Unified gain formula for control diagrams with matrix-based transfer functions.

The conventional Mason's gain formula is limited to single-input single-output (SISO) systems and is inadequate for coupled multi-input multi-output (MIMO) systems as it does not consider matrix operations. To fill this gap, this paper introduces a novel unified gain formula for control diagrams with matrix-based transfer functions. The formula is developed using partitioned matrix theory and feature extraction methods. Applying the partitioned matrix concept, the transfer function between any input and output of three type control diagrams is derived. A unified gain formula for input u to output y of the control diagram is then proposed based on feature extraction. Subsequently, the unified gain formula for any input and output of the control system is proposed based on the deduced equivalent relationship between the original and moved disturbance input nodes. Lastly, case studies demonstrate the effectiveness of the proposed unified gain formula, verifying its potential for use in both SISO and MIMO systems.

Synthesis and Biological Evaluation of 1-(2-(6-Methoxynaphthalen-2-yl)-6-methylnicotinoyl)-4-Substituted Semicarbazides/Thiosemicarbazides as Anti-Tumor Nur77 Modulators.

Nur77 is an orphan nuclear receptor that participates in the occurrence and development of a variety of tumors. Many agonists of Nur77 have been reported to have significant anticancer effects. Our previous studies have found that the introduction of bicyclic aromatic rings, such as naphthalyl and quinoline groups, into the N'-methylene position of indoles' Nur77 modulators can effectively improve the anti-tumor activity of the target compounds. Following our previous studies, a series of novel 1-(2-(6-methoxynaphthalen-2-yl)-6-methylnicotinoyl)-4-substituted semicarbazide/thiosemicarbazide derivatives 9a-9w were designed and synthesized in four steps from 6-methoxy-2-acetonaphthone and N-dimethylformamide dimethylacetal. All compounds were characterized by 1H-NMR, 13C-NMR and HRMS, and their anti-tumor activity on various cancer cell lines such as A549, HepG2, HGC-27, MCF-7 and HeLa are also evaluated. From the series of compounds, 9h exhibited the most potent anti-proliferative activity against several cancer cells. Colony formation and cell cycle experiments showed that compound 9h inhibited cell growth and arrested the cell cycle. Additionally, 9h leads to the cleavage of PARP. We initially explored the mechanism of 9h-induced apoptosis and found that compound 9h can upregulate Nur77 expression and triggered Nur77 nuclear export, indicating the occurrence of Nur77-mediated apoptosis. These results suggested that 9h may be a promising anti-tumor leading compound for the further research.

Light-driven decarboxylative deuteration enabled by a divergently engineered photodecarboxylase.

Despite the well-established chemical processes for C-D bond formation, the toolbox of enzymatic methodologies for deuterium incorporation has remained underdeveloped. Here we describe a photodecarboxylase from Chlorella variabilis NC64A (CvFAP)-catalyzed approach for the decarboxylative deuteration of various carboxylic acids by employing D2O as a cheap and readily available deuterium source. Divergent protein engineering of WT-CvFAP is implemented using Focused Rational Iterative Site-specific Mutagenesis (FRISM) as a strategy for expanding the substrate scope. Using specific mutants, several series of substrates including different chain length acids, racemic substrates as well as bulky cyclic acids are successfully converted into the deuterated products (>40 examples). In many cases WT-CvFAP fails completely. This approach also enables the enantiocomplementary kinetic resolution of racemic acids to afford chiral deuterated products, which can hardly be accomplished by existing methods. MD simulations explain the results of improved catalytic activity and stereoselectivity of WT CvFAP and mutants.

Substrate Engineering in Lipase-Catalyzed Selective Polymerization of d-/l-Aspartates and Diols to Prepare Helical Chiral Polyester.

The synthesis of optically pure polymers is one of the most challenging tasks in polymer chemistry. Herein, Novozym 435 (Lipase B from Candida antarctica, immobilized on Lewatit VP OC 1600)-catalyzed polycondensation between d-/l-aspartic acid (Asp) diester and diols for the preparation of helical chiral polyesters was reported. Compared with d-Asp diesters, the fast-reacting l-Asp diesters easily reacted with diols to provide a series of chiral polyesters containing N-substitutional l-Asp repeating units. Besides amino acid configuration, N-substituent side chains and the chain length of diols were also investigated and optimized. It was found that bulky acyl N-substitutional groups like N-Boc and N-Cbz were more favorable for this polymerization than small ones probably due to competitively binding of these small acyl groups into the active site of Novozym 435. The highest molecular weight can reach up to 39.5 × 103 g/mol (Mw, D = 1.64). Moreover, the slow-reacting d-Asp diesters were also successfully polymerized by modifying the substrate structure to create a "nonchiral" condensation environment artificially. These enantiocomplementary chiral polyesters are thermally stable and have specific helical structures, which was confirmed by circular dichroism (CD) spectra, scanning electron microscope (SEM), and molecular calculation.

The mutagenesis of a single site for enhancing or reversing the enantio- or regiopreference of cyclohexanone monooxygenases.

The mutagenesis of a "second sphere" switch residue of CHMOAcineto could control its enantio- and regiopreference. Replacing phenylalanine (F) at position 277 of CHMOAcineto into larger tryptophan (W) enabled a significant enhancement of enantio- or regioselectivity toward structurally diverse substrates, moreover, a complete reversal of enantio- or regiopreference was realized by mutating F277 into a range of smaller amino acids (A/C/D/E/G/H/I/K/L/M/N/P/Q/R/S/T/V).

Double Enzyme-Catalyzed One-Pot Synthesis of Enantiocomplementary Vicinal Fluoro Alcohols.

A double-enzyme-catalyzed strategy for the synthesis of enantiocomplementary vicinal fluoro alcohols through a one-pot, three-step process including lipase-catalyzed hydrolysis, spontaneous decarboxylative fluorination, and subsequent ketoreductase-catalyzed reduction was developed. With this approach, ß-ketonic esters were converted to the corresponding vicinal fluoro alcohols with high isolated yields (up to 92%) and stereoselectivities (up to 99%). This new cascade process addresses some issues in comparison with traditional methods such as environmentally hazardous reaction conditions and low stereoselectivity outcome.

Enzymatic Synthesis and Stereocomplex Formation of Chiral Polyester Containing Long-Chain Aliphatic Alcohol Backbone.

Herein we demonstrated a novel lipase-catalyzed synthesis of isotactic D-/L-poly(aspartate-octanediol) ester containing long chain alcohols backbone and discovered their stereocomplex feature with an increased Tm for the first time. Simple design of monomer structures not only overcomes the inherent selectivity limitation of enzyme used, but also achieves totally isotactic polyester products. By crystallizing the mixed enantiopure isotactic polyesters in different solvents, the formation of amorphous mixture, homocrystallites or stereocomplex crystallites were observed, respectively. This study is expected to open up a new way to prepare various stereocomplex polyesters containing a long-chain aliphatic alcohol backbone and a wide variety of functional groups.

Exploiting Cofactor Versatility to Convert a FAD-Dependent Baeyer-Villiger Monooxygenase into a Ketoreductase.

Cyclohexanone monooxygenases (CHMOs) show very high catalytic specificity for natural Baeyer-Villiger (BV) reactions and promiscuous reduction reactions have not been reported to date. Wild-type CHMO from Acinetobacter sp. NCIMB 9871 was found to possess an innate, promiscuous ability to reduce an aromatic α-keto ester, but with poor yield and stereoselectivity. Structure-guided, site-directed mutagenesis drastically improved the catalytic carbonyl-reduction activity (yield up to 99 %) and stereoselectivity (ee up to 99 %), thereby converting this CHMO into a ketoreductase, which can reduce a range of differently substituted aromatic α-keto esters. The improved, promiscuous reduction activity of the mutant enzyme in comparison to the wild-type enzyme results from a decrease in the distance between the carbonyl moiety of the substrate and the hydrogen atom on N5 of the reduced flavin adenine dinucleotide (FAD) cofactor, as confirmed using docking and molecular dynamics simulations.

Light-Driven Kinetic Resolution of α-Functionalized Carboxylic Acids Enabled by an Engineered Fatty Acid Photodecarboxylase.

Chiral α-functionalized carboxylic acids are valuable precursors for a variety of medicines and natural products. Herein, we described an engineered fatty acid photodecarboxylase (CvFAP)-catalyzed kinetic resolution of α-amino acids and α-hydroxy acids, which provides the unreacted R-configured substrates with high yields and excellent stereoselectivity (ee up to 99 %). This efficient light-driven process requires neither NADPH recycling nor prior preparation of esters, which were required in previous biocatalytic approaches. The structure-guided engineering strategy is based on the scanning of large amino acids at hotspots to narrow the substrate binding tunnel. To the best of our knowledge, this is the first example of asymmetric catalysis by an engineered CvFAP.

Stereodivergent Protein Engineering of a Lipase To Access All Possible Stereoisomers of Chiral Esters with Two Stereocenters.

Enzymatic stereodivergent synthesis to access all possible product stereoisomers bearing multiple stereocenters is relatively undeveloped, although enzymes are being increasingly used in both academic and industrial areas. When two stereocenters and thus four stereoisomeric products are involved, obtaining stereodivergent enzyme mutants for individually accessing all four stereoisomers would be ideal. Although significant success has been achieved in directed evolution of enzymes in general, stereodivergent engineering of one enzyme into four highly stereocomplementary variants for obtaining the full complement of stereoisomers bearing multiple stereocenters remains a challenge. Using Candida antarctica lipase B (CALB) as a model, we report the protein engineering of this enzyme into four highly stereocomplementary variants needed for obtaining all four stereoisomers in transesterification reactions between racemic acids and racemic alcohols in organic solvents. By generating and screening less than 25 variants of each isomer, we achieved >90% selectivity for all of the four possible stereoisomers in the model reaction. This difficult feat was accomplished by developing a strategy dubbed "focused rational iterative site-specific mutagenesis" (FRISM) at sites lining the enzyme's binding pocket. The accumulation of single mutations by iterative site-specific mutagenesis using a restricted set of rationally chosen amino acids allows the formation of ultrasmall mutant libraries requiring minimal screening for stereoselectivity. The crystal structure of all stereodivergent CALB variants, flanked by MD simulations, uncovered the source of selectivity.

"Top" or "bottom" switches of a cyclohexanone monooxygenase controlling the enantioselectivity of the sandwiched substrate.

Single mutation at the switch residues F432 (F432I/L) or L435 (L435A/G) efficiently reversed the inherent enantiopreference of WT CHMOAcineto in the Baeyer-Villiger oxidation of various 4-phenyl-cyclohexanone derivatives and 4-alkyl-cyclohexanones, producing a series of substituted lactones with inversed configuration (up to 99% ee and 99% conversion).

Enantiocomplementary Chiral Polyhydroxyenoate: Chemoenzymatic Synthesis and Helical Structure Control.

Here, we present the chemoenzymatic synthesis of two pairs of configuration-customized unsaturated chiral polyesters and discover that they are able to self-assemble into a helical superstructure. The chiral (R)- or (S)-polyesters with a polar unsaturated main-chain and an apolar side chain were designed to be stereoregular and amphiphilic-like. The solvent polarity, stereoregularity, unsaturated bond in the backbone and the structure of side chains were found to be the key factors to affect the self-assembly performance of the chiral polyesters. As the solvent polarity increased, the nanostructures of stereoregular unsaturated polyesters transformed from spheres to helical fibers, while there was no such transformation for the racemic or saturated polyesters.

Dual-Enzyme-Catalyzed Synthesis of Enantiocomplementary Polyesters.

Herein, a series of enantiocomplementary polyesters with either (S)- or (R)-configurations were successfully prepared by applying a dual-enzyme biocatalytic system. In the step of Baeyer-Villiger oxidation, cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMOAcineto) was engineered rationally to tailor the enantiopreference of mutants, providing (S)- and (R)-lactones, respectively, with high optical purities (up to 99% ee) as polymeric precursors. By subsequent enzymatic ring-opening polymerization of the enantiopure monomers, enantiocomplementary polyesters with high molecular weight (up to 21.8 kDa Mn) were synthesized by lipase CALB/MML. Our research offers an environmentally friendly synthesis route for the production of optically pure lactones and chiral polyesters, which are of particular significance for their application in organic syntheis or biomedical materials.

Effect of Additives on the Selectivity and Reactivity of Enzymes.

Enzymes have been widely used as efficient, eco-friendly, and biodegradable catalysts in organic chemistry due to their mild reaction conditions and high selectivity and efficiency. In recent years, the catalytic promiscuity of many enzymes in unnatural reactions has been revealed and studied by chemists and biochemists, which has expanded the application potential of enzymes. To enhance the selectivity and activity of enzymes in their natural or promiscuous reactions, many methods have been recommended, such as protein engineering, process engineering, and media engineering. Among them, the additive approach is very attractive because of its simplicity to use and high efficiency. In this paper, we will review the recent developments about the applications of additives to improve the catalytic performances of enzymes in their natural and promiscuous reactions. These additives include water, organic bases, water mimics, cosolvents, crown ethers, salts, surfactants, and some particular molecular additives.

Antitumor gemcitabine conjugated micelles from amphiphilic comb-like random copolymers.

Gemcitabine is an important pyrimidine antimetabolite that inhibits cellular DNA synthesis. However, the therapeutic efficacy and clinical benefit of gemcitabine are severely compromised due to its rapid plasma metabolism and low selectivity towards tumor tissues. To overcome these limitations, we prepared novel PEGylated gemcitabine-contained comb-like copolymers poly(monomethoxyl PEG350 methylacrylate -co- 5'-O-vinyladipyl- gemcitabine) (poly(mPEG350MA-co-VAG) and (poly(mPEG1000MA-co-VAG), which could self-assemble into micelles and displayed enhanced antitumor activity. The copolymers and the formed micelles were well characterized for their structure, critical aggregation concentration (CAC), morphology, cellular uptake, cell cytotoxicity, and controlled drug release. Cellular uptake and in vitro cytotoxicity assays against human lung cancerous cells (A549) demonstrated that these micelles could be effectively internalized and induced cell apoptosis. These micelles efficiently inhibited tumor growth when injected intravenously into A549 cell derived xenograft tumor bearing Balb/C nude mice using a dose of 10mg/kg in terms of reduced tumor volume compared to free gemcitabine. In conclusion, PEGylated micelles could protect gemcitabine from rapid plasma metabolism, provided a sustained release and showed enhanced antitumor activity, thus have the potential to be used as novel anticancer drug delivery vehicle.

Solvent-Free Lipase-Catalyzed Synthesis: Unique Properties of Enantiopure D- and L- Polyaspartates and Their Complexation.

Amino acids are attractive monomers for the large-scale preparation of chiral polyamides. For enzymatic polymerization of amino acids using protease in aqueous environment as the catalysis system, one main restriction is oligomer formation, usually along with other displayed advantages. Herein we developed an efficient solvent-free lipase-catalyzed polymerization of diethyl D- or L-aspartate, providing chiral D- and L-polyaspartates with an average degree of polymerization (DPavg) up to 60 and having about 96% ß-linkages. Additionally, their distinct chemical and physical properties were characterized by circular dichroism (CD) spectra, X-ray powder diffraction (XRD), microscopic observation, and thermal analysis. Poly(ß-D-AspEt) and Poly(ß-L-AspEt) showed vertically mirrored negative and positive CD signals, high crystallinity, and entirely different microscopic morphology. They are thermal stable while having different decomposition (Td), melting (Tm), and cold crystallization temperatures (Tcc), respectively. Our results also showed that the complexation of enantiopure D- and L-polyaspartates was not stereocomplex but homocomplex.

Mapping inhibitor response to the in-frame deletions, insertions and duplications of epidermal growth factor receptor (EGFR) in non-small cell lung cancer.

Human epidermal growth factor receptor (EGFR) has become a well-established target for the treatment of non-small cell lung cancer (NSCLC). However, a large number of in-frame deletion, insertion and duplication mutations in the EGFR tyrosine kinase (TK) domain have been observed to alter drug response to such a kinase target. Thus, a systematic investigation of the intermolecular interactions between the clinical small-molecule agents and various EGFR in-frame mutants would help to establish a complete picture of drug response to kinase mutations in lung cancer, and to design new EGFR inhibitors with high potency and selectivity to target drug-resistant mutants. Here, we describe a combined pipeline to explore the drug response of five representative EGFR inhibitors, including three FDA-approved agents (gefitinib, erlotinib and lapatinib) and two compounds under clinical development (AEE788 and TAK-285) to a number of clinically relevant EGFR in-frame mutations, aiming at a comprehensive understanding of molecular mechanism and biological implication underlying drug resistance and sensitivity to EGFR in-frame mutations. It was found that the insertion and duplication mutations in exon 20 can generally cause drug resistance to EGFR due to the reduced size of kinase's active pocket, while deletion mutations in exon 19 associate closely with increased inhibitor sensitivity to EGFR by establishing additional non-bonded interactions across complex interface, including hydrogen bonds, cation-π interactions and hydrophobic contacts.

Two Enzyme Cooperatively Catalyzed Tandem Polymerization for the Synthesis of Polyester Containing Chiral (R)- or (S)-Ibuprofen Pendants.

An interesting cooperation between Candida antarctica Lipase B (CAL-B) and alkaline protease from Bacillus subtilis (BSP) in the copolymerization of bulky ibuprofen-containing hydroxyacid methyl ester (HAEP) and ε-caprolactone (ε-CL) is observed. This cooperation improved the M¯n of the polymers from 3130 (CAL-B) to 9200 g mol-1 (CAL-B/BSP). Experimental results clearly indicate that CAL-B mainly catalyzes the ring-opening polymerization (ROP) of ε-CL under the initiation of HAEP to form the homopolymer of ε-CL, while BSP catalyzes the subsequent polycondensation of the ROP product to yield the copolymer with increased molecular weight. Furthermore, using suitable chemo-enzymatic methods, valuable polyesters with chiral (R)- or (S)-ibuprofen pendants can be tailor-made.

D-Aminoacylase-initiated cascade Aldol condensation/Robinson annulation for synthesis of substituted cyclohex-2-enones from simple aldehydes and acetone.

As an important building block, developing efficient and green synthesis strategy of cyclohex-2-enones is of great importance. In this present work, a general approach to the mild synthesis of substituted cyclohex-2-enones derivatives starting fro m simple aldehydes and acetone have been achieved via D-aminoacylase-initiated Aldol condensation/Robinson annulation cascade reaction using imidazole as an additive in organic media. The influences of reaction conditions including solvents, enzyme concentration, additives type, molar ratio of enzyme to additive, and substrate scopes were systematically investigated. Furthermore, some experiments were designed to explore the catalytic roles of D-aminoacylase and imidazole in the multistep cascade process, and one possible mechanism was proposed.

Enzymatic enantioselective aldol reactions of isatin derivatives with cyclic ketones under solvent-free conditions.

Nuclease p1 from Penicillium citrinum was observed to directly catalyze the asymmetric aldol reactions between isatin derivatives and cyclic ketones with high isolated yields (up to 95%) and moderate to good stereoselectivity (dr up to >99/1, ee up to 82%). A series of reaction conditions were investigated in detail, and the addition of deionized water had a big influence upon the enzyme activity. This case of biocatalytic promiscuity not only widens the applicability of nuclease p1 to new chemical transformation in organic synthesis, but also provides a potentially valuable method to construct pharmaceutically active compounds in medicinal chemistry.