Bioconversion of organosilicon compounds by horse liver alcohol dehydrogenase: the role of the silicon atom in enzymatic reactions

Summary Bioconversion of three organosilicon compounds of different chain length between the silicon atom and the hydroxyl group (Me₃Si(CH₂)_nOH, n = 1–3) by horse liver alcohol dehydrogenase (HLADH, EC 1.1.1.1.) was studied. Furthermore, the effect of the silicon atom on the HLADH-catalysed reaction was examined in comparison with the corresponding carbon compounds. HLADH could catalyse the dehydrogenation of trimethylsilyeethanol (n = 2) and trimethylsilylpropanol (n = 3). Trimethylsilylethanol was a better substrate than both its carbon analogue, 3,3-dimethylbutanol, and ethanol. The improved activity of HLADH on trimethylsilylethanol could be accounted for by a higher affinity toward HLADH and a lower activation energy of the reaction by HLADH than those of the carbon counterpart. These are derived from physical properties of the silicon atom, that is, the lower electronegativity and the bigger radius than those of the carbon atom. In contrast, HLADH showed no activity on trimethylsilylmethanol (n = 1), whereas it catalysed the dehydrogenation of the carbon analogue, 2,2-dimethylpropanol, fairly well. The reason for the inactivity of HLADH in the case of trimethylsilylmethanol based on the electric effect of the silicon atom is also discussed. Offsprint requests to: A. Tanaka     

Efficient kinetic resolution of organosilicon compounds by stereoselective esterification with hydrolases in organic solvent

Stereoselective esterification of three isomers of trimethylsilylpropanol, 1-trimethylsilyl-2-propanol, 1-trimethylsilyl-1-propanol, and 2-trimethylsilyl-1-propanol, was systematically studied with five kinds of hydrolases in an organic solvent system in connection with the structure of the compounds. The hydrolases were found to be able to esterify these organosilicon compounds, even -hydroxyalkylsilanes, which are unstable under the conditions of acid-catalysed esterification, and the highly optically active organosilicon compounds were successfully prepared with the selected hydrolases. Even a primary alcohol, 2-trimethylsilyl-1-propanol, was stereoselectively esterified by lipase. Furthermore, comparative studies were made by using their carbon counterparts. The silicon atom in the substrates was found to enhance the enzyme stereoselectivity in some cases, but its effect on the substrate reactivity was dependent on the structure of the substrates. These results are discussed based on the specific characters of the silicon atom. Correspondence to: A. Tanaka     

Enantioselective dehydrogenation of β-hydroxysilanes by horse liver alcohol dehydrogenase with a novel in-situ NAD+ regeneration system

Dehydrogenation of 2-trimethylsilyl-1-propanol (1) was carried out with horse liver alcohol dehydrogenase (HLADH, EC 1.1.1.1). It was found that the hydrogenation of 1 proceeded enantioselectively with only HLADH and a catalytic amount of NAD⁺ due to in-situ NAD⁺ regeneration based on a specific property of -carbonylsilanes. That is, (+)-1 was enantioselectively dehydrogenated by HLADH to 2-trimethylsilyl-1-propanal, which was spontaneously degraded by addition of water into trimethylsilanol and n-propanal. Then, NAD⁺ was regenerated through HLADH-catalyzed reduction of n-propanal to n-propanol. On the other hand, dehydrogenation of the carbon analogue of 1 was negligible with a catalytic amount of NAD⁺, indicating that the in-situ NAD⁺ regeneration was not available without the specific property of organosilicon compounds. Other primary -hydroxysilanes having different substituents on the chiral center or on the silicon atom were also found to serve as substrates in enantioselective dehydrogenation by HLADH with this novel NAD⁺ regeneration system. Chiral recognition of HLADH toward primary alcohols is also discussed.     

底物中的硅原子对酶反应的影响

在酶工程学的研究史上,人们一方面不断地研制开发新的酶种;一方面利用固定化、酶分子改造和修饰等技术来提高酶的活性和稳定性;另一方面,则不断地开拓酶的新用途。酶催化非天然化合物的生物合成和转化（正是这一方面研究的新进展）。由于有机硅化合物在有机合成,尤其...     

Enantioselective Bioconversion of Non-Natural Compounds

Enantioselective bioconversion of organosilicon compounds was successfully carried out with hydrolases and a dehydrogenase. Substituents on silicon atom were found to affect the efficiency of the reactions. In many cases, the characteristics of silicon atom reflected the reactivity.     

Long-chained 1-mercapto-n-alkanes as potent inhibitors toward liver alcohol dehydrogenase

Long-chained 1-mercapto-n-alkanes showed potent inhibitory effects on horse liver alcohol dehydrogenase (HLADH). The inhibitory effect of the thiols was enhanced by increasing the number of the alkyl carbon atoms up to 10-12 and steeply lowered by further increase in the carbon number. The HLADH activity was almost completely inhibited in competitive manner by an equivalent concentration of 1-mercapto-n-decane or -n-dodecane to that of the subunit of the dimeric zinc enzyme; inhibition constant Ki was 0.55 nM for the former. The present study strongly suggests that the thiols interact simultaneously with at least two sites of HLADH; the primary one could be the zinc atom in the active site of the enzyme, interacting with the sulfhydryl groups, and the other a hydrophobic binding site for the their alkyl carbons.     

Immobilized horse liver alcohol dehydrogenase as an on‐line high‐performance liquid chromatographic enzyme reactor for stereoselective synthesis

Horse liver alcohol dehydrogenase (HLADH) has been non‐covalently immobilized on an immobilized artificial membrane (IAM) high‐performance liquid chromatography (HPLC) stationary phase. The resulting IAM‐HLADH retained the reductive activity of native HLADH as well as the enzyme's enantioselectivity and enantiospecificity. HLADH was also immobilized in an IAM HPLC stationary phase prepacked in a 13 × 4.1 mm ID column to create an immobilized enzyme reactor (HLADH‐IMER). The reactor was connected through a switching valve to a column containing a chiral stationary phase (CSP) based upon p‐methylphenylcarbamate derivatized cellulose (Chiralcel OJR‐CSP). The results from the combined HLADH‐IMER/CSP and chromatographic system demonstrate that the enzyme retained its activity and stereoselectivity after immobilization in the column and that the substrate and products from the enzymatic reduction could be transferred to a second column for analytical or preparative separation. The combined HLADH‐IMER/CSP system is a prototype for the preparative on‐line use of cofactor‐dependent enzymes in large‐scale chiral syntheses. Chirality 11:39–45, 1999. © 1999 Wiley‐Liss, Inc.     

Synthesis of silica particles and their application as supports for alcohol dehydrogenases and cofactor immobilizations: Conformational changes that lead to switch in enzyme stereoselectivity

FTIR, circular dichroism (CD) and fluorescence spectroscopies were used to characterize conformational changes in horse liver alcohol dehydrogenase (HLADH) and ketoreductase (KRED 117) upon physical and covalent immobilizations on silica particles (functionalized with amino, epoxy and thiol groups) of different sizes. Conformational changes for immobilized enzymes were associated with high and low frequency shifts of the amide I and II bands. CD spectra of native HLADH and KRED 117 characterized with a negative peak at 222nm indicating a α-helical structure. The disappearance of the negative peak in the CD spectra of immobilized enzymes and appearance of a positive peak at 222nm supported these observations. These findings demonstrated unfolding of folded enzymes and exposure of the amino acid residues during denaturation with a red shift in tryptophan fluorescence. The decrease in specific activities (by 60-70% in all cases) for both immobilized enzymes was correlated to those of conformational changes. Silica-attached enzyme-NADH systems were evaluated for enantioselective reduction of 1-(p-methoxyphenyl)-propan-2-one. Conformational changes enhanced the enantioselectivity of immobilized HLADH with a switch in its stereoselectivity. In the case of immobilized KRED 117, kinetic values (V(max) and K(m)) were lower than that of the free enzyme, without enhancing enzyme enantio- and stereoselectivity.     

Enantiotopic differentiation in horse-liver alcohol-dehydrogenase-catalyzed oxidoreduction studied with novel substrates having organometallic moieties

Horse-liver alcohol-dehydrogenase-catalyzed oxidation of 1,2-bis(hydroxymethyl)ferrocene (1) gave (1R)-(+)-1-formyl-2-hydroxymethylferrocene (3) (86 +/- 2% enantiomeric excess, e.e.), while the reduction of the corresponding dialdehyde 1,2-diformylferrocene (2) gave the antipode (1S)-(-)-3 (94 +/- 2% e.e.). This fact indicates that the pro-R group in both 1 and 2 was preferentially converted by the enzyme. When one of two substituents on the substrate was replaced by a methyl group or moved to the beta-site, the stereoselectivity in the reaction decreased as evidenced by the enantiomeric purity of the products (5-64% e.e.). Treatment of racemic 1-hydroxyethylferrocene (14) with horse-liver alcohol dehydrogenase (HLADH) gave optically pure (R)-(-)-14 together with acetylferrocene. The reduction of 2 with HLADH, NAD and (2H6)ethanol gave (-)-(1S,2R)-1-formyl-2-[(R)-hydroxy(2H1)methyl]ferrocene and that of 1,2-di[(2H)formyl]ferrocene with HLADH, NAD and ethanol gave (-)-(1S,2R)-1-(2H)formyl-2-[(S)-hydroxy(2H1)methyl]ferrocene. These configurations indicate that the enzymic reduction occurred on the re-face of pro-R formyl group. The re-face selectivity was also found in the enzymic reduction of (eta 6-benzaldehyde)tricarbonylchromium and its (2H)formyl analogue. Docking of 2 into the active site of HLADH was examined using computer graphics. It has been suggested that the enantioselectivity to the pro-R side in the oxidoreduction of 1 and 2 by HLADH is a natural consequence of the re-face selectivity, which is caused by a steric interaction between the ligand and the side chain of Phe-93 or the Zn complex and strengthened by an interaction between the unreactive polar alpha-substituent and the protein, probably by hydrogen bond formation.     

Pyrrolidine based chiral organocatalyst for efficient asymmetric Michael addition of cyclic ketones to β-nitrostyrenes

An efficient asymmetric Michael addition of cyclic ketones to β-nitrostyrenes using secondary diamine as an organocatalyst derived from l-proline and (R)-α-methylbenzyl amine has been described. This pyrrolidine based catalyst 1 was found to be very effective to synthesize various γ-nitrocarbonyl compounds in good yield (up to 81%) with excellent stereoselectivity (up to >99:1 dr and >99% ee).     

Chiral aggregates and asymmetric induction of the reduction of prochiral ketones

The aim of this work was to establish structure-reactivity relationships between chiral aggregates (micelles, vesicles, and "pseudo-micelles" of amphiphilic dendrimers) and asymmetric induction. In water, micelles or vesicles formed with sugar-headed surfactants gave less than 10% ee in the reduction of prochiral ketones by NaBH(4), in contrast with dendrimers bearing the same types of sugar moieties, which gave more than 50% ee under the same reaction conditions. Moreover, in the presence of neoglycodendrimers in THF we have been able to improve reduction of prochiral ketones to give very high stereoselectivity, near 100% in many cases. Comparison of these results suggests that to improve high stereoselectivity it is necessary to work with rigid, well-organized colloidal objects. Copyright 2000 Wiley-Liss, Inc.     

Evaluation of Alcaligenes eutrophus cells as an NADH regenerating catalyst in organic-aqueous two-phase system

A soluble NAD-dependent hydrogenase contained in Alcaligenes eutrophus was evaluated as a coenzyme regenerating catalyst in an organic-aqueous two-phase (predominantly organic) system. The horse-liver alcohol-dehydrogenase (HLADH) catalyzed reduction of cyclohexanone to cyclohexanol was used as a model reaction. The impact of different solvents (selected to span a large variety of principal properties) on the stability and activity of the HLADH, using substrate-driven regeneration, was studied. Solvents suitable for the HLADH were then selected for an evaluation of the hydrogenase-driven coenzyme regeneration. Hydrophobic solvents such as heptane, toluene, and 1,1,1-trichloroethane were found to be suitable for the coupled reactions catalyzed by HLADH and hydrogenase. Nonimmobilized cells, permeabilized with cetyl-trimethyl-ammonium bromide, were the most efficient preparation for the regeneration of NADH. The use of this preparation in heptane (10% water) was optimized with respect to the yield obtained in the HLADH-catalyzed reduction of cyclohexanone. Using the optimized conditions, yields of 99% cyclohexanol were obtained.     

Comparative thermostability of mesophilic and thermophilic alcohol dehydrogenases: Stability-determining roles of proline residues and loop conformations

The present study demonstrates the comparative thermal, conformational and kinetic stabilities of the three closely related enzymes; the mesophilic yeast alcohol dehydrogenase (YADH), horse liver alcohol dehydrogenase (HLADH), and the extreme-thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH). The mid-point unfolding temperatures for TBADH and HLADH were at least 10 °C and 6 °C higher, respectively, than that of YADH. When YADH was completely inactivated by thermal stress, the residual activities of HLADH and TBADH were 70% and 100%, respectively. The optimum temperature (T_opt) activities of HLADH and TBADH were at least 40 °C and 55 °C higher, respectively, than that of YADH. Due to the higher rigidity of HLADH and TBADH, the enzymatic activation energies of HLADH and TBADH were higher than that of YADH. Geometric X-ray analysis indicated a comparatively higher coil (turn and loop) percentage in TBADH and HLADH than in YADH. Pairwise alignment for TBADH/HLADH exhibited a similarity score approximately 2.5-fold greater than that of the TBADH/YADH pair. Multiple alignments made with ClustalW revealed a higher number of conserved proline residues in the two most stable enzymes (HLADH/TBADH). These extra prolines tend to occur in surface loops and are likely to be responsible for the increased stability of TBADH and HLADH, by loop rigidification.     

Readily available pyridine- and quinoline-N-oxides as new organocatalysts for the enantioselective allylation of aromatic aldehydes with allyl(trichloro)silane

The straightforward synthesis of a series of enantiomerically pure pyridine- and quinoline-N-oxides and their use as new organocatalysts for the enantioselective allylation of aromatic aldehydes with allyl(trichloro)silane is reported. The catalysts were readily assembled by combining commercially available enantiopure diamines with heterocyclic carboxylic acid N-oxides. The obtained compounds showed moderate to good chemical efficiency (up to 73% chemical yield) and satisfactory stereoselectivity (up to 50% ee). Tentative models of stereoselection were proposed to account for the stereochemical outcome of the reaction and to explain how the structural features of the catalyst control the stereoselctivity.     

Efficient Repeated Use of Alcohol Dehydrogenase with NAD + Regeneration in an Aqueous-organic Two-phase System

Bioconversion of cinnamyl alcohol to cinnamaldehyde was carried out in an aqueous-organic two-phase reaction system by the repeated use of horse liver alcohol dehydrogenase (HLADH) and NAD + with coenzyme regeneration. Both HLADH and the coenzyme were efficiently entrapped in the aqueous phase, while the substrate was supplied successively from the organic phase and the product was accumulated in the organic phase. Optimum conditions for cinnamaldehyde production in the aqueous-organic two-phase system were also examined, including substrate concentration, pH, and organic solvent type. Under suitable conditions, both HLADH and NAD + in the aqueous-organic two-phase system could be reused, and NAD + cycling numbers of 3040 were obtained after repeated operation for 40 &#117 h.     

利用完整细胞不对称合成R-苯乙醇胺的研究

Effects of various factors on asymmetric synthesis of R-phenylaminoethanol from aminoacetophenone by the whole cells of Arachnia sp. P163 producing alcohol dehydrogenase for phenylethanol amine was investigated. It found that, although the reduction was inhibited by the substrate and the product, but it has the very high stereoselectivity. The reduction was normaly carried out with 2% glucose for reproduction of coenzyme in the reaction system without oxygen. The conversion yield and ee value of the product achieved 65% and 100%, respectively.     

Efficient Repeated Use of Alcohol Dehydrogenase with NAD + Regeneration in an Aqueous-organic Two-phase System

Bioconversion of cinnamyl alcohol to cinnamaldehyde was carried out in an aqueous-organic two-phase reaction system by the repeated use of horse liver alcohol dehydrogenase (HLADH) and NAD + with coenzyme regeneration. Both HLADH and the coenzyme were efficiently entrapped in the aqueous phase, while the substrate was supplied successively from the organic phase and the product was accumulated in the organic phase. Optimum conditions for cinnamaldehyde production in the aqueous-organic two-phase system were also examined, including substrate concentration, pH, and organic solvent type. Under suitable conditions, both HLADH and NAD + in the aqueous-organic two-phase system could be reused, and NAD + cycling numbers of 3040 were obtained after repeated operation for 40 λh.     

Stereoselective oxidation of sulfides by cloned naphthalene dioxygenase

Washed-cell preparations of recombinant Escherichia coli JM109(pDTG141), engineered to express the naphthalene dioxygenase (NDO) gene from Pseudomonas sp. NCIB 9816-4, have been used to biooxidise a range of aryl alkyl-, dialkyl- and bicyclic sulfides. A series of 16 phenyl alkyl sulfides was oxidised to equivalent sulfoxides, typically with moderate to high (>90%) yield and high enantioselectivity (>85% ee), the (S)-enantiomer being the predominant product, with little if any further oxidation. The addition of some electron-donating or electron-withdrawing groups to the phenyl ring decreased yield and/or stereoselectivity of the NDO-catalysed biotransformation, whereas increasing the size of the alkyl chain (nC₃H₇, iC₃H₇ and nC₄H₉) resulted in a notable inversion in selectivity to yield (R)-series sulfoxides (>74% ee) as the predominant products. The addition of one or more methylene groups between the phenyl ring and sulfur atom resulted in notable reductions in both the yield and stereoselectivity of the (S)-predominant sulfoxidations. With the exception of cyclohexyl- and n-hexyl methyl sulfide which both gave (S)-sulfoxides with good stereoselectivity and yield, other dialkyl- and bicyclic sulfides were poor substrates for sulfoxidation by NDO. Both the close agreement with data obtained using purified NDO and the absence of stereoselective sulfoxidation in equivalent controls with the E. coli JM109 host support the contribution made by the cloned NDO carried on the pDTG141 plasmid.     

Enantioselective reduction of acetyldimethylphenylsilane by Trigonopsis variabilis (DSM 70714)

Summary Growing and resting cells of the yeast Trigonopsis variabilis (DSM 70714) can be used for the enantioselective reduction of the organosilicon compound acetyldimethylphenylsilane (1) to give optically active (R)-(1-hydroxyethyl)dimethylphenylsilane [(R)-2] in good yields. The enantiomeric purity of the isolated product was determined to be 62–86% ee depending on the substrate concentration used. Both substrate and product caused an inhibition of the reaction at concentrations higher than 0.35 and 0.5 g/l, respectively. Besides, higher substrate and product concentrations led to increased formation of the by-product 1,1,3,3-tetramethyl-1,3-diphenyldisiloxane. Considering the limiting substrate and product concentrations, it was possible to use the same biomass at least 5 times without significant loss of enzyme activity. 3-Methyl-3-phenyl-2-butanone (5) and acetyldimethylphenylgermane (7), which represent carbon and germanium analogues of 1, were also found to be accepted as substrates by Trigonopsis variabilis (DSM 70714). The reduction rates of the silicon (1) and germanium compound (7) were much higher than the transformation rate of the corresponding carbon analogue 5.     

Scalable biocatalytic synthesis of optically pure ethyl (R)-2-hydroxy-4-phenylbutyrate using a recombinant E. coli with high catalyst yield

Ethyl (R)-2-hydroxy-4-phenylbutanoate [(R)-HPBE] is a versatile and important chiral intermediate for the synthesis of angiotensin-converting enzyme (ACE) inhibitors. Recombinant E. coli strain coexpressing a novel NADPH-dependent carbonyl reductase gene iolS and glucose dehydrogenase gene gdh from Bacillus subtilis showed excellent catalytic activity in (R)-HPBE production by asymmetric reduction. IolS exhibited high stereoselectivity (>98.5% ee) toward α-ketoesters substrates, whereas fluctuant ee values (53.2–99.5%) for β-ketoesters with different halogen substitution groups. Strategies including aqueous/organic biphasic system and substrate fed-batch were adopted to improve the biocatalytic process. In a 1-L aqueous/octanol biphasic reaction system, (R)-HPBE was produced in 99.5% ee with an exceptional catalyst yield (gproduct/gcatalyst) of 31.7 via bioreduction of ethyl 2-oxo-4-phenylbutyrate (OPBE) at 330 g/L.