New dsDNA‐Binding Hybrid Molecules Combining an Unnatural Peptide and an Intercalating Moiety

In our search for new dsDNA‐binding ligands, combinatorial chemistry was first applied to select unnatural oligopeptides with moderate affinity for dsDNA. To enhance the binding affinity of a heptapeptide lead structure, Ac‐Arg‐Ual‐Sar‐Chi‐Chi‐Tal‐Arg‐NH₂ (K_d=4.9?10^?4 M ), the compound was conjugated to different heteropolyaromatic moieties by means of a variety of linker arms. Glycine, β‐alanine, glycyl‐glycine, glycyl‐β‐alanine, γ‐aminobutyric acid, and 6‐aminocaproic acid were used as spacers, representing different lengths and/or flexibilities. The intercalators coupled to the oligopeptide were acridine, fluorenone, anthracene, anthraquinone, and 3,8‐diamino‐5‐methyl‐6‐phenylphenantridinium (methidium). The binding capacities of these new hybrid molecules to dsDNA have been investigated by gel retardation and footprinting assays. The results show that, by conjugating the unnatural oligopeptide to intercalators, the affinity for dsDNA could be enhanced more than 100‐fold. For methidium‐β‐alanyl‐glycyl‐Arg‐Ual‐Sar‐Chi‐Chi‐Tal‐Arg‐NH₂ (K_d of 2.1?10^?6 M ), the interaction with dsDNA was dominated by the intercalator in such a way that the sequence specificity of the heptapeptide was changed. The interaction with dsDNA of hybrid molecules of other intercalators was mainly governed by the oligopeptide, since the sequence selectivity of the heptapeptide was conserved. In general, the linker arm glycine (shortest spacer) and glycine‐β‐alanine were preferred over β‐alanine, glycyl‐glycine and the more‐flexible spacers γ‐aminobutyric acid and 6‐aminocaproic acid. This way new hybrid molecules endowed with dsDNA affinities of ca. 10^?6 M and displaying different sequence selectivities have been developed. Therefore, combinations of such unnatural peptides with intercalators can be used to broaden the knowledge about the sequence‐selective recognition of dsDNA.     

Selection of New Sequence‐Selective Unnatural Peptides Binding to Double‐Stranded Deoxyribonucleic Acids (dsDNA) by Means of a Gel‐Retardation Experiment for Library Analysis

Previously, we developed a methodology for the solid‐phase screening of peptide libraries for interaction with double‐stranded deoxyribonucleic acids (dsDNA). In the search for new and more‐potent DNA ligands, we investigated the strategy of solution‐phase screening of chemical libraries consisting of unnatural oligopeptides. After synthesis of the selected amino acid building blocks, libraries were constructed with the general structure Ac‐Arg‐Ual‐Sar‐X¹‐X²‐X³‐Arg‐NH₂, where X represents each of twelve unnatural or natural amino acids. Optimization of the sequence of binding peptides was performed with an iterative deconvolution procedure. Selection of interacting peptides was carried out in solution by means of gel‐retardation experiments, starting with libraries of 144 compounds. A 14‐base‐pair double‐stranded DNA fragment was chosen as the target. After several cycles of synthesis and screening of libraries and individual peptides, an oligopeptide was selected with an apparent dissociation constant of 9⋅10⁻⁵ M , as determined by gel‐retardation experiments. This peptide was studied by NMR spectroscopy. A certain degree of conformational pre‐organization of the peptides was shown by temperature‐dependent circular‐dichroism experiments. Finally, DNase‐I‐footprinting studies indicated a preferential interaction with a 6‐base‐pair mixed sequence 5′‐CTGCAT‐3′. This study demonstrates that gel‐shift experiments can be used for the solution‐phase screening of library mixtures of peptides against dsDNA. In general, this technique allows the selection of new sequence‐selective dsDNA‐interacting molecules. Furthermore, novel dsDNA‐binding unnatural oligopeptides were developed with affinities in the 0.1 mM range.     

The Coordination of NiII and CuII Ions to the Polyhistidyl Motif of Hpn Protein: Is It as Strong as We Think?

Hpn, one of Helicobacter pylori′s nickel‐accessory proteins, is an amazingly peculiar protein: Almost half of its sequence consists of polyhistidyl (poly‐His) residues. Herein, we try to understand the origin of this naturally occurring sequence, thereby shedding some light on the bioinorganic chemistry of Hpn′s numerous poly‐His repeats. By using potentiometric, mass spectrometric, and various spectroscopic techniques, we studied the Ni^II‐ and Cu^II complexes of the wild‐type Ac‐THHHHYHGG‐NH₂ fragment of Hpn and of its six analogues, in which consecutive residues (His or Tyr) were replaced by Ala (Ala‐substitution or Ala‐scan approaches), thereby resulting in Ac‐TAHHHYHGG‐NH₂, Ac‐THAHHYHGG‐NH₂, Ac‐THHAHYHGG‐NH₂, Ac‐THHHAYHGG‐NH₂, Ac‐THHHHAHGG‐NH₂, and Ac‐THHHHYAGG‐NH₂ peptides. We found that the His4 residue is critical for both Ni^II‐ and Cu^II‐ion binding and the effectiveness of binding varies even if the substituted amino acid does not take part in the direct binding interactions.     

Molecular‐Recognition‐Assisted pKa Shifts and Metal‐Ion‐Induced Fluorescence Regeneration in p‐Sulfonatocalix[6]arene‐Encapsulated Acridine

The host–guest interactions of cationic (AcH⁺) and neutral (Ac) forms of the dye acridine with the macrocyclic host p‐sulfonatocalix[6]arene (SCX6) were investigated by using ground‐state absorption, steady‐state and time‐resolved fluorescence, and NMR measurements. The cationic form undergoes significant complexation with SCX6 (K_eq=2.5×10⁴ M ^?1), causing a sharp decrease in the fluorescence intensity and severe quenching in the excited‐state lifetime of the dye. The strong binding of the AcH⁺ form of the dye with SCX6 is attributed to ion–ion interactions involving the sulfonato groups (SO₃^?) of SCX6 and the positively charged AcH⁺ at pH of approximately 4.3. Whereas, the neutral Ac form of the dye undergoes weak complexation with SCX6 (K_eq=0.9×10³ M ^?1) and the binding constant is lowered by one order of magnitude compared with that of the SCX6–AcH⁺ system. The strong affinity of SCX6 to the protonated form leads to a large upward pK_a shift (≈2 units) in the dye. In contrast, strong emission quenching upon SCX6 interaction and the regeneration of fluorescence intensity of the dye in the presence of Gd³⁺ through competitive binding have also been demonstrated.     

Electrochemical and Spectroscopic Studies on the Interaction of Gallocyanine with DNA

The interaction of gallocyanine (GC) with double‐stranded DNA (dsDNA) in pH 3.5 Tris‐HCl buffer solution was investigated by electrochemical methods and spectrophotometric methods as well. In the potential scan range of ‐0.25 ? +0.18 V(vs. SCE), GC had a couple of well‐defined redox peaks at ‐0.022 V and ‐0.069 V on a cyclic voltammogram at the scan rate of 100.0 mV/s, respectively. After the addition of dsDNA into the GC solution, the redox‐peak currents decreased obviously and the peak potentials shifted positively. The results demonstrated that GC binding to DNA was caused by intercalation. Electrochemical parameters such as the electron number (n), the charge transfer coefficient (α) and the electrochemical reaction standard rate constant (k_s) were calculated and compared in the absence and presence of dsDNA. Almost unchanged values of the electrochemical parameters after adding dsDNA showed that non‐electroactive complexes were formed when GC interacted with DNA. The results indicated that the decrease of the redox‐peak currents was caused by the decrease of the free concentration of GC in the reaction solution. The binding constant and binding ratio were investigated by spectrophotometric methods. DNA concentration can be determined by the decrease of the peak current of GC. The linear range for dsDNA was in the range of 1.45 × 10^?7 ? 1.45 × 10^?6mol/Land 1.45 × 10^?6 ? 1.45 × 10^?5 mol/L, respectively with the linear regression equation as Δi_P (10^?7 A) = 0.037 + 0.018C (10^?7mol/L), and Δi_P (10^?7 A) = 0.25 + 0.041C (10^?6mol/L), respectively, and the detection limit (3σ) was 1.13 × 10^?7 mol/L.     

Ab initio chemical kinetics for the NH2 + HNOx reactions,part II: Kinetics and mechanism for NH2 + HONO

The kinetics and mechanism for the reaction of NH₂ with HONO have been investigated by ab initio calculations with rate constant prediction. The potential energy surface of this reaction has been computed by single‐point calculations at the CCSD(T)/6‐311+G(3df, 2p) level based on geometries optimized at the CCSD/6‐311++G(d, p) level. The reaction producing the primary products, NH₃ + NO₂, takes place via precomplexes, H₂N???c‐HONO or H₂N???t‐HONO with binding energies, 5.0 or 5.9 kcal/mol, respectively. The rate constants for the major reaction channels in the temperature range of 300–3000 K are predicted by variational transition state theory or Rice–Ramsperger–Kassel–Marcus theory depending on the mechanism involved. The total rate constant can be represented by k_total = 1.69 × 10^?20 × T^2.34 exp(1612/T) cm³ molecule^?1 s^?1 at T = 300–650 K and 8.04 × 10^?22 × T^3.36 exp(2303/T) cm³ molecule^?1 s^?1 at T = 650–3000 K. The branching ratios of the major channels are predicted: k₁ + k₃ producing NH₃ + NO₂ accounts for 1.00–0.98 in the temperature range 300–3000 K and k₂ producing OH + H₂NNO accounts for 0.02 at T > 2500 K. The predicted rate constant for the reverse reaction, NH₃ + NO₂ → NH₂ + HONO represented by 8.00 × 10^?26 × T^4.25 exp(?11,560/T) cm³ molecule^?1 s^?1, is in good agreement with the experimental data. © 2009 Wiley Periodicals, Inc. Int J Chem Kinet 41: 678–688, 2009     

Ab initio chemical kinetics for the NH2 + HNOx reactions,part III: Kinetics and mechanism for NH2 + HONO2

The kinetics and mechanism for the reaction of NH₂ with HONO₂ have been investigated by ab initio calculations with rate constant prediction. The potential energy surface of this reaction has been computed by single‐point calculations at the CCSD(T)/6‐311+G(3df, 2p) level based on geometries optimized at the B3LYP/6‐311+G(3df, 2p) level. The reaction producing the primary products, NH₃ + NO₃, takes place via a precursor complex, H₂N…HONO₂ with an 8.4‐kcal/mol binding energy. The rate constants for major product channels in the temperature range 200–3000 K are predicted by variational transition state or variational Rice–Ramsperger–Kassel–Marcus theory. The results show that the reaction has a noticeable pressure dependence at T < 900 K. The total rate constants at 760 Torr Ar‐pressure can be represented by k_total = 1.71 × 10^?3 × T^?3.85 exp(?96/T) cm³ molecule^?1 s^?1 at T = 200–550 K, 5.11 × 10^?23 × T^+3.22 exp(70/T) cm³ molecule^?1 s^?1 at T = 550–3000 K. The branching ratios of primary channels at 760 Torr Ar‐pressure are predicted: k₁ producing NH₃ + NO₃ accounts for 1.00–0.99 in the temperature range of 200–3000 K and k₂ + k₃ producing H₂NO + HONO accounts for less than 0.01 when temperature is more than 2600 K. The reverse reaction, NH₃ + NO₃ → NH₂ + HONO₂ shows relatively weak pressure dependence at P < 100 Torr and T < 600 K due to its precursor complex, NH₃…O₃N with a lower binding energy of 1.8 kcal/mol. The predicted rate constants can be represented by k_?1 = 6.70 × 10^?24 × T^+3.58 exp(?850/T) cm³ molecule^?1 s^?1 at T = 200–3000 K and 760 Torr N₂ pressure, where the predicted rate at T = 298 K, 2.8 × 10^?16 cm³ molecule^?1 s^?1 is in good agreement with the experimental data. The NH₃ + NO₃ formation rate constant was found to be a factor of 4 smaller than that of the reaction OH + HONO₂ producing the H₂O + NO₃ because of the lower barrier for the transition state for the OH + HONO₂. © 2009 Wiley Periodicals, Inc. Int J Chem Kinet 42: 69–78, 2010     

Mass Spectrometric Strategies to Improve the Identification of Pt(II)-Modification Sites on Peptides and Proteins

To further explore the binding chemistry of cisplatin (cis-Pt(NH₃)₂Cl₂) to peptides and also establish mass spectrometry (MS) strategies to quickly assign the platinum-binding sites, a series of peptides with potential cisplatin binding sites (Met(S), His(N), Cys(S), disulfide, carboxyl groups of Asp and Glu, and amine groups of Arg and Lys, were reacted with cisplatin, then analyzed by electron capture dissociation (ECD) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS). Radical-mediated side-chain losses from the charge-reduced Pt-binding species (such as CH₃S^? or CH₃SH from Met, SH^? from Cys, CO₂ from Glu or Asp, and NH₂ ^? from amine groups) were found to be characteristic indicators for rapid and unambiguous localization of the Pt-binding sites to certain amino acid residues. The method was then successfully applied to interpret the top-down ECD spectrum of an inter-chain Pt-crosslinked insulin dimer, insulin?+?Pt(NH₃)₂?+?insulin (>10 kDa). In addition, ion mobility MS shows that Pt binds to multiple sites in Substance P, generating multiple conformers, which can be partially localized by collisionally activated dissociation (CAD). Platinum(II) (Pt(II)) was found to coordinate to amine groups of Arg and Lys, but not to disulfide bonds under the conditions used. The coordination of Pt to Arg or Lys appears to arise from the migration of Pt(II) from Met(S) as shown by monitoring the reaction products at different pH values by ECD. No direct binding of cisplatin to amine groups was observed at pH 3?~?10 unless Met residues were present in the sequence, but noncovalent interactions between cisplatin hydrolysis and amination [Pt(NH₃)₄]²⁺ products and these peptides were found regardless of pH.

Helix Nucleation by the Smallest Known α‐Helix in Water

Cyclic pentapeptides (e.g. Ac‐(cyclo‐1,5)‐[KAXAD]‐NH₂; X=Ala, 1 ; Arg, 2 ) in water adopt one α‐helical turn defined by three hydrogen bonds. NMR structure analysis reveals a slight distortion from α‐helicity at the C‐terminal aspartate caused by torsional restraints imposed by the K(i)–D(i+4) lactam bridge. To investigate this effect on helix nucleation, the more water‐soluble 2 was appended to N‐, C‐, or both termini of a palindromic peptide ARAARAARA (≤5 % helicity), resulting in 67, 92, or 100 % relative α‐helicity, as calculated from CD spectra. From the C‐terminus of peptides, 2 can nucleate at least six α‐helical turns. From the N‐terminus, imperfect alignment of the Asp5 backbone amide in 2 reduces helix nucleation, but is corrected by a second unit of 2 separated by 0–9 residues from the first. These cyclic peptides are extremely versatile helix nucleators that can be placed anywhere in 5–25 residue peptides, which correspond to most helix lengths in protein–protein interactions.     

Ab initio chemical kinetics for the NH2 + HNOx Reactions,Part I: Kinetics and Mechanism for NH2 + HNO

The kinetics and mechanism for the reaction of NH₂ with HNO have been investigated by ab initio calculations with rate constant prediction. The potential energy surface of this reaction has been computed by single‐point calculations at the CCSD(T)/6‐311+G(3df, 2p) level based on geometries optimized at the CCSD/6‐311++G(d, p) level. The major products of this reaction were found to be NH₃ + NO formed by H‐abstraction via a long‐lived H₂N???HNO complex and the H₂NN(H)O radical intermediate formed by association with 26.9 kcal/mol binding energy. The rate constants for formation of primary products in the temperature range of 300–3000 K were predicted by variational transition state or RRKM theories. The predicted total rate constants at the 760 Torr Ar pressure can be represented by k_total = 3.83 × 10^?20 × T^+2.47exp(1450/T) at T = 300–600 K; 2.58 × 10^?22 × T^+3.15 exp(1831/T) cm³ molecule^?1 s^?1 at T = 600?3000 K. The branching ratios of major channels at 760 Torr Ar pressure are predicted: k₁ + k₃ + k₄ producing NH₃ + NO accounts for 0.59–0.90 at T = 300–3000 K peaking around 1000 K, k₂ accounts for 0.41–0.03 at T = 300–600 K decreasing with temperature, and k₅ accounts for 0.07–0.27 at T > 600 K increasing gradually with temperature. The NH₃ + NO formation rate constant was found to be a factor of 3–10 smaller than that of the isoelectronic reaction CH₃ + HNO producing CH₄ + NO, which has been shown to take place by barrierless H‐abstraction without involving a hydrogen‐bonding complex as in the NH₂ case. © 2009 Wiley Periodicals, Inc. Int J Chem Kinet 41: 677–677, 2009     

Synthesis and Cytotoxic Activity of Macromolecular Prodrug of Cisplatin Using Poly(ethylene glycol) with Galactose Residues or Antennary Galactose Units

To provide a macromolecular prodrug with recognition ability for hepatoma cells, we synthesized new conjugates of cisplatin (CDDP) and poly(ethylene glycol) (PEG) with galactose residues or antennary galactose units (Gal4A, four branched galactose residues) at the chain terminus, Gal‐PEG‐DA/CDDP or Gal4A‐PEG‐DA/CDDP conjugates. An antennary (branched) structure of Gal4A was designed based on the fact that saccharide clusters with branched structures show highly effective binding with saccharide receptors, a phenomenon known as the ‘cluster effect’. The cytotoxic activity of the conjugates was investigated against HepG2 human hepatoma cells in vitro and compared with a control conjugate without galactose, MeO‐PEG‐DA/CDDP. Gal‐PEG‐DA/CDDP and Gal4A‐PEG‐DA/CDDP conjugates showed lower IC₅₀ values (3.1×10^–4 and 2.3×10^–4 M , respectively) than the MeO‐PEG‐DA/CDDP conjugate (10.5×10^–4 M ). The cytotoxic activities of these conjugates with galactose residues or antennary galactose units were inhibited as a result of the addition of galactose and strongly inhibited by the addition of Gal4A, however the inclusion of a methoxy group (the MeO‐PEG‐DA/CDDP conjugate) did not affect the activity. These results suggest that the Gal4A unit introduced to the conjugate has effective recognition ability against HepG2 human hepatoma cells.     

Low‐Background,Highly Sensitive DNA Biosensor by Using an Electrically Neutral Cobalt(II) Complex as the Redox Hybridization Indicator

An electrically neutral cobalt complex, [Co(GA)₂(phen)] (GA=glycollic acid, phen=1,10‐phenathroline), was synthesized and its interactions with double‐stranded DNA (dsDNA) were studied by using electrochemical methods on a glassy carbon electrode (GCE). We found that [Co(GA)₂(phen)] could intercalate into the DNA duplex through the planar phen ligand with a high binding constant of 6.2(±0.2)×10⁵ M ^?1. Surface studies showed that the cobalt complex could electrochemically accumulate within the modified dsDNA layer, rather than within the single‐stranded DNA (ssDNA) layer. Based on this feature, the complex was applied as a redox‐active hybridization indicator to detect 18‐base oligonucleotides from the CaMV35S promoter gene. This biosensor presented a very low background signal during hybridization detection and could realize the detection over a wide kinetic range from 1.0×10^?14 M to 1.0×10^?8 M , with a low detection limit of 2.0 fM towards the target sequences. The hybridization selectivity experiments further revealed that the complementary sequence, the one‐base‐mismatched sequence, and the non‐complementary sequence could be well‐distinguished by the cobalt‐complex‐based biosensor.     

Evaluation of the Oxo‐bridged Dinuclear Ruthenium Ammine Complex as Redox Mediator in an Electrochemical Biosensor

The mediation of electron‐transfer by oxo‐bridged dinuclear ruthenium ammine [(bpy)₂(NH₃)Ru^III(µ‐O)Ru^III(NH₃)(bpy)₂]⁴⁺ for the oxidation of glucose was investigated by cyclic voltammetry. These ruthenium (III) complexes exhibit appropriate redox potentials of 0.131–0.09 V vs. SCE to act as electron‐transfer mediators. The plot of anodic current vs. the glucose concentration was linear in the concentration range between 2.52×10^?5 and 1.00×10^?4 mol L^?1. Moreover, the apparent Michaelis‐Menten kinetic (K_M^app) and the catalytic (K_cat) constants were 8.757×10^?6 mol L^?1 and 1,956 s^?1, respectively, demonstrating the efficiency of the ruthenium dinuclear oxo‐complex [(bpy)₂(NH₃)Ru^III(µ‐O)Ru^III(NH₃)(bpy)₂]⁴⁺ as mediator of redox electron‐transfer.     

Interactions of Dissolved dsDNA with Intercalating Drug by Anodic Voltammetry and Spectroscopy. Influence of pH

The interactions of C‐1305 (5‐dimethylaminopropylamino‐8‐hydroxy‐6H‐v‐triazolo[4,5,1‐de]acridin‐6‐one) with DNA were studied using differential pulse voltammetry and UV‐vis spectroscopy. C‐1305 interacts with dsDNA in two ways: by intercalation and by binding to the minor‐groove. For the intercalation at physiological pH (7.4) the values of the binding constant, K₁, and the binding‐site size, n₁, equal 3.36×10⁵ M^?1 and 2.5, respectively. For the weak interactions the K₂ and n₂ parameters equal 0.18×10⁵ M^?1 and 4. In the presence of excess NaCl the weak interactions do not vanish, therefore they are assigned to the minor groove binding. Substantial and complex is the influence of pH.     

Electrocatalytic Oxidation and Voltammetric Determination of Nitrite on Hydrophobic Ionic Liquid‐Carbon Nanotube Gel‐Chitosan Composite Modified Electrodes

A novel room temperature ionic liquid (i.e., 1‐octyl‐3‐methylimidazolium hexafluorophosphate, OMIMPF₆)‐multiwall carbon nanotube (MWNT) gel‐chitosan (Chi) composite modified glassy carbon electrode (GCE) was fabricated and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), infrared spectroscopy (IR), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). The OMIMPF₆‐MWNT gel‐Chi composite showed good conductivity, stability, and extraction effect due to the synergic action of OMIMPF₆, MWNT, and Chi. Furthermore, it was found that the OMIMPF₆‐MWNT gel‐Chi composite had strong electrocatalytic effect on the oxidation of nitrite and at the OMIMPF₆‐MWNT gel‐Chi/GCE nitrite could produce a very sensitive anodic peak. Under optimized conditions, the peak current was linear to nitrite concentration from 2.0×10^?8 to 6.0×10^?5 M. The detection limit was 1.0×10^?8 M. The electrode also exhibited acceptable stability, repeatability and selectivity. It was used successfully for the determination of nitrite in soil, sewage and sausage samples.     

Nitrate Anion Recognition in Organic–Aqueous Solvent Mixtures by a Bis(triazolium)acridine‐Containing [2]Rotaxane

The synthesis of a novel [2]rotaxane host system containing a bis(triazolium)acridine‐based axle component is reported. ¹H NMR anion‐binding titrations reveal that the rotaxane is able to recognise selectively the NO₃^? anion over a range of more basic oxoanions (AcO^?, HCO₃^? and H₂PO₄^?) in a competitive organic–aqueous solvent mixture.     

Nucleic Acid Biosensor for Detection of Human Immunodeficiency Virus Using Aquabis(1,10‐phenanthroline)copper(II) Perchlorate as Electrochemical Indicator

The electrochemical behavior of aquabis(1,10‐phenanthroline)copper(II) perchlorate [Cu(H₂O)(phen)₂]·2ClO₄, where phen=1,10‐phenanthroline, on binding to DNA at a glassy carbon electrode (GCE) and in solution, was described. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) results showed that [Cu(H₂O)(phen)₂]²⁺ had excellent electrochemical activity on the GCE with a couple of quasi‐reversible redox peaks. The interaction mode between [Cu(H₂O)(phen)₂]²⁺ and double‐strand DNA (dsDNA) was identified to be intercalative binding. An electrochemical DNA biosensor was developed with covalent immobilization of human immunodeficiency virus (HIV) probe for single‐strand DNA (ssDNA) on the modified GCE. Numerous factors affecting the probe immobilization, target hybridization, and indicator binding reactions were optimized to maximize the sensitivity and speed of the assay. With this approach, a sequence of the HIV could be quantified over the range from 7.8×10^?9 to 3.1×10^?7 mol·L^?1 with a linear correlation of γ=0.9987 and a detection limit of 1.3×10^?9 mol·L^?1.     

New Insight in Copper‐Ion Binding to Human Islet Amyloid: The Contribution of Metal‐Complex Speciation To Reveal the Polypeptide Toxicity

Type‐2 diabetes (T2D) is considered to be a potential threat on a global level. Recently, T2D has been listed as a misfolding disease, such as Alzheimer's and Parkinson's diseases. Human islet amyloid polypeptide (hIAPP) is a molecule cosecreted in pancreatic β cells and represents the main constituent of an aggregated amyloid found in individuals affected by T2D. The trace‐element serum level is significantly influenced during the development of diabetes. In particular, the dys‐homeostasis of Cu²⁺ ions may adversely affect the course of the disease. Conflicting results have been reported on the protective role played by complex species formed by Cu²⁺ ions with hIAPP or its peptide fragments in vitro. The histidine (His) residue at position 18 represents the main binding site for the metal ion, but contrasting results have been reported on other residues involved in metal‐ion coordination, in particular those toward the N or C terminus. Sequences that encompass regions 17–29 and 14–22 were used to discriminate between the two models of the hIAPP coordination mode. Due to poor solubility in water, poly(ethylene glycol) (PEG) derivatives were synthesized. A peptide fragment that encompasses the 17–29 region of rat amylin (rIAPP) in which the arginine residue at position 18 was substituted by a histidine residue was also obtained to assess that the PEG moiety does not alter the peptide secondary structure. The complex species formed by Cu²⁺ ions with Ac‐PEG‐hIAPP(17–29)‐NH₂, Ac‐rIAPP(17–29)R18H‐NH₂, and Ac‐PEG‐hIAPP(14–22)‐NH₂ were studied by using potentiometric titrations coupled with spectroscopic methods (UV/Vis, circular dichroism, and EPR). The combined thermodynamic and spectroscopic approach allowed us to demonstrate that hIAPP is able to bind Cu²⁺ ions starting from the His18 imidazole nitrogen atom toward the N‐terminus domain. The stability constants of copper(II) complexes with Ac‐PEG‐hIAPP(14–22)‐NH₂ were used to simulate the different experimental conditions under which aggregate formation and oxidative stress of hIAPP has been reported. Speciation unveils: 1) the protective role played by increased amounts of Cu²⁺ ions on the hIAPP fibrillary aggregation, 2) the effect of adventitious trace amounts of Cu²⁺ ions present in phosphate‐buffered saline (PBS), and 3) a reducing fluorogenic probe on H₂O₂ production attributed to the polypeptide alone.     

Electrochemical Determination and in silico Studies of Fludarabine on NH2 Functionalized Multiwalled Carbon Nanotube Modified Glassy Carbon Electrode

A sensitive voltammetric technique has been developed for the determination of Fludarabine using amine‐functionalized multi walled carbon nanotubes modified glassy carbon electrode (NH₂‐MWCNTs/GCE). Molecular dynamics simulations, an in silico technique, were employed to examine the properties including chemical differences of Fludarabine‐ functionalized MWCNT complexes. The redox behavior of Fludarabine was examined by cyclic, differential pulse and square wave voltammetry in a wide pH range. Cyclic voltammetric investigations emphasized that Fludarabine is irreversibly oxidized at the NH₂‐MWCNTs/GCE. The electrochemical behavior of Fludarabine was also studied by cyclic voltammetry to evaluate both the kinetic (k_s and E_a) and thermodynamic (ΔH, ΔG and ΔS) parameters on NH₂‐MWCNTs/GCE at several temperatures. The mixed diffusion‐adsorption controlled electrochemical oxidation of Fludarabine revealed by studies at different scan rates. The experimental parameters, such as pulse amplitude, frequency, deposition potential optimized for square‐wave voltammetry. Under optimum conditions in phosphate buffer (pH 2.0), a linear calibration curve was obtained in the range of 2×10^?7 M–4×10^?6 M solution using adsorptive stripping square wave voltammetry. The limit of detection and limit of quantification were calculated 2.9×10^?8 M and 9.68×10^?8 M, respectively. The developed method was applied to the simple and rapid determination of Fludarabine from pharmaceutical formulations.     

Preparation and characterization of conductive chitosan–ionic liquid composite membranes

Anhydrous conductive membranes composing of a composite of chitosan (CS) and ionic liquids with symmetrical carboxyl groups were explored. Scanning electron microscope images revealed that porous composite membranes could be obtained by combining CS with different amounts of 1,4‐bis(3‐carboxymethyl‐imidazolium)‐1‐yl butane chloride ([CBIm]Cl). Fourier transform infrared and proton nuclear magnetic resonance confirmed that the formation of ammonium salts after CS was combined with [CBIm]Cl. The thermal property of CS–ionic liquid composite membranes was studied through thermogravimetric analysis. The anhydrous ionic conductivities of CS–[CBIm]X (X = Cl, Ac, BF₄, and I) composite membranes were measured using ac impedance spectroscopy at room temperature in N₂ atmosphere. The conductivities (0.4–0.7 × 10^?4 Scm^?1), found to be in the same range as semiconductors, were significantly higher than those of pure CS membrane (<10^?8 Scm^?1). In addition, the anhydrous conductivity of composite membrane based on CS–[CBIm]I at room temperature reached a level as high as 0.91 × 10^?2 Scm^?1 when iodine was doped. Copyright © 2011 John Wiley & Sons, Ltd.