Christian Bijani

Iron(II) binding to amyloid-β, the Alzheimer's peptide.

Iron has been implicated in Alzheimers disease, but until now no direct proof of Fe(II) binding to the amyloid-β peptide (Aβ) has been reported. We used NMR to evidence Fe(II) coordination to full-length Aβ40 and truncated Aβ16 peptides at physiological pH and to show that the Fe(II) binding site is located in the first 16 amino-acid residues. Fe(II) caused selective broadening of some NMR peaks that was dependent on the Fe:Aβ stoichiometry and temperature. Analysis of Fe(II) broadening effect in the (1)H, (13)C, and 2D NMR data established that Asp1, Glu3, the three His, but not Tyr10 nor Met35 are the residues mainly involved in Fe(II) coordination.

X‐ray and Solution Structures of CuIIGHK and CuIIDAHK Complexes: Influence on Their Redox Properties

The Gly-His-Lys (GHK) peptide and the Asp-Ala-His-Lys (DAHK) sequences are naturally occurring high-affinity copper(II) chelators found in the blood plasma and are hence of biological interest. A structural study of the copper complexes of these peptides was conducted in the solid state and in solution by determining their X-ray structures, and by using a large range of spectroscopies, including EPR and HYSCORE (hyperfine sub-level correlation), X-ray absorption and (1)H and (13)C NMR spectroscopy. The results indicate that the structures of [Cu(II)(DAHK)] in the solid state and in solution are similar and confirm the equatorial coordination sphere of NH(2), two amidyl N and one imidazole N. Additionally, a water molecule is bound apically to Cu(II) as revealed by the X-ray structure. As reported previously in the literature, [Cu(II)(GHK)], which exhibits a dimeric structure in the solid state, forms a monomeric complex in solution with three nitrogen ligands: NH(2), amidyl and imidazole. The fourth equatorial site is occupied by a labile oxygen atom from a carboxylate ligand in the solid state. We probe that fourth position and study ternary complexes of [Cu(II)(GHK)] with glycine or histidine. The Cu(II) exchange reaction between different DAHK peptides is very slow, in contrast to [Cu(II)(GHK)], in which the fast exchange was attributed to the presence of a [Cu(II)(GHK)(2)] complex. The redox properties of [Cu(II)(GHK)] and [Cu(II)(DAHK)] were investigated by cyclic voltammetry and by measuring the ascorbate oxidation in the presence of molecular oxygen. The measurements indicate that both Cu(II) complexes are inert under moderate redox potentials. In contrast to [Cu(II)(DAHK)], [Cu(II)(GHK)] could be reduced to Cu(I) around -0.62 V (versus AgCl/Ag) with subsequent release of the Cu ion. These complete analyses of structure and redox activity of those complexes gave new insights with biological impact and can serve as models for other more complicated Cu(II)-peptide interactions.

Copper(II) Coordination to Amyloid β: Murine versus Human Peptide

In Alzheimer s disease (AD), the amyloid b (Ab) peptide seems to play a causative role. Ab is the major constituent of amyloid plaques, a hallmark of AD. According to the amyloid cascade hypothesis, in AD, the aggregation of Ab leads to the formation of toxic species, which induce neuronal cell death. It has been proposed that reactive oxygen species (ROS) are produced, and that these species mediate cell toxicity. 2] Although still under debate, a large body of evidence suggests that metallic ions (copper, zinc, and iron) play a role in the etiology of AD. For example, amyloid plaques extracted from human brains contain high amounts of Cu and Zn ions bound to the Ab peptide. 9] Chelators were able to partially solubilize the plaques, and studies on neuronal cell culture and transgenic mice supported the involvement of ions in Ab metabolism. 11] Copper(II) can be released in the synaptic cleft and can reach concentrations up to 15 mm. This value is in line with the possibility of Cu binding to Ab in vivo, since a dissociation constant in the picomolar range has been determined for the Cu–Ab species. Furthermore, in vitro aggregation of the Ab peptide can be modulated by Cu and Zn ions, and because of its redox nature, Cu may play a role in ROS production. These observations and hypotheses explain the intensive research on the modulation of metal-ion homeostasis as a therapeutic approach. 16] A better understanding of the AD mechanisms requires investigations on mouse and rat animal models. 18] However, these animals, whose peptide differs from the human Ab peptide by three point mutations, do not show amyloid deposition. Consequently, studies are performed on transgenic mice or rats that produce the human Ab (hAb) peptide in addition to their own peptide (mAb). Cu coordination to murine and human peptides has been proposed to differ. Thus, in the present study, to explain the distinct Cu coordination to hAb and mAb, we used complementary spectroscopic techniques to determine the crucial mutation(s). We also propose Cu–mAb structural models. Finally, we discuss possible consequences of such differences in Cu coordination with respect to the use of mice or rats as AD animal models. We examined the coordination of Cu to six peptides: hAb (DAEFRHDSGYEVHHQK; see Scheme S1 in the Supporting Information), Y10F-hAb, H13R-hAb, R5GhAb, mAb (DAEFGHDSGFEVRHQK; see Scheme S2 in the Supporting Information), and F10Y-mAb (or R5G-H13RhAb). These shorter 16-residue peptides were used as valuable models of Cu binding to the full-length peptides. Indeed, no differences in spectroscopic signature, binding affinity, or ROS production have been observed between the truncated and full-length hAb peptides. Two peptide families can be distinguished from the spectroscopic signatures of their Cu complexes (see Figures S1–S5 and Table S1 in the Supporting Information): hAb, Y10F-hAb, and H13R-hAb (humanlike family), and mAb, F10Y-mAb, and R5G-hAb (murine-like family). Thus, the key mutation between the hAb and mAb peptides with regard to Cu binding is the R5G mutation. For both families, two Cu complexes that differ in the protonation state of the peptide are present near the physiological pH value, namely, components I and II. Figure 1 shows the differences between the CD and EPR spectroscopic signatures of Cu–hAb and Cu–mAb solutions at pH 6.7 and 5.4, at which I is predominant, and at pH 8.7 and 7.6, at which II is predominant. The pKa(I/II) values are close to pH 7.7 for Cu complexes of the humanlike peptides and close to pH 6.2 for the murine-like family (see Figures S3 and S5 and Table S1 in the Supporting Information). We previously described copper(II)-induced modification of the peptide NMR spectroscopic signature to determine the Cu-binding sites of hAb. The results obtained were in line with most previous studies 22, 26, 27] and showed that the equatorial binding site of component I is formed by the NH2 group of Asp1, two of the three imidazole rings of His6, His13, and His14, and a CO function. At higher pH values, deprotonation of the Asp1 Ala2 peptide bond leads to the replacement of one imidazole ring with the Asp1 Ala2 deprotonated amide (amidyl) ligand. In this study, we used NMR spectroscopy to gain more insight into Cu coordination to the mAb peptide. We recorded H, C, and 2D NMR spectra of mAb peptide at pH 5.4 and 7.6 with or without a substoichiometric amount of Cu ions (see Figures S6–S14 in the Supporting Information). At pH 5.4 and in the presence of Cu, the side chains of Asp, [*] H. Eury, Dr. C. Bijani, Prof. Dr. P. Faller, Dr. C. Hureau CNRS, LCC (Laboratoire de Chimie de Coordination) 205, route de Narbonne, 31077 Toulouse (France) and Universit de Toulouse, UPS, INPT, LCC 31077 Toulouse (France) Fax: (+ 33)5-6155-3003 E-mail: christelle.hureau@lcc-toulouse.fr

pH-Dependent Cu(II) Coordination to Amyloid-β Peptide: Impact of Sequence Alterations, Including the H6R and D7N Familial Mutations.

Copper ions have been proposed to intervene in deleterious processes linked to the development of Alzheimers disease (AD). As a direct consequence, delineating how Cu(II) can be bound to amyloid-β (Aβ) peptide, the amyloidogenic peptide encountered in AD, is of paramount importance. Two different forms of [Cu(II)(Aβ)] complexes are present near physiological pH, usually noted components I and II, the nature of which is still widely debated in the literature, especially for II. In the present report, the phenomenological pH-dependent study of Cu(II) coordination to Aβ and to ten mutants by EPR, CD, and NMR techniques is described. Although only indirect insights can be obtained from the study of Cu(II) binding to mutated peptides, they reveal very useful for better defining Cu(II) coordination sites in the native Aβ peptide. Four components were identified between pH 6 and 12, namely, components I, II, III and IV, in which the predominant Cu(II) equatorial sites are {-NH(2), CO (Asp1-Ala2), N(im) (His6), N(im) (His13 or His14)}, {-NH(2), N(-) (Asp1-Ala2), CO (Ala2-Glu3), N(im)}, {-NH(2), N(-) (Asp1-Ala2), N(-) (Ala2-Glu3), N(im)} and {-NH(2), N(-) (Asp1-Ala2), N(-) (Ala2-Glu3), N(-) (Glu3-Phe4)}, respectively, in line with classical pH-induced deprotonation of the peptide backbone encountered in Cu(II) peptidic complexes formation. The structure proposed for component II is discussed with respect to another coordination model reported in the literature, that is, {CO (Ala2-Glu3), 3 N(im)}. Cu(II) binding to the H6R-Aβ and D7N-Aβ peptides, where the familial H6R and D7N mutations have been linked to early onset of AD, has also been investigated. In case of the H6R mutation, some different structural features (compared to those encountered in the native [Cu(II)(Aβ)] species) have been evidenced and are anticipated to be important for the aggregating properties of the H6R-Aβ peptide in presence of Cu(II).

Taxonomy and chemical characterization of new antibiotics produced by Saccharothrix SA198 isolated from a Saharan soil

Actinomycete strain SA198, isolated from a Saharan soil sample of Algeria, exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria, and phytopathogenic and toxinogenic fungi. The morphological and chemotaxonomic characteristics of the strain were consistent with those of the genus Saccharothrix. Analysis of the 16S rRNA gene sequence of strain SA198 showed a similarity level ranging between 97.2 and 98.8% within Saccharothrix species, S. australiensis being the most closely related. Two new active products were isolated by reverse HPLC using a C18 column. The ultraviolet-visible (UV-VIS), infrared (IR), mass, and (1)H and (14)C nuclear magnetic resonance (NMR) spectra showed that these products were new bioactive compounds. The minimum inhibitory concentrations of these antibiotics showed a strong activity against fungi and moderate activities against Gram-positive and Gram-negative bacteria.

Copper Coordination to Native N-Terminally Modified versus Full-Length Amyloid-β: Second-Sphere Effects Determine the Species Present at Physiological pH

Alzheimers disease is characterized by senile plaques in which metallic ions (copper, zinc, and iron) are colocalized with amyloid-β peptides of different sequences in aggregated forms. In addition to the full-length peptides (Aβ1-40/42), N-terminally truncated Aβ3-40/42 forms and their pyroglutamate counterparts, Aβp3-40/42, have been proposed to play key features in the aggregation process, leading to the senile plaques. Furthermore, they have been shown to be more toxic than the full-length Aβ, which made them central targets for therapeutic approaches. In order to better disentangle the possible role of metallic ions in the aggregation process, copper(II) coordination to the full-length amyloid peptides has been extensively studied in the last years. However, regarding the N-terminally modified forms at position 3, very little is known. Therefore, copper(I) and copper(II) coordination to those peptides have been investigated in the present report using a variety of complementary techniques and as a function of pH. Copper(I) coordination is not affected by the N-terminal modifications. In contrast, copper(II) coordination is different from that previously reported for the full-length peptide. In the case of the pyroglutamate form, this is due to preclusion of N-terminal amine binding. In the case of the N-terminally truncated form, alteration in copper(II) coordination is caused by second-sphere effects that impact the first binding shell and the pH-dependent repartition of the various [Cu(peptide)] complexes. Such second-sphere effects are anticipated to apply to a variety of metal ions and peptides, and their importance on changing the first binding shell has not been fully recognized yet.

Saccharothrix sp. PAL54, a new chloramphenicol-producing strain isolated from a Saharan soil

An actinomycete strain designated PAL54, producing an antibacterial substance, was isolated from a Saharan soil in Ghardaïa, Algeria. Morphological and chemical studies indicated that this strain belonged to the genus Saccharothrix. Analysis of the 16S rDNA sequence showed a similarity level ranging between 96.9 and 99.2% within Saccharothrix species, with S. longispora DSM 43749T, the most closely related. DNA–DNA hybridization confirmed that strain PAL54 belonged to Saccharothrix longispora. It showed very strong activity against pathogenic Gram-positive and Gram-negative bacteria responsible for nosocomial infections and resistant to multiple antibiotics. Strain PAL54 secreted the antibiotic optimally during mid-stationary and decline phases of growth. One antibacterial compound was isolated from the culture broth and purified by HPLC. The active compound was elucidated by uv-visible and NMR spectroscopy and by mass spectrometry. The results showed that this compound was a d(−)-threo chloramphenicol. This is the first report of chloramphenicol production by a Saccharothrix species.

Probing highly selective H/D exchange processes with a ruthenium complex through neutron diffraction and multinuclear NMR studies.

Deuterium labeling is a powerful way to gain mechanistic information in biology and chemistry. However, selectivity is hard to control experimentally, and labeled sites can be difficult to assign both in solution and in the solid state. Here we show that very selective high-deuterium contents can be achieved for the polyhydride ruthenium phosphine complex [RuH2(H2)2(PCyp3)2] (1) (PCyp3 = P(C5H9)3). The selectivity of the H/D exchange process is demonstrated by multinuclear NMR and neutron diffraction analyses. It has also been investigated through density functional theory (DFT) calculations. The reactions are performed under mild conditions at room temperature, and the extent of deuterium incorporation, involving selective C-H bond activation within the cyclopentyl rings of the phosphine ligands, can easily be tuned (solvent effects, D2 pressure). It is shown that D2 gas can inhibit the C-H/C-D exchange process.

A Highly Effective Ruthenium System for the Catalyzed Dehydrogenative Cyclization of Amine–Boranes to Cyclic Boranes under Mild Conditions

We recently disclosed a new ruthenium-catalyzed dehydrogenative cyclization process (CDC) of diamine-monoboranes leading to cyclic diaminoboranes. In the present study, the CDC reaction has been successfully extended to a larger number of diamine-monoboranes (4-7) and to one amine-borane alcohol precursor (8). The corresponding NB(H)N- and NB(H)O-containing cyclic diaminoboranes (12-15) and oxazaborolidine (16) were obtained in good to high yields. Multiple substitution patterns on the starting amine-borane substrates were evaluated and the reaction was also performed with chiral substrates. Efforts have been spent to understand the mechanism of the ruthenium CDC process. In addition to a computational approach, a strategy enabling the kinetic discrimination on successive events of the catalytic process leading to the formation of the NB(H)N linkage was performed on the six-carbon chain diamine-monoborane 21 and completed with a (15) N NMR study. The long-life bis-σ-borane ruthenium intermediate 23 possessing a reactive NHMe ending was characterized in situ and proved to catalyze the dehydrogenative cyclization of 1, ascertaining that bis σ-borane ruthenium complexes are key intermediates in the CDC process.

Antimicrobial activity of saquayamycins produced by Streptomyces spp. PAL114 isolated from a Saharan soil

A new strain of actinomycete designated PAL114, producing antimicrobial compounds, was isolated from a Saharan soil in Ghardaïa, Algeria. Morphological and chemical studies showed that this strain belonged to the genus Streptomyces. Two bioactive compounds, named P41A and P41B, were extracted by dichloromethane from the cell-free supernatant broth of strain PAL114 and were purified by HPLC. Minimum inhibitory concentrations of the pure antibiotics were determined against yeasts, filamentous fungi and bacteria, most of which are pathogenic or toxigenic for human and multiresistant to antibiotics. The strongest activities were observed against Candida albicans M3 and Bacillus subtilis ATCC 6633. The chemical structures of the compounds were determined by spectroscopic analysis of UV-visible and 1H and 13C NMR spectra and spectrometric analysis of mass spectrum. The compounds P41A and P41B were identified as saquayamycins A and C, respectively. These compounds belong to the aquayamycin-group antibiotics, which are known in the literature for their anticancer and antibacterial activities.