面包酵母催化羰基不对称还原合成手性醇的研究

以2-辛酮和4-氯乙酰乙酸乙酯(COBE)为模型底物分别考察了酵母细胞对直链甲基酮和陆羰基酯中的羰基不对称还原情况。实验发现不对称还原2-辛酮的产物主要是S型的2-辛醇,且对映体选择性很高。不对称还原COBE生成的主要是S(D)-型产物,反应COBE的转化率、光学选择性都比较高。同时发现COBE的浓度和产物对不对称还原都有一定负面的影响。     

Enzymatic hydrogen transfer reduction of α-chloro aromatic ketones catalyzed by a hyperthermophilic alcohol dehydrogenase

An alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus (PFADH) effectively catalyzed the reductions of various substituted α-chloroacetophenones to furnish the corresponding (R)-configurated α-chlorohydrins with excellent enantiomeric purity. The co-factor NADH could be recycled with d-glucose dehydrogenase/d-glucose system or in a coupled substrate approach using iso-propanol as the hydrogen donor. The hydrogen transfer mode should be more cost-effective. Thus, the PFADH-catalyzed hydrogen transfer reductions of some substrates were carried out on the preparative scale, demonstrating that this enzyme would be a valuable biocatalyst for the preparation of chiral chlorohydrins of pharmaceutical interest.     

Fusarium roseum and Aspergillus oryzae-mediated enantioselective reduction of benzils to benzoins

Aspergillus oryzae OUT5048 and Fusarium roseum OUT4019 were found to be effective biocatalysts in the reduction of benzils to optically active benzoins. Easily available symmetrical benzil derivatives were reduced to the corresponding benzoins [(S)-2-hydroxy-1,2-diphenylethanones] by A. oryzae OUT5048 with up to 94% ee and by F. roseum OUT4019 with up to 98% ee, respectively. In this work, first general method for whole-cell-mediated selective reduction of benzils to benzoins is reported. It is also shown that this method is applicable for benzils with both electron-withdrawing and electron-donating groups.     

Baker’s yeast mediated asymmetric reduction of cinnamaldehyde derivatives

The enantioselective reduction of cinnamaldehyde derivatives is an attractive strategy to prepare various optically active multifunctional molecules that can be used as chiral building blocks for the synthesis of some HIV-protease inhibitors. The asymmetric reduction with pH adjusted to 5.5 of α-substituted-cinnamaldehydes (Br, N₃) mediated by baker’s yeast (Saccharomyces cerevisiae) yielded α-substituted-3-phenyl-1-propanol in excellent enantiomeric excesses and yields.     

Transglucosylation of hydroquinone catalysed by α-glucosidase from baker's yeast

Hydroquinone α-isomaltoside and hydroquinone α-glucoside were synthesized by transglucosylation in an aqueous system with baker's yeast α-glucosidase from hydroquinone and maltose as a glucosyl donor. Only one phenolic group was glucosylated, with α-selectivity, and the nature of the reaction products was governed by the concentration of hydroquinone. The optimal conditions for synthesis of glycosides were 9 mM hydroquinone and 1.5 M maltose in a 100 mM sodium citrate/phosphate buffer at pH 5.0 and 30 °C for 20 h. Under these conditions both hydroquinone α-glycosides were obtained in nearly equimolar amounts with a total molar yield of 28% with respect to hydroquinone and a total glycoside concentration of 1 mg/mL in the reaction mixture.     

Enantiofacial selective reduction of 2-allyl-2-carboethoxy-cyclopentanone mediated by baker's yeast

Enantioselective reduction of 2-allyl-2-carboethoxy-cyclopentanone (2) was accomplished in high enantiomeric excess ( > 99%), using baker's yeast in the presence of CuO, to obtain the (+)-2-allyl-2-carboethoxy-cyclopentanol derivative (6). This methodology also provides an entry to corresponding β-keto ester (-)-(2), representing an important strategy to prepare chiral functionalized 2-oxabicyclic[3.3.0]octane and 2-oxabicyclic[4.4.0]nonane derivatives, useful synthons to access new bioactive compounds. © 1996 Wiley-Liss, Inc.     

Baker's yeast mediated reduction of β-keto esters and β-keto amides in an organic solvent system

The baker's yeast mediated reduction of four β-keto esters in petroleum ether indicated that the size of the group attached to the keto carbon affected their reactivity. Ethyl 3-phenyl-3-oxopropanoate (1), which has a phenyl group directly attached to the keto carbon, is incompletely reduced using 20 g yeast/mmol substrate, ethyl 4-phenyl-3-oxobutanoate (2), which has one methylene group between the phenyl and keto carbon, was also incompletely reduced using 20 g yeast/mmol, although the extent of reduction was about double that of (1), ethyl 5-phenyl-3-oxopentanoate (3), which has two methylene groups between the phenyl and keto carbon, is completely reduced using 10 g yeast/mmol and ethyl 3-oxobutanoate (4), which has a methyl group attached to the keto carbon shows complete reduction using only 1 g yeast/mmol. The corresponding β-keto amides are considerably less reactive than the corresponding β-keto esters with only the amides derived from ethyl 3-oxobutanoate indicating any significant reduction using 20 g yeast/mmol.     

Hydrogen transfer pathways of the asymmetric reduction of alpha,beta-unsaturated ketone mediated by baker's yeast

The hydrogen transfer mechanism of cofactor reduction and recycling processes in the yeast reduction of alpha,beta-unsaturated ketone was studied by using quantitative isotope tracing close to natural abundance measured by (2)H NMR. In the reaction, the active cofactor is NADPH. The cofactor-transferred hydride attacks the beta sp(2) carbon of the enone carbonyl while water hydrogen is transferred to the alpha position. The reductant involved in the reaction depends on the quantity of yeast. When the amount of yeast is very large, the enzymes use preferentially certain unidentified substance stored in the yeast cells rather than the added glucose as electron donor. In this case, the hydrogen transferred by the cofactor is mainly of water origin. When the yeast amount is low, the added glucose is more efficiently used by the enzymes as electron donor and its hydrogen atoms bound to C-1 and C-3 are delivered to the substrate.     

Identification of alpha-tubulin as an hsp105alpha-binding protein by the yeast two-hybrid system

Hsp105alpha is a mammalian stress protein that belongs to the HSP105/110 family. Hsp105alpha prevents stress-induced apoptosis in neuronal cells and binds to Hsp70/Hsc70 and suppresses the Hsp70 chaperone activity in vitro. In this study, to further elucidate the function of Hsp105alpha, we searched for Hsp105alpha-binding proteins by screening a mouse FM3A cell cDNA library with full-length Hsp105alpha using the yeast two-hybrid system and obtained alpha-tubulin as an Hsp105alpha-binding protein. Hsp105alpha bound directly to alpha-tubulin both in vitro and in vivo. Indirect immunofluorescence analysis with anti-Hsp105 and anti-alpha-tubulin antibodies indicated that Hsp105alpha was colocalized with microtubules. Furthermore, the disorganization of microtubules induced by heat shock was prevented in Hsp105alpha-overexpressing COS-7 cells. These findings suggested that Hsp105alpha associates with alpha-tubulin and microtubules in cells and plays a role in protection of microtubules under conditions of stress.     

Characterizing cellular systems by means of dielectric spectroscopy

The dielectric behavior of a suspension of synchronized, spherical cells has been investigated in relation to the electrical parameters of certain cell structures. In the quasistatic approximation, Poisson's equations are solved for the respective diffusive media, and the local charge distributions are derived by taking into account the continuity equations. The results describe both α and β dispersion and reduce, in the corresponding limiting cases, to previous reports. The dependence of suspension permittivity in α-and β-dispersion ranges on the diffusive effects, the conductivity, and the permittivity of cytoplasm, of membrane, and of culture medium as well as on membrane thickness is pointed out. The possibility is pointed out of characterizing cellular behavior by means of the evolution of certain electrical and morphological parameters during cell cycle progression as well the effects of different stimuli on cellular systems derived by fast dielectric spectroscopy. © 1996 Wiley-Liss, Inc.     

Reduction in post-prandial hyperglycemic excursion through alpha-glucosidase inhibition by beta-acetamido carbonyl compounds

A series of β-acetamido carbonyl compounds (S₁–S₇) were prepared using Dakin-West reaction from different substituted aldehyde and acetophenone in the presence of lanthanum triflate as a solid catalyst. All the compounds were tested for their α-glucosidase inhibitory potential against rat intestinal α-glucosidase. The most potent rat intestinal α-glucosidase inhibitors S₅ and S₇ were tested for their antihyperglycemic activity following carbohydrate tolerance test. Both the compounds displayed antihyperglycemic activity equivalent to the standard drug acarbose.     

Mutator alleles of yeast DNA polymerase zeta

The yeast REV3 gene encodes the catalytic subunit of DNA polymerase zeta (pol zeta), a B family polymerase that performs mutagenic DNA synthesis in cells. To probe pol zeta mutagenic functions, we generated six mutator alleles of REV3 with amino acid replacements for Leu979, a highly conserved residue inferred to be at the pol zeta active site. Replacing Leu979 with Gly, Val, Asn, Lys, Met or Phe resulted in yeast strains with elevated UV-induced mutant frequencies. While four of these strains had reduced survival following UV irradiation, the rev3-L979F and rev3-L979M strains had normal survival, suggesting retention of pol zeta catalytic activity. UV mutagenesis in the rev3-L979F background was increased when photoproduct bypass by pol eta was eliminated by deletion of RAD30. The rev3-L979F mutation had little to no effect on mutagenesis in an ogg1Delta background, which cannot repair 8-oxo-guanine in DNA. UV-induced can1 mutants from rev3-L979F and rad30Deltarev3-L979F strains primarily contained base substitutions and complex mutations, suggesting error-prone bypass of UV photoproducts by L979F pol zeta. Spontaneous mutation rates in rev3-L979F and rev3-L979M strains are elevated by about two-fold overall and by two- to eight-fold for C to G transversions and complex mutations, both of which are known to be generated by wild-type pol zetain vitro. These results indicate that Rev3p-Leu979 replacements reduce the fidelity of DNA synthesis by yeast pol zetain vivo. In conjunction with earlier studies, the data establish that the conserved amino acid at the active site location occupied by Leu979 is critical for the fidelity of all four yeast B family polymerases. Reduced fidelity with retention of robust polymerase activity suggests that the homologous rev3-L979F allele may be useful for analyzing pol zeta functions in mammals, where REV3 deletion is lethal.     

Kinetics of the diacetyl and 2,3-pentanedione reduction by diacetyl reductase (α-diketone reductase (NAD)) from Staphylococcus aureus

The kinetic mechanism of diacetyl and 2,3-pentanedione reduction by diacetyl reductase from Staphylococcus aureus was investigated. The shape of the primary double reciprocal plots, the product inhibition pattern, and the features of the inhibition by a substrate analogue (acetone) show that diacetyl is reduced via an Ordered Bi-Bi mechanism, and 2,3-pentanedione by an Ordered Bi-Bi or Theorell-Chance mechanism. NADH is the leading substrate in both reactions.Affinity constants for the coenzyme and the substrates and inhibition constants for NAD, acetoin, and acetone were also calculated. This enzyme has a high affinity for NADH; K_m (31–50 μM) and K_s (20–27 μM) for this compound are around one-tenth of the NADH intracellular concentration. Therefore, it must operate in vivo saturated with the coenzyme. This condition is not adequate to play the role, formerly proposed for diacetyl reductases, of regulating the equilibrium between oxidized and reduced forms of pyridine-nucleotides.     

Integration of enzyme, strain and reaction engineering to overcome limitations of baker's yeast in the asymmetric reduction of alpha-keto esters

We report on the development of a whole-cell biocatalytic system based on the popular host Saccharomyces cerevisiae that shows programmable performance and good atom economy in the reduction of alpha-keto ester substrates. The NADPH-dependent yeast reductase background was suppressed through the combined effects of overexpression of a biosynthetic NADH-active reductase (xylose reductase from Candida tenuis) to the highest possible level and the use of anaerobic reaction conditions in the presence of an ethanol co-substrate where mainly NADH is recycled. The presented multi-level engineering approach leads to significant improvements in product optical purity along with increases in the efficiency of alpha-keto ester reduction and co-substrate yield (molar ratio of formed alpha-hydroxy ester to consumed ethanol). The corresponding alpha-hydroxy esters were obtained in useful yields (>50%) with purities of > or =99.4% enantiomeric excess. The obtained co-substrate yield reached values of greater than 1.0 with acetate as the only by-product formed.     

Binding of rat α1-inhibitor-3-methylamine to the α2-macroglobulin signaling receptor induces second messengers

Binding of receptor-recognized forms of tetrameric human α₂-macroglobulin (α₂M*) to a macrophage signaling receptor induces cAMP synthesis, increases in inositol 1,4,5-triphosphate (IP₃) synthesis, and a concomitant rise in cytosolic free calcium ([Ca²⁺]_i). The α₂M* signaling receptor is coupled to a pertussis-toxin insensitive G protein. Binding of α₂M* also occurs to the low density lipoprotein receptor-related protein/α₂M receptor (LRP/α₂MR), but this binding does not induce signal transduction. Rat α₁-inhibitor-3 (α₁I₃) is a monomeric member of the α-macroglobulin/complement superfamily. Like α₂M, it can react with proteinases or methylamine which induces a conformational change causing activated α₁I₃ to bind to LRP/α₂MR. We now report that α₁I₃-methylamine binds to the macrophage α₂M* signaling receptor inducing a rapid rise in the synthesis of IP₃ with a subsequent 1.5- to 3-fold rise in [Ca²⁺]_i. α₁I₃-methylamine binding to macrophages also caused a statistically significant elevation in cAMP. Native α₁I₃, like α₂M, was unable to induce signal transduction. α₁I₃ forms a complex with α₁-microglobulin, which has a distinct conformation from α₁I₃ and is recognized by LRP/α₂MR. This complex also induces an increase in [Ca²⁺]_i comparable to the effect of α₁I₃-methylamine on macrophages. It is concluded that activation of α₁I₃ by methylamine or binding of α₁-microglobulin causes similar conformational changes in the inhibitor, exposing the receptor recognition site for the α₂M* signaling receptor, as well as for LRP/α₂MR. © 1996 Wiley-Liss, Inc.     

Stereoselective anti-Prelog reduction of ketones by whole cells of Comamonas testosteroni in a ‘substrate-coupled’ approach

Lyophilized cells of the open accessible bacterium Comamonas testosteroni DSM 1455 proved to be an excellent catalyst for the asymmetric reduction of different α-azido, α-bromo, and α-nitro ketones at elevated substrate concentrations (16 g/L) in a ‘substrate-coupled’ approach using 20% (v/v) of 2-propanol as hydrogen donor. Excellent anti-Prelog stereoselectivity was obtained, which is less common found in nature.     

Proteolytically stable peptides by incorporation of α-Tfm amino acids

A series of model peptides containing α-trifluoromethyl-substituted amino acids in five different positions relative to the predominant cleavage site of the serine protease α-chymotrypsin was synthesized by solution methods to investigate the influence of α-Tfm substitution on the proteolytic stability of peptides. Proteolysis studies demonstrated absolute stability of peptides substituted in the P₁ position and still considerable proteolytic stability for peptides substituted at the P₂ and P′₂ positions compared with the corresponding unsubstituted model peptide. Comparison with peptides containing the fluorine-free disubstituted amino acid α-aminoisobutyric acid allowed to separate electronic from steric effects. Furthermore, the absolute configuration of the α-Tfm-substituted amino acid was found to exert considerable effects on the proteolytic stability, especially in P′₁ substituted peptides. Investigations of this phenomenon using empirical force field calculations revealed that in the (S,R,S)-diasteromer the steric constraints exhibited by the α-Tfm group can be outweighed by an advantageous interaction of the fluorine atoms with the serine side chain of the enzyme. In contrast, a favourable interaction between substrate and enzyme is impossible for the (S,S,S)-diastereomer. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

15β-hydroxysteroids (Part IV). Steroids of the human perinatal period: The synthesis of 3α,15β,17α-trihydroxy-5α-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15β-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3α,15β,17α-trihydroxy-5β-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3ξ,5ξ-isomers, namely 3α,15β,17α-trihydroxy-5α-pregnan-20-one (3), 3β,15β,17α-trihydroxy-5α-pregnan-20-one (7) and 3β,15β,17α-trihydroxy-5β-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3β,15β-Diacetoxy-17α-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15β,17α-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15β-acetoxy-3β,17α-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15β-acetoxy-3β,17α-dihydroxy-5α-pregnan-20-one (13) a common intermediate for the synthesis of the 3β(and α),5α-isomers. Hydrolysis of the 15β-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15β-acetoxy-17α-hydroxy-5α-pregnan-3,20-dione (14) which on reduction with -Selectride and hydrolysis of the 15β-acetate gave 3. Finally, hydrogenation of 4 gave 15β,17α-dihydroxy-5β-pregnan-3,20-dione (10) which on reduction with -Selectride gave 8.     

Antidiabetic potential: in vitro inhibition effects of some natural phenolic compounds on α‐glycosidase and α‐amylase enzymes

α‐Glycosidase is a catalytic enzyme and it destroys the complex carbohydrates into simple absorbable sugar units. The natural phenolic compounds were tested for their antidiabetic properties as α‐glycosidase and α‐amylase inhibitors. The phenolic compounds investigated in this study have been used as antidiabetic common medicines. This paper aimed to consider their capability to inhibit α‐amylase and α‐glycosidase, two significant enzymes defined in serum glucose adjustment. These examination recorded impressive inhibition profiles with IC₅₀ values in the range of 137.36–737.23 nM against α‐amylase and 29.01–157.96 nM against α‐glycosidase.     

Enzymatic synthesis of ethyl glucoside lactate in non-aqueous system

Ethyl glucoside lactate, a novel α-hydroxy acid derivative, was synthesized by transesterification in non-aqueous phase using immobilized lipase as biocatalyst. Parameters such as solvent type, substrate concentration, reaction temperature, and enzyme concentration were investigated to optimize the lipase-catalyzed transesterification. In solvent-free system with butyl lactate as both acyl donor and solvent, a 71% conversion was achieved. In order to investigate the effect of initial water content, the reactions were carried out in the mediums treated with molecular sieves. The results showed that conversion and initial rate decreased with the increase of water content. The conversion and initial rate reached to 95% and 67.4 mM/h, respectively, by carrying out the reaction under reduced pressure, which was employed to eliminate butanol and the initial water.