Structural Snapshots of α‐1,3‐Galactosyltransferase with Native Substrates: Insight into the Catalytic Mechanism of Retaining Glycosyltransferases

Glycosyltransferases (GTs) are a key family of enzymes that catalyze the synthesis of glycosidic bonds in all living organisms. The reaction involves the transfer of a glycosyl moiety and can proceed with retention or inversion of the anomeric configuration. To date, the catalytic mechanism of retaining GTs is a topic of great controversy, particularly for those enzymes containing a putative nucleophilic residue in the active site, for which the occurrence of a double‐displacement mechanism has been suggested. We report native ternary complexes of the retaining glycosyltransferase α‐1,3‐galactosyltransferase (α3GalT) from Bos taurus , which contains such a nucleophile in the active site, in a productive mode for catalysis in the presence of its sugar donor UDP‐Gal, the acceptor substrate lactose, and the divalent cation cofactor. This new experimental evidence supports the occurrence of a front‐side substrate‐assisted S_Ni‐type reaction for α3GalT, and suggests a conserved common catalytic mechanism among retaining GTs.     

A Native Ternary Complex Trapped in a Crystal Reveals the Catalytic Mechanism of a Retaining Glycosyltransferase

Glycosyltransferases (GTs) comprise a prominent family of enzymes that play critical roles in a variety of cellular processes, including cell signaling, cell development, and host–pathogen interactions. Glycosyl transfer can proceed with either inversion or retention of the anomeric configuration with respect to the reaction substrates and products. The elucidation of the catalytic mechanism of retaining GTs remains a major challenge. A native ternary complex of a GT in a productive mode for catalysis is reported, that of the retaining glucosyl‐3‐phosphoglycerate synthase GpgS from M. tuberculosis in the presence of the sugar donor UDP‐Glc, the acceptor substrate phosphoglycerate, and the divalent cation cofactor. Through a combination of structural, chemical, enzymatic, molecular dynamics, and quantum‐mechanics/molecular‐mechanics (QM/MM) calculations, the catalytic mechanism was unraveled, thereby providing a strong experimental support for a front–side substrate‐assisted S_Ni‐type reaction.     

Kinetics of Iterative Carbohydrate Transfer to Polysaccharide Catalyzed by Chondroitin Polymerase on a Highly Sensitive Flow‐Type 27 MHz Quartz‐Crystal Microbalance

Using a highly sensitive flow‐type 27 MHz quartz crystal microbalance, we could detect a small mass change during stepwise and alternating one‐sugar transfer of glucuronic acid (GlcA) and N‐acetylgalactosamine (GalNAc) to an acceptor, catalyzed by chondroitin polymerase from Escherichia coli strain K4 (K4CP), and analyze the elongation mechanism of K4CP. K4CP was found to bind strongly to a chondroitin acceptor (K_d=0.97 μM ). Although the binding affinity and the catalytic rate constant for each monomer were considerably different, the apparent catalytic efficiency (k_cat/K_m) was similar (6.3×10⁴ M ^?1 s^?1 for GlcA transfer and 3.4×10⁴ M ^?1 s^?1 for the GalNAc transfer). This is reasonable for the smooth alternating elongation of GlcA and GalNAc on the acceptor. This is the first study to report the determination of kinetic parameters for enzymatic, alternated, sugar elongation.     

Expression,Purification and Characterization of a Bifunctional α‐L‐Arabinofuranosidase/β‐D‐Xylosidase from Trichoderma Koningii G‐39

A gene of α‐L ‐arabinofuranosidase (Abf) from Trichoderma koningii G‐39 was successfully expressed in Pichia pastoris. The recombinant enzyme was purified to > 90% homogeneity by a cation‐exchanged chromatography. The purified enzyme exhibits both α‐L ‐arabinofuranosidase and β‐D ‐xylosidase (Xyl) activities with p‐nitrophenyl‐α‐L ‐arabionfuranoside (pNPAF) and 2,4‐dinitrophenyl‐β‐D ‐xylopyanoside (2,4‐DNPX) as substrate, respectively. The stability and the catalytic feature of the bifunctional enzyme were characterized. The enzyme was stable for at least 2 h at pH values between 2 and 8.3 at room temperature when assayed for Abf and Xyl activities. Enzyme activity decreased dramatically when the pH exceeded 9.5 or dropped below 1.5. The enzyme lost 35% of Abf activity after incubation at 55 °C for 2 h, but retained 95% of Xyl activity, with 2,4‐DNXP as substrate, under the same conditions. Further investigation of the active site topology of both enzymatic functions was performed with the inhibition study of enzyme activities. The results revealed that methyl‐α‐L ‐arabinofuranoside inhibition is noncompetitive towards 2,4‐DNPX as substrate but competitive towards pNPAF. Based on the thermal stability and the inhibition studies, we suggest that the enzymatic reactions of Abf and Xyl are performed at distinct catalytic sites. The recombinant enzyme possesses both the retaining transarabinofuranosyl and transxylopyranosyl activities, indicating both enzymatic reactions proceed through a two‐step, double displacement mechanism.     

A QM/MM Investigation of the Catalytic Mechanism of Metal‐Ion‐Independent Core 2 β1,6‐N‐Acetylglucosaminyltransferase

β1,6‐GlcNAc‐transferase (C2GnT) is an important controlling factor of biological functions for many glycoproteins and its activity has been found to be altered in breast, colon, and lung cancer cells, in leukemia cells, in the lymhomonocytes of multiple sclerosis patients, leukocytes from diabetes patients, and in conditions causing an immune deficiency. The result of the action of C2GnT is the core 2 structure that is essential for the further elongation of the carbohydrate chains of O‐glycans. The catalytic mechanism of this metal‐ion‐independent glycosyltransferase is of paramount importance and is investigated here by using quantum mechanical (QM) (density functional theory (DFT))/molecular modeling (MM) methods with different levels of theory. The structural model of the reaction site used in this report is based on the crystal structures of C2GnT. The entire enzyme–substrate system was subdivided into two different subsystems: the QM subsystem containing 206 atoms and the MM region containing 5914 atoms. Three predefined reaction coordinates were employed to investigate the catalytic mechanism. The calculated potential energy surfaces discovered the existence of a concerted S_N2‐like mechanism. In this mechanism, a nucleophilic attack by O6 facilitated by proton transfer to the catalytic base and the separation of the leaving group all occur almost simultaneously. The transition state for the proposed reaction mechanism at the M06‐2X/6‐31G** (with diffuse functions on the O1′, O5′, O_Glu, and O6 atoms) level was located at C1? O6=1.74 Å and C1? O1=2.86 Å. The activation energy for this mechanism was estimated to be between 20 and 29 kcal mol^?1, depending on the method used. These calculations also identified a low‐barrier hydrogen bond between the nucleophile O6H and the catalytic base Glu320, and a hydrogen bond between the N‐acetamino group and the glycosidic oxygen of the donor in the TS. It is proposed that these interactions contribute to a stabilization of TS and participate in the catalytic mechanism.     

Revised mechanism of carboxylation of ribulose‐1,5‐biphosphate by rubisco from large scale quantum chemical calculations

Here, we describe a computational approach for studying enzymes that catalyze complex multi‐step reactions and apply it to Ribulose 1,5‐bisphosphate carboxylase–oxygenase (Rubisco), the enzyme that fixes atmospheric carbon dioxide within photosynthesis. In the 5‐step carboxylase reaction, the substrate Ribulose‐1,5‐bisphosphate (RuBP) first binds Rubisco and undergoes enolization before binding the second substrate, CO₂. Hydration of the RuBP.CO₂ complex is followed by C C bond scission and stereospecific protonation. However, details of the roles and protonation states of active‐site residues, and sources of protons and water, remain highly speculative. Large‐scale computations on active‐site models provide a means to better understand this complex chemical mechanism. The computational protocol comprises a combination of hybrid semi‐empirical quantum mechanics and molecular mechanics within constrained molecular dynamics simulations, together with constrained gradient minimization calculations using density functional theory. Alternative pathways for hydration of the RuBP.CO₂ complex and associated active‐site protonation networks and proton and water sources were investigated. The main findings from analysis of the resulting energetics advocate major revision to existing mechanisms such that: hydration takes place anti to the CO₂; both hydration and C C bond scission require early protonation of CO₂ in the RuBP.CO₂ complex; C C bond scission and stereospecific protonation reactions are concerted and, effectively, there is only one stable intermediate, the C3‐gemdiolate complex. Our main conclusions for interpreting enzyme kinetic results are that the gemdiolate may represent the elusive Michaelis–Menten‐like complex corresponding to the empirical K_m (=K_c) with turnover to product via bond scission concerted with stereospecific protonation consistent with the observed catalytic rate. © 2018 Wiley Periodicals, Inc.     

Three one‐dimensional coordination polymers based on 1,1′‐bis(pyridin‐4‐ylmethyl)‐2,2′‐bi‐1H‐benzimidazole and HgX2 (X = Cl,Br and I)

A new 2,2′‐bi‐1H‐benzimidazole bridging organic ligand, namely 1,1′‐bis(pyridin‐4‐ylmethyl)‐2,2′‐bi‐1H‐benzimidazole, C₂₆H₂₀N₆, L or (I), has been synthesized and used to create three new one‐dimensional coordination polymers, viz.catena‐poly[[dichloridomercury(II)]‐μ‐1,1′‐bis(pyridin‐4‐ylmethyl)‐2,2′‐bi‐1H‐benzimidazole], [HgCl₂(C₂₆H₂₀N₆)]_n, (II), and the bromido, [HgBr₂(C₂₆H₂₀N₆)]_n, (III), and iodido, [HgI₂(C₂₆H₂₀N₆)]_n, (IV), analogues. Free ligand L crystallizes with two symmetry‐independent half‐molecules in the asymmetric unit and each L molecule resides on a crytallographic inversion centre. In structures (II)–(IV), the L ligand is also positioned on a crystallographic inversion centre, whereas the Hg centre resides on a crystallographic twofold axis. Compound (I) adopts an anti conformation in the solid state and forms a two‐dimensional network in the crystallographic bc plane viaπ–π and C—H...π interactions. The three Hg^II coordination complexes, (II)–(IV), have one‐dimensional zigzag chains composed of L and HgX₂ (X = Cl, Br and I), and the Hg^II centres are in a distorted tetrahedral [HgX₂N₂] coordination geometry. Complexes (III) and (IV) are isomorphous, whereas complex (II) displays an interesting conformational difference from the others, i.e. a twist in the flexible bridging ligand.     

Carbonic Anhydrase‐Mimetic Bolaamphiphile Self‐Assembly for CO2 Hydration and Sequestration

A biomimetic catalyst was prepared through the self‐assembly of a bolaamphiphilic molecule with histidine moieties for the sequestration of carbon dioxide. The histidyl bolaamphiphilic molecule bis(N‐α‐amidohistidine)‐1,7‐heptane dicarboxylate has been synthesized and self‐assembled to produce analogues of the active sites of carbonic anhydrase (CA) after association with Zn²⁺ ions. Spectroscopic analysis demonstrated the coordination of the Zn²⁺ ions with histidine imidazole moieties, which is the core conformation of CA active sites. The Zn‐associated self‐assembly worked as a CA‐mimetic catalyst that shows catalytic activity for CO₂ hydration. Evaluation of the kinetics of using para‐nitrophenylacetate revealed that the kinetic parameters of the CA‐mimetic catalyst were maximized at the optimal Zn concentration and that excess Zn ions resulted in deteriorated catalytic activity. The performance of the CA‐mimetic catalyst was enhanced by changing the pH value and temperature of the reaction, which implies that the hydrolysis of the substrate is the rate‐determining step. The catalyst‐assisted sequestration of CO₂ was demonstrated by CaCO₃ precipitation upon the addition of Ca²⁺ ions. This study offers an easy way to prepare enzyme analogues for CO₂ sequestration through the self‐assembly of bolaamphiphile molecules with designer biochemical moieties.     

The Catalytic Mechanism of the Marine‐Derived Macrocyclase PatGmac

Cyclic peptides are a class of compounds with high therapeutic potential, possessing bioactivities including antitumor and antiviral (including anti‐HIV). Despite their desirability, efficient design and production of these compounds has not been achieved to date. The catalytic mechanism of patellamide macrocyclization by the PatG macrocyclase domain has been computationally investigated by using quantum mechanics/molecular mechanics methodology, specifically ONIOM(M06/6‐311++G(2d,2p):ff94//B3LYP/6‐31G(d):ff94). The mechanism proposed herein begins with a proton transfer from Ser783 to His 618 and from the latter to Asp548. Nucleophilic attack of Ser783 on the substrate leads to the formation of an acyl–enzyme covalent complex. The leaving group Ala‐Tyr‐Asp‐Gly (AYDG) of the substrate is protonated by the substrate's N terminus, leading to the breakage of the P1?P1′ bond. Finally, the substrate's N terminus attacks the P1 residue, decomposing the acyl–enzyme complex forming the macrocycle. The formation and decomposition of the acyl–enzyme complex have the highest activation free energies (21.1 kcal mol^?1 and 19.8 kcal mol^?1 respectively), typical of serine proteases. Understanding the mechanism behind the macrocyclization of patellamides will be important to the application of the enzymes in the pharmaceutical and biotechnological industries.     

Kinetics and Protein‐Inhibitor Docking Studies of Enantiomers of exo‐2‐Norbornyl‐N‐n‐butylcarbamates as Pseudomonas Lipase Inhibitors to Probe the Enantioselectivity of the Enzyme

The Pseudomonas species lipase inhibition shows enantioselectivity for R‐enantiomer over S‐enantiomer of exo‐2‐norbornyl‐N‐n‐butylcarbamates. R‐, S‐, and racemic‐exo‐2‐norbornyl‐N‐n‐butylcarbamates are all characterized as pseudo substrate inhibitors of the enzyme. Thus, the mechanism for Pseudomonas species lipase‐catalyzed hydrolysis of the inhibitor is formation of the first enzyme‐inhibitor Michaelis complex via nucleophilic attack of the active site serine to the inhibitor (K_i step) then formation of the butylcarbamyl enzyme intermediate from this complex (k₂ step). Comparison of bimolecular rate constants (k_i = k₂ / K_i) of the inhibitors indicates that R‐enantiomer is 1.8 times more potent than S‐enantiomer. Thus, Pseudomonas species lipase shows enantioselectivity of 1.8 for R‐exo‐2‐norbornyl‐N‐n‐butyl‐carbamate over S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate. Protein‐ligand interaction studies on both enantiomers of exo‐2‐norbornyl‐N‐n‐butylcarbamate as inhibitors of Pseudomonas species lipase using AutoDock suggest that R‐enantiomer binds more tightly into the active site of the enzyme than S‐enantiomer. The norbornyl ring of S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate is repulsive to Ser 82 and His 251 of the catalytic triad as well as to Met 16 of the oxyanion hole. These repulsions may create few unfavorable interactions between S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate and the enzyme and make this inhibitor a less potent one.     

Synthesis and structure of a µ‐oxo diiron(III) complex with an N‐pyridylmethyl‐N,N‐bis(4‐methylbenzimidazol‐2‐yl)amine ligand and its catalytic property for hydrocarbon oxidation

A µ‐oxo diiron(III) complex [{Fe(pbba)Cl}₂(µ‐O)]Cl₂ (1, pbba = N‐pyridylmethyl‐N,N‐bis(4‐methylbenzimidazol‐2‐yl)amine) bearing multi‐imidazolyl motifs was synthesized and characterized by X‐ray crystallography to closely mimic the structural features of methane monooxygenase. As shown by its X‐ray crystal structure, complex 1 is a centrosymmetric dimer with an Fe? O? Fe angle of 180° , and pseudo‐octahedral around each iron(III) center. The catalytic ability of title compound in the oxidation of alkane and alkene is investigated by employing tert‐butylhydroperoxide and m‐chloroperbenzoic acid as oxidants under mild conditions. The catalytic oxidation results showed that radical intermediate dominates the oxidation process. Copyright © 2008 John Wiley & Sons, Ltd.     

An Organic Sol‐Gel Film as Modifier to Construct Biosensor

A new amperometric biosensor for hydrogen peroxide (H₂O₂) was developed by adsorbing hemoglobin (Hb) on an organic sol‐gel film. The organic sol‐gel was prepared using resorcinol and formaldehyde as monomers. This sol‐gel film shows a biocompatible microenvironment for retaining the native activity of the adsorbed Hb. The direct electron transfer between Hb and electrode is achieved. Hb adsorbed on the film shows an enzyme‐like catalytic activity for the reduction of H₂O₂. The reduction peak currents are proportional linearly to the concentration of hydrogen peroxide in the range of 6×10^?8 to 3.6×10^?6 M, with a detection limit of 2.4×10^?8 M (S/N=3). This research enlarges the applications of organic sol‐gel materials in biosensor field.     

A TEMPO‐Free Copper‐Catalyzed Aerobic Oxidation of Alcohols

The copper‐catalyzed aerobic oxidation of primary and secondary alcohols without an external N‐oxide co‐oxidant is described. The catalyst system is composed of a Cu/diamine complex inspired by the enzyme tyrosinase, along with dimethylaminopyridine (DMAP) or N‐methylimidazole (NMI). The Cu catalyst system works without 2,2,6,6‐tetramethyl‐l‐piperidinoxyl (TEMPO) at ambient pressure and temperature, and displays activity for un‐activated secondary alcohols, which remain a challenging substrate for catalytic aerobic systems. Our work underscores the importance of finding alternative mechanistic pathways for alcohol oxidation, which complement Cu/TEMPO systems, and demonstrate, in this case, a preference for the oxidation of activated secondary over primary alcohols.     

In situ green synthesis of Cu‐Ni bimetallic nanoparticles supported on reduced graphene oxide as an effective and recyclable catalyst for the synthesis of N‐benzyl‐N‐aryl‐5‐amino‐1H‐tetrazoles

In the present work, for the first time we have designed a novel approach for the synthesis of N‐benzyl‐N‐aryl‐5‐amino‐1H‐tetrazoles using reduced graphene oxide (rGO) decorated with Cu‐Ni bimetallic nanoparticles (NPs). In situ synthesis of Cu/Ni/rGO nanocomposite was performed by a cost efficient, surfactant‐free and environmentally benign method using Crataegus azarolus var. aronia L. leaf extract as a stabilizing and reducing agent. Phytochemicals present in the extract can be used to reduce Cu²⁺ and Ni²⁺ ions and GO to Cu NPs, Ni NPs and rGO, respectively. Analyses by means of FT‐IR, UV–Vis, EDS, TEM, FESEM, XRD and elemental mapping confirmed the Cu/Ni/rGO formation and also FT‐IR, NMR, and mass spectroscopy as well as elemental analysis were used to characterize the tetrazoles. The Cu/Ni/rGO nanocomposite showed the superior catalytic activity for the synthesis of N‐benzyl‐N‐aryl‐5‐amino‐1H‐tetrazoles within a short reaction time and high yields. Furthermore, this protocol eliminates the need to handle HN₃.     

Synthesis of metal‐free poly(1,4‐dioxan‐2‐one) by enzyme‐catalyzed ring‐opening polymerization

To avoid the harmful effects of metallic residues in poly(1,4‐dioxan‐2‐one) (PPDO) for medical applications, the enzymatic polymerization of 1,4‐dioxan‐2‐one (PDO) was carried out at 60 °C for 15 h with 5 wt % immobilized lipase CA. The lipase CA, derived from Candida antarctica, exhibited especially high catalytic activity. The highest weight‐average molecular weight (M_w = 41,000) was obtained. The PDO polymerization by the lipase CA occurred because of effective enzyme catalysis. The water component appeared to act not only as a substrate of the initiation process but also as a chain cleavage agent. A slight amount of water enhanced the polymerization, but excess water depressed the polymerization. PPDO prepared by enzyme‐catalyzed polymerization is a metal‐free polyester useful for medical applications. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 1560–1567, 2000     

Homology Modeling and Molecular Docking Studies of (S)‐Scoulerine 9‐O‐Methyltransferase from Coptis chinensis

(S)‐Scoulerine 9‐O‐methyltransferase (SMT), belonging to the S‐adenosyl‐L‐methionine (SAM)‐dependent O‐methyltransferase family, is an essential enzyme in the berberine biosynthetic pathways. In order to study the interactions of SMT with its substrate and further to understand the catalytic mechanism and substrate specificity, a three dimensional model of SMT from Coptis chinensis was constructed by homology modeling using the crystal structure of caffeic acid/5‐hydroxyferulic acid 3/5‐O‐methyltransferase (COMT) as a template. The three dimensional structure of SMT, which was mainly composed of α‐helices and some β‐sheets, was similar to that of COMT. In contrast with COMT, the non‐conserved residues in the substrate binding pocket of SMT might be responsible for their differences in the substrate specificity. Val119 and Asp254 in SMT were the key residues for orienting substrate for methylation as both residues had H‐bonds with (S)‐scoulerine. The methylation of (S)‐scoulerine involved deprotonation of the 9‐hydroxyl group by His253 and Asp254 in SMT followed by a nucleophilic attack on the SAM‐methyl resulting in the product, (S)‐tetrahydrocolumbamine.     

N‐(2‐benzimidazolylquinolin‐8‐yl)benzamidate half‐titanocene chlorides: Synthesis,characterization and their catalytic behavior toward ethylene polymerization

A series of N‐(2‐benzimidazolyquinolin‐8‐yl)benzamidate half‐titanocene chlorides, Cp′TiLCl ( C1 – C8 : Cp′ = C₅H₅, MeC₅H₄, or C₅Me₅; L = N‐(benzimidazolyquinolin‐8‐yl)benzamides)), was synthesized by the KCl elimination reaction of half‐titanocene trichlorides with the correspondent potassium N‐(2‐benzimidazolyquinolin‐8‐yl)benzamide. These half‐titanocene complexes were fully characterized by elemental and NMR analyses, and the molecular structures of complexes C2 and C8 were determined by the single‐crystal X‐ray diffraction. The high stability of the pentamethylcyclopentadienyl complex ( C8 ) was evident by no decomposing nature of its solution in air for one week. The oxo‐bridged dimeric complex ( C9 ) was isolated from the solution of the corresponding cyclopentadienyl complex ( C3 ) solution in air. Complexes C1 – C8 exhibited good to high catalytic activities toward ethylene polymerization and ethylene/α‐olefin copolymerization in the presence of methylaluminoxane (MAO) cocatalyst. In the typical catalytic system of C1/ MAO, the polymerization productivities were enhanced with either elevating reaction temperature or increasing the ratio of MAO to titanium precursor. In general, it was observed that higher the catalytic activity of the catalytic system lower the molecular weight of polyethylene. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 3154–3169, 2009     

Synthesis of 3‐Alkoxybenzo[c]thiophen‐1(3H)‐ones by Hydrolysis of N‐Substituted 3‐Alkoxybenzo[c]thiophen‐1(3H)‐imines Derived from 1‐Bromo‐2‐(dialkoxymethyl)benzenes and Isothiocyanates

A convenient procedure for the preparation of a new type of thiophthalides, 3‐alkoxybenzo[c]thiophen‐1(3H)‐ones 4 and 9 has been developed. Thus, 1‐(dialkoxymethyl)‐2‐lithiobenzenes, generated by Br/Li exchange between 2‐bromo‐1‐(dialkoxymethyl)benzenes 1 and 6 , and BuLi, react with isothiocyanates to afford N‐substituted 2‐(dialkoxymethyl)benzothioamides 2 and 7 , which, on treatment with a catalytic amount of TsOH?H₂O, give N‐substituted 3‐alkoxybenzo[c]thiophen‐1(3H)‐imines 3 and 8 . The latter are hydrolyzed under acidic conditions to the desired products 4 and 9 , respectively.     

Quantum Mechanics/Molecular Mechanics Study on the Oxygen Binding and Substrate Hydroxylation Step in AlkB Repair Enzymes

AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N¹‐methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)–oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate‐determining hydrogen‐atom abstraction on competitive σ‐ and π‐pathways on a quintet spin‐state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen‐bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained.     

Synthesis of 2′‐Deoxy‐1′‐homo‐N‐nucleosides with Anti‐Influenza Activity by Catalytic Methyltrioxorhenium (MTO)/H2O2 Oxyfunctionalization

This paper describes a new route for the synthesis of 1′‐homo‐N‐nucleoside derivatives by means of either methyltrioxorhenium (MTO) or supported MTO catalysts, with H₂O₂ as the primary oxidant. Under these selective conditions, the oxyfunctionalization of the heterocyclic ring and the N heteroatom oxidation were operative processes, regardless of the type of substrate used, that is, purine or pyrimidine derivatives. In addition, the oxidation of 1′‐homo‐N‐thionucleosides, showed the occurrence of site‐specific oxidative nucleophilic substitutions of the heterocyclic ring. The MTO/H₂O₂ system showed, in general, high reactivity under both homogeneous and heterogeneous conditions, affording the final products with high conversion values of substrates and from medium to high yields. Many of the novel 1′‐homo‐N‐nucleoside analogues were active against the influenza A virus, without any cytotoxic effects, retaining their activity in both protected and unprotected forms.