Extended Scope and Understanding of Zinc-Dependent Alcohol Dehydrogenases for Reduction of Cyclic α-Diketones

Alcohol dehydrogenases (ADH) are important tools for generating chiral α-hydroxyketones. Previously, only the ADH of Thauera aromatica was known to convert cyclic α-diketones with appropriate preference. Here, we extend the spectrum of suitable enzymes by three alcohol dehydrogenases from Citrifermentans bemidjiense (CibADH), Deferrisoma camini (DecADH), and Thauera phenylacetica (ThpADH). Of these, DecADH is characterized by very high thermostability; CibADH and ThpADH convert α-halogenated cyclohexanones with increased activity. Otherwise, however, the substrate spectrum of all four ADHs is highly conserved. Structural considerations led to the conclusion that conversion of diketones requires not only the expansion of the active site into a large binding pocket, but also the circumferential modification of almost all amino acid residues that form the first shell of the binding pocket. The constellation appears to be overall highly specific for the relative positioning of the carbonyl functions and the size of the C-ring.     

Secondary Alcohol Dehydrogenases from Thermoanaerobacter pseudoethanolicus and Thermoanaerobacter brockii as Robust Catalysts

Alcohol dehydrogenases (ADHs) are an important type of enzyme that have significant applications as biocatalysts. Secondary ADHs from Thermoanaerobacter pseudoethanolicus (TeSADH) and Thermoanaerobacter brockii (TbSADH) are well-known as robust catalysts. However, like most other ADHs, these enzymes suffer from their high substrate specificities (i. e., limited substrate scope), which to some extent restricts their use as biocatalysts. This minireview discusses recent efforts to expand the substrate scope and tune the enantioselectivity of TeSADH and TbSADH by using site-directed mutagenesis and directed evolution. Various examples of asymmetric synthesis of optically active alcohols using both enzymes are highlighted. Moreover, the unique thermal stability and organic solvent tolerance of these enzymes is illustrated by their concurrent inclusion with other interesting reactions to synthesize optically active alcohols and amines. For instance, TeSADH has been used in quantitative non-stereoselective oxidation of alcohols to deracemize alcohols via cyclic deracemization and in the racemization of enantiopure alcohols to accomplish a bienzymatic dynamic kinetic resolution.     

Asymmetric reduction of aliphatic and cyclic ketones with secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus: effects of substrate

The reduction of aliphatic ketones catalyzed by a secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus affords (S) -alcohols in high enantiomeric purities, when the chain has six or more carbons. With 2-pentanone, the reduction gives (S)-2-pentanol, and (R)-stereoselectivity is observed in the case of 2-butanone at 37°C. The rate of the reduction of aliphatic methyl ketones decreases about three-fold for each additional methylene increment. A temperature dependent reversal of enantiospeficity is observed in the oxidation of enantiomers of 2-butanol. A linear dependence of RTInE with temperature is observed for 2-butanol, 2-pentanol, and 2-hexanol. The cofactor analogues, thionicotinamide adenine dinucleotide phosphate (SNADP) and acetylpyridine adenine dinucleotide phosphate (APADP) gave higher enantioselectivity in the reduction of 2-butanone to (R) -2-butanol. For the reduction of cyclic ketones, SADH is enantiospecific for (S) isomers of cyclic alkyl ketones, and the transfer of hydrogen is stereoselective for the Re face to give (1S) cyclic alcohols. The facial stereospecificity of SADH for hydride transfer to NADP was determined by NMR, and it was found to be re-specific ( ‘A face’ ). In order to explain the stereoselectivity of SADH catalyzed reductions, a model is proposed that emphasizes the importance of the stability of substrate conformation and the steric interaction between substrate, enzyme and coenzyme.     

Investigation of Structural Determinants for the Substrate Specificity in the Zinc‐Dependent Alcohol Dehydrogenase CPCR2 from Candida parapsilosis

Zinc‐dependent alcohol dehydrogenases (ADHs) are a class of enzymes applied in different biocatalytic processes ranging from lab to industrial scale. However, one drawback is the limited substrate range, necessitating a whole array of different ADHs for the relevant substrate classes. In this study, we investigated structural determinants of the substrate spectrum in the zinc‐dependent ADH carbonyl reductase 2 from Candida parapsilosis (CPCR2), combining methods of mutational analysis with in silico substrate docking. Assigned active site residues were genetically randomized, and the resulting mutant libraries were screened with a selection of challenging carbonyl substrates. Three variants (C57A, W116K, and L119M) with improved activities toward different substrates were detected at neighboring positions in the active site. Thus, all possible combinations of the mutations were generated and characterized for their substrate specificity, yielding several improved variants. The most interesting were a C57A variant, with a 27‐fold increase in specific activity for 4′‐acetamidoacetophenone, and the double mutant CPCR2 B16‐(C57A, L119M), with a 45‐fold improvement in the k_cat?K_M^?1 value. The obtained variants were further investigated by in silico docking experiments. The results indicate that the mentioned residues are structural determinants of the substrate specificity of CPCR2, being major players in the definition of the active site. Comparison of these results with closely related enzymes suggests that these might even be transferred to other ADHs.     

Structures of Alcohol Dehydrogenases from Ralstonia and Sphingobium spp. Reveal the Molecular Basis for Their Recognition of ‘Bulky–Bulky’ Ketones

Alcohol dehydrogenases (ADHs) are applied in industrial synthetic chemistry for the production of optically active secondary alcohols. However, the substrate spectrum of many ADHs is narrow, and few, for example, are suitable for the reduction of prochiral ketones in which the carbonyl group is bounded by two bulky and/or hydrophobic groups; so-called ‘bulky–bulky’ ketones. Recently two ADHs, RasADH from Ralstonia sp. DSM 6428, and SyADH from Sphingobium yanoikuyae DSM 6900, have been described, which are distinguished by their ability to accept bulky–bulky ketones as substrates. In order to examine the molecular basis of the recognition of these substrates the structures of the native and NADPH complex of RasADH, and the NADPH complex of SyADH have been determined and refined to resolutions of 1.5, 2.9 and 2.5 Å, respectively. The structures reveal hydrophobic active site tunnels near the surface of the enzymes that are well-suited to the recognition of large hydrophobic substrates, as determined by modelling of the bulky–bulky substrate n-pentyl phenyl ketone. The structures also reveal the bases for NADPH specificity and (S)-stereoselectivity in each of the biocatalysts for n-pentyl phenyl ketone and related substrates.     

Aldehyde‐Catalyzed Transition Metal‐Free Dehydrative β‐Alkylation of Methyl Carbinols with Alcohols

Different to the borrowing hydrogen strategy in which alcohols were activated by transition metal‐catalyzed anaerobic dehydrogenation, the direct addition of aldehydes was found to be an effective but simpler way of alcohol activation that can lead to efficient and green aldehyde‐catalyzed transition metal‐free dehydrative C‐alkylation of methyl carbinols with alcohols. Mechanistic studies revealed that the reaction proceeds via in situ formation of ketones by Oppenauer oxidation of the methyl carbinols by external aldehydes, aldol condensation, and Meerwein–Ponndorf–Verley (MPV)‐type reduction of α,β‐unsatutated ketones by substrate alcohols, affording the useful long chain alcohols and generating aldehydes and ketones as the by‐products that will be recovered in the next condensation to finish the catalytic cycle.     

Access to Enantiopure α‐Alkyl‐β‐hydroxy Esters through Dynamic Kinetic Resolutions Employing Purified/Overexpressed Alcohol Dehydrogenases

α‐Alkyl‐β‐hydroxy esters were obtained via dynamic kinetic resolution (DKR) employing purified or crude E. coli overexpressed alcohol dehydrogenases (ADHs). ADH‐A from R. ruber, CPADH from C. parapsilosis and TesADH from T. ethanolicus afforded syn‐(2R,3S) derivatives with very high selectivities for sterically not impeded ketones (‘small‐bulky’ substrates), while ADHs from S. yanoikuyae (SyADH) and Ralstonia sp. (RasADH) could also accept bulkier keto esters (‘bulky‐bulky’ substrates). SyADH also provided preferentially syn‐(2R,3S) isomers and RasADH showed in some cases good selectivity towards the formation of anti‐(2S,3S) derivatives. With anti‐Prelog ADHs such as LBADH from L. brevis or LKADH from L. kefir, syn‐(2S,3R) alcohols were obtained with high conversions and diastereomeric excess in some cases, especially with LBADH. Furthermore, due to the thermodynamically favoured reduction of these substrates, it was possible to employ just a minimal excess of 2‐propanol to obtain the final products with quantitative conversions.     

Organocatalytic Asymmetric Aldol Reaction of Ketones with β,γ‐Unsaturated α‐Keto Esters: An Efficient Access to Chiral Tertiary Alcohol Skeletons

This update describes a highly efficient organocatalytic aldol reaction of ketones and β,γ‐unsaturated α‐keto esters for constructing the chiral tertiary alcohol motif. With the application of 9‐amino(9‐deoxy)epi‐Cinchona alkaloid and an acidic additive as catalysts, both acyclic and cyclic ketones react with β,γ‐unsaturated α‐keto esters smoothly to afford aldol adducts in good to excellent yields and asymmetric induction. This protocol offers a new pathway for the construction of adjacent chiral carbon centers and the synthesis of chiral β‐hydroxy carbonyl compounds.     

Stereoselective Biocatalyzed Reductions of Ginger Active Components Recovered from Industrial Wastes

Ginger is among the most widespread and widely consumed traditional medicinal plants around the world. Its beneficial effects, which comprise e. g. anticancer and anti-inflammatory activities as well as gastrointestinal regulatory effects, are generally attributed to a family of non-volatile compounds characterized by an arylalkyl long-chained alcohol, diol, or ketone moiety. In this work, ginger active components have been successfully recovered from industrial waste biomass of fermented ginger. Moreover, their recovery has been combined with the first systematic study of the stereoselective reduction of gingerol-like compounds by isolated alcohol dehydrogenases (ADHs), obtaining the enantioenriched sec-alcohol derivatives via a sustainable biocatalytic path in up to >99 % conversions and >99 % enantiomeric/diastereomeric excesses.     

Biocatalytic Properties and Structural Analysis of Eugenol Oxidase from Rhodococcus jostii RHA1: A Versatile Oxidative Biocatalyst

Eugenol oxidase (EUGO) from Rhodococcus jostii RHA1 had previously been shown to convert only a limited set of phenolic compounds. In this study, we have explored the biocatalytic potential of this flavoprotein oxidase, resulting in a broadened substrate scope and a deeper insight into its structural properties. In addition to the oxidation of vanillyl alcohol and the hydroxylation of eugenol, EUGO can efficiently catalyze the dehydrogenation of various phenolic ketones and the selective oxidation of a racemic secondary alcohol—4‐(1‐hydroxyethyl)‐2‐methoxyphenol. EUGO was also found to perform the kinetic resolution of a racemic secondary alcohol. Crystal structures of the enzyme in complexes with isoeugenol, coniferyl alcohol, vanillin, and benzoate have been determined. The catalytic center is a remarkable solvent‐inaccessible cavity on the si side of the flavin cofactor. Structural comparison with vanillyl alcohol oxidase from Penicillium simplicissimum highlights a few localized changes that correlate with the selectivity of EUGO for phenolic substrates bearing relatively small p‐substituents while tolerating o‐methoxy substituents.     

Screening,Molecular Cloning,and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β‐Hydroxy‐β‐trifluoromethyl Ketone

The stereoselective synthesis of chiral 1,3‐diols with the aid of biocatalysts is an attractive tool in organic chemistry. Besides the reduction of diketones, an alternative approach consists of the stereoselective reduction of β‐hydroxy ketones (aldols). Thus, we screened for an alcohol dehydrogenase (ADH) that would selectively reduce a β‐hydroxy‐β‐trifluoromethyl ketone. One potential starting material for this process is readily available by aldol addition of acetone to 2,2,2‐trifluoroacetophenone. Over 200 strains were screened, and only a few yeast strains showed stereoselective reduction activities. The enzyme responsible for the reduction of the β‐hydroxy‐β‐trifluoromethyl ketone was identified after purification and subsequent MALDI‐TOF mass spectrometric analysis. As a result, a new NADP⁺‐dependent ADH from Pichia pastoris (PPADH) was identified and confirmed to be capable of stereospecific and diastereoselective reduction of the β‐hydroxy‐β‐trifluoromethyl ketone to its corresponding 1,3‐diol. The gene encoding PPADH was cloned and heterologously expressed in Escherichia coli BL21(DE3). To determine the influence of an N‐ or C‐terminal His‐tag fusion, three different recombinant plasmids were constructed. Interestingly, the variant with the N‐terminal His‐tag showed the highest activity; consequently, this variant was purified and characterized. Kinetic parameters and the dependency of activity on pH and temperature were determined. PPADH shows a substrate preference for the reduction of linear and branched aliphatic aldehydes. Surprisingly, the enzyme shows no comparable activity towards ketones other than the β‐hydroxy‐β‐trifluoromethyl ketone.     

Polarographically Active Structural Fragments of Lignin. I. Monomeric Model Compounds

Abstract

Electrochemical properties of compounds modeling typical structural units of lignin were determined by differential pulse polarography in dimethylsulfoxide with tetrabutylammonium perchlorate as a supporting electrolyte. p-Quinonemethides; derivatives of cinnamyl aldehyde and cinnamic acid; aromatic aldehydes, ketones, diketones, and carboxylic acids; isoeugenol; and coniferyl alcohol were studied. Structural effects on the reduction potentials of lignin units were estimated. The potentials, which correspond to the first polarographic peak, varied from -0.8 (p-quinonemethide) to -2.8 V (coniferyl alcohol). The compounds containing nonconjugated carbonyl or carboxyl groups were not polarographically active. The autoprotonation reaction had a profound effect on the electrochemical reduction of compounds containing hydroxyl groups.     

Evaluation of Ligands for Ketone Reduction by Asymmetric Hydride Transfer in Water by Multi‐Substrate Screening

Various ligands for the ruthenium‐catalyzed enantioselective reduction of ketones in water have been investigated. Multi‐substrate reactions have been carried out for the comparison of various proline amides and aminoalcohol ligands. Two sets of six aromatic ketones have been selected in order to evaluate the enantiomeric excesses of all the resulting alcohols by a single chromatographic analysis. The proline amide derivative prepared from (1R,2S)‐cis‐aminoindanol revealed as the best ligand for most of the ketones used in the multi‐substrate reductions. This ligand has been employed for the enantioselective reduction of a variety of other aromatic ketones and in all cases the enantiomeric excesses were improved compared to those obtained with phenylprolineamide used in our previous work.     

Lewis acid induced additions to unsaturated fatty compounds IV: Synthesis of cyclopentenones from Friedel- Crafts acylation products of unsaturated fatty compounds with α,β-unsaturated acyl chlorides

The ethylaluminium dichloride induced Friedel- Crafts acylation of unsaturated fatty compounds such as oleic acid ( 1a ), methyl oleate ( 1b ) and 10-undecenoic acid ( 9b ) and furthermore of 1-octene ( 9a ) with α,β-unsaturated acyl chlorides e.g. crotonic acid chloride ( 2a ) and acrylic acid chloride ( 2b ) gave the corresponding allyl vinyl ketones. Nazarov cyclizations of the acylation products 3a/4a, 3b/4b, 10a and 10b afforded the alkyl substituted 2-cyclopentenones 5a/6a, 5b/6b, 11a/12a and 11b/12b . Catalytic hydrogenation of 5b/6b and 11b/12b gave the respective saturated cyclic products 7b/8b and 13b/14b as diastereomeric mixtures.     

Decoupled Roles for the Atypical,Bifurcated Binding Pocket of the ybfF Hydrolase

Serine hydrolases have diverse intracellular substrates, biological functions, and structural plasticity, and are thus important for biocatalyst design. Amongst serine hydrolases, the recently described ybfF enzyme family are promising novel biocatalysts with an unusual bifurcated substrate‐binding cleft and the ability to recognize commercially relevant substrates. We characterized in detail the substrate selectivity of a novel ybfF enzyme from Vibrio cholerae (Vc‐ybfF) by using a 21‐member library of fluorogenic ester substrates. We assigned the roles of the two substrate‐binding clefts in controlling the substrate selectivity and folded stability of Vc‐ybfF by comprehensive substitution analysis. The overall substrate preference of Vc‐ybfF was for short polar chains, but it retained significant activity with a range of cyclic and extended esters. This broad substrate specificity combined with the substitutional analysis demonstrates that the larger binding cleft controls the substrate specificity of Vc‐ybfF. Key selectivity residues (Tyr116, Arg120, Tyr209) are also located at the larger binding pocket and control the substrate specificity profile. In the structure of ybfF the narrower binding cleft contains water molecules prepositioned for hydrolysis, but based on substitution this cleft showed only minimal contribution to catalysis. Instead, the residues surrounding the narrow binding cleft and at the entrance to the binding pocket contributed significantly to the folded stability of Vc‐ybfF. The relative contributions of each cleft of the binding pocket to the catalytic activity and folded stability of Vc‐ybfF provide a valuable map for designing future biocatalysts based on the ybfF scaffold.     

Effect of aliphatic diacyl adipic dihydrazides on the crystallization of poly(lactic acid)

A series of aliphatic diacyl adipic dihydrazides (ADHs) with different alkyl moieties were synthesized by the reaction between adipic dihydrazide and acyl chloride. Then these ADHs were solution blended with PLA respectively and were evaluated as nucleating agents. Through the investigation of nonisothermal and isothermal crystallization, it was found that both the crystallization rate and the crystallinity of PLA could be enhanced by adding only 1 wt % of ADHs. Especially for ADH‐Oc (ADH‐Octyl), the crystallization rate of PLA increased about 4 times at 105°C. Optical morphology showed that and the size of PLA spherulites decreased and the nucleation density increased with the existence of ADH‐Oc. Meanwhile, the crystal structure of PLA were not discerniblly affected after the addition of ADHs as found by wide‐angle X‐ray diffraction. Thus, this study suggested these ADHs compounds are simple and potential nucleating agents to enhance crystallization ability of PLA. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42028.     

Electrocatalytic carboxylation of aromatic ketones with carbon dioxide in ionic liquid 1-butyl-3-methylimidazoliumtetrafluoborate to α-hydroxy-carboxylic acid methyl ester

A new electrochemical procedure for the electrocatalytic carboxylation of aromatic ketones with carbon dioxide in ionic liquid, 1-butyl-3-methylimidazolium tetrafluoborate (BMIMBF₄), to α-hydroxycarboxylic acid methyl ester was investigated for the first time. The electrochemical behavior of acetophenone in BMIMBF₄ was studied by cyclic voltammetry with a reduction peak at −1.9 V (vs. Ag). The electrolyses experiments were carried out in an undivided cell under mild conditions without any toxic solvents, catalysts and supporting electrolytes, followed by addition of an alkylating agent, affording the α-hydroxycarboxylic acid methyl ester in a moderate yield (62%). The results showed that the yields were strongly affected by various factors: temperature, current density, charge passed, electrode material and substrate concentration. Moreover, the ionic liquid was successfully recycled for this reaction.     

Computationally Supported Inversion of Ketoreductase Stereoselectivity

Whereas directed evolution and rational design by structural inspection are established tools for enzyme redesign, computational methods are less mature but have the potential to predict small sets of mutants with desired properties without laboratory screening of large libraries. We have explored the use of computational enzyme redesign to change the enantioselectivity of a highly thermostable alcohol dehydrogenase from Thermus thermophilus in the asymmetric reduction of ketones. The enzyme reduces acetophenone to (S)-1-phenylethanol. To invert the enantioselectivity, we used an adapted CASCO workflow which included Rosetta for enzyme design and molecular dynamics simulations for ranking. To correct for unrealistic binding modes, we used Boltzmann weighing of binding energies computed by a linear interaction energy approach. This computationally cheap method predicted four variants with inverted enantioselectivity, each with 6–8 mutations around the substrate-binding site, causing only modest reduction (2- to 7-fold) of k_cat/K_M values. Laboratory testing showed that three variants indeed had inverted enantioselectivity, producing (R)-alcohols with up to 99 % enantiomeric excess. The broad substrate range allowed reduction of acetophenone derivatives with full conversion to highly enantioenriched alcohols. The results demonstrate the use of computational methods to control ketoreductase stereoselectivity in asymmetric transformations with minimal experimental screening.     

Modeling-Assisted Design of Thermostable Benzaldehyde Lyases from Rhodococcus erythropolis for Continuous Production of α-Hydroxy Ketones

Enantiopure α-hydroxy ketones are important building blocks of active pharmaceutical ingredients (APIs), which can be produced by thiamine-diphosphate-dependent lyases, such as benzaldehyde lyase. Here we report the discovery of a novel thermostable benzaldehyde lyase from Rhodococcus erythropolis R138 (ReBAL). While the overall sequence identity to the only experimentally confirmed benzaldehyde lyase from Pseudomonas fluorescens Biovar I (PfBAL) was only 65 %, comparison of a structural model of ReBAL with the crystal structure of PfBAL revealed only four divergent amino acids in the substrate binding cavity. Based on rational design, we generated two ReBAL variants, which were characterized along with the wild-type enzyme in terms of their substrate spectrum, thermostability and biocatalytic performance in the presence of different co-solvents. We found that the new enzyme variants have a significantly higher thermostability (up to 22 °C increase in T₅₀) and a different co-solvent-dependent activity. Using the most stable variant immobilized in packed-bed reactors via the SpyCatcher/SpyTag system, (R)-benzoin was synthesized from benzaldehyde over a period of seven days with a stable space-time-yield of 9.3 mmol ⋅ L^-1 ⋅ d⁻¹. Our work expands the important class of benzaldehyde lyases and therefore contributes to the development of continuous biocatalytic processes for the production of α-hydroxy ketones and APIs.     

Ring-Opening Amino Esterification of Cyclic Ketones to Access Distal Amino Acid Derivatives

This study developed a method of synthesizing distal amino acid derivatives by the ring-opening reaction of cyclic ketones, following the amino esterification functionalization of both terminals. To achieve this goal, we performed the ring-opening reaction of cyclic ketones with an aminating reagent and alcohol under metal- and photocatalysis-free conditions in a single step. The method directly afforded distal amino acid derivatives bearing C4−C6, C8, and C12 carbon main chains, such as γ, δ, and ϵ-amino esters. The obtained amino esters were simply transformed into amino acids by hydrolysis.