Cell-wall engineering of living bacteria

The cell walls of living bacteria were chemically modified by adding cell-wall precursors. As the precursors to be incorporated into the cell wall, UDP-MurNAc pentapeptide, lipid I, and lipid II derivatives were synthesized. The aimed compounds were attached to the amine residue of lysine at the pentapeptide moiety. Fluorescein-attached UDP-MurNAc pentapeptide was efficiently incorporated into both Gram-positive and Gram-negative bacteria. In the case of Gram-negative bacteria, such as Escherichia coli, the permeability of the outer membrane (lipopolysaccharide layer) was enhanced by EDTA treatment before the incorporation. For Gram-positive bacteria, UDP-MurNAc derivatives were incorporated in the cell wall without EDTA treatment due to the lack of the lipopolysaccharide layer. Furthermore, instead of dyes, a ketone group was attached to the UDP-MurNAc pentapeptide. The ketone group was also delivered to the bacterial cell wall of lactic acid bacteria, giving a platform to attach large molecules on the surface.     

Chemoenzymatic synthesis of Park’s nucleotide: toward the development of high-throughput screening for MraY inhibitors

An efficient chemoenzymatic synthesis of UDP-N-acetylmuramyl-l-alanyl-γ-d-glutamyl-meso-diaminopimelyl-d-alanyl-d-alanine (Park’s nucleotide) is reported. UDP-MurNAc is efficiently synthesized by a minimum number of protecting strategies. One-pot amino acid ligation reactions catalyzed by MurC, D, E, and F enzymes are amenable to scale-up production.     

Ring-closing metathesis for the synthesis of side chain knotted pentapeptides inspired by vancomycin

A versatile method for the synthesis of bicyclic side chain knotted peptides inspired by vancomycin is described. The synthetic approach is based on the incorporation of a central amino acid derivative having two allylic groups-introduced by a Stille coupling-into pentapeptide 8 containing two allylated serine residues. Treatment of this bis-ring-closing metathesis precursor with 2nd generation Grubbs catalyst results in the formation of a bicyclic pentapeptide with the correct side chain to side chain connectivity pattern as observed in vancomycin: i- 2 --> i, i --> i + 2. Modelling studies using MacroModel hint at a cavity-like structure of the bicyclic pentapeptide which may bind suitable ligands.     

Enhanced peptide beta-sheet affinity by metal to ligand coordination

A histidine-coordinating metal complex substituted with methoxypyrrole amino acids (MOPAS) as a peptide beta-sheet binder shows high affinity for a H(2)N-His-Leu-Leu-Val-Phe-OMe pentapeptide in DMSO solution. Within the complex, a beta-sheet conformation is induced into the pentapeptide by weak intra-assembly interactions with the MOPAS units.     

Asymmetric synthesis of the C(1)---C(47) backbone of antitumour antibiotic A83586C

Protected pentapeptide 9 has been converted to hexapeptide 3 and this chemoselectively coupled to activated ester 2 to give 1 after glycal hydration during SiO₂ flash chromatography.     

Total synthesis of modified pentapeptide,caldoramide

Total synthesis of modified pentapeptide, caldoramide, a cytotoxic linear pentapeptide from the marine cyanobacterium Caldora penicillate is described. Notable features of the route include the efficient preparation of three key fragments and final assembly to the natural product via sequential amide couplings. The spectral data of the synthetic compound was found to be in comparison with that of the isolated natural product.     

Isolation, structure determination, and synthesis of galaxamide, a rare cytotoxic cyclic pentapeptide from a marine algae Galaxaura filamentosa

Galaxamide (1), a rare cyclic pentapeptide, was isolated from the marine algae Galaxaura filamentosa. A preliminary bioassay of Galaxamide showed remarkable in vitro antiproliferative activities against GRC-1 and HepG2 cell lines. The first total synthesis of the cyclic peptide was achieved for further biological evaluation.     

Efficient Access to Peptidyl‐RNA Conjugates for Picomolar Inhibition of Non‐ribosomal FemXWv Aminoacyl Transferase

Peptidyl–RNA conjugates have various applications in studying the ribosome and enzymes participating in tRNA‐dependent pathways such as Fem transferases in peptidoglycan synthesis. Herein a convergent synthesis of peptidyl–RNAs based on Huisgen–Sharpless cycloaddition for the final ligation step is developed. Azides and alkynes are introduced into tRNA and UDP‐MurNAc‐pentapeptide, respectively. Synthesis of 2′‐azido RNA helix starts from 2′‐azido‐2′‐deoxyadenosine that is coupled to deoxycytidine by phosphoramidite chemistry. The resulting dinucleotide is deprotected and ligated to a 22‐nt RNA helix mimicking the acceptor arm of Ala‐tRNA^Ala by T4 RNA ligase. For alkyne UDP‐MurNAc‐pentapeptide, meso‐cystine is enzymatically incorporated into the peptidoglycan precursor and reduced, and L ‐Cys is converted to dehydroalanine with O‐(mesitylenesulfonyl)hydroxylamine. Reaction of but‐3‐yne‐1‐thiol with dehydroalanine affords the alkyne‐containing UDP‐MurNAc‐pentapeptide. The Cu^I‐catalyzed azide alkyne cycloaddition reaction in the presence of tris[(1‐hydroxypropyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amine provided the peptidyl‐RNA conjugate, which was tested as an inhibitor of non‐ribosomal FemX_Wv aminoacyl transferase. The bi‐substrate analogue was found to inhibit FemX_Wv with an IC₅₀ of (89±9) pM , as both moieties of the peptidyl–RNA conjugate contribute to high‐affinity binding.     

A heterocyclic analog of leucine-enkephalin

A series of di-, tri-, tetra- and pentapeptide analogs of leucine-enkephalin have been prepared in which the initial tyrosinylglycine fragment has been replaced by the 5,6-dihydro-5,5-dimethyl-2-oxo-3-phenyl-1(2H)-pyrazineacetic acid moiety.     

Macrocyclic β-sheet peptides that inhibit the aggregation of a tau-protein-derived hexapeptide

This paper describes studies of a series of macrocyclic β-sheet peptides 1 that inhibit the aggregation of a tau-protein-derived peptide. The macrocyclic β-sheet peptides comprise a pentapeptide "upper" strand, two δ-linked ornithine turn units, and a "lower" strand comprising two additional residues and the β-sheet peptidomimetic template "Hao". The tau-derived peptide Ac-VQIVYK-NH(2) (AcPHF6) aggregates in solution through β-sheet interactions to form straight and twisted filaments similar to those formed by tau protein in Alzheimer's neurofibrillary tangles. Macrocycles 1 containing the pentapeptide VQIVY in the "upper" strand delay and suppress the onset of aggregation of the AcPHF6 peptide. Inhibition is particularly pronounced in macrocycles 1a, 1d, and 1f, in which the two residues in the "lower" strand provide a pattern of hydrophobicity and hydrophilicity that matches that of the pentapeptide "upper" strand. Inhibition varies strongly with the concentration of these macrocycles, suggesting that it is cooperative. Macrocycle 1b containing the pentapeptide QIVYK shows little inhibition, suggesting the possibility of a preferred direction of growth of AcPHF6 β-sheets. On the basis of these studies, a model is proposed in which the AcPHF6 amyloid grows as a layered pair of β-sheets and in which growth is blocked by a pair of macrocycles that cap the growing paired hydrogen-bonding edges. This model provides a provocative and appealing target for future inhibitor design.     

Application of Pac Ester in Thioester Method for the Synthesis of Cyclopentapeptides

Thioester method for the synthesis of cyclopeptides is improved by using Pac (Pac = phenacyl, CH2COC6Hs) ester as a protecting group of 3-mercaptopropionic acid. The Pac group is easy to be removed from C-terminal with zinc in acetic acid. The protected glycine thioester and peptide thioesters synthesized by the improved method, are easy to be purified, so the final linear peptides are pure enough for the following cyclization. Furthermore, this method is flexible for peptide chain elongation,either from C-termlnal or from N.terminal. So it is an efficient and practical method for synthesis of bioactive peptides. Two N-protected pentapeptide thioesters, Boc-Pro-Tyr-Leu-Ala-GIySCH2CH2COOPac and Boc-Ala-Tyr-Leu-Ala-Gly-SCH2CH2COOPac were synthesized by the improved thloester method.After deprotecting Pac ester with zinc in aqueous acetic acid and Boc group with trifluoroacetic acid in CH2C12, two free pentapeptide tldoesters were obtained. Ag^ -assisted cyclization in acetate buffered solution afforded two cyclic pentapeptides c(Pro-Tyr-Leu-Ala-Gly) and c(Ala-Tyr-Leu-Ala-Gly).Effects of different buffer pH, different Ag^ concentrations, etc. on the cyclization were studied.     

Intramolecular Transfer of Excitation Energy,Complexation with Metal Ions,and Interactions with Liposomes of Cyclo [tryptophyl-sarcosyl-sarcosyl-Nϵ-dansyllysyl-prolyl]

A cyclic pentapeptide containing an energy donor group and an energy acceptor group within the molecule, cyclo [Trp-Sar-Sar-Lys (DNS)-Pro], was synthesized. Its conformation, complexation with metal ions, intramolecular energy transfer, and interaction with liposomes were investigated. The precursor, cyclo [Trp-Sar-Sar Lys ( Z )-Pro], contained one cis-sarcosyl peptide bond and a β-turn in which the Pro-residue occupied the 3rd position. This cyclic pentapeptide formed a complex selectively with Ba²⁺ and its binding constant in 95% aqueous methanol solution was 2.0 × 10³ 1 · mol^?1. The intramolecular energy-transfer efficiency of cyclo [Trp-Sar-Sar-Lys (DNS)-Pro] was almost constant between ? 60° and 40° in ethanol, while it increased at temperatures lower than 34° in liposomes as a result of the pre-transition of the lipid assembly. On the other hand, the intramolecular energy transfer efficiency of cyclo [Trp-Sar-Sar-Lys (DNS)-Pro] decreased upon complexation with Ba²⁺.     

Asymmetric synthesis of (2S,3R)-capreomycidine and the total synthesis of capreomycin IB

A 27 step total synthesis of the tuberculostatic macrocyclic peptide antibiotic capreomycin IB has been accomplished. The synthesis features the use of an enolate-aldimine condensation between a chiral glycine aluminum enolate and the benzyl imine of 3-tert-butyldimethylsiloxy-propanal as a means of preparing the cyclic guanidine amino acid (2S,3R)-capreomycidine. Additionally, a Hofmann rearrangement was exacted on a late-stage pentapeptide in order to transform an asparagine residue into a diaminopropanoic acid residue.     

Amino acids and peptides. XXXIII. Synthesis of N-terminal epitope peptides of mammalian metallothioneins (MTs).

In order to determine the fine structure of the mammalian metallothionein (MT) epitope to a monoclonal anti-rat Zn-MT-II antibody (MT 189-14-7), N-terminal peptides of various lengths of mammalian metallothioneins (MTs) were synthesized by a conventional solution method using the newly developed beta-2-adamantylaspartate, and their immunological properties were examined. It was found that the N-terminal acetyl group was indispensable for the reaction with the monoclonal antibody and the N-terminally acetylated pentapeptide, Ac-Met-Asp-Pro-Asn-Cys-OH, was the smallest peptide which exhibited a significant reactivity with the antibody.     

Asymmetric synthesis of the C(1)C(47) backbone of antitumour antibiotic A83586C

Protected pentapeptide 9 has been converted to hexapeptide 3 and this chemoselectively coupled to activated ester 2 to give 1 after glycal hydration during SiO₂ flash chromatography.     

Application of tandem Ugi multi-component reaction/ring closing metathesis to the synthesis of a conformationally restricted cyclic pentapeptide

A conformationally restricted cyclic pentapeptide, containing an unsaturated 9-membered lactam as a semi-rigid scaffold, was prepared in a very convergent manner, through tandem Ugi reaction/ring closing metathesis.     

Matrix-free laser desorption/ionization of ions landed on plasma-treated metal surfaces

We report new experiments in which laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF-MS) was applied to detection and characterization of gramicidin S and IgG pentapeptide (DSDPR) that were reactively landed on plasma-treated stainless steel surfaces. The distributions of [M + H](+), [M + Na](+) and [M + K](+) ion species in LDI-TOF for gramicidin S and IgG pentapeptide (DSDPR) were found to be markedly different from those in conventional MALDI-TOF spectra of the same samples. LDI-TOF mass spectra showed a strong preference for [M + K](+) adducts even in the presence of a large excess of sodium cations, or following surface treatment with trifluoroacetic acid. Alkali metal cations (K(+) and Cs(+)) can be exchanged in reactively landed peptide samples to provide the corresponding cationized peptide ions by LDI. Multiple charged trypsin cations were reactively landed into a layer of 2-(4-hydroxyphenylazo)benzoic acid and ionized by LDI. The ionization mechanisms for LDI of surface-deposited peptides are briefly discussed. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Conformational preference and potential templates of N-methylated cyclic pentaalanine peptides

Systematic N-methylation of all peptide bonds in the cyclic pentapeptide cyclo(-D-Ala-Ala(4)-) has been performed yielding 30 different N-methylated derivatives, of which only seven displayed a single conformation on the NMR time scale. The conformation of these differentially N-methylated peptides was recently reported by us (J. Am. Chem. Soc. 2006, 128, 15 164-15 172). Here we present the conformational characterization of nine additional N-methylated peptides from the previous library which are not homogeneous but exist as a mixture in which at least one conformation is preferred by over 80 %. The structures of these peptides are investigated employing various 2D-NMR techniques, distance geometry calculations and further refined by molecular dynamics simulations in explicit DMSO. The comparison of the conformation of these nine peptides and the seven conformationally homogeneous peptides allow us to draw conclusions regarding the influence of N-methylation on the peptide backbone of cyclic pentapeptide of the class cyclo(-D-Ala-Ala(4)-). Here we present the different conformational classes of the peptides arising from the definitive pattern of N-methylation which can eventually serve as templates for the design of bioactive peptides.     

In Silico and Experimental Characterization of Chimeric Bacillus thermocatenulatus Lipase with the Complete Conserved Pentapeptide of Candida rugosa Lipase

Lipases are one of the highest value commercial enzymes as they have broad applications in detergent, food, pharmaceutical, and dairy industries. To provide chimeric Bacillus thermocatenulatus lipase (BTL2), the completely conserved pentapeptide (¹¹²Ala-His-Ser-Gln-Gly¹¹⁶) was replaced with similar sequences (²⁰⁷Gly-Glu-Ser-Ala-Gly²¹¹) of Candida rugosa lipase (CLR) at the nucleophilic elbow region. For this purpose, three mutations including A112G, H113E, and Q115A were inserted in the conserved pentapeptide sequence of btl2 gene. Based on the crystal structures of 2W22, the best structure of opened form of the chimeric lipases were garnered using the MODELLER v9.10 software. The native and chimeric lipases were docked to a set of ligands, and a trial version of Molegro Virtual Docker (MVD) software was used to obtain the energy values. Docking results confirmed chimeric lipase to be better than the native lipase. Following the in silico study, cloning experiments were conducted and expression of native and chimeric btl2 gene in Pichia pastoris was performed. The native and chimeric lipases were purified, and the effect of these mutations on characteristics of chimeric lipase studied and then compared with those of native lipase. Chimeric lipase exhibited 1.6-fold higher activity than the native lipase at 55 °C. The highest percentage of both lipases activity was observed at 60 °C and pH of 8.0. The ion Ca²⁺ slightly inhibited the activity of both lipases, whereas the organic solvent enhanced the lipase stability of chimeric lipase as compared with the native lipase. According to the results, the presence of two glycine residues at the conserved pentapeptide region of this chimeric lipase (¹¹² Gly-Glu-Ser-Ala-Gly ¹¹⁶) may increase the flexibility of the nucleophilic elbow region and affect the enzyme activity level.     

Biomimetic formation of gramicidin S by dimerization-cyclization of pentapeptide precursor on solid support

The biomimetic formation of gramicidin S, cyclo(-d-Phe-Pro-Val-Orn-Leu-)₂, by the dimerization and cyclization of pentapeptide precursor without the protection of δ-amino group of the Orn residue was examined on a solid support. The cyclization of H-d-Phe-Pro-Val-Orn-Leu-oxime on a resin with an oxime group of 0.62 mmol/g in 1,4-dioxane directly gave gramicidin S in a 50% yield. The dimerization-cyclization mode on the solid support was similar to that of the biosynthesis of gramicidin S on an enzyme.