Peptide bond modifications

Conradi RA, AR Hilgers, NFH Ho, PS Burton. (1992). The influence of peptide structure on transport across Caco-2 cells. II. Peptide bond modification which results in improved permeability. Pharm Res 9 435-439. 

Extensive studies of enzyme-substrate complexes by resonance Raman spectroscopy (RR) have prompted the synthesis of new peptide bond modifications such as thionoesters and dithioesters (Scheme l7)t82-83l within simple model substrates. The resulting acyl-enzyme complexes are especially amenable to RR analysis with cysteine proteases such as papain due to formation of the transient dithioester intermediates. 

This chapter aims to present an overview on the main peptide bond modifications which have been proposed in the recent years. The solution- and solid-phase synthesis of the new synthetic oligomers, the methods for their characterization, and their biological and folding properties will be discussed. Particular emphasis will be given to those modifications which are suitable for combinatorial chemistry applications and developments. 

Single Position Peptide Bond Modifications 7.9.1 Thioamide Pseudopeptides... 

One of the most subtle peptide bond modifications is the insertion of a thioamide /[CSNH] group, in which the carbonyl oxygen has been replaced by a sulfur atom [125, 126], Thioamide formations became more easily accessible with the introduction of the thionating Lawesson s reagent. 

This same species subsequently yielded callipeltins J-M, which are related to callipeltins C and B by breakage of peptide bonds, modification of an amino acid or internal cydization (D Auria et al., 2007). 

Post-translation modification Changes that occur to proteins after peptide-bond formation has occurred, e.g. glycosylation and acylation. 

In view of these constraints, we recently suggested a different strategy for the improvement of the material properties of synthetic poly (amino acids) (12). Our approach is based on the replacement of the peptide bonds in the backbone of synthetic poly(amino acids) by a variety of "nonamide" Linkages. "Backbone modification," as opposed to "side chain modification," represents a fundamentally different approach that has not yet been explored in detail and that can potentially be used to prepare a whole family of structurally new polymers. 

Our interest in the synthesis of poly (amino acids) with modified backbones is based on the hypothesis that the replacement of conventional peptide bonds by nonamide linkages within the poIy(amino acid) backbone can significantly alter the physical, chemical, and biological properties of the resulting polymer. Preliminary results (see below) point to the possibility that the backbone modification of poly(amino acids) circumvents many of the limitations of conventional poly(amino acids) as biomaterials. It seems that backbone-modified poly (amino acids) tend to retain the nontoxicity and good biocompatibility often associated with conventional poly (amino acids)... 

In mammalian cells, the two most common forms of covalent modification are partial proteolysis and ph osphorylation. Because cells lack the ability to reunite the two portions of a protein produced by hydrolysis of a peptide bond, proteolysis constitutes an irreversible modification. By contrast, phosphorylation is a reversible modification process. The phosphorylation of proteins on seryl, threonyl, or tyrosyl residues, catalyzed by protein kinases, is thermodynamically spontaneous. Equally spontaneous is the hydrolytic removal of these phosphoryl groups by enzymes called protein phosphatases. 

The modification of amino acids in proteins and peptides by oxidative processes plays a major role in the development of disease and in aging (Halliwell and Gutteridge, 1989, 1990 Kim et al., 1985 Tabor and Richardson, 1987 Stadtman, 1992). Tissue damage through free radical oxidation is known to cause various cancers, neurological degenerative conditions, pulmonary problems, inflammation, cardiovascular disease, and a host of other problems. Oxidation of protein structures can alter activity, inhibit normal protein interactions, modify amino acid side chains, cleave peptide bonds, and even cause crosslinks to form between proteins. 

Certain bifunctional metal chelating agents have been used to investigate protein interactions by virtue of their ability to generate reactive oxygen species that affects protein structure in the immediate vicinity of their modification site. The following sections discuss two applications of such chelate labels, one of which cleaves peptide bonds while the other one causes covalent crosslinks to occur between interacting protein structures. 

Transition metals such as iron can catalyze oxidation reactions in aqueous solution, which are known to cause modification of amino acid side chains and damage to polypeptide backbones (see Chapter 1, Section 1.1 Halliwell and Gutteridge, 1984 Kim et al., 1985 Tabor and Richardson, 1987). These reactions can oxidize thiols, create aldehydes and other carbonyls on certain amino acids, and even cleave peptide bonds. The purposeful use of metal-catalyzed oxidation in the study of protein interactions has been done to map interaction surfaces or identify which regions of biomolecules are in contact during specific affinity binding events. 

The mechanism by which serine peptidases, particularly serine endopep-tidases (EC 3.4.21), hydrolyze peptide bonds in peptides and proteins has been extensively investigated by X-ray crystallography, site-directed mutagenesis, detection of intermediates, chemical modification, H-NMR spectroscopy, and neutron diffraction [2-14], These studies revealed that all serine peptidases possess a catalytic triad, composed of a serine, a histidine, and an aspartate residue, and a so-called oxyanion hole formed by backbone NH groups. 

The tripeptides in Fig. 6.17 underwent a few breakdown reactions (N-ter-minus elimination, Qm formation, peptide bond hydrolysis), some of which will be considered later in this section. Of relevance here was that, of the two amidated tripeptides, the amide at the C-terminus underwent deamidation predominantly (Fig. 6.17, Reaction a), which, perhaps, explains the somewhat lesser stability compared to the free carboxylic acid forms. While the hexapeptide (6.52, Fig. 6.17) followed a different pattern of decomposition [76b], deamidation was also a predominant hydrolytic reaction at all pH values. Thus, the procedure to extrapolate results from small model peptides to larger medicinal peptides appears to be an uncertain one, since small modifications in structure can cause large differences in reactivity. 

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