Cross-coupling of hydroxycinnamyl aldehydes into lignins

Pathways for hydroxycinnamyl aldehyde incorporation into lignins are revealed by examining transgenic plants deficient in cinnamyl alcohol dehydrogenase, the enzyme that converts hydroxycinnamyl aldehydes to the hydroxycinnamyl alcohol lignin monomers. In such plants the aldehydes incorporate into lignins via radical coupling reactions. As diagnostically revealed by long-range (13)C-(1)H correlative NMR, sinapyl aldehyde (3, 5-dimethoxy-4-hydroxy-cinnamaldehyde) 8-O-4-cross-couples with both guaiacyl (3-methoxy-4-hydroxyphenyl-propanoid) and syringyl (3, 5-dimethoxy-4-hydroxyphenyl-propanoid) units, whereas coniferyl aldehyde cross-couples only with syringyl units.     

Synthesis and Characterization of New 5‐Linked Pinoresinol Lignin Models

Pinoresinol structures, featuring a β‐β′‐linkage between lignin monomer units, are important in softwood lignins and in dicots and monocots, particularly those that are downregulated in syringyl‐specific genes. Although readily detected by NMR spectroscopy, pinoresinol structures largely escaped detection by β‐ether‐cleaving degradation analyses presumably due to the presence of the linkages at the 5 positions, in 5‐5′‐ or 5‐O‐4′‐structures. In this study, which is aimed at helping better understand 5‐linked pinoresinol structures by providing the required data for NMR characterization, new lignin model compounds were synthesized through biomimetic peroxidase‐mediated oxidative coupling reactions between pre‐formed (free‐phenolic) coniferyl alcohol 5‐5′‐ or 5‐O‐4′‐linked dimers and a coniferyl alcohol monomer. It was found that such dimers containing free‐phenolic coniferyl alcohol moieties can cross‐couple with the coniferyl alcohol producing pinoresinol‐containing trimers (and higher oligomers) in addition to other homo‐ and cross‐coupled products. Eight new lignin model compounds were obtained and characterized by NMR spectroscopy, and one tentatively identified cross‐coupled β‐O‐4′‐product was formed from a coniferyl alcohol 5‐O‐4′‐linked dimer. It was demonstrated that the 5‐5′‐ and 5‐O‐4′‐linked pinoresinol structures could be readily differentiated by using heteronuclear multiple‐bond correlation (HMBC) NMR spectroscopy. With appropriate modification (etherification or acetylation) to the newly obtained model compounds, it would be possible to identify the 5‐5′‐ or 5‐O‐4′‐linked pinoresinol structures in softwood lignins by 2D HMBC NMR spectroscopic methods. Identification of the cross‐coupled dibenzodioxocin from a coniferyl alcohol 5‐5′‐linked moiety suggested that thioacidolysis or derivatization followed by reductive cleavage (DFRC) could be used to detect and identify whether the coniferyl alcohol itself undergoes 5‐5′‐cross‐linking during lignification.     

Xylem-specific and tension stress-responsive expression of cellulose synthase genes from aspen trees

Genetic improvement of cellulose biosynthesis in woody trees is one of the major goals of tree biotechnology research. Yet, progress in this field has been slow owing to (1) unavailability of key genes from tree genomes, (2) the inability to isolate active and intact cellulose synthase complexes and, (3) the limited understanding of the mechanistic processes involved in the wood cellulose development. Here I report on the recent advances in molecular genetics of cellulose synthases (CesA) from aspen trees. Two different types of cellulose synthases appear to be involved in cellulose deposition in primary and secondary walls in aspen xylem. The three distinct secondary CesAs from aspen—PtrCesA1, PtrCesA2, and PtrCesA3—appear to be aspen homologs of Arabidopsis secondary CesAs AtCesA8, AtCesA7, and AtCesA4, respectively, based on their high identity/similarity (>80%). These aspen CesA proteins share the transmembrane domain (TMD) structure that is typical of all known “true” CesA proteins: two TMDs toward the N-terminal and six TMDs toward the C-terminal. The putative catalytic domain is present between TMDs 2 and 3. All signature motifs of processive glycosyltransferases are also present in this catalytic domain. In a phylogenetic tree based on various predicted CesA proteins from Arabidopsis and aspen, aspen CesAs fall into families similar to those seen with Arabidopsis CesAs, suggesting their functional similarity. The coordinate expression of three aspen secondary CesAs in xylem and phloem fibers, along with their simultaneous tension stress-responsive upregulation, suggests that these three CesAs may play a pivotal role in biosynthesis of better-quality cellulose in secondary cell walls of plants. These results are likely to have a direct impact on genetic manipulation of trees in the future.     

Purification and Characterization of a Zinc-Dependent Cinnamyl Alcohol Dehydrogenase from Leucaena leucocephala,a Tree Legume

A cinnamyl alcohol dehydrogenase (CAD) from the secondary xylem of Leucaena leucocephala has been purified to homogeneity through successive steps of ammonium sulfate fractionation, DEAE cellulose, Sephadex G-75, and Blue Sepharose CL-6B affinity column chromatographies. CAD was purified to 514.2 folds with overall recovery of 13 % and specific activity of 812. 5 nkat/mg. Native and subunit molecular masses of the purified enzyme were found to be ～76 and ～38 kDa, respectively, suggesting it to be a homodimer. The enzyme exhibited highest catalytic efficiency (Kcat/Km 3.75 μM^?1 s^?1) with cinnamyl aldehyde among all the substrates investigated. The pH and temperature optima of the purified CAD were pH 8.8 and 40 °C, respectively. The enzyme activity was enhanced in the presence of 2.0 mM Mg²⁺, while Zn²⁺ at the same concentration exerted an inhibitory effect. The inclusion of 2.0 mM EDTA in the assay system activated the enzyme. The enzyme was inhibited with caffeic acid and ferulic acid in a concentration-dependent manner, while no inhibition was observed with salicylic acid. Peptide mass analysis of the purified CAD by MALDI-TOF showed a significant homology to alcohol dehydrogenases of MDR superfamily.     

肿瘤酸度响应性聚(L-赖氨酸)-阿霉素键合药的制备与表征

用丁二酸酐(SA)和顺式乌头酸酐(CA)分别对阿霉素(DOX)进行修饰, 得到非酸响应的SA-DOX(SAD)和酸响应的CA-DOX(CAD). 通过SAD或CAD、端羧基化的聚乙二醇单甲醚(mPEG-COOH)与聚(L-赖氨酸)(PLL)的缩合反应, 制得非酸响应的PLL-g-mPEG/SAD和酸响应的PLL-g-mPEG/CAD键合药. 通过核磁共振氢谱和红外光谱表征键合药的化学结构, 并通过紫外-可见分光光度计测定药物键合量. 动态激光光散射研究结果表明, 两亲性的PLL-DOX键合药可以在pH=7.4的磷酸缓冲溶液中自组装形成稳定的纳米微粒. 体外释放实验及噻唑蓝检测结果表明, PLL-g-mPEG/SAD在实验pH范围和时间段内只释放出少量DOX, 不具有酸响应特性, 且对HeLa细胞增殖抑制作用较小. 而PLL-g-mPEG/CAD在生理条件(pH=7.4)下相对稳定, 在弱酸性条件(pH=5.3, 6.8)下, CAD中酸响应的酰胺键能快速水解并释放出DOX, 表现出较强的HeLa细胞增殖抑制效果.     

Differential effects of the Zn-His-Bkb vs Zn-His-[Asp/Glu] triad on Zn-core stability and reactivity

The most common partner of the Zn-bound His is the Asp/Glu carboxylate side chain in catalytic Zn sites and the backbone (Bkb) carbonyl group in structural Zn sites. To elucidate the factors governing the selection of the second-shell partner of the Zn-bound His in structural/catalytic Zn sites, systematic studies using density functional theory and continuum dielectric calculations were performed to determine the relative contributions of the second-shell Bkb carbonyl and the Asp/Glu carboxylate to the Zn-core stability and reactivity. The results show that the contributions of the second-shell Bkb carbonyl and Asp/Glu carboxylate to the Zn-core stability depend mainly on the solvent accessibility of the Zn-site and the composition of the Zn-core. They reveal the advantage of a second-shell Bkb carbonyl in anionic Zn cavities: it stabilizes anionic, buried Zn-cores more than the corresponding negatively charged Asp/Glu carboxylate, thus explaining the absence of the Zn-His-Asp/Glu triad in structural [Zn(Cys)3(His)]- cores. They also reveal the advantage of a second-shell Asp/Glu carboxylate in catalytic Zn-cores: relative to a Bkb carbonyl group, it increases (i) the HOMO energy of the cationic/neutral zinc core, (ii) the reactivity of the attacking Zn-bound OH-, (iii) electron transfer to the substrate, and (iv) the stability of the metal complex upon electron transfer. Furthermore, a second-shell Asp/Glu carboxylate could facilitate product release in the common cationic catalytic cores, by acting as a proton acceptor of the Zn-bound His creating an Asp...His- dyad that stabilizes the zinc dication more than the respective Bkb...His0 dyad.     

电分析化学研究锌在乳酸脱氨酶和谷氨酸脱氢酶体系中的作用

酶是生物催化剂，在数以千计的酶中，第一大类以烟酰胺腺嘌呤核苷酸（ＮＡＤ／ＮＡＤＨ）为辅酶的多种氧化－还原酶需要金属离子作为辅助因子而发生独特的催化功能。其中含锌的醇脱氨酶（ＡＤＨ）是目前研究得最多的一种。本文用电化学分析法研究稀土离子对乳酸脱氢酶（ＬＤＨ）和谷氨酸脱氢酶（ＧＤＨ）两体系的作用时也探讨了Ｚｎ２＋在此两体系中的作用。结果指出在ＬＤＨ体系中不需要 Ｚｎ２＋；在 ＧＤＨ（牛肝中提取）体系中， Ｚｎ２＋是一种强抑制剂，Ｅｕ３＋可以缓解它的抑制作用，但是在黄瓜根系提取的粗ＧＤＨ体系中，Ｚｎ２＋必需存在于反介质中起激活酶的催化作用。     

Non-enzymatic reduction of quinone methides during oxidative coupling of monolignols: implications for the origin of benzyl structures in lignins

Lignin is believed to be synthesized by oxidative coupling of 4-hydroxyphenylpropanoids. In native lignin there are some types of reduced structures that cannot be explained solely by oxidative coupling. In the present work we showed via biomimetic model experiments that nicotinamide adenine dinucleotide (NADH), in an uncatalyzed process, reduced a beta-aryl ether quinone methide to its benzyl derivative. A number of other biologically significant reductants, including the enzyme cellobiose dehydrogenase, failed to produce the reduced structures. Synthetic dehydrogenation polymers of coniferyl alcohol synthesized (under oxidative conditions) in the presence of the reductant NADH produced the same kind of reduced structures as in the model experiment, demonstrating that oxidative and reductive processes can occur in the same environment, and that reduction of the in situ-generated quinone methides was sufficiently competitive with water addition. In situ reduction of beta-beta-quinone methides was not achieved in this study. The origin of racemic benzyl structures in lignins therefore remains unknown, but the potential for simple chemical reduction is demonstrated here.     

Synthesis and structure of [Zn(OMe)(L)] x [Zn(OH)(L)] x 2(BPh4), L = cis,cis-1,3,5-tris[(E,E)-3-(2-furyl)acrylideneamino]cyclohexane: structural models of carbonic anhydrase and liver alcohol dehydrogenase

Direct reaction of [Zn(OH)(L)]+, L = cis,cis-1,3,5-tris[(E,E)-3-(2-furyl)acrylideneamino]cyclohexane, with methanol gives a mixture of the starting material and [Zn(OMe)(L)]+; structural analysis of the complexes shows that they are models of reactive intermediates in the catalytic cycles of the zinc enzymes carbonic anhydrase and liver alcohol dehydrogenase.     

2,2-二(4-羟基-3-氨基)苯基丙烷的合成

在乙醇介质中,以Fe(OH)3／C为催化剂,用80％水合肼将2,2-二(4-羟基-3-硝基)苯基丙烷还原为2,2-二(4-羟基-3-氨基)苯基丙烷。产率99．0％,纯度98．5％。考察了10种金属离子对催化剂活性的影响,结果发现Pb^2 会引起催化剂中毒;Mg^2 ,Cu^2 和Zn^2 钝化了催化剂的催化活性;Ba^2 和Cr^3 不影响催化剂的活性;Al^3 ,Ni^2 ,Ti^3 和Ti^4 能活化催化剂,使反应速度加快,但它们单独使用时无催化活性。     

硫堇-甲苯二异氰酸酯衍生物修饰的玻碳基乙醇传感器的研究

硫堇－甲苯二异氰酸酯衍生物修饰的玻碳基乙醇传感器的研究俞爱民,韩吉林,杨可盛,陈洪渊（南京大学化学系,南京,２１００９３）关键词硫堇－ＴＤＩ衍生物,ＮＡＤ~＋,ＡＤＨ,乙醇传感器经典的测定乙醇含量的方法有气相色谱法［１］、分光光度法［２］．近年来,随?..     

Polarographic study of zinc bound to alcohol dehydrogenase

The differential pulse polarogram of native liver alcohol dehydrogenase consists of two peaks which correspond to the reduction of zinc associated with the enzyme. On the basis of its electrochemical properties, peak I (with peak potential at −1.0 V) was attributed to the signal of zinc ions liberated from the enzyme adsorbed at the electrode surface. Peak II (with peak potential at −1.1V) probably corresponds to the reduction of zinc still bound to the enzyme molecules adsorbed at the electrode surface. The possible difference between the contributions of catalytic and structural zinc of liver alcohol dehydrogenase to the observed currents is discussed. The polarographic behaviour of the enzyme of animal origin is compared with that of yeast alcohol dehydrogenase which yields only one peak at −1.0 V.The denatured alcohol dehydrogenases give a single signal at −1.0 V which corresponds to the zinc liberated from the enzymes in the solution. The direct denaturation of liver alcohol dehydrogenase in the polarographic cell provides a quick and simple method for determination of the zinc content of the enzyme. In addition, the normal pulse polarograms of native and denatured liver alcohol dehydrogenase show considerable differences.     

486—Polarographic study of zinc bound to alcohol dehydrogenase

The differential pulse polarogram of native liver alcohol dehydrogenase consists of two peaks which correspond to the reduction of zinc associated with the enzyme. On the basis of its electrochemical properties peak I (with peak potential at ? 1.0 V) was attributed to the signal of zinc ions liberated from the enzyme adsorbed at the electrode surface. Peak II (with peak potential at ? 1.1 V) probably corresponds to the reduction of zinc still bound to the enzyme molecules adsorbed at the electrode surface. The possible difference between the contributions of catalytic and structural zinc of liver alcohol dehydrogenase to the observed currents is discussed. The polarographic behaviour of the enzyme of animal origin is compared with that of yeast alcohol dehydrogenase which yields only one peak at ? 1.0 V.The denatured alcohol dehydrogenases give a single signal at ?1.0 V which corresponds to the zinc liberated from the enzymes in the solution. The direct denaturation of liver alcohol dehydrogenase in the polarographic cell provides a quick and simple method for determination of the zinc content of the enzyme. In addition, the normal pulse polarograms of native and denatured liver alcohol dehydrogenase show considerable differences.     

Arylpropane-1,3-diols in lignins from normal and CAD-deficient pines

Significant quantities of arylopropane-1,3-diols have been identified in lignins isolated from a CAD-deficient pine mutant; smaller amounts are also present in lignins from normal pine. They arise from dihydroconiferyl alcohol via the action of peroxidases which are responsible for the radical generation steps of lignification. The structures in the complex lignin polymers are proven using 2D and 3D NMR of isolated lignin fractions.     

Factors governing the protonation state of Zn-bound histidine in proteins: a DFT/CDM study

We have performed systematic theoretical studies to elucidate the factors governing the His protonation/deprotonation state in Zn-binding sites, especially those containing the ubiquitous Zn-His-Asp/Glu triad. Specifically, we have addressed the following three questions: (1) How does the transfer of the Zn-bound His imidazole proton to the second-shell Asp/Glu carboxylate oxygen depend on the composition of the other first-shell ligands and the solvent accessibility of the metal-binding site? (2) Can any second-shell ligand with a proton acceptor group such as the backbone carbonyl oxygen also act as a proton acceptor? (3) What is the effect of the Asp/Glu in the Zn-His-Asp/Glu triad on the Zn-bound water protonation state? To address these questions, we used a combination of quantum mechanical and continuum dielectric methods to compute the free energies for deprotonating a Zn-bound imidazole/water in various Zn complexes. The calculations show that whether the Zn-bound His is protonated or deprotonated depends on (1) the solvent accessibility of the metal-binding site, and (2) the Lewis acid ability of Zn, which is indirectly determined by both the first- and the second-shell Zn ligands. The calculations also show that the effect of the Zn-His-Asp/Glu interaction on the nucleophilicity of the Zn-bound water depends on the solvent accessibility of the catalytic Zn site. Furthermore, they show that the Asp/Glu side chain in the Zn-His-Asp/Glu triad can increase the negative charge of its partner, His, and create an anionic hole that may stabilize a cation in buried cavities, provided that the Zn complex is cationic/neutral. The findings of this work are in accord with available experimental data.     

Zn(II)/amine-catalyzed coupling reaction of alkylidenemalonates with propargyl alcohol: a one-pot synthesis of methylenetetrahydrofurans

[reaction: see text] A metal-catalyzed tandem 1,4-addition/cyclization between propargyl alcohol and a Michael acceptor, such as alkylidene malonate, has been developed. In the presence of catalytic amounts of zinc triflate [Zn(OTf)(2)] and triethylamine (Et(3)N), various 2-alkylidene-1,3-dicarbonyl compounds reacted with propargyl alcohol to give 3- or 4-methylene tetrahydrofurans in excellent yields.     

Synthesis of seven-membered lactones via nickel- and zinc-catalyzed highly regio- and stereoselective cyclization of 2-iodobenzyl alcohols with propiolates

A new class of substituted seven-membered lactones 3 were conveniently synthesized via cyclization of o-iodobenzyl alcohol 1 (o-IC(6)H(4)CH(2)OH) with various propiolates 2 (RC triple bond CCOOMe) in the presence of Ni(dppe)Br(2) and Zn powder in acetonitrile at 80 degrees C. The catalytic reaction is highly regio- and stereoselective affording seven-membered lactones in moderate to good yields. This methodology can be successfully extended to various substituted o-iodobenzyl alcohols. An intermediate 7 was obtained from the reaction of 1a with methyl 2-octynoate (2a) in the presence of Ni(dppe)Br(2) and Zn at room temperature. A mechanism involving an unusual E/Z isomerization of the carbon-carbon double bond of 7 prior to lactone formation is proposed to account for the catalytic reaction.     

Catalytic mechanism and metal specificity of bacterial peptide deformylase: a density functional theory QM/MM study

Bacterial peptide deformylase (PDF) represents a novel class of mononuclear iron peptidase, and has an intriguing metal preference different from most other metalloproteases. Using a hybrid density functional theory (B3LYP) QM/MM method, we have theoretically investigated its catalytic mechanism and metal specificity by studying both Fe2+-PDF and Zn2+-PDF. In both forms of PDF, the conserved Glu133 residue is protonated in the reactant complex, and acts as a general acid during the reaction. The initial reaction step is the nucleophilic attack of the metal-bound hydroxide on the carbonyl carbon of the substrate. Our calculations indicate that the metal ion in Fe2+-PDF is always pentacoordinated during the reaction process, while that in Zn2+-PDF is only tetrahedrally coordinated and not bound to the substrate in the reactant complex. This difference in their metal coordination is suggested to account for the lower activity of Zn2+-PDF in comparison with Fe2+-PDF.     

Enzymatic method for the determination of inorganic and organic inhibitors of alcohol dehydrogenase

The inhibiting effect of heavy metal ions, organic nitrogen-containing heterocyclic compounds, and amino acids on the catalytic activity of yeast alcohol dehydrogenase (ADH) in the reaction of ethanol oxidation by nicotinamide adenine dinucleotide was detected. Sensitive procedures were developed for the determination of the most effective ADH inhibitors [mercury (II), silver(I), zinc(II), copper(II), 1,10-phenanthroline, 2,2′-dipyridyl, 8-hydroxyquinoline, quinoline, 3,4-dimethylimidazole, benzimidazole, 1,2,3-benzotriazole, histidine, tryptophan, proline, and histamine] with c_L = 5x 10^-10-1 x 10^-4M (RSD = 1–12%).     

Modulation of the reactivity, stability and substrate- and enantioselectivity of an epoxidation catalyst by noncovalent dynamic attachment of a receptor functionality--aspects on the mechanism of the Jacobsen-Katsuki epoxidation applied to a supramolecular system

The synthesis of the components of the dynamic supramolecular hydrogen-bonded catalytic system 2 + 3 is described. The catalytic performance and substrate- and enantioselectivity of Mn(salen) catalyst 2 were investigated in the presence and absence of the Zn(porphyrin) receptor unit 3. The effects of pyridine and pyridine N-oxide donor ligands were also studied. Some aspects on the mechanism of the Jacobsen-Katsuki epoxidation, based on literature observations, are introduced as a means to analyse the behaviour of 2 and its modulation by the formation of macrocycle 1 with 3. A complete association model of the metal-free system 4 + 5 refutes the earlier assumption that macrocycle 1 is the predominant form of catalyst 2 under the standard epoxidation reaction conditions with 2 + 3. Evidence are provided that receptor-binding substrates and nonbinding substrates, respectively, are epoxidised by two different catalytic species, or two distinct distributions of species in competitive epoxidations using catalytic system 2 + 3. The two species are assigned to the endo and exo faces of the Mn(salen) catalyst in macrocycle 1, and to equivalently folded oligomeric structures with monomers 2 and 3 in adjacent positions.