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.     

Influence of the Nucleophile on the Candida antarctica Lipase B‐Catalysed Resolution of a Chiral Acyl Donor

The resolution of methyl (±)‐3‐hydroxypentanoate catalysed by Candida antarctica lipase B has been performed by using ammonia and benzyl amine as nucleophiles. In all cases, the lipase reacts faster with the R enantiomer of the ester, but when benzyl amine is used, the enantiomeric ratio is approximately three times as high as that measured for ammonia. The analysis of the molecular dynamics simulations carried out over the corresponding deacylation transition state analogues indicated specular binding modes between enantiomers that vary greatly upon the nucleophile used. For the case of ammonia, an intramolecular hydrogen bond between the β‐hydroxyl group and the protons of the nucleophile is established. However, the presence of the substituent in benzyl amine disrupts this interaction. Instead, the acyl chain binds to a more restrictive area of the protein where the higher number of contacts established with the side chains of Thr40, Gln157 and Ile189 have been identified as the reason for the higher enantioselectivity observed in the aminolysis reaction.     

N‐Octanoyldimethylglycine Trifluoroethyl Ester,an Acyl Donor Leading to Highly Enantioselective Protease‐Catalysed Kinetic Resolution of Amines

The use of N‐octanoyldimethylglycine trifluoroethyl ester as acyl donor in the kinetic resolution of aliphatic amines catalysed by proteases led to enantiomeric ratios >200 in most cases. The resolutions mediated by Protex 6L were shown to be much faster than the resolutions achieved with the most efficient commercially available serine proteases, i.e., alkaline protease, Properase 1600L, and Subtilisin     

Molecular Basis for the Enantioselective Ring Opening of β‐Lactams Catalyzed by Candida antarctica Lipase B

Lipase B from Candida antarctica (CAL‐B) catalyzes the slow, but highly enantioselective (E>200), ring‐opening alcoholysis of two bicyclic and two 4‐aryl‐substituted β‐lactams. Surprisingly, the rate of the reaction varies with the nature of the alcohols and was fastest with either enantiomer of 2‐octanol. A 0.5‐g scale reaction with 2‐octanol as the nucleophile in diisopropyl ether at 60 °C yielded the unreacted β‐lactam in 39–46% yield (maximum yield is 50%) with ≥96% ee. The product β‐amino acid esters reacted further by polymerization (not isolated or characterized) or by hydrolysis due to small amounts of water in the reaction mixture yielding β‐amino acids (7–11% yield, ≥96% ee). The favored enantiomer of all four β‐lactams had similar 3‐D orientation of substituents, as did most previously reported β‐lactams and β‐lactones in similar ring‐opening reactions. Computer modeling of the ring opening of 4‐phenylazetidin‐2‐one suggests that the reaction proceeds via an unusual substrate‐assisted transition state, where the substrate alcohol bridges between the catalytic histidine and the nitrogen of the β‐lactam. Computer modeling also suggested that the molecular basis for the high enantioselectivity is a severe steric clash between Ile189 in CAL‐B and the phenyl substituent on the slow‐reacting enantiomer of the β‐lactam.     

Candida antarctica Lipase B (CAL‐B)‐Catalyzed Carbon‐Sulfur Bond Addition and Controllable Selectivity in Organic Media

A novel enzymatic, promiscuous protocol for Candida antarctica lipase B (CAL‐B)‐catalyzed carbon‐sulfur bond addition is described. Some control experiments have been designed to demonstrate the catalytic specificity of CAL‐B. Selectivity between anti‐Markovnikov addition and Markovnikov addition was achieved in different organic media. A series of thioether‐containing ester functional groups was synthesized under the catalysis of CAL‐B at 50 °C. All the products were characterized by spectroscopic methods (IR, NMR, ESI‐MS).     

Understanding the Plasticity of the α/β Hydrolase Fold: Lid Swapping on the Candida antarctica Lipase B Results in Chimeras with Interesting Biocatalytic Properties

The best of both worlds . Long molecular dynamics (MD) simulations of Candida antarctica lipase B (CALB) confirmed the function of helix α5 as a lid structure. Replacement of the helix with corresponding lid regions from CALB homologues from Neurospora crassa and Gibberella zeae resulted in new CALB chimeras with novel biocatalytic properties. The figure shows a snapshot from the MD simulation.

     

Remarkable Activation of an Enzyme by (R)‐Pyrrolidine‐ Substituted Imidazolium Alkyl PEG Sulfate

The chiral pyrrolidine‐substituted imidazolium cetyl‐PEG10‐sulfate (D ‐ProMe) derived from D ‐proline worked as an excellent activating agent of Burkholderia cepacia lipase; it is particularly interesting that the D ‐isomer of the imidazolium salt worked better than the L ‐isomer. This suggests that the imidazolium cation group directly interacts with the enzyme protein and causes preferable modification of the reactivity.     

Dynamic Kinetic Resolution of Primary Alcohols with an Unfunctionalized Stereogenic Center in the β‐Position

Primary alcohols with an unfunctionalized stereogenic center in the β‐position undergo an enzyme‐ and metal‐catalyzed dynamic kinetic resolution (DKR). The in situ racemization of the primary alcohol, required for the DKR, takes place via: (i) ruthenium‐catalyzed dehydrogenation of the alcohol, (ii) enolization of the aldehyde formed, and (iii) ruthenium‐catalyzed readdition of hydrogen to the aldehyde. The present method widens the scope of metal‐ and enzyme‐catalyzed DKR, which has so far been limited to α‐chiral alcohol and amine derivatives.     

Fully Enzymatic Resolution of Chiral Amines: Acylation and Deacylation in the Presence of Candida antarctica Lipase B

A fully enzymatic methodology for the resolution of chiral amines has been demonstrated. Candida antarctica lipase B (CaLB)‐catalyzed acylation with N‐methyl‐ and N‐phenylglycine, as well as analogues having the general formula R¹ X CH₂CO₂R² (R¹=Me, Ph; X=O, S) afforded the corresponding enantioenriched amides, which were subsequently enzymatically hydrolyzed. Surprisingly, CaLB also proved to be the catalyst of choice for this latter step. The heteroatom in the acyl donor profoundly influences both the enzymatic acylation and deacylation; the O‐substituted reagents performed best with regard to enantioselectivity as well as reaction rate in synthesis and hydrolysis.     

Direct Asymmetric Dynamic Kinetic Resolution by Combined Lipase Catalysis and Nitroaldol (Henry) Reaction

The asymmetric synthesis of β‐nitroalkanol derivatives was simply achieved by a combined nitroaldol (Henry) reaction with lipase‐catalyzed transesterification in high yield and enantiomeric purity (up to 92% and 99% ee) through a direct one‐pot procedure.     

Mutant Lipase‐Catalyzed Kinetic Resolution of Bulky Phenyl Alkyl sec‐Alcohols: A Thermodynamic Analysis of Enantioselectivity

The size of the stereoselectivity pocket of Candida antarctica lipase B limits the range of alcohols that can be resolved with this enzyme. These steric constrains have been changed by increasing the size of the pocket by the mutation W104A. The mutated enzyme has good activity and enantioselectivity toward bulky secondary alcohols, such as 1‐phenylalkanols, with alkyl chains up to eight carbon atoms. The S enantiomer was preferred in contrast to the wild‐type enzyme, which has R selectivity. The magnitude of the enantioselectivity changes in an interesting way with the chain length of the alkyl moiety. It is governed by interplay between entropic and enthalpic contributions and substrates with long alkyl chains are resolved best with E values higher than 100. The enantioselectivity increases with temperature for the small substrates, but decreases for the long ones.     

Enantioselective Hydrolysis of d,l‐Menthyl Benzoate to L‐(–)‐Menthol by Recombinant Candida rugosa Lipase LIP1

The stereoselective synthesis of L ‐menthol is an attractive process in the flavor and fragrance industry. One promising way to obtain optically pure menthol is the enantioselective hydrolysis of menthol esters under enzymatic catalysis. We developed an effective and highly enantioselective method for the synthesis of L ‐(−)‐menthol (>99% EE) by hydrolyzing the key industrial starting compound, d, l ‐menthyl benzoate. The enzyme of choice was the lipase from Candida rugosa (CRL). While commercially available preparations of this lipase showed only minor selectivity (E=15), excellent enantiomeric purity (E>100) was achieved using the heterologously expressed isoenzyme LIP1.     

A water molecule in the stereospecificity pocket of Candida antarctica lipase B enhances enantioselectivity towards pentan-2-ol

The effect of water activity on enzyme-catalyzed enantioselective transesterification was studied by using a solid/gas reactor. The experimental results were compared with predictions from molecular modelling. The system studied was the esterification of pentan-2-ol with methylpropanoate as acyl donor and lipase B from Candida antarctica as catalyst. The data showed a pronounced water-activity effect on both reaction rate and enantioselectivity. The enantioselectivity increased from 100, at water activity close to zero, to a maximum of 320, at a water activity of 0.2. Molecular modelling revealed how a water molecule could bind in the active site and obstruct the binding of the slowly reacting enantiomer. Measurements of enantioselectivity at different water-activity values and temperatures showed that the water molecule had a high affinity for the stereospecificity pocket of the active site with a binding energy of 9 kJ mol-1, and that it lost all its degrees of rotation, corresponding to an entropic energy of 37 J mol-1 K-1.     

Epoxide Hydrolase‐Catalysed Kinetic Resolution of a Spiroepoxide,a Key Building Block of Various 11‐Heterosteroids

Two microbial epoxide hydrolases – i.e., Aspergillus niger (AnEH) and Rhodococcus erythropolis (the so‐called “Limonene EH”: LEH) were used to achieve, for the first time, the biocatalysed hydrolytic kinetic resolution (BHKR) of spiroepoxide rac‐ 1 . This compound is a strategic key building block allowing the synthesis of 11‐heterosteroids. Interestingly enough, the two enzymes exhibited opposite and therefore complementary enantioselectivity allowing us to isolate the residual (R,R)‐ 1 (from AnEH) and the residual (S,S)‐ 1 (from LEH) in nearly enantiopure forms (>98 %). Their absolute configurations were determined by X‐ray crystallography. An opposite regioselectivity of the oxirane ring opening for both enantiomers of substrate 1 , determined using H₂¹⁸O labelling and chiral GC‐MS analysis, was also observed, corresponding to an attack at the less substituted carbon atom using AnEH, and at the most substituted carbon atom using LEH. A chemical process‐improving methodology was also developed. This allowed us to obtain both enantiomers of the substrate in high enantiomeric purity (99 %) and optimised quantity. In the case of the AnEH, the use of a biphasic (water/isooctane) reaction medium allowed us to increase the global substrate concentration up to 200 g/ L. The preparation of both enantiomers of 1 clearly paves the way to the preparative scale synthesis and biochemical evaluation of the corresponding 11‐heterosteroid enantiomers.     

Organocatalysis in Enantioselective α‐Functionalization of 2‐Cyanoacetates

Recent progress in asymmetric organocatalysis has led to the development of several asymmetric transformations that employ various substrates. Among these, cyanoacetates have emerged as excellent nucleophiles in conjugate addition, alkylation, Mannich and α‐heterofunctionalization reactions. In this review we discuss the enantioselective functionalization of 2‐cyanoacetates through organocatalytic reactions.

     

Bioreduction of α‐Acetoxymethyl Enones: Proposal for an SN2′ Mechanism Catalyzed by Enereductase

(Z)‐3‐Acetoxymethyl‐4‐R‐3‐buten‐2‐ones (R=aryl, alkyl) and (Z)‐3‐methyl‐4‐R‐3‐buten‐2‐ones (R=aryl) were synthesized and submitted to reduction by the yeast Saccharomyces cerevisiae producing the (R)‐ and (S)‐4‐R‐3‐methybutan‐2‐ones, respectively. This stereochemistry control strategy was applied in the syntheses of (R)‐ and (S)‐Tropional® with moderate to high enantiomeric excesses. Other (Z)‐3‐acyloxymethyl‐4‐phenyl‐3‐buten‐2‐ones showed similar behavior to the (Z)‐3‐acetoxymethyl counterpart, and the acylated Morita–Baylis–Hillman adduct 1‐acetoxy‐2‐methylene‐1‐phenylbutan‐3‐one produced a mixture of products, with and without the acetoxy group, via three different reaction pathways. In addition to experiments employing whole cells, those in which isolated enereductases were used suggested that the main pathway through which the loss of the acetoxy group occurs during the biocatalytic cascade is an S_N2′‐type reaction, rather than formal hydrogen addition followed by acetic acid elimination. Finally, related ethyl enones were reduced enantioselectively by the yeast Candida albicans, producing both (R)‐ and (S)‐reduction products, depending on the presence of the acetoxy group in the starting material.

     

Enantiopure N‐Benzyloxycarbonyl‐β2‐amino Acid Allyl Esters from Racemic β‐Lactams by Dynamic Kinetic Resolution using Candida antarctica Lipase B

The dynamic kinetic resolution of α‐substituted racemic β‐lactams by alcoholytic ring‐opening, catalyzed by immobilized lipase B from Candida antarctica is described. With this process, a variety of racemic α‐substituted N‐Cbz‐azetidinones (Cbz=benzyloxycarbonyl) was transformed to the corresponding N‐Cbz‐protected β²‐amino acid allyl esters with high enantioselectivity (up to 99%) and high yields (up to quantitative) at room temperature.

     

A Protection Strategy Substantially Enhances Rate and Enantioselectivity in ω‐Transaminase‐Catalyzed Kinetic Resolutions

The kinetic resolution of 3‐aminopyrrolidine (3AP) and 3‐aminopiperidine (3APi) with ω‐transaminases was facilitated by the application of a protecting group concept. 1‐N‐Cbz‐protected 3‐aminopyrrolidine could be resolved with >99% ee at 50% conversion, the resolution of 1‐N‐Boc‐3‐aminopiperidine yielded 96% ee at 55% conversion. The reaction rate was up to 50‐fold higher by using protected substrates. Most importantly, enantioselectivity increased remarkably after carbamate protection compared to the unprotected substrates (86 vs. 99% ee). Surprisingly, benzyl protection of 3AP had no influence on enantioselectivity. A possible explanation for this observation could be the different flexibility of the benzyl‐ or carbamate‐protected 3AP as confirmed by NMR spectroscopy.     

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.