The ability of peptide nucleic acids (PNA) to enter and to cross filter-grown MDCK, HEK and CHO cells was studied by means of a protocol based on capillary electrophoresis combined with laser-induced fluorescence detection. The used approach avoided possible errors encountered in protocols based on confocal laserscanning microscopy and FACS analysis. In contradiction to the commonly anticipated unability of PNA to cross biomembranes, extensive translocation of unmodified PNA into and across the investigated cell types was found. The transport mode comprised a variety of energy dependent and -independent as well as temperature sensitive mechanisms being probably destined to natural substrates and hijacked by PNA. The presented results suggest active as well as passive export mechanisms rather than poor penetration into cells to be responsible for the only weak biological activity of unmodified PNA.

The N-dicyclopropylmethyl (Dcpm) residue, introduced into amino acids via reaction of dicyclopropylmethanimine hydrochloride with an amino acid ester followed by sodium cyanoborohydride or triacetoxyborohydride reduction, can be used as an amide bond protectant for peptide synthesis. Examples which demonstrate the amelioration of aggregation effects include syntheses of the alanine decapeptide and the prion peptide (106-126). Avoidance of cyclization to the aminosuccinimide followed substitution of Fmoc-(Dcpm)Gly-OH for Fmoc-Gly-OH in the assembly of sequences containing the sensitive Asp-Gly unit.

Introduction Most of the pharmaceuticals today target at G protein-coupled receptors (GPCRs) [1] that transmit extracellular signals into cells. GPCRs are promiscuous, that means a single receptor can activate different signalling events, presumably via different G protein subtypes. Functional selectivity of GPCR ligands has been observed and points at a new orientation in pharmaceutical research. However, the structural characteristics of ligands that produce this selectivity are far from known [2]. The polypeptide hormone urocortin I (Ucn) activates both Gs and Gi proteins via the corticotropin-releasing factor receptor type 1 (CRF1). We have recently developed an easy method for the separate measurement of Gs and Gi activation at HEK 293 cells stably transfected with cDNA coding for CRF1 [3,4]. The aim of this study was to search for structural determinants of the peptide agonist Ucn (DDPPLSIDLT FHLLRTLLEL ARTQSQRERA EQNRIIFDSV-NH2) that direct the signalling to Gs and Gi, respectively. For this purpose, the effect of single replacements by bulky amino acids, benzoyl-phenylalanine (Bpa) and naphthyl alanine (Nal), on Gs and Gi signalling pathways was measured in HEK-CRF1 cells, using receptor binding, [S]-GTPγS binding stimulation, and cAMP accumulation assays.

The instability of the undecapeptide substance P (SP), a neuropeptide implicated in several physiological processes, was occasionally observed when the peptide was stored in the solid state or in solution. The aim of the present study was to identify the decomposition products of SP stored as lyophilized peptide or in aqueous neutral solution. The main pathway of the decomposition of SP acetate consists of the subsequent release of N-terminal dipeptides via their diketopiperazines, cyclo(Arg-Pro) and cyclo(Lys-Pro). In contrast to the decomposition of the acetate of SP, the hydrochloride and trifluoroacetate salts were found to be considerably more stable. Under the studied conditions the release of N-terminal dipeptides dominates over other possible routes of spontaneous modifications, such as S-oxidation and deamidation.

Toll-like receptor 2 (TLR2) initiates inflammation in response to bacterial lipopeptide (BLP). However, the molecular mechanisms enabling the detection of BLP by TLR2 are unknown. Here we investigated the interaction of BLP with human serum proteins and identified vitronectin as a BLP-recognition molecule. Vitronectin and its receptor, integrin beta(3), were required for BLP-induced TLR2-mediated activation of human monocytes. Furthermore, monocytes from patients with Glanzmann thrombasthenia, which lack integrin beta(3), were completely unresponsive to BLP. In addition, integrin beta(3) formed a complex with TLR2 and this complex dissociated after BLP stimulation. Notably, vitronectin and integrin beta(3) coordinated responses to other TLR2 agonists such as lipoteichoic acid and zymosan. Our findings show that vitronectin and integrin beta(3) contribute to the initiation of TLR2 responses.

A promising application of the depsipeptide technique has recently been proposed to provide ideal conditions for segment condensation, in that coupling of peptides bearing a C ‐terminal depsipeptide unit occurs without giving rise to epimerization at the activated amino acid. This is due to the low tendency of the activated depsipeptide units, in contrast to the corresponding peptide segments, to form optically labile oxazolones. In this work we demonstrate that coupling of depsipeptides via base‐assisted activation using HBTU occurs not only without loss of configuration, but even much faster than the coupling of the corresponding all‐amide segments. Nevertheless, when the coupling of long depsipeptide segments proceeds slowly, we uncovered the occurrence of β‐elimination at the activated depsipeptide unit, in an extent dependent on the presence of base in the system and on the type of the solvent. Beta‐elimination was completely suppressed by using carbodiimide/HOBt activation in a non‐polar solvent (DCM), and in more polar media it was limited by substituting TMP for DIEA during HBTU activation, or using particular solvent mixtures (such as DMSO/toluene) for activation via carbodiimide. Finally, we show the application of C ‐terminal pseudoprolines, in comparison with that of depsipeptide units, to segment coupling. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.

The first solid-phase synthesis of cotransin-a cyclic depsipeptide having high pharmacological potential-was achieved, by a proper choice of coupling reagents and use of either TBAF or DBU for Fmoc removal to suppress the otherwise dominating, sequence-derived diketopiperazine formation. Starting the assembly from C-terminal lactic acid allowed fast and epimerization-free cyclization in solution. Novel conditions for orthogonal use of the Fmoc/Bsmoc-protection system were discovered, and an unexpected nucleophilic behavior of DBU was observed.

The synthesis of a 23-kDa protein that mimics the ligand-binding extracellular part of a G-protein-coupled receptor shows the potential of a combined recombinant, enzymatic, and chemical synthesis (CRECS) strategy. The mimic of the corticotropin-releasing factor receptor, synthesized from single domains by chemical ligation and sortase A-mediated coupling, has a high affinity for natural ligands. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2008/z705718_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Die Synthese eines 23-kDa-Proteins als Mimetikum des ligandbindenden, extrazellulären Teils eines G-Protein-gekoppelten Rezeptors verdeutlicht das Potenzial einer Kombination von molekularbiologischen, enzymatischen und chemischen Synthesemethoden. Das vorgestellte Mimetikum des Rezeptors des Corticotropin-releasing-Faktors, das aus gereinigten Einzeldomänen durch chemische Ligation und Sortase-A-katalysierte Kupplung hergestellt wurde, zeigte eine hohe Affinität für natürliche Liganden. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2001/2008/z705718_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Cyclization of R‐ and W‐rich hexapeptides has been found to enhance specifically the antimicrobial activity against Gram‐negative Escherichia coli . To gain insight into the role of the bacterial outer membrane in mediating selectivity, we assayed the activity of cyclic hexapeptides derived from the parent sequence c‐(RRWWRF) against several E. coli strains and Bacillus subtilis, L‐form bacteria, and E. coli lipopolysaccharide (LPS) mutant strains, and we also investigated the peptide‐induced permeabilization of the outer and inner membrane of E. coli . Wall‐deficient L‐form bacteria were distinctly less susceptible than the wild type strain. The patterns of peptide‐induced permeabilization of the outer and inner E. coli membranes correlated well with the antimicrobial activity, confirming that membrane permeabilization is a detrimental effect of the peptides upon bacteria. Truncation of LPS had no influence on the activity of the cyclic parent peptide, but the highly active c‐(RRWFWR), with three adjacent aromatic residues, required the complete LPS for maximal activity. Furthermore, differences in the activity of the parent peptide and its all‐D sequence indicated stereospecific interactions with the LPS mutant strains. We suggest that, depending on the primary sequence of the peptides, either hydrophobic interactions with the fatty acid chains of lipid A, or electrostatic interactions disturbing the polar core region and interference with saccharide‐saccharide interactions prevail in the barrier‐disturbing effect upon the outer membrane and thereby provide peptide accessibility to the inner membrane. The results underline the importance of tryptophan and arginine residues and their relative location for a high antimicrobial effect, and the activity‐modulating function of the outer membrane of E. coli . In addition to membrane permeabilization, the data provided evidence for the involvement of other mechanisms in growth inhibition and killing of bacteria. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.

The synthesis of the lipophilic chiral amino acid 1 bearing the bicyclo[1.1.1] pentane moiety is described. Linear and cyclic hexapeptides of the type Arg-Arg-Xaa-Yaa-Arg-Phe containing 1 instead of one or two tryptophan residues are prepared by solid phase peptide synthesis and the antimicrobial and hemolytic activity of the peptides obtained are discussed.

Sortase A is a transpeptidase that cleaves at a pentapeptide-motif and subsequently transfers the acyl component to a nucleophile containing N-terminal oligoglycines. We investigate the reaction conditions of the sortase-mediated ligation and demonstrate a useful application by the synthesis of a peptide nucleic acid-cell-penetrating peptide chimera, the reaction equilibrium of which can be shifted in favor of the product by dialyzing out the low molecular weight byproduct. The synthesized conjugate exhibits dose-dependent antisense activity.

This protocol for solid-phase peptide synthesis (SPPS) is based on the widely used Fmoc/tBu strategy, activation of the carboxyl groups by aminium-derived coupling reagents and use of PEG-modified polystyrene resins. A standard protocol is described, which was successfully applied in our lab for the synthesis of the corticotropin-releasing factor (CRF), >400 CRF analogs and a countless number of other peptides. The 41-mer peptide CRF is obtained within ∼80 working hours. To achieve the so-called difficult sequences, special techniques have to be applied in order to reduce aggregation of the growing peptide chain, which is the main cause of failure for peptide chemosynthesis. Exemplary application of depsipeptide and pseudoproline units is shown for synthesizing an extremely difficult sequence, the Asn(15) analog of the WW domain FBP28, which is impossible to obtain using the standard protocol.

We have established a combination of fluorescence‐spectroscopic uptake, release, and dilution experiments as a powerful tool for studying the translocation of fluorescent compounds across lipid membranes, demonstrating this through intrinsic tryptophan fluorescence for the interaction of the cell‐penetrating peptide penetratin with phospholipid membranes, for which conflicting results have been reported. We found that penetratin is not membrane‐permeant under the conditions used here. To confirm this finding and to validate the approach, we also employed an established titration‐calorimetric method, the results of which were in excellent agreement with a thermodynamic analysis of the fluorescence‐spectroscopic experiments. Further support was provided by a comparison with published data obtained under similar conditions by using a variety of techniques. Unlike these methods, however, the new approach allows consistent and simultaneous assessment of membrane binding and transbilayer movement without depending on extrinsic labels attached to the molecule of interest or on reporter moieties inserted into the lipid membrane.

Background and purpose: Most of the pharmaceuticals target G-protein-coupled receptors (GPCRs) which can generally activate different signalling events. The aim of this study was to achieve functional selectivity of corticotropin-releasing factor receptor type 1 (CRF1) ligands.Experimental approach: We systematically substituted urocortin, a natural peptide agonist of CRF1, with bulky amino acids (benzoyl-phenylalanine, naphthylalanine) and determined the effect of the analogues on coupling of CRF1 to Gs-and G(i)-protein in human embryonic kidney cells, using receptor binding, [S-35]-GTPgS binding stimulation, and cAMP accumulation assays.Key results: Native ligands stimulated G(s) and G(i) activation through CRF1, resulting in stimulation and then inhibition of cAMP accumulation. Single replacements in urocortin at positions 6 -15 led, dependent on the position and nature of the substituent, to ligands that conserved G(s) activity, but were devoid of G(i) activity, only stimulating cAMP accumulation, and competitively antagonized the G(i) activation by sauvagine. In contrast, analogues with substitutions outside this sequence non-selectively activated G(s) and G(i), as urocortin did.Conclusions and implications: Modifications in a specific region, which we have called the signalling domain, in the polypeptide agonist urocortin resulted in analogues that behaved as agonists and, at the same time, antagonists for the activation of different G-proteins by CRF1. This finding implies significant differences between active conformations of the receptor when coupled to different G-proteins. A similar structural encoding of signalling information in other polypeptide hormone receptor ligands would result in a general concept for the development of signalling-selective drug candidates.