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).

Rhodium carbenoid O-H insertion reactions with phenols; a facile method for the synthesis of trialkyl 2-aryloxyphosphonoacetates and their use in the preparation of 2-aryloxy-3-phenylpropanoates

Substituted phenols undergo a facile Rh carbenoid-mediated O-H insertion reaction with (EtO)2P(O)C(:N2)CO2R (I; R = Et, Me) to give 44-86% 2-aryloxyphosphonoacetates II (R1 = e.g., H, 4-Me, 4-Cl, 2-OH, 4-PhCH2O). Phenols contg. strongly electron withdrawing groups, bulky ortho-substituents or certain ortho-heteroatom substituents show reduced or variable yields. Catechol affords a mono-adduct which cyclizes to lactate III. Aniline inserts preferentially and exclusively over phenol in a competition reaction with I (R = Et) to give (EtO)2P(O)CH(NHPh)CO2Et. II are versatile intermediates in a prepn. of 2-aryloxy-3-phenylpropenoates IV by Wadsworth-Emmons reaction with benzaldehydes R2C6H4CHO (R2 = PhCH2O, 2-Cl, H). Dissolving Mg metal redn. provides a mild method for the conversion of propenoates IV into the corresponding propanoates.

Structure, stereochemistry and synthesis of enantiopure cyclohexenone cis-diol bacterial metabolites derived from phenols

Biotransformation of 3-substituted and 2,5-disubstituted phenols, using whole cells of P. putida UV4, yielded cyclohexenone cis-diols as single enantiomers; their structures and absolute configurations have been determined by NMR and ECD spectroscopy, X-ray crystallography, and stereochemical correlation involving a four step chemoenzymatic synthesis from the corresponding cis-dihydrodiol metabolites. An active site model has been proposed, to account for the formation of enantiopure cyclohexenone cis-diols with opposite absolute configurations.

The Unexpected Preference for the fac-Isomer with the Tris(5-ester-substituted-2,2′-bipyridine) Complexes of Ruthenium(II)

The synthesis of a number of new 2,2'-bipyridine ligands, functionalized with bulky ester side groups is reported (L2 - L8). Their reaction with [Ru(DMSO)4Cl2] gives rise to tris-chelate ruthenium(II) metal complexes which show an unusually high proportion of the fac-isomer, as judged by 1H NMR following conversion to the ruthenium(II) complex of 2,2'-bipyridine-5-carboxylic acid methyl ester (L1). The initial reaction appears to have thermodynamic control with the steric bulk of the ligands causing the third ligand to be labile under the reaction conditions used, giving rise to disappointing yields and allowing rearrangement to the more stable facial form. DFT studies indicate that this does not appear to be as a consequence of a metal centered electronic effect. The two isomers of [Ru(L1)3](PF6)2 were separated into the two individual forms using silica preparative plate chromatographic procedures, and the photophysical characteristics of the two forms compared. The results appear to indicate that there is no significant difference in both their room temperature electronic absorption and emission spectra or their excited state lifetimes at 77K.

Tandem enzyme-catalysed oxidations of alkyl phenyl sulfides and alkyl benzenes: enantiocomplementary routes to chiral phenols.

Dioxygenase-catalysed trioxygenation of alkyl phenyl sulfides and alkyl benzenes yields enantiopure cis-dihydrodiol sulfoxides and triols respectively; naphthalene cis-dihydrodiol dehydrogenase-catalysed aromatisation of these diastereoisomers gives enantiopure catechols of either configuration.

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.

Biotransformation of Substituted Pyridines with Dioxygenase-containing Microorganisms.

A series of 2-, 3- and 4-substituted pyridines was metabolised using the mutant soil bacterium Pseudomonas putida UV4 which contains a toluene dioxygenase (TDO) enzyme. The regioselectivity of the biotransformation in each case was determined by the position of the substituent. 4-Alkylpyridines were hydroxylated exclusively on the ring to give the corresponding 4-substituted 3-hydroxypyridines, while 3-alkylpyridines were hydroxylated stereoselectively on C-1 of the alkyl group with no evidence of ring hydroxylation. 2-Alkylpyridines gave both ring and side-chain hydroxylation products. Choro- and bromo-substituted pyridines, and pyridine itself, while being poor substrates for P. putida UV4, were converted to some extent to the corresponding 3-hydroxypyridines. These unoptimised biotransformations are rare examples of the direct enzyme-catalysed oxidation of pyridine rings and provide a novel synthetic method for the preparation of substituted pyridinols. Evidence for the involvement of the same TDO enzyme in both ring and side-chain hydroxylation pathways was obtained using a recombinant strain of Escherichia coli (pKST11) containing a cloned gene for TDO. The observed stereoselectivity of the side-chain hydroxylation process in P. putida UV4 was complicated by the action of an alcohol dehydrogenase enzyme in the organism which slowly leads to epimerisation of the initial (R)-alcohol bioproducts by dehydrogenation to the corresponding ketones followed by stereoselective reduction to the (S)-alcohols.

A comparative study of the synthesis of 3-substituted catechols using an enzymatic and a chemoenzymatic method

A series of cis-dihydrodiol metabolites, available from the bacterial dioxygenase-catalysed oxidation of monosubstituted benzene substrates using Pseudomonas putida UV4, have been converted to the corresponding catechols using both a heterogeneous catalyst (Pd/C) and a naphthalene cis-diol dehydrogenase enzyme present in whole cells of the recombinant strain Escherichia coli DH5 alpha(pUC129: nar B). A comparative study of the merits of both routes to 3-substituted catechols has been carried out and the two methods have been found to be complementary. A similarity in mechanism for catechol formation under both enzymatic and chemoenzymatic conditions, involving regioselective oxidation of the hydroxyl group at C-1, has been found using deuterium labelled toluene cis-dihydrodiols. The potential, of combining a biocatalytic step (dioxygenase-catalysed cis-dihydroxylation) with a chemocatalytic step (Pd/C-catalysed dehydrogenation), into a one-pot route to catechols, from the parent substituted benzene substrates, has been realised.

Synthesis of Piano Stool Complexes Employing the Pentafluorophenyl Substituted Diphosphine (C6F5)2PCH2P(C6F5)2 and the Effect of Phosphine Modifiers on Hydrogen Transfer Catalysis.

Ruthenium, rhodium, and iridium piano stool complexes of the pentafluorophenyl-substituted diphosphine (C6F5)2PCH2P(C6F5)2 (2) have been prepared and structurally characterized by single-crystal X-ray diffraction. The Cp-P tethered complex [{(C5Me4CH2C6F4(C6F5)CH2P(C6F5)2}RhCl2] (9), in which only one phosphorus is coordinated to the rhodium, was prepared by thermolysis of a slurry of [Cp*RhCl(-Cl)]2 and 2 and was structurally characterized by single-crystal X-ray diffraction. The tethering occurs by intramolecular dehydrofluorinative coupling of the pentamethylcyclopentadienyl ligand and P,P-coordinated 2. The geometric changes that occur on tethering force dissociation of one of the phosphorus atoms. The effects of introducing phosphine ligands to the coordination sphere of piano stool hydrogen transfer catalysts have been studied. The complexes of fluorinated phosphine complexes are found to transfer hydrogen at rates that compare favorably with leading catalysts, particularly when the phosphine and cyclopentadienyl functionalities are tethered. The highly chelating Cp-PP complex [(C5Me4CH2-2-C5F3N-4-PPhCH2CH2PPh2)RhCl]BF4 (1) was found to outperform all other complexes tested. The mechanism of hydrogen transfer catalyzed by piano stool phosphine complexes is discussed with reference to the trends in activity observed.

Characterisation and biological activity of Glu(3) amino acid substituted GIP receptor antagonists

Glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal hormone, which regulates insulin release and glucose homeostasis, but is rapidly inactivated by enzymatic N-terminal truncation. Here we report the enzyme resistance and biological activity of several Glu(3) -substituted analogues of GIP namely; (Ala(3))GIP, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))GIP. Only (Lys(3))- GIP demonstrated moderately enhanced resistance to DPP-IV (p <0.05 to p <0.01) compared to native GIP. All analogues demonstrated a decreased potency in cAMP production (EC50 1.47 to 11.02 nM; p <0.01 to p <0.001) with (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated cAMP production (p <0.05). In BRIN-BD11 cells, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))- GIP did not stimulate insulin secretion with both (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated insulin secretion (p <0.05). Injection of each GIP analogue together with glucose in oblob mice significantly increased the glycaemic excursion compared to control (p <0.05 to p <0.001). This was associated with lack of significant insulin responses. (Ala(3))GIP, (Phe(3))GIP and (Tyr(3))GIP, when administered together with GIP, significantly reduced plasma insulin (p <0.05 top <0.01) and impaired the glucose-lowering ability (p <0.05 to p <0.01) of the native peptide. The DPP-IV resistance and GIP antagonism observed were similar but less pronounced than (Pro(3))GIP. These data demonstrate that position 3 amino acid substitution of GIP with (Ala(3)), (Phe(3)), (Tyr(3)) or (Pro(3)) provides a new class of functional GIP receptor antagonists. (C) 2007 Elsevier Inc. All rights reserved.