Henrik Franzyk

Chemotaxonomy of Plantago. Iridoid glucosides and caffeoyl phenylethanoid glycosides

Data for 34 species of Plantago (Plantaginaceae), including subgen. Littorella (= Littorella uniflora), have been collected with regard to their content of iridoid glucosides and caffeoyl phenylethanoid glycosides (CPGs). In the present work, 21 species were investigated for the first time and many known compounds were found together with three new iridoid glucosides. Of these, arborescoside and arborescosidic acid, both of the uncommon type with an 8,9-double bond, were present in several species, while 6-deoxymelittoside was found only in P. subulata. The known compounds deoxyloganic acid, caryoptoside and rehmannioside D were isolated from the genus for the first time. The earlier reported occurrence of sorbitol in the family was confirmed, and this compound was shown by NMR spectroscopy to be the main sugar in the three species investigated for this. The combined data show that CPGs are present in all species investigated. With regard to the iridoids, the distribution patterns showed a good correlation with the classification of Rahn. Thus, aucubin is typical for the whole genus, while bartsioside and catalpol as well as 5-substituted iridoids are each characteristic for a subgenus in the family. Finally, the close relationship between Plantago and Veronica suggested by chloroplast DNA sequence analysis. could be corroborated by the common occurrence of the rare 8,9-unsaturated iridoids in these two genera.

Antimicrobial, Hemolytic, and Cytotoxic Activities of β-Peptoid–Peptide Hybrid Oligomers: Improved Properties Compared to Natural AMPs

While natural host-defense antimicrobial peptides (AMPs) and analogues thereof have been investigated intensely in the last two decades with the purpose of combating the still increasing threat from emerging multiresistant pathogenic microbes, 2] compounds with peptidomimetic backbones have received considerable attention due to their superior stability against proteolytic enzymes. 4] Typically, studies of peptidomimetic AMP analogues have involved a brief microbiological evaluation of an array of oligomers, and only occasionally has testing been performed across a broader range of microorganisms or involved systematic structure–activity relationship (SAR) studies. Such investigations have proven fruitful for a-peptidic AMPs, 18] and might reveal unexpected lead structures and selectivity profiles when applied to peptidomimetics as well. Therefore, we have performed a more rigorous microbiological evaluation as well as toxicity profiling of a series of oligomers based on our b-peptoid–peptide hybrid backbone architecture. 20] Antimicrobial activities were determined alongside the archetypal cationic AMP magainin-2 and its clinically tested derivative pexiganan against a series of five important pathogens belonging to different classes. The obtained SAR data were subsequently correlated with various measurements of toxicity towards mammalian cells. Thereby we were able to derive useful trends for the future design of antibacterial and antifungal peptidomimetic constructs with potential for enhanced selectivity. Three subclasses 1 a–3 d (Scheme 1) were originally designed to address the general effects of length, type of cationic side chains, and presence of a-chirality in the b-peptoid residues. These series had previously been confirmed to possess membrane activity, as indicated by testing for hemolytic and prehemolytic effects, as well as by calcein release experiments with model liposomes, albeit the interaction of these compounds with intracellular targets cannot be ruled out based on our data. The all-aliphatic compound 4 and the mixed aromatic–aliphatic chimera 5 were included to address the importance of lipophilicity and type of cationic residue. Finally, we included three 5/6-carboxyfluorescein-labeled oligomers (6–8) to assess the influence of the presence of this widely used fluorophore on the antimicrobial activity, which might have important implications for confocal fluorescence microscopy studies of the interaction of labeled compounds with live bacteria. The chimeras 4–8 were of dodecamer length to minimize undesired mammalian cell toxicity that might be observed with increasing length. This compound collection was tested against a variety of clinically relevant pathogens and human red blood cells (Table 1). Scheme 1. Chemical structures of the examined hybrid oligomers. The abbreviations used for the b-peptoid units were adapted from the abbreviations commonly used for peptoids (i.e. , N-alkylglycines), 7] by adding the b-prefix. bNspe = N-(S)-1-phenylethyl-balanine, bNphe =b-N-phenylalanine, bNsce = N-(S)-1-cyclohexylethyl-b-alanine, hArg = homoarginine, CF = 5/6-carboxyfluoresceinoyl.

Biosynthesis of secoiridoid glucosides in Oleaceae

Abstract The biosynthesis of the oleoside-type of glucosides (oleosides) has been investigated in two species of Oleaceae, namely Fraxinus excelsior and Syringa josikaea . Deuterated analogues of deoxyloganic acid, 8-epi-deoxyloganic acid, 7-epi-loganic acid and loganic acid were prepared. On administration of the above compounds high incorporations of deoxyloganic acid as well as of loganic acid and its 7-epimer were obtained, strongly implying that 7-ketologanic acid is an intermediate in the biosynthesis of oleosides. The incorporations were measured by 2 H NMR in several isolated secoiridoids all belonging to the oleosides. In addition, the content of acidic iridoids (both carbocyclic and seco-type) in two additional species of Oleaceae, namely Ligustrum ovalifolium and Phillyrea latifolia , were examined. Based on these results, a plausible biosynthetic route from deoxyloganic acid, 7-epi-loganic acid, 7-ketologanic acid, 8-epi-kingisidic acid, oleoside 11-methyl ester, 7-β-1- d -glucopyranosyl 11-methyl oleoside and ligstroside to oleuropein can now be presented.

Antimicrobial and cell-penetrating properties of penetratin analogs: Effect of sequence and secondary structure

Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) show great potential as drug delivery vectors and new antibiotic drug entities, respectively. The current study deals with the properties of a variety of peptide analogs derived from the well-known CPP penetratin as well as octaarginine and different Tat sequences. The effects of peptide length, guanidinium content, and sequence of non-cationic residues were assessed in mammalian and bacterial cells. The arginine (Arg) content in the penetratin analogs was found to influence eukaryotic cell uptake efficiency, antimicrobial activity towards both Gram-positive and Gram-negative bacteria as well as eukaryotic cell viability. All examined analogs retained the ability to cross eukaryotic membranes giving rise to a distribution within the vacuolar apparatus. Interestingly, a series of shuffled analogs of penetratin with the cationic residues in conserved positions, attain the same α-helical conformation as native penetratin in the presence of cholesterol-containing liposomes, while conformational differences were observed in the presence of highly anionic liposomes. While the antibacterial effect of the two groups of peptides was similar, the eukaryotic cellular uptake of the shuffled analogs was noticeably lower than for native penetratin. Moreover, a point substitution of Met to Leu in native penetratin had no influence on eukaryotic cellular uptake and antimicrobial effect, and only a minor effect on cytotoxicity, in contrast to the fact that the same substitution in the shuffled analog gave rise to reduced eukaryotic cellular uptake while increasing the antibacterial effect and cytotoxicity.

Chemotaxonomy and evolution of Plantago L.

Abstract.In continuation of our investigations of the genus Plantago L. (Plantaginaceae), sixteen species were investigated with respect to water-soluble glycosides. The iridoids auroside, strictoloside and globularicisin, as well as poliumoside, 3-[(4-β-D-glucopyranosyloxy)phenyl]propionic acid and 2-[4-(β-D-glucopyranosyloxy)phenyl]acetic acid were isolated from Plantago for the first time. The latter compound has not previously been isolated as a natural product. Sorbitol was the main carbohydrate in all the species investigated. The distribution of iridoids correlates well with the morphological classification of Rahn and also with a recently published molecular phylogenetic study of nuclear ribosomal and plastid DNA sequences. A new chemotaxonomic finding is an abundance of iridoid glucosides present in one of the two groups within subgenus Coronopus section Coronopus, while the other group lacks iridoids, supporting a subdivision of this section. Moreover, in subgenus Albicans, 10-benzoylcatalpol is a characteristic constituent of section Gnaphaloides, while the corresponding cinnamoyl ester globularin occurs in section Lanceifolia. In biosynthetic experiments, labelled epideoxyloganic acid and deoxygeniposidic acid were incorporated into aucubin and geniposidic acid in Plantago ovata, consistent with earlier findings, but no incorporation into asperuloside was observed. The evolution of biosynthetic pathways in Plantago is discussed.

Excelsioside, a secoiridoid glucoside from Fraxinus excelsior

Abstract A new secoiridoid named excelsioside has been isolated from the leaves of Fraxinus excelsior along with several known secoirioids and verbascoside. The compounds were identified by NMR, and the structure of excelsioside was finally assigned on the basis of a long-range selective proton decoupling (LSPD) 13 C NMR experiment.

Cellular uptake and membrane-destabilising properties of α-peptide/β-peptoid chimeras: lessons for the design of new cell-penetrating peptides

Novel peptidomimetic backbone designs with stability towards proteases are of interest for several pharmaceutical applications including intracellular delivery. The present study concerns the cellular uptake and membrane-destabilising effects of various cationic chimeras comprised of alternating N-alkylated beta-alanine and alpha-amino acid residues. For comparison, homomeric peptides displaying octacationic functionalities as well as the Tat(47-57) sequence were included as reference compounds. Cellular uptake studies with fluorescently labelled compounds showed that guanidinylated chimeras were taken up four times more efficiently than Tat(47-57). After internalisation, the chimeras were localised primarily in vesicular compartments and diffusively in the cytoplasm. In murine NIH3T3 fibroblasts, the chimeras showed immediate plasma membrane permeabilising properties, which proved highly dependent on the chimera chain length, and were remarkably different from the effects induced by Tat(47-57). Finally, biophysical studies on model membranes showed that the chimeras in general increase the permeability of fluid phase and gel phase phosphatidylcholine (PC) vesicles without affecting membrane acyl chain packing, which suggests that they restrict lateral diffusion of the membrane lipids by interaction with phospholipid head groups. The alpha-peptide/beta-peptoid chimeras described herein exhibit promising cellular uptake properties, and thus represent proteolytically stable alternatives to currently known cell-penetrating peptides.

Antimicrobial Activity of Peptidomimetics against Multidrug-Resistant Escherichia coli: A Comparative Study of Different Backbones

Novel remedies in the battle against multidrug-resistant bacterial strains are urgently needed, and one obvious approach involves antimicrobial peptides and mimics hereof. The impact of α- and β-peptoid as well as β(3)-amino acid modifications on the activity profile against β-lactamase-producing Escherichia coli was assessed by testing an array comprising different types of cationic peptidomimetics obtained by a general monomer-based solid-phase synthesis protocol. Most of the peptidomimetics possessed high to moderate activity toward multidrug-resistant E. coli as opposed to the corresponding inactive peptides. Nevertheless, differences in hemolytic activities indicate that a careful choice of backbone design constitutes a significant parameter in the search for effective cationic antimicrobial peptidomimetics targeting specific bacteria.

Incorporation of a synthetic mycobacterial monomycoloyl glycerol analogue stabilizes dimethyldioctadecylammonium liposomes and potentiates their adjuvant effect in vivo

The combination of delivery systems such as cationic liposomes and immunopotentiating molecules is a promising approach for the rational design of vaccine adjuvants. In this study, a synthetic analogue of the mycobacterial lipid monomycoloyl glycerol (MMG), referred to as MMG-1, was synthesized and combined with the cationic surfactant dimethyldioctadecylammonium (DDA). The purpose of the study was to provide a thorough pharmaceutical characterization of the resulting DDA/MMG-1 binary system and to evaluate how incorporation of MMG-1 affected the adjuvant activity of DDA liposomes. Thermal analyses demonstrated that MMG-1 was incorporated into the DDA lipid bilayers, and cryo-transmission electron microscopy (TEM) confirmed that liposomes were formed. The particles had a polydisperse size distribution and an average diameter of approximately 400 nm. Evaluation of the colloidal stability indicated that at least 18 mol% MMG-1 was required to stabilize the DDA liposomes as the average particle size remained constant during storage for 6 months. The improved colloidal stability is most likely caused by increased hydration of the lipid bilayer. This was demonstrated by studying Langmuir-Blodgett monolayers of DDA and MMG-1 which revealed an increased surface pressure in the presence of high concentrations of MMG-1 when the DDA/MMG-1 monolayers were fully compressed, indicating an increased interaction with water due to enhanced hydration of the lipid head groups. Finally, immunization of mice with the tuberculosis fusion antigen Ag85B-ESAT-6 and DDA/MMG-1 liposomes induced a strong cell-mediated immune response characterized by a mixed Th1/Th17 profile and secretion of IgG1 and IgG2c antibodies. The Th1/Th17-biased immunostimulatory effect was increased in an MMG-1 concentration-dependent manner with maximal observed effect at 31 mol% MMG-1. Thus, incorporation of 31 mol% MMG-1 into DDA liposomes results in an adjuvant system with favorable physical as well as immunological properties.

Antiplasmodial and Prehemolytic Activities of α-Peptide–β-Peptoid Chimeras

Malaria, especially the type caused by Plasmodium falciparum parasite strains, is a major tropical disease, estimated by the World Health Organization (WHO) to infect 300 to 500 million people per year, with a mortality rate of 1.1 to 2.7 million deaths per year. The advancement of resistance towards known drugs calls for the discovery of novel classes of antiparasitic compounds. Recently, the antiplasmodial activity of ACHTUNGTRENNUNGantimicrobial peptides of fungal and amphibian origin as well as of peptides from a phage-display library has been reported. Although some of these peptides contain nonproteinogenic amino acids, they are all a-peptides and are as such susceptible to proteolytic degradation. However, natural products that contain multiple N-methylated amino-acid residues have also been shown to have antiplasmodial activity. 6] Hence, we found it would be interesting to test whether the recently ACHTUNGTRENNUNGreported antimicrobial a-peptide–b-peptoid chimeras would display antiparasitic activity. Novel peptidomimetic backbone designs are of interest as a means of expanding the diversity of biomimetic polymers with respect to folding and biological properties. The chimeric a-peptide–b-peptoid constructs (Scheme 1), which have been found to possess antimicrobial activity without lysing human red blood cells in the active concentration range, are resistant against proteolysis. These chimeras are readily assembled by solid-phase peptide synthesis protocols by using building blocks that are efficiently prepared on the gram scale. 10] Here, we describe the discovery of promising biomimetic antiplasmodial oligomers with this backbone as well as the profiling of their membrane activity. The molecular design chosen here is based on alternating repeats of N-alkylated b-alanine (b-peptoid) units and aamino acids. The rationale behind this design was to gain structure-promoting effects and lipophilicity from the unnatural chiral b-peptoid residues (for circular dichroism studies, see refs. [7] , [11], and [12]), while the a-amino acids provide the side-chain functionality and intramolecular hydrogen bonding