Synthesis of peptide aldehydes.

The functionalization of peptides and proteins by aldehyde groups has become the subject of intensive research since the discovery of the inhibition properties of peptide aldehydes towards various enzymes. Furthermore, peptide aldehydes are of great interest for peptide backbone modification or ligation reactions. This review focuses upon their synthesis, which has been developed following two main strategies. The first strategy consists of prior synthesis of the peptide, followed by the introduction of the aldehyde function. The second possible strategy uses alpha-amino aldehydes as starting materials. After protection of the aldehyde, peptide elongation occurs. At the end of the synthesis, the aldehyde function can be unmasked.     

Pepsin as a catalyst of peptide synthesis. Enzyme co-precipitation with emerging peptide products.

Pepsin successfully catalyzed the synthesis of several peptide derivatives from N-protected di- or tripeptides and amino acid or peptide esters or p-nitroanilides in dimethylformamide-water solutions at pH 4.6. An optimal substrates:pepsin ratio depended on the structure of starting peptides, especially their fit to the substrate binding sites of the enzyme. For hexapeptide Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 formation, an equilibrium yield was attained at 1:3.10(5) enzyme-substrates ratio that indicated high efficiency of pepsin in synthesis reactions. In the course of the equilibrium peptide synthesis, pepsin gradually disappeared from the liquid phase due to its entrapment within a gel, formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin, so far as inert proteins, lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin, an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.     

Aib-based peptide backbone as scaffolds for helical peptide mimics.

Helical peptides that can intervene and disrupt therapeutically important protein-protein interactions are attractive drug targets. In order to develop a general strategy for developing such helical peptide mimics, we have studied the effect of incorporating alpha-amino isobutyric acid (Aib), an amino acid with strong preference for helical backbone, as the sole helix promoter in designed peptides. Specifically, we focus on the hdm2-p53 interaction, which is central to development of many types of cancer. The peptide corresponding to the hdm2 interacting part of p53, helical in bound state but devoid of structure in solution, served as the starting point for peptide design that involved replacement of noninteracting residues by Aib. Incorporation of Aib, while preserving the interacting residues, led to significant increase in helical structure, particularly at the C-terminal region as judged by nuclear magnetic resonance and circular dichroism. The interaction with hdm2 was also found to be enhanced. Most interestingly, trypsin cleavage was found to be retarded by several orders of magnitude. We conclude that incorporation of Aib is a feasible strategy to create peptide helical mimics with enhanced receptor binding and lower protease cleavage rate.     

N-terminal peptide sequence of goat-beta-lactoglobulin.

Three cyanogen bromide peptides from native goat beta-lactoglobulin have been isolated by gel-filtration. The N-terminal fragment has been identified and its sequence was determined to be: Ile-Val-Thr-Gln-Thr-. The results are compared with the N-terminal region of cow beta-lactoglobulins A and B.     

"Joining peptide" of pro-opiomelanocortin. II. Interspecies heterogeneity of the joining peptide fragment

The use of an antiserum raised against the joining peptide sequence -23 to -14 of bovine pro-opiomelanocortin (POMC) enabled the detection of related immunoreactive sequences of peptides in bovine, porcine, mouse and guinea-pig pituitaries, as well as in mouse brain and cerebral cortex, guinea-pig cerebral cortex, and bovine hypothalamus. Gel chromatography of pituitary extracts (Sephadex G-75 and Bio-Gel P-4) indicated the presence of several immunoreactive joining peptide fragments ranging in the molecular weight range (Mr) of 1,500 to 2,300. Furthermore, high molecular weight (Mr greater than 22,500) immunoreactive-precursor from bovine anterior pituitary was readily digested with trypsin into an immunoreactive fragment of approximately Mr 1,500. Analyses of these immunoreactive peptides by reverse-phase high-performance liquid chromatography (HPLC) led to their resolution into six distinct peptides. The only apparent correspondence in the elution profiles of immunoreactive peptide profiles between different mammalian species was the identification of a similar fragment (Mr 2,000) from bovine and guinea-pig pituitaries. Thus, we conclude that immunoreactivity to the joining peptide region of POMC from various mammalian species exhibits a degree of heterogeneity in its composition. The relatively low levels of immunoreactivity in comparison to that of ACTH also suggest that the joining peptide domain may be further processed. The hormonal status of the joining peptide region remains to be determined.     

Gastrointestinal peptide hormone analogues.

All derivatives of the C-terminal tetrapeptide fragment of gastrins with 17 amino acids elicit every physiological effect of the gastrin. This recognation led to detailed studies on the structure-activity relationships. In the case of cholecystokinin-pancreozymin, a substance consisting of 33 amino acids, the smallest sequence which possesses biological activity is the C-terminal octapeptide fragment. Synthesis of analogous derivatives of this fragment allowed the determination of the structural elements necessary to evoke the biological action.     

Methods and strategies of peptide ligation.

This review focuses on the concept, methods, and strategies of orthogonal peptide ligation. It updates our previous review in 1999 on the same subject matter in Biopolymers (Peptide Science, 1999, Vol. 51, p. 311). Orthogonal peptide ligation is an amino terminal specific method to couple chemically unprotected peptides or proteins derived from synthetic or biosynthetic sources. Unlike conventional chemical methods, peptide ligation methods do not require coupling reagents or protection schemes, but are achieved through a variable chemoselective capture step and then an invariable intramolecular acyl transfer reaction. It is also a convergent method with the fewest steps. More than a dozen orthogonal ligation methods have been developed based on captures by either imine or thioester chemistries to afford native and unusual amino acids at ligation sites of linear, branched, or cyclic peptides. The ligation strategies for multiple segments including sequential and tandem ligations are also discussed.     

The proteinase-catalysed synthesis of peptide hydrazides.

1. We report the trypsin-catalysed conversion, in high yield, of peptides to peptide hydrazides, t-butyloxycarbonylhydrazides and phenylhydrazides. The substitution is at the alpha-carboxy group. 2. We discuss the relative merits of carrying out the conversion either simultaneously with tryptic cleavage of the parent protein or after such cleavage. 3. We report analogous results with chymotrypsin, elastase and subtilisin. 4. We propose the use of such products in protein semi-synthesis and in the preparation of specific proteinase inhibitors.     

Affinity enhancement of complementary peptide recognition.

A peptide hydropathically complementary to Big Endothelin [Big ET] residues 16-29 has been synthesized in a multimeric form starting from an octadentate polylysine core, essentially in a way similar to the procedure used for the production of multiple antigenic peptides [MAP's]. Interaction between the multimeric complementary peptide [8 delta ET] and the Big ET fragment 16-32 containing the target complementary region, also synthesized in a multimeric form [8ET], was evaluated by analytical high performance affinity chromatography and solid phase binding assays. While the binding interaction between the monomerics peptide pair was in the micromolar range, the recognition between the corresponding multimeric form was characterized by enhanced binding affinity of at least two orders of magnitude. In solution, complex formation between multimeric complementary peptide and target Big ET sequence in the monomeric and multimeric form was accompanied by precipitation at concentrations higher than 0.5 mg/mL and 0.1 mg/mL, respectively. Polyclonal antibodies raised against the multimeric target sequence recognized multimeric and monomeric ET target sequences with binding affinities similar to binding affinities exhibited by the multimeric complementary peptide. Multimerization of hydropathically complementary peptides could provide an improved opportunity to measure and thus probe quantitative binding properties of complementary peptides.     

Controlled disassembly of peptide amphiphile fibres.

In this paper, the introduction of both a methionine residue and a nitrobenzyl derivative as a labile linker between the peptide part and the hydrophobic alkyl chain of a peptide amphiphile are presented. These modifications are shown not to inhibit the formation of structured assemblies that analogous peptide amphiphiles lacking the linkers are able to form. Moreover, the introduction of either labile linker allows removal of the peptide amphiphile's stabilizing hydrophobic moieties to initiate a controlled disassembly of fibre aggregates. This is achieved by either treatment with CNBr or UV irradiation, respectively. These disassembly mechanisms could be the starting point for methodology that allows further manipulation of self-assembled peptide amphiphile architectures.