Biotransformation of l‐menthol by soil‐borne plant pathogenic fungi (Rhizoctonia solani)

The biotransformation of l‐menthol was investigated by using nine isolates of Rhizoctonia solani (AG‐1‐IA Rs24, Joichi‐2, RRG97‐1; AG‐1‐IB TR22, R147, 110.4; AG‐1‐IC F‐1, F‐4 and P‐1) as a biocatalyst. In the cases of Rhizoctonia solani F‐1, F‐4 and P‐1, almost all of the substrate was consumed in 3 days and the major metabolite increased rapidly for the first of 3 days incubation. The structure of the major metabolite was elucidated on the basis of its spectral data. The major metabolite was determined to be (?)‐(1S,3R,4S,6S)‐6‐hydroxymenthol which indicated that l‐menthol was hydroxylated at the C‐6 position. From the main component analysis, the nine isolates of Rhizoctonia solani were divided into two groups based on their ability to transform l‐menthol to (?)‐(1S,3R,4S,6S)‐6‐hydroxymenthol. This is the first report on the biotransformation of l‐menthol by Rhizoctonia solani. © 2001 Society of Chemical Industry     

Biotransformation of three aromadendrane‐type sesquiterpenoids by Aspergillus wentii

BACKGROUND: The biotransformation of sesquiterpenoids, which are a large class of naturally occurring compounds, using microorganisms as a biocatalyst to produce useful novel organic compounds was investigated. The biotransformation of sesquiterpenoids, (+)‐aromadendrene ( 1 ), (−)‐alloaromadendrene ( 2 ) and (+)‐ledene ( 3 ) has been investigated using Aspergillus wentii as a biocatalyst. Results: Compound 1 was converted to (−)‐(10S,11S)‐10,13,14‐trihydroxyaromadendrane ( 4 ). Compound 2 was converted to (+)‐(1S,11S)‐1,13‐dihydroxyaromadendrene ( 5 ) and (−)‐5,11‐epoxycadin‐1(10)‐en‐14‐ol ( 6 ). Compound 3 was converted to compound 6 , (+)‐(10R,11S)‐10,13‐dihydroxyaromadendr‐1‐ene ( 7 ) and (+)‐(10S,11S)‐10,13‐dihydroxyaromadendr‐1‐ene ( 8 ). The structure of the metabolic products has been elucidated on the basis of their spectral data. CONCLUSION: Compound 1 gave only one product that was hydroxylated at C‐10, C‐13 and C‐14. By contrast, compounds 2 and 3 gave a number of products, one of which was common. The differences in oxidation of 1–3 are due to the configuration of the C‐1 position. Compounds 4–8 were new compounds. Copyright © 2008 Society of Chemical Industry     

Biological stereoselective reduction of 2,6‐ and 3,5‐dimethylcyclohexanones by Glomerella cingulata

The microbial transformations of 2,6‐ and 3,5‐dimethylcyclohexanone were investigated using the plant pathogenic fungus, Glomerella cingulata. With this organism 2,6‐ and 3,5‐dimethylcyclohexanone gave the corresponding 2,6‐ and 3,5‐dimethylcyclohexanol. The metabolites from 2,6‐dimethylcyclohexanone indicated enantioselective reduction by specific optical rotation of the products. The enantiomeric excesses of the microbiological reduction products were determined by ¹H‐NMR spectra of (+)‐MTPA‐esters of the alcohols produced. The reduction of 2,6‐dimethylcyclohexanone was stereospecific, with the (2R,6R)‐ketone being converted to the corresponding (2R,6R)‐(−)‐2,6‐dimethylcyclohexanol; absolute configuration, 70% ee. On the other hand, 3,5‐dimethylcyclohexanone gave the (1α,3α,5α)‐3,5‐dimethylcyclohexanol (74%) and (1α,3β,5β)‐3,5‐dimethylcyclohexanol (26%). © 1999 Society of Chemical Industry     

Approaches to the Preparation of Enantiomerically Pure (2R,2′R)‐(+)‐threo‐Methylphenidate Hydrochloride

Various approaches to the preparation of enantiomerically pure (2R,2′R)‐(+)‐threo‐methylphenidate hydrochloride ( 1 ) are reviewed. These approaches include synthesis using enantiomerically pure precursors obtained by resolution, classical and enzyme‐based resolution approaches, enantioselective synthesis approaches, and approaches based on enantioselective synthesis of (2S,2′R)‐erythro‐methylphenidate followed by epimerization at the 2‐position. 1 Introduction 2 Methods for the Enhancement of Enantiomeric Purity of 1 3 Approaches Using Enantiomerically Pure Precursors Obtained by Resolution 4 Classical Resolution Approaches 4.1 Resolution of Amide and Acid Derivatives 4.2 Resolution of (±)‐threo‐Methylphenidate 5 Enzyme‐Based Resolution Approaches 6 Enantioselective Synthesis Approaches 7 Approaches Based on Enantioselective Synthesis of (2S,2′R)‐erythro‐Methylphenidate and Epimerization 8 Conclusions     

Efficient separation of (R)‐(‐)‐mandelic acid biosynthesized from (R,S)‐mandelonitrile by nitrilase using ion‐exchange process

BACKGROUND: (R)‐(‐)‐Mandelic acid (R‐MA) is an important intermediate and chiral regent with broad uses. An efficient method for the separation of R‐MA from the bioreaction mixture with high yield is of great importance, thus, the main objective of this work is to investigate the recovery of R‐MA using an ion‐exchange process. RESULTS: The equilibrium isotherms for the separation of R‐MA by resin HZ202 were obtained in the pH range 5.0–9.0 and temperature range 25–35 °C. The equilibrium data are well fitted by the Langmuir isotherm. Batch kinetic experiments showed that the mobility of R‐MA^? in solution was rapid and the R‐MA^?/OH^? ion‐exchange process reached equilibrium after about 60 min. Adsorption kinetics were analyzed by a linear driving force mass‐transfer model, yielding good prediction of the kinetic behavior. In fixed bed column experiments, the breakthrough curves of R‐MA from the solution on resin HZ202 were determined at different flow rates and R‐MA was eluted with different concentrations of HCl. A favorable breakthrough curve and optimal eluant concentration were obtained. The results were used for the separation of R‐MA biosynthesized from (R,S)‐mandelonitrile with nitrilase, and separation was successfully achieved with above 90% recovery yield. CONCLUSION: Resin HZ202 presents favorable behavior for the recovery of R‐MA, in terms of capacity, kinetics, affinity, and susceptibility to regeneration. The results of this study provide an efficient method for R‐MA recovery from bioreaction mixture and could potentially be used in industry. Copyright © 2010 Society of Chemical Industry     

Chemoenzymatic Preparation of Enantiopure Homoadamantyl β‐Amino Acid and β‐Lactam Derivatives

Racemic cis‐10‐azatetracyclo[7.2.0.1^2,6.1^4,8]tridecan‐11‐one was prepared from homoadamant‐4‐ene by chlorosulfonyl isocyanate addition. The transformation of the β‐lactam to the corresponding β‐amino ester followed by Candida antarctica lipase A‐catalyzed enantioselective (E>>200) N‐acylation with 2,2,2‐trifluoroethyl butanoate afforded methyl (1R,4R,5S,8S)‐5‐aminotricyclo[4.3.1.1^3,8]undecane‐4‐carboxylate and the (1S,4S,5R,8R)‐butanamide with>99% ee at 50% conversion. Alternatively, transformation of the β‐lactam to the corresponding N‐hydroxymethyl‐β‐lactam and the following Pseudomonas cepacia (currently Burkholderia cepacia) lipase‐catalyzed enantioseletive O‐acylation provided the (1S,4S,6R,9R)‐alcohol (ee=87%) and the corresponding (1R,4R,6S,9S)‐butanoate (ee>99%). In the latter method, competition for the enzyme between the (1R,4R,6S,9S)‐butanoate, 2,2,2‐trifluoroethyl butanoate and the hydrolysis product, butanoic acid, tended to stop the reaction at about 45% conversion and finally gave racemization in the (1S,4S,6R,9R)‐alcohol with time.     

Cerebrosides from a Far‐Eastern Glass Sponge Aulosaccus sp.

Nine new cerebrosides 1a–d , 2a , 2b , 3a–c were found in the extract of a Far‐Eastern glass sponge Aulosaccus sp. (class Hexactinellida). These β‐d ‐glucopyranosyl‐(1 → 1)‐ceramides contain sphingoid bases (2S,3S,4R,11Z)‐2‐aminoeicos‐11‐ene‐1,3,4‐triol (in 1a – d ), (2S,3S,4R,13Z)‐2‐aminoeicos‐13‐ene‐1,3,4‐triol (in 2a , b ) and (2S,3S,4R,13S*,14R*)‐2‐amino‐13,14‐methylene‐eicosane‐1,3,4‐triol (in 3a – c ), which are N‐acylated by (2R,15Z)‐2‐hydroxydocos‐15‐enoic (in 1a , 2a , 3a ), (2R,16Z)‐2‐hydroxytricos‐16‐enoic (in 1b , 2b , 3b ), (2R,17Z)‐2‐hydroxytetracos‐17‐enoic (in 1d ) and (2R)‐2‐hydroxydocosanoic (in 1c , 3c ) acids. The monoenoic and cyclopropane‐containing sphingoid bases of compounds 1a–d , 2a , 2b , 3a–c have not been found previously in any sphingolipids. The structures of the cerebrosides were elucidated on the basis of ¹H‐, ¹³C‐NMR spectroscopy, mass spectrometry, optical rotation data and chemical transformations. A simplified method for the assignment of the absolute configuration of 2‐hydroxy fatty acids by GC analysis of their (2R)‐ and (2S)‐oct‐2‐yl esters was proposed.     

Yeast‐mediated enantioselective synthesis of chiral R‐(+)‐ and S‐(−)‐1‐phenyl‐1‐butanol from prochiral phenyl n‐propyl ketone in hexane–water biphasic culture

BACKGROUND: The hydrophobic phenyl n‐propyl ketone was used as a model compound to examine alcohol dehydrogenase activity in Saccharomyces cerevisiae mediated cell culture. Parameters such as pH, hexane‐to‐water volume percentage, and the amount of cofactor Zn²⁺ ion for either cell growth or reduction were studied to see their effect on the enantioselectivity toward the product R‐(+)‐ or S‐(?)‐1‐phenyl‐1‐butanol. RESULTS: The pH for cell growth in aqueous culture was 7.0, while the pH for reduction in the aqueous portion of the biphasic culture was 5.0. Without Zn²⁺ ion the biphasic cultures of middle to high hexane‐to‐water volume percentage exhibited an R‐(+)‐1‐phenyl‐1‐butanol enantiomeric excess of 53.7% to > 99%. Without Zn²⁺ ion the biphasic cultures at low hexane‐to‐water volume percentage possessed an S‐(?)‐1‐phenyl‐1‐butanol enantiomeric excess of 14.5–46.5%. Exclusively, the enantioselectivity for biphasic cultures containing Zn²⁺ ion was an S‐(?)‐1‐phenyl‐1‐butanol enantiomeric excess of 27.5% to > 99%. Reduction mediated in aqueous culture with varied amount of Zn²⁺ ion by the yeast Candida utilis also showed an S‐(?)‐1‐phenyl‐1‐butanol enantiomeric excess of 79.2–95.4%. CONCLUSION: The enantioselectivity of S. cerevisiae mediated biphasic culture reduction of phenyl n‐propyl ketone can be manipulated through the cofactor Zn²⁺ ion and the hexane volume percentage of the biphasic culture. Copyright © 2008 Society of Chemical Industry     

Biological enantioselective reduction of methylcyclohexanones by Glomerella cingulata

Biological reduction of alkylcyclohexanones by Glomerella cingulata was studied. With this organism regioisomeric 2-, 3- or 4-methylcyclohexanone gave the corresponding cis- and trans-methylcyclohexanols. The major metabolites of (±)-2- and (±)-3-methylcyclohexanone were cis-2- and cis-3-methylcyclohexanol. On the other hand, 4-methylcyclohexanone yielded mainly the trans-4-methylcyclohexanol. In addition, the metabolites from (±)-2- and (±)-3-methylcyclohexanone indicated enantioselective reduction by specific optical rotation of the products. The enantiomeric excesses of the microbiological reduction products were determined by NMR spectra of (+)-MTPA-esters of the alcohols produced. The reduction of (±)-2-methylcyclohexanone was stereospecific, with the (2R)-ketone being converted to the corresponding (+)-cis-2-methylcyclohexanol (1S-2R); absolute configuration, 92% e.e. On the other hand, the enantiomeric excess of the major metabolite of (±)-3-methylcyclohexanone was (−)-cis-3-methylcyclohexanol (1S-3R); absolute configuration, 33% e.e.     

Enzymatic enantioselective hydrolysis of acyloxyferrocenophanes using lipases and esterases

Enantioselective hydrolysis of racemic acetate or butyrates of 1-hydroxy [3](1,1′) ferrocenophane and endo-1-hydroxy [4](1,2) ferrocenophane using lipases, pig liver esterase and horse liver esterase resulted in the formation of (R)-alcohols and (S)-esters.     

Chemical Synthesis of the Epimeric (23R)‐ and (23S)‐Fluoro Derivatives of Bile Acids via Horner–Wadsworth–Emmons Reaction

A method for the synthesis of two (23R)‐ and (23S)‐epimeric pairs of 23‐fluoro‐3α,7α,12α‐trihydroxy‐5β‐cholan‐24‐oic acid and 23‐fluoro‐3α,7α‐dihydroxy‐5β‐cholan‐24‐oic acid is described. The key intermediates, 23,24‐dinor‐22‐aldehyde peracetates were prepared from cholic and chenodeoxycholic acids via the 24‐nor‐22‐ene, 24‐nor‐22ξ,23‐epoxy, and 23,24‐dinor‐22‐aldehyde derivatives. The Horner–Wadsworth–Emmons reaction of the 23,24‐dinor‐22‐aldehydes using triethyl 2‐fluoro‐2‐phosphonoacetate in the presence of LiCl and 1,8‐diazabicyclo[5,4,0]undec‐7‐ene (DBU), and subsequent hydrogenation of the resulting 23ξ‐fluoro‐22‐ene ethyl esters, followed by hydrolysis, gave a mixture of the epimeric (23R)‐ and (23S)‐fluorinated bile acids which were resolved efficiently by preparative RP‐HPLC. The stereochemical configuration of the fluorine atom at C‐23 in the newly synthesized compounds was confirmed directly by the X‐ray crystallographic data. The ¹H and ¹³C NMR spectral differences between the (23R)‐ and (23S)‐epimers were also discussed.     

Lower critical solution temperatures of thermo‐responsive poly(N‐isopropylacrylamide) copolymers with racemate or single enantiomer groups

BACKGROUND: Thermo‐responsive copolymers with racemate or single enantiomer groups are attracting increasing attention due to their fascinating functional properties and potential applications. However, there is a lack of systematic information about the lower critical solution temperature (LCST) of poly(N‐isopropylacrylamide)‐based thermo‐responsive chiral recognition systems. In this study, a series of thermo‐responsive chiral recognition copolymers, poly[(N‐isopropylacrylamide)‐co‐(N‐(S)‐sec‐butylacrylamide)] (PN‐S‐B) and poly[(N‐isopropylacrylamide)‐co‐(N‐(R,S)‐sec‐butylacrylamide)] (PN‐R,S‐B), with different molar compositions, were prepared. The effects of heating and cooling processes, optical activity and amount of chiral recognition groups in the copolymers on the LCSTs of the prepared copolymers were systematically studied. RESULTS: LCST hysteresis phenomena are found in the phase transition processes of PN‐S‐B and PN‐R,S‐B copolymers in a heating and cooling cycle. The LCSTs of PN‐S‐B and PN‐R,S‐B during the heating process are higher than those during the cooling process. With similar molar ratios of N‐isopropylacrylamide groups in the copolymers, the LCST of the copolymer containing a single enantiomer (PN‐S‐B) is lower than that of the copolymer containing racemate (PN‐R,S‐B) due to the steric structural difference. The LCSTs of PN‐R,S‐B copolymers are in inverse proportion to the molar contents of the hydrophobic R,S‐B moieties in these copolymers. CONCLUSION: The results provide valuable guidance for designing and fabricating thermo‐responsive chiral recognition systems with desired LCSTs. Copyright © 2008 Society of Chemical Industry     

Biotransformation of (+)‐ and (−)‐bornyl acetate using the plant parasitic fungus Glomerella cingulata as a biocatalyst

The microbial transformations of (+)‐ and (?)‐bornyl acetate were investigated using the plant parasitic fungus, Glomerella cingulata. As a result, (+)‐ and (?)‐bornyl acetate were converted to (+)‐ and (?)‐5‐exo‐hydroxybornyl acetate, (+)‐ and (?)‐5‐oxobornyl acetate and (+)‐ and (?)‐borneol respectively. The structures of the metabolic products were determined by spectroscopic data. © 2001 Society of Chemical Industry     

Asymmetric Sharpless epoxidation of 13S‐hydroxy‐ 9Z, 11E‐octadecadienoic acid (13S‐HODE)

The asymmetric Sharpless epoxidation of methyl 13S‐hydroxy‐9Z, 11E‐octadeca‐dienoate (13S‐HODE, 1 ) with tert‐butyl hydroperoxide (TBHP) catalysed by titanium tetraisopropoxide {Ti(ⁱOPr)₄} in the presence of L(+)‐diisopropyl tartrate (L‐DIPT) gave methyl 13S‐hydroxy‐11S, 12S‐epoxy‐9Z‐octadecenoate 2 (erythro isomer) in 84% diastereomeric excess (de). The epoxidation of 1 with TBHP catalysed by Ti(ⁱOPr)₄ in the presence of D(‐)‐DIPT yielded methyl 13S‐hydroxy‐11RR12R‐epoxy‐9Z‐octadecenoate (threo isomer) 3 in 76% de.     

Experimental and Computation Studies on Candida antarctica Lipase B‐Catalyzed Enantioselective Alcoholysis of 4‐Bromomethyl‐β‐lactone Leading to Enantiopure 4‐Bromo‐3‐hydroxybutanoate

Both enantiomers of optically pure 4‐bromo‐3‐hydroxybutanoate, which is an important chiral building block in the syntheses of various biologically active compounds including statins, were synthesized from rac‐4‐bromomethyl‐β‐lactone through kinetic resolution. Candida antarctica lipase B (CAL‐B) enantioselectively catalyzes the ring opening of the β‐lactone with ethanol to yield ethyl (R)‐4‐bromo‐3‐hydroxybutanoate with high enantioselectivity (E>200). The unreacted (S)‐4‐bromomethyl‐β‐lactone was converted to ethyl (S)‐4‐bromo‐3‐hydroxybutanoate (>99% ee), which can be further transformed to ethyl (R)‐4‐cyano‐3‐hydroxybutanoate, through an acid‐catalyzed ring opening in ethanol. Molecular modeling revealed that the stereocenter of the fast‐reacting enantiomer, (R)‐bromomethyl‐β‐lactone, is ∼2 Å from the reacting carbonyl carbon. In addition, the slow‐reacting enantiomer, (S)‐4‐bromomethyl‐β‐lactone, encounters steric hindrance between the bromo substituent and the side chain of the Leu278 residue, while the fast‐reacting enantiomer does not have any steric clash.     

Enzymatic Synthesis of Chiral Phenylalanine Derivatives by a Dynamic Kinetic Resolution of Corresponding Amide and Nitrile Substrates with a Multi‐Enzyme System

Mutant α‐amino‐ε‐caprolactam (ACL) racemase (L19V/L78T) from Achromobacter obae with improved substrate specificity toward phenylalaninamide was obtained by directed evolution. The mutant ACL racemase and thermostable mutant D ‐amino acid amidase (DaaA) from Ochrobactrum anthropi SV3 co‐expressed in Escherichia coli (pACLmut/pDBFB40) were utilized for synthesis of (R)‐phenylalanine and non‐natural (R)‐phenylalanine derivatives (4‐OH, 4‐F, 3‐F, and 2‐F‐Phe) by dynamic kinetic resolution (DKR). Recombinant E. coli with DaaA and mutant ACL racemase genes catalyzed the synthesis of (R)‐phenylalanine with 84% yield and 99% ee from (RS)‐phenylalaninamide (400 mM) in 22 h. (R)‐Tyrosine and 4‐fluoro‐(R)‐phenylalanine were also efficiently synthesized from the corresponding amide compounds. We also co‐expresed two genes encoding mutant ACL racemase and L ‐amino acid amidase from Brevundimonas diminuta in E. coli and performed the efficient production of various (S)‐phenylalanine derivatives. Moreover, 2‐aminophenylpropionitrile was converted to (R)‐phenylalanine by DKR using a combination of the non‐stereoselective nitrile hydratase from recombinamt E. coli and mutant ACL racemase and DaaA from E. coli encoding mutant ACL racemase and DaaA genes.     

Blends of bacterial poly[(R)‐3‐hydroxybutyrate] with oligo[(R,S)‐3‐hydroxybutyrate]‐diol

The miscibility, melting and crystallization behaviour of poly[(R)‐3‐hydroxybutyrate], PHB, and oligo[(R,S)‐3‐hydroxybutyrate]‐diol, oligo‐HB, blends have been investigated by differential scanning calorimetry: thermograms of blends containing up to 60 wt% oligo‐HB showed behaviour characteristic of single‐phase amorphous glasses with a composition dependent glass transition, T_g, and a depression in the equilibrium melting temperature of PHB. The negative value of the interaction parameter, determined from the equilibrium melting depression, confirms miscibility between blend components. In parallel studies, glass transition relaxations of different melt‐crystallized polymer blends containing 0–20 wt% oligo‐HB were dielectrically investigated between ?70 °C and 120 °C in the 100 Hz to 50 kHz range. The results revealed the existence of a single α‐relaxation process for blends, indicating the miscibility between amorphous fractions of PHB and oligo‐HB. © 2002 Society of Chemical Industry     

Design of gem‐Difluoro‐bis‐Tetrahydrofuran as P2 Ligand for HIV‐1 Protease Inhibitors to Improve Brain Penetration: Synthesis,X‐ray Studies,and Biological Evaluation

The structure‐based design, synthesis, biological evaluation, and X‐ray structural studies of fluorine‐containing HIV‐1 protease inhibitors are described. The synthesis of both enantiomers of the gem‐difluoro‐bis‐THF ligands was carried out in a stereoselective manner using a Reformatskii–Claisen reaction as the key step. Optically active ligands were converted into protease inhibitors. Two of these inhibitors, (3R,3aS,6aS)‐4,4‐difluorohexahydrofuro[2,3‐b]furan‐3‐yl(2S,3R)‐3‐hydroxy‐4‐((N‐isobutyl‐4‐methoxyphenyl)sulfonamido)‐1‐phenylbutan‐2‐yl) carbamate ( 3 ) and (3R,3aS,6aS)‐4,4‐difluorohexahydrofuro[2,3‐b]furan‐3‐yl(2S,3R)‐3‐hydroxy‐4‐((N‐isobutyl‐4‐aminophenyl)sulfonamido)phenylbutan‐2‐yl) carbamate ( 4 ), exhibited HIV‐1 protease inhibitory K_i values in the picomolar range. Both 3 and 4 showed very potent antiviral activity, with respective EC₅₀ values of 0.8 and 3.1 nM against the laboratory strain HIV‐1_LAI. The two inhibitors exhibited better lipophilicity profiles than darunavir, and also showed much improved blood–brain barrier permeability in an in vitro model. A high‐resolution X‐ray structure of inhibitor 4 in complex with HIV‐1 protease was determined, revealing that the fluorinated ligand makes extensive interactions with the S2 subsite of HIV‐1 protease, including hydrogen bonding interactions with the protease backbone atoms. Moreover, both fluorine atoms on the bis‐THF ligand formed strong interactions with the flap Gly 48 carbonyl oxygen atom.     

Synthesis and Pharmacological Evaluation of α4β2 Nicotinic Ligands with a 3‐Fluoropyrrolidine Nucleus

Nicotinic acetylcholine receptors (nAChRs) play an important role in many central nervous system disorders such as Alzheimer’s and Parkinson’s diseases, schizophrenia, and mood disorders. The α₄β₂ subtype has emerged as an important target for the early diagnosis and amelioration of Alzheimer’s disease symptoms. Herein we report a new class of α₄β₂ receptor ligands characterized by a basic pyrrolidine nucleus, the basicity of which was properly decreased through the insertion of a fluorine atom at the 3‐position, and a pyridine ring carrying at the 3‐position substituents known to positively affect affinity and selectivity toward the α₄β₂ subtype. Derivatives 3‐(((2S,4R)‐4‐fluoropyrrolidin‐2‐yl)methoxy)‐5‐(phenylethynyl)pyridine ( 11 ) and 3‐((4‐fluorophenyl)ethynyl)‐5‐(((2S,4R)‐4‐fluoropyrrolidin‐2‐yl)methoxy)pyridine ( 12 ) were found to be the most promising ligands identified in this study, showing good affinity and selectivity for the α₄β₂ subtype and physicochemical properties predictive of a relevant central nervous system penetration.     

Regio‐ and Stereoselective Biohydroxylations with a Recombinant Escherichia coli Expressing P450pyr Monooxygenase of Sphingomonas Sp. HXN‐200

A recombinant Escherichia coli expressing P450_pyr monooxygenase of Sphingomonas sp. HXN‐200 was developed as a useful biocatalyst for regio‐ and stereoselective hydroxylations, with no side reaction and easy cell growth. The resting E. coli cells showed an activity of 4.1 U/g cdw and 9.9 U/g cdw for the hydroxylation of N‐benzylpyrrolidin‐2‐one 1 and N‐benzyloxycarbonylpyrrolidine 3 , respectively, being as active as the wide‐type strain. Biohydroxylation of N‐benzylpyrrolidin‐2‐one 1 with the resting cells gave (S)‐N‐benzyl‐4‐hydroxypyrrolidin‐2‐one 2 in >99% ee and 10.8 mM, a 2.6 times increase of product concentration in comparison with the wild‐type strain. Biohydroxylation of N‐tert‐butoxycarbonylpiperidin‐2‐one 5 , N‐benzylpiperidine 7 and N‐tert‐butoxycarbonylazetidine 9 with the E. coli cells afforded the corresponding 4‐hydroxypiperidin‐2‐one 6 , 4‐hydroxypiperidine 8 , and 3‐hydroxyazetidine 10 in 14 mM, 17 mM, and 21 mM, respectively. Moreover, hydroxylation of (−)‐β‐pinene 11 with the recombinant E. coli cells showed excellent regio‐ and stereoselectivity and gave (1R)‐trans‐pinocarveol 12 in 82% yield and 4.1 mM, which is over 200 times higher than that obtained with the best biocatalytic system known thus far. The recombinant strain was also able to hydroxylate other types of substrates with unique selectivity: biohydroxylation of norbornane 13 gave exo‐norbornaeol 14 , with exo/endo selectivity of 95%; tetralin 15 and 6‐methoxytetralin 17 were hydroxylated at the non‐activated 2‐position, for the first time, with regioselectivities of 83–84%.