Tetracycline-Cu(II) photo-induced fragmentation of serum albumin

The protein-damaging potential of photosensitized tetracycline hydrochloride alone and in combination with the metal ion Cu(II) was assessed using serum albumin as a model protein. Exposure of tetracycline to white light in an aqueous solution triggered the generation of significant amounts of reactive oxygen species (ROS) and engendered substantial protein damage. The appearance of distinct low-molecular-mass protein bands on 10% SDS-polyacrylamide gel ascertained the tetracycline concentration-dependent fragmentation of albumin. Photoexcited tetracycline in combination with 100 microM Cu(II) enhanced the protein fragmentation process with concurrent increase in free radical production. The significant release of acid-soluble amino groups and carbonyl groups from treated albumin provided quantitative estimation of protein fragmentation at 0-1.0 mM concentrations of tetracycline. Cu(II) ions per se did not cause any perceptible protein damage. The results with free radical quenchers suggested the role of hydroxyl radicals (*OH) in tetracycline-Cu(II)-induced protein fragmentation, as no superoxide dismutase (SOD)-mediated quenching effect was noted. The generation of free radicals upon tetracycline photoexcitation and consequent protein fragmentation may be considered as important factors in augmentation of tetracycline-induced phototoxic responses.     

Molecular Beam Mass Spectrometry With Tunable Vacuum Ultraviolet (VUV) Synchrotron Radiation

Tunable soft ionization coupled to mass spectroscopy is a powerful method to investigate isolated molecules, complexes and clusters and their spectroscopy and dynamics^1-4. Fundamental studies of photoionization processes of biomolecules provide information about the electronic structure of these systems. Furthermore determinations of ionization energies and other properties of biomolecules in the gas phase are not trivial, and these experiments provide a platform to generate these data. We have developed a thermal vaporization technique coupled with supersonic molecular beams that provides a gentle way to transport these species into the gas phase. Judicious combination of source gas and temperature allows for formation of dimers and higher clusters of the DNA bases. The focus of this particular work is on the effects of non-covalent interactions, i.e., hydrogen bonding, stacking, and electrostatic interactions, on the ionization energies and proton transfer of individual biomolecules, their complexes and upon micro-hydration by water^{1, 5-9}.We have performed experimental and theoretical characterization of the photoionization dynamics of gas-phase uracil and 1,3-dimethyluracil dimers using molecular beams coupled with synchrotron radiation at the Chemical Dynamics Beamline¹⁰ located at the Advanced Light Source and the experimental details are visualized here. This allowed us to observe the proton transfer in 1,3-dimethyluracil dimers, a system with pi stacking geometry and with no hydrogen bonds¹. Molecular beams provide a very convenient and efficient way to isolate the sample of interest from environmental perturbations which in return allows accurate comparison with electronic structure calculations^{11, 12}. By tuning the photon energy from the synchrotron, a photoionization efficiency (PIE) curve can be plotted which informs us about the cationic electronic states. These values can then be compared to theoretical models and calculations and in turn, explain in detail the electronic structure and dynamics of the investigated species ^{1, 3}.     

Hexachlororhenate(IV) salts of organic radical cations

The ionic salts (p-rad)₂[ReCl₆] (1) and (m-rad)₂[ReCl₆] (2) (p/m-rad = 2-(4/3-N-methylpyridinium)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazol-1-oxyl-3-N-oxide) have been prepared and their crystal structures determined by single-crystal X-ray diffraction. The nitronyl nitroxide cations in compound 1 show a layered disposition, whereas the [ReCl₆]²⁻ units are placed between these layers. The nitronyl nitroxide cations in compound 2 adopt an hexagonal array but they do not result in layers. Bulk magnetic properties of 1 and 2 have been investigated in the temperature range 2-300 K. Both compounds show weak but significant intermolecular antiferromagnetic interactions.     

The optical properties of tyrosine-containing cyclic dipeptides

The chiroptical properties of the cyclic dipeptides cyclo (L -alanyl-L -tyrosine) and cyclo(L -tyrosyl-L -tyrosine) have been investigated as a function of molecular conformation. Theoretical optical calculations and conformational energy calculations have been carried out as a function of the side-chain dihedral angles χ¹ and χ², and as a function of the angle of fold of the cyclic dipeptide backbone. The results of these theoretical calculations have been compared with experimental circular dichroism (CD) data. Theoretical predictions are in very good agreement with experiment for c(L -Tyr-L -Tyr). Agreement was not quite as good for c(L -Ala-L -Tyr), although the signs of all of the Cotton effects were apparently predicted correctly except for that associated with the lowest energy tyrosine absorption band.     

Conformation of cyclic dipeptides from empirical energy calculations

Empirical energy calculations on cyclo-Gly-X with X- Phe, Tyr, Val, and Leu as a function of the side-chain torsion angles χ indicate that the conformation of minimum energy are characterized by χ₁ = 60°, χ² = 90° for Phe and Try, χ¹ = ?60° for Val and χ¹ = ?60°, χ² = 180° and χ¹ = 60° and χ² = 150° for Leu. The minimum energy conformation of cyclo-Gly-Phe and cyclo-Gly-Val have the side chains of Phe and Val stacked over the poperazinedione ring as suggested by NMR and found for cyclo-Gly-Tyr crystal structure. In contrast, the Leu side chain is expected to exist in an extended or a quasi-folded form.     

Fast repair of deoxynucleotide radical cations by phenylpropanoid glycosides (PPGs) and their analogs

The repair effects on deoxynucleotide radical cations of phenylpropanoid glycosides (PPGs) and their analogs, isolated from a Chinese folk medicinal herb, were studied using the pulse radiolysis technique. The radical cations of deoxynucleotides were formed by the reaction of SO4*- with deoxynucleotides. On pulse irradiation of a nitrogen saturated deoxynucleotide aqueous solution containing 20 mM K2S2O8, 200 mM t-BuOH and one of the PPGs or their analogs, the transient absorption spectra of the radical cations of nucleotide decayed with the formation of those of the radical cation of PPGs or their analogs within several tens of microseconds after electron pulse irradiation. The result indicates that deoxynucleotide radical cations can be repaired by PPGs or their analogs. The rate constants of the repair reactions were determined to be 0.48-1.1 x 10(9), 0.64-1.80 x 10(9) and 2.12-4.4 x 10(9) M(-1) s(-1) for dAMP, dGMP and dCMP radical cations respectively. It is obvious that the rate constants of the repair reaction depend on the number of phenolic hydroxyl groups contained in the PPGs and their analogs. A deeper understanding of this new repair mechanism will undoubtedly help researchers design strategies to prevent and/or intervene more effective in free radical related diseases.     

Spin-spin interactions in iron(III) porphyrin radical cations with ruffled and saddled structure

Oxidation of essentially pure intermediate-spin iron(III) porphyrinates such as ruffled Fe(TⁱPrP)ClO₄ and saddled Fe(OETPP)ClO₄ produces the corresponding six-coordinate iron(III) porphyrin(por) radical cations [Fe(Por)(ClO₄)₂], where TⁱPrP and OETPP are dianions of 5,10,15,20-tetraisopropylporphyrin and 2,3,7,8,12,13,17,18-octaethyl-5,10,15,20-tetraphenylporphyrin, respectively.Spin-spin interactions in these complexes are very much different; while ruffled [Fe(TⁱPrP)(ClO₄)₂] exhibits no antiferromagnetic coupling, saddled [Fe(OETPP)(ClO₄)₂] does exhibit it. The difference in magnetic behaviors has been explained in terms of the deformation mode and electron configuration of these complexes.     

Kinetic studies of reactions of iron(IV)-oxo porphyrin radical cations with organic reductants

Iron(IV)-oxo porphyrin radical cations are observed intermediates in peroxidase and catalase enzymes, where they are known as Compound I species, and the putative oxidizing species in cytochrome P450 enzymes. In this work, we report kinetic studies of reactions of iron(IV)-oxo porphyrin radical cations that can be compared to reactions of other metal-oxo species. The iron(IV)-oxo radical cations studied were those produced from 5,10,15,20-tetramesitylporphryinato-iron(III) perchlorate (1), 5,10,15,20-tetramesitylporphryinato-iron(III) chloride (2), both in CH(3)CN solvent, and that from 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinato-iron(III) perchlorate (3) in CH(2)Cl(2) solvent. The substrates studied were alkenes and activated hydrocarbons diphenylmethane and ethylbenzene. For a given organic reductant, various iron(IV)-oxo porphyrin radical cations react in a relatively narrow kinetic range; typically the second-order rate constants vary by less than 1 order of magnitude for the oxidants studied here and the related oxidant 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinato-iron(IV)-oxo porphyrin radical cation in CH(3)CN solvent. Charge transfer in the transition states for epoxidation reactions of substituted styrenes by oxidants 1 and 2, rho(+) values of -1.9 and -0.9, respectively, mirrors results found previously for related species. Competition kinetic reactions with a catalytic amount of porphyrin iron(III) species and a terminal oxidant give relative rate constants for oxidations of competing substrates that are somewhat smaller than the ratios of absolute rate constants. Water in CH(3)CN solutions has an apparent modest stabilizing effect on oxidant 1 as indicated in slightly reduced rate constants for oxidation reactions. The iron(IV)-oxo porphyrin radical cations are orders of magnitude less reactive than porphyrin-manganese(V)-oxo cations and a corrole-iron(V)-oxo species. The small environment effects found here suggest that high energy demanding hydrocarbon oxidation reactions catalyzed by cytochrome P450 enzymes might require highly reactive iron(V)-oxo transients as oxidants instead of the more stable, isomeric iron(IV)-oxo porphyrin radical cations.     

Synthesis and activity evaluation of the cyclic dipeptides arylidene N-alkoxydiketopiperazines

A series of arylidene N-alkoxydiketopiperazines was designed and stereoselectively synthesized via oxime-ether formation and intramolecular acylation. Possible cyclization and acid-catalyzed rearrangement-fragmentation mechanisms were discussed. The crystal structure of the novel diketopiperazine further confirmed the rearrangement mechanism. Most compounds exhibited antitumor activity. Several compounds were more potent against caspase-3. Specifically, compounds 6e, 6g, and 6f inhibited caspase-3 at IC₅₀ values lying within the low micromolar range and demonstrated good selectivity. The binding modes of alkoxydiketopiperazines in the active center of caspase-3 were also discussed based on the molecular docking results.     

Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid

We have isolated a Lactobacillus plantarum strain (MiLAB 393) from grass silage that produces broad-spectrum antifungal compounds, active against food- and feed-borne filamentous fungi and yeasts in a dual-culture agar plate assay. Fusarium sporotrichioides and Aspergillus fumigatus were the most sensitive among the molds, and Kluyveromyces marxianus was the most sensitive yeast species. No inhibitory activity could be detected against the mold Penicillium roqueforti or the yeast Zygosaccharomyces bailii. An isolation procedure, employing a microtiter well spore germination bioassay, was devised to isolate active compounds from culture filtrate. Cell-free supernatant was fractionated on a C(18) SPE column, and the 95% aqueous acetonitrile fraction was further separated on a preparative HPLC C(18) column. Fractions active in the bioassay were then fractionated on a porous graphitic carbon column. The structures of the antifungal compounds cyclo(L-Phe-L-Pro), cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid (L/D isomer ratio, 9:1), were determined by nuclear magnetic resonance spectroscopy, mass spectrometry, and gas chromatography. MIC values against A. fumigatus and P. roqueforti were 20 mg ml(-1) for cyclo(L-Phe-L-Pro) and 7.5 mg ml(-1) for phenyllactic acid. Combinations of the antifungal compounds revealed weak synergistic effects. The production of the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) by lactic acid bacteria is reported here for the first time.     

Enzymic isoprenylation of tryptophanyl cyclic dipeptides by Aspergillus amstelodami

Cyclo-l-prolyl-l-tryptophanyl, a component of several metabolites of Aspergillus ustus and cyclo-l-alanyl-l-tryptophanyl were compared as co-substrates with 3-methyl-2-butenyl-[1-³H]-1-pyrophosphate for an enzyme from A. amstelodami which previously had been described to isoprenylate cyclo-l-alanyl-l-tryptophanyl. Both compounds were equally active as isoprene acceptors using the A. amstelodami enzyme. The mass spectrum of the isoprenylated cyclo-l-prolyl-l-tryptophanyl indicated that the product was a monoisoprenylated derivative of the starting cyclic dipeptide. The most probable structure for this enzymic product is cyclo-l-prolyl-2(1,1-dimethylallyl)-l-tryptophanyl. The cyclo-pentylidene analogue of 3-methyl-2-butenyl-1-pyrophosphate did not serve as an alkylating agent when cyclo-l-alanyl-l-tryptophanyl was used as co-substrate.     

On the taste of stereoisomeric cyclic dipeptides containing a proline residue

An investigation of the taste character and intensity of sixproline-containing cylic dipeptides demonstrates that thesecompounds are differentiated largley on the basis of their chemicalconfiguration. ^x/ Permanent address: Technical University, 80–952 Gdask,Poland     

Inhibitory effect against Akt of cyclic dipeptides isolated from Bacillus sp

Among thirteen strains of the genus Bacillus isolated from Shrimp-jeotkal in our laboratory, a strain BA34 showing good antifungal activity against Phytophthora infestans in a previous experiment was tested for the inhibitory effect against Akt, protein kinase B. Since Akt is known to play an important role in controlling apoptosis, its inhibitors can be used as potential apoptosis-inducing agents in the treatment of cancer. Two active compounds were isolated and their structures were determined. They have similar structures, despite showing different inhibitory effects. In order to elucidate the reasons for these different effects, three-dimensional studies were carried out.     

Anti-biofilm and anti-adherence properties of novel cyclic dipeptides against oral pathogens

Microorganisms embedded in a biofilm are significantly more resistant to antimicrobial agents and the defences of the human immune system, than their planktonic counterpart. Consequently, compounds that can inhibit biofilm formation are of great interest for novel therapeutics. In this study, a screening approach was used to identify novel cyclic dipeptides that have anti-biofilm activity against oral pathogens. Five new active compounds were identified that prevent biofilm formation by the cariogenic bacterium Streptococcus mutans and the pathogenic fungus Candida albicans. These compounds also inhibit the adherence of microorganisms to a hydroxylapatite surface. Further investigations were conducted on these compounds to establish the structure–activity relationship, and it was deduced that the common cleft pattern is required for these molecules to act effectively against biofilms.     

Theoretical pi-pi* absorption and circular dichroic spectra of cyclic dipeptides

The dipole interaction model, treated by the partially dispersive normal mode method, is used to calculate circular dichroic spectra of cyclo(Gly-Gly), cyclo (Ala-Gly), cyclo(Ala-Ala), cyclo(Pro-Gly), cyclo(Pro-Ala), cyclo(Pro-Val), cyclo (Pro-D-Val), and cyclo(Pro-Pro) in the amide pi-pi* absorption band near 190 nm. Assuming a standard backbone geometry, spectra which are in fair to good agreement with experiment are obtained for these molecules. The spectra are predicted to be sensitive to conformations of Pro and Val side chains. The effects of dipeptide ring folding on calculated CD spectra are mostly consistent with those found by other workers, except that it is found that a planar ring conformation of cyclo (Ala-Ala) and cyclo (Ala-Gly) gives predicted spectra comparable to experiment. The same model gives theoretical absorption spectra consistent with available experimental data.     

Synthesis and structural study of cyclic 5-aminovaleric acid-linked beta-Ala-beta-Ala dipeptides

5-Aminovaleric acid and ornithine were evaluated as linkers for the cyclization of beta-dipeptides. Two linked examples of beta-Ala-beta-Ala were prepared by standard coupling methods and their conformations probed by NMR, CD, and computational means. The data suggest that these non- or monosubstituted versions of the target compounds are flexible in solution.