Cooperative Tertiary Amine/Palladium-Catalyzed Sequential [4 + 3] Cyclization/[1,3]-Rearrangement for Stereoselective Synthesis of Spiro [Methylenecyclopentane-1,3'-oxindolines].

A cooperative tertiary amine/palladium-catalyzed sequential reaction process, proceeding via a [4 + 3] cyclization of isatin-derived Morita-Baylis-Hillman Expansion (MBH) carbonates and tert-butyl 2-(hydroxymethyl)allyl carbonates followed by a [1,3]-rearrangement, has been found and developed. A range of structurally diverse spiro[methylene cyclopentane-1,3'-oxindolines] bearing two adjacent ß,γ-acyl quaternary carbon stereocenters, which are difficult to obtain by conventional strategies, were obtained in good yields. Further synthetic utility of this protocol is highlighted by its excellent regio- and stereocontrol as well as the large-scale synthesis and diverse functional transformations of the synthetic compounds. Moreover, the control experiments probably established the plausible mechanism for this sequential [4 + 3] cyclization/[1,3]-rearrangement process.

Enantiocomplementary synthesis of ß-adrenergic blocker precursors via biocatalytic nitration of phenyl glycidyl ethers.

Enantiopure ß-nitroalcohols, as an important class of nitro-containing compounds, are essential building blocks in pharmaceutical and organic chemistry, particularly for the synthesis of ß-adrenergic blockers. In this study, we present the successful protein engineering of halohydrin dehalogenase HHDHamb for the enantioselective bio-nitration of various phenyl glycidyl ethers to the corresponding chiral ß-nitroalcohols, using the inexpensive, commercially available, and safer nitrite as a nitrating agent. The chiral (R)- and (S)-1-nitro-3-phenoxypropan-2-ols were synthesized by the several enantiocomplementary HHDHamb variants through the whole-cell biotransformation, which showed good catalytic efficiency (up to 43% isolated yields) and high optical purity (up to >99% ee). In addition, we also demonstrated that the bio-nitration method was able to tolerate the substrate at a high concentration of 1000 mM (150 g/L). Furthermore, representative synthesis of two optically active enantiomers of the ß-adrenergic blocker metoprolol was successfully achieved by utilizing the corresponding chiral ß-nitroalcohols as precursors.

Copper-Catalyzed [5 + 1] Cyclization of o-Pyrrolo Anilines and Heterocyclic N-Tosylhydrazones for Access to Spiro-dihydropyrrolo[1,2-a]quinoxaline Derivatives.

An inexpensive copper-catalyzed sequential reaction process, proceeding via a nucleophilic attack of amine to Cu-carbene generated in situ from heterocyclic N-tosylhydrazone precursors followed by a 1,2-H shift/oxidative cyclization cascade of N-ylides, has been described, smoothly generating the corresponding structurally various spiro-dihydropyrrolo[1,2-a]quinoxaline derivatives. Furthermore, the significance of this protocol can be also highlighted by its diverse conversions of the synthetic compounds to the potentially bioactive molecules such as the 2-substituted pyrrolo[1,2-a]quinoxalins.

Combined photoredox/engineered P450 enzymatic direct dioxygen-functionalization of arylalkanes to chiral acyloins.

To date, the examples of difunctionalization of alkanes to directly incorporate two functional groups are very limited. In this study, we combined photoorgano redox catalysis and P450 biocatalysts to obtain dioxygen-functionalization of α/ß-C-H bonds of arylalkanes in a straightforward manner. The synthesis of enantiomerically chiral acyloins through a one-pot two-step photoredox/P450-catalyzed cascade reaction is described. Two P450 mutants with stereocomplementary bio-oxidation were obtained using mutagenesis technology and were able to asymmetrically hydroxylate ketones to acyloins with excellent ee values, which were further proved to be efficient on a wide range of substrates. Moreover, a photoredox synthesis of ketones in situ was developed by the direct carbonylation of aromatic methyl C-H bonds and subsequently combined with the aerobic P450-biocatalytic enantioselective hydroxylation of intermediate ketones, thus providing a green and sustainable approach towards optically pure acyloins.

Identification and Structure Analysis of an Unusual Halohydrin Dehalogenase for Highly Chemo-, Regio- and Enantioselective Bio-Nitration of Epoxides.

We report the discovery of an unusual halohydrin dehalogenase, HHDHamb, that can work under relatively low acidic conditions and extremely low temperatures for the bio-nitration of epoxides using nitrite as a nitrating agent. The bio-nitration strategy exhibits high chemo-, regio-, and enantioselectivity, catalyzing the kinetic resolution of various epoxides to enantiopure ß-nitroalcohols with nitro-bearing stereocenters in up to 41 % isolated yield and >99 % enantiomeric excess (ee). Additionally, the bio-nitration method displays a high reaction efficiency and can be performed on a gram scale. We also solved the crystal structure of HHDHamb to understand the possible structural determinants of chemoselectivity control in the bio-nitration reaction.

Biocatalytic Thionation of Epoxides for Enantioselective Synthesis of Thiiranes.

Expanding the enzymatic toolbox for the green synthesis of valuable molecules is still of high interest in synthetic chemistry and the pharmaceutical industry. Chiral thiiranes are valuable sulfur-containing heterocyclic compounds, but relevant methods for their enantioselective synthesis are limited. Herein, we report a biocatalytic thionation strategy for the enantioselective synthesis of thiiranes, which was developed based on the halohydrin dehalogenase (HHDH)-catalyzed enantioselective ring-opening reaction of epoxides with thiocyanate and a subsequent nonenzymatic rearrangement process. A novel HHDH was identified and engineered for enantioselective biocatalytic thionation of various aryl- and alkyl-substituted epoxides on a preparative scale, affording the corresponding thiiranes in up to 43 % isolated yield and 98 % ee. Large-scale synthesis and useful transformations of chiral thiiranes were also performed to demonstrate the utility and scalability of the biocatalytic thionation strategy.

Palladium-catalyzed asymmetric allylic alkylation of 3-aminooxindoles to access chiral homoallylic aminooxindoles.

An organometal catalytic conversion of 3-aminooxindoles for the diastereo- and enantioselective synthesis of homoallylic aminooxindoles has been described. The asymmetric allylic alkylation of 3-aminooxindoles with allyl carboxylates proceeded smoothly to afford a series of chiral 3-allyl-3-aminooxindoles. This work offers an alternative route to build these scaffolds. The application of this protocol is also highlighted by a significant conversion of products to the potential applicable spiro[indoline-3,2'-pyrrolidin]-2-one derivatives.

Characterization of a Self-Sufficient Cytochrome P450 Monooxygenase from Deinococcus apachensis for Enantioselective Benzylic Hydroxylation.

A self-sufficient cytochrome P450 monooxygenase from Deinococcus apachensis (P450DA) was identified and successfully overexpressed in Escherichia coli BL21(DE3). P450DA would be a member of the CYP102D subfamily and assigned as CYP102D2 according to the phylogenetic tree and sequence alignment. Purification and characterization of the recombinant P450DA indicated both NADH and NADPH could be used by P450DA as a reducing cofactor. The recombinant E. coli (P450DA) strain was functionally active, showing excellent enantioselectivity for benzylic hydroxylation of methyl 2-phenylacetate. Further substrate scope studies revealed that P450DA is able to catalyze benzylic hydroxylation of a variety of compounds, affording the corresponding chiral benzylic alcohols in 86-99 % ee and 130-1020 total turnover numbers.

Synthesis of chromone-containing polycyclic compounds via palladium-catalyzed [2 + 2 + 1] annulation.

A palladium-catalyzed [2 + 2 + 1] domino annulation of 3-iodochromones, α-bromo carbonyl compounds, and tetracyclododecene (TCD) is described. This approach provides a facile, efficient and atom-economical route to a variety of chromone-containing polycyclic compounds bearing fused/bridged-ring systems in good yields (up to 81%) with excellent diastereoselectivities (99 : 1 dr in all cases).

A Protocol for the Synthesis of CF2H-Containing Pyrazolo[1,5- c]quinazolines from 3-Ylideneoxindoles and in Situ Generated CF2HCHN2.

Herein is disclosed a selective and facile approach for the construction of CF2H-containing pyrazolo[1,5- c]quinazolines from easily accessible 3-ylideneoxindoles and in situ generated CF2HCHN2. The reaction involving a [3 + 2] cycloaddition/1,3-H shift/rearrangement/dehydrogenation cascade proceeded smoothly at room temperature in the absence of catalyst and additive. Moreover, this metal-free process along with mild conditions is desirable and valuable for the pharmaceutical industry. Importantly, this reaction features a broad substrate scope, good functional group tolerance, and gram-scale synthesis.

Asymmetric [3 + 2] Cycloaddition Reaction of Isatin-Derived MBH Carbonates with 3-Methyleneoxindoles: Enantioselective Synthesis of 3,3'-Cyclopentenyldispirooxindoles Incorporating Two Adjacent Quaternary Spirostereocenters.

A highly regio- and stereoselective [3 + 2] cycloaddition reaction for constructing novel 3,3'-cyclopentenyldispirooxindoles incorporating two adjacent quaternary spirostereocenters is reported. Under the mild conditions, the asymmetric annulation of isatin-derived MBH carbonates with 3-methyleneoxindoles involving a chiral tertiary amine catalyst provides the corresponding dispirooxindole frameworks with an extraordinary level of enantioselective control. Further synthetic utility of this method was demonstrated by the gram-scale experiment and simple transformation of the obtained product. Moreover, a plausible mechanism for this annulation reaction was also proposed on the basis of the control experiments.

A novel and simple spectrophotometric method for detection of nitrite in water.

A novel and simple spectrophotometric method is developed for the determination of nitrite in water by using the self-coupling diazotization reagent 2-amino-6-chlorobenzoic acid and UV light illumination. The method only involves one reagent and one step, which can be easily applied to the determination of nitrite and tolerates many foreign anions' effects under acidic conditions.

Identification and characterization of a highly S-enantioselective halohydrin dehalogenase from Tsukamurella sp. 1534 for kinetic resolution of halohydrins.

Halohydrin dehalogenases are remarkable enzymes which possess promiscuous catalytic activity and serve as potential biocatalysts for the synthesis of chiral halohydrins, epoxides and ß-substituted alcohols. The enzyme HheC exhibits a highly R enantioselectivity in the processes of dehalogenation of vicinal halohydrins and ring-opening of epoxides, which attracts more attentions in organic synthesis. Recently dozens of novel potential halohydrin dehalogenases have been identified by gene mining, however, most of the characterized enzymes showed low stereoselectivity. In this study, a novel halohydrin dehalogenase of HheA10 from Tsukamurella sp. 1534 has been heterologously expressed, purified and characterized. Substrate spectrum and kinetic resolution studies indicated the HheA10 was a highly S enantioselective enzyme toward several halohydrins, which produced the corresponding epoxides with the ee (enantiomeric excess) and E values up to >99% and >200 respectively. Our results revealed the HheA10 was a promising biocatalyst for the synthesis of enantiopure aromatic halohydrins and epoxides via enzymatic kinetic resolution of racemic halohydrins. What's more important, the HheA10 as the first individual halohydrin dehalogenase with the highly S enantioselectivity provides a complementary enantioselectivity to the HheC.

Synthesis of 2,3'-spirobi[indolin]-2-ones enabled by a tandem nucleophilic benzylation/C(sp2)-N cross-coupling reaction sequence.

An efficient complementary strategy for the construction of spiro[pyrrolidin-3,2'-oxindole] derivatives has been described. With the sequential nucleophilic benzylation and copper-catalyzed intramolecular C(sp2)-N cross-coupling reaction of 3-aminooxindoles with 2-bromobenzyl bromides, a wide range of 2,3'-spirobi[indolin]-2-ones were smoothly obtained in moderate to good yields. A plausible catalytic cycle for this tandem reaction process was proposed based on the control experiments. This study represents a new perspective for the synthesis of structurally diverse spirocyclic oxindoles by employing 3-aminooxindole substrates.

Enantioselective synthesis of 1,2,3,4-tetrahydroquinoline-4-ols and 2,3-dihydroquinolin-4(1H)-ones via a sequential asymmetric hydroxylation/diastereoselective oxidation process using Rhodococcus equi ZMU-LK19.

A cascade biocatalysis system involving asymmetric hydroxylation and diastereoselective oxidation was developed using Rhodococcus equi ZMU-LK19, which gave chiral 2-substituted-1,2,3,4-tetrahydroquinoline-4-ols (2) (up to 57% isolated yield, 99 : 1 dr, and >99% ee) and chiral 2-substituted-2,3-dihydroquinolin-4(1H)-ones (3) (up to 25% isolated yield, and >99% ee) from (±)-2-substituted-tetrahydroquinolines (1). In addition, a possible mechanism for this cascade biocatalysis was tentatively proposed.

An enantioselective synthesis of spiro-oxindole-based 3,4-dihydropyrroles via a Michael/cyclization cascade of 3-aminooxindoles with 2-enoylpyridines.

An organocatalytic and highly diastereo- and enantioselective reaction of 3-aminooxindoles with 2-enoylpyridines for the synthesis of chiral spiro[pyrrolidin-3,2'-oxindole] derivatives has been achieved. With the cinchonidine-based thiourea catalyst, the asymmetric Michael/cyclization reaction sequence of 3-aminooxindoles with 2-enoylpyridines followed by the dehydration and deprotection with concentrated hydrochloric acid provided a wide range of chiral 3',4'-dihydrospiro[indoline-3,2'-pyrrol]-2-ones, bearing two adjacent tri- and tetrasubstituted stereocenters, in moderate to good yields with overall excellent stereoselectivities. The synthetic application of this methodology was presented by the scale-up experiment and transformation of product 5a into the spiro-oxindole-based pyrrolidine 6. Furthermore, a plausible transition state for this cascade reaction sequence was also proposed. And this work will provide a new way to access chiral spiro[pyrrolidin-3,2'-oxindole] derivatives.

Diastereoselective [3 + 2] cycloaddition of 3-ylideneoxindoles with in situ generated CF2HCHN2: syntheses of CF2H-containing spirooxindoles.

An efficient [3 + 2] cycloaddition of 3-ylideneoxindoles with in situ generated CF2HCHN2 for the syntheses of spirooxindoles has been developed. This methodology gives access to a range of relatively complex spirooxindoles featuring a CF2H group and three contiguous stereogenic centers in up to 84% yield and 99 : 1 trans/cis.

An efficient high-throughput screening assay for rapid directed evolution of halohydrin dehalogenase for preparation of ß-substituted alcohols.

Halohydrin dehalogenases (HHDHs) are an important class of enzymes for preparing optically active haloalcohols, epoxides, and ß-substituted alcohols. However, natural HHDHs rarely meet the requirements of industrial applications. Here, a novel high-throughput screening (HTS) methodology for directed evolution of HHDH was developed based on the colorimetric determination of azide. In this method, azide was involved in the HHDH-catalyzed ring-opening process and the decrease of azide was used to quantitatively evaluate HHDH activity. The HTS methodology was simple and sensitive (ε460 = 1.2173 × 10(4) L mol(-1) cm(-1)) and could be performed in a microplate format using whole cells. To verify the efficiency of the HTS methodology, it was adopted to engineer a HHDH (HHDH-PL) from Parvibaculum lavamentivorans DS-1, which was applied in the process for ethyl (R)-4-cyano-3-hydroxybutanoate (HN) by the conversion of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE)). A random mutant library containing 2500 colonies was screened using the HTS methodology, and three beneficial mutants F176M, A187R, and A187S were obtained. By combining the beneficial mutated residues, the variant F176M/A187R was identified with 2.8-fold higher catalytic efficiency for preparation of HN. The high-throughput colorimetric assay would be very useful for directed evolution of HHDH for preparing ß-substituted alcohols.

Biocatalytic Synthesis of Metaxalone and Its Analogues.

A biocatalytic approach for the synthesis of metaxalone and its analogues was developed based on the reaction of epoxides and cyanate catalyzed by halohydrin dehalogenase. Gram-scale synthesis of chiral and racemic metaxalone was achieved with 44% (98% ee) and 81% yields, respectively, by protein engineering of the halohydrin dehalogenase HHDHamb from Acidimicrobiia bacterium. Additionally, various metaxalone analogues were synthesized at 28-40% yields (90-99% ee) for chiral forms and 77-92% yields for racemic forms.

Palladium-Catalyzed Domino Reaction for the Assembly of Norbornane-Containing Chromones with Dimethyl Squarate as the Solid C1 Source.

Reported herein is a novel palladium-catalyzed [2 + 2 + 1] domino annulation of 3-iodochromones, bridged olefins, and dimethyl squarate allowing the construction of chromone-containing polycyclic compounds in good to high yields. Importantly, dimethyl squarate is first employed as the solid C1 source in organic synthesis. Gram-scale experiments, late-stage modification of natural products, as well as transformations of products show potential for further synthetic elaborations.