Production of (S)-β-Nitro Alcohols by Enantioselective C−C Bond Cleavage with an R-Selective Hydroxynitrile Lyase

Hydroxynitrile lyase (HNL)-catalysed stereoselective synthesis of β-nitro alcohols from aldehydes and nitroalkanes is considered an efficient biocatalytic approach. However, only one S-selective HNL—Hevea brasiliensis (HbHNL)—exists that is appropriate for the synthesis of (S)-β-nitro alcohols from the corresponding aldehydes. Further, synthesis catalysed by HbHNL is limited by low specific activity and moderate yields. We have prepared a number of (S)-β-nitro alcohols, by kinetic resolution with the aid of an R-selective HNL from Arabidopsis thaliana (AtHNL). Optimization of the reaction conditions for AtHNL-catalysed stereoselective C−C bond cleavage of racemic 2-nitro-1-phenylethanol (NPE) produced (S)-NPE (together with benzaldehyde and nitromethane, largely from the R enantiomer) in up to 99 % ee and with 47 % conversion. This is the fastest HNL-catalysed route known so far for the synthesis of a series of (S)-β-nitro alcohols. This approach widens the application of AtHNL for the synthesis not only of (R)- but also of (S)-β-nitro alcohols from the appropriate substrates. Without the need for the discovery of a new enzyme, but rather by use of a retro-Henry approach, it was used to generate a number of (S)-β-nitro alcohols by taking advantage of the substrate selectivity of AtHNL.     

Asymmetric Synthesis of Optically Pure Pharmacologically Relevant Amines Employing ω‐Transaminases

Various ω‐transaminases were tested for the synthesis of enantiomerically pure amines from the corresponding ketones employing D ‐ or L ‐alanine as amino donor and lactate dehydrogenase to remove the side‐product pyruvate to shift the unfavourable reaction equilibrium to the product side. Both enantiomers, (R)‐ and (S)‐amines, could be prepared with up to 99% ee and >99% conversions within 24 h at 50 mM substrate concentration. The activity and stereoselectivity of the amination reaction depended on the ω‐transaminase and substrate employed; furthermore the co‐solvent significantly influenced both the stereoselectivity and activity of the transaminases. Best results were obtained by employing ATA‐117 to obtain the (R)‐enantiomer and ATA‐113 or ATA‐103 to access the (S)‐enantiomer with 15% v v⁻¹ DMSO.     

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.     

Influence of Reaction Conditions on Enzymatic Enantiopreference: the Curious Case of HEwT in the Synthesis of THF-Amine

Enzymatic enantiopreference is one of the key advantages of biocatalysis. While exploring the synthesis of small cyclic (chiral amines) such as 3-aminotetrahydrofuran (THF-amine), using the (S)-selective transaminase from Halomonas elongata (HEwT), inversion of the enantiopreference was observed at increasing substrate loadings. In addition, the enantiopreference could be altered by variation of the ionic strength, or of the co-solvent content in the reaction mixture. For example, using otherwise identical reaction conditions, the presence of 2 M sodium chloride gave (R)-THF-amine (14 % ee), while the addition of 2.2 M isopropyl alcohol gave the (S)-enantiomer in 30 % ee. While the underlying cause is not currently understood, it appears likely that subtle changes in the structure of the enzyme cause the shift in enantiopreference and are worth exploring further.     

Highly Enantioselective Friedel–Crafts Reaction of Indole with Alkylidenemalonates Catalyzed by Heteroarylidene Malonate‐Derived Bis(oxazoline) Copper(II) Complexes

A series of cheap and easily accessible heteroarylidenemalonate‐derived bis(oxazoline) ligands 1 and 2 were synthesized and their copper(II) complexes were applied to the catalytic Friedel–Crafts reaction between indoles and diethyl alkylidenemalonates, Excellent asymmetric enantioselectivities were afforded for the S‐enantiomer (up to >99% ee) in isobutyl alcohol, and the R‐enantiomer (up to 96.5% ee) in dichloromethane.     

Regio‐ and Enantioselective Sequential Dehalogenation of rac‐1,3‐Dibromobutane by Haloalkane Dehalogenase LinB

The hydrolytic dehalogenation of rac‐1,3‐dibromobutane catalyzed by the haloalkane dehalogenase LinB from Sphingobium japonicum UT26 proceeds in a sequential fashion: initial formation of intermediate haloalcohols followed by a second hydrolytic step to produce the final diol. Detailed investigation of the course of the reaction revealed favored nucleophilic displacement of the sec‐halogen in the first hydrolytic event with pronounced R enantioselectivity. The second hydrolysis step proceeded with a regioselectivity switch at the primary position, with preference for the S enantiomer. Because of complex competition between all eight possible reactions, intermediate haloalcohols formed with moderate to good ee ((S)‐4‐bromobutan‐2‐ol: up to 87 %). Similarly, (S)‐butane‐1,3‐diol was formed at a maximum ee of 35 % before full hydrolysis furnished the racemic diol product.     

MOP‐Type Binaphthyl Phosphite and Diamidophosphite Ligands and Their Application in Catalytic Asymmetric Transformations

Monodentate phosphite and diamidophosphite ligands have been developed based on O‐methyl‐BINOL. These chiral ligands are easy to prepare from readily accessible phosphorylating reagents – (S_a or R_a)‐2‐chlorodinaphtho[2,1‐d:1′,2′‐f][1,3,2]dioxaphosphepine and (2R,5S)‐2‐chloro‐3‐phenyl‐1,3‐diaza‐2‐phosphabicyclo[3.3.0]octane. The new ligands have demonstrated excellent enantioselectivity in the palladium‐catalysed allylic substitution reactions of (E)‐1,3‐diphenylallyl acetate with sodium p‐toluenesulfinate (up to 99 % ee), pyrrolidine (up to 97 % ee), dipropylamine (up to 95 % ee) and dimethyl malonate (up to 99 % ee). In the palladium‐catalysed deracemization of ethyl (E)‐1,3‐diphenylallyl carbonate, up to 96 % enantioselectivity has been achieved. The diamidophosphite ligands have exhibited very good enantioselectivity in the Rh‐catalysed asymmetric hydrogenation of dimethyl itaconate (up to 90 % ee).     

Hidden Specificities in Enzyme Catalysis: Structural Basis of Substrate Structure-Selectivity Relationship of a Ketoreductase

Enzymes often convert both physiological and non-physiological substrates with high stereoselectivity; yet, for some enzymes, opposite product chirality is observed. A possible explanation is the existence of hidden specificities becoming apparent when non-physiological substrates confer different substrate–enzyme interactions than the physiological substrate. To test this hypothesis, a series of α-methylated β-keto esters were converted with Tyl-KR1, a ketoreductase from polyketide synthesis in Streptomyces fradiae. The conversions of six substrates with different physicochemical properties exhibited enantioselectivities ranging from 84 % ee for R,R to 84 % ee for S,S, yet high and uniform diastereoselectivity (anti, d.r.>9:1). The exchange of a single atom, namely an oxygen ester instead of a thioester, led to almost complete loss of enantioselectivity (<5 % ee). An additional S,S-selective binding mode as a hidden specificity in Tyl-KR1 has been identified through molecular modeling and site-directed mutagenesis.     

Stereoselectivity of Four (R)‐Selective Transaminases for the Asymmetric Amination of Ketones

Four (R)‐ω‐transaminases originating from Hyphomonas neptunium (HN‐ωTA), Aspergillus terreus (AT‐ωTA) and Arthrobacter sp. (ArR‐ωTA), as well as an evolved transaminase (ArRmut11‐ωTA) were successfully employed for the amination of prochiral ketones leading to optically pure (R)‐amines. The first three transaminases displayed perfect stereoselectivity for the amination of all substrates tested (ee >99%). Furthermore, the transaminase AT‐ωTA led in most cases to better conversion than ArR‐ωTA and HN‐ωTA using D ‐alanine as amine donor. α‐Tetralone, which was the only substrate not accepted by HN‐ωTA, ArR‐ωTA, and AT‐ωTA, was successfully transformed with perfect enantioselectivity (ee >99%) into the corresponding optically pure amine employing the variant ArRmut11‐ωTA.     

Enantioselective Reduction of Citral Isomers in NCR Ene Reductase: Analysis of an Active‐Site Mutant Library

A deeper understanding of the >99 % S‐selective reduction of both isomers of citral catalyzed by NCR ene reductase was achieved by active‐site mutational studies and docking simulation. Though structurally similar, the E/Z isomers of citral showed a significantly varying selectivity response to introduced mutations. Although it was possible to invert (E)‐citral reduction enantioselectivity to ee 46 % (R) by introducing mutation W66A, for (Z)‐citral it remained ≥88 % (S) for all single‐residue variants. Residue 66 seems to act as a lever for opposite binding modes. This was underlined by a W66A‐based double‐mutant library that enhanced the (E)‐citral derived enantioselectivity to 63 % (R) and significantly lowered the S selectivity for (Z)‐citral to 44 % (S). Formation of (R)‐citronellal from an (E/Z)‐citral mixture is a desire in industrial (?)‐menthol synthesis. Our findings pave the way for a rational enzyme engineering solution.     

Enhancement of enantioselectivity and reaction rate on the synthesis of (S)‐ketoprofen hydroxyalkyl ester in organic solvents via isopropanol‐dried immobilized lipase

Lipase‐catalyzed enantioselective esterification between (R,S)‐ketoprofen and alkanediol in organic solvents was developed to produce (S)‐ketoprofen hydroxyalkyl esters. The acyl acceptor of 1,6‐hexanediol for the resolution of (R,S)‐ketoprofen yielded only the enantioselectivity (the enantiomeric ratio of initial rate for (S)‐ketoprofen to that of (R)‐ketoprofen) V_S/V_R = 8, when crude Lipase MY originating from Candida rugosa was used. However, isopropanol‐dried immobilized lipases (IPA‐dried IM‐lipase) effectively enhanced the enantioselectivity to greater than 20 in the esterification of (R,S)‐ketoprofen when 1,4‐butanediol, 1,5‐pentanediol or 1,6‐hexanediol was employed. IPA‐dried IM‐lipase and isooctane were selected to use for optimally immobilized lipase and reaction medium, respectively. The IPA‐dried IM‐lipase exhibited the highest enantioselectivity, E = 26.7, to the (S)‐enantiomer with 1,5‐pentanediol and the best enzyme activity to the (S)‐enantiomer with 1,4‐butanediol. The finding indicates that the carbon chain length of the alkanediol strongly affected the enzyme activity and enantioselectivity of lipase‐catalyzed esterification. A maximum enantioselectivity of 37 at 27 °C was generated by IPA‐dried IM‐lipase for the enantioselective esterification of racemic ketoprofen with 1,4‐butanediol. IPA‐dried IM‐lipase can effectively increase the enantioselectivity of lipase. Copyright © 2005 Society of Chemical Industry     

Hydrogen‐Bonding‐Driven Enantioselective Resolution against the Kazlauskas Rule To Afford γ‐Amino Alcohols by Candida rugosa Lipase

Most lipases resolve secondary alcohols in accordance with the “Kazlauskas rule” to give the R enantiomers. In a similar manner to other lipases, Candida rugosa lipase (CRL) exhibits R enantioselectivity towards heptan‐2‐ol, although the enantiomeric ratio (E) is low (E=1.6). However, unexpected enantioselectivity (i.e., S enantioselectivity, E=58) of CRL towards 4‐(tert‐butoxycarbonylamino)butan‐2‐ol, which has a similar chain length to heptan‐2‐ol, has been observed. To develop a deeper understanding of the molecular basis for this unusual enantioselectivity, we have conducted a series of molecular modeling and substrate engineering experiments. The results of these computational and experimental analyses indicated that a hydrogen bond between the Ser450 residue and the nitrogen atom of the carbamate group is critical to stabilize the transition state of the S enantiomer.     

Enantiomerically Pure Quinoline-Based κ-Opioid Receptor Agonists: Chemoenzymatic Synthesis and Pharmacological Evaluation

Racemic _K-opioid receptor (KOR) agonist 2-(3,4-dichlorophenyl)-1-[(4aRS,8SR,8aSR)-8-(pyrrolidin-1-yl)-3,4,4a,5,6,7,8,8a-octahydroquinolin-1(2H)-yl]ethan-1-one ((±)- 4 ) was prepared in a diastereoselective synthesis. The first key step of the synthesis was the diastereoselective hydrogenation of the silyl ether of 1,2,3,4-tetrahydroquinoin-8-ol ((±)- 9 ) to afford cis,cis-configured perhydroquinoline derivative (±)- 10 . Removal of the TBDMS protecting group led to a β-aminoalcohol that reacted with SO₂Cl₂ to form an oxathiazolidine. Nucleophilic substitution with pyrrolidine resulted in the desired cis,trans-configured perhydroquinoline upon inversion of the configuration. In order to obtain enantiomerically pure KOR agonists 4 (99.8 % ee) and ent- 4 (99.0 % ee), 1,2,3,4-tetrahydroquinolin-8-ols (R)- 8 (99.1 % ee) and (S)- 8 (98.4 % ee) were resolved by an enantioselective acetylation catalyzed by Amano lipase PS-IM. The absolute configuration was determined by CD spectroscopy. The 4aR,8S,8aS-configured enantiomer 4 showed sub-nanomolar KOR affinity (K_i=0.81 nM), which is more than 200 times higher than the KOR affinity of its enantiomer ent- 4 . In the cAMP assay and the Tango β-arrestin-2 recruitment assay, 4 behaved as a KOR agonist. Upon incubation of human macrophages, human dendritic cells, and mouse myeloid immune cells with 4 , the number of cells expressing co-stimulatory receptor CD86 and proinflammatory cytokines interleukin 6 and tumor necrosis factor α was significantly reduced; this indicates the strong anti-inflammatory activity of 4 . The anti-inflammatory effects correlated well with the KOR affinity: (4aR,8S,8aS)- 4 was slightly more potent than the racemic mixture (±)- 4 , and the distomer ent- 4 was almost inactive.     

Molecular Basis for the Stereoselective Ammoniolysis of N‐Alkyl Aziridine‐2‐Carboxylates Catalyzed by Candida antarctica Lipase B

Candida antarctica lipase B catalyzed the stereoselective ammoniolysis of N‐alkyl aziridine‐2‐carboxylates in tBuOH saturated with ammonia and yielded the (2S)‐aziridine‐2‐carboxamide and unreacted (2R)‐aziridine‐2‐carboxylate. Varying the N‐1 substituent on the aziridine ring changed the rate and stereoselectivity of the reaction. Substrates with a benzyl substituent or a (1′R)‐1‐phenylethyl substituent reacted approximately ten times faster than substrates with a (1′S)‐1‐phenylethyl substituent. Substrates with a benzyl substituent showed little stereoselectivity (E=5–7) while substrates with either a (1′R)‐ or (1′S)‐1‐phenylethyl substituent showed high stereoselectivity (D>50). Molecular modeling by using the current paradigm for enantioselectivity—binding of the slow enantiomer by an exchange‐of‐substituents orientation—could not account for the experimental results. However, modeling an umbrella‐like‐inversion orientation for the slow enantiomer could account for the experimental results. Steric hindrance between the methyl in the (1′S)‐1‐phenylethyl substituent and Thr138 and Ile189 in the acyl‐binding site likely accounts for the slow reaction. Enantioselectivity likely stems from an unfavorable interaction of the methine hydrogen with Thr40 for the slow enantiomer and from subtle differences in the orientations of the other three substituents. This success in rationalizing the enantioselectivity supports the notion that an umbrella‐like‐inversion orientation can contribute to enantioselectivity in lipases.     

Palladium(II) Complexes of C2‐Bridged Chiral Diphosphines: Application to Enantioselective Carbonyl‐Ene Reactions

(11bR,11′bR)‐4,4′‐(1,2‐Phenylene)bis[4,5‐dihydro‐3H‐dinaphtho[2,1‐c:1′,2′‐e]phosphepin] [abbreviated as (R)‐BINAPHANE], (3R,3′R,4S,4′S,11bS,11′bS)‐4,4′‐bis(1,1‐dimethylethyl)‐4,4′,5,5′‐tetrahydro‐3,3′‐bi‐3H‐dinaphtho[2,1‐c:1′,2′‐e]phosphepin [(S)‐BINAPINE], (1S,1′S,2R,2′R)‐1,1′‐bis(1,1‐dimethylethyl)‐2,2′‐biphospholane [(S,S,R,R)‐TANGPHOS] and (2R,2′R,5R,5′R)‐1,1′‐(1,2‐phenylene)bis[2,5‐bis(1‐methylethyl)phospholane] [(R,R)‐i‐Pr‐DUPHOS] are C₂‐bridged chiral diphosphines that form stable complexes with palladium(II) and platinum(II) containing a five‐membered chelate ring. The Pd(II)‐BINAPHANE catalyst displayed good to excellent enantioselectivities with ee values as high as 99.0% albeit in low yields for the carbonyl‐ene reaction between phenylglyoxal and alkenes. Its Pt(II) counterpart afforded improved yields while retaining satisfactory enantioselectivity. For the carbonyl‐ene reaction between ethyl trifluoropyruvate and alkenes, the Pd(II)‐BINAPHANE catalyst afforded both good yields and extremely high enantioselectivities with ees as high as 99.6%. A comparative study on the Pd(II) catalysts of the four C₂‐bridged chiral diphosphines revealed that Pd(II)‐BINAPHANE afforded the best enantioselectivity. The ee values derived from Pd(II)‐BINAPHANE are much higher than those derived from the other three Pd(II) catalysts. A comparison of the catalyst structures shows that the Pd(II)‐BINAPHANE catalyst is the only one that has two bulky (R)‐binaphthyl groups close to the reaction site. Hence it creates a deep chiral space that can efficiently control the reaction behavior in the carbonyl‐ene reactions resulting in excellent enantioselectivity.     

New Pathway to C2‐Symmetric Atropoisomeric Bipyridine N,N′‐Dioxides and Solvent Effect in Enantioselective Allylation of Aldehydes

The [2+2+2] cyclotrimerization of 1,7,9,15‐hexadecatetrayne with nitriles catalyzed by dicarbonylcyclopentadienylcobalt(I) opened a new pathway for the synthesis of C₂‐symmetrical bis(tetrahydroisoquinolines) that were used as starting material for the preparation of axially chiral bipyridine N,N′‐dioxides. The N,N′‐dioxides (1 mol%) were found to be highly catalytically active and enantioselective (up to 83% ee) for the asymmetric allylation of aldehydes with allyl(trichloro)silane in various solvents. In addition, a dramatic solvent effect was observed where the use of different solvents induced opposite chiralities of the product with the same enantiomer of the catalyst, e.g., 65% ee (S) in acetonitrile (MeCN) vs. 82% ee (R) in chlorobenzene.     

Enantioselective Cascade Biocatalysis for Deracemization of Racemic β-Amino Alcohols to Enantiopure (S)-β-Amino Alcohols by Employing Cyclohexylamine Oxidase and ω-Transaminase

Optically active β-amino alcohols are very useful chiral intermediates frequently used in the preparation of pharmaceutically active substances. Here, a novel cyclohexylamine oxidase (ArCHAO) was identified from the genome sequence of Arthrobacter sp. TYUT010-15 with the R-stereoselective deamination activity of β-amino alcohol. ArCHAO was cloned and successfully expressed in E. coli BL21, purified and characterized. Substrate-specific analysis revealed that ArCHAO has high activity (4.15 to 6.34 U mg⁻¹ protein) and excellent enantioselectivity toward the tested β-amino alcohols. By using purified ArCHAO, a wide range of racemic β-amino alcohols were resolved, (S)-β-amino alcohols were obtained in >99 % ee. Deracemization of racemic β-amino alcohols was conducted by ArCHAO-catalyzed enantioselective deamination and transaminase-catalyzed enantioselective amination to afford (S)-β-amino alcohols in excellent conversion (78–94 %) and enantiomeric excess (>99 %). Preparative-scale deracemization was carried out with 50 mM (6.859 g L⁻¹) racemic 2-amino-2-phenylethanol, (S)-2-amino-2-phenylethanol was obtained in 75 % isolated yield and >99 % ee.     

Enantioselective Carbonyl‐Ene Reactions of Arylglyoxals with a Chiral Palladium(II)‐BINAP Catalyst

The palladium(II)‐BINAP‐catalyzed enantioselective carbonyl‐ene reactions between ten arylglyoxals and five alkenes were systematically investigated and demonstrated good to excellent enantioselectivities with high ee values of up to 93.8 %. The results showed that both arylglyoxals and alkenes exert evident effects on the enantioselectivity. Particularly, the ortho‐methyl substituents of the substrates could increase the enantioselectivity. The achieved excellent enantioselectivities may be due to the corresponding substrate matches well fitting the chiral space created by the chiral palladium(II)‐BINAP catalyst. The ortho‐methyl substituents may improve the fitting of the substrate match to the chiral space created by the chiral catalyst, hence the enantioselectivity is improved. When using dienes (1,4‐diisopropenylbenzene and 1,3‐diisopropenylbenzene) as substrates in this reaction, only one of the two carbon‐carbon double bonds participated into the reaction affording tetrafunctional organic compounds with moderate enantioselectivities of up to 83.8 % ee. The chiral Lewis acid palladium(II) catalyst incorporating (R)‐BINAP, which is a conformationally restricted chiral ligand, is very stable in ionic liquids and could be recycled 21 times with retention of the high enantioselectivity.     

Enhancing Effect of Calix[4]arene Amide Derivatives on Lipase Performance in Enantioselective Hydrolysis of Racemic Arylpropionic Acid Methyl Esters

Calix[4]arene amide derivatives were employed as new additives within the sol-gel encapsulation of lipase from Candida rugosa (CRL) to improve its catalytic properties. Evaluation of catalytic activity of the encapsulated lipases was acheived by enantioselective hydrolysis of both racemates, Naproxen methyl ester and 2-phenoxypropionic acid methyl ester, in aqueous buffer solution/isooctane reaction system. Results show that enantioselectivity was improved by using calix[4]arene amide derivatives-based encapsulated lipases. The reaction of naproxen methyl ester resulted in 47.6% conversion (x) in 24 h with 88.9% enantiomeric excess of substrate (ee_s), analogous to an enantioselectivity (E) value of 297 (E = 137 for the encapsulated free enzyme). The conversion of 2-phenoxypropionic acid methyl ester, obtained was 48.4% with E value of 327, enantiomeric excess of substrate (ee_s) of 92% for the reaction time of 1 h (E = 211 for the encapsulated free enzyme).     

Selective Enantioseparation of Racemic Amlodipine by Biphasic Recognition Chiral Separation System

The biphasic recognition chiral extraction process was developed and applied to separate amlodipine enantiomer. The chiral extraction system contained tartaric acid derivatives in the organic phase and β-cyclodextrin derivatives in the aqueous phase. The effect of extraction equilibrium time and the influence of different types of tartaric acids, types of β-cyclodextrin derivatives, organic solvents, and buffer pH were investigated. The results indicated that hydroxypropyl-β-cyclodextrin (HP-β-CD) showed a higher recognition ability toward (S)-amlodipine than (R)-amlodipine while dibenzoyl-D-tartrate demonstrated the strongest ability among tartaric acid derivatives to bind with (R)-amlodipine. The distribution ratios for (S)-amlodipine (k_S) and (R)-amlodipine (k_R) gave optimum values at pH 5.0 of 16.54 and 0.78, respectively. Biphasic chiral recognition extraction has great significance for preparative separation of (S)-amlodipine. It can also be used to design and apply the enantioseparation process.