Synthesis of gramicidin S and its analogues via an on-resin macrolactamization assisted by a predisposed conformation of the linear precursors

A simple and efficient preparation of gramicidin S and its analogues is described. It involves solid-phase peptide synthesis and on-resin macrolactamization without side chain protection, affording cyclic products in high yield and high purity. The high specificity of the cyclization reaction was shown to originate in the formation of a pre-organized conformation of the linear biosynthetic precursor of gramicidin S. This facile method will provide convenient access to the analogues of the natural product for functional optimization to counter microbial resistance.     

Ion mobility-mass spectrometry applied to cyclic peptide analysis: Conformational preferences of gramicidin S and linear analogs in the gas phase

In this paper, we present an investigation of the gas-phase structural differences between cyclic and linear peptide ions by matrix-assisted laser desorption ionization-ion mobility-mass spectrometry. Specifically, data is shown for gramicidin S (cyclo-VOLFPVOLFP where phenylalanines are D rather than L-type amino acids and the O designates the non-standard amino acid ornithine) and five linear gramicidin S analogues. Results are interpreted as evidence for a beta-sheet (or beta-hairpin) conformational preference in both linear-protonated and sodiated-cyclic gramicidin S gas-phase peptides, and a preference for the protonated-cyclic peptide to adopt a collapsed, random coil-type conformation. A comparison with solution-phase circular dichroism measurements is performed, and structures similar to those observed in the gas phase appear to be favored in low-dielectric solvents such as 2,2,2-triflouroethanol. The utility of ion mobility-mass spectrometry (IM-MS) as a means of rapidly distinguishing between linear and cyclic peptide forms in also discussed.     

Synthesis and Controlled Polymerisation of a Novel Gramicidin S Analogue

Summary: The controlled polymerisation of a bulky, peptide‐based monomer was investigated. The cyclic β‐sheet forming decapeptide gramicidin S was modified with a methacrylate handle and subsequently polymerised via atom transfer radical polymerisation (ATRP), to yield a well‐defined gramicidin‐S‐containing polymer. The secondary structure of the peptide moiety was retained within the resulting polymer, as indicated by IR spectroscopy. This is the first example of the use of ATRP to create a synthetic polymer with a cyclic peptide as a side chain.

The gramicidin S based monomers synthesised here were then polymerised by ATRP.     

Inhibiting and Reversing Amyloid‐β Peptide (1–40) Fibril Formation with Gramicidin S and Engineered Analogues

In Alzheimer’s disease, amyloid‐β (Aβ) peptides aggregate into extracellular fibrillar deposits. Although these deposits may not be the prime cause of the neurodegeneration that characterizes this disease, inhibition or dissolution of amyloid fibril formation by Aβ peptides is likely to affect its development. ThT fluorescence measurements and AFM images showed that the natural antibiotic gramicidin S significantly inhibited Aβ amyloid formation in vitro and could dissolve amyloids that had formed in the absence of the antibiotic. In silico docking suggested that gramicidin S, a cyclic decapeptide that adopts a β‐sheet conformation, binds to the Aβ peptide hairpin‐stacked fibril through β‐sheet interactions. This may explain why gramicidin S reduces fibril formation. Analogues of gramicidin S were also tested. An analogue with a potency that was four‐times higher than that of the natural product was identified.     

Formylation domain: an essential modifying enzyme for the nonribosomal biosynthesis of linear gramicidin

Formylation is an important part of ribosomal peptide synthesis of prokaryotes. In nonribosomal peptide synthesis, however, N-formylation is rather unusual and therefore so far unexplored. In this work, the first module of the linear gramicidin nonribosomal peptide synthetase, LgrA1, consisting of a hypothetical formylation domain, an adenylation, and a peptidyl carrier protein domain was tested for formyltransferase activity in vitro. We demonstrate here that the putative formylation domain does indeed transfer the formyl group of formyltetrahydrofolate (fH4F) onto the first amino acid valine using both cofactors N10- and N5-fH4F, respectively. Most important, the necessity of the formylated starter unit formyl-valine for the initiation of the gramicidin biosynthesis was tested by elongation assays with the bimodular system from LgrA. By omitting the formyl group donor, no condensation product of valine with the subsequent building block glycine was detected, whereas the dipeptide formyl-valyl-glycine was found when assayed in the presence of either formyl donor. The proven formylation activity of the first domain of LgrA represents a novel tailoring enzyme in nonribosomal peptide synthesis.     

Biomimetic synthesis of gramicidin s and analogues by enzymatic cyclization of linear precursors on solid support

[reaction: see text] Gramicidin S is a potent decapeptide antibiotic with high hemolytic activity but is unlikely to provoke microbial resistance. Here we demonstrate that gramicidin thioesterase (GrsB TE) correctly cyclizes immobilized linear decapeptide precursors into head-to-tail products, indicating its suitability for parallel solid-phase synthesis of gramicidin analogues from linear precursors on solid support. This chemoenzymatic method will enable the optimization of the therapeutic index of the natural product to fight microbial resistance.     

Orthogonal time-of-flight secondary ion mass spectrometric analysis of peptides using large gold clusters as primary ions

Secondary ion mass spectrometry (SIMS) for biomolecular analysis is greatly enhanced by the instrumental combination of orthogonal extraction time-of-flight mass spectrometry with massive gold cluster primary ion bombardment. Precursor peptide molecular ion yield enhancements of 1000, and signal-to-noise improvements of up to 20, were measured by comparing SIMS spectra obtained using Au(+) and massive Au(400) (4+) cluster primary ion bombardment of neat films of the neuropeptide fragment dynorphin 1-7. Remarkably low damage cross-sections were also measured from dynorphin 1-7 and gramicidin S during prolonged bombardment with 40 keV Au(400) (4+). For gramicidin S, the molecular ion yield increases slightly as a function of Au(400) (4+) beam fluence up to at least 2 x 10(13) Au(400) (4+)/cm(2). This is in marked contrast to the rapid decrease observed when bombarding with ions such as Au(5) (+) and Au(9) (+). When gramicidin S is impinged with Au(5) (+), the molecular ion yield decreases by a factor of 10 after a fluence of only 8 x 10(12) ions/cm(2). Comparison of these damage cross-sections implies that minimal surface damage occurs during prolonged Au(400) (4+) bombardment. Several practical analytical implications are drawn from these observations.     

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.     

Allylic Amines as Key Building Blocks in the Synthesis of (E)-Alkene Peptide Isosteres

Nucleophilic imine additions with vinyl organometallics have developed into efficient, high yielding, and robust methodologies to generate structurally diverse allylic amines. We have used the hydrozirconation-transmetalation-imine addition protocol in the synthesis of allylic amine intermediates for peptide bond isosteres, phosphatase inhibitors, and mitochondria-targeted peptide mimetics. The gramicidin S-derived XJB-5-131 and JP4-039 and their analogs have been prepared on up to 160 g scale for preclinical studies. These (E)-alkene peptide isosteres adopt type II' β-turn secondary structures and display impressive biological properties, including selective reactions with reactive oxygen species (ROS) and prevention of apoptosis.     

Conformation and Interaction of a D,L-alternating peptide with a bilayer membrane: x-ray reflectivity, CD, and FTIR spectroscopy.

Peptides with alternating amino acid configuration provide helical secondary structures that are especially known from the membrane channel and pore-forming gramicidin A. In analogy to this natural D,L-alternating pentadecapeptide, the potential of D,L-alternating peptides for membrane insertion is investigated using the model dodecamer peptide H-(Phe-Tyr)(5)-Trp-Trp-OH. This aromatic peptide is introduced as a novel pore-forming synthetic analogue of gramicidin A. It forms a well-organized homodimer similar to one of the gramicidin A transmembrane motifs. X-ray reflectivity measurements are performed on solid-supported peptide-lipid complexes to obtain information about the influence of the artificial dodecamer peptide on the bilayer parameters. In addition, Fourier-transform infrared (FTIR) and circular dichroism (CD) spectroscopic studies determine the conformational state of H-(Phe-Tyr)(5)-Trp-Trp-OH within the model membrane. Site-specific iodine labeling assists in determining the topology of the membrane-embedded peptide by pinpointing the position of the iodine label within the bilayers.     

An Adamantyl Amino Acid Containing Gramicidin S Analogue with Broad Spectrum Antibacterial Activity and Reduced Hemolytic Activity

The cyclic cationic antimicrobial peptide gramicidin S (GS) is an effective topical antibacterial agent that is toxic for human red blood cells (hemolysis). Herein, we present a series of amphiphilic derivatives of GS with either two or four positive charges and characteristics ranging between very polar and very hydrophobic. Screening of this series of peptide derivatives identified a compound that combines effective antibacterial activity with virtually no toxicity within the same concentration range. This peptide acts against both Gram‐negative and Gram‐positive bacteria, including several MRSA strains, and represents an interesting lead for the development of a broadly applicable antibiotic.     

Stereochemistry of protected ornithine side chains of gramicidin S derivatives: X-ray crystal structure of the bis-Boc-tetra-N-methyl derivative of gramicidin S

An X-ray crystallographic analysis of the bis-Ndelta-Boc-tetra-Nalpha-methyl derivative of gramicidin S, cyclo(-Val-MeOrn(Boc)-Leu-d-MePhe-Pro-)2, was undertaken successfully (R-factor = 0.088). As expected, the main chain adopts an antiparallel pleated beta-sheet conformation, but the pleated sheet is slightly twisted, and the sense of twisting is opposite to that found in the reported crystal structures of the gramicidin S-urea complex and the bis-Ndelta-(trichloroacetyl) and bis-Ndelta-(m-bromobenzoyl) derivatives of gramicidin S. In agreement with the observed resistance toward N-methylation, the urethane NH groups of the protected Orn side chains are hydrogen bonded to the carbonyl groups of the d-Phe residues. However, the side-chain-main-chain hydrogen bonding is in the i --> i - 3 mode, although hydrogen bonding in the i --> i + 2 mode was deduced from a 1H NMR study of protected gramicidin S derivatives and was actually found in the crystal structures of the diacylated gramicidin S.     

Trisubstituted (E)-alkene dipeptide isosteres as beta-turn promoters in the gramicidin S cyclodecapeptide scaffold

[reaction: see text] A concise synthesis of a gramicidin S analogue with trisubstituted (E)-alkene dipeptide isostere (TEADI) replacements at both d-Phe-Pro positions was realized. Conformational analysis demonstrated that TEADIs can serve as type II beta-turn promoters in a cyclic scaffold and successfully mimic a proline residue.     

Combined electrochemistry and surface-enhanced infrared absorption spectroscopy of gramicidin a incorporated into tethered bilayer lipid membranes

Support from the support: Tethered bilayer lipid membranes containing the cation-channel-forming peptide gramicidin?A were assembled on nanostructured Au films. The combination of surface-enhanced infrared absorption (SEIRA) and electrochemical impedance spectroscopy (EIS) was used for the in situ structural and functional characterization of gramicidin?A in the same device.     

Synthesis and biological evaluation of gramicidin S dimers

The design and synthesis of analogues of the cyclic beta-sheet gramicidin S (GS), having additional functionalities in their turn regions, is reported. The monomeric GS analogues were transformed into dimers and their activities towards biological membranes, through antimicriobial and hemolytic assays, were evaluated. Finally, conductivity measurements have been performed to elucidate ion channel forming properties.     

Multivariate data analysis for enhanced interpretation of electrochemical impedance spectra of gramicidin-ion interactions in phospholipid monolayers

This article describes a multifrequency electrochemical impedance study of phospholipid monolayers on a mercury drop electrode in solutions containing electrolytes and gramicidin derivatives: gramicidin A (gA), gramicidin-BOC (g-BOC), and desformylgramicidin (g-des). The impedance spectra have been studied individually (univariate approach) and also transformed using a multivariate data reduction method (multivariate approach). It was shown that the two approaches are complementary. Thus the formation of K+-conducting channels is observed in gA only, and these channels can be distinguished from an interaction of all gramicidin derivatives with Mg2+. An unknown peptide interaction in the monolayer was observed on a slow time scale.     

An unusual reverse turn structure adopted by a furanoid sugar amino acid incorporated in gramicidin S

A new reverse turn, replacing one of the native type II' beta-turns in the cyclic peptide antibiotic gramicidin S, induced by a furanoid sugar amino acid is revealed. The C3-hydroxyl function plays a pivotal role by acting as a H-bond acceptor, consequently flipping the amide bond between residues i and i + 1, as was established by NMR and X-ray crystallographic analysis.     

Synthese von [2,7-Cystin]-Gramicidin S,einem künstlichen homodet-heterodet-bicyclischen Decapeptid

The first synthesis of a homodetic-heterodetic-bicyclic polypeptide, [2,7-cystine]-gramicidin S, is described. For the protection of the C-terminal carboxyl and the cysteine sulfhydryl functions, the 2-(toluene-p-sulfonyl)-ethyl- (Tsa) and the acetamidomethyl- (Acm) groups, respectively, were used. Stepwise synthesis from the C-terminus, using N^α Boc-amino acids, and selective removal of protecting groups yielded the two pentapeptide derivatives: Boc · Val-Cys-(Acm)-Lem)-Leu-phe-Pro · OH and H · Val-Cys(Acm)-Leu-phe-Pro · OTsa. They were condensed with dicyclohexyl-carbodiimide and 1-hydroxybenzotriazole to give the crystalline decapeptide Boc · [Val-Cys(Acm)-Leu-phe-Pro]₂ · OTsa. Removal of the Tsa group by β-elimination at pH 11.5 yielded the crystalline free acid, which was further converted (by treatment with di-p-nitrophenyl sulfite followed by TFA) to TFA, H · [Val-Cys(Acm)-Leu-phe-Pro]₂ · ONp. Cyclization of the active ester in warm pyridine gave a mixture of (2,7-bis-S-acetamidomethyl-cysteine]-gramicidin S (27% yield) and (2,7-cysteine)-gramicidin S (4%). The former compound was readily converted to the latter by treatment with I₂ in MeOH. In the bicyclic peptide, the decapeptide ring is contained in a β-type secondary structure, identical with that in gramicidin S; the disulfide bridge shows P-helical chirality and gives rise to a negative Cotton effect at 271 nm [3].     

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.     

Substrate recognition and selection by the initiation module PheATE of gramicidin S synthetase.

The initiation module of non-ribosomal peptide synthetases (NRPS) selects and activates the first amino acid and serves as the aminoacyl donor in the first peptide bond-forming step of the NRPS assembly line. The gramicidin S synthetase initiation module (PheATE) is a three-domain subunit, recognizing L-phenylalanine (L-Phe) and activating it (by adenylation domain) as tightly bound L-phenylalanyl-adenosine-5'-monophosphate diester (L-Phe-AMP), transferring it to the HS-phosphopantetheine arm of the holo-thiolation (holo-T) domain, and then epimerizing it (by epimerization domain) to the D-Phe-S-4'-Ppant-acyl enzyme. In this study, we have assayed the selectivity of the PheATE adenylation domain with a number of proteinogenic amino acids and observed that three additional amino acids, L-Tyr, L-Trp, and L-Leu, were activated to the aminoacyl-AMPs and transferred to the HS-phosphopantetheine arm of the holo-T domain. Hydrolytic editing of noncognate aminoacyl-AMPs and/or aminoacyl-S-4'-Ppant-acyl enzymes by the enzyme was not observed by three different assays for adenylation domain function. The microscopic reaction rates and thermodynamic equilibrium constants obtained from single-turnover studies of reactions of L-Phe, L-Trp, L-Tyr, and L-Leu with holoPheATE allowed us to construct free energy profiles for the reactions, revealing the kinetic and thermodynamic basis for substrate recognition and selection. In particular, the rates of epimerization of the L-aminoacyl-S-enzyme to the D-aminoacyl-S-enzyme intermediate showed reductions of 245-, 300-, and 540-fold for L-Trp, L-Tyr, and L-Leu respectively, suggesting that the epimerization domain is an important gatekeeper for generation of the D-Phe-S-enzyme that starts gramicidin S chain growth.