Synthesis of DNA-sequence-selective hairpin polyamide platinum complexes

Two DNA-sequence-selective hairpin polyamide platinum(II) complexes, containing pyrrole and imidazole heterocyclic rings, have been synthesised by different methods. A six-ring complex, selective for (A/T)GGG(A/T) DNA sequences, was made by using solid-phase synthesis, whilst an eight-ring complex, selective for (A/T)CCTG(A/T) DNA sequences, was made by utilising standard wet chemistry. Solid-phase synthesis resulted in a significantly higher yield, required less purification and is more efficient than the wet synthesis; as such, it is the preferred method for further work. The metal complexes were characterised by (1)H and (195)Pt NMR spectroscopy and ESI mass spectrometry. The two compounds provide a foundation for the synthesis of more complex molecules containing multiple hairpins and/or platinum groups.     

Design of a peptide hairpin containing a central three-residue loop

The construction of a designed beta-hairpin structure, containing a central three-residue loop has been successfully achieved in the synthetic nonapeptide Boc-Leu-Phe-Val-(D)Pro-(L)Pro-(D)Ala-Leu-Phe-Val-OMe (2). The design is based on expanding the two-residue loop established in the peptide beta-hairpin Boc-Leu-Phe-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (1). Characterization of the registered beta-hairpins in peptides 1 and 2 is based on the observation of key nuclear Overhauser effects (NOEs) in CDCl(3) and CD(3)OH. Solvent titration and temperature dependence of NH chemical shifts establish the identity of NH groups involved in interstrand hydrogen bonding. In peptide 2, the antiparallel registry is maintained, with the formation of a (D)Pro-(L)Pro-(D)Ala loop, stabilized by a 5-->1 hydrogen bond between Val3 CO and Leu7 NH groups (C(13), alpha-turn) and a 3-->1 hydrogen bond between (D)Pro4 CO and (d)Ala6 NH groups (C(7), gamma-turn). NMR derived structures suggest that in peptide 2, (d)Ala(6) adopts an alpha(L) conformation. In peptide 1, the (D)Pro-(L)Pro segment adopts a type II' beta-turn. Replacement of (D)Ala (6) in peptide 2 by (L)Ala in peptide 3 yields a beta-hairpin conformation, with a central (D)Pro-(L)Pro two-residue loop. Strand slippage at the C-terminus results in altered registry of the antiparallel strands.     

Folding of the GB1 hairpin peptide from discrete path sampling

The discrete path sampling technique is used to calculate folding pathways of the 16-amino acid beta hairpin-forming sequence from residues 41-56 of the B1 domain of protein G. The folding time is obtained using master equation dynamics and kinetic Monte Carlo simulations, and the time evolution of different order parameters and occupation probabilities of groups of minima are calculated and used to characterize intermediates on the folding pathway.     

Cyclic peptide inhibitors of staphylococcal virulence prepared by Fmoc-based thiolactone peptide synthesis

Virulence factor production in Staphylococcus aureus is largely under the control of the accessory gene regulator (agr) quorum sensing system. There are four agr groups, all of which exhibit bacterial interference: each agr type synthesizes a cyclic autoinducing peptide (AIP) with a distinct sequence that activates its cognate AgrC receptor and inhibits activation of others. To better understand inhibitory AIP-AgrC interactions, we aimed to identify the minimal molecular determinants required to inhibit both non-cognate and cognate receptors. This minimization of the AIP pharmacophore also may have therapeutic relevance as the use of native AIPs to block virulence of non-cognate agr strains can prevent the establishment of an infection in vivo. We synthesized and evaluated the inhibitory activities of 10 AIP derivatives based on a truncated AIP analogue that inhibits all four agr types. To carry out the rapid, parallel synthesis of these peptides, we employed a new linker for Fmoc-based thioester peptide synthesis. Our results identify key structural elements that are necessary for AgrC inhibition and reveal key differences between non-cognate and cognate inhibitory requirements.     

Comparative reactivity of ferric-superoxo and ferryl-oxo species in heme and non-heme complexes

Ferryl-oxo species have been recognized as a key oxidant in many heme and non-heme enzymes. Recently, less-characterized ferric-superoxo species have been found or suggested to be another electrophilic oxidant. Reactivity of several vital ferryl-oxo and ferric-superoxo model complexes was examined by DFT calculations. Reactivity is found to correlate well with thermodynamic driving force and can increase with higher electrophilicity of the oxidant. Reactivity of the ferric-superoxo oxidants generally is not "superior" to the ferryl-oxo ones. Compared to the high-spin non-heme ferric-superoxo, the lower reactivity of low-spin heme ferric-superoxo, seldom utilized in nature, can be attributed to lower electrophilicity and more pronounced quenching of anti-ferromagnetic coupling between the ferric and superoxo parts. The present comparison should shed some light on mechanistic strategies in heme and non-heme enzymes and provide clues to rational design of ferric-superoxo oxidants.     

Cyclic voltammetry of iron dimine complexes in acetonitrile

The electrochemical behavior of iron diimine complexes, (H₃C?N=C(R)?C(R′)=N?CH₃)₃Fe(II) (R, R′=H,H;H, CH₃; CH₃, CH₃), and (C₅H₄N?C(R₁)=N(R₂))₃Fe(II) (R₁, R₂=H, CH₃; CH₃, CH₃) on a platinum working electrode in acetonitrile is described, and compared to that of the parent aromatic complex, tris-(2,2′-bipyridine)Fe(II). One-electron reversible oxidations were found for all the compounds studied. The electrochemical reductions show 2–3 reduction waves in the potential range studied. Only for the complexes of mixed diimine ligands or 2,2′-bipyridine, a pre-adsorption wave is also observed. It is possible to stabilize low valence states with all ligands studied. A formal iron(I) state is described for the first time for all aliphatic diimine complexes, thus showing that the acceptor properties of the diimine complexes do not depend on the presence of the aromatic rings, but on the iron-diimine chromophore.     

Cyclic boronium and borenium cations derived from borabenzene-pyridine complexes

A new class of boronium and borenium cations are prepared by reaction of pyridinium hydrochloride with pyridine stabilized borabenzene derivatives.     

Fast photoreduction of a heme peptide encapsulated in nanostructured materials

Microperoxidase-11 has been immobilized on siliceous materials MCM-41 and SBA-15 and on amino-functionalized SBA-15. Resonance Raman spectroscopy has provided solid evidence that the exogenous species occupy the pores of the mesoporous silica materials. Photoreduction of the microperoxidase-11 Fe(III) center has been observed to occur in the immobilized samples and results in a long-lived stable reduced heme. Reoxidation of the heme occurs upon addition of oxygen, and the redox cycle can be repeated numerous times. The source of the electron resulting in reduction of the heme is proposed to originate from the silica matrix, and functionalization of silica surface is suggested to facilitate electron transfer to the heme.     

Analysis of hairpin polyamide complexes having DNA binding sites in close proximity

The binding of two hairpin polyamide ligands at adjacent sites on DNA has been studied using NMR spectroscopy. The ligands ImPyPy-gamma-PyPyPy-Gly-Dp and Ac-ImPyPy-gamma-PyPyPy-Gly-Dp were studied binding to oligomers containing one or two matched binding sites: 5'-XGTTA-3' and 5'-TAACXNGTTA-3', where X is G, C, or A and N = 0, 1 or 2. At these sites the C-terminal ring shows an equilibrium between normal and inverted conformations. Better binding was observed with the ligand running 5' to 3' along the contacted strand than in the opposite direction. Complexes of DNAs with two binding sites indicated that at least one spacing base pair was required, and that the identity of this base pair was not critical. Binding with 5' to 3' contact is again preferred. Demonstrated binding at adjacent sites indicates that it may be possible to engineer cooperative binding for enhanced specificity or affinity.     

Electronic structure of ferric heme nitrosyl complexes with thiolate coordination

The effect of trans thiolate ligation on the coordinated nitric oxide in ferric heme nitrosyl complexes as a function of the thiolate donor strength, induced by variation of NH-S(thiolate) hydrogen bonds, is explored. Density functional theory (DFT) calculations (BP86/TZVP) are used to define the electronic structures of corresponding six-coordinate ferric [Fe(P)(SR)(NO)] complexes. In contrast to N-donor-coordinated ferric heme nitrosyls, an additional Fe-N(O) sigma interaction that is mediated by the dz2/dxz orbital of Fe and a sigma*-type orbital of NO is observed in the corresponding complexes with S-donor ligands. Experimentally, this is reflected by lower nu(N-O) and nu(Fe-N) stretching frequencies and a bent Fe-N-O moiety in the thiolate-bound case.     

Efficient photodissociation of O2 from synthetic heme and heme/M (M = Fe, Cu) complexes

Single wavelength excitation (lambdaex = 355 or 532 nm) of low-temperature stabilized (198 K) synthetic heme-dioxygen and heme-dioxygen/M complexes, where M = copper or iron in a non-heme environment, results in the dissociation of dioxygen as indicated by the generation of the ferrous heme (Soret band, 427 nm) and the bleaching of the ferric-superoxide (FeIII(O2-)) 410-nm Soret band in the transient absorption difference spectrum. Dioxygen rebinds to the four heme complexes studied with comparable rate constants ( approximately 6-9 x 105 M-1 s-1). However, the quantum yield for complete dissociation of O2 from our simplest heme-O2 complex (F8)FeIII(O2-) (phi = 0.60) is higher than the other complexes measured (phi = approximately 0.2-0.3) as well as that for oxy-myoglobin (phi = 0.3).     

Cyclic peptide conjugate of curcumin and doxorubicin as an anticancer agent

The hydrophobicity of curcumin creates hurdle towards its use in the anticancer therapy. Herein, we synthesized a curcumin-doxorubicin conjugated cyclic peptide scaffold to improve the solubility of curcumin and create a conjugate containing two anticancer agents. A solid-phase Fmoc/tBu solid phase methodology was used to synthesize a cell-penetrating nuclear targeting peptide with free thiol and amine groups, which was coupled with the activated doxorubicin (Dox) and curcumin, affording Dox-peptide-curcumin conjugate (DPCC) (10). The antiproliferative activity of the conjugate was evaluated in human leukemia carcinoma cell (CCRF-CEM), human ovarian carcinoma cell (SKOV-3), and normal kidney cell line (LLCPK). Cyclic peptide-doxorubicin conjugate (7) and DPCC (10) did not inhibit the proliferation of normal kidney LLCPK cells after 72?h incubation, but were cytotoxic in CCRF-CEM (73% and 41%, respectively) and SKOV-3 (55% and 30%, respectively) cells while Dox was cytotoxic (60–79%) in all three cell lines under similar conditions, suggesting selectivity of these compounds towards cancer cells.     

组氨酸和碘代组氨酸钴氧合作用的循环伏安研究

本文利用循环伏安法研究了组氨酸钴和碘代组氨酸钴配合物在不同条件下的氧合过程 ,通过与光度法和电位滴定法的研究比较 ,探讨了以循环伏安法研究这两种配合物的氧合过程的可行性 .     

Fluorescence study of peptide and protein containing interpolyelectrolyte complexes

Four polyelectrolyte (PE)-protein and PE-peptide systems were selected for fluorescence experiments. The mode of binding of proteins to PE and structure of forming polycomplexes depends upon the ratio of components and chemical composition of polymer macromolecules. Complex formation of proteins with oppositely charged polycations is realized by the self-assembly of non-stoichiometric polycomplex particles with characteristic composition. These polycomplex particles have a rigid rod-like carcass structures in which protein molecules are practically exposed to the solvent. Two types of ternary polyanion-Cu²⁺-protein polycomplex particles are formed depending on the monomer composition of the copolymer. At higher content of hydrophobic N-isopropylacrylamide monomer unites, the protein globules in the structure of ternary polycomplex particles are densely covered by the shell of a polymer coil and practically “fenced of” from the water environment. At higher content of acrylic acid unites, polycomplex particles have more friable structures in which protein molecules are practically exposed to the solution. The PE-peptide conjugate species can be represented rather as a macromolecule of a segmented (block) copolymer in which the hydrophobic blocks, i.e. the sequences of copolymer and peptide unit pairs, which have formed the covalent and salt bonds alternate with hydrophilic ones, i.e. the sequences of the copolymer chain not participating in the formation of double strand blocks.     

Site-specific relaxation kinetics of a tryptophan zipper hairpin peptide using temperature-jump IR spectroscopy and isotopic labeling

Two antiparallel beta-strands connected by a turn make beta-hairpins an ideal model system to analyze the interactions and dynamics of beta-sheets. Site-specific conformational dynamics were studied by temperature-jump IR spectroscopy and isotopic labeling in a model based on the tryptophan zipper peptide, Trpzip2, developed by Cochran et al. (Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5578). The modified Trpzip2C peptides have nearly identical equilibrium spectral behavior as Trpzip2 showing that they also form well-characterized beta-hairpin conformations in aqueous solution. Selective introduction of 13C=O groups on opposite strands lead to distinguishable cross-strand coupling of the labeled residues as monitored in the amide I' band. These frequency patterns reflect theoretical predictions, and the coupled 13C=O band loses intensity with increase in temperature and unfolding of the hairpin. Thermal relaxation kinetics were analyzed for unlabeled and cross-strand isotopically labeled variants. T-jumps of approximately 10 degrees C induce relaxation times of a few microseconds that decrease with increase of the peptide temperature. Differences in kinetic behavior for the loss of beta-strand and gain of disordered structure can be used to distinguish localized structure dynamics by comparison of nonlabeled and labeled amide I' components. Analysis of the data supports multistate dynamic and equilibrium behavior, but because of this process it is not possible to clearly define a folding and unfolding rate. Nonetheless, site-specific relaxation kinetics could be seen to be consistent with a hydrophobic collapse hypothesis for hairpin folding.