Dioxygenase-catalyzed cis-dihydroxylation of pyridine-ring systems

Toluene dioxygenase-catalyzed dihydroxylation, in the carbocyclic rings of quinoline, 2-chloroquinoline, 2-methoxyquinoline, and 3-bromoquinoline, was found to yield the corresponding enantiopure cis-5,6- and -7,8-dihydrodiol metabolites using whole cells of Pseudomonas putida UV4. cis-Dihydroxylation at the 3,4-bond of 2-chloroquinoline, 2-methoxyquinoline, and 2-quinolone was also found to yield the heterocyclic cis-dihydrodiol metabolite, (+)-cis-(3S,4S)-3,4-dihydroxy-3,4-dihydro-2-quinolone. Heterocyclic cis-dihydrodiol metabolites, resulting from dihydroxylation at the 5,6- and 3,4-bonds of 1-methyl 2-pyridone, were isolated from bacteria containing toluene, naphthalene, and biphenyl dioxygenases. The enantiomeric excess (ee) values (>98%) and the absolute configurations of the carbocyclic cis-dihydrodiol metabolites of quinoline substrates (benzylic R) and of the heterocyclic cis-diols from quinoline, 2-quinolone, and 2-pyridone substrates (allylic S) were found to be in accord with earlier models for dioxygenase-catalyzed cis-dihydroxylation of carbocyclic arenes. Evidence favouring the dioxygenase-catalyzed cis-dihydroxylation of pyridine-ring systems is presented.

Selectivity studies of the benzene cis-dihydrodiol dehydrogenase enzyme from Pseudomonas putida ML2 with Vicinal diol substrates

The enantiopure (1S, 2S)-cis-dihydrodiol metabolites 2B-5B have been obtained in low yield from the corresponding monosubstituted halobenzene substrates 2A-5A, using a wild-type strain of Pseudomonas putida (ML2) containing benzene dioxygenase (BDO). Benzene cis-dihydrodiol dehydrogenase (BCD) from P. putida ML2 and naphthalene cis-dihydrodiol dehydrogenase (NCD) from P. putida 8859 were purified and used in a comparative study of the stereoselective biotransformation of cis-dihydrodiol enantiomers 2B-5B. The BCD and NCD enzymes were found to accept cis-dihydrodiol enantiomers of monosubstituted benzene cis-dihydrodiol substrates 2B-5B of opposite absolute configuration. The acyclic alkene 1,2-diols 10-17 were also found to be acceptable substrates for BCD.

Dioxygenase-catalysed mono-, di- and tri-oxygenation of dialkyl sulfides and thioacetals: chemoenzymatic synthesis of enantiopure cis-diol sulfoxides

Toluene dioxygenase (TDO)-catalysed monooxygenation of methylsulfanylmethyl phenyl sulfide 1 and methylsulfanylmethyl 2-pyridyl sulfide 4, using whole cells of Pseudomonas putida UV4, occurred exclusively at the alkyl aryl sulfur centre to yield the alkyl aryl sulfoxides 2 and 5 respectively. These sulfoxides, accompanied by the dialkyl sulfoxides 3 and 6, were also obtained from naphthalene dioxygenase (NDO)-catalysed sulfoxidation of thioacetals 1 and 4 using intact cells of P. putida NCIMB 8859. Enzymatic oxidation of methyl benzyl sulfide 7, 2-phenyl-1,3-dithiane 19, and 2-phenyl-1,3-dithiolane 23, using TDO, gave the corresponding dialkyl sulfoxides 8, 20 and 24 as minor bioproducts. TDO-catalysed dioxygenation of the alkyl benzyl sulfides 7, 15 and 17 and the thioacetals 19 and 23, with P. putida UV4, yielded the corresponding enantiopure cis-dihydrodiols 9, 16, 18, 21 and 25 as major metabolites and cis-dihydrodiol sulfoxides 14, 22 and 26 as minor metabolites, resulting from a tandem trioxygenation of substrates 7, 19 and 23 respectively. Chemical oxidation, of the enantiopure cis-dihydrodiol sulfides 9, 16, 18 and 21 with dimethyldioxirane (DMD), gave separable mixtures of the corresponding pairs of cis-dihydrodiol sulfoxide diastereoisomers 14 and 27, 28 and 29, 30 and 31, 22 and 32. While dialkyl sulfoxide bioproducts 3, 6, 20 and 24 were of variable enantiopurity (27-greater than or equal to 98% ee), alkyl aryl monosulfoxides 2 and 5, cis-dihydrodiols 9, 16, 18, 21 and 25 and cis-dihydrodiol sulfoxide bioproducts 14, 22 and 26 were all single enantiomers (greater than or equal to 98% ee). The absolute configurations of the products, obtained from enzyme-catalysed (TDO and NDO) and chemical (DMD) oxidation methods, were determined by stereochemical correlation, circular dichroism, and X-ray crystallographic methods.

Stereochemical and mechanistic aspects of dioxygenase-catalysed benzylic hydroxylation of indene and chromane substrates

Toluene dioxygenase (TDO)-catalysed benzylic hydroxylation of indene substrates (8, 16 and 17), using whole cell cultures of Pseudomonas putida UV4, was found to yield inden-1-ol (14 and 22) and indan-1-one bioproducts (15 and 23). The formation of these bioproducts is consistent with the involvement of carbon-centred radical intermediates. TDO-catalysed oxidation of indenes 8 and 16 also gave cis-diols 13 and 18 respectively. TDO and naphthalene dioxygenase (NDO), used as both whole-cell preparations and as purified enzymes, were found to catalyse the benzylic hydroxylation of chromane 30, deuteriated (+/-)-chromane 30(D) and enantiomers (4S)-30(D) and (4R)-30(D) to yield (4R)- and (4S)-chroman-4-ols 31/31(D) respectively. The mechanism of benzylic hydroxylation of chromane 30/30(D) involves the stereoselective abstraction of a pro-R (with TDO) or a pro-S (with NDO) hydrogen atom at C-4 and a marked preference for retention of configuration.

Metal-Directed Synthesis of Enantiomerially Pure Dimetallic Lanthanide Luminescent Triple-Stranded Helicates

The synthesis and photophysical evaluation of two enatiomerially pure dimetallic lanthanide luminescent triple-stranded helicates is described. The two systems, formed from the chiral (R,R) ligand 1 and (S,S) ligand 2, were produced as single species in solution, where the excitation of either the naphthalene antennae or the pyridiyl units gave rise to Eu(III) emission in a variety of solvents. Excitation of the antennae also gave rise to circularly polarized Eu(III) luminescence emissions for Eu2:13 and Eu2:23 that were of equal intensity and opposite sign, confirming their enantiomeric nature in solution providing a basis upon which we were able to assign the absolute configurations of Eu2:13 and Eu2:23.

Formation of Novel Di-Nuclear Lanthanide Luminescent Sm(III), Eu(III) and Tb(III) Triple Stranded Helicates from a C2 Symmetrically 2,6-Dicarboxylic Amide based 1,3-Xylene Ligand in CH3CN

The synthesis of the C2-symmetrical ligand 1 consisting of two naphthalene units connected to two pyridine-2,6-dicarboxamide moieties linked by a xylene spacer and the formation of LnIII-based (Ln1/4 Sm, Eu, Tb, and Lu) dimetallic helicates [Ln2 · 13] in MeCN by means of a metal-directed synthesis is described. By analyzing the metal-induced changes in the absorption and the fluorescence of 1, the formation of the helicates, and the presence of a second species [Ln2 · 12] was confirmed by nonlinear- regression analysis. While significant changes were observed in the photophysical properties of 1, the most dramatic changes were observed in the metal-centred lanthanide emissions, upon excitation of the naphthalene antennae. From the changes in the lanthanide emission, we were able to demonstrate that these helicates were formed in high yields (ca. 90% after the addition of 0.6 equiv. of LnIII), with high binding constants, which matched well with that determined from the changes in the absorption spectra. The formation of the LuIII helicate, [ Lu2 · 13 ] , was also investigated for comparison purposes, as we were unable to obtain accurate binding constants from the changes in the fluorescence emission upon formation of [Sm2 · 13], [Eu2 · 13], and [Tb2 · 13].

Lanthanide(III) nitrobenzenesulfonates as new nitration catalysts: The role of the metal and of the counterion in the catalytic efficiency

Lanthanide(III) complexes of p-nitrobenzenesulfonic acid, Ln(p-NBSA)(3), m-nitrobenzenesulfonic acid, Ln(m-NBSA)(3), and 2,4-nitrobenzenesulfonic acid, Ln(2,4-NBSA)(3), were prepared, characterized and examined as catalyst for the nitration of benzene, toluene, xylenes, naphthalene, bromobenzene and chlorobenzene. The initial screening of the catalysts showed that lanthanum(III) complexes were more effective than the corresponding ytterbium(III) complexes, and that catalysts containing the bulky 2,4-NBSA ligand were less effective than the catalyst containing p-NBSA (nosylate) or m-NBSA ligands. Examination of a series of Ln(p-NBSA)(3) and Ln(m-NBSA)(3) catalysts revealed that there is a clear correlation between the ionic radii of the lanthanide(III) ions and the yields of nitration, with the lighter lanthanides being more effective. The X-ray single crystal structure of Yb(m-NBSA)(3).6H(2)O shows that two m-NBSA ligands are directly bound to the metal centre while the third ligand is not located in the first coordination sphere, but it is hydrogen bonded to one of the water molecules which is coordinated to ytterbium(III). NMR studies suggest that this structure is preserved under the conditions used in the nitration reaction. The structure of Yb(m-NBSA)(3) is markedly different from the structure of the well-known ytterbium(III) triflate catalyst. The coordination of the nitrobenzenesulfonate counterion to the lanthanide(III) ion suggests that steric effects might play an important role in determining the efficiency of these novel nitration catalysts. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004).

Resolving genetic functions within microbial populations: In situ analyses using rRNA and mRNA stable isotope probing coupled with single-cell raman-fluorescence in situ hybridization

Prokaryotes represent one-half of the living biomass on Earth, with the vast majority remaining elusive to culture and study within the laboratory. As a result, we lack a basic understanding of the functions that many species perform in the natural world. To address this issue, we developed complementary population and single-cell stable isotope (C-13)-linked analyses to determine microbial identity and function in situ. We demonstrated that the use of rRNA/mRNA stable isotope probing (SIP) recovered the key phylogenetic and functional RNAs. This was followed by single-cell physiological analyses of these populations to determine and quantify in situ functions within an aerobic naphthalene-degrading groundwater microbial community. Using these culture-independent approaches, we identified three prokaryote species capable of naphthalene biodegradation within the groundwater system: two taxa were isolated in the laboratory (Pseudomonas fluorescens and Pseudomonas putida), whereas the third eluded culture (an Acidovorax sp.). Using parallel population and single-cell stable isotope technologies, we were able to identify an unculturable Acidovorax sp. which played the key role in naphthalene biodegradation in situ, rather than the culturable naphthalene-biodegrading Pseudomonas sp. isolated from the same groundwater. The Pseudomonas isolates actively degraded naphthalene only at naphthalene concentrations higher than 30 mu M. This study demonstrated that unculturable microorganisms could play important roles in biodegradation in the ecosystem. It also showed that the combined RNA SIP-Raman-fluorescence in situ hybridization approach may be a significant tool in resolving ecology, functionality, and niche specialization within the unculturable fraction of organisms residing in the natural environment.

A crystallographic and modelling study of a human telomeric RNA (TERRA) quadruplex

DNA telomeric repeats in mammalian cells are transcribed to guanine-rich RNA sequences, which adopt parallel-stranded G-quadruplexes with a propeller-like fold. The successful crystallization and structure analysis of a bimolecular human telomeric RNA G-quadruplex, folded into the same crystalline environment as an equivalent DNA oligonucleotide sequence, is reported here. The structural basis of the increased stability of RNA telomeric quadruplexes over DNA ones and their preference for parallel topologies is described here. Our findings suggest that the 2'-OH hydroxyl groups in the RNA quadruplex play a significant role in redefining hydration structure in the grooves and the hydrogen bonding networks. The preference for specific nucleotides to populate the C3'-endo sugar pucker domain is accommodated by alterations in the phosphate backbone, which leads to greater stability through enhanced hydrogen bonding networks. Molecular dynamics simulations on the DNA and RNA quadruplexes are consistent with these findings. The computations, based on the native crystal structure, provide an explanation for RNA G-quadruplex ligand binding selectivity for a group of naphthalene diimide ligands as compared to the DNA G-quadruplex.

Targeting Human Gastrointestinal Stromal Tumor Cells with a Quadruplex-Binding Small Molecule

Most of human gastrointestinal stromal tumors (GIST) are driven by activating mutations in the protooncogene KIT, a tyrosine kinase receptor. Clinical treatment with imatinib targets the kinase domain of KIT, but tumor regrowth occurs as a result of them development of resistant mutations in the kinase active site. An alternative small-molecule approach to GIST therapy is described, in which the KIT gene is directly targeted, and thus, kinase resistance may be circumvented. A naphthalene diimide derivative has been used to demonstrate the concept of dual quadruplex targeting. This compound strongly stabilizes both telomeric quadruplex DNA and quadruplex sites in the KIT promoter in vitro. It is shown here that the compound is a potent inducer of growth arrest in a patient-derived GIST cell line at a concentration (similar to 1 mu M) that also results in effective inhibition of telomerase activity and almost complete suppression of KIT mRNA and KIT protein expression. Molecular modeling studies with a telomeric quadruplex have been used to rationalize aspects of the experimental quadruplex melting data.

Artemisinins act through at least two targets in a yeast model

Artemisinin and related compounds are potent and widely used antimalarial drugs but their biochemical mode of action is not clear. There is strong evidence that ATP-dependent calcium transporters are a key target in the malarial parasite. However, work using Saccharomyces cerevisiae suggests that disruption of mitochondrial function is critical in the cell killing activity of these compounds. Here it is shown that, in the absence of reducing agents, artemisinin and artesunate targeted the S. cerevisiae calcium channels Pmr1p and Pmc1p. Both compounds affected the growth of yeast on fermentable and nonfermentable media. This growth inhibition was not seen in a yeast strain in which the genes encoding both calcium channels were deleted. In the presence of reducing agents, which break the endoperoxide bridge in the drugs, growth inhibition was only observed in nonfermentable media. This inhibition could be partially relieved by the addition of a free radical scavenger. These results suggest that the drugs have two biochemical modes of action - one acting by specific binding to calcium channels and one involving free radical production in the mitochondria.

Bacterial dioxygenase- and monooxygenase-catalysed sulfoxidation of benzo[b]thiophenes

Asymmetric heteroatom oxidation of benzo[b]thiophenes to yield the corresponding sulfoxides was catalysed by toluene dioxygenase (TDO), naphthalene dioxygenase (NDO) and styrene monooxygenase (SMO) enzymes present in P. putida mutant and E. coli recombinant whole cells. TDO-catalysed oxidation yielded the relatively unstable benzo[b] thiophene sulfoxide; its dimerization, followed by dehydrogenation, resulted in the isolation of stable tetracyclic sulfoxides as minor products with cis-dihydrodiols being the dominant metabolites. SMO mainly catalysed the formation of enantioenriched benzo[b] thiophene sulfoxide and 2-methyl benzo[b] thiophene sulfoxides which racemized at ambient temperature. The barriers to pyramidal sulfur inversion of 2- and 3-methyl benzo[b] thiophene sulfoxide metabolites, obtained using TDO and NDO as biocatalysts, were found to be ca.: 25-27 kcal mol(-1). The absolute configurations of the benzo[b] thiophene sulfoxides were determined by ECD spectroscopy, X-ray crystallography and stereochemical correlation. A site-directed mutant E. coli strain containing an engineered form of NDO, was found to change the regioselectivity toward preferential oxidation of the thiophene ring rather than the benzene ring.

Contrasting effects of a nonionic surfactant on the biotransformation of polycyclic aromatic hydrocarbons to cis-dihydrodiols by soil bacteria

The biotransformation of the polycyclic aromatic hydrocarbons (PAHs) naphthalene and phenanthrene was investigated by using two dioxygenase-expressing bacteria, Pseudomonas sp. strain 9816/11 and Sphingomonas yanoikuyae B8/36, under conditions which facilitate mass-transfer limited substrate oxidation. Both of these strains are mutants that accumulate cis-dihydrodiol metabolites under the reaction conditions used. The effects of the nonpolar solvent 2,2,4,4,6,8,8-heptamethylnonane (HMN) and the nonionic surfactant Triton X-100 on the rate of accumulation of these metabolites were determined. HMN increased the rate of accumulation of metabolites for both microorganisms, with both substrates. The enhancement effect was most noticeable with phenanthrene, which has a lower aqueous solubility than naphthalene. Triton X-100 increased the rate of oxidation of the PAHs with strain 9816/11 with the effect being most noticeable when phenanthrene was used as a substrate. However, the surfactant inhibited the biotransformation of both naphthalene and phenanthrene with strain B8/36 under the same conditions. The observation that a nonionic surfactant could have such contrasting effects on PAH oxidation by different bacteria, which are known to be important for the degradation of these compounds in the environment, may explain why previous research on the application of the surfactants to PAH bioremediation has yielded inconclusive results. The surfactant inhibited growth of the wild-type strain S. yanoikuyae B1 on aromatic compounds but did not inhibit B8/36 dioxygenase enzyme activity in vitro.

Isolation of Rhodococcus rhodochrous NCIMB13064 derivatives with new biodegradative abilities

Rhodococcus rhodochrous NCIMB13064 can dehalogenate and utilise a number of halogenated aliphatic compounds as sole carbon and energy source. Mutants of NCIMB13064 can be easily isolated with an enlarged range of 1-chloroalkane utilising ability. Dehalogenation of 1-chlorononane, 1-chlorodecane and short-chain 1-chloroalkanes (C-3-C-8) is encoded by the same plasmid pRTL1. However, a different genetic element(s) is required for the dehalogenation of 3-chloropropionic acid. Two derivatives (P200 and P400) of R. rhodochrous NCIMB13064 were isolated which had acquired the ability to utilise naphthalene as sole carbon and energy source. Both strains lost the ability to utilise short-chain 1-chloroalkanes and underwent some rearrangements associated with pRTL1 plasmid.

Characterising cement-superplasticiser interaction using zeta potential measurements

The zeta potential generated at the interface between cement particle surfaces adsorbed with superplasticisers have been studied using electroacoustic technique, which is capable of measuring zeta potential at high concentrated suspensions. The study has been undertaken to examine the differences in the magnitude of the zeta potential for ordinary Portland cement (OPC) and Portland pozzolanic (fly ash) cement (PPC) pastes along with the differential impacts of different types of superplasticisers on both the varieties of cement pastes. In the latter context, the effects of three different types of superplasticisers namely Ligno Sulphonate (LS), Sulphonated Melamine Formaldehyde (SMF) and Sulphonated Naphthalene Formaldehyde (SNF) have been specifically studied. The results show that the cement pastes with PPC shows better dispersion when compared with the OPC. The paper also endeavors to unfold the relationship and significance of cement interaction with three different superplasticisers.