Fluorescence Properties of Y-Nucleoside Derivatives

Abstract

Modified-base and modified N³-(β-D_-ribofuranoside) derivatives of the Y base exhibit subnanosecond fluorescence decays. Attachment of groups to the “unnatural” N¹ base position produces compounds showing dominant 7 - 10 ns decays. These spectral properties have been compared with those of the free Y base and suggest that the free Y base exists mainly as the N¹-H tautomer.     

Reactivity of [Fe] and [Ni-Fe-Se] hydrogenases with their oxido-reduction partner: the tetraheme cytochrome c3.

In order to understand the electron transfer mechanisms for the [Fe] and [Ni-Fe] hydrogenases, a kinetic study of cytochrome c3 reduction has been undertaken. Cyclic voltammetry and controlled-potential amperometry techniques have been used to investigate the intermolecular electron-transfer reaction between cytochrome c3 and [Fe] hydrogenase from Desulfovibrio vulgaris Hildenborough. Electron-transfer cross-reactions between [Fe] or [Ni-Fe-Se] hydrogenase and cytochrome c3 from Desulfovibrio vulgaris Hildenborough or Desulfovibrio desulfuricans Norway have been studied. Some structural implications are considered from these experimental data.     

Ordered Water Structure and Enhanced Reactivity

Analogies are drawn between the ordered structure of the colloidal water pool of reversed micelles, water present in the micropores of dense cellulose acetate films and the water vicinal to interfaces in biological systems. Chemical reactivity is enhanced in the solvent which is entirely different from normal water. In particular, it is possible to use the stretched water present in the cellulose acetate films to synthesise biochemicals.     

Ordered Water Structure and Enhanced Reactivity

Analogies are drawn between the ordered structure of the colloidal water pool of reversed micelles, water present in the micropores of dense cellulose acetate films and the water vicinal to interfaces in biological systems. Chemical reactivity is enhanced in the solvent which is entirely different from normal water. In particular, it is possible to use the stretched water present in the cellulose acetate films to synthesise biochemicals.     

Reactivity of nitric oxide with the [4Fe-4S] cluster of dihydroxyacid dehydratase from Escherichia coli

Although the NO (nitric oxide)-mediated modification of iron-sulfur proteins has been well-documented in bacteria and mammalian cells, specific reactivity of NO with iron-sulfur proteins still remains elusive. In the present study, we report the first kinetic characterization of the reaction between NO and iron-sulfur clusters in protein using the Escherichia coli IlvD (dihydroxyacid dehydratase) [4Fe-4S] cluster as an example. Combining a sensitive NO electrode with EPR (electron paramagnetic resonance) spectroscopy and an enzyme activity assay, we demonstrate that NO is rapidly consumed by the IlvD [4Fe-4S] cluster with the concomitant formation of the IlvD-bound DNIC (dinitrosyl-iron complex) and inactivation of the enzyme activity under anaerobic conditions. The rate constant for the initial reaction between NO and the IlvD [4Fe-4S] cluster is estimated to be (7.0+/-2.0)x10(6) M(-2) x s(-1) at 25 degrees C, which is approx. 2-3 times faster than that of the NO autoxidation by O2 in aqueous solution. Addition of GSH failed to prevent the NO-mediated modification of the IlvD [4Fe-4S] cluster regardless of the presence of O2 in the medium, further suggesting that NO is more reactive with the IlvD [4Fe-4S] cluster than with GSH or O2. Purified aconitase B [4Fe-4S] cluster from E. coli has an almost identical NO reactivity as the IlvD [4Fe-4S] cluster. However, the reaction between NO and the endonuclease III [4Fe-4S] cluster is relatively slow, apparently because the [4Fe-4S] cluster in endonuclease III is less accessible to solvent than those in IlvD and aconitase B. When E. coli cells containing recombinant IlvD, aconitase B or endonuclease III are exposed to NO using the Silastic tubing NO delivery system under aerobic and anaerobic conditions, the [4Fe-4S] clusters in IlvD and aconitase B, but not in endonuclease III, are efficiently modified forming the protein-bound DNICs, confirming that NO has a higher reactivity with the [4Fe-4S] clusters in IlvD and aconitase B than with O2 or GSH. The results suggest that the iron-sulfur clusters in proteins such as IlvD and aconitase B may constitute the primary targets of the NO cytotoxicity under both aerobic and anaerobic conditions.     

Methylation of a Mesoionic Tricyclic Nucleoside Related to Wyosine

Abstract

4-Desmethylwyosine/2/, a formal precursor of a hyper-modified nucleoside wyosine /1/ was modified by N-1 benzylation. Mesoionic character of the resulting compound 3b, despite the similarities to wyosine in electron density distribution as shown by 13c NMR, does not enforce the change of methylation direction towards the desired N-4 position.     

Structure and Reactivity of Chiral Fenchone Based Organozinc Catalysts

The propensity of zinc alkoxides to dimerize arises from the polarity of Zn⁽⁺⁾-O^(-) units, which can be visualized by electrostatic potential plots, e. g. of the zinc chelate complex {methylzinc(1R,2R,4S)-2-endo-oxido-2-exo-(2-methoxyphenyl)-1,3,3-trimethylbicyclo[2.2.1] heptane}. Monomer-dimer equilibria of this fenchone-based methylzinc chelate complex and its derivatives determine catalyst reactivity in dialkylzinc additions to aldehydes and were computed [ONIOM (RHF/LanL2DZ:UFF)] to assess the relative reactivities of the catalysts. Increased monomer formation, i. e. increased catalyst reactivity, is predicted in ligand systems with bulky t-butyl and Si(CH₃)₃ ortho-substituents but not for the methyl derivative. Geometrical aspects of dimeric zinc chelate complexes, such as interring C_aryl-C_aryl distances, the dimer forming and internal Zn-O bond distances and the (H₃C)-O-C_aryl-C_aryl dihedral angles were found to correspond with the relative stabilities of the dimeric complexes. These geometrical criteria are promising structural probes to assess catalyst reactivity and hence are helpful tools for a rational catalyst design.Electronic Supplementary Material available.     

Structure of unfolded and refolded recombinant derived [Ala125]interleukin 2

Naturally occurring interleukin 2 (IL-2) contains an odd number (three) of cysteinyl residues and thus is susceptible to the formation of a variety of intramolecular and intermolecular disulfide bonds. The cysteine at residue 125 has been replaced with an alanine residue by site-directed mutagenesis, and hence, this analogue can form only one intrachain disulfide bond. When expressed at high levels in Escherichia coli, this recombinant DNA derived IL-2 analogue is insoluble, reduced, and inactive. The protein was solubilized by denaturants and, after purification, was oxidized to form an intramolecular disulfide bond. Circular dichroism (CD) has been used to investigate the effects of various denaturants on the unfolding-refolding process of the purified, oxidized protein. A similar conformation is obtained when [Ala125]interleukin 2 [IL-2(Ala-125)] is refolded from 6 M guanidine hydrochloride, 8 M urea, or 5% acetic acid. The resultant protein, refolded from these denaturants, is monomeric and has activity comparable to or greater than that reported for naturally derived IL-2. In addition to this form, aggregates, as evidenced from gel filtration, are obtained. The specific activities of these are greatly reduced, and CD spectra indicated that they have much less helical content than the monomeric form of the protein. CD spectra also showed that the tertiary structure of IL-2(Ala-125) is entirely different in the presence of sodium dodecyl sulfate (SDS) from that of the monomeric form in the absence of SDS.     

On the feeding habits ofRhynchopsilopa [Dip.: Ephydridae]

Further evidence for the myrmecophagous habits of adultRhynchopsilopa flies is supplied. The genus probably evolved in association with ants of the genusCrematogaster. The feeding behaviour ofR. nitidissima Hendel on workers ofCrematogaster is described. Choice experiments show an absolute preference forCrematogaster over 3 other and genera. The mouth parts ofR. nitidissima are briefly described.

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Résumé Un témoignage supplémentaire des habitudes myrmécophages des adultes de DiptèresRhynchopsilopa est fourni. Le genre a probablement évolué en association avec les fourmis du genreCrematogaster. Le comportement alimentaire deR. nitidissima sur les ouvrières deCrematogaster est décrit. Les choix expérimentaux montrent la préférence absolue pour le genreCrematogaster à l'exclusion des 3 autres genres de fourmis. Les pièces buccales deR. nitidissima sont brièvement décrites.

     

On the species-specific biotransformation of dibenzo[a,l]pyrene

We were aimed at investigating the activation of the carcinogenic polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P) in Chinese hamster V79 cells that express single human, rat or fish cytochrome P450 (CYP) enzymes. DB[a,l]P is detectable in environmental samples and has been characterized as the most potent carcinogenic species among all PAHs as yet tested in rodent bioassays. Metabolite profiles and metabolite-dependent cytotoxic and clastogenic activities were monitored. The total turnover of CYP-mediated transformation of DB[a,l]P was as follows: human CYP1B1>fish CYP1A1 approximately human CYP1A1>rat CYP1A2>rat CYP1A1. By contrast, enzyme forms that are not classified as being members of family CYP1, such as CYP2A6, 2E1, 2B1, and 3A4, failed to catalyze any detectable conversion of this substrate. All CYP1A1 enzymes tested formed both the K-region trans-8,9- and the trans-11,12-dihydrodiol, whereas human CYP1B1 failed to catalyze K-region activation. In cells expressing human or fish CYP1A1, human CYP1B1, and rat CYP1A2, the (-)-trans-11,12-dihydrodiol was formed enantiospecifically. DB[a,l]P-dependent cytotoxicities (EC(50)) were found in the following order: human CYP1A1 (12 nM)>fish CYP1A1 (30 nM)>human CYP1B1 (45 nM)>other forms. In addition, an appreciable micronuclei formation was detected in human CYP1A1- and 1B1-expressing cells during exposure to DB[a,l]P. Our study demonstrates that human CYP1A1, 1B1 and fish CYP1A1 are able to transform DB[a,l]P into genotoxic derivatives in appreciable amounts. In contrast, CYP enzymes from rat predominantly target the K-region of DB[a,l]P and thus are serving more a rather protective route of biotransformation. Together our data suggest that humans might be more susceptible to DB[a,l]P-induced carcinogenicity than rats.     

Synthesis of new aromatic pyrrolo[2,1-c] [1,4]benzodiazepines and pyrrolo[1,2-a]thieno[3,2-e] [1,4]diazepines as anti-tumoral agents

Diazepine analogs of thieno[2,3-b]pyrrolizin-8-ones were synthesized by aromatization of 2-hydroxypyrrolo[1,2-a]thieno[3,2-e][1,4]diazepines. These compounds were evaluated in vitro for their antiproliferative activity against the L1210 leukemia cell line. The activity of these compounds was in the micromolar range, the best result being for the mixture of the isomers 5 and 6 which showed a 0.35 microM IC50 against cell growth.     

Methylation of desmethyl analogue of Y nucleosides. Wyosine from guanosine.

Wyosine la, one of the fluorescent hypermodified Y nucleosides found in tRNAsPhe, was synthesized chemically from its biogenetic precursor guanosine 2. The route involved transformation of 2 into the tricyclic structure 3a and subsequent methylation at N-4. The major products of various methylation procedures were isomers of wyosine, methylated at N-5 (3b) or at N-1 (4). Mesoionic compound 4 is a new analogue of 7-methylguanosine 5, modified nucleoside occurring in the unique positions in transfer, messenger and ribosomal RNAs. The chromatographic and spectral characteristics of wyosine and its isomers is given.