Au Nanoparticles for Applications in Analysis of Cellular and Biomolecular Recognitions

Au nanoparticles (AuNPs) have attracted a great interest in fabrication of various biosensor systems for analysis of cellular and biomolecular recognitions. In conjunction with vast conjugation chemistry available, the materials are easily coupled with biomolecules such as nucleic acids, antigens or antibodies in order to achieve their many potential applications as ligand carriers or transducing platforms for preparation, detection and quantification purposes. Furthermore, the nanoparticles possess easily tuned and unique optical/ physical/ chemical characteristics, and high surface areas, making them ideal candidates to this end. In this topic, sensing mechanisms based on localized surface plasmon resonance (LSPR), particle aggregation, catalytic property, and Fluorescence Resonance Energy Transfer (FRET) of AuNPs as well as barcoding technologies including DNA biobarcodes will be discussed.

Synthesis of N3- and 2-NH2-substituted 6,7-diphenylpterins and their use as intermediates for the preparation of oligonucleotide conjugates designed to target photooxidative damage on single-stranded DNA representing the bcr-abl chimeric gene

Two 17-mer oligodeoxynucleotide-5'-linked-(6,7-diphenylpterin) conjugates, 2 and 3, were prepared as photosensitisers for targeting photooxidative damage to a 34-mer DNA oligodeoxynucleotide (ODN) fragment 1 representing the chimeric bcr-abl gene that is implicated in the pathogenesis of chronic myeloid leukaemia (CML). The base sequence in the 17-mer was 3'G G T A G T T A T T C C T T C T T5'. In the first of these ODN conjugates (2) the pterin was attached at its N3 atom, via a -(CH2)3OPO(OH)- linker, to the 5'-OH group of the ODN. Conjugate 2 was prepared from 2-amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone 10, using phosphoramidite methodology. Starting material 10 was prepared from 5-amino-7-methylthiofurazano[3,4-d]pyrimidine 4 via an unusual highly resonance stabilised cation 8, incorporating the rare 2H,6H-pyrimido[6,1-b][1,3]oxazine ring system. In the characterisation of 10 two pteridine phosphazenes, 15 and 29, were obtained, as well as new products containing two uncommon tricyclic ring systems, namely pyrimido[2,1-b]pteridine (20 and 24) and pyrimido[1,2-c]pteridine (27). In the second ODN conjugate the linker was -(CH2)5CONH(CH2)6OPO(OH)- and was attached to the 2-amino group of the pterin. In the preparation of 3, the N-hydroxysuccinimide ester 37 of 2-(5-carboxypentylamino)-6,7-diphenyl-4(3H)-pteridinone was condensed with the hexylamino-modified 17-mer. Excitation of 36 with near UV light in the presence of the single-stranded target 34-mer, 5'T G A C C A T C A A T A A G14 G A A G18 A A G21 C C C T T C A G C G G C C3' 1 caused oxidative damage at guanine bases, leading to alkali-labile sites which were monitored by polyacrylamide gel electrophoresis. Cleavage was observed at all guanine sites with a marked preference for cleavage at G14. In contrast, excitation of ODN-pteridine conjugate 2 in the presence of 1 caused oxidation of the latter predominantly at G18, with a smaller extent of cleavage at G15 and G14 (in the double-stranded portion) and G21. These results contrast with our previous observation of specific cleavage at G21 with ruthenium polypyridyl sensitisers, and suggest that a different mechanism, probably one involving Type 1 photochemical electron transfer, is operative. Much lower yields were found with the ODN-pteridine conjugate 3, perhaps as a consequence of the longer linker between the ODN and the pteridine in this case.

A concise and convergent (formal) total synthesis of huperzine A

The first convergent synthesis of the tricyclic skeleton of huperzine A is described and includes, as the key step, an efficient regioselective intramolecular Heck reaction of 2-(tert-butyldimethylsillyoxymethyl)-6-(2-methoxy-5-bromopyridin-6-yl)methylcyclohex-2-enol.

Dioxygenase-catalysed Oxidation of Disubstituted Benzene Substrates: Benzylic Monohydroxylation versus Aryl cis-Dihydroxylation and the Meta Effect

Biotransformations of a series of ortho-, meta- and para-substituted ethylbenzene and propylbenzene substrates have been carried out, using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). The ortho- and para-substituted alkylbenzene substrates yielded, exclusively, the corresponding enantiopure cis-dihydrodiols of the same absolute configuration. However, the meta isomers, generally, gave benzylic alcohol bioproducts, in addition to the cis-dihydrodiols (the meta effect). The benzylic alcohols were of identical (R) absolute configuration but enantiomeric excess values were variable. The similar (2R) absolute configurations of the cis-dihydrodiols are consistent with both the ethyl and propyl groups having dominant stereodirecting effects over the other substituents. The model used earlier, to predict the regio- and stereo-chemistry of cis-dihydrodiol bioproducts derived from substituted benzene substrates has been refined, to take account of non-symmetric subsituents like ethyl or propyl groups. The formation of benzylic hydroxylation products, from meta-substituted benzene substrates, without further cis-dihydroxylation to yield triols provides a further example of the meta effect during toluene dioxygenase-catalysed oxidations.

Stereoselective reductase-catalysed deoxygenation of sulfoxides in aerobic and anaerobic bacteria

Direct and indirect evidence, Of unexpected stereoselective reductase-catalysed deoxygenations of sulfoxides, was found. The deoxygenations proceeded simultaneously, with the expected dioxygenase-catalysed asymmetric sulfoxidation of sulfides, during some biotransformations with the aerobic bacterium Pseudomonas putida UV4. Stereoselective reductase-catalysed asymmetric deoxygenation of racemic alkylaryl, dialkyl and phenolic sulfoxides was observed, without evidence of the reverse sulfoxidation reaction, using anaerobic bacterial strains. A purified dimethyl sulfoxide reductase, obtained from the intact cells of the anaerobic bacterium Citrobacter braakii DMSO 11, yielded, from the corresponding racemates, enantiopure alkylaryl sulfoxide and thiosulfinate samples.

syn-Benzene dioxides: chemoenzymatic synthesis from 2,3-cis-dihydrodiol derivatives of monosubstituted benzenes and their application in the synthesis of regioisomeric 1,2- and 3,4-cis-dihydrodiols and 1,4-dioxocins

cis-2,3-Dihydrodiol metabolites of monosubstituted halobenzenes and toluene have been used as synthetic precursors of the corresponding 3,4-cis-dihydrodiols. Enantiopure syn-benzene dioxide intermediates were reduced to the 3,4-cis-dihydrodiols and thermally racemised via the corresponding 1,4-dioxocins. The syn-benzene dioxide-1,4-dioxocin valence tautomeric equilibrium ratio was found to be dependent on the substituent position. The methodology has also been applied to the synthesis of both enantiomers of the 1,2-(ipso)- and 3,4-cis-dihydrodiols of toluene. This chemoenzymatic approach thus makes available, for the first time, all three possible cis-dihydrodiol regioisomers of a monosubstituted benzene.

Enzyme-catalysed synthesis and reactions of benzene oxide/oxepine derivatives of methyl benzoates

A series of twelve benzoate esters was metabolised, by species of the Phellinus genus of wood-rotting fungi, to yield the corresponding benzyl alcohol derivatives and eight salicylates. The isolation of a stable oxepine metabolite, from methyl benzoate, allied to evidence of the migration and retention of a carbomethoxy group ( the NIH Shift), during enzyme-catalysed ortho-hydroxylation of alkyl benzoates to form salicylates, is consistent with a mechanism involving an initial arene epoxidation step. This mechanism was confirmed by the isolation of a remarkably stable, optically active, substituted benzene oxide metabolite of methyl 2-( trifluoromethyl) benzoate, which slowly converted into the racemic form. The arene oxide was found to undergo a cycloaddition reaction with 4-phenyl-1,2,4-triazoline-3,5-dione to yield a crystalline cycloadduct whose structure and racemic nature was established by X-ray crystallography. The metabolite was also found to undergo some novel benzene oxide reactions, including epoxidation to give an anti-diepoxide, base-catalysed hydrolysis to form a trans-dihydrodiol and acid-catalysed aromatisation to yield a salicylate derivative via the NIH Shift of a carbomethoxy group.

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.

Tuberculosis among Iron Age individuals from Tyva, South Siberia: palaeopathological and biomolecular findings

The study focuses on the evidence for tuberculosis apparent in an Iron Age population recovered from the cemetery of Aymyrlyg, Tyva (Tuva), South Siberia. A recent wholly molecular study of five of the cases confirmed the presence of Mycobacterium tuberculosis (MTB) complex DNA in four of the individuals. In all cases the disease was caused by strains of Mycobacterium bovis rather than Mycobacterium tuberculosis and represents the first positive identification of the bovine form of the disease in archaeological human remains. Details of the palaeopathological characteristics of the cases are provided in the current paper, while the molecular observations are extended to include a quantitative evaluation of the surviving mycobacterial DNA using real-time PCR. The observation that bovine tuberculosis was the pathogen responsible is discussed in terms of current understanding of the evolution of the MTB complex as well as the implications for future ancient DNA studies in this area.

Regioselectivity and stereoselectivity of dioxygenase catalysed cis-dihydroxylation of mono- and tri-cyclic azaarene substrates

cis-Dihydrodiol metabolites were obtained from dioxygenase-catalysed asymmetric dihydroxylations of. five monocyclic (azabiphenyl) and four tricyclic (azaphenanthrene) azaarene substrates. Enantiopurity values and absolute configuration assignments were determined using a combination of stereochemical correlation, X-ray crystallography and spectroscopy methods. The degree of regioselectivity found during cis-dihydroxylation of monocyclic azaarenes (2,3 bond >> 3,4 bond) and of tricyclic azaarenes (bay region > non-bay region bonds) was dependent on the type of dioxygenase used. The cis-dihydrodiol metabolite from an azaarene (3-phenylpyridine) was utilised in the chemoenzymatic synthesis of the corresponding trans-dihydrodiol.