Emulation of racemase activity by employing a pair of stereocomplementary biocatalysts

Racemization is the key step to turn a kinetic resolution process into dynamic resolution. A general strategy for racemization under mild reaction conditions by employing stereoselective biocatalysts is presented, in which racemization is achieved by employing a pair of stereocomplementary biocatalysts that reversibly interconvert an sp3 to a sp2 center. The formal interconversion of the enantiomers proceeds via a prochiral sp2 intermediate the formation of which is catalyzed either by two stereocomplementary enzymes or by a single enzyme with low stereoselectivity. By choosing appropriate reaction conditions, the amount of the prochiral intermediate is kept to a minimum. This general strategy, which is applicable to redox enzymes (e.g., by acting on R2CHOH and R2CHNHR groups) and lyase-catalyzed addition-elimination reactions, was proven for the racemization of secondary alcohols by employing alcohol dehydrogenases. Thus, enantiopure chiral alcohols were used as model substrates and were racemized either with highly stereoselective biocatalysts or by using (rarely found) non-selective enzymes.     

α-氰醇立体选择性合成新进展

总结了近十年来立体选择性合成α-氰醇的最新进展, 包括金属配合物催化的醛、酮类底物的立体选择性氢氰化和硅氰化反应, 不含金属的有机小分子催化剂催化的醛、酮类底物的立体选择性硅氰化反应以及生物催化的立体选择性氢氰化反应. 另外对部分反应中涉及的机理也作了介绍.     

High throughput screening and QSAR-3D/CoMFA: useful tools to design predictive models of substrate specificity for biocatalysts

After a hierarchical microbial screening process, new microorganisms have been discovered that act as biocatalysts for the stereoselective oxidation of secondary alcohols or for ketone reduction. Oxidation activity is more widespread in yeasts and bacteria, while actinomycetes, filamentous fungi and yeasts present the highest reduction activities. QSAR-3D/CoMFA is an adequate technique to design predictive models of the biocatalysts' activity. In this paper CoMFA models are designed to compare the activities of the biocatalysts selected for the oxidation of alcohols and for the reduction of ketones, starting from the results obtained during the screening process. These models are useful for learning about the activity of these microorganisms and to compare the substrate specificity requirements between alcohol oxidation and ketone reduction biocatalysts.     

Creation of a pair of stereochemically complementary biocatalysts

N-Acetylneuraminic acid lyase (NAL) exhibits poor facial selectivity during carbon-carbon formation, and as such, its utility as a catalyst for use in synthetic chemistry is limited. For example, the NAL-catalyzed condensation between pyruvate and (2R,3S)-2,3-dihydroxy-4-oxo-N,N-dipropylbutyramide yields ca. 3:1 mixtures of diastereomeric products under either kinetic or thermodynamic control. Engineering the stereochemical course of NAL-catalyzed reactions could remove this limitation. We used directed evolution to create a pair of stereochemically complementary variant NALs for the synthesis of sialic acid mimetics. The E192N variant, a highly efficient catalyst for aldol reactions of (2R,3S)-2,3-dihydroxy-4-oxo-N,N-dialkylbutyramides, was chosen as a starting point. Initially, error-prone PCR identified residues in the active site of NAL that contributed to the stereochemical control of an aldolase-catalyzed reaction. Subsequently, an intense structure-guided program of saturation and site-directed mutagenesis was used to identify a complementary pair of variants, E192N/T167G and E192N/T167V/S208V, which were approximately 50-fold selective toward the cleavage of the alternative 4S- and 4R-configured condensation products, respectively. It was shown that wild-type NAL could not be used for the highly stereoselective synthesis of a 6-dipropylamide sialic acid mimetic because the 4S-configured product was only approximately 3-fold kinetically favored and only approximately 3-fold thermodynamically favored over the alternative 4R-configured product. However, the complementary 4R- and 4S-selective variants allowed the highly (>98:<2) diastereoselective synthesis of both 4S- and 4R-configured products under kinetic control from the same starting materials. Conversion of an essentially nonselective aldolase into a pair of complementary biocatalysts will be of enormous interest to synthetic chemists. Furthermore, since residues identified as critical for stereoselectivity are conserved among members of the NAL superfamily, the approach might be extended to the evolution of other useful biocatalysts for the stereoselective synthesis of biologically active molecules.     

Individual Surface‐Engineered Microorganisms as Robust Pickering Interfacial Biocatalysts for Resistance‐Minimized Phase‐Transfer Bioconversion

A powerful strategy for long‐term and diffusional‐resistance‐minimized whole‐cell biocatalysis in biphasic systems is reported where individually encapsulated bacteria are employed as robust and recyclable Pickering interfacial biocatalysts. By individually immobilizing bacterial cells and optimizing the hydrophobic/hydrophilic balance of the encapsulating magnetic mineral shells, the encased bacteria became interfacially active and locate at the Pickering emulsion interfaces, leading to dramatically enhanced bioconversion performances by minimizing internal and external diffusional resistances. Moreover, in situ product separation and biocatalyst recovery was readily achieved using a remote magnetic field. Importantly, the mineral shell effectively protected the entire cell from long‐term organic‐solvent stress, as shown by the reusability of the biocatalysts for up to 30 cycles, while retaining high stereoselective catalytic activities, cell viabilities, and proliferative abilities.     

Serine hydroxymethyl transferase from Streptococcus thermophilus and L-threonine aldolase from Escherichia coli as stereocomplementary biocatalysts for the synthesis of beta-hydroxy-alpha,omega-diamino acid derivatives

A novel serine hydroxymethyl transferase from Streptococcus thermophilus (SHMT) and a L-threonine aldolase from Escherichia coli (LTA) were used as stereocomplementary biocatalysts for the aldol addition of glycine to N-Cbz amino aldehydes and benzyloxyacetaldehyde (Cbz=benzyloxycarbonyl). Both threonine aldolases were classified as low-specific L-allo-threonine aldolases, and by manipulating reaction parameters, such as temperature, glycine concentration, and reaction media, SHMT yielded exclusively L-erythro diastereomers in 34-60 % conversion, whereas LTA gave L-threo diastereomers in 30:70 to 16:84 diastereomeric ratios and with 40-68 % conversion to product. SHMT is among the most stereoselective L-threonine aldolases described. This is due, among other things, to its activity-temperature dependence: at 4 degrees C SHMT has high synthetic activity but negligible retroaldol activity on L-threonine. Thus, the kinetic L-erythro isomer was largely favored and the reactions were virtually irreversible, highly stereoselective, and in turn, gave excellent conversion. It was also found that treatment of the prepared N-Cbz-gamma-amino-beta-hydroxy-alpha-amino acid derivatives with potassium hydroxide (1 m) resulted in the spontaneous formation of 2-oxazolidinone derivatives of the beta-hydroxyl and gamma-amino groups in quantitative yield. This reaction might be useful for further chemical manipulations of the products.     

Highly stereoselective biocatalytic reduction of alpha-halo ketones

The use of recombinant carbonyl reductase biocatalysts for the reduction of alpha-halo ketone intermediates to their corresponding alpha-halo alcohols has been investigated. The alpha-halo alcohol is obtained in good yield from the corresponding ketone in a stereoselective manner.     

Stereochemistry of reactions in the 1,2-dimethylsilacyclopentane ring system. II. Stereoselective transformation

A number of stereoselective reactions of 1-substituted-1,2-dimethylsilacyclopentanes are described. Reactions of the silyl chloride (II) with ZnF₂ and with alcohols catalyzed by amines are stereoselective as a result of rapid isomerization of II. Alcoholysis of silicon hydride (I) catalyzed by transition metals is apparently an inversion reaction regardless of the nature of the catalyst, but can appear to be stereoselective because of isomerization of alkoxysilane product. Reduction of silyl fluoride (IV) by lithium aluminum hydride is nonstereoselective, a result which is proposed to arise through rapid isomerization of intermediates with expanded coordination.     

A concise and stereoselective total synthesis of L-783,290

A stereoselective total synthesis of L-783,290 has been achieved involving Alder–Rickert reaction, ring closing metathesis, and chemoselective oxidation as key steps. This approach has successfully demonstrated a linear sequence of reactions, which are highly stereoselective for the construction of the resorcylic acid unit.     

Co‐immobilized Phosphorylated Cofactors and Enzymes as Self‐Sufficient Heterogeneous Biocatalysts for Chemical Processes

Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co‐immobilizing phosphorylated cofactors (PLP, FAD⁺, and NAD⁺) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self‐sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD⁺‐dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.     

Co-immobilized Phosphorylated Cofactors and Enzymes as Self-Sufficient Heterogeneous Biocatalysts for Chemical Processes

Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co-immobilizing phosphorylated cofactors (PLP, FAD⁺, and NAD⁺) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self-sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD⁺-dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.     

Highly stereoselective and enantiodivergent synthesis of cyclopropylphosphonates with engineered carbene transferases

Organophosphonate compounds have represented a rich source of biologically active compounds, including enzyme inhibitors, antibiotics, and antimalarial agents. Here, we report the development of a highly stereoselective strategy for olefin cyclopropanation in the presence of a phosphonyl diazo reagent as carbene precursor. In combination with a ‘substrate walking’ protein engineering strategy, two sets of efficient and enantiodivergent myoglobin-based biocatalysts were developed for the synthesis of both (1R,2S) and (1S,2R) enantiomeric forms of the desired cyclopropylphosphonate ester products. This methodology enables the efficient transformation of a broad range of vinylarene substrates at a preparative scale (i.e. gram scale) with up to 99% de and ee. Mechanistic studies provide insights into factors that contribute to make this reaction inherently more challenging than hemoprotein-catalyzed olefin cyclopropanation with ethyl diazoacetate investigated previously. This work expands the range of synthetically useful, enzyme-catalyzed transformations and paves the way to the development of metalloprotein catalysts for abiological carbene transfer reactions involving non-canonical carbene donor reagents.

Two enantiocomplementary myoglobin-based carbene transfer biocatalysts were developed for the synthesis of cyclopropylphosphonate esters with high diastereo- and enantioselectivity and in high yields.     

New Generation of Biocatalysts for Organic Synthesis

The use of enzymes as catalysts for the preparation of novel compounds has received steadily increasing attention over the past few years. High demands are placed on the identification of new biocatalysts for organic synthesis. The catalysis of more ambitious reactions reflects the high expectations of this field of research. Enzymes play an increasingly important role as biocatalysts in the synthesis of key intermediates for the pharmaceutical and chemical industry, and new enzymatic technologies and processes have been established. Enzymes are an important part of the spectrum of catalysts available for synthetic chemistry. The advantages and applications of the most recent and attractive biocatalysts—reductases, transaminases, ammonia lyases, epoxide hydrolases, and dehalogenases—will be discussed herein and exemplified by the syntheses of interesting compounds.     

Crotonase catalysis enables flexible production of functionalized prolines and carbapenams

The biocatalytic versatility of wildtype and engineered carboxymethylproline synthases (CMPSs) is demonstrated by the preparation of functionalized 5-carboxymethylproline derivatives methylated at C-2, C-3, C-4, or C-5 of the proline ring from appropriately substituted amino acid aldehydes and malonyl-coenzyme A. Notably, compounds with a quaternary center (at C-2 or C-5) were prepared in a stereoselective fashion by engineered CMPSs. The substituted-5-carboxymethyl-prolines were converted into the corresponding bicyclic β-lactams using a carbapenam synthetase. The results demonstrate the utility of the crotonase superfamily enzymes for stereoselective biocatalysis, the amenability of carbapenem biosynthesis pathways to engineering for the production of new bicyclic β-lactam derivatives, and the potential of engineered biocatalysts for the production of quaternary centers.     

生物催化用于C—C键的立体选择性合成

C—C键的立体选择性形成是有机合成化学的重要方面. 生物催化剂的立体选择性是它们的主要优势之一, 用酶催化C—C键形成已引起了广泛关注. 总结了生物转化中C—C键形成的最新应用, 着重讨论了醛缩酶和转酮醇酶生物催化剂的应用.     

Phenylalanine Ammonia Lyase Catalyzed Synthesis of Amino Acids by an MIO‐Cofactor Independent Pathway

Phenylalanine ammonia lyases (PALs) belong to a family of 4‐methylideneimidazole‐5‐one (MIO) cofactor dependent enzymes which are responsible for the conversion of L ‐phenylalanine into trans‐cinnamic acid in eukaryotic and prokaryotic organisms. Under conditions of high ammonia concentration, this deamination reaction is reversible and hence there is considerable interest in the development of PALs as biocatalysts for the enantioselective synthesis of non‐natural amino acids. Herein the discovery of a previously unobserved competing MIO‐independent reaction pathway, which proceeds in a non‐stereoselective manner and results in the generation of both L ‐ and D ‐phenylalanine derivatives, is described. The mechanism of the MIO‐independent pathway is explored through isotopic‐labeling studies and mutagenesis of key active‐site residues. The results obtained are consistent with amino acid deamination occurring by a stepwise E₁cB elimination mechanism.     

Immobilization of a Bacterial Cytochrome P450 Monooxygenase System on a Solid Support

Bacterial cytochrome P450s (P450s), which catalyze regio‐ and stereoselective oxidations of hydrocarbons with high turnover rates, are attractive biocatalysts for fine chemical production. Enzyme immobilization is needed for cost‐effective industrial manufacturing. However, immobilization of P450s is difficult because electron‐transfer proteins are involved in catalysis and anchoring these can prevent them from functioning as shuttle molecules for carrying electrons. We studied a heterotrimeric protein‐mediated co‐immobilization of a bacterial P450, and its electron‐transfer protein and reductase. Fusion with subunits of a heterotrimeric Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) enabled immobilization of the three proteins on a solid support. The co‐immobilized enzymes catalyzed monooxygenation because the electron‐transfer protein fused to PCNA via a single peptide linker retained its electron‐transport function.     

Carbohydrates as Chiral Auxiliaries in Stereoselective Synthesis. New Synthetic Methods (90)

Carbohydrates are inexpensive natural products in which numerous functional groups and stereogenic centers are combined in one molecule. By directed regio-and stereoselective formation of derivatives they can be converted into efficient chiral auxiliaries for controlling asymmetric syntheses. Stereoelectronic effects and pre-orientation of the reactive and shielding groups through formation of complexes can often be used for effective diastereofacial differentiation. In aldol reactions and alkylations on carbohydrate ester enolates intramolecular complexation promotes simultaneous elimination with formation of ketene. The steric, stereoelectronic, and coordinating properties of carbohydrate templates can also be used selectively to attain high levels of asymmetric induction in processes such as Diels–Alder reactions, hetero-Diels–Alder reactions, [2 + 2] cycloadditions, cyclopropanations, and Michael additions. It was possible with bicyclic, strongly stereodifferentiating carbohydrate auxiliaries to achieve a diastereoselective synthesis of carboxylic acid derivatives branched in the β position by a new 1,4-addition of alkylaluminum halides to α,β-unsaturated N-acylurethanes, in which methylaluminum halides and higher alkyl- or arylaluminum compounds behave mechanistically in a strikingly different manners. As complex ligands in chiral reagents and promoters, carbohydrates allow highly stereoselective reductions and aldol reactions that lead, amongst others, to chiral alcohols and β-hydroxy-α-amino acids in excellent enantiomeric excesses. Glycosylamines offer the possibility of versatile stereoselective applications: in the presence of Lewis acids the corresponding aldimines permit high-yielding syntheses of enantiomerically pure α-amino acids by Strecker and Ugi reactions, controlled by steric and stereoelectronic effects and by complex formation. They can be used with equal efficiency for asymmetric syntheses of chiral homoallylamines and for asymmetric Mannich syntheses of β-amino acids and chiral heterocycles, for example alkaloids.     

Discovery, application and protein engineering of Baeyer-Villiger monooxygenases for organic synthesis

Baeyer-Villiger monooxygenases (BVMOs) are useful enzymes for organic synthesis as they enable the direct and highly regio- and stereoselective oxidation of ketones to esters or lactones simply with molecular oxygen. This contribution covers novel concepts such as searching in protein sequence databases using distinct motifs to discover new Baeyer-Villiger monooxygenases as well as high-throughput assays to facilitate protein engineering in order to improve BVMOs with respect to substrate range, enantioselectivity, thermostability and other properties. Recent examples for the application of BVMOs in synthetic organic synthesis illustrate the broad potential of these biocatalysts. Furthermore, methods to facilitate the more efficient use of BVMOs in organic synthesis by applying e.g. improved cofactor regeneration, substrate feed and in situ product removal or immobilization are covered in this perspective.     

Photoenzymatic Hydrosulfonylation for the Stereoselective Synthesis of Chiral Sulfones

Chiral sulfones are recurrent motifs in pharmaceuticals and bioactive molecules. Although chemical methods have been developed to afford α- or β- chiral sulfones, these protocols rely heavily on the pre-synthesis of structurally complicated starting materials and chiral metal complexes. Herein, we described a photoenzymatic approach for the radical-mediated stereoselective hydrosulfonylation. Engineered variants of ene reductases provide efficient biocatalysts for this transformation, enabling to achieve a series of β-chiral sulfonyl compounds with high yields (up to 92 %) and excellent e.r. values (up to 99 : 1).