72 research outputs found
Protolytic properties of polyamine wasp toxin analogues studied by 13C NMR spectroscopy
No full textAcid-base properties of the natural polyamine wasp toxin PhTX-433 (1) and seven synthetic analogues [PhTX-343 (2), PhTX-334 (3), PhTX-443 (4), PhTX-434 (5), PhTX-344 (6), PhTX-444 (7), and PhTX-333 (8)], each having four protolytic sites, were characterized by (13)C NMR spectroscopy. Nonlinear, multiparameter, simultaneous fit of all chemical shift data obtained from the NMR titration curves yielded macroscopic pK(a) values as well as intrinsic chemical shift data of all differently protonated macrospecies. Analyses of the chemical shift data demonstrated strong interactions between all four sites and provided information about complex relationships between chemical shift values and protonation state. Deprotonation of fully protonated forms starts at the central amino group of the polyamine moiety, and the extent of this trend depends on the distance to the flanking, protonated amino groups. The pK(a1) values of 1-8 are in the range 8.2-9.4. Hence, some of the toxins are incompletely protonated at the pH and ionic strength conditions used for assessment of their interactions with ionotropic glutamate and nicotinic acetylcholine receptors, and the degree of protonation is expected to have pharmacological importance in the ion-channel binding event
Inoculum effect of antimicrobial peptides
No full textThe activity of many antibiotics depends on the initial density of cells used in bacterial growth inhibition assays. This phenomenon, termed the inoculum effect, can have important consequences for the therapeutic efficacy of the drugs, because bacterial loads vary by several orders of magnitude in clinically relevant infections. Antimicrobial peptides are a promising class of molecules in the fight against drug-resistant bacteria because they act mainly by perturbing the cell membranes rather than by inhibiting intracellular targets. Here, we report a systematic characterization of the inoculum effect for this class of antibacterial compounds. Minimum inhibitory concentration values were measured for 13 peptides (including all-D enantiomers) and peptidomimetics, covering more than seven orders of magnitude in inoculated cell density. In most cases, the inoculum effect was significant for cell densities above the standard inoculum of 5 × 105 cells/mL, while for lower densities the active concentrations remained essentially constant, with values in the micromolar range. In the case of membrane-active peptides, these data can be rationalized by considering a simple model, taking into account peptide-cell association, and hypothesizing that a threshold number of cell-bound peptide molecules is required in order to cause bacterial killing. The observed effect questions the clinical utility of activity and selectivity determinations performed at a fixed, standardized cell density. A routine evaluation of the dependence of the activity of antimicrobial peptides and peptidomimetics on the inoculum should be considered
Water-membrane partition and the mutant selection window of antimicrobial peptides:insights from liposome studies
No full textThe mutant selection window (MSW) is a range of antimicrobial concentrations, where some bacteria are killed, while others survive. Within this interval resistance may develop. Antimicrobial peptides (AMPs) are a promising class of antimicrobials that generally act by perturbing the integrity of bacterial membranes. Their MSW is typically narrower than that of traditional antibiotics, but it still encompasses about one order of magnitude of peptide concentrations. Phenotypic or genetic differences between individual cells may cause this heterogeneous bacterial response to AMPs. Therefore, we minimized the system complexity by investigating pore formation in liposomes with homogeneous size and composition. Surprisingly, the AMPs novicidin, P9-4, and Sub3 formed pores only in a fraction of vesicles, over a wide range of total peptide concentrations. By characterizing the water/membrane partition equilibrium of these three AMPs, we were able to report the vesicle-perturbing activity as a function of the membrane-bound peptide concentration. In this case, the curves became essentially step functions with well-defined (bound) concentration thresholds at which pores were formed in all liposomes. Therefore, the apparent heterogeneous effects of AMPs on vesicles were actually determined by variations in the fraction of membrane-bound peptides under different conditions, due to water-membrane partition. Unexpectedly, the thresholds coincided for all peptides in terms of bound amino acids per lipid (∼0.4), suggesting that the mechanism of pore formation primarily depends on the surface coverage by the AMPs.</p
Inoculum effect of antimicrobial peptides
No full textThe activity of many antibiotics depends on the initial density of cells used in bacterial growth inhibition assays. This phenomenon, termed the inoculum effect, can have important consequences for the therapeutic efficacy of the drugs, because bacterial loads vary by several orders of magnitude in clinically relevant infections. Antimicrobial peptides are a promising class of molecules in the fight against drug-resistant bacteria because they act mainly by perturbing the cell membranes rather than by inhibiting intracellular targets. Here, we report a systematic characterization of the inoculum effect for this class of antibacterial compounds. Minimum inhibitory concentration values were measured for 13 peptides (including all-D enantiomers) and peptidomimetics, covering more than seven orders of magnitude in inoculated cell density. In most cases, the inoculum effect was significant for cell densities above the standard inoculum of 5 × 105 cells/mL, while for lower densities the active concentrations remained essentially constant, with values in the micromolar range. In the case of membrane-active peptides, these data can be rationalized by considering a simple model, taking into account peptide-cell association, and hypothesizing that a threshold number of cell-bound peptide molecules is required in order to cause bacterial killing. The observed effect questions the clinical utility of activity and selectivity determinations performed at a fixed, standardized cell density. A routine evaluation of the dependence of the activity of antimicrobial peptides and peptidomimetics on the inoculum should be considered.</p
Iridoid glycoside biosynthesis in Penstemon secundiflorus. Another H-5, H-9 trans iridoid glycoside.
No full textIsolation and characterisation of the new iridoid 10-hydroxy-(5alphaH)-6-epidihydrocornin from Penstemon secundiflorus (Scrophulariaceae) is described. In biosynthetic experiments, deoxyloganic acid was incorporated into the trans-fused iridoid glycosides (5alphaH)-6-epidihydrocornin and 10-hydroxy-(5alphaH)-6-epidihydrocornin in P. secundiflorus. Formation of the trans-fused compounds is therefore a late event in the biosynthesis and does not occur during iridoid formation by cyclization of the open chain monoterpene precursor. In the same plant, 8-epideoxyloganic acid was not incorporated into the trans-iridoids. Deoxyloganic acid was also incorporated into 10-hydroxyhastatoside (which bears an 8beta-methyl group), while 8-epideoxyloganic acid was incorporated into penstemoside (with an 8alpha-methyl group). Thus, iridoid biosynthetic pathways leading from both deoxyloganic acid and 8-epideoxyloganic acid were found in the same plant.Isolation and characterization of the new iridoid 10-hydroxy-(5 alpha H)-6-epidihydrocornin from Penstemon secundiflorus (Scrophulariaceae) is described. In biosynthetic experiments, deoxyloganic acid was incorporated into the transfused iridoid glycosides (5 alpha H)-6-epidihydrocornin and 10-hydroxy-(5 alpha H)-6-epidihydrocornin in P. secundiflorus. Formation of the trans-fused compounds is therefore a late event in the biosynthesis and does not occur during iridoid formation by cyclization of the open chain monoterpene precursor. In the same plant, 8-epideoxyloganic acid was not incorporated into the trans-iridoids. Deoxyloganic acid was also incorporated into 10-hydroxyhastatoside (which bears an 8 beta-methyl group), while 8-epideoxyloganic acid was incorporated into penstemoside (with an 8 alpha-methyl group). Thus, iridoid biosynthetic pathways leading from both deoxyloganic acid and 8-epideoxyloganic acid were found in the same plant. (C) 1998 Elsevier Science Ltd. All rights reserved
Evaluation of 1<i>H</i>-Triazole-1-[<i>N,N'</i>-Bis(tert-butoxycarbonyl)]carboxamidine in Solution-Phase and On-Resin Guanidinylation
No full textSeveral guanidines and guanidinylated peptides have substantial potential as therapeutics, but efficient guanidinylation reagents are vital for easy access to these compounds. Presently, pyrazole-1-carboxamidine type reagents are commonly used in the transformations of amines into corresponding guanidines. Here, we report a comparative study of the utility of 1H-triazole-1-[N,N'-bis(tert-butoxycarbonyl)]carboxamidine, which was synthesized in two steps and readily upscaled to gram amounts. It exhibited excellent performance in solution-phase reactions, rapidly converting a set of representative aliphatic primary and unhindered secondary amines as well as aniline into the corresponding bis(tert-butoxycarbonyl)-protected guanidines. To enable a direct assessment of the reactivity of guanidinylation reagents, conversions were performed in deuterated solvents (d7-DMF or d8-THF), allowing for continuous analysis of the reaction mixtures by 1H and 13C NMR. Likewise, 1H-triazole-1-[N,N'-bis(tert-butoxycarbonyl)]carboxamidine proved to be a versatile reagent in solid-phase conversions, for example, a resin-bound test peptide (KFFKFFK) was fully guanidinylated in only 2 h by using 2 equivalents of the reagent per free amino group. Also, 1H-triazole-1-[N,N'-bis(tert-butoxycarbonyl)]carboxamidine proved capable of completely guanidinylating more sterically hindered N-terminal residues (e.g., N-methyl amino acids or a peptoid) in resin-bound peptides. Its superior reactivity and stability demonstrated under heating conditions make 1H-triazole-1-[N,N'-bis(tert-butoxycarbonyl)]carboxamidine a valuable guanidinylation reagent both in solution- and solid-phase synthesis.</p
Iridoid glucosides from Penstemon secundiflorus and P. grandiflorus: Revised structure of 10-hydroxy-8-epihastatoside
No full textThorough large scale investigations of Penstemon secundiflorus ssp. lavendulus and of P. grandiflorus resulted in the isolation of the new iridoid glucoside 5,6beta-dihydroxyadoxoside. The structure of 10-hydroxyepihastatoside has been revised to 10-hydroxyhastatoside using a chemical correlation. Also, the known iridoids (5alphaH)-6-epidihydrocornin, cornin, hastatoside, 8-epihastatoside, beta-dihydrohastatoside, penstemoside, (5alphaH)-10-hydroxy-6-epidihydrocornin, 10-hydroxycornin, and catalpol were isolatedThorough large scale investigations of Penstemon secundiflorus ssp. lavendulus and of P. grandiflorus resulted in the isolation of the new iridoid glucosides 5, 6 beta-dihydroxyadoxoside. The structure of 10-hydroxyepihastatoside has been revised to 10-hydroxyhastatoside: using a chemical correlation. Also, the known iridoids (5 alpha H)-6-epidihydrocornin, cornin, hastatoside, 8-epihastatoside, beta-dihydrohastatoside, penstemoside, (5 alpha H)-10-hydroxy-6-epidihydrocornin, 10-hydroxycornin and catalpol were isolated. (C) 1998 Elsevier Science Ltd. All rights reserved
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