Enzymatic Assembly of Diverse Lactone Structures: An Intramolecular C-H Functionalization Strategy.

Lactones are cyclic esters with extensive applications in materials science, medicinal chemistry, and the food and perfume industries. Nature's strategy for the synthesis of many lactones found in natural products always relies on a single type of retrosynthetic strategy, a C-O bond disconnection. Here, we describe a set of laboratory-engineered enzymes that use a new-to-nature C-C bond-forming strategy to assemble diverse lactone structures. These engineered "carbene transferases" catalyze intramolecular carbene insertions into benzylic or allylic C-H bonds, which allow for the synthesis of lactones with different ring sizes and ring scaffolds from simple starting materials. Starting from a serine-ligated cytochrome P450 variant previously engineered for other carbene-transfer activities, directed evolution generated a variant P411-LAS-5247, which exhibits a high activity for constructing a five-membered ε-lactone, lactam, and cyclic ketone products (up to 5600 total turnovers (TTN) and >99% enantiomeric excess (ee)). Further engineering led to variants P411-LAS-5249 and P411-LAS-5264, which deliver six-membered δ-lactones and seven-membered ε-lactones, respectively, overcoming the thermodynamically unfavorable ring strain associated with these products compared to the γ-lactones. This new carbene-transfer activity was further extended to the synthesis of complex lactone scaffolds based on fused, bridged, and spiro rings. The enzymatic platform developed here complements natural biosynthetic strategies for lactone assembly and expands the structural diversity of lactones accessible through C-H functionalization.

Enantio- and Diastereoenriched Enzymatic Synthesis of 1,2,3-Polysubstituted Cyclopropanes from (Z/E)-Trisubstituted Enol Acetates.

In nature and synthetic chemistry, stereoselective [2 + 1] cyclopropanation is the most prevalent strategy for the synthesis of chiral cyclopropanes, a class of key pharmacophores in pharmaceuticals and bioactive natural products. One of the most extensively studied reactions in the organic chemist's arsenal, stereoselective [2 + 1] cyclopropanation, largely relies on the use of stereodefined olefins, which can require elaborate laboratory synthesis or tedious separation to ensure high stereoselectivity. Here, we report engineered hemoproteins derived from a bacterial cytochrome P450 that catalyze the synthesis of chiral 1,2,3-polysubstituted cyclopropanes, regardless of the stereopurity of the olefin substrates used. Cytochrome P450BM3 variant P411-INC-5185 exclusively converts (Z)-enol acetates to enantio- and diastereoenriched cyclopropanes and in the model reaction delivers a leftover (E)-enol acetate with 98% stereopurity, using whole Escherichia coli cells. P411-INC-5185 was further engineered with a single mutation to enable the biotransformation of (E)-enol acetates to α-branched ketones with high levels of enantioselectivity while simultaneously catalyzing the cyclopropanation of (Z)-enol acetates with excellent activities and selectivities. We conducted docking studies and molecular dynamics simulations to understand how active-site residues distinguish between the substrate isomers and enable the enzyme to perform these distinct transformations with such high selectivities. Computational studies suggest the observed enantio- and diastereoselectivities are achieved through a stepwise pathway. These biotransformations streamline the synthesis of chiral 1,2,3-polysubstituted cyclopropanes from readily available mixtures of (Z/E)-olefins, adding a new dimension to classical cyclopropanation methods.

Enantio- and Diastereoenriched Enzymatic Synthesis of 1,2,3-Polysubstituted Cyclopropanes from (Z/E)-Trisubstituted Enol Acetates.

In nature and synthetic chemistry, stereoselective [2+1] cyclopropanation is the most prevalent strategy for the synthesis of chiral cyclopropanes, a class of key pharmacophores in pharmaceuticals and bioactive natural products. One of the most extensively studied reactions in the organic chemist's arsenal, stereoselective [2+1] cyclopropanation, largely relies on the use of stereodefined olefins, which require elaborate laboratory synthesis or tedious separation to ensure high stereoselectivity. Here we report engineered hemoproteins derived from a bacterial cytochrome P450 that catalyze the synthesis of chiral 1,2,3-polysubstituted cyclopropanes, regardless of the stereopurity of the olefin substrates used. Cytochrome P450 BM3 variant IC-G3 exclusively converts ( Z )-enol acetates to enantio- and diastereoenriched cyclopropanes and in our model reaction delivers a leftover ( E )-enol acetate with 98% stereopurity, using whole Escherichia coli cells. IC-G3 was further engineered with a single mutation to enable the biotransformation of ( E )-enol acetates to α -branched ketones with high levels of enantioselectivity while simultaneously catalyzing the cyclopropanation of ( Z )-enol acetates with excellent activities and selectivities. We conducted docking studies and molecular dynamics simulations to understand how active-site residues distinguish between the substrate isomers and enable the enzyme to perform these distinct transformations with such high selectivities. Computational studies suggest the observed enantio- and diastereoselectivities are achieved through a stepwise pathway. These biotransformations streamline the synthesis of chiral 1,2,3-polysubstituted cyclopropanes from readily available mixtures of ( Z/E )-olefins, adding a new dimension to classical cyclopropanation methods.

Chemodivergent C(sp3)-H and C(sp2)-H Cyanomethylation Using Engineered Carbene Transferases.

The ubiquity of C-H bonds presents an attractive opportunity to elaborate and build complexity in organic molecules. Methods for selective functionalization, however, often must differentiate among multiple chemically similar and, in some cases indistinguishable, C-H bonds. An advantage of enzymes is that they can be finely tuned using directed evolution to achieve control over divergent C-H functionalization pathways. Here, we demonstrate engineered enzymes that effect a new-to-nature C-H alkylation with unparalleled selectivity: two complementary carbene C-H transferases derived from a cytochrome P450 from Bacillus megaterium deliver an α-cyanocarbene into the α-amino C(sp3)-H bonds or the ortho-arene C(sp2)-H bonds of N-substituted arenes. These two transformations proceed via different mechanisms, yet only minimal changes to the protein scaffold (nine mutations, less than 2% of the sequence) were needed to adjust the enzyme's control over the site-selectivity of cyanomethylation. The X-ray crystal structure of the selective C(sp3)-H alkylase, P411-PFA, reveals an unprecedented helical disruption which alters the shape and electrostatics in the enzyme active site. Overall, this work demonstrates the advantages of enzymes as C-H functionalization catalysts for divergent molecular derivatization.

Docosahexaenoic acid alleviates the excessive degradation of extracellular matrix in the nucleus pulposus by reducing the content of lncRNA NEAT1 to prevent the progression of intervertebral disc degeneration.

The pathogenesis of intervertebral disc degeneration (IVDD), as a multifactorial disease, has not been fully elucidated. However, damage to the stress-bearing system in the intervertebral disc (IVD) mediated by the excessive decomposition of extracellular matrix (ECM) in nucleus pulposus (NP) cells caused by local stimulation is widely considered the core pathological process underlying IVDD. Docosahexaenoic acid (DHA) plays a protective role in various chronic diseases. However, whether it can have such effects in IVDD has not been clearly reported. In recent years, in-depth research on the role of long non-coding RNA (lncRNA) nuclear-enriched transcript 1 (NEAT1) in various diseases has continuously emerged, but such research in the field of IVD is not sufficient. In this study, tert-butyl hydroperoxide (TBHP) was used to induce oxidative stress in human NP cells and construct a cell model of excessive ECM decomposition in vitro. A plasmid over-expressing lncRNA NEAT1 was introduced into human NP cells to establish an NP cell model. For this specific experiment, Cell Counting Kit 8 was used to explore the timing and concentration of DHA and TBHP activity. A common gene chip platform was also used to select potential lncRNAs. Western blot and immunofluorescence assays were used to detect the expression of ECM-related proteins in NP cells in each group. Quantitative real-time polymerase chain reaction was used to detect the expression of lncRNA NEAT1 in NP cells in each group. On this basis, we proved that DHA alleviates excessive degradation of the ECM in NP cells in response to oxidative stress by reducing the content of lncRNA NEAT1. In conclusion, our study reveals the mechanism through which DHA relieves excessive ECM decomposition in NP cells and provides a potential new idea for the treatment of IVDD in clinical practice.

Visible-light-promoted 3,5-dimethoxyphenyl glycoside activation and glycosylation.

A new glycosylation method promoted by visible light with 3,5-dimethoxyphenyl glycoside as the donor was developed. This protocol delivers both O-glycosides and N-glycosides in moderate to excellent yields using a wide range of O-nucleophiles and nucleobases as the glycosyl acceptors.

Photocatalytic Decarboxylative Coupling of Aliphatic N-hydroxyphthalimide Esters with Polyfluoroaryl Nucleophiles.

Polyfluoroarenes are an important class of compounds in medical and material chemistry. The synthesis of alkylated polyfluoroarenes remains challenging. Here we describe a decarboxylative coupling reaction of N-hydroxyphthalimide esters of aliphatic carboxylic acids with polyfluoroaryl zinc reagents (Zn-ArF ) via synergetic photoredox and copper catalysis. This method readily converts primary and secondary alkyl carboxylic acids into the corresponding polyfluoroaryl compounds, which could have a wide range of F-content (2F-5F) and variable F-substitution patterns on the aryl groups. Broad scope and good functional group compatibility were achieved, including on substrates derived from natural products and pharmaceuticals. Mechanistic study revealed that a [Cu-(ArF )2 ] species could be responsible for the transfer of polyfluoroaryl groups to the alkyl radicals.

Copper-Catalyzed Intermolecular Functionalization of Unactivated C(sp3)-H Bonds and Aliphatic Carboxylic Acids.

Intermolecular functionalization of C(sp3)-H bonds and aliphatic carboxylic acids enables the efficient synthesis of high value-added organic compounds from readily available starting materials. Although methods involving hydrogen atom transfer have been developed for such functionalization, these methods either work for only activated C(sp3)-H bonds or bring in a narrow set of functional groups. Here we describe a Cu-catalyzed process for the diverse functionalization of both unactivated C(sp3)-H bonds and aliphatic carboxylic acids. The process is enabled by the trapping of alkyl radicals generated through hydrogen atom abstraction by arylsulfonyl-based SOMO-philes, which introduces a large array of C, N, S, Se, and halide-based functional groups. The chemoselectivity can be switched from C-H functionalization to decarboxylative functionalization by matching the bond dissociation energy of the hydrogen atom transfer reagent with that of the target C-H or O-H bond.

Enantioselective C(sp3)-C(sp3) cross-coupling of non-activated alkyl electrophiles via nickel hydride catalysis.

Cross-coupling of two alkyl fragments is an efficient method to produce organic molecules rich in sp3-hybridized carbon centres, which are attractive candidate compounds in drug discovery. Enantioselective C(sp3)-C(sp3) coupling is challenging, especially of alkyl electrophiles without an activating group (aryl, vinyl, carbonyl). Here, we report a strategy based on nickel hydride addition to internal olefins followed by nickel-catalysed alkyl-alkyl coupling. This strategy enables the enantioselective cross-coupling of non-activated alkyl halides with alkenyl boronates to produce chiral alkyl boronates. Employing readily available and stable olefins as pro-chiral nucleophiles, the coupling proceeds under mild conditions and exhibits broad scope and high functional-group tolerance. Applications for the functionalization of natural products and drug molecules, as well as the synthesis of chiral building blocks and a key intermediate to (S)-(+)-pregabalin, are demonstrated.

Tandem Photoredox and Copper-Catalyzed Decarboxylative C(sp3)-N Coupling of Anilines and Imines Using an Organic Photocatalyst.

An organic photoredox catalyst, 4CzIPN, was used in combination with a copper catalyst, CuCl, to effect decarboxylative C(sp3)-N coupling. The coupling worked with both anilines and imines as nitrogen sources and could be used to prepare a variety of alkyl amines from readily available alkyl carboxylic acids.

Deoxygenative trifluoromethylthiolation of carboxylic acids.

Here we describe a deoxygenative trifluoromethylthiolation method that yields trifluoromethyl thioesters from readily available carboxylic acids. The method is built upon an "umpolung" strategy where triphenylphosphine is used to first activate an electrophilic trifluoromethylthiolating reagent and then serves as an oxygen acceptor for the deoxygenation. The method is mild, efficient, broad-scope, and tolerant. It can be applied for the late-stage functionalization of numerous natural products and drug molecules containing a carboxylic acid group. The trifluoromethyl thioesters can be converted into trifluoromethyl thioethers by Pd-catalyzed decarbonylation.

Analysis and Evaluation of Eye Behavior for Marine Operation Training - A Pilot Study.

This paper presents a new analysis approach for evaluating situation awareness in marine operation training. Taking advantage of eye tracking technology, the situation awareness reflected by visual attention can be visualized and analyzed. A scanpath similarity comparison method that allows group-wise comparisons is proposed. The term 'Expert zone' is introduced to evaluate the performance of novice operator based on expert operators' eye movement. It is used to evaluate performance of novice operators in groups in certain segment of marine operation. A pilot study of crane lifting experiment was carried out. Two target stages of operation for the load descending until total immersion to the seabed were selected and analyzed for both novice and expert operators. The group-wise evaluation method is proven to be able to access the performance of the operator. Besides that, from data analysis of fixation-related source and scanpath, the similarities and dissimilarities of eye behavior between novice and expert is concluded with the scanpath mode in target segment.

Decarboxylative C(sp3 )-O Cross-Coupling.

Alkyl aryl ethers are an important class of compounds in medicinal and agricultural chemistry. Catalytic C(sp3 )-O cross-coupling of alkyl electrophiles with phenols is an unexplored disconnection strategy to the synthesis of alkyl aryl ethers, with the potential to overcome some of the major limitations of existing methods such as C(sp2 )-O cross-coupling and SN 2 reactions. Reported here is a tandem photoredox and copper catalysis to achieve decarboxylative C(sp3 )-O coupling of alkyl N-hydroxyphthalimide (NHPI) esters with phenols under mild reaction conditions. This method was used to synthesize a diverse set of alkyl aryl ethers using readily available alkyl carboxylic acids, including many natural products and drug molecules. Complementarity in scope and functional-group tolerance to existing methods was demonstrated.

Cross-Coupling of Alkyl Redox-Active Esters with Benzophenone Imines: Tandem Photoredox and Copper Catalysis.

Alkyl amines are an important class of organic compounds in medicinal and materials chemistry. Until now very have been very few methods for the synthesis of alkyl amines by metal-catalyzed cross-coupling of alkyl electrophiles with nitrogen nucleophiles. Described here is an approach to employ tandem photoredox and copper catalysis to enable the cross-coupling of alkyl N-hydroxyphthalimide esters, readily derived from alkyl carboxylic acids, with benzophenone-derived imines. Hydrolysis of the coupling products furnish alkylated primary amines. Primary, secondary, and tertiary alkyl groups can be transferred, and the coupling tolerates a diverse set of functional groups. The method allows rapid functionalization of natural products and drugs, and can be used to expedite syntheses of pharmaceuticals from readily available chemical feedstocks.

Visible Light Photoredox-Catalyzed O-Sialylation Using Thiosialoside Donors.

An efficient protocol for the O-sialylation using thiosialoside donors under visible light photocatalysis was developed. Thiosialosides were activated under the irradiation with blue light in the presence of Ru(bpy)3(PF6)2 as photocatalyst, Umemoto's reagent as CF3 radical source and Cu(OTf)2 as an additive in acetonitrile/dichloromethane at -30 °C, and the subsequent reaction with glycosyl acceptors generally produced the desired sialosides in good to excellent yields with the satisfactory α-selectivity.

Photoinduced C-S Bond Cleavage of Thioglycosides and Glycosylation.

A glycosyl coupling reaction via photoinduced direct activation of thioglycosides and subsequent O-glycosylation in the absence of photosensitizer was developed for the first time. This reaction underwent a selectively homolytic cleavage of a C-S bond to generate a glycosyl radical, which was oxidized to an oxacarbenium ion by Cu(OTf)2, and a sequential O-glycosylation. A wide range of glycosides were synthesized in moderate to excellent yield using sugars, amino acids, or cholesterol as the acceptors.

Pd(II)-catalyzed one-pot, three-step route for the synthesis of unsymmetrical acridines.

Unsymmetric acridines are synthesized via a one-pot amination/cyclization/aromatization reaction for the first time. With Pd(OAc)2-X-Phos as the catalyst, a series of unsymmetric acridines are obtained in moderate to excellent yields (up to 99% yield). Meanwhile, the diphenylamine intermediate could be isolated, which is evidence of the domino process.

Synthesis of N-(2-chloro purin-6-yl) aza-18-crown-6 and its interaction with human serum albumin.

The synthesis of novel purine nucleosides-linked azacrown ethers in the C6 position, N-(2-chloro purin-6-yl) aza-18-crown-6 (NCPAC), was described. This new nucleoside analogue can be prepared from a series of N9-modified nucleosides and the method allows for new and easy modification of the nucleosides. The interaction between NCPAC and human serum albumin (HSA) was studied using molecular docking and fluorescence techniques. Thermodynamics revealed that the interaction was entropy driven with predominantly hydrophobic forces. From the observed Föster's-type fluorescence resonance energy transfer, the donor (Trp 214 in HSA) to acceptor (NCPAC) distance was calculated to be 3.6 nm. The conformational changes of HSA due to the interaction were investigated qualitatively from synchronous fluorescence spectra. Molecular docking studies were performed to obtain information on the possible residues involved in the interaction process.