Enhanced cellular uptake with a cobaltacarborane-porphyrin-HIV-1 Tat 48-60 conjugate

A series of four porphyrin-cobaltacarborane conjugates have been synthesized, containing three or four cobaltabisdicarbollide anions linked by O(CH(2)CH(2)O)(2) groups to the porphyrin macrocycle and one of them containing a HIV-1 Tat 48-60 peptide sequence linked via a low molecular weight poly(ethylene glycol) (PEG) spacer. The cellular uptake, cytotoxicity, and preferential sites of intracellular localization of the conjugates were evaluated in human HEp2 cells. All conjugates are nontoxic in the dark at the concentrations studied. Upon exposure to low light dose (1 J cm(-)(2)) only the porphyrin-cobaltacarborane-HIV-1 Tat 48-60 conjugate showed 30% inhibition of cell proliferation at a concentration of 10 microM. The cellular uptake was dependent on the number of carborane cages and was significantly enhanced by the presence of the cell penetrating peptide sequence HIV-1 Tat 48-60. All conjugates preferentially localized in the cell lysosomes.     

Synthesis and spectroelectrochemistry of N-cobaltacarborane porphyrin conjugates

N-Substituted porphyrins are well-known for the distortion they exhibit of the porphyrin plane through the sp(3) hybridization of one of the pyrrolenic units. They have served as model compounds in investigations of many biochemical processes. In this paper, we developed an efficient route to N-substituted porphyrins, and report the synthesis of a series of new N-substituted cobaltacarborane-porphyrins containing one or two cobaltabisdicarbollide anions linked by (CH(2)CH(2)O)(2) chains to either the core porphyrin nitrogens or to a meso-aminophenyl group. These conjugates show different degrees of distortion of the porphyrin macrocycle, which affect their spectroscopic and electrochemical properties. In particular, the core N-substituted conjugates show significant fluorescence quenching in comparison with the noncore substituted macrocycles. The X-ray structures of two targeted core N-cobaltacarborane porphyrin conjugates are presented. The electrochemical and spectroelectrochemical properties of these porphyrin conjugates were investigated; while the peripheral N-substituted cobaltacarboranylporphyrins undergo three reversible reductions and three reversible oxidations (two attributed to the porphyrin and one to the Co(III) cluster), the core N-substituted porphyrins exhibit complicated electrochemical behavior with coupled chemical reactions.     

Synthesis and characterization of positively charged porphyrin-peptide conjugates

The total syntheses of 14 porphyrin conjugates containing one to four positively charged amino acids and two distinct linkers are described. All conjugates were fully characterized using spectroscopic methods, and the X-ray structure of a porphyrin isothiocyanate precursor was obtained. In vitro studies using HEp2 cells show that these conjugates have low cytotoxicity (IC50 > 250 microM) and that the extent of their cellular uptake depends significantly on the number, nature, and sequence of amino acids in the peptide, and on the presence of a centrally chelated metal ion. Metal-free conjugates bearing three consecutive arginine residues accumulated the most within cells. On the other hand, the preferential sites of subcellular localization were found to be independent from the number, nature, and sequence of amino acids in the conjugate, the linker, and coordinated metal ion; it is suggested, based on theoretical calculations, that the peptides in these conjugates fold over the porphyrin macrocycle in order to maximize intramolecular hydrophobic interactions.     

Single diastereomers of unsymmetrical tris-spirocyclic cyclotriphosphazenes based on 1,1'-bi-2-naphthol--synthesis and structures

Diastereoselective synthesis and characterization of chiral unsymmetrical tris-spirocyclic cyclotriphosphazenes based on chiral 1,1'-bi-2-naphthol (BINOL) are reported. Specifically, the chiral compounds (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)Cl(2) [(-)-4] and (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](OCH(2)CH(2)NMe)(2) [(-)-5] are prepared by starting with the chiral mono-spiro compound (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)]Cl(4) [(-)-3]. Synthesis of four other chiral spirocyclics, N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](OCH(2)CH(2) NMe)(O-2,2'C(6)H(4)-C(6)H(4)O)[(-)-6 and (+)-6], N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](NMe(2))(4) [(-)-7], N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)(NMeCH(2)CH(2)OH)(2) [(-)-8 and (+)-8], and N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)[NHCH(2)CH(2)CH(2)Si(OEt)(3)](2) (9) is also reported herein. Compounds 4-6 are obtained in the solid state diastereoselectively and their X-ray structures have been determined and discussed. The diastereoselectivity is also shown by structural characterization of two distinct isomers in the case of 6 [(-)-6 and (+)-6, respectively] by starting with precursor of 3 having (R) or (S)-BINOL residue. The (1)H NMR spectra of 7 and 8 exhibit doublets with virtual coupling for the methyl protons, consistent with the chiral nature of the binaphthoxy residue. The potential of 9, which hydrolyzes readily in CDCl(3) solution, as a useful precursor for chiral polymer applications is highlighted.     

Carboranyl peptide-antibody conjugates for neutron-capture therapy: preparation, characterization, and in vivo evaluation.

Two model peptides rich in boron and prepared by Merrifield syntheses, dansyl.(nido-CB)2, (1) and dansyl.(nido-CB)10.Lys.Ac (2), where nido-CB represents the alpha-amino acid [nido-7-CH3-8-(CH2)3CH-(NH2)COOH-7,8-C2B9H10]-, were conjugated with the anti-CEA mAb T84.66 using peptide active ester reagents. The dansyl groups provided a means of fluorimetric analysis of mAb conjugates which was augmented by conventional amino acid analyses for nido-CB. The conjugate of 1 contained an average of 63 B atoms per mAb molecule. The mAb conjugate of 2 was chromatographically separated into a strongly fluorescent high molecular weight aggregated fraction (HMW) and a less intensely fluorescent monomeric fraction. Both fractions retained immunoreactivity. The HMW species contained an average of ca. 490 B atoms/mAb molecule, as determined by amino acid analysis. Biodistribution data were collected using nude mice bearing LS174T xenografts and 125I-labeled mAb conjugates. While the lightly B-loaded dipeptide conjugate gave biodistribution results which resembled those of native T84.66 mAb, the undecapeptide conjugate displayed greatly enhanced liver uptake and decreased tumor accretion. These results suggest that as the boron-containing burden on the supporting immunoprotein is greatly increased, as in the case of the T84.66-2 conjugate, loss of circulating conjugate to liver effectively competes with the desired tumor localization. Means which might be taken to circumvent this difficulty have been described elsewhere (ref 15).     

Synthesis and antimalarial activities of rhenium bioorganometallics based on the 4-aminoquinoline structure

A bioorganometallic approach to malaria therapy led to the discovery of ferroquine (FQ, SSR97193). To assess the importance of the electronic properties of the ferrocenyl group, cyclopentadienyltricarbonylrhenium analogues related to FQ, were synthesized. The reaction of [N-(7-chloro-4-quinolinyl)-1,2-ethanodiamine] with the cyrhetrenylaldehyde complexes (η(5)-C(5)H(4)CHO)Re(CO)(3) and [η(5)-1,2-C(5)H(3)(CH(2)OH)(CHO)]Re(CO)(3) produces the corresponding imine derivatives [η(5)-1,2-C(5)H(3)(R)(CHN-CH(2)CH(2)NH-QN)]Re(CO)(3) R=H 3a; R=CH(2)OH 3b; QN=N-(7-Cl-4-quinolinyl). Reduction of 3a and 3b with sodium borohydride in methanol yields quantitatively the amine complexes [η(5)-1,2-C(5)H(3)(R)(CH(2)-NH-CH(2)CH(2)NH-QN)]Re(CO)(3) R=H 4a; R=CH(2)OH 4b. To establish the role of the cyrethrenyl moiety in the antimalarial activity of this series, purely organic parent compounds were also synthesized and tested. Evaluation of antimalarial activity measured in vitro against the CQ-resistant strains (W2) and the CQ-susceptible strain (3D7) of Plasmodium falciparum indicates that these cyrhetrene conjugates are less active compared to their ferrocene and organic analogues. These data suggest an original mode-of-action of FQ and ferrocenyl analogues in relationship with the redox pharmacophore.     

O6-ethylguanine carcinogenic lesions in DNA: an NMR study of O6etG.T pairing in dodecanucleotide duplexes

High-resolution two-dimensional NMR studies are reported on the self-complementary d-(C1-G2-C3-O6etG4-A5-G6-C7-T8-T9-G10-C11-G12) duplex (designated O6etG.T 12-mer) containing two symmetrically related O6etG.T lesion sites located four base pairs in from either end of the duplex. Parallel studies were undertaken on a related sequence containing O6meG.T lesion sites (designated O6meG.T 12-mer) in order to evaluate the influence of the size of the alkyl substituent on the structure of the duplex and were undertaken on a related sequence containing G.T mismatch sites (designated G.T 12-mer duplex), which served as the control duplex. The exchangeable and nonexchangeable proton and the phosphorus nuclei have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOE) and correlated spectra of the O6etG.T 12-mer, O6meG.T 12-mer, and G.T 12-mer duplexes in H2O and D2O solutions. The distance connectivities observed in the NOESY spectra of the O6alkG.T 12-mer duplexes establish that the helix is right-handed and all of the bases adopt an anti conformation of the glycosidic torsion angle including the O6alkG4 and T9 bases at the lesion site. The imino proton of T9 at the O6alkG.T lesion sites resonates at 8.85 ppm in the O6etG.T 12-mer duplex and at 9.47 ppm in the O6meG.T 12-mer duplex. The large upfield shift of the T9 imino proton resonance at the O6alkG4.T9 lesion site relative to that of the same proton in the G4.T9 wobble pair (11.99 ppm) and the A4.T9 Watson-Crick pair (13.95 ppm) in related sequences establishes that the hydrogen bonding of the imino proton of T9 to O6alkG4 is either very weak or absent. The imino proton of T9 develops NOEs to the CH3 protons of the O6etG and O6meG alkyl groups across the base pair, as well as to the imino and H5 protons of the flanking C3.G10 base pair and the imino and CH3 protons of the flanking A5.T8 base pair in the O6alkG.T 12-mer duplexes. These observations establish that the O6alkG4 and T9 residues are stacked into the duplex and that the O6CH3 and O6CH2CH3 groups of O6alkG4 adopt a syn orientation with respect to the N1 of the alkylated guanine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of hydrophobic structure on the catalysis of nitric oxide release from zwitterionic diazeniumdiolates in surfactant and liposome media.

The effect of phospholipid liposomes and surfactant micelles on the rate of nitric oxide release from zwitterionic diazeniumdiolates, R1R2N[N(O)NO]-, with significant hydrophobic structure, has been explored. The acid-catalyzed dissociation of NO has been examined in phosphate-buffered solutions of sodium dodecylsulfate (SDS) micelles and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-[phospho-(1-glycerol)] sodium salt (DPPG) phospholipid liposomes. The reaction behavior of dibenzylamine-, monobenzylamine-, and dibutylamine-derived substrates [1]: R1 = C6H5CH2, R2 = C6H5CH2 NH2+(CH2)2, 2: R1 = C6H5CH2, R2 = NH3+(CH2)2, and 3: R1 = n-butyl, R2 = n-butyl-NH2+(CH2)6] has been compared with that of SPER/NO, 4: R1 = H2N(CH2)3, R2 = H2N(CH2) 3NH2+(CH2)4]. Catalysis of NO release is observed in both micellar and liposome media. Hydrophobic interactions contribute to micellar binding for 1-3 and appear to be the main factor facilitating catalysis by charge neutral DPPC liposomes. Binding constants for the association of 1 and 3 with SDS micelles were 3-fold larger than those previously obtained with comparable zwitterionic substrates lacking their hydrophobic structure. Anionic DPPG liposomes were much more effective in catalyzing NO release than either DPPC liposomes or SDS micelles. DPPG liposomes (at 10 mM total lipid) induced a 30-fold increase in the NO dissociation rate of SPER/NO compared to 12- and 14-fold increases in that of 1 and 3.     

O6-ethylguanine carcinogenic lesions in DNA: an NMR study of O6etG.C pairing in dodecanucleotide duplexes

The pairing of O6etG with C located four base pairs in from either end of the self-complementary d(C1-G2-C3-O6etG4-A5-G6-C7-T8-C9-G10-C11-G12) duplex (designated O6etG.C 12-mer) has been investigated from an analysis of proton and phosphorus two-dimensional NMR experiments. The structural consequences of increasing the alkyl group size were elucidated from a comparative study of the pairing of O6meG4 with C9 in a related sequence (designated O6meG.C 12-mer). The NMR parameters for both O6alkG-containing dodecanucleotides are also compared with those of the control sequence containing G4.C9 base pairs (designated G.C 12-mer). The NOE cross-peaks detected in the two-dimensional NOESY spectra of the O6alkG.C 12-mer duplexes in H2O solution establish that the O6etG4/O6meG4 and C9 bases at the lesion site stack into the helix between the flanking C3.G10 and A5.T8 Watson-Crick base pairs. The amino protons of C9 at the O6alkG4-C9 lesion site resonate as an average resonance at 7.78 and 7.63 ppm in the O6etG.C 12-mer and O6meG.C 12-mer duplexes, respectively. The observed NOEs between the amino protons of C9 and the CH3 protons of O6alkG4 establish a syn orientation of the O6-alkyl group with respect to the N1 of alkylated guanine. A wobble alignment of the O6alkG4.C9 base pair stablized by two hydrogen bonds, one between the amino group of C9 and N1 of O6alkG and the other between the amino group of O6alkG and N3 of C9, is tentatively proposed on the basis of the NOEs between the amino protons of C9 at the lesion site and the imino protons of flanking Watson-Crick base pairs. The proton and phosphorus chemical shift differences between the O6etG.C 12-mer and O6meG.C 12-mer duplexes are small compared to the differences between these O6alkG-containing duplexes and the control G.C 12-mer duplex.     

Synthesis and antiamoebic activity of new cyclooctadiene ruthenium(II) complexes with 2-acetylpyridine and benzimidazole derivatives

Reaction of [Ru(eta4-C8H12) (CH3CN)2 Cl2] with 2-(2-pyridyl) benzimidazole or Schiff bases derived from 2-acetylpyridine and S-methyldithiocarbazate, S-benzyldithiocarbazate and thiosemicarbazide leads to form new complexes of the type [Ru(eta4-C8H12)(L)Cl2] (where L=ligand). In vitro, most of the compounds exhibited potent activity and the Ru derivatives 1a [Ru(eta4-C8H12)(2-Acpy-SMDT)Cl2], 2a [Ru(eta4-C8H12)(2-Acpy-SBDT)Cl2] and 3a [Ru(eta4-CsH12)(2-Acpy-TSC)Cl2] were found more active than metronidazole against (HK-9) strain of Entamoeba histolytica.     

Effect of stocking rate on soil-atmosphere CH4 flux during spring freeze-thaw cycles in a northern desert steppe, China

BACKGROUND: Methane (CH(4)) uptake by steppe soils is affected by a range of specific factors and is a complex process. Increased stocking rate promotes steppe degradation, with unclear consequences for gas exchanges. To assess the effects of grazing management on CH(4) uptake in desert steppes, we investigated soil-atmosphere CH(4) exchange during the winter-spring transition period. METHODOLOGY/MAIN FINDING: The experiment was conducted at twelve grazing plots denoting four treatments defined along a grazing gradient with three replications: non-grazing (0 sheep/ha, NG), light grazing (0.75 sheep/ha, LG), moderate grazing (1.50 sheep/ha, MG) and heavy grazing (2.25 sheep/ha, HG). Using an automatic cavity ring-down spectrophotometer, we measured CH(4) fluxes from March 1 to April 29 in 2010 and March 2 to April 27 in 2011. According to the status of soil freeze-thaw cycles (positive and negative soil temperatures occurred in alternation), the experiment was divided into periods I and II. Results indicate that mean CH(4) uptake in period I (7.51 μg CH(4)-C m(-2) h(-1)) was significantly lower than uptake in period II (83.07 μg CH(4)-C m(-2) h(-1)). Averaged over 2 years, CH(4) fluxes during the freeze-thaw period were -84.76 μg CH(4)-C m(-2) h(-1) (NG), -88.76 μg CH(4)-C m(-2) h(-1) (LG), -64.77 μg CH(4)-C m(-2) h(-1) (MG) and -28.80 μg CH(4)-C m(-2) h(-1) (HG). CONCLUSIONS/SIGNIFICANCE: CH(4) uptake activity is affected by freeze-thaw cycles and stocking rates. CH(4) uptake is correlated with the moisture content and temperature of soil. MG and HG decreases CH(4) uptake while LG exerts a considerable positive impact on CH(4) uptake during spring freeze-thaw cycles in the northern desert steppe in China.     

Chemical properties of water-soluble porphyrins. 5. Reactions of some manganese (III) porphyrins with the superoxide and other reducing radicals

Solution properties of three manganese porphyrins, in monomeric form, were investigated. These were the 'picket-fence-like' porphyrin Mn(III)-alpha,alpha,alpha,beta- tetra-ortho(N-methylisonicotinamidophenyl)porphyrin (Mn(III)PFP) and two 'planar unhindered' porphyrins, the Mn(III)TMPyP (tetrakis (4-N-methylpyridyl)porphyrin) and Mn(III)TAP (tetra(4-N,N,N-trimethylanilinium)porphyrin). The porphyrin properties studied were: the absorption spectra in their manganic and manganous forms; acid/base properties of the aquo complexes; the effect of potential axial ligands (up to a concentration of 0.1 mol dm-3) and their one electron reduction potentials. Knowing these properties, the reaction of the Mn(III) porphyrins with the superoxide radical and other reducing radicals were studied using the pulse radiolysis technique. The second-order reaction rate constant of O2- with the Mn(III) porphyrins, which governs the catalytic efficiency of the metalloporphyrins upon the disproportionation of the superoxide radical, was 5.1 X 10(7) to 4.0 X 10(5) dm3 mol-1 s-1, depending on the pH and the nature of the metalloporphyrin. These values are at least one order of magnitude lower than found for Fe(III)TMPyP. One electron reduction of the three Mn(III) porphyrins by eaq-, CO2-, CH2OH and (CH3)2COH had similar second-order rate constants (10(9)-10(10) dm3 mol-1 s-1). That for (CH3)2(CH2)COH was about 10(5) dm3 mol-1 s-1. Reduction in all cases produced the corresponding Mn(II) porphyrin and no intermediate was found. The oxidation reaction of the Mn(II) porphyrins by O2- was approximately two orders of magnitude faster when compared to the reduction of Mn(III) porphyrins with the same radical. Since the reactivities of O2- towards the three manganese (III) compounds follow their reduction potentials, it is suggested that these reactions are governed by an outer-sphere mechanism. This suggestion is corroborated by the finding that water molecules acting as axial ligands, in these aqueous solution systems, are not replaced by another potential ligand when the latter is in the concentration range of 100 mM or less.     

Cobalt(IV) corroles as catalysts for the electroreduction of O2: reactions of heterobimetallic dyads containing a face-to-face linked Fe(III) or Mn(III) porphyrin

A series of heterobinuclear cofacial porphyrin-corrole dyads containing a Co(IV) corrole linked by one of four different spacers in a face-to-face arrangement with an Fe(III) or Mn(III) porphyrin have been examined as catalysts for the electroreduction of O(2) to H(2)O and/or H(2)O(2) when adsorbed on the surface of a graphite electrode in air-saturated aqueous solutions containing 1M HClO(4). The examined compounds are represented as (PCY)M(III)ClCo(IV)Cl where P is a porphyrin dianion, C is a corrole trianion and Y is a biphenylene (B), 9,9-dimethylxanthene (X), dibenzofuran (O) or anthracene (A) spacer. The catalytic behavior of the seven investigated dyads in the two heterobimetallic (PCY)MClCoCl series of catalysts is compared on one hand to what was previously reported for related dyads with a single Co(III) corrole macrocycle linked to a free-base porphyrin with the same set of linking bridges, (PCY)H(2)Co, and on the other hand to dicobalt porphyrin-corrole dyads of the form (PCY)Co(2) which were shown to efficiently electrocatalyze the four electron reduction of O(2) at a graphite electrode in acid media. Comparisons between the four series of porphyrin-corrole dyads, (PCY)Co(2), (PCY)H(2)Co, (PCY)FeClCoCl and (PCY)MnClCoCl, show that in all cases the biscobalt dyads catalyze O(2) electroreduction at potentials more positive by an average 110mV as compared to the related series of compounds containing a Co(III) or Co(IV) corrole macrocycle linked to a free-base metalloporphyrin or a metalloporphyrin with an Fe(III) or Mn(III) central metal ion. The data indicates that the E(1/2) values where electrocatalysis is initiated is related to the initial site of electron transfer, which is the Co(III)/Co(II) porphyrin reduction process in the case of (PCY)Co(2) and the Co(IV)/Co(III) corrole reduction in the case of (PCY)MnClCoCl, (PCY)FeClCoCl and (PCY)H(2)Co. The overall data also suggests that the catalytically active form of the biscobalt dyad in (PCY)Co(2) contains a Co(II) porphyrin and a Co(IV) corrole.     

Low frequency resonance Raman spectra of isolated alpha and beta subunits of hemoglobin and their deuterated analogues

In an attempt to gain further insight into the nature of the low frequency vibrational modes of hemoglobin and its isolated subunits, a comprehensive study of several different isotopically labeled analogues has been undertaken and is reported herein. Specifically, the resonance Raman spectra, between 200 and 500 cm(-1), are reported for the deoxy and ligated (CO and O2) forms of the isolated alpha and beta subunits containing the natural abundance or various deuterated analogues of protoheme. The deuterated protoheme analogues studied include the 1,3,5,8-C2H3-protoheme (d12- protoheme), the 1,3-C2H3-protoheme (1,3-d6-protoheme), the 5,8-C2H3-protoheme (5,8-d6-protoheme), and the meso-C2H4-protoheme (d4-protoheme). The entire set of acquired spectra has been analyzed using a deconvolution procedure to help correlate the shifted modes with their counterparts in the spectra of the native forms. Interestingly, modes previously associated with so-called vinyl bending modes or propionate deformation modes are shown to be quite sensitive to deuteration of the peripheral methyl groups of the macrocycle, shifting by up to 12-15 cm(-1), revealing their complex nature. Of special interest is the fact that shifts observed for the 1,3-d6- and 5,8-d6-protoheme analogues confirm the fact that certain modes are associated with a given portion of the macrocycle; i.e., only certain modes shift upon deuteration of the 1 and 3 methyl groups, while others shift upon deuteration of the 5 and 8 methyl groups. Compared with the spectra previously reported for the corresponding myoglobin derivatives, the data reported here reveal the appearance of several additional features that imply splitting of modes associated with the propionate groups or that are indicative of greater distortion of the heme prosthetic groups.     

Effect of carboxylate and Na ions on the tautomeric status of 9-methylguanine

Interactions of 9-methylguanine (m9Gua) with carboxylate ion of acetic acid (CH3COO-) and Na+ were studied by 1H NMR spectroscopy and ab initio quantum chemical calculations of the B3LYP/6-31++G(d,p) and B3LYP/6-311++G(d,p) levels of theory. Changes in the m9Gua 1H NMR spectrum in the presence of the equimolar amount of sodium acetate (NaAc), which in anhydrous DMSO dissociates to CH3COO- and Na+, were interpreted as a consequence of a complex formation of m9Gua in the amino-keto-N1H tautomeric form (m9GuaN1H) with carboxylate ions via two H-bonds involving amino and N1H-imino protons. Quantum chemical calculations of interactions of the m9GuaN1H ground-state tautomer and the m9GuaN3H high energy one with relative energy 20.01 kcal/mol show that the ground state tautomer forms the ground-state complex CH3COO-:mgGuaN1H, by 5.57 kcal/mol more stable than the CH3COO-:m9GuaN3H complex, and coordination of Na+ with the O6 and N7 atoms reduces this energy difference to 2.57 kcal/mol. Such a coordination of Na+ with tautomer m9GuaN3H therewith decreases its relative energy only to 13.31 kcal/mol. Non-additivity of the two ligands contributions to the 8-times reduction of the relative energy of the high energy tautomer in the CH3COO-:m9GuaN3H:Na+(O6,N7) triple complex was concluded, the role of CH3COO- being dominant. Besides, coordination with Na+ resulted in an iminoproton transfer from the base to CH3COO- in the triple complexes of both tautomers, according to calculations in vacuum. Biological significance of the results is noticed.     

Fungitoxicity of 1,4-naphthoquinones to Candida albicans and Trichophyton mentagrophytes.

Twenty-one substituted 1,4-naphthoquinones and five 8-quinolinols and copper(II) chelates were tested for antifungal activity against Candida albicans and Trichophyton mentagrophytes. Compounds containing electron-releasing or weak electron-withdrawing groups in the 2 and 3 positions of the 1,4-naphthoquinone ring were the most active against C. albicans at pH 7.0 in the presence of beef serum in the following order: 2-CH3O = 2,3-(CH3O)2 greater than 2-CH3 greater than 2-CH3S greater than 2-NH2 greater than 2,6-(CH3)2. For T. mentagrophytes under the same conditions the inhibitory 1,4-naphthoquinones contained the substituents 2-CH3O greater than 2,3-(CH3O)2 greater than 2-CH2S greater than 2-CH3 greater than 2-CH3(NaHSO3) greater than 2-NH2 greater than 2-C2H5S, 3-CH3 greater than 2,6-(CH3)2 greater than 2,3-CL2 greater than 5,8-(OH)2.     

Synthesis and in vitro biological evaluation of lipophilic cation conjugated photosensitizers for targeting mitochondria

Mitochondria-specific photosensitizers were designed by taking advantage of the preferential localization of delocalized lipophilic cations (DLCs) in mitochondria. Three DLC-porphyrin conjugates: CMP-Rh (a core modified porphyrin-rhodamine B cation), CMP-tPP (a core modified porphyrin-mono-triphenyl phosphonium cation), CMP-(tPP)₂ (a core modified porphyrin-di-tPP cation) were prepared. The conjugates were synthesized by conjugating a monohydroxy core modified porphyrin (CMP-OH) to rhodamine B (Rh B), or either one or two tPPs, respectively, via a saturated hydrocarbon linker. Their ability for delivering photosensitizers to mitochondria was evaluated using dual staining fluorescence microscopy. In addition, to evaluate the efficiency of the conjugates as photosensitizers, their photophysical properties and in vitro biological activities were studied in comparison to those of CMP-OH. Fluorescence imaging study suggested that CMP-Rh specifically localized in mitochondria. On the other hand, CMP-tPP and CMP-(tPP)₂ showed less significant mitochondrial localization. All conjugates were capable of generating singlet oxygen at rates comparable to CMP-OH. Interestingly, all cationic conjugates showed dramatic increase in cellular uptake and phototoxicity compared to CMP-OH. This improved photodynamic activity might be primarily due to an enhanced cellular uptake. Our study suggests that Rh B cationic group is better at least for CMP than tPP as a mitochondrial targeting vector.     

Antioxidant defense systems of two lipidopteran insect cell lines

Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines were found to contain unique assemblages of antioxidant enzymes. Specifically, the Sf-9 insect cell line contained Manganese and Copper-Zinc superoxide dismutase (MnSOD and CuZnSOD) for reducing the superoxide radical (O(2)(*-)) to hydrogen peroxide (H(2)O(2)) and ascorbate peroxidase (APOX) for reducing the resulting H(2)O(2) to H(2)O. Approximately one third of the total SOD activity was found to be MnSOD. The Tn-5B1-4 cells were also found to contain MnSOD (approximately two thirds of the total SOD activity), CuZnSOD and APOX activities. However, the Tn-5B1-4 cell line, in contrast to the Sf-9 cell line, contained catalase (CAT) activity for reducing H(2)O(2) to H(2)O. Both the Sf-9 and Tn-5B1-4 cell lines contained glutathione reductase and dehydroascorbic acid reductase activities for regenerating the reduced forms of glutathione and ascorbic acid, respectively. In addition, both cell lines contained glutathione S-transferase peroxidase activity towards hydroperoxides other than H(2)O(2). Finally, neither cell line contains the glutathione peroxidase activity that is ubiquitous in mammalian cells.     

Kinetics of nitroxyl radical reactions. A pulse-radiolysis conductivity study.

Absolute rate-constants for the reaction of the nitroxyl free radicals TAN and TMPN with radiation-chemically-formed radicals and ions have been determined. k(TAN + X) (in M(-1) sec(-1)=4-0 X 10(9) (for X = OH-), 2-9 X 10(10) (eaq-), 8-0 X 10(9) (H-), 7-2 X 10(8) (-CH2OH), 4-0 X 10(8) (CH3CHOH), 4-3 X 10(8) ((CH3)2COH) 2-8 X 10(8) (-CH2(CH3)2COH), 5-9 X 10(7) (glucose radical), 4-0 X 10(8) (c-C5H9-), and k(TMPN + X)=3-4 X 10(9) (OH-), 7-8 X 10(9) (eq-), 4-9 X 10(9) (H-), 4-4 X 10(8) (-CH2OH), 4-9 X 10(8) (CH3CHOH), 3-6 X 10(8) ((CH3)2COH), 1-5 X 10(8) (-CH2(CH3)2COH), 4-9 X 10(7) (glucose radical), 4-3 X 10(8) (c-C5H9-). Direct measurements by means of a pulse-radiolysis conductivity technique were based on the formation and destruction of charged species in these reactions within certain pH ranges. It is indicated that the radiosensitizing nitroxyls undergo both redox and addition reactions.     

Design of peptide oxytocin antagonists with strikingly higher affinities and selectivities for the human oxytocin receptor than atosiban.

The peptide oxytocin (OT) antagonist atosiban, approved for tocolytic use in Europe (under the tradename Tractocile), represents an important new therapeutic advance for the treatment of premature labor. This paper presents some new peptide OT antagonists which offer promise as superior tocolytics. The solid phase synthesis is reported of four pairs of L and D-2-naphthylalanine (L/D-2Nal) position-2 modified analogs of the following four oxytocin (OT) antagonists: des-9-glycinamide [1-(beta-mercapto-beta,beta-pentamethylene propionic acid), 2-O-methyltyrosine, 4-threonine]ornithine-vasotocin (desGly-NH(2),d(CH(2))(5)[Tyr(Me)(2),Thr(4)]OVT) (A); the Tyr-NH(2) (9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2) (9)]OVT (B); the Eda(9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)]OVT (C); and the retro COCH(2)Ph(4-0H)(10) modified analog of (C), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT (D). The eight new analogs of A-D are (1) desGly-NH(2),d(CH(2))(5)[D-2Nal(2),Thr(4)]OVT, (2) desGly-NH(2),d(CH(2))(5)[2-Nal(2),Thr(4)]OVT, (3) d(CH(2))(5)[D-2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (4) d(CH(2))(5)[2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (5) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)]OVT, (6) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)]OVT, (7) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT, (8) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-OH)(10)]OVT. Peptides 1-8 were evaluated for agonistic and antagonistic activities in in vitro and in vivo rat bioassays, in rat OT receptor (rOTR) binding assays and in human OT receptor (hOTR) and human vasopressin (VP) vasopressor (V(1a)) receptor (hV(1a)R) binding assays. Also reported are the hOTR and hV(1a)R affinity data for atosiban and for B. None of the eight peptides exhibit oxytocic or vasopressor agonism. Peptides 1-8 exhibit weak antidiuretic agonism (activities in the range 0.014-0.21 U/mg). Peptides 1-6 exhibit potent in vitro (no Mg(2+)) OT antagonism (anti-OT pA(2) values range from 7.63 to 8.08). Peptides 7 and 8 are weaker OT antagonists. Peptides 1-6 are all OT antagonists in vivo (estimated in vivo anti-OT pA(2) values in the range 6.94-7.23). Peptides 1-8 exhibit vasopressor antagonism, anti-V(1a) pA(2) values in the range 5.1-7.65. Peptides 1-8 exhibit high affinities for the rOTR (K(i) values = 0.3-7.8 nM). Peptides 1-4 and B exhibit surprisingly very high affinities for the hOTR; their K(i) values are 0.17, 0.29, 0.07, 0.14 and 0.59 nM, respectively. Peptides 1-4 and B exhibit respectively 449, 263, 1091, 546 and 129 times greater affinity for the hOTR than atosiban (K(i) = 76.4 nM). Peptides 1-4 exhibit high affinities for the hV(1a)R (K(i)s = 1.1 nM, 1.3 nM, 0.19 nM and 0.54 nM, all higher than the hV1(a)R affinities exhibited by atosiban (K(i) = 5.1 nM) and by B (K(i) = 5.26 nM). Because of their strikingly higher affinities for the hOTR than atosiban, peptides 1-4 and B exhibit gains in anti hOT/anti hV(1a) receptor selectivity compared with atosiban of 93, 64, 39, 56 and 127, respectively. These OT antagonists are thus promising candidates for development as potential new tocolytic agents.