figure 4.7 Fragment condensation of short, prebiotically formed, oligopeptides. The asterisk indicates the catalytically active peptide, which can induce the fragment condensation by reverse peptide hydrolysis n peptides (e.g., ten residues long) react with each other to build ideally 20-peptides, and of these, m react further (to build ideally 40-peptides, of course in practice all possible mixtures may be present), and m are eliminated because of being unusable (e.g., insoluble) under the contingent conditions - and so on.

The experiment is used to identify the subspectra of isolated spin system.s and is most often used for molecules which consist of rows or networks of similar fragments with no coupling interactions between them, e.g. oligosaccharides, oligopeptides or oligonucleotides. The experiment is usually combined with a COSY experiment, where it helps to overcome problems of overlapping cross peaks that can arise in the latter case. 

Any number of amino acids can be joined by successive peptide linkages, forming a polypeptide chain. The polypeptide chain, like the dipeptide, has a directional sense. One end, called the N-terminal, or amino-terminal, end, has a free a-amino group, whereas the other end, the C-terminal, or carboxyl-terminal, end, has a free a-carboxyl group. The sequence of main-chain atoms from the N-terminal end to the C-terminal end is N—Ca—C—N and so on, and in the opposite direction it is C—Ca—N—C and so on. Short polypeptide chains, up to a length of about 20 amino acids, are called peptides or oligopeptides if they are fragments of whole polypeptide chains. A small protein mole-... 

Proteolysis of peptides and proteins commences in the stomach when pepsin is present. As a result, protein or peptide drugs will be hydrolyzed into smaller fragments like amino acids or oligopeptides, which are absorbed through the mucosa either by diffusion or by a carrier-mediated transport [18], In an average individual, 94-98% of the total protein is completely digested and absorbed [19], Proteolysis continues in the intestine with pancreatic enzymes like trypsine and brush-border enzymes. 

While chemical synthesis is mostly an art, with specialized reactions for both inorganic and inorganic synthesis, the complexities of biochemistry have nurtured specialized instruments that can split or assemble biomolecules. An automated solid-phase peptide synthesizer was introduced by Merrifield15 in 1963 this allows for the facile synthesis of oligopeptides (up to 100 amino acid units) [2], The enzyme DNA polymerase I was discovered by Komberg16 in 1957 this allowed the assembly of DNA from fragments [3]. 

Accurate 13C-15N interatomic distances have been measured by means of rotational echo double resonance (REDOR) experiments for oligopeptides [50]. The interatomic 13C-15N distance in the hydrogen bonded fragment was measured to be 4.5 0.1 A in five different samples studied. This finding is consistent with an a-helix structure, in agreement with conformation-dependent 13C chemical shift data. 

Studies of the synthesis of Fe(II) complexes of oligopeptides containing the two invariant Cys-X-Y-Cys fragments have been carried out to... 

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