Kinetics of hydrolysis and self condensation reactions of silanes by NMR spectroscopy

The hydrolysis of four alkoxy-silane coupling agents, 3-methacryloxypropyl trimethoxy silane (MPMS), 3-mercaptopropyl trimethoxy silane (MRPMS), octyl triethoxy silane (OES) and 3-aminopropyl triethoxy silane (APES) was carried out in an ethanol/water 80/20 (w/w) solution under acidic, alkaline and neutral conditions and followed by ¹H, ¹³C and ²⁹Si NMR spectroscopy. It was found that the kinetic rate of the hydrolysis of the silanes under neutral conditions was very low, except for APES, which displayed the fastest reaction speed. The addition of TEA catalyzed both silane hydrolysis and self condensation reactions. Acidic conditions enhanced the hydrolysis and the ensuing silanol entities were quite stable. In fact, these conditions slowed down the rate of the self condensation reactions, as deduced from in situ ¹H and ¹³C NMR. Thanks to in situ ²⁹Si NMR spectroscopy, the nature of the intermediary species versus reaction time was established.     

N‐Terminal Protein Modification by Substrate‐Activated Reverse Proteolysis

Although site‐specific incorporation of artificial functionalities into proteins is an important tool in both basic and applied research, it can be a major challenge to protein chemists. Enzymatic protein modification is an attractive goal due to the inherent regio‐ and stereoselectivity of enzymes, yet their specificity remains a problem. As a result of the intrinsic reversibility of enzymatic reactions, proteinases can in principle catalyze ligation reactions. While this makes them attractive tools for site‐specific protein bioconjugation, competing hydrolysis reactions limits their general use. Here we describe the design and application of a highly specific trypsin variant for the selective modification of N‐terminal residues of diverse proteins with various reagents. The modification proceeds quantitatively under native (aqueous) conditions. We show that the variant has a disordered zymogen‐like activation domain, effectively suppressing the hydrolysis reaction, which is converted to an active conformation in the presence of appropriate substrates.     

Asymmetrical nonbridgehead nitrogen-XXVI: Synthesis,configurational stability,and resolution of N,N-dialkoxyamines into antipodes

Alkoxyamines with tertiary N-alkyl substituents were chlorinated to N-chloro-N-alkoxyamines whose reaction with alcohols enabled synthesis of N,N-dialkoxyamines. The DNMR method was used to determine the barriers of inversion of these compounds. Alkaline hydrolysis (13) followed by subsequent reactions with R-(+)- and S-(?)-α-phenylethylamine yielded diastereomeric salts (+29 and ?29) whose crystallization and subsequent esterification resulted in optically active acyclic amines (?13 and +13) with the asymmetric center only at the N atom in the open chain.     

Substrate‐Guided Front‐Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide N‐Acetylgalactosaminyltransferase 2

The retaining glycosyltransferase GalNAc‐T2 is a member of a large family of human polypeptide GalNAc‐transferases that is responsible for the post‐translational modification of many cell‐surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum‐mechanics/molecular‐mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic‐electronic level of detail. Our study provides a detailed structural rationale for an ordered bi–bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front‐face S_Ni‐type reaction in which the substrate N‐acetyl sugar substituent coordinates efficient glycosyl transfer.     

Structural insights of a cellobiose dehydrogenase enzyme from the basidiomycetes fungus Termitomyces clypeatus

Filamentous fungi secrete various oxidative enzymes to degrade the glycosidic bonds of polysaccharides. Cellobiose dehydrogenase (CDH) (E.C.1.1.99.18) is one of the important lignocellulose degrading enzymes produced by various filamentous fungi. It contains two stereo specific ligand binding domains, cytochrome and dehydrogenase - one for heme and the other for flavin adenine dinucleotide (FAD) respectively. The enzyme is of commercial importance for its use in amperometric biosensor, biofuel production, lactose determination in food, bioremediation etc. Termitomyces clypeatus, an edible fungus belonging to the basidiomycetes group, is a good producer of CDH. In this paper we have analyzed the structural properties of this enzyme from T. clypeatus and identified a distinct carbohydrate binding module (CBM) which is not present in most fungi belonging to the basidiomycetes group. In addition, the dehydrogenase domain of T. clypeatus CDH exhibited the absence of cellulose binding residues which is in contrast to the dehydrogenase domains of CDH of other basidiomycetes. Sequence analysis of cytochrome domain showed that the important residues of this domain were conserved like in other fungal CDHs. Phylogenetic tree, constructed using basidiomycetes and ascomycetes CDH sequences, has shown that very surprisingly the CDH from T. clypeatus, which is classified as a basidiomycetes fungus, is clustered with the ascomycetes group. A homology model of this protein has been constructed using the CDH enzyme of ascomycetes fungus Myricoccum thermophilum as a template since it has been found to be the best match sequence with T. clypeatus CDH. We also have modelled the protein with its substrate, cellobiose, which has helped us to identify the substrate interacting residues (L354, P606, T629, R631, Y649, N732, H733 and N781) localized within its dehydrogenase domain. Our computational investigation revealed for the first time the presence of all three domains - cytochrome, dehydrogenase and CBM - in the CDH of T. clypeatus, a basidiomycetes fungus. In addition to discovering the unique structural attributes of this enzyme from T. clypeatus, our study also discusses the possible phylogenetic status of this fungus.     

A New α-Galactosidase from Thermoacidophilic Alicyclobacillus sp. A4 with Wide Acceptor Specificity for Transglycosylation

An α-galactosidase gene (gal36A4) of glycosyl hydrolase family 36 was identified in the genome of Alicyclobacillus sp. A4. It contains an ORF of 2,187 bp and encodes a polypeptide of 728 amino acids with a calculated molecular mass of 82.6 kDa. Deduced Gal36A4 shows the typical GH36 organization of three domains—the N-terminal β-sheets, the catalytic (β/α)₈-barrels, and the C-terminal antiparallel β-sheet. The gene product was produced in Escherichia coli and showed both hydrolysis and transglycosylation activities. The optimal pH for hydrolysis activity was 6.0, and a stable pH range of 5.0–11.0 was found. The enzyme had a temperature optimum of 60 °C. It is specific for α-1,6-glycosidic linkages and had a K _m value of 1.45 mM toward pNPGal. When using melibiose as both donor and acceptor of galactose, Gal36A4 showed the transfer ratio of 23.25 % at 96 h. With respect to acceptor specificity, all tested monosaccharides, disaccharides, and oligosaccharides except for D-xylose and L-arabinose were good acceptors for transglycosylation. Thus, Gal36A4 may find diverse applications in industrial fields, especially in the food industry.     

Yeast GH30 Xylanase from Sugiyamaella lignohabitans Is a Glucuronoxylanase with Auxiliary Xylobiohydrolase Activity

Xylanases are the enzymes that catalyze the breakdown of the main hemicellulose present in plant cell walls. They have attracted attention due to their biotechnological potential for the preparation of industrially interesting products from lignocellulose. While many xylanases have been characterized from bacteria and filamentous fungi, information on yeast xylanases is scarce and no yeast xylanase belonging to glycoside hydrolase (GH) family 30 has been described so far. Here, we cloned, expressed and characterized GH30 xylanase SlXyn30A from the yeast Sugiyamaella lignohabitans. The enzyme is active on glucuronoxylan (8.4 U/mg) and rhodymenan (linear β-1,4-1,3-xylan) (3.1 U/mg) while its activity on arabinoxylan is very low (0.03 U/mg). From glucuronoxylan SlXyn30A releases a series of acidic xylooligosaccharides of general formula MeGlcA²Xyl_n. These products, which are typical for GH30-specific glucuronoxylanases, are subsequently shortened at the non-reducing end, from which xylobiose moieties are liberated. Xylobiohydrolase activity was also observed during the hydrolysis of various xylooligosaccharides. SlXyn30A thus expands the group of glucuronoxylanases/xylobiohydrolases which has been hitherto represented only by several fungal GH30-7 members.     

Unraveling Synergism between Various GH Family Xylanases and Debranching Enzymes during Hetero-Xylan Degradation

Enzymes classified with the same Enzyme Commission (EC) that are allotted in different glycoside hydrolase (GH) families can display different mechanisms of action and substrate specificities. Therefore, the combination of different enzyme classes may not yield synergism during biomass hydrolysis, as the GH family allocation of the enzymes influences their behavior. As a result, it is important to understand which GH family combinations are compatible to gain knowledge on how to efficiently depolymerize biomass into fermentable sugars. We evaluated GH10 (Xyn10D and XT6) and GH11 (XynA and Xyn2A) β-xylanase performance alone and in combination with various GH family α-l-arabinofuranosidases (GH43 AXH-d and GH51 Abf51A) and α-d-glucuronidases (GH4 Agu4B and GH67 AguA) during xylan depolymerization. No synergistic enhancement in reducing sugar, xylose and glucuronic acid released from beechwood xylan was observed when xylanases were supplemented with either one of the glucuronidases, except between Xyn2A and AguA (1.1-fold reducing sugar increase). However, overall sugar release was significantly improved (≥1.1-fold reducing sugar increase) when xylanases were supplemented with either one of the arabinofuranosidases during wheat arabinoxylan degradation. Synergism appeared to result from the xylanases liberating xylo-oligomers, which are the preferred substrates of the terminal arabinofuranosyl-substituent debranching enzyme, Abf51A, allowing the exolytic β-xylosidase, SXA, to have access to the generated unbranched xylo-oligomers. Here, it was shown that arabinofuranosidases are key enzymes in the efficient saccharification of hetero-xylan into xylose. This study demonstrated that consideration of GH family affiliations of the carbohydrate-active enzymes (CAZymes) used to formulate synergistic enzyme cocktails is crucial for achieving efficient biomass saccharification.     

Computational design of an amidase by combining the best electrostatic features of two promiscuous hydrolases

While there has been emerging interest in designing new enzymes to solve practical challenges, computer-based options to redesign catalytically active proteins are rather limited. Here, a rational QM/MM molecular dynamics strategy based on combining the best electrostatic properties of enzymes with activity in a common reaction is presented. The computational protocol has been applied to the re-design of the protein scaffold of an existing promiscuous esterase from Bacillus subtilis Bs2 to enhance its secondary amidase activity. After the alignment of Bs2 with a non-homologous amidase Candida antarctica lipase B (CALB) within rotation quaternions, a relevant spatial aspartate residue of the latter was transferred to the former as a means to favor the electrostatics of transition state formation, where a clear separation of charges takes place. Deep computational insights, however, revealed a significant conformational change caused by the amino acid replacement, provoking a shift in the pK_a of the inserted aspartate and counteracting the anticipated catalytic effect. This prediction was experimentally confirmed with a 1.3-fold increase in activity. The good agreement between theoretical and experimental results, as well as the linear correlation between the electrostatic properties and the activation energy barriers, suggest that the presented computational-based investigation can transform in an enzyme engineering approach.

A computational strategy, based on combining the best electrostatic properties of enzymes with activity on a common reaction, is presented and applied to the re-design of the protein scaffold of an promiscuous esterase to enhance its secondary amidase activity.

The application of enzymes for desired chemical transformations has been demonstrated by the report of novel and functional designed structures.^1–5 Recent advances in molecular biology and screening technologies have enabled the creation of enzymes via directed evolution. By mimicking the process of natural evolution, iterative cycles of (semi-)random mutations facilitate the improvement of proteins in the laboratory through screening and selection, and hence the identification of active variants.^6–12 Minimal structural information is needed for this strategy and distal sites critical for enzyme catalysis can also be identified. Nevertheless, directed evolution is limited by the fact that, even with the most efficient high-throughput system, only a fraction of all the possible mutants of a given enzyme can be sampled within a set timeframe.¹³ Furthermore, the development of an efficient screening system for a tailored reaction remains challenging. Recently machine-learning (ML) methods have been proposed to expedite evolution and expand the number of properties that can be optimized.^14,15 However, in order to create enzymes with novel reactivities by means of ML methods, protein engineers will have to use proteins with sequences not assigned to the designated reaction or with properties other than those of specific interest, which currently is a technical challenge. Sequence–function data from engineering experiments must be collected to catalogue the natural diversity of proteins in order to convert ML into a useful tool.¹⁵An alternative approach is a rational design, a technique that modifies selected residues at specific positions of an already existing protein scaffold through the analysis of existing mechanistic and structural data.¹⁶ To reveal the structures of the protein in the full catalytic process under physiological conditions, including metastable transition state (TS) structures, computer simulations are essential. Among all the computer-assisted design strategies, two philosophies can be identified: the redesign of the active site of an existing substrate-promiscuous enzyme and the de novo design that constructs an enzyme “from scratch”. The use of promiscuous enzymes is found to be a very promising starting point for the design of new and highly efficient biocatalysts.^17,18 However, the knowledge about the particular molecular mechanisms that allow enzymes to catalyze more than one chemical reaction is still under debate.^19–22Because both the enzyme redesign and the de novo design approach require knowledge of the TS of the reaction to be catalyzed, quantum mechanical (QM) calculations offer crucial complementary information that accelerates the development of novel designed reactions. Moreover, multiscale methods are the only tool that can offer a detailed atomistic picture of the reactions in the active site of the enzyme, which can be dramatically different from that in the gas phase or solution. In multiscale methods, electrons of the reacting fragments are explicitly described by QM methods and the large and complex interacting environment (the fully solvated protein) is described by molecular mechanics (MM) force fields. The mechanism of a reaction in the active site of an enzyme can be determined within these hybrid QM/MM methods through the extensive exploration of the Free Energy Surface (FES). This allows the determination of the rate-limiting step in a multi-step process and, within the framework of Transition State Theory (TST),²³ the prediction of rate constants directly comparable with experiments. Previous studies combining computer simulations with experimental kinetic measurements have demonstrated the good agreement that can be achieved,^24,25 which obviously depends on the quality of both simulations and experiments. In this regard, the error in the determination of activation free energies associated with the use of computational methods such as the umbrella sampling method,^26,27 employed in the present study, is usually accepted to be within 1 kcal mol⁻¹.²⁸Optimizing the secondary activity of promiscuous enzymes is a non-trivial challenge as can be illustrated by analysing the Bacillus subtilis esterase Bs2. While Bs2 is recognized as a serine hydrolase whose primary reaction is the hydrolysis of esters, it can also catalyze the hydrolysis of the amide bond of N-(4-nitrophenyl)-butyramide as a secondary reaction (Fig. 1).^22,29 Previously directed evolution experiments by Arnold and co-workers resulted in a 7-mutation variant with a 100-fold enhancement of the esterase activity (using para-nitrobenzyl butyrate as the substrate).³⁰ Bornscheuer and co-workers used a combination of directed evolution and rational design based on docking and classical energy minimization to get a 3-fold increase of the amidase activity of Bs2 after two single mutations.²² In a larger context, despite the successes of different computer-assisted designs of new enzymes, it has been argued that the high activities of the best artificial enzymes have been largely due to directed evolution and the contribution of computation was comparatively modest.³¹Open in a separate windowFig. 1Schematic representation of the reaction mechanism of the hydrolysis of N-(4-nitrophenyl)-butyramide catalyzed by Bs2. (a) Acylation step: the nucleophilic addition of Ser189 to the carbonyl followed by the breaking of the C–N bond is triggered by His399-assisted proton shuffling, and the leaving group, in this case, is 4-nitroaniline. (b) Hydrolysis step: the nucleophilic addition of a water molecule followed by the resolution of the acyl–enzyme complex is triggered by His399-assisted protein shuffling, yielding butyric acid as a product.We envisaged that creating mutations to optimize the preorganization of the protein environment will result in a variant that exhibits improved activity for the desired reaction.³² Based on our recent QM/MM studies of different enzymatic reactions, we have quantified and shown how the reactivity of different proteins can be rationalized from their electrostatic properties,^{24,25,33–36} as the pioneering studies reported by Warshel and co-workers.^37–39 The computed changes of the electrostatic potential or the electric field exerted by the studied proteins on the key atoms of the substrates reflect that there is a small reorganization of these entities when evolving from the reactant state (RS) to the TS at the lowest energy cost.^24,33–36 The electrostatic effects within the active site of the enzyme, therefore, appear to be critical for the electronic reorganization of the reactants during chemical transformations. These studies support the idea that the electrostatic properties of enzymes are the origin of their catalytic features;⁴⁰ consequently, we view that a detailed understanding of the molecular mechanism, including the evolution of electrostatic potential generated in the active site of the enzyme, could be useful in future computer-assisted protein design methods.To engineer enzymes with optimal electrostatic preorganization, comparative analysis between unrelated natural enzymes that catalyze the same chemical reactions can be a reliable strategy. In previous studies, we have shown that Candida antarctica lipase B (CALB) also displays amidase activity similar to that of Bs2, though being non-homologous with each other.^24,25 QM/MM studies of the amidase reaction catalyzed by wild-type Bs2 and CALB enzymes were previously conducted.^24,25 We expect that the favorable features of each enzyme could be isolated and combined to create a redesigned enzyme with improved catalytic activity for the secondary amidase reaction. In the present paper, based on our knowledge derived from previous comparative studies, and by applying the concept of electrostatic pre-organization,^{24,33–36,40–43} a variant with improved activity for the designated amidase reaction was generated. After overlapping the structures of both proteins in one of the located TSs, through the use of a rotation quaternion around selected atoms of the substrate, a catalytically improved Bs2 variant was delineated. In particular, residues of Bs2 with an unfavorable electrostatic effect on catalysis were substituted by those placed in an equivalent spatial position in CALB with a favorable effect, as explained in detail below. The QM/MM FES of the full catalytic reaction in the proposed variant, combined with the experimental characterization, will be used to propose a general computer-based strategy that can be potentially used to design new enzymes.     

Regio- and enantioselective hydrolysis of phenyloxiranes catalyzed by soluble epoxide hydrolase

The regio- and enantioselective hydrolysis of several phenyloxiranes catalyzed by soluble epoxide hydrolase (sEH) was investigated using recombinant human, mouse or cress sEH. Results indicate that human and mouse sEH enantioselectively hydrolyze (S,S)-alkyl-phenyloxiranes faster than the (R,R)-alkyl-phenyloxiranes investigated in this study, while cress sEH displayed opposite enantioselectivity. Preparation of pure (2R,3R)-3-phenylglycidol from the racemic mixture was achieved with a 31% yield using human sEH as catalyst. The sEH enzymes were found to be regioselective at the benzylic carbon of the phenyloxiranes, supporting the proposed mechanism in which one or more tyrosine residues in the active site of the enzyme act as a general acid catalyst in the alkylation half reaction.     

Biodegradation and hydrolysis rate of aliphatic aromatic polyester

The biodegradation and hydrolysis rates of an aliphatic aromatic copolyester were measured in manure, food, and yard compost environments and in phosphate buffer solution (pH = 8.0) and vermiculite at 58 °C. Mineralization, molecular weight reduction, and structural changes determined by DSC, FTIR, and ¹H NMR were used as indicators of the biodegradation and hydrolysis rates. Poly(butylene adipate-co-terephthalate), PBAT, film biodegraded at distinctive rates in manure, food, and yard compost environments having different microbial activities. The highest biodegradation rate was found in manure compost, which had the highest CO₂ emissions and lowest C/N ratio. The possible presence of extracellular enzymes in manure and food composts may facilitate the hydrolytic reaction since greater molecular weight reduction rates were observed in these composts. ¹H NMR and thermal analysis revealed that, while PBAT is a semi-crystalline copolyester with cocrystallization of BT and BA dimers, the soft aliphatic domain (BA) and the amorphous region are more susceptible to hydrolysis and biodegradation than the rigid aromatic domain (BT) and the crystalline region.     

Determination of the modes of action and synergies of xylanases by analysis of xylooligosaccharide profiles over time using fluorescence‐assisted carbohydrate electrophoresis

The structure of xylan, which has a 1,4‐linked β‐xylose backbone with various substituents, is much more heterogeneous and complex than that of cellulose. Because of this, complete degradation of xylan needs a large number of enzymes that includes GH10, GH11, and GH3 family xylanases together with auxiliary enzymes. Fluorescence‐assisted carbohydrate electrophoresis (FACE) is able to accurately differentiate unsubstituted and substituted xylooligosaccharides (XOS) in the heterogeneous products generated by different xylanases and allows changes in concentrations of specific XOS to be analyzed quantitatively. Based on a quantitative analysis of XOS profiles over time using FACE, we have demonstrated that GH10 and GH11 family xylanases immediately degrade xylan into sizeable XOS, which are converted into smaller XOS in a much lower speed. The shortest substituted XOS produced by hydrolysis of the substituted xylan backbone by GH10 and GH11 family xylanases were MeGlcA²Xyl₃ and MeGlcA²Xyl₄, respectively. The unsubstituted xylan backbone was degraded into xylose, xylobiose, and xylotriose by both GH10 and GH11 family xylanases; the product profiles are not family‐specific but, instead, depend on different subsite binding affinities in the active sites of individual enzymes. Synergystic action between xylanases and β‐xylosidase degraded MeGlcA²Xyl₄ into xylose and MeGlcA²Xyl₃ but further degradation of MeGlcA²Xyl₃ required additional enzymes. Synergy between xylanases and β‐xylosidase was also found to significantly accelerate the conversion of XOS into xylose.     

Functional group-selective ion-molecule reactions of ethylene glycol and its monomethyl and dimethyl ethers

The selective methylation and methylene substitution reactions of dimethyl ether ions with ethylene glycol, ethylene glycol monomethyl ether, and ethylene glycol dimethyl ether were investigated in a quadrupole ion trap mass spectrometer. Whereas the reactions of ethylene glycol and ethylene glycol monomethyl ether with the methoxymethylene cation 45⁺ gave only [M + 13]⁺ product ions, the reaction of ethylene glycol dimethyl ether with the same reagent ion yielded exclusively [M + 15]⁺ ions. The relative rates of formation of these products and those from competing reactions were examined and rationalized on the basis of structural and electronic considerations. The heats of formation for various relevant species were estimated by computational methods and showed that the reactions leading to the [M + 13]⁺ ions were more energetically favorable than those leading to the [M + 15]⁺ products for cases in which both reactions are possible. Finally, the collision-induced dissociation behavior of the [M + H]⁺, [M + 13]⁺, and [M + 15]⁺ ions indicated that the and [M + H]⁺ rons dissociated by analogous pathways and were thus structurally similar, whereas the [M + 13]⁺ ions possessed distinctly different structural characteristics.     

Purification and Analytical Application of <Emphasis Type="Italic">Vigna mungo</Emphasis> Chitinase for Determination of Total Fungal Load of Stored Cereals

Chitinases are glycosyl hydrolases that catalyze the hydrolysis of β-(1,4)-glycosidic bonds in chitin, the major structural polysaccharide presented in the cuticle and gut peritrophic matrix of insects. Two aspartate residues (D143, D145) and one tryptophan (W146) in the Lymantria dispar chitinase are highly conserved residues observed within the second conserved motif of the family 18 chitinase catalytic region. In this study, a chitinase cDNA, LdCht5, was cloned from L. dispar, and the roles of the three residues were investigated using site-directed mutagenesis and substituting them with three other amino acids. Seven mutant proteins, D143E, D145E, W146G, D143E/D145E, D143E/W146G, D145E/W146G, and D143E/D145E/W146G, as well as the wild-type enzyme, were produced using the baculovirus-insect cell line expression system. The enzymatic and kinetic properties of these mutant enzymes were measured using the oligosaccharide substrate MU-(GlcNAc)₃. Among the seven mutants, the D145E, D143E/D145E, and D145E/W146G mutations kept some extant catalytic activity toward MU-(GlcNAc)₃, while the D143E, W146G, D143E/W146G, and D143E/D145E/W146G mutant enzymes were inactivated. Compared with the mutant enzymes, the wild-type enzyme had higher values of k _cat and k _cat / K _m . A study of the multiple point mutations in the second conserved catalytic region would help to elucidate the role of the critical residues and their relationships.     

Donor Promiscuity of a Thermostable Transketolase by Directed Evolution: Efficient Complementation of 1‐Deoxy‐d‐xylulose‐5‐phosphate Synthase Activity

Enzymes catalyzing asymmetric carboligation reactions typically show very high substrate specificity for their nucleophilic donor substrate components. Structure‐guided engineering of the thermostable transketolase from Geobacillus stearothermophilus by directed in vitro evolution yielded new enzyme variants that are able to utilize pyruvate and higher aliphatic homologues as nucleophilic components for acyl transfer instead of the natural polyhydroxylated ketose phosphates or hydroxypyruvate. The single mutant H102T proved the best hit toward 3‐methyl‐2‐oxobutyrate as donor, while the double variant H102L/H474S showed highest catalytic efficiency toward pyruvate as donor. The latter variant was able to complement the auxotrophic deficiency of Escherichia coli cells arising from a deletion of the dxs gene, which encodes for activity of the first committed step into the terpenoid biosynthesis, offering the chance to employ a growth selection test for further enzyme optimization.     

Chemoenzymatic preparation of optically active 4-aryl-5-carboxy-6-methyl-3,4-dihydro-2(1H)-pyridone derivatives

A series of racemic 4-aryl-5-(tert-butoxycarbonyl)-6-methyl-3,4-dihydro-2(1H)-pyridones have been prepared by means of a modified Hantzsch reaction using commercially available starting materials. An easy removal of the tert-butyl group of these pyridones and subsequent reaction with cesium carbonate and chloromethyl 2-methylpropanoate provided us suitable substrates (±)-5 to be used in lipase-catalyzed hydrolysis reactions. Lipase B from Candida antarctica (CAL-B) was the most adequate lipase in the hydrolysis of (±)-5. Despite the low enantioselectivity values obtained (E≤12), several optically active pyridone derivatives were finally isolated with high enantiomeric excesses (ee≥91%) and moderate yields.     

High throughput screening of O-glycosylation conditions

We report a novel methodology for rapid and quantitative screening of O-glycosylation reactions of application to the analysis of parallel reaction systems. Our system exploits perdeuterated benzyl (Bn-d₇) ether, and stereoselectivity and yield are evaluated by ¹H NMR and MALDI-TOF MS, respectively. This paper summarizes over 240 screenings of 1 → 3 linkage formation between glucose residues targeting the α-isomer in high yield.     

Hydridosilazanes hydrolysis-condensation reactions studied by <Superscript>1</Superscript>H and <Superscript>29</Superscript>Si liquid NMR spectroscopy

Hydridosilazane compounds containing Si–N and Si–H bonds can be used as precursors of SiO_x materials. The hydrolysis-condensation reactions of tetramethyldisilazane, as a polyhydridosilazane model compound, were investigated by ¹H and ²⁹Si liquid NMR spectroscopy. These reactions were carried out at room temperature for up to 120 min in presence of water. The identified products are short linear siloxane species (hydride terminated polydimethylsiloxanes M^HD_xM^H) and cyclosiloxanes. Silicon hydride persistence in the reactional mixture suggested that silazane group is more sensitive to hydrolysis reaction than silicon hydride group. Moreover, additional experiments evidenced that the low steric hindrance of the silicon hydride influences the silazane hydrolysis kinetic. Hence the presence of ammonia released during silazane hydrolysis reaction was demonstrated to be a catalyst of the silicon hydride hydrolysis reaction.     

Kinetic study of a complex triangular reaction system in alkaline aqueous-ethanol medium using on-line transmission FTIR spectroscopy and BTEM analysis

The kinetics of the base-catalyzed reaction of methyl 4-hydroxybenzoate in aqueous-ethanol solvent medium was studied and analyzed via combined on-line transmission FTIR spectroscopy and Band-Target Entropy Minimization (BTEM) technique. This reaction is considered complex since it involves simultaneous hydrolysis and ethanolysis reactions of methyl 4-hydrozybenzoate (MP) to form ethyl 4-hydroxybenzoate (EP) as an intermediate and sodium 4-hydroxybenzoate as a final product. The pure component spectra of the reactive species involved in the reaction were reconstructed using BTEM technique. Their corresponding real concentrations were calculated and subsequently used for analyzing the kinetics of this triangular reaction system. The effects of temperature and solvent mixture compositions were studied. In general, the results show that the rates of both hydrolysis and ethanolysis reactions increase with temperature. Addition of ethanol to the solvent mixture also reduces the rates of the hydrolysis reactions. The effect of solvent mixture on the rate of ethanolysis reaction is more complex and influenced by at least two competing factors, namely the concentration of ethoxide ion in the solution and the stabilization effect on the reactant. The enthalpy and entropy activation parameters, ΔH^‡ and ΔS^‡, of both the hydrolysis and ethanolysis reactions were determined using the Eyring equation and the activation parameters confirm the associative nature in the elementary steps in these reactions. Finally, it is shown that the dominant synthetic pathway in this triangular system changes from direct hydrolysis of methyl 4-hydrozybenzoate to the indirect pathway via ethanolysis and then hydrolysis depending on the solvent mixture composition.