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.

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.

Regio- and stereo-selective dioxygenase-catalysed cis-dihydroxylation of fjord-region polycyclic arenes

Bacterial dioxygenase-catalysed cis-dihydroxylation of the tetracyclic arenes benzo[c]phenanthrene 2, and the isosteric compounds benzo[b]naphtho[1,2-d]furan 8, and benzo[b]naphtho[1,2-d]thiophene 9, has been found to occur exclusively at fjord-region bonds. The resulting cis-dihydrodiols 7, 10 and 11 were found to be enantiopure and of similar absolute configuration. cis-Dihydroxylation was also observed in the pseudo-fjord region of the 8,9,10,11-tetrahydro-precursors (12 and 13) of benzo[b]naphtho[1,2-d]furan 8, and benzo[b]naphtho[1,2-d]thiophene 9, to yield the corresponding enantiopure hexahydro cis-diols 14 and 15. A novel tandem cis-dihydroxylation and bis-desaturation of the tetrahydro-substrate, tetrahydrobenzo[b]naphtho[1,2-d]thiophene 13, catalysed by biphenyl dioxygenase, was found to yield the fjord-region cis-dihydrodiol 17 of benzo[b]naphtho[1,2-d]thiophene 9.

Kinetic analysis of the cleavage of human protease-activated receptor-1/2/3 and 4 using quenched-fluorescent peptide substrates

Protease-activated receptors [PARs] are a family of G-protein-coupled seven-transmembrane domain receptors that are activated by proteolytic cleavage of their amino-terminal exodomain. To characterize the cleavage rate of human PAR-1 / 2 / 3 and 4 by trypsin and thrombin, four synthetic quenched-fluorescent peptide substrates have been synthesized. Each substrate consisted of a ten-residue peptide spanning the receptor activation cleavage site and using progress-curve kinetics, k(cat)/K-m values were determined.

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.

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.

New families of enantiopure cyclohexenone cis-diol, o-quinol dimer and hydrate metabolites from dioxygenase-catalysed dihydroxylation of phenols

Toluene dioxygenase-catalysed cis-dihydroxylation of phenols has led to the discovery of new enantiopure cyclohexenone cis-diol, o-quinol dimer and phenol hydrate metabolites having synthetic potential.

Dioxygenase-catalysed cis-dihydroxylation of meta-substituted phenols to yield cyclohexenone cis-diol and derived enantiopure cis-triol metabolites

cis-Dihydroxylation of meta-substituted phenol (m-phenol) substrates, to yield the corresponding cyclohexenone cis-diol metabolites, was catalysed by arene dioxygenases present in mutant and recombinant bacterial strains. The presence of cyclohexenone cis-diol metabolites and several of their cyclohexene and cyclohexane cis-triol derivatives was detected by LC-TOFMS analysis and confirmed by NMR spectroscopy. Structural and stereochemical analyses of chiral ketodiol bioproducts, was carried out using NMR and CD spectroscopy and stereochemical correlation methods. The formation of enantiopure cyclohexenone cis-diol metabolites is discussed in the context of postulated binding interactions of the m-phenol substrates at the active site of toluene dioxygenase (TDO).

Sequence-Specific and Strand-Specific Cleavage In Oligodeoxyribonucleotides and DNA Containing 3'-Thiothymidine

Oligonucleotides containing a 3'-thiothymidine residue (T3's) at the cleavage site for the EcoRV restriction endonuclease (between the central T and A residues of the sequence GATATC) have been prepared on an automated DNA synthesizer using 5'-O-monomethoxytritylthymidine 3'-S-(2-cyanoethyl N,N-di-isopropylphosphorothioamidite). The self-complementary sequence GACGAT3'sATCGTC was completely resistant to cleavage by EcoRV, while the heteroduplex composed of 5'-TCTGAT3'sATCCTC and 5'-GAGGATATCAGA (duplex 4) was cleaved only in the unmodified strand (5'-GAGGATATCAGA). In contrast, strands containing a 3'-S-phosphorothiolate linkage could be chemically cleaved specifically at this site with Ag+. A T3's residue has also been incorporated in the (-) strand of double-stranded closed circular (RF IV) M13mp18 DNA at the cleavage site of a unique EcoRV recognition sequence by using 5'-pCGAGCTCGAT3'sATCGTAAT as a primer for polymerization on the template (+) strand of M13mp18 DNA. On treatment of this substrate with EcoRV, only one strand was cleaved to produce the RF II or nicked DNA. Taken in conjunction with the cleavage studies on the oligonucleotides, this result demonstrates that the 3'-S-phosphorothiolate linkage is resistant to scission by EcoRV. Additionally, the phosphorothiolate-containing strand of the M13mp18 DNA could be cleaved specifically at the point of modification using iodine in aqueous pyridine. The combination of enzymatic and chemical techniques provides, for the first time, a demonstrated method for the sequence-specific cleavage of either the (+) or (-) strand.

Synthesis and Phosphorus Sulfur Bond-Cleavage Of 3'- Thiothymidylyl(3'5')Thymidine

The title compound is readily prepared from 5'-O-monomethoxytrityl-3'-thiothymidine (5); cleavage of the P–S bond can be accomplished by mild oxidative hydrolysis.

Sequential C-H and C-Ru bond formation and cleavage during the thermally induced rearrangement of aryl ruthenium(II) complexes with [C6H3(CH2NMe2)(2)-2,6](-) as a bidentate eta(2)-C,N coordinated ligand. The crystal structures of the isomeric pairs [RuCl{eta(6)-C10H14}{eta(2)-C,N-C6H3(CH2NMe2)(2)-2,n}] (n = 4 or 6) and [Ru(eta(5)-C5H5){(eta(2)-C,N-C6H3(CH2NMe2)(2)-2,n} (PPH3)] (n = 4 or 6)

New air-stable ruthenium(II) complexes that contain the aryldiamine ligand [C6H3(CH2-NMe2)(2)-2,6](-) (NCN) are described. These complexes are [RuCl{eta(2)-C,N-C6H3(CH2NMe2)(2)-2,6}(eta(6)-C10H14)] (2; C10H14 = p-cymene = C6H4Me-Pr-i-4), [Ru{eta(2)-C,N-C6H3(CH2NMe2)(2)-2,6}(eta(5)-C5H5)(PPh3)] (5), and their isomeric forms [RuCl{eta(2)-C,N-C6H3(CH2NMe2)(2)-2,4}(eta(6)-C10H14)] (3) and [Ru{eta(2)-C,N-C6H3(CH2NMe2)(2)-2,4}(eta(5)-C5H5)(PPh3)] (6), respectively. Complex 2 has been prepared from the reaction of [Li(NCN)](2) with [RuCl2(eta(6)-C10H14)](2), whereas complex 5 has been prepared by the treatment of [RuCl{eta(3)-N,C,N-C6H3(CH2NMe2)(2)-2,6}(PPh3)] (4) with [Na(C5H5)](n). Both 2 and 5 are formally 18-electron ruthenium(II) complexes in which the monoanionic potentially tridentate coordinating ligand NCN is eta(2)-C,N-bonded, In solution (halocarbon solvent at room temperature or in aromatic solvents at elevated temperature), the intramolecular rearrangements of 2 and 5 afford complexes 3 and 6, respectively. This is a result of a shift of the metal-C-aryl bond from position-1 to position-3 on the aromatic ring of the NCN ligand. The mechanism of the isomerization is proposed to involve a sequence of intramolecular oxidative addition and reductive elimination reactions of both aromatic and aliphatic C-H bonds. This is based on results from deuterium labeling, spectroscopic studies, and some kinetic experiments. The mechanism is proposed to contain fully reversible steps in the case of 5, but a nonreversible step involving oxidative addition of a methyl NCH2-H bond in the case of 2. The solid-state structures of complexes 2, 3, 5, and 6 have been determined by single-crystal X-ray diffraction. A new dinuclear 1,4-phenylene-bridged bisruthenium(II) complex, [1,4-{RuCl(eta(6)-C10H14)}(2){C-6(CH2NMe2)(4)-2,3,5,6-C,N,C',N'}] (9) has also been prepared from the dianionic ligand [C-6(CH2NMe2)(4)-2,3,5,6](2-) (C2N4). The C2N4 ligand is in an eta(2)-C,N-eta(2)-C',N'-bis(bidentate) bonding mode. Compound 9 does not isomerize in solution (halocarbon solvent), presumably because of the absence of an accessible C-aryl-H bond. Complex 9 could not be isolated in an analytically pure form, probably because of its high sensitivity to air and very low solubility, which precludes recrystallization.

Double cyclometalation via carbon-silicon bond cleavage by palladium(II) acetate. X-ray structure of a cationic 1,4-dipalladated benzene ring and selective synthesis of heterobimetallic 1,4-phenylene-bridged platinum(II)-palladium(II) complexes

The disilylated compound 1,4-bis(trimethylsilyl)-2,3,5,6-tetrakis((dimethylamino)methyl)benzene, (Me(3)Si)(2)C2N4, 4, can be electrophilically palladated selectively at the C-Si bonds to afford the neutral 1,4-bis(palladium) complex [(AcOPd)(2)(C2N4)], from which the dicationic [(LPd)(2)(C2N4)](2+) (L = MeCN) organometallic species are accessible. The monosilylated species (Me(3)Si)(H)C2N4, 5, can be used for the preparation of the dicationic heterodinuclear platinum(II)-palladium(II) species [(LPd)(LPt)(C2N4)](2+) (L = MeCN) via a sequence of transmetalation of the organolithium derivative of 5 with [PtCl2(SEt(2))(2)], followed by a C-Si bond palladation reaction.