Peptide ligation through click chemistry for the generation of assembled and scaffolded peptides

The synthesis of [1,2,3]-triazoles through copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition was examined for its utility to generate assembled and scaffolded peptides from peptide and scaffold precursors, which were N-terminally modified with azido and alkyne moieties, respectively.     

Messenger RNA-programmed incorporation of multiple N-methyl-amino acids into linear and cyclic peptides

Natural peptide products often contain N-methylated backbones, and such a modification plays a crucial role in making natural peptides peptidase resistant and membrane permeable. Here, we demonstrate the ribosomal synthesis of N-methyl-peptides by means of genetic code reprogramming. Two key technologies, a ribozyme-based de novo tRNA acylation (flexizyme) system and an E. coli reconstituted cell-free translation (PURE) system, were used in order to reassign arbitrarily chosen codons to N(alpha)-methylated amino acids ((Me)aa). Using this combination, we determined the general structural requirement of "accessible"(Me)aa and demonstrated their multiple incorporations into the nascent peptide chain according to the assignments made on mRNA, giving linear and cyclic N-methyl-peptides in high purities. This platform technology offers a convenient tool for the construction of N-methyl-peptide libraries, potentially leading to the discovery of therapeutic peptides.     

Rotaxanes of cyclic peptides

The synthesis of rotaxanes derived from the synthetic peptide macrocycles cyclo(l-ProGly)4 and cyclo(l-ProGly)5 and diammonium threads is described. [2]Rotaxanes are formed in good yields (56-63%), despite the disruption of internal amide-amide hydrogen bonding in the macrocycles.     

Structure of electron-capture dissociation fragments from charge-tagged peptides probed by tunable infrared multiple photon dissociation

In this study, we propose the first spectroscopic structural characterization of c-type ions produced by ECD of a peptide. The structure of c-type ions formed by electron capture dissociation and the overall mechanism leading to their formation are still a question of debate. Depending on the mechanism, c-type ions have been proposed to have either an enol-imine structure (-C(OH)NH) or an amide one (-C(O)-NH2). Since these ions are isomeric, mass spectrometry only cannot discriminate between them, but infrared spectroscopy can bring experimental evidence and help determine which scheme is operative. Using the coupling between a tunable free electron laser and a FT-ICR mass spectrometer, we show that c-type ions have an amide structure, characterized by an IR signature of the C=O stretch at 1731 cm(-1). This result is particularly interesting from the perspective of the elucidation of the ECD mechanism.     

beta-turn mimetic: synthesis of cyclic thioenamino peptides

[reaction: see text] Following the discovery of callynormine A, a marine metabolite of a new class, the cyclic endiamino peptides, and the synthesis of compounds of this group, we have now prepared an analogue group of compounds, i.e., cyclic thioenamine peptides. The latter peptides contain the alpha-amino-beta-thioacrylamide functionality, a potential new type of beta-turn mimic. The superiority of the SH group over the NH(2) group in the reaction with enol-tosylates was demonstrated.     

Synthesis of reversible cyclic peptides

A novel approach for the synthesis of cyclic peptides that can exist in either linear or cyclized conformations is described. Synthesis of the peptides was achieved via a modified solid phase methodology. The reversible linear/cyclized (i.e., open/closed) states are controlled via the reduction/oxidation of a disulfide bond incorporated into the backbone of the peptide chain.     

Synthesis of cyclic endiamino peptides

Several building blocks for endiamino peptides, as well as several cyclic endiamino peptides themselves, and pyrazin-6-one, which embodies the endiamino group, were prepared. The variety of synthesized compounds shows the potential of this synthesis in the preparation of many different groups of compounds.     

Fragmentation of biotinylated cyclic peptides

Electrospray ionization coupled with tandem mass spectrometry (MS/MS) was used to determine the preferred binding site(s) of biotin NHS ester with a series of cyclic peptides with antibiotic properties. The peptides investigated are polymyxins, cyclic peptides produced by Bacillus polymyxa. In spite of the 1:1 stoichiometry used in the labeling reaction, multiple biotin molecules were incorporated into intact polymyxin peptides. Given the amine specificity of the activated biotin and the large number of amino acids with primary amines in the polymyxins, it was not clear by inspection which binding sites were more reactive than others. MS/MS was used to characterize the structure of the biotinylated peptides. MS/MS spectra of cyclic peptides often lead to ambiguous structure determinations due to the potential for multiple ring openings which result in the generation of multiple ion series. The MS/MS spectra of polymyxin peptides are especially difficult to characterize due to the lack of variety in their amino acids; however, the added complexity of the biotin aided the elucidation of the fragmentation pathways. MS/MS spectra of the species with biotin additions were used to rationalize the preferential binding sites of these molecules.     

Computer-assisted structure generation from a gross formula: II. Multiple bond unsaturated and cyclic compounds. Employment of fragments

A general method for the generation of molecular structures from a gross formula is described. This method allows the construction of different classes of structures: saturated, multiple bond, conjugated, as well as cyclic and acyclic, in a uniform way. The employment of fragments within this approach is provided.For part 1, see ref. [1].     

Kinetics of comet formation in single‐cell gel electrophoresis: Loops and fragments

We investigated the mechanisms of DNA exit during single‐cell gel electrophoresis (the comet assay) by measuring the kinetics of the comet tail formation. In the neutral comet assay, the rate of DNA exit was found to be dependent on the topological state of DNA, which was influenced by either ethidium bromide or a low radiation dose. The results clearly show that the comet tail is formed by extended DNA loops: the loop extension, being reversible when the DNA torsional constraint remains in the loops, is favored when the constraint is relaxed. The kinetics of the comet formation in the case of a high radiation dose points out that accumulation of the single‐strand breaks causes DNA fragmentation. In contrast to the neutral comet assay, the alkaline comet assay is not related to the chromatin loops. Our results imply that the alkaline treatment induces detachment of the loops from the nuclear matrix, and the comet tail is formed by ssDNA fragments, the ends of which are pulled out from the comet head by electric force. We suggest that the kinetic approach can be considered as an important improvement of the comet assay.     

Travelling fingers in two-frequency cholesteric liquid crystals: fragments, loops and spirals

We review the different dynamical patterns that cholesteric fingers of the first type (CF-1) and of the second type (CF-2) form in an a.c. electric field near the coexistence line with the homeotropic nematic phase. Videomicroscopy and computer image analysis were used for investigation of the patterns in polarized light. We show that CF-1s can form stable rectilinear fragments that crawl at constant velocity along their axes, whereas CF-2s form only unstable curved fragments that drift perpendicularly to their axes. Observations of CF-2 staple-shaped fragments which continuously lengthen in their centres are also reported. Finally, we describe in detail the experimental conditions in which CF-2 loops and spirals grow, collapse and destabilize.     

Travelling fingers in two-frequency cholesteric liquid crystals: fragments,loops and spirals

We review the different dynamical patterns that cholesteric fingers of the first type (CF-1) and of the second type (CF-2) form in an a.c. electric field near the coexistence line with the homeotropic nematic phase. Videomicroscopy and computer image analysis were used for investigation of the patterns in polarized light. We show that CF-1s can form stable rectilinear fragments that crawl at constant velocity along their axes, whereas CF-2s form only unstable curved fragments that drift perpendicularly to their axes. Observations of CF-2 staple-shaped fragments which continuously lengthen in their centres are also reported. Finally, we describe in detail the experimental conditions in which CF-2 loops and spirals grow, collapse and destabilize.     

Nonribosomal cyclic peptides: specific markers of fungal infections

Some cyclic peptides and depsipeptides are synthesized in microorganisms by large multienzymes called nonribosomal peptide synthetases. The structures of peptide products originating in this way are complex and diverse and are microorganism-specific. This work proposes the use of fungal cyclic peptides and depsipeptides as extremely specific markers of fungal infections. Since a reliable molecular tool for diagnosing fungal infections at an early stage is still missing, we present mass spectrometry as a new, modern, broadband (with respect to fungal strain) and specific tool for clinical mycologists. More than 40 different fungal species can be rapidly characterized according to specific families of cyclic peptides, and in some cases, a particular fungal strain can be identified on the basis of its cyclopeptide profile. This paper is also aimed at initiating discussion on the biological role of these secondary metabolites, especially of those synthesized by medically important strains. Proven cytotoxic, anti-inflammatory or immunosuppressive activities of some cyclic peptides indicate that these molecules may contribute to the synergistic array of fungal virulence factors and support microbial invasion during fungal infection. In addition to an overview on recent mass spectrometric protocols for cyclic peptide sequencing, the structures of new peptides from Paecilomyces and Pseudallescheria are presented.     

Exploring privileged structures: the combinatorial synthesis of cyclic peptides

Head-to-tail cyclic peptides have been reported to bind to multiple, unrelated classes of receptor with high affinity. They may therefore be considered to be privileged structures. This review outlines the strategies by which both macrocyclic cyclic peptides and cyclic dipeptides or diketopiperazines have been synthesised in combinatorial libraries. It also briefly outlines some of the biological applications of these molecules, thereby justifying their inclusion as privileged structures.     

Use of antibody fragments in immunoaffinity chromatography. Comparison of FV fragments, VH fragments and paralog peptides.

Some new antibody fragments have recently been described: FV fragments (Mr 25,000), VH fragments or "dAbs" (12,500) and paralog peptides (1000-2000). FV fragments, VH fragments and a paralog peptide that had been derived from a parent antibody with a specificity for hen lysozyme were produced. All three reagents were immobilized on Sepharose and evaluated for their ability to recover hen lysozyme from "spiked" serum and to separate hen lysozyme from turkey lysozyme. The FV column had excellent specificity for hen lysozyme, the VH column had significantly reduced specificity and the paralog peptide column did not bind lysozyme at all.     

Synthesis of cyclic peptides by ring-closing metathesis

The synthesis of a series of "amide to amide" cyclized peptides by ring-closing metathesis (RCM) as well as a convenient synthesis for the linear precursors is described. In addition, the influence of the length of the alkene substituents and the influence of the peptide sequence is investigated, leading to a set of general rules to obtain "amide to amide" cyclized peptides by RCM.     

Acyclic and cyclic thioenamino peptides: solution- and solid-phase synthesis

Seven- and 10-membered cyclic thioenamino peptides, that is, 1,4-thiazepinone (11) and cyclic thioenamino peptide 9 (which represents a potential γ-turn mimetic), were synthesized, and the structure of 11 was secured by X-ray diffraction analysis of its TFA salt. The aforementioned compounds were prepared in solution and by solid-phase synthesis. Additionally, we have prepared thioenamino diketopiperazine synthon 16.     

DFT study on reaction mechanisms of cyclic dipeptide generation

In this work, three possible reaction pathways (Path 1, Path 2 and Path 3) for the generation process of cyclic dipeptide from amino acid have been investigated in detail using density functional theory. Path 1 and Path 2 are the intramolecular reaction processes, while Path 3 involves the intermolecular reaction process that assisted with water molecule. Our calculated results indicate that Path 3 is more energy favorable than Path 1 and Path 2. There are four steps in Path 3 proceed from the amino acid to cyclic dipeptide. The first step is two adjacent amino acids to form precursor of dipeptide, the second step is the removal of water molecule of precursor of dipeptide for the formation of the linear dipeptide, the third step is generation of precursor of cyclic dipeptide associated with other hydrogen atom transfer, and the last step is another dehydration process to generate the final product of cyclic dipeptide. Moreover, the obtained results indicate that the generation mechanisms of different cyclic dipeptides are similar, and the energy barrier of the rate-determined step influenced somewhat by the hydrophilic or hydrophobic group linked to the C_α atom. Additionally, the potential energy profiles suggest that the generation reactions of the studied nine cyclic dipeptides are exothermic processes. The detailed mechanisms should be helpful for people to understanding the title reaction at the molecular level, and the proposed novel intermolecular process might provide valuable insights on rational improve reaction condition for this type of reaction.