Structural studies of copper(II)-amine terminated dendrimer complexes by EXAFS

The three-dimensional branched nature of dendritic macromolecules provides many potential sites per molecule for the complexation of metal ions. Therefore, dendrimers may act as hosts for metals with coordination potentially occurring at the periphery, the interior, or both. To understand further the complexation of dendrimers with metal ions EXAFS experiments were carried out. In this work, the interaction of amine-terminated polyamido(amine), PAMAM, dendrimer with copper(II) ions determined by EXAFS is reported. It was found that a model consisting of the copper(II) ion forming five- and six-membered rings by chelating with the primary amine, amide, and tertiary amine nitrogen donors of the PAMAM dendrimer could describe the experimental EXAFS data well. Corroborative evidence for binding to amide nitrogen donors comes from the broadening of NMR resonances of a copper(Il)-PAMAM mixture revealing the presence of paramagnetic copper(II) ions at these sites. The significance of the results presented in this paper is that copper(II) ions form complexes within the dendrimer structure and not just at the periphery. The current study may have implications for the use of PAMAM dendrimers as effective ligands in sensing systems.

Facile ketene-ketene and ketene-ketenimine rearrangements: A study of the 1,3-migration of alpha-substituents interconverting alpha-imidoylketenes and alpha-oxoketenimines, a pseudopericyclic reaction

Theoretical calculations (B3LYP/6-311+G(3df,2p)//B3LYP/6-31G*) of the 1,3 migration of NR2 transforming alpha-oxoketenimines 1 to alpha-imidoylketenes 3 and vice versa indicate that this process is a pseudo-pericyclic reaction with a low activation energy (NH2 97 kJ mol(-1), N(CH3)(2) 62 kJ mol(-1)). The oxoketenimines were found to be more stable (by 18-35 kJ mol(-1)) which is in line with experimental observations. The hindered amine rotation in the amide and amidine moieties adjacent to the cumulenes are important in the migration of the NR2 group, as one of the rotation transition states is close to the 1,3 migration pathway. This gives an interesting potential energy surface with a valley-ridge inflection (VRI) between the orthogonal hindered amine rotation and 1,3 migration transition states. The imidoylketene may also undergo ring closure to an azetinone 5; however, this is metastable, and under the conditions that allow the 1,3-migration, the oxoketenimine 1 will be favored. The imine NH E/Z-interconversion of the ketenimine group takes place by inversion and has a low activation barrier (similar to40 kJ mol(-1)). In all the amidines examined the E/Z-interconversion of the imine function was predicted to be by rotation with a high barrier (>80 kJ mol(-1)), in contrast to all other reported imine E/Z-interconversions which are by inversion.

Structure of thermolysin cleaved microcin J25: Extreme stability of a two-chain antimicrobial peptide devoid of covalent links

sThe structure of a two-chain peptide formed by the treatment of the potent antimicrobial peptide microcin J25 (MccJ25) with thermolysin has been characterized by NMR spectroscopy and mass spectrometry. The native peptide is 21 amino acids in size and has the remarkable structural feature of a ring formed by linkage of the side chain of Glu8 to the N-terminus that is threaded by the C-terminal tail of the peptide. Thermolysin cleaves the peptide at the Phe10-Val11 amide bond, but the threading of the C-terminus through the N-terminal ring is so tight that the resultant two chains remain associated both in the solution and in the gas phases. The three-dimensional structure of the thermolysin-cleaved peptide derived using NMR spectroscopy and simulated annealing calculations has a well-defined core that comprises the N-terminal ring and the threading C-terminal tail. In contrast to the well-defined core, the newly formed termini at residues Phe10 and Val11 are disordered in solution. The C-terminal tail is associated to the ring both by hydrogen bonds stabilizing a short beta-sheet and by hydrophobic interactions. Moreover, unthreading of the tail through the ring is prevented by the bulky side chains of Phe19 and Tyr20, which flank the octapeptide ring. This noncovalent two-peptide complex that has a remarkable stability in solution and in highly denaturing conditions and that survives in the gas phase is the first example of such a two-chain peptide lacking disulfide or interchain covalent bonds.

Chemical and functional identification and characterization of novel sulfated alpha-conotoxins from the cone snail Conus anemone

An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the -conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3/beta2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr(16)]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC50 0.6 nM) and alpha7(IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC50 367 nM) than at the alpha3beta2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.

Structure-function studies of the plant cyclotides: The role of a circular protein backbone

The traditional idea of proteins as linear chains of amino acids is being challenged with the discovery of miniproteins that contain a circular backbone. The cyclotide family is the largest group of circular proteins and is characterized by an amide-circularized protein backbone and six conserved cysteine residues. These conserved cysteines are paired to form a knotted network of disulfide bonds. The combination of the circular backbone and a cystine knot, known as the cyclic cystine knot (CCK) motif, confers exceptional stability upon the cyclotides. This review discusses the role of the circular backbone based on studies of both the oxidative folding of kalata B1, the prototypical cyclotide, and a comparison of the structure and activity of kalata B1 and its acyclic permutants.

A versatile synthetic approach to peptidyl privileged structures using a "safety-catch" linker

Peptidyl privileged structures have been widely used by many groups to discover biologically active molecules. In this context, privileged substructures are used as hydrophobic anchors, to which peptide functionality is appended to gain specificity. Utilization of this concept has led to the discovery of many different active compounds at a wide range of biological receptors. A synthetic approach to these compounds has been developed on a safety-catch linker that allows rapid preparation of large libraries of these molecules. Importantly, amide bond formation/cleavage through treatment with amines is the final step; it is a linker strategy that allows significant diversification to be easily incorporated, and it only requires the inclusion of an amide bond. In addition, chemistry has been developed that permits the urea moiety to be inserted at the N-terminus of the peptide, allowing the same set of amines (either privileged substructures or amino acid analogues) to be used at both the N- and C-termini of the molecule. To show the robustness of this approach, a small library of peptidyl privileged structures were synthesized, illustrating that large combinatorial libraries can be synthesized using these technologies.

Flexible synthesis of enantiomerically pure 2,8-dialkyl-1,7-dioxaspiro[5.5]undecanes and 2,7-dialkyl-1,6-dioxaspiro[4.5]decanes from propargylic and homopropargylic alcohols

A new approach to enantiomerically pure 2,8-dialkyl-1,7-dioxaspiro[5.5]undecanes and 2,7-dialkyl-1,6-dioxaspiro [4.5] decanes is described and utilizes enantiomerically pure homopropargylic alcohols obtained from lithium acetylide opening of enantiomerically pure epoxides, which are, in turn, acquired by hydrolytic kinetic resolution of the corresponding racemic epoxides. Alkyne carboxylation and conversion to the Weinreb amide may be followed by triple-bond manipulation prior to reaction with a second alkynyllithium derived from a homo- or propargylic alcohol. In this way, the two ring components of the spiroacetal are individually constructed, with deprotection and cyclization affording the spiroacetal. The procedure is illustrated by acquisition of (2S,5R,7S) and (2R,5R,7S)-2-n-butyl-7-methyl-1,6-dioxaspiro[4.5]-decanes (1), (2S,6R,8S)-2-methyl-8-n-pentyl-1,7-dioxaspiro[5.5]undecane (2), and (2S,6R,8S)-2-methyl-8-n-propyl-1,7-dioxaspiro[5.5]undecane (3). The widely distributed insect component, (2S,6R,8S)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (4), was acquired by linking two identical alkyne precursors via ethyl formate. In addition, [H-2(4)]-regioisomers, 10,10,11,11-[H-2(4)] and 4,4,5,5-[H-2(4)] of 3 and 4,4,5,5-[H-2(4)]-4, were acquired by triple-bond deuteration, using deuterium gas and Wilkinson's catalyst. This alkyne-based approach is, in principle, applicable to more complex spiroacetal systems not only by use of more elaborate alkynes but also by triple-bond functionalization during the general sequence.

Effects of nitrogen source and ectomycorrhizal association on growth and delta N-15 of two subtropical Eucalyptus species from contrasting ecosystems

Ectomycorrhizal (EM) associations facilitate plant nitrogen (N) acquisition, but the contribution of EM associations to tree N nutrition is difficult to ascertain in ecosystems. We studied the abilities of subtropical EM fungi and nutritionally contrasting Eucalyptus species, Eucalyptus grandis W. Hill ex Maiden and Eucalyptus racemosa Cav, to use N sources in axenic and soil cultures, and determined the effect of EM fungi on plant N use and plant N-15 natural abundance (delta N-15). As measured by seedling growth, both species showed little dependence on EM when growing in the N-rich minerotrophic soil from E. grandis rainforest habitat or in axenic culture with inorganic N sources. Both species were heavily dependent on EM associations when growing in the N-poor, organotrophic soil from the E. racemosa wallum habitat or in axenic culture with organic N sources. In axenic culture, EM associations enabled both species to use organic N when supplied with amide-, peptide- or protein-N. Grown axenically with glutamine- or protein-N, delta N-15 of almost all seedlings was lower than source N. The delta N-15 of all studied organisms was higher than the N source when grown on glutathione. This unexpected N-15 enrichment was perhaps due to preferential uptake of an N moiety more N-15-enriched than the bulk molecular average. Grown with ammonium-N, the delta N-15 of non-EM seedlings was mostly higher than that of source N. In contrast, the delta N-15 of EM seedlings was mostly lower than that of source N, except at the lowest ammonium concentration. Discrimination against N-15 was strongest when external ammonium concentration was high. We suggest that ammonium assimilation via EM fungi may be the cause of the often observed distinct foliar delta N-15 of EM and non-EM species, rather than use of different N sources by species with different root specialisations. In support of this notion, delta N-15 of soil and leaves in the rainforest were similar for E. grandis and co-occurring non-mycorrhizal Proteaceae. In contrast, in wallum forest, E. racemosa leaves and roots were strongly N-15-depleted relative to wallum soil and Proteaceae leaves. We conclude that foliar delta N-15 may be used in conjunction with other ecosystem information as a rapid indicator of plant dependency on EM associations for N acquisition.

Efficient -tensor determination and NH assignment of paramagnetic proteins

Anisotropic magnetic susceptibility tensors chi of paramagnetic metal ions are manifested in pseudocontact shifts, residual dipolar couplings, and other paramagnetic observables that present valuable long-range information for structure determinations of protein-ligand complexes. A program was developed for automatic determination of the chi-tensor anisotropy parameters and amide resonance assignments in proteins labeled with paramagnetic metal ions. The program requires knowledge of the three-dimensional structure of the protein, the backbone resonance assignments of the diamagnetic protein, and a pair of 2D N-15-HSQC or 3D HNCO spectra recorded with and without paramagnetic metal ion. It allows the determination of reliable chi-tensor anisotropy parameters from 2D spectra of uniformly N-15-labeled proteins of fairly high molecular weight. Examples are shown for the 185-residue N-terminal domain of the subunit epsilon from E. coli DNA polymerase III in complex with the subunit theta and La3+ in its diamagnetic and Dy3+, Tb3+, and Er3+ in its paramagnetic form.

Enhancement of drug affinity for cell membranes by conjugation with lipoamino acids II. Experimental and computational evidence using biomembrane models

Lipoamino acids (LAAs) are promoieties able to enhance the amphiphilicity of drugs, facilitating their interaction with cell membranes. Experimental and computational studies were carried out on two series of lipophilic amide conjugates between a model drug (tranylcypromine, TCP) and LAA or alkanoic acids containing a short, medium or long alkyl side chain (C-4 to C-16). The effects of these compounds were evaluated by monolayer surface tension analysis and differential scanning calorimetry using dimyristoylphosphatidylcholine nnonolayers and liposomes as biomembrane models. The experimental results were related to independent calculations to determine partition coefficient and blood-brain partitioning. The comparison of TCP-LAA conjugates with the related series of TCP alkanoyl amides confirmed that the ability to interact with the biomembrane models is not due to the mere increase of lipophilicity, but mainly to the amphipatic nature and the kind of LAA residue. (C) 2005 Elsevier B.V. All rights reserved.

Synthesis of 'difficult' peptides free of aspartimide and related products, using peptoid methodology

We developed an efficient, cost effective strategy for Fmoc-based solid phase synthesis of 'difficult' peptides and/or peptides containing Asp/Asn-Gly sequences, free of aspartimide and related products, using a peptoid methodology for the preparation of N-substituted glycines.

VIP and PACAP potentiation of nicotinic ACh-evoked currents in rat parasympathetic neurons is mediated by G-protein activation

The effects of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP27 and PACAP38) on isolated parasympathetic neurons of rat intracardiac and submandibular ganglia were examined under voltage clamp using whole-cell patch-clamp recording techniques. VIP and PACAP (less than or equal to 10 nm) selectively and reversibly increased the affinity of nicotinic acetylcholine receptor channels (nAChRs) for their agonists resulting in a potentiation of acetylcholine (ACh)-evoked whole-cell currents at low agonist concentrations. VIP-induced potentiation was observed with either ACh or nicotine as the cholinergic agonist. The VIP- but not the PACAP-induced potentiation of ACh-evoked currents was inhibited by [Ac-Tyr(1), D-Phe(2)]-GRF 1-29, amide (100 nm), a selective antagonist of VPAC(1) and VPAC(2) receptors; whereas the PACAP38- but not the VIP-induced potentiation was inhibited by 100 nm PACAP6-38, a PAC(1) and VPAC(2) receptor antagonist. The signal transduction pathway mediating VIP- and PACAP-induced potentiation of nicotinic ACh-evoked currents involves a pertussis toxin (PTX)-sensitive G-protein. Intracellular application of 200 mu m GTP gamma S or GDP beta S inhibited VIP-induced potentiation of ACh-evoked whole-cell currents. GTP gamma S alone potentiated ACh- and nicotine-evoked currents and the magnitude of these currents was not further increased by VIP or PACAP. The G-protein subtype modulating the neuronal nAChRs was examined by intracellular dialysis with antibodies directed against alpha(o), alpha(i-1,2), alpha(i-3) or beta G-protein subunits. Only the anti-G alpha(o) and anti-G beta antibodies significantly inhibited the effect of VIP and PACAP on ACh-evoked currents. The potentiation of ACh-evoked currents by VIP and PACAP may be mediated by a membrane-delimited signal transduction cascade involving the PTX-sensitive G(o) protein.