廣谷 功

Six possible diastereomers of conduritols have been synthesized diastereoselectively in an integrated manner starting from a single chiral precursor, which served as a synthetic equivalent of chiral cis-1,4-dihydroxycyclohexa-2,5-diene.

A concise diastereo- and enantio-controlled synthesis of (-)-epibatidine, the potent analgesic alkaloid from an Ecuadoran poison frog, Epipedobates tricolor, has been developed starting with a synthetic equivalent of chiral cis-cyclohexadiene- 1,4-diol.

Both enantiomeric tricyclic enones 2 and 3 serving as the synthetic equivalents of chiral cyclohexadienone and cycloheptadienone have been prepared by lipase-mediated kinetic eater exchange reaction as the key step. The absolute configuration of the latter has been determined by transformation of the (-)-enantiomer 3 into the known (-)-6-methyl-2-cycloheptenone 16 and the cytotoxic marine natural product (-)-clavularin B 19. However, it has been found that the seven-membered enone 3 exhibits lower diastereoselectivity and less favorable thermal stability than the five-membered and six-membered analogues.

(-)-Quinic acid and (-)-shikimic acid, both recognized as the key intermediates in the shikimate pathway in plants and microorganisms, have been synthesized concisely in an enantio- and diastereo-controlled manner starting from a synthetic equivalent of (R)-4-hydroxycyclohex-2-enone.

A meso-1,4-enediol bis-silyl ether 2 having bicyclo[2.2.1]heptene background has been transformed diastereo- and enantioselectively into (-)-malyngolide 1, an antibiotic isolated from the blue-green marine algae, Lyngbya majuscula, via Rh(I)-(R)BINAP-catalyzed asymmetrization and diastereoselective modification of the optically active product thus obtained. (C) 1997 Elsevier Science Ltd.

Stereocontrolled syntheses of (+)-uleine and (+)-dasycarpidone are achieved for the first time in an enantiocontrolled way starting from (+)-norcamphor.

Two naturally occurring polyoxygenated cyclohexenemethanols, (+)-epiepoxydon and (-)-phyllostine, have been first synthesized in stereo- and enantio-controlled manner using a chiral cyclohexadienol synthon prepared by catalytic asymmetrization of a meso substrate. The synthesis has verified the proposed absolute structures of these natural products which have been deduced by CD measurements.

A key intermediate for the synthesis of (+)-vincamine, the major alkaloid of Vinca minor and an important cerebral vasodilatory agent, has been synthesized in an enantioconvergent way from either (R)- or (S)-enantiomer of 2-carbethoxy-2-cyclopenten-1-ol obtained by lipase-mediated resolution.

Catalytic asymmetrization of meso-3,7-bis-tert-butyldimethylsiloxycycloheptene occurs in the presence of a chiral rhodium(I) binap catalyst to give optically active 4-tert-butyldimethylsiloxycycloheptanone in 70% ee after hydrolytic workup, the (R)-enantiomer of which has been transformed into (-)-(S)-physoperuvine, the major alkaloid of Physalis peruviana.

Diastereofacial selectivity in an enantiospecific intramolecular 1,3-dipolar addition is controlled by adjusting the size of the tether:between the dipole and the dipolarophile to give 2,3-disubstituted pyrrolidines enantiomeric with respect to the newly generated stereogenic 2,3-centres depending on the tether size; this leads to stereocontrolled synthesis bf both enantiomers of the; necine base dihydroxyheliotridane from chiral O-benzylglycidol.

Three structural analogues of a potent non-opiate analgesic alkaloid epibatidine have been synthesized in optically pure forms in enantio- and diastereo-controlled ways using a chiral 2,5-cyclohexadiene-1,4-diol synthon.

Diastereomerically pure trans- and cis-4-tert-butylcyclohexanemethanols have been obtained by kinetic acylation of the diastereomeric alcohol in an organic medium and by kinetic deacylation of the diastereomeric acetate in an aqueous medium both in the presence of the same lipase (lipase PS, Pseudomonas sp., Amano). The reactions take place preferentially with the trans-isomers both in organic and aqueous media to give the trans-acetate with recovery of the cis-alcohol on acylation in an organic medium, and the trans-alcohol with recovery of the cis-acetate on deacylation in an aqueous medium.

Diastereomerically pure tranexamic acid has been prepared via a diastereomeric separation of a trans-lcis-mixture of 1,4-cyclohexanedimethanol using lipase PS (Pseudomonas sp. Amano).

An expeditious route to 3-formylfuran from cis-2-butene-1,4-diol hits been developed in 42% overall yield in a sequence of five-stage transformations.

Dehydrotremetone, a toxic ketone isolated from the weeds Eupatorium urticaefolium and Aplopappus heterophyllus, has been synthesized from isovanillin via palladium-mediated cross-coupling reaction and lithium chloride-mediated concurrent demethylation-benzofuran formation reaction. The present procedure also allows a simple preparation of three other non-heterocyclic acetylenic phytotoxic compounds isolated from the culture medium of Eutypa lata.

The effect of cytochrome b(5) on the activity of cytochrome P450 1A2 (P450 1A2) and three site-directed mutants designed to alter surface charges has been investigated. Cytochrome b(5) increased the rate of oxidation of methanol and/or 7-ethoxycoumarin 2- to 10-fold in both wild type and mutant P450. Cytochrome b(5) also increased the coupling of electron transfer to substrate hydroxylation as opposed to the uncoupling reaction which leads to peroxide production. The effect of cytochrome bs on both wild type and the Lys99Glu and Lys401Glu mutants are similar. In contrast, turnover numbers in the His163Glu mutant did not change in the presence of cytochrome b(5). The reduction potential of the His163Glu mutant decreased approximately -40 mV while the Lys99Glu and Lys401Glu mutants exhibited little change. The rate of photoreduction decreased from 1.1 X 10(-1) min(-1) to 8.3 X 10(-3) min(-1) in the His163Glu mutant while the rate of electron transfer from ferrous P450 to ferric cytochrome b(5) increased from 0.02 min(-1) to > 5 min(-1). Overall, the present study suggests that His163 is important to keep an appropriate redox potential of P450 1A2 for optimum electron transfer to occur from cytochrome b(5). Based on the P450 101 crystal structure, His163 is not expected to directly contact the heme. Therefore, the resulting change in redox properties in the His163Glu mutant is probably not the result of a direct electrostatic change in the heme environment. (C) 1994 Academic Press, Inc.

The kinetic values for 7-ethoxycoumarin (7-EC) hydroxylation have been obtained in both the NADPH-cytochrome P450 reductase- and tert-butyl hydroperoxide (TBHP)-supported systems for several Glu318 and Thr319 mutants of cytochrome P450 1A2. The results with the reductase-supported system suggest that Glu318 is important for both substrate binding and catalysis, whereas Thr3 19 is critical for neither, although the size of the residue at position 319 influences catalytic activity. In contrast, neither Glu318 nor Thr319 appears to be important for catalytic turnover in the TBHP-supported system despite the fact that the size of the amino acid at position 319 affects the binding of TBHP and 7-EC in opposite manners. The roles of these two distal amino acids in the cytochrome P450 1A2-catalyzed oxidation of 7-EC therefore differ for the reactions supported by cytochrome P450 reductase and TBHP. (C) 1994 Academic Press, Inc.

It has been revealed that the generation of 2-cyclohexenones from cis-l,4-dihydroxycyclohexene derivatives under PdCl2(PPh(3))(2)-HCO2NH4 system takes place in an intramolecular pathway involving unprecedented mode of suprafacial 1,4-hydrogen migration across the 1,4-allylic centers.

Catalytic efficiencies, percentages of rates of product formation per NADPH oxidized, and rates of product formation per O2 consumed of ionic mutants of cytochrome P450 1A2 (P450 1A2) were studied. Efficiencies of Lys99Glu, Lys453Glu, and Arg455Glu mutants for the hydroxylation reaction toward 7-ethoxycoumarin in the reconstituted system were much lower than that of the wild type (less than 17%), which corresponds to lower turnover numbers for these mutants. In contrast, the catalytic efficiencies for the hydroxylation reaction toward methanol of the three mutants were more than 45% that of the wild type in spite of these mutants' lower turnover numbers. Turnover numbers and catalytic efficiencies of Arg137Leu and Lys401Glu mutants toward both substrates were comparable to those of the wild type. The electron-transfer rate from the reductase to the heme of P450 1A2 was decreased by 30% upon addition of excess methanol, while it was not influenced by addition of excess 7-ethoxycoumarin. The turnover numbers toward both 7-ethoxycoumarin and methanol as well as the rate constant of electron transfer were decreased by 25-40% by raising the concentration of KCl from 0 to 300 mM in the reconstituted system containing 50 mM potassium phosphate buffer. The turnover numbers toward both substrates of the above-mentioned five ionic mutants caused by tert-butyl hydroperoxide in the absence of the reductase and NADPH were comparable to those of the wild type. The effect of phospholipid constituents on the catalytic activity toward 7-ethoxycoumarin of the wild type was also studied. From these findings it is suggested that (1) the electron transfer from the reductase to P450 1A2 and/or the interaction between P450 1A2 and the reductase are modulated by the substrates and thus are different between 7-ethoxycoumarin and methanol systems; (2) Lys401 of P450 1A2 is not directly involved in the interaction with the reductase; (3) the interaction between P450 1A2 and the reductase is partially ionic; and (4) mutations of ionic amino acids such as Lys99, Arg137, Lys401, Lys453, and Arg455 of P450 1A2 do not largely change the heme active site and the substrate-binding site(s).

Phenyldiazene reacts with rat liver CYP1A2 expressed in Saccharomyces cerevisiae to give a phenyl-iron complex that rearranges to a mixture (N(B):N(A):N(C):N(D) = 12:54:14:20, subscript indicates pyrrole ring) of N-phenyl-PPIX (PPIX = protoporphyrin) regioisomers. The same isomer pattern is obtained in each instance when the purified or microsomal enzyme reacts with phenyldiazene, indicating that the active site topology is not altered by removal of the protein from the membrane. Reaction of the enzyme with biphenylhydrazine gives a similar distribution of N-biphenyl-PPIX isomers, but reaction with (2-naphthyl)-hydrazine only gives the N(C) and N(D) regioisomers and a trace of the N(A) isomer of N-(2-naphthyl)-PPIX. The mutations E318D, E318A, and E318V cause relatively minor changes in the observed regioisomer ratios. In contrast, the mutations T319A, T319V, and T319S suppress formation of the N(C) and N(D) isomers of N-phenyl-PPIX. The reaction of T319A with biphenylhydrazine yields major amounts of the N(B) adduct rather than the small amounts observed with CYP1A2 and the Glu-318 mutants, but does not give the N(C) and N(D) regioisomers. Other, less dramatic, changes in the isomer ratios are also observed. The results indicate that the active site of CYP1A2 is open above all four quadrants of the heme group including, to some extent, the region above pyrrole ring B. Pyrrole ring B is completely inaccessible in most cytochrome P450 enzymes. Mutations of Glu-318 cause relatively minor changes in the active site topology, as expected if it is on the periphery of the active site, but mutations of Thr-319 open up the region above pyrrole rings A and B while constricting the region above pyrrole rings C and D. The results suggest a large active site for CYP1A2 in which the ''I-helix'' is displaced away from pyrrole ring A, as it is in cytochrome P450BM-3 but not in cytochrome P450cam.

Effects of mutations at the putative distal site of cytochrome P450 1A2 on chiral discrimination for binding (R)-(+)- and (S)-(-)-1-(1-naphthyl)ethylamine (ligand I), (R)-(-)- and (S)-(+)-1-cyclohexylethylamine (ligand II), and (R)-(+)- and (S)-(-)-1-(4-pyridyl)ethanol (ligand III) were studied by optical absorption spectra. The wild-type P450 1A2 exhibited different dissociation constants (K(d)) for the R- and S-enantiomers of these ligands. The R/S ratios of the K(d) values for ligands I and II were 5.2 and 2.9, respectively, and the S/R ratio for ligand III was 6.0. Mutations at the putative distal site, such as Glu318 Asp and Glu318Ala, remarkably enhanced the discrimination: the R/S ratio of the K(d) values for ligand I increased from 5.2 to 20-60, while the R/S ratio for ligand II decreased from 2.9 to 0.8-0.9. These remarkable changes in the R/S ratios were not observed with Glu318Asp mutation for ligand III binding, whereas affinities for both enantiomers of ligand III were markedly decreased by the Glu318Ala mutation. Mutation Thr319Ala increased the R/S ratio of the K(d) values for ligand I slightly but markedly decreased the R/S ratio of ligand II (from 2.9 to 0.8) and the S/R ratio of ligand III (from 6.0 to 1.0). Similar enhancements of the chiral discriminations were observed with the mutation Lys250Leu at another putative substrate-recognition site. Differences between the R- and S-enantiomers of the standard enthalpy and entropy of ligand III binding were changed most remarkably by the Thr319Ser mutation. From these findings, together with other spectral data, it is suggested that (1) Glu318 and Thr319 play important roles in the chiral recognition of asymmetrical axial ligands, (2) Thr319 contributes thermodynamically to the discriminations of those chiral axial ligands, and (3) Lys250 is important in the chiral recognition and may be located close to a ligand access channel and/or a substrate-recognition site of this enzyme.

Most microsomal P450s have a conserved "threonine cluster" composed of three Thrs (Thr3l9, Thr321, Thr322 for P450d) at a putative distal site. An ionic amino acid at 318 is also well conserved as Glu or Asp for most P450s. To understand the role of these conserved polar amino acids at the putative distal site in the catalytic function of microsomal P450, we studied how mutations at this site of P450d influence the activation of molecular oxygen in the reconstituted system. Catalytic activity (0.02 min-1) toward 7-ethoxycoumarin of the Glu318Ala mutant of P450d was just 6% of that (0.33 min-1) of the wild type, while those of Glu318Asp, Thr319Ala, and Thr322Ala were comparable to or even higher than that of the wild type. Consumption rates of O2 and formation rates of H2O2 of those mutants varied in accord with the catalytic activities. Especially, the efficiency (0.5%) of incorporated oxygen atom to the substrate versus produced H2O2 for the Glu318Ala mutant was much lower than that (3.7%) of the wild type, while that (58.8%) for the mutant Glu318Asp was 16-fold higher than that of the wild type. In addition, the autoxidation [Fe(II) --> Fe(III)] rate (0.074 s-1) of the Glu318Ala mutant was much lower than those (0.374-0.803 s-1) of the wild type and other mutants. Thus, we strongly suggest that Glu318 plays an important role in the catalytic function toward 7-ethoxycoumarin of microsomal P450d.

Polar amino acids in the (putative) distal site are well conserved in P450s. For example, Glu318 for P450d is well conserved as either Glu or Asp for P450s, and Thr319 for P450d is also conserved for P450s. We have studied how mutations at Glu318 and Thr319 of P450d influence the catalytic activity toward methanol associated with the activation of O2. Catalytic activities of Glu318Asp, Glu318Ala, and Thr319Ala mutants toward methanol were 60, 25, and 38%, respectively, compared with that of the wild type. O2 consumption and NADPH oxidation rates of each mutants varied corresponding to the catalytic activities. However, surprisingly, efficiency (16-40%) of incorporated O to the substrate vs. consumed O2 for the Glu318Ala and Thr319Ala mutants were higher than that (9%) of the wild type. In addition, H2O2, which is produced from uncoupling for the wild-type P450d, was not observed for reaction of the Glu318Ala and Thr319Ala mutants. It seemed that consumed O2 was partially reduced to 2 mol of H2O by 4-electron transfer from NADPH for the wild-type and Thr319Ala mutant. However, for the two Glu318 mutants, it appeared that the consumed O2 was not reduced in the same way. It was thus suggested that the conserved Glu318 and Thr319 of P450d are not essential for the activation Of O2 in the methanol oxidation. Role of the water molecule or the methanol molecule in the catalytic function was implied.

Interactions of phenyl isocyanide (PheNC) with purified engineered cytochrome P450d wild type and putative distal mutants, Glu318Asp and Glu318Ala, were studied with optical absorption spectra. The wild type and the mutant Glu318Asp were purified as the high-spin state, while the mutant Glu318Ala was purified as the oxygen-bound low-spin form. Thus, it is suggested that Glu318 is important to make the appropriate heme environment of P450d. SpeCtral dissociation constants (0.19-0.39 mM) of the ligand for the ferric mutants were lower than that (0.74 mM) of the wild type. These dissociation constants were changed by adding a substrate, 7-ethoxycoumarin. The reduced wild type-PheNC complex showed a Soret peak at 451 nm, while the reduced mutant-PheNC complexes showed two peaks at 451 and 423 nm. The 451-nm peak of the complexes decreased with the concomitant increase of a new peak at 433 nm at room temperature. Thus, it was suggested that P450d can take two conformationally different forms from the characteristic spectral features. The Soret spectral conversions which followed the first-order kinetics were analyzed by changing the temperature. The activation energy (69 kcal/mol) for the conversion for the wild type was higher than those (37-50 kcal/mol) for the mutants. The activation energy for the wild type further increased (by 55%) by adding the substrate, while those for the mutants were essentially unchanged by adding the substrate. We discuss the important role of Glu3l8 at the putative distal site of P450d in the packing or the conformational stability of the putative distal site of the P450d molecule.

The catalytic activity, 6-beta-hydroxylation, toward testosterone of engineered cytochrome P-450d was three-times increased by Thr319Ala mutation at the putative distal site, while that was abolished by mutations Ala315Ser and Thr322Ala at the same site, suggesting the important role of this region in catalytic activity toward the steroid of cytochrome P-450d.

A zinc tetraazaoctaphenylporphyrin (ZnTAP) and benzene or naphthalene ring-fused unsymmetrcal ZnTAPs are synthesized and characterized by UV-visible absorption, emission, excitation, and magnetic circular dichroism (MCD) spectroscopy together with cyclic and differential pulse voltammetry. With lowering of the molecular symmetry, both the energy differences, between the Soret and Q bands and the splitting of the Q_0-0 band increase while the quantum yield (φ_F) of the fluorescence decreases.