Coordination abilities of N-terminal fragments of alpha-synuclein towards copper(II) ions: a combined potentiometric and spectroscopic study

Copper(II) complexes of the 1-17 (MDVFMKGLSKAKEGVVA-NH(2)), 1-28 (MDVFMKGLSKAKEGVVAAAEKTKQGVAE-NH(2)), 1-39 (MDVFMKGLSKAKEGVVAAAEKTKQGVAEAPGKTKEGVLY-NH(2)) and 1-39 (A30P) fragments of alpha-synuclein were studied by potentiometric, UV-Vis (UV-visible), CD (circular dichroism) and EPR (electron paramagnetic resonance) spectroscopic methods to determine the stoichiometry, stability constants and coordination modes of the complexes formed. The beta-carboxylate group of Asp residue in second position of the peptide chain coordinates strongly to Cu(II) ion over the pH range 4-9.5 to give unusually stable 2N complex with {NH(2), N(-), beta-COO(-), H(2)O} coordination mode. At pH above 7 the results suggest the formation of 2N, 3N, 4N complexes (in equatorial plane) and the involvement of the lateral NH(2) group of Lys residue in the axial coordination of Cu(II) ion. In CD spectra sigma (epsilon-NH(2)-Lys)-->Cu(II) charge transfer transition is observed. Addition of the 18-28 and 18-39 fragments to the 1-17 peptide does not change the coordination mode and the 1-39 fragment forms the Cu(II) complexes with higher stabilities compared to those of the 1-17, 1-28 and 1-39(A30P) fragments of alpha-synuclein.     

Iron(III)- and copper(II) complexes of an asymmetric, pentadentate salen-like ligand bearing a pendant carboxylate group

The equilibrium and solution structural properties of the iron(III) and copper(II) complexes of an asymmetric salen-like ligand (N,N'-bis(2-hydroxybenzyl)-2,3-diamino-propionic acid, H(3)bhbdpa) bearing a pendant carboxylate group were characterized in aqueous solution by potentiometric, pH-dependent electron paramagnetic resonance (EPR) and UV-Vis (UV-Visible) measurements. In the equimolar systems the pentadentate ligand forms very stable, differently protonated mononuclear complexes with both metal ions. In the presence of iron(III) {NH, PhO(-), COO(-)}, {2NH, 2PhO(-), COO(-)} and {2NH, 2PhO(-), COO(-), OH(-)} coordinated complexes are dominant. The EPR titrations reflected the presence of microscopic complex formation pathways, leading to the formation of binding isomers in case of Cu(H(2)bhbdpa)(+), Cu(Hbhbdpa) and Cu(bhbdpa)(-). The {2NH, 2PhO(-)+COO(-)/H(2)O} coordinated Cu(bhbdpa) is the only species between pH 6-11. At twofold excess of metal ion dinuclear complexes were detected with both iron(III) and copper(II). In presence of iron(III) a mu-carboxylato-mu-hydroxo-bridged dinuclear complex (Fe(2)(bhbdpa)(OH)(3)) is formed from Fe(H(2)bhbdpa)(2+) through overlapping proton release processes, providing one of the rare examples for the stabilization of an endogenous carboxylate bridged diiron core in aqueous solution. The complex Cu(2)(bhbdpa)(+) detected in the presence of copper(II) is a paramagnetic (S=1) species with relatively weakly coupled metal ions.     

Acid–base and metal ion binding properties of 2-thiocytidine in aqueous solution

The thionucleoside 2-thiocytidine (C2S) occurs in nature in transfer RNAs; it receives attention in diverse fields like drug research and nanotechnology. By potentiometric pH titrations we measured the acidity constants of H(C2S)(+) and the stability constants of the M(C2S)(2+) and M(C2S-H)(+) complexes (M(2+) = Zn(2+), Cd(2+)), and we compared these results with those obtained previously for its parent nucleoside, cytidine (Cyd). Replacement of the (C2)=O unit by (C2)=S facilitates the release of the proton from (N3)H(+) in H(C2S)(+) (pK (a) = 3.44) somewhat, compared with H(Cyd)(+) (pK (a) = 4.24). This moderate effect of about 0.8 pK units contrasts with the strong acidification of about 4 pK units of the (C4)NH(2) group in C2S (pK (a) = 12.65) compared with Cyd (pK (a) approximately 16.7); the reason for this result is that the amino-thione tautomer, which dominates for the neutral C2S molecule, is transformed upon deprotonation into the imino-thioate form with the negative charge largely located on the sulfur. In the M(C2S)(2+) complexes the (C2)S group is the primary binding site rather than N3 as is the case in the M(Cyd)(2+) complexes, though owing to chelate formation N3 is to some extent still involved in metal ion binding. Similarly, in the Zn(C2S-H)(+) and Cd(C2S-H)(+) complexes the main metal ion binding site is the (C2)S(-) unit (formation degree above 99.99% compared with that of N3). However, again a large degree of chelate formation with N3 must be surmised for the M(C2S-H)(+) species in accord with previous solid-state studies of related ligands. Upon metal ion binding, the deprotonation of the (C4)NH(2) group (pK (a) = 12.65) is dramatically acidified (pK (a) approximately 3), confirming the very high stability of the M(C2S-H)(+) complexes. To conclude, the hydrogen-bonding and metal ion complex forming capabilities of C2S differ strongly from those of its parent Cyd; this must have consequences for the properties of those RNAs which contain this thionucleoside.     

Transition metal complexes of terminally protected peptides containing histidyl residues

Histidine-containing peptide fragments of prion protein are efficient ligands to bind various transition metal ions and they have high selectivity in metal binding. The metal ion affinity follows the order: Pd(II)>Cu(II)>Ni(II)Zn(II)>Cd(II) approximately Co(II)>Mn(II). The high selectivity of metal binding is connected to the involvement of both imidazole and amide nitrogen atoms in metal binding for Pd(II), Cu(II) and Ni(II), while only the monodentate N(im)-coordination is possible with the other metal ions. The stoichiometry and binding mode of palladium(II) complexes show great variety depending on the metal ion to ligand ratio, pH and especially the presence of coordinating donor atoms in the side chains of peptide fragments. It is also clear from our data that the peptide fragments containing histidine outside the octarepeat (His96, His111 and His187) are more efficient ligands than the monomer peptide fragments of the octarepeat domain.     

Preparation of platinum(IV) complexes with dipeptide and diimine. X-ray crystal structure and 195Pt NMR spectra

We prepared platinum(IV) complexes containing dipeptide and diimine or diamine, the [PtCl(dipeptide-N,N,O)(diimine or diamine)]Cl complex, where -N,N,O means dipeptide coordinated as a tridentate chelate, dipeptide=glycylglycine (NH(2)CH(2)CON(-)CH(2)COO(-), digly, where two protons of dipeptide are detached when the dipeptide coordinates to metal ion as a tridentate chelate), glycyl-L-alanine (NH(2)CH(2)CON(-)CHCH(3)COO(-), gly-L-ala), L-alanylglycine (NH(2)CH CH(3)CON(-)CH(2)COO(-), L-alagly), or L-alanyl-L-alanine (NH(2)CHCH(3)CON(-)CHCH(3)COO(-), dil-ala), and diimine or diamine=bipyridine (bpy), ethylenediamine (en), N-methylethylenediamine (N-Me-en), or N,N'-dimethylethylenediamine (N,N'-diMe-en). In the complexes containing gly-L-ala or dil-ala, two separate peaks of the (195)Pt NMR spectra of the [PtCl(dipeptide-N,N,O)(diimine or diamine)]Cl complexes appeared in, but in the complexes containing digly or L-alagly, one peak which contained two overlapped signals appeared. One of the two complexes containing gly-L-ala and bpy, [PtCl(gly-L-ala-N,N,O)(bpy)]NO(3), crystallized and was analyzed. This complex has the monoclinic space group P2(1)2(1)2(1) with unit cell dimensions of a=9.7906(3)A, b=11.1847(2)A, c=16.6796(2)A, Z=4. The crystal data revealed that this [PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex has the near- (Cl, CH(3)) configuration of two possible isomers. Based on elemental analysis, the other complex must have the near- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) configuration. The (195)Pt NMR chemical shifts of the near- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex and the far- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex are 0 ppm and -19 ppm, respectively (0 ppm for the Na(2)[PtCl(6)] signal). The additive property of the (195)Pt NMR chemical shift is discussed. The (195)Pt NMR chemical shifts of [PtCl(dipeptide-N,N,O)(bpy)]Cl appeared at a higher field when the H attached to the dipeptide carbon atom was replaced with a methyl group. On the other hand, the (195)Pt NMR chemicals shifts of [PtCl(dipeptide-N,N,O)(diamine)] appeared at a lower field when the H attached to the diamine nitrogen atom was replaced with a methyl group, in the order of [PtCl(digly-N,N,O)(en)]Cl, [PtCl(digly-N,N,O)(N-Me-en)]Cl, and [PtCl(digly-N,N,O)(N,N'-diMe-en)]Cl.     

Copper (II) complexes with Ac-HXH-NHMe (X=Gly, Ala and Aib) peptide motifs: influence of increasing CH(3) groups at C(alpha) of residue X on the coordination in solution

The interaction of Cu(II) ion with small peptides has been an interesting subject to clarify the role of copper in detail. As various Cu(II)-oligopeptide complexes can also be good models for the active centers of metalloenzymes, complexes of tripeptide and tetrapeptides are frequently investigated instead of the complexes of large peptides. The histidine side-chains of various metalloproteins frequently take part in the copper(II) coordination. Accordingly, we studied the coordination of Cu(II) to the N and C terminal protected tripeptide ligands L(A) (Ac-HisGlyHis-NHMe), L(B) (Ac-HisAlaHis-NHMe) and L(C) (Ac-HisAibHis-NHMe) in aqueous solution potentiometrially in order to determine the effect of C(alpha) methyl groups at middle residue acid on the ligation of the backbone NH and also on histidine's N(im) of coordination. Species distribution curves indicates that in acidic pH, all three peptides behave as bidentate ligands and a macrochelate forms on the metal coordination with the two histidine imidazolyl N. This coordination remains unaffected with the +I effect of increasing CH(3) groups at C(alpha) of middle residue. In the pH range 4-8, the tridentate coordination from the peptide is seen in ligand L(A) and L(B) while it is absent in L(C) due to +I effect of two C(alpha) methyl groups at middle residue as they makes N-terminal NH deprotonation difficult in this pH range and it takes place along with C terminal NH and only 4N coordinated species formed at higher pH. These 4N (N(im), N(-), N(-), N(im)) coordinated species are formed by all the three ligands at higher pH values.     

Acid-base properties and copper(II) complexes of dipeptides containing histidine and additional chelating bis(imidazol-2-yl) residues

Copper(II) complexes of dipeptides of histidine containing additional chelating bis(imidazol-2-yl) agent at the C-termini (PheHis-BIMA [N-phenylalanyl-histidyl-bis(imidazol-2-yl)methylamine] and HisPhe-BIMA [N-histidyl-phenylalanyl-bis(imidazol-2-yl)methylamine]) were studied by potentiometric, UV-Visible and Electron Paramagnetic Resonance (EPR) techniques. The imidazole nitrogen donor atoms of the bis(imidazol-2-yl)methyl group are described as the primary metal binding sites forming stable mono- and bis(ligand) complexes at acidic pH. The formation of a ligand-bridged dinuclear complex [Cu2L2]4+ is detected in equimolar solutions of copper(II) and HisPhe-BIMA. The coordination isomers of the dinuclear complex are described via the metal binding of the bis(imidazol-2-yl)methyl, amino-carbonyl and amino-imidazole(His) functions. In the case of the copper(II)-PheHis-BIMA system the [NH2, N-(amide), N(Im)] tridentate coordination of the ligand is favoured and results in the formation of di- and trinuclear complexes [Cu2H(-1)L]3+ and [Cu3H(-2)L2]4+ in equimolar solutions. The presence of these coordination modes shifts the formation of "tripeptide-like" ([NH2, N-, N-, N(Im)]-coordinated) [CuH(-2)L] complexes into alkaline pH range as compared to other dipeptide derivatives of bis(imidazol-2-yl) ligands. Although there are different types of imidazoles in these ligands, the deprotonation and coordination of the pyrrole-type N(1)H groups does not occur below pH 10.     

Interaction of Cu(2+) with His-Val-His and of Zn(2+) with His-Val-Gly-Asp, two peptides surrounding metal ions in Cu,Zn-superoxide dismutase enzyme

His-Val-His and His-Val-Gly-Asp are two naturally occurring peptide sequences, present at the active site of Cu,Zn-superoxide dismutase (Cu,Zn-SOD). The interactions of His-Val-His=A (copper binding site) with Cu(II) and of His-Val-Gly-Asp=B (zinc binding site) with Zn(II) have been studied by using both potentiometric and spectroscopic methods (visible, EPR, NMR). The stoichiometry, stability constants and solution structure of the complexes formed have been determined. The binding modes of the species [CuAH](2+) and [CuA](+) were characterized by histamine type of coordination. [CuA](+) is further stabilized by the formation of a macrochelate with the involvement of the imidazole of the C-terminal histidine. The existence of macrochelate results in a slight distortion of the coordination geometry providing good base for the development of enzyme models. The enhanced stability of the macrochelate suppresses the formation of bis-complexes as well as the amide deprotonation. This process, however, takes place at higher pH resulting in the formation of the 4 N(-) coordinated [NH(2),N(-),N(-),N(im)] species [CuAH(2-)](-). On the other hand, in the case of the Zn(II)-His-Val-Gly-Asp system, coordination takes place at the terminal carboxylate in species [ZnBH(2)](2+). Monodentate binding occurs via the N-terminal imidazole in [ZnBH](+) while histamine type of coordination is possible in [ZnB], [ZnB(2)H](-) and [ZnB(2)](2-) species. Amide deprotonation does not take place in the case of Zn(2+), hydroxo-complexes are formed instead.     

Pyrazolyl derivatives as bifunctional chelators for labeling tumor-seeking peptides with the fac-[M(CO)3]+ moiety (M = 99mTc, Re): synthesis, characterization, and biological behavior

Radiolabeling of biologically active molecules with the [(99m)Tc(CO)(3)](+) unit has been of primary interest in recent years. With this in mind, we herein report symmetric (L(1)) and asymmetric (L(2)-L(5)) pyrazolyl-containing chelators that have been evaluated in radiochemical reactions with the synthon [(99m)Tc(H(2)O)(3)(CO)(3)](+) (1a). These reactions yielded the radioactive building blocks [(99m)Tc(CO)(3)(k(3)-L)](+) (L = L(1)-L(5), 2a-6a), which were identified by RP-HPLC. The corresponding Re surrogates (2-6) allowed for macroscopic identification of the radiochemical conjugates. Complexes 2a-6a, with log P(o/w) values ranging from -2.35 to 0.87, were obtained in yields of > or =90% using ligand concentrations in the 10(-5-)10(-4) M range. Challenge studies with cysteine and histidine revealed high stability for all of these radioactive complexes, and biodistribution studies in mice indicated a fast rate of blood clearance and high rate of total radioactivity excretion, occurring primarily through the renal-urinary pathway. Based on the framework of the asymmetric chelators, the novel bifunctional ligands 3,5-Me(2)-pz(CH(2))(2)N((CH(2))(3)COOH)(CH(2))(2)NH(2) (L(6)) and pz(CH(2))(2)N((CH(2))(3)COOH)(CH(2))(2)NH(2) (L(7)) have been synthesized and their coordination chemistry toward (NEt(4))(2)[ReBr(3)(CO)(3)] (1) has been explored. The resulting complexes, fac-[Re(CO)(3)(k(3)-L)]Br (L(6)(7), L(7)(8)), contain tridentate ancillary ligands that are coordinated to the metal center through the pyrazolyl and amine nitrogen atoms, as observed for the other related building blocks. L(6) and L(7) were coupled to a glycylglycine ethyl ester dipeptide, and the resulting functionalized ligands were used to prepare the model complexes fac-[Re(CO)(3)(kappa(3)-3,5-Me(2)-pz(CH(2))(2)N(glygly)(CH(2))(2)NH(2))](+) (9/9a) and fac-[Re(CO)(3)(kappa(3)-pz(CH(2))(2)N(CH(2))(3)(glygly)(CH(2))(2)NH(2))](+) (10/10a) (M = Re, (99m)Tc). These small conjugates have been fully characterized and are reported herein. On the basis of the in vitro/in vivo behavior of the model complexes (2a-6a, 9a, 10a), we chose to evaluate the in vitro/in vivo biological behavior of a new tumor-seeking Bombesin pyrazolyl conjugate, [(L(6))-G-G-G-Q-W-A-V-G-H-L-M-NH(2)], that has been labeled with the [(99m)Tc(CO)(3)](+) metal fragment. Stability, in vitro cell binding assays, and pharmacokinetics studies in normal mice are reported herein.     

Quantitative aspects of metal ion binding to certain transfer RNA anticodon loop modified nucleosides

Magnesium and manganese ions bind strongly to the unusual transfer RNA anticodon loop nucleotides, N-[(9-beta-D-ribofuranosyl-9H-purin-6-yl)carbamoyl]-L-threonine 5'-monophosphate (pt6A) and uridine-5-oxyacetic acid 5'-monophosphate (pV). Potentiometric measurements have shown that the delta G for metal ion-pt6A complex formation is 2-3-times more exothermic than for AMP. Electron-nuclear longitudinal dipolar relaxation data yielded manganese-ligand atom distances which permit a three-dimensional construct of the complex in which metal is coordinated to the phosphate, carboxylate of the threonine side-chain (with the nucleotide in the anti glycosidic conformation) and N7 of the adenine ring. Similarly, manganese binds strongly to pV, involving phosphate and carboxylate functions. It is possible that a facet of the functional role of these unusual residues is to chelate magnesium ions and in so doing permit optimum anticodon loop conformational stability and stability of tRNA-mRNA-ribosome complexes.     

1H NMR spectroscopic characterization of binary and ternary complexes of cobalt(II) carboxypeptidase A with inhibitors

The binding of L- and D-phenylalanine and carboxylate inhibitors to cobalt(II)-substituted carboxypeptidase A, Co(II)CPD (E), in the presence and absence of pseudohalogens (X = N3-, NCO-, and NCS-) has been studied by 1H NMR spectroscopy. This technique monitors the proton signals of histidine residues bound to cobalt(II) and is therefore sensitive to the interactions of inhibitors that perturb the coordination sphere of the metal. Enzyme-inhibitor complexes, E.I, E.I2, and E.I.X, each with characteristic NMR features, have been identified. Thus, for example, L-Phe binds close to the metal ion to form a 1:1 complex, whereas D-Phe binds stepwise, first to a nonmetal site and then to the metal ion to form a 2:1 complex. Both acetate and phenylacetate also form 2:1 adducts stepwise with the enzyme, but beta-phenylpropionate gives a 2:1 complex without any detectable 1:1 intermediate. N3-, NCO-, and NCS- generate E.I.X ternary complexes directly with Co(II)CPD.L-Phe and indirectly with the D-Phe and carboxylate inhibitor 2:1 complexes by displacing the second moiety from its metal binding site. The NMR data suggest that when the carboxylate group of a substrate or inhibitor binds at the active site, a conformational change occurs that allows a second ligand molecule to bind to the metal ion, altering its coordination sphere and thereby attenuating the bidentate behavior of Glu-72. The 1H NMR signals also reflect alterations in the histidine interactions with the metal upon inhibitor binding. Isotropic shifts in the signals for the C-4 (c) and N protons (a) of one of the histidine ligands are readily observed in all of these complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and oxidation of carboxylate-bridged diiron(II) complexes with substrates tethered to primary alkyl amine ligands

The synthesis and crystallographic characterization of a series of diiron(II) complexes with sterically hindered terphenyl carboxylate ligands and alkyl amine donors are presented. The compounds [Fe(2)(mu-O(2)CAr(Tol))(4)(L)(2)] (L=NH(2)(CH(2))(2)SBn (1); NH(2)(CH(2))(3)SMe (2); NH(2)(CH(2))(3)CCH (3)), where (-)O(2)CAr(Tol) is 2,6-di(p-tolyl)benzoate, and [Fe(2)(mu-O(2)CAr(Xyl))(2)(O(2)CAr(Xyl))(2)(L)(2)] (L=NH(2)(CH(2))(3)SMe (4); NH(2)(CH(2))(3)CCH (5)), where (-)O(2)CAr(Xyl) is 2,6-di(3,5-dimethylphenyl)benzoate, were prepared as small molecule mimics of the catalytic sites of carboxylate-bridged non-heme diiron enzymes. The compounds with the (-)O(2)CAr(Tol) carboxylate form tetrabridged structures, but those containing the more sterically demanding (-)O(2)CAr(Xyl) ligand have only two bridging ligands. The ancillary nitrogen ligands in these carboxylate-rich complexes incorporate potential substrates for the reactive metal centers. Their oxygenation chemistry was studied by product analysis of the organic fragments following decomposition. Compound 1 reacts with dioxygen to afford PhCHO in approximately 30% yield, attributed to oxidative dealkylation of the pendant benzyl group. Compound 3 decomposes to form Fe(II)Fe(III) and Fe(III)Fe(IV) mixed-valence species by established bimolecular pathways upon exposure to dioxygen at low temperatures. Upon decomposition, the alkyne-substituted amine ligand was recovered quantitatively. When the (-)O(2)CAr(Tol) carboxylate was replaced by the (-)O(2)CAr(Xyl) ligand in 5, different behavior was observed. The six-coordinate iron(III) complex with one bidentate and two monodentate carboxylate ligands, [Fe(O(2)CAr(Xyl))(3)(NH(2)(CH(2))(3)CCH)(2)] (6), was isolated from the reaction mixture following oxidation.     

DNA, protein binding, cytotoxicity, cellular uptake and antibacterial activities of new palladium(II) complexes of thiosemicarbazone ligands: effects of substitution on biological activity

The coordination propensities of 4(N,N')-diethylaminosalicylaldehyde-4(N)-substituted thiosemicarbazones (H(2)L(1-4)) were investigated by reacting with an equimolar amount of [PdCl(2)(PPh(3))(2)]. The new complexes were characterized by various spectroscopic techniques. The structure determination of the complexes [Pd(DeaSal-tsc)(PPh(3))] (1), [Pd(DeaSal-mtsc)(PPh(3))] (2) and [Pd(DeaSal-etsc)(PPh(3))] (3) by X-ray crystallography showed that ligands are coordinated in a dibasic tridentate ONS donor fashion forming stable five and six membered chelate rings. The binding ability of complexes (1-4) to calf-thymus DNA (CT DNA) has been explored by absorption and emission titration methods. Based on the observations, an electrostatic and an intercalative binding mode have been proposed. The protein binding studies have been monitored by quenching of tryptophan and tyrosine residues in the presence of complexes using lysozyme as a model protein. As determined by MTT assays, complex 3 exhibited a higher cytotoxic effect towards human lung cancer cell line (A549) and liver cancer cells (HepG2). LDH, NO assay and cellular uptake of the complexes have been studied. Further, antibacterial activity studies of the complexes have been screened against the pathogenic bacteria such as Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, MIC50 values of the complexes showed that the complexes exhibited significant activity against the pathogens and among the complexes, 3 exhibited higher activity.     

Binding of distamycin A and netropsin to the 12mer DNA duplexes containing mixed AT.GC sequences with at most five or three successive AT base pairs

Circular dichroism (CD), isothermal calorimetric titrations (ITC), and temperature-dependent UV spectroscopy were used to investigate binding of the minor groove-directed ligands distamycin A (Dst) and netropsin (Net) to the following duplexes: d(GTTAGTATTTGG). d(CCAAATACTAAC), d(GTTAGTATATGG).d(CCATATACTAAC), d(GTTAGTACTTGG). d(CCAAGTACTAAC), and d(GTTAGTAGTTGG).d(CCAACTACTAAC). Our results reveal that Dst binds within the minor grooves of these dodecamers that contain five-AT and/or four-AT.GC binding sites exclusively in a dimeric high-affinity 2:1 binding mode (K approximately 10(16) M(-)(2)). By contrast, Net exhibits high-affinity binding only when it binds in a 1:1 mode (K(1) approximately 10(9) M(-)(1)) to the two duplexes that contain five-AT sites (5'-TATTT-3' and 5'-TATAT-3'). Its further binding to these two duplexes occurs in a low-affinity mode (K(2) approximately 10(6) M(-)(1)) and results in the formation of 2:1 Net-DNA complexes. To the other two duplexes that contain sequences with at most three AT consecutive base pairs Net binds in two distinctive low-affinity 1:1 binding modes (K(1) approximately 10(7) M(-)(1), K(2) approximately 10(6) M(-)(1)). Competition experiments (CD and ITC titrations) reveal that Dst entirely displaces Net from its 1:1 and 2:1 complexes with any of the four duplexes. We discuss and interpret our optical and calorimetric results in the context of the available structural information about the complexes between DNA and the sequence-specific minor groove binders Dst and Net.     

X-ray photoelectron spectroscopy of DNA-Pt complexes. Evidence of O6 (Gua)-N7 (Gua) chelation of DNA with cis- dichlorodiamine platinum(II).

The binding energies of nitrogen, oxygen, phosphorus, chlorine and Pt in several DNA - Pt (II) complexes are reported and discussed. The nitrogen band of DNA is slightly shifted upon complexation with Pt. Oxygen binding energies in the complexes studied clearly show that cis-Pt(NH3)2Cl2 forms a specific chelate N7(Gua) - O6 (Gua) with DNA as opposed to trans-Pt(NH3)2Cl2 and the other Pt compounds which react only with the N7(Gua) site of DNA.     

A theoretical model for the binding of cis-Pt(NH3)2(+2) to DNA

The binding of cis-Pt(NH3)2B1B2 to the bases B1 and B2, i.e., guanine (G), cytosine (C), adenine (A), and thymine (T), of DNA is studied theoretically. The components of the binding are analyzed and a model structure is proposed for the intrastrand binding to the dB1pdB2 sequence of a kinked double helical DNA. Quantum mechanical calculations of the ligand binding energy indicates that cis-Pt(NH3)2(+2) (cis-PDA) binds to N7(G), N3(C), O2(C), O6(G), N3(A), N7(A), O4(T) and O2(T) in order of decreasing binding energy. Conformational analysis provides structures of kinked DNA in which adjacent bases chelate to cis-PDA. Only bending toward the major groove allows the construction of acceptable square planar complexes. Examples are presented for kinks of -70 degrees and -40 degrees at the receptor site to orient the base pairs for ligand binding to B1 and B2 to form a nearly square planar complex. The energies for complex formation of cis-PDA to the various intra-strand base sites in double stranded DNA are estimated. At least 32 kcal/mole separates the energetically favorable dGpdG.cis-PDA chelate from the dCpdG.cis-PDA chelate. All other possible chelate structures are much higher in energy which correlates with their lack of observation in competition with the preferred dGpdG chelate. The second most favorable ligand energy occurs with N3(C). A novel binding site involving dC(N3)pdG(N7) is examined. Denaturation can result in an anti----syn rotation of C about its glycosidic bond to place N3(C) in the major groove for intrastrand binding in duplex DNA. This novel intrastrand dCpdG complex and the most favored dGpdG structure are illustrated with stereographic projections.     

The effect of histidyl residues on the complexation of bis(imidazolyl) containing tripeptides with copper(II) ion

Copper(II) complexes of tripeptide derivatives of bis(imidazol-2-yl) group have been studied by potentiometric, UV-visible and EPR spectroscopic methods. The peptide molecules correspond to the amino acid sequence of collagen containing histidyl residues in different locations and were connected to the bis(imidazol-2-yl) group either on the C-termini (BOC-Pro-Leu-His-BIMA, BOC-His-Leu-Gly-BIMA) or on the N-termini (BIP-His-Ala-Gly-OEt, BIP-Ile-Ala-His-OMe). It was concluded that the imidazole nitrogen donor atoms of the bis(imidazol-2-yl) moiety are the primary metal binding sites, but the histidyl imidazole nitrogens in the side chains have also some effect on the stability and the coordination mode of the complexes. All ligands can coordinate tridentately to copper(II) ion forming a six-membered chelate and a macrochelate in the [CuL]2+ complexes, which results in a slight distortion in the coordination geometry of [CuL2]2+ complexes. The deprotonation and coordination of amide nitrogens, however, were not observed in any cases.     

13C NMR studies of D- and L-phenylalanine binding to cobalt(II) carboxypeptidase A

13C NMR T1 and T2 measurements have been performed on cobalt(II) substituted carboxypeptidase A in the presence of carboxylate-13C-enriched L- and D-phenylalanine. Upon binding to the cobalt enzyme, the longitudinal and transverse relaxation rates T1p-1 and T2p-1 of these inhibitors are enhanced significantly compared to the zinc enzyme, allowing both determination of an affinity constant for inhibitor binding, K, and calculation of the metal-13C carboxylate distances. The L-and D- Phe concentration dependence of T2p-1 yields affinity constants of 290 +/- 60M-1 and 670 +/- 90M-1. The distance measurements calculated for Co-13C from T1p-1 are 0.39 +/- 0.04 and 0.42 +/- 0.04 nm for L-Phe and D-Phe. Both values are too great for direct coordination of their carboxylate groups to the metal atom. Upon formation of their respective ternary enzyme.Phe.N3- complexes, the distances are essentially unaltered. In conjunction with electronic absorption studies on these complexes it can be concluded that N3-, but not the amino acid carboxylate, is bound to the metal.     

Model studies on the coordination of copper in biological systems. The deprotonated peptide nitrogen as a potential binding site for copper(II)

1. A large number of potentially bidentate and tridentate amides, X-Y-CONH-Z, were used as model ligands to investigate the complex formation of Cu(II) with the deprotonated peptide nitrogen in biological molecules. A combination of potentiometric titration, spectrophotometry and electron paramagnetic resonance was applied to analyse the structure of the Cu(II) chelates formed at neurtal and basic pH. 2. By systematic variation of the primary binding function X, the ring size of the chelate, and the spatial properties of the C-terminal and N-terminal substituents, three classes of amide ligands could be established with reference to their capacity for Cu(II)-induced deprotonation of NHCO and metal binding. 3. Under physiological conditions of pH, peptide (class A) chelates are only formed by those bidentate amide ligands with X being an imidazole (sp2) nitrogen or a terminal (sp3) amino nitrogen. Mercaptide sulfur must also be considered to belong in this group of strong copper(II)-binding sites, but in our low-molecular-weight model ligands the redox equilibrium 2 Cu(II) + 2 RSH in equilibrium or formed from 2 CU(II) + RSSR + 2 H+ interferes, yielding insoluble Cu(I)-S polymers above pH 4. In addition to the unidentate binding strength of X, entropy effects play an important role. Depending on whether X is an imidazole or amino nitrogen, only five-membered or six-membered monocyclic chelate structures respectively cause coordination of the deprotonated peptide function. 4. Biuret (class B) Cu(II) chelates are only formed under non-physiological conditions at pH > 11.5 producing the well known violet chromophores CuIIN4(-). In general these complexes, which also include the Cu(II) biguanides, show a clearly resolved electron paramagnetic resonance spectrum with nitrogen superhyperfine structure. 5. A third class of peptide model ligands (class C) consists of those amides where the CuII-X bond does not provide enough thermodynamic stability. The binding site of these class C amides includes functional groups such as carboxylate (COO-), methionine sulfur (RSR'), aliphatic or aromatic hydroxyl (OH) and amide nitrogen (NHCO) itself. When X is a pyridine (sp2) nitrogen or an amino (sp3) nitrogen, NHCO deprotonation is only promoted in five-membered but not six-membered ring chelates. On the other hand, a combination of COO- and NH2, as in asparagine, will allow deprotonation of NHCO in the presence of Cu(II). And third, despite a pronounced unidentate affinity of the imidazole nitrogen for Cu(II), N-acetylhistamine acts as a class C amine, in contrast to imidazolylacetamide, which forms a stable Cu(II) peptide chelate. This difference in Cu binding is explained on the basis of space-filling models. These clearly demonstrate that in the case of the 2:1 complex of Cu(II) with N-acetylhistamine, the planarity of the ionised peptide function can not be retained in a square planar arrangement of the two amide ligands around the copper center.