Extent of Intramolecular π-Stacks in Aqueous Solution in Mixed-Ligand Copper(II) Complexes Formed by Heteroaromatic Amines and Several 2-Aminopurine Derivatives of the Antivirally Active Nucleotide Analog 9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA)

The acidity constants of twofold protonated, antivirally active, acyclic nucleoside phosphonates (ANPs), H(2) (PE)(±) , where PE(2-) =9-[2-(phosphonomethoxy)ethyl]adenine (PMEA(2-) ), 2-amino-9-[2-(phosphonomethoxy)ethyl]purine (PME2AP(2-) ), 2,6-diamino-9-[2-(phosphonomethoxy)ethyl]purine (PMEDAP(2-) ), or 2-amino-6-(dimethylamino)-9-[2-(phosphonomethoxy)ethyl]purine (PME(2A6DMAP)(2-) ), as well as the stability constants of the corresponding ternary Cu(Arm)(H;PE)(+) and Cu(Arm)(PE) complexes, where Arm=2,2'-bipyridine (bpy) or 1,10-phenanthroline (phen), are compared. The constants for the systems containing PE(2-) =PMEDAP(2-) and PME(2A6DMAP)(2-) have been determined now by potentiometric pH titrations in aqueous solution at I=0.1M (NaNO(3) ) and 25°; the corresponding results for the other ANPs were taken from our earlier work. The basicity of the terminal phosphonate group is very similar for all the ANP(2-) species, whereas the addition of a second amino substituent at the pyrimidine ring of the purine moiety significantly increases the basicity of the N(1) site. Detailed stability-constant comparisons reveal that, in the monoprotonated ternary Cu(Arm)(H;PE)(+) complexes, the proton is at the phosphonate group, that the ether O-atom of the ?CH(2) ?O?CH(2) ?P(O)$\rm{{_{2}^{-}}}$(OH) residue participates, next to the P(O)$\rm{{_{2}^{-}}}$(OH) group, to some extent in Cu(Arm)(2+) coordination, and that π?π stacking between the aromatic rings of Cu(Arm)(2+) and the purine moiety is rather important, especially for the H?PMEDAP(-) and H?PME(2A6DMAP)(-) ligands. There are indications that ternary Cu(Arm)(2+) -bridged stacks as well as unbridged (binary) stacks are formed. The ternary Cu(Arm)(PE) complexes are considerably more stable than the corresponding Cu(Arm)(R?PO(3) ) species, where R?PO$\rm{{_{3}^{2-}}}$ represents a phosph(on)ate ligand with a group R that is unable to participate in any kind of intramolecular interaction within the complexes. The observed stability enhancements are mainly attributed to intramolecular-stack formation in the Cu(Arm)(PE) complexes and also, to a smaller extent, to the formation of five-membered chelates involving the ether O-atom present in the ?CH(2) ?O?CH(2) ?PO$\rm{{_{3}^{2-}}}$ residue of the PE(2-) species. The quantitative analysis of the intramolecular equilibria involving three structurally different Cu(Arm)(PE) isomers shows that, e.g., ca. 1.5% of the Cu(phen)(PMEDAP) system exist with Cu(phen)(2+) solely coordinated to the phosphonate group, 4.5% as a five-membered chelate involving the ether O-atom of the ?CH(2) ?O?CH(2) ?PO$\rm{{_{3}^{2-}}}$ residue, and 94% with an intramolecular π?π stack between the purine moiety of PMEDAP(2-) and the aromatic rings of phen. Comparison of the various formation degrees of the species formed reveals that, in the Cu(phen)(PE) complexes, intramolecular-stack formation is more pronounced than in the Cu(bpy)(PE) species. Within a given Cu(Arm)(2+) series the stacking intensity increases in the order PME2AP(2-) 相似文献   

Inheritance of chilling and heat requirements for flowering in almond and QTL analysis

The chilling and heat requirements and flowering time were studied, for 2 years, in an almond progeny from the cross between the late-flowering French selection “R1000” and the early-flowering Spanish “Desmayo Largueta”. These three temperature-dependent traits showed quantitative inheritance, although for chilling requirements and flowering time a major gene could be involved, modified by other minor genes. The results indicate that flowering time is mainly a consequence of the chilling requirements; heat requirements having a smaller effect. In agreement with the genetic findings, a significant Quantitative Trait Loci (QTL) for chilling requirements was found in G4 together with other minor QTLs in G1, G3, and G7. For heat requirements, two QTLs in G2 and G7 were identified. The results also show the high influence of temperature in the expression of the three traits and their QTL analyses. In addition, QTL analysis for flowering time allowed the identification of one significant QTL in linkage group 4 (G4) that explained most of the phenotypic variation together with other minor QTLs located in G1, G6, and G7.     

Cladonia subturgida and C. iberica (Cladoniaceae) form a single, morphologically and chemically polymorphic species

Cladonia subturgida and C. iberica constitute the whole of a Mediterranean problematic species, which shows great morphological polymorphism. A study was carried out in order to delimit the extant taxa within this group. The variability of the group was studied morphologically, chemically and phylogenetically. The phylogeny was based on three loci (ITS rDNA, rpb2 and mtLSU), using Maximum Parsimony and Bayesian analyses. Six chemotypes were identified, the most common one containing atranorin and protolichesterinic acid. Our results prove that C. subturgida and C. iberica constitute a single, morphologically and chemically polymorphic species. The taxonomic rank of C. turgida var. corsicana was also studied based on analyses of morphological, chemical and ITS rDNA data. The new nomenclatural combination, C. corsicana, is proposed.     

Identification of a New Borrelia Species among Small Mammals in Areas of Northern Spain Where Lyme Disease Is Endemic

The role of small mammals as reservoir hosts for Borrelia burgdorferi was investigated in several areas where Lyme disease is endemic in northern Spain. A low rate of infestation by Ixodes ricinus nymphs was found in the small mammal populations studied that correlated with the near-absence of B. burgdorferi sensu lato in 184 animals tested and with the lack of transmission of B. burgdorferi sensu lato to I. ricinus larvae that fed on them. In contrast, questing ticks collected at the same time and in the same areas were found to carry a highly variable B. burgdorferi sensu lato repertoire (B. burgdorferi sensu stricto, Borrelia garinii, Borrelia valaisiana, and Borrelia afzelii). Interestingly, the only isolate obtained from small mammals (R57, isolated from a bank vole) grouped by phylogenetic analyses with other Borrelia species but in a separate clade from the Lyme disease and relapsing fever organisms, suggesting that it is a new species. This new agent was widely distributed among small mammals, with infection rates of 8.5 to 12% by PCR. Moreover, a high seroprevalence to B. burgdorferi sensu lato was found in the animal sera, suggesting cross-reactivity between B. burgdorferi sensu lato and R57. Although small mammals do not seem to play an important role as reservoirs for B. burgdorferi sensu lato in the study area, they seem to be implicated in the maintenance of spirochetes similar to R57.     

Selectivity of the enzymatic synthesis of ampicillin by E. coli PGA in the presence of high concentrations of substrates

Penicillin G acylase (PGA) catalyzes the synthesis/hydrolysis of acyl derivatives of phenylacetic acid through the formation of a covalent intermediate (the acyl–enzyme complex). When used for the kinetically controlled synthesis of β-lactam antibiotics, this enzyme promotes two undesired side reactions: the hydrolysis of the acyl side-chain precursor and of the antibiotic. Therefore, a high selectivity (synthesis/hydrolysis, S/H ratio) is very important for the process economics. Here, the enzymatic synthesis of ampicillin from d-phenylglycine methyl ester (PGME) and 6-aminopenicillic acid (6-APA), using PGA from Escherichia coli (EC 3.5.1.11) is studied. Kinetic assays provided S/H for high concentrations of substrates (up to 200 mM of 6-APA and 500 mM of PGME), using soluble PGA, at 25 °C, pH 6.5. S/H increased with 6-APA concentration, in accordance with the literature. However, when the concentration of 6-APA approached saturation, the rate of enzymatic hydrolysis tended towards zero (i.e., S/H tended to infinity). On the other hand, when the concentration of ester was augmented, S/H consistently decreased. This behavior, to the best of our knowledge still not reported, indicates that the acylation step may occur with 6-APA already positioned for the nucleophilic attack.     

Dual mechanism of bacterial lethality for a cationic sequence-random copolymer that mimics host-defense antimicrobial peptides

Flexible sequence-random polymers containing cationic and lipophilic subunits that act as functional mimics of host-defense peptides have recently been reported. We used bacteria and lipid vesicles to study one such polymer, having an average length of 21 residues, that is active against both Gram-positive and Gram-negative bacteria. At low concentrations, this polymer is able to permeabilize model anionic membranes that mimic the lipid composition of Escherichia coli, Staphylococcus aureus, or Bacillus subtilis but is ineffective against model zwitterionic membranes, which explains its low hemolytic activity. The polymer is capable of binding to negatively charged vesicles, inducing segregation of anionic lipids. The appearance of anionic lipid-rich domains results in formation of phase-boundary defects through which leakage can occur. We had earlier proposed such a mechanism of membrane disruption for another antimicrobial agent. Experiments with the mutant E. coli ML-35p indicate that permeabilization is biphasic: at low concentrations, the polymer permeabilizes the outer and inner membranes; at higher polymer concentrations, permeabilization of the outer membrane is progressively diminished, while the inner membrane remains unaffected. Experiments with wild-type E. coli K12 show that the polymer blocks passage of solutes into the intermembrane space at high concentrations. Cell membrane integrity in E. coli K12 and S. aureus exhibits biphasic dependence on polymer concentration. Isothermal titration calorimetry indicates that the polymer associates with the negatively charged lipopolysaccharide of Gram-negative bacteria and with the lipoteichoic acid of Gram-positive bacteria. We propose that this polymer has two mechanisms of antibacterial action, one predominating at low concentrations of polymer and the other predominating at high concentrations.