Copper(II) complexes of rat amylin fragments

The fragments of rat amylin rIAPP(17-29) (Ac-VRSSNNLGPVLPP-NH(2)), rIAPP(17-22) (Ac-VRSSNN-NH(2)), rIAPP(19-22) (Ac-SSNN-NH(2)) and rIAPP(17-20) (Ac-VRSS-NH(2)) together with the related mutant peptides (Ac-VASS-NH(2) and Ac-VRAA-NH(2)) have been synthesized and their copper(II) complexes studied by potentiometric, UV-Vis, CD and EPR spectroscopic methods. Despite the lack of any common strongly coordinating donor functions some of these fragments are able to bind copper(II) ions in the physiological pH range. The longest fragment rat amylin(17-29) keeps one equivalent copper(II) ion in solution in the whole pH range, while two other peptides Ac-VRSSNN-NH(2) and Ac-SSNN-NH(2) are also able to interact with copper(II) ions in the slightly alkaline pH range. According to the spectral parameters of the complexes, the peptides can be classified into two different categories: (i) the tetrapeptides Ac-VRSS-NH(2), Ac-VASS-NH(2) and Ac-VRAA-NH(2) can interact with copper(II) only under strongly alkaline conditions (pH > 10.0) and the formation of only one species with four amide nitrogen coordination can be detected; (ii) the peptides Ac-VRSSNNLGPVLPP-NH(2), Ac-VRSSNN-NH(2) and Ac-SSNN-NH(2) can form complexes above pH 6.0 with the major stoichiometries [CuH(-2)L], [CuH(-3)L](-) and [CuH(-4)L](2-). These data support that rIAPP(17-29) can interact with copper(II) ions under physiological conditions and the SSNN tetrapeptide fragment can be considered as the shortest sequence responsible for metal binding. Density functional theory (DFT) calculations provide some information on the possible coordination modes of Ac-SSNN-NH(2) towards the copper(II) ion and suggest that for [CuH(-2)L], [CuH(-3)L](-) and [CuH(-4)L](2-), the binding of two, three and four deprotonated amide nitrogens, with NH(-) of the side chain of asparagine as anchoring group, is probable. Moreover, these data reveal that peptides can be effective metal binding ligands even in the absence of anchoring groups, if more polar side chains are present in a specific sequence.     

Mechanism of the Tyrosine Ammonia Lyase Reaction—Tandem Nucleophilic and Electrophilic Enhancement by a Proton Transfer

Quantum mechanics/molecular mechanics calculations in tyrosine ammonia lyase (TAL) ruled out the hypothetical Friedel–Crafts (FC) route for ammonia elimination from L ‐tyrosine due to the high energy of FC intermediates. The calculated pathway from the zwitterionic L ‐tyrosine‐binding state (0.0 kcal mol^?1) to the product‐binding state ((E)‐coumarate+H₂N? MIO; ?24.0 kcal mol^?1; MIO=3,5‐dihydro‐5‐methylidene‐4H‐imidazol‐4‐one) involves an intermediate (IS, ?19.9 kcal mol^?1), which has a covalent bond between the N atom of the substrate and MIO, as well as two transition states (TS1 and TS2). TS1 (14.4 kcal mol^?1) corresponds to a proton transfer from the substrate to the N1 atom of MIO by Tyr300? OH. Thus, a tandem nucleophilic activation of the substrate and electrophilic activation of MIO happens. TS2 (5.2 kcal mol^?1) indicates a concerted C? N bond breaking of the N‐MIO intermediate and deprotonation of the pro‐S β position by Tyr60. Calculations elucidate the role of enzymic bases (Tyr60 and Tyr300) and other catalytically relevant residues (Asn203, Arg303, and Asn333, Asn435), which are fully conserved in the amino acid sequences and in 3D structures of all known MIO‐containing ammonia lyases and 2,3‐aminomutases.     

The effect of point mutations on copper(II) complexes with peptide fragments encompassing the 106–114 region of human prion protein

Abstract  

The tetrapeptides Ac-SKHM-NH₂, Ac-TKHM-NH₂, Ac-MKHS-NH₂, Ac-S(OMe)KHM-NH₂, and Ac-MKHS(OMe)-NH₂ and the nonapeptides Ac-KTNSKHMAG-NH₂ and Ac-KTNMKHSAG-NH₂ were synthesized and their copper(II) complexes were studied by potentiometric, UV–Vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopic methods. These peptides mimic the 109–112 and 106–114 residues of the sequence of human prion protein. The imidazole-N donor atoms of histidyl residues were found to be the primary metal binding sites of all peptide fragments. This binding mode provides a good possibility for the cooperative deprotonation and metal ion coordination of two amide functions preceding histidine. The (N_im,N⁻,N⁻)-bonded species predominate in the pH range 5.5–7.0 and the free coordination sites of these species make possible the metal binding of weakly coordinating side chains. The comparison of the potentiometric and spectroscopic results revealed the stabilizing role of the oxygen donors of seryl, threonyl, or methoxyseryl residues of Ac-SKHM-NH₂, Ac-TKHM-NH₂, Ac-S(OMe)KHM-NH₂, and Ac-KTNSKHMAG-NH₂ containing the mutations in position 109. These interactions were, however, not observed in the peptides containing the specific amino acids in other locations of the peptide sequence.     

Mechanism of the tyrosine ammonia lyase reaction-tandem nucleophilic and electrophilic enhancement by a proton transfer

Quantum mechanics/molecular mechanics calculations in tyrosine ammonia lyase (TAL) ruled out the hypothetical Friedel-Crafts (FC) route for ammonia elimination from L-tyrosine due to the high energy of FC intermediates. The calculated pathway from the zwitterionic L-tyrosine-binding state (0.0?kcal mol(-1)) to the product-binding state ((E)-coumarate+H(2)N-MIO; -24.0?kcal mol(-1); MIO = 3,5-dihydro-5-methylidene-4H-imidazol-4-one) involves an intermediate (IS, -19.9?kcal mol(-1)), which has a covalent bond between the N atom of the substrate and MIO, as well as two transition states (TS1 and TS2). TS1 (14.4?kcal mol(-1)) corresponds to a proton transfer from the substrate to the N1 atom of MIO by Tyr300-OH. Thus, a tandem nucleophilic activation of the substrate and electrophilic activation of MIO happens. TS2 (5.2?kcal mol(-1)) indicates a concerted C-N bond breaking of the N-MIO intermediate and deprotonation of the pro-S β position by Tyr60. Calculations elucidate the role of enzymic bases (Tyr60 and Tyr300) and other catalytically relevant residues (Asn203, Arg303, and Asn333, Asn435), which are fully conserved in the amino acid sequences and in 3D structures of all known MIO-containing ammonia lyases and 2,3-aminomutases.     

VO2+ complexation by bioligands showing keto-enol tautomerism: a potentiometric, spectroscopic, and computational study

The interaction of VO(2+) ion with ligands of biological interest that are present in important metabolic pathways--2-oxopropanoic acid (pyruvic acid, pyrH), 3-hydroxy-2-oxopropanoic acid (3-hydroxypyruvic acid, hydpyrH), oxobutanedioic acid (oxalacetic acid, oxalH(2)), (S)-hydroxybutanedioic acid (l-malic acid, malH(2)), and 2,3-dihydroxy-(E)-butanedioic acid (dihydroxyfumaric acid, dhfH(2))--was described. Their complexing capability was compared with that of similar ligands: 3-hydroxy-2-butanone (hydbut) and 3,4-dihydroxy-3-cyclobutene-1,2-dione (squaric acid, squarH(2)). All of these ligands (except l-malic acid) exhibit keto-enol tautomerism, and the presence of a metal ion can influence such an equilibrium. The different systems were studied with electron paramagnetic resonance (EPR) and UV-vis spectroscopies and with pH potentiometry. Density functional theory (DFT) methods provide valuable information on the relative energy of the enol and keto forms of the ligands both in the gas phase and in aqueous solution, on the geometry of the complexes, and on EPR and electronic absorption parameters. The results show that most of the ligands behave like α-hydroxycarboxylates, forming mono- and bis-chelated species with (COO(-), O(-)) coordination, demonstrating that the metal ion is able to stabilize the enolate form of some ligands. With dihydroxyfumaric acid, the formation of a non-oxidovanadium(IV) complex, because of rearrangement of dihydroxyfumaric to dihydroxymaleic acid (dhmH(2)), can be observed. With 3-hydroxy-2-butanone and 3,4-dihydroxy-3-cyclobutene-1,2-dione, complexation of VO(2+) does not take place and the reason for this behavior is explained by chemical considerations and computational calculations.     

Eine Methode zur Bestimmung des Isopropylalkohols in Gegenwart anderer Alkohole

Zusammenfassung Nach einer Literaturübersicht wird ein Schnellverfahren für die Untergrundkorrektion und die gleichzeitige Auswertung beschrieben. Dies erfolgt mit Hilfe eines mit einem Spektralprojektor projizierten Graphikons, einer Indexlinie verÄnderlicher Winkelstellung und mit einer auf den Projektionsschirm gelegten Y- und log c-Skala. Die Genauigkeit des Verfahrens wird unter Berechnung des Ablesungsfehlers und auf Grund praktischer Bestimmungen festgestellt. Die erreichbare Genauigkeit erweist sich als genügend. Die gute Verwendbarkeit der Methode wird an praktischen analytischen Beispielen gezeigt.     

Determination of Chiral Impurity of Naproxen in Different Pharmaceutical Formulations Using Polysaccharide-Based Stationary Phases in Reversed-Phased Mode

A novel, validated, reversed-phase (RP), chiral high performance liquid chromatography (HPLC) method was developed for the enantiopurity control analysis of naproxen, a frequently used non-steroidal anti-inflammatory agent using polysaccharide-type chiral stationary phase (CSP). In the screening phase of method development, seven columns were tested in polar organic (PO) mode using mobile phases consisting of 0.1% acetic acid in methanol, ethanol, 2-propanol, and acetonitrile. Enantiorecognition was observed only in five cases. The best enantioseparation was observed on a Lux Amylose-1 column with 0.1% (v/v) acetic acid in ethanol with a resolution (R_s) of 1.24. The enantiomer elution order was unfavorable, as the distomer eluted after the eutomer. When the ethanolic mobile phase was supplemented with water, enantiomer elution order reversal was observed, indicating a difference in the enantiorecognition mechanism upon switching from PO to RP mode. Furthermore, by changing ethanol to methanol, not only lower backpressure, but also higher resolution was obtained. Subsequent method optimization was performed using a face-centered central composite design (FCCD) to achieve higher chiral resolution in a shorter analysis time. Optimized parameters offering baseline separation were as follows: Lux Amylose-1 stationary phase, thermostated at 40 °C, and a mobile phase consisting of methanol:water:acetic acid 85:15:0.1 (v/v/v), delivered with 0.65 mL/min flow rate. Using these optimized parameters, a R_s = 3.21 ± 0.03 was achieved within seven minutes. The optimized method was validated according to the ICH guidelines and successfully applied for the analysis of different pharmaceutical preparations, such as film-coated tablets and gel, as well as fixed-dose combination tablets, containing both naproxen and esomeprazole.