棕色固氮菌变种UW45缺辅基钼铁蛋白的分离及性质研究(Ⅰ)

应用DEAE-11#纤维素柱层析,结合无氧制备电泳从棕色固氮菌变种uw45无细胞抽提液中分离得到了一种电泳纯蛋白质,回收率为18.4%。该蛋白与FeMoco重组可以催化乙炔还原成乙烯,比活可达5.85nM乙烯/分·毫克蛋白,比其无细胞抽提液活性提高22.7倍。该蛋白与FeMoco重组之后,电泳迁移率明显变慢,更接近棕色固氮菌钼铁蛋白,含铁量增加一倍,井含钼,分子量22万,可见光谱与棕色固氮菌钼铁蛋白的相同。因此,我们认为uw45缺辅基铜铁蛋白与FeMoco重组之后形成了类似棕色固氮菌钼铁蛋白的活性蛋白。     

基于ESIPT机理香豆素荧光化合物的合成和光学性质

以3-碘苯酚为原料,经过甲酰化反应、Knoevenagel缩合反应、Sonogashira偶联和还原反应得到以香豆素为母体的目标化合物7-（甲氧基苯乙炔基）香豆素-3-甲醇(5)。化合物5的结构经过¹H NMR, ¹³C NMR和MS(ESI)分析表征确认。并对化合物5的光学性质进行了研究,结果表明：化合物5具有典型的 ESIPT 发光机理,可以构建比率计量型双光子荧光探针。      

MoFe_3S_4单立方烷自兜合成及(Et_4 N)(MoFe_3S(Et_2dtc)_5]·MeCN的晶体结构

<正> 具有MoFe_3S_4单立方烷簇骼的化合物与固氮酶的钼铁蛋白及铁钼辅基存在着某些类似性,从而引起人们的极大注意。1973年卢嘉锡提出的固氮酶活性中心模型—福州模型(Ⅰ)—就具有网兜状缺口MoFe_3S_3单立方烷结构。迄今,还没有见到用简单化合物一步合成,即所谓自兜(Spontaneous self-assembly)合成单立方烷的报导。     

第四周期过渡金属钼磷三元杂多化合物的氧化还原性质研究

研究了Keggin结构钼磷杂多化合物Na₅[PM(H₂O)Mo₁₁O₃₉]·nH₂O(M=Mn²⁺,Co²⁺,Ni²⁺,Cu²⁺,Zn²⁺在溶液中的氧化还原性质,发现环境的改变可对杂多阴离子的极谱半波电位产生影响,其影响程度的大小决定于过渡金属离子本身的性质。取代后的钼磷杂多阴离子的半波电位顺序为Ni²⁺>Co²⁺>Zn²⁺>Cu²⁺>Mn²⁺,pH值的变化影响氧化还原性质,并阐述了变价金属Cu²⁺对杂多阴离子氧化还原性质的影响.     

五苯基苯基硅化合物及其原料苯乙炔基硅化合物的合成

近年来,我们从事多苯基芳基类有机硅化合物的合成。并发现这类化合物对有机硅高聚物的热稳定性有一定的影响^[1.2]。本文研究了五苯基苯基硅化合物的合成。其主要方法是以苯乙炔基有机硅化合物与四苯基环戊二烯酮为原料,通过Diels-Alder反应来合成的。有的文献认为苯乙炔基硅烷由于电子效应与位阻关系,除苯乙炔基三甲基硅烷外,一般不容易与四苯基环戊二烯酮进行缩合^[3]。根据我们的实验却得到了较好产率的各种五苯基苯基有机硅化合物,而且产物也易于分离。同时我们还对原料苯乙炔基甲基二乙氧基硅烷的合成进行了研究。     

柄型茂金属化合物(Ⅲ)──四甲基二硅桥连二(1-茚基和四氢茚基)钛和锆化合物的合成及催化乙烯聚合

1,2二(1茚基)四甲基二硅烷相继与丁基锂及MCl₄·2THF作用,生成四甲基二硅桥连二(1茚基)钛和锆化合物(Me₂SiSiMe₂)[Ind]₂MCl₂[M=Ti(1),Zr(2)].对其进行催化氢化,得到相应的四氢茚基化合物(Me₂SiSiMe₂)[IndH₄]₂MCl₂[M=Ti(3),Zr(4)].通过元素分析、MS和1HNMR谱表征了化合物的分子结构,并研究了在MAO(MethylAluminoxane)的助催化下,化合物3和4对乙烯聚合的催化性能.同锆化合物4相比,钛化合物3活性较低,但得到聚乙烯的分子量却相当高.催化剂的活性和聚乙烯的分子量都具有明显的温度效应.     

钼铁硫簇合物催化乙炔还原反应的研究

钼铁辅基的分离及其EXAFS(外延X射线精细结构)的测定结果表明,固氮酶活性中心是一种Mo-Fe-S原子簇结构.为模拟固氮酶活性中心结构及研究其性能,人们开展了钼铁硫簇合物的合成、结构和性质的研究.迄今已经合成了上百种钼铁硫簇合物,从而形成了钼铁硫簇合物化学. 固氮酶是由钼铁蛋白和铁蛋白组成的,活性中心在钼铁蛋白中,铁蛋白的作用是传递外部还原剂提供的电子.固氮酶的基本功能是具有固氮活性.生物学家在研究固氮酶的     

含吡唑酮基团的喹唑啉衍生物的合成及其作为EGFR/VEGFR-2双靶标酪氨酸激酶抑制剂

双靶标酪氨酸激酶抑制剂在克服药物抗性和减少药物毒副作用方面具有重要作用,本文设计并合成了含有吡唑酮基团的喹唑啉衍生物作为EGFR/VEGFR-2双靶标酪氨酸激酶抑制剂。目标化合物由喹唑啉中间体和吡唑酮中间体通过亲核取代反应合成。喹唑啉中间体以2,3,4-三羟基苯甲酸为原料,通过酯化、硝化、还原、氯化和环化等反应合成;吡唑酮中间体以4-取代苯基肼盐酸盐为原料,通过甲基化和氧化等反应合成。目标化合物通过¹H NMR、¹³C NMR和HR-MS进行结构鉴定。分别采用ADP-Glo激酶活性检测方法和CCK-8法测定了目标化合物对EGFR和VEGFR-2的抑制活性以及对Hela细胞、A549细胞、HUVEC细胞的抗增殖活性,其对EGFR和VEGFR-2抑制活性IC₅₀值为10～899 nM, 15～712 nM;对部分在分子水平测定表现出较高活性的化合物进行了抗增殖活性测定,所选定的化合物对人肺癌A549细胞的半抑制浓度IC₅₀值为10～267 nM,对人脐静脉内皮细胞HUVEC的半抑制浓度IC_50...     

新型二茂铁茚基化合物的设计、合成及电化学性质研究

利用Sonogashira偶联反应,以1,1¢-二碘二茂铁(I-C₅H₄FeC₅H₄-I),1,1¢-二碘联二茂铁(I-C₅H₄FeC₅H₄-C₅H₄FeC₅H₄-I)和2-乙炔茚(Ind-C≡CH)为原料,设计合成了6种新型二茂铁茚基化合物I-C₅H₄FeC₅H₄-C≡C-Ind(1,Ind为茚基), Ind-C≡C-C₅H₄FeC₅H₄-C≡C-Ind(2), I-C₅H₄FeC₅H₄-C₅H₄FeC₅H₄-C≡C-Ind(3), Ind-C≡C-C₅H₄FeC₅H₄-C₅H₄FeC₅H₄-C≡C-Ind(4), C₅H₅FeC₅H₄-C≡C-C₅H₄FeC₅H₄-C≡C-Ind(5), Fc-C≡C-C₅H₄FeC₅H₄-C₅H₄FeC₅H₄-C≡C-Ind(6,Fc为二茂铁基),其结构经¹H NMR, ¹³C NMR, MS(ESI)和X-射线单晶衍射表征,其中化合物1~3的晶体结构为首次报道。采用循环伏安法研究了化合物1~6的电化学性质。结果表明：取代基对调控二茂铁茚基衍生物二茂铁基之间的电子交互作用显著。单二茂铁茚基衍生物1和2中分别引入不对称和对称的共轭取代基团后,二茂铁基的氧化电位值升高。联二茂铁茚基衍生物3和4二茂铁基之间的电子交互作用基本不受取代基的影响。在乙炔桥相连接的取代二茂铁茚基衍生物5中,受金属-金属间距增加的影响,两个二茂铁基之间的电子交互作用减弱。不对称地引入二茂铁乙炔和乙炔茚取代基后,联二茂铁茚基衍生物6的两个二茂铁基之间的电子交互作用明显减弱。      

含钼(钨)锡键的四元金属有机杂环化合物的合成与反应研究

通过三苯基锡基-双(3,5-二甲基吡唑)甲烷与羰基钼的反应合成了钼锡键合的四元金属杂环化合物CH(3,5- Me₂Pz)₂Mo(CO)₃SnPh₃ (Pz＝1-吡唑基). 当用叔丁基异腈处理该化合物及其钨类似物时, 伴随着有机锡结构单元的丢失, 金属钼(钨)上发生还原消除并分解得到化合物CH₂(3,5-Me₂Pz)₂M(CO)₃(CNBu-t) (M为Mo或W). 另外, 该化合物与三苯基膦或亚磷酸甲酯及异丙酯反应时, 只发生羰基取代反应得到化合物CH(3,5-Me₂Pz)₂Mo(CO)₂(PR₃)SnPh₃ (R为苯基、甲氧基及异丙氧基). 而用亚磷酸苯酯与之反应, 除得到羰基取代产物外, 还伴随着P-O/C键的交换, 得到金属钼上还原消除的产物(PhO)₂PCH(3,5-Me₂Pz)₂Mo(CO)₃.     

Model for acetylene reduction by nitrogenase derived from density functional theory

The catalytic cycle of acetylene reduction at the FeMo cofactor of nitrogenase has been investigated on the basis of density functional theory. C2H2 binds to the same site as N2, but it binds to a less reduced state of the cofactor. In a manner similar to that of N2 binding, one of the sulfur bridges opens during acetylene binding. The model explains the strong noncompetitive inhibition of N2 reduction by C2H2 and the weak competitive inhibition of C2H2 reduction by N2. Our proposed mechanism is consistent with experimentally observed stereoselectivity and the ability of C2H2 to suppress H2 production by nitrogenase.     

优化固氮活性模型信息研究—双齿配体DPPE和DPPM对固氮酶促反应的影响

报道了某些双齿配体对固氮酶促反应影响的研究结果．发现１，２－双（二苯基磷）－乙烷（ＤＰＰＥ）对固氮酶酶促乙炔还原反应有促进作用，但同时又抑制了固氮酶的放氢反应。而比ＤＰＰＥ少一个－ＣＨ２－链的双（二苯基磷）－甲烷（ＤＰＰＭ）却不能表现出对固氮酶促乙炔还原活力的促进作用．对照固氮酶ＭｏＦｅ－蛋白Ｘ－光衍射结构分析结果和量子化学近似计算所导出的固氮酶活性中心结构模型提出了ＤＰＰＥ促进酶促乙炔还原反应的一种可能的解释．     

固氮酶的固氮机理和其人工模拟问题的探讨

固氮酶将N₂ 还原为NH₃ 的过程是自然界实现氮循环的重要环节。本文着重对固氮酶的固氮机理和其活性中心FeMo 辅基的人工模拟合成进行探讨, 其中包括FeMo蛋白中的质子和电子的传递, FeMo 辅基对N₂ 的活化方式,Mo 原子的作用, 固氮活性的测试。最后还就固氮酶的活性中心FeMo 辅基的人工模拟合成进行了探讨。     

Intermediates trapped during nitrogenase reduction of N triple bond N, CH3-N=NH, and H2N-NH2

A high population intermediate has been trapped on the nitrogenase active site FeMo cofactor during reduction of N2. In addition, intermediates have been trapped during reduction of CH3-N=NH by the alpha-195Gln variant and during reduction of H2N-NH2 by the alpha-70Ala/alpha-195Gln variant. Each of these trapped states shows an EPR signal arising from an S = 1/2 state of the FeMo cofactor. 15N ENDOR shows that each intermediate has a nitrogenous species bound to the FeMo cofactor, with a single type of N seen for each bound intermediate. The g tensors are unique to each intermediate, g(e) = [2.084, 1.993, 1.969], g(m) = [2.083, 2.021, 1.993], g(l) = [2.082, 2.015, 1.987], as are the 15N hyperfine couplings at g1, which suggests that three distinct stages of NN reduction may have been trapped. The 1H ENDOR spectra show that the N2 intermediate is at a distinct and earlier stage of reduction from the other two, so at least two stages of NN reduction have been trapped. Some possible structures of the hydrazine intermediate are presented.     

Modeling a central ligand in the nitrogenase FeMo cofactor

In very recent work by Einsle et al. (Science 2002, 297, 1696), a new X-ray crystallographic structure of the FeMo cofactor of nitrogenase with a central ligand was presented. The central ligand is a light atom (N, O, or C), and Einsle et al. suggest that it is nitrogen. We present density functional calculations on the FeMo cofactor, and we investigate N, O, and C as central ligands. We show that both N and O lead to energetically stable FeMo cofactor structures, whereas C is energetically unfavorable. By comparison of bond geometries with the crystallographically determined values, we show that the central ligand is most likely nitrogen.     

Studies on the hydrogenation steps of the nitrogen molecule at the Azotobacter vinelandii nitrogenase site

We follow the initial activation of the nitrogen molecule at the FeMo cofactor of nitrogenase and subsequently model the hydrogenation of N₂ up to the fourth protonation step using the intermediate neglect of differential overlap quantum-chemical model. The results obtained favor a reaction mechanism going through hydrazido intermediates on the 4-Fe surfaces, externally to the FeMo cofactor. Calculations using density functional theory on smaller model systems also support the suggested mechanism over other possible schemes that involve early release of the first molecule of ammonia as a product of the enzymatic reaction. We also demonstrate that dielectric stabilization due to the protein around the cofactor could lower markedly the barrier for the product release as an ammonium ion. © 1998 John Wiley & Sons, Inc. Int J Quant Chem 70: 1159–1168, 1998     

The consequences of an interstitial N atom in the FeMo cofactor of nitrogenase

The atom-centred FeMo cofactor of nitrogenase most likely contains N, resting at the [NMoFe7]18+ redox level, inserted from N2, and subsequently restricting the modes of binding of substrate to the NFe6 core.     

Enzymatic and catalytic reduction of dinitrogen to ammonia: Density functional theory characterization of alternative molybdenum active sites

We used density functional calculations to model dinitrogen reduction by a FeMo cofactor containing a central nitrogen atom and by a Mo‐based catalyst. Plausible intermediates, reaction pathways, and relative energetics in the enzymatic and catalytic reduction of N₂ to ammonia at a single Mo center are explored. Calculations indicate that the binding of N₂ to the Mo atom and the subsequent multiple proton–electron transfer to dinitrogen and its protonated species involved in the conversion of N₂ are feasible energetically. In the reduction of N₂ the Mo atom experiences a cycled oxidation state from Mo(IV) to Mo(VI) by nitrogenase and from Mo(III) to Mo(VI) by the molybdenum catalyst, respectively, tuning the gradual reduction of N₂. Such a wide range of oxidation states exhibited by the Mo center is crucial for the gradual reduction process via successive proton–electron transfer. Present results suggest that the Mo atom in the N‐centered FeMo cofactor is a likely alternative active site for dinitrogen binding and reduction under mild conditions once there is an empty site available at the Mo site. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005     

对棕色固氮菌-230固氮酶动力学的研究

对于棕色固氮菌-230固氮酶进行了动力学研究,发现:固氮酶是一种别构酶;ATP是固氮酶的正效应剂;ADP是固氮酶的负效应剂;ATP结合部位是固氮酶的调节部位;乙炔还原活性部位是固氮酶的催化部位;Av1与Av2之间有两个或两个以上的结合部位,这些结合部位之间存在着正协同效应,Av1与Av2的结合部位对乙炔还原活性部位有正协同效应.     

FeMo cofactor of nitrogenase: a density functional study of states M(N), M(OX), M(R), and M(I).

The M(N) S = (3)/(2) resting state of the FeMo cofactor of nitrogenase has been proposed to have metal-ion valencies of either Mo(4+)6Fe(2+)Fe(3+) (derived from metal hyperfine interactions) or Mo(4+)4Fe(2+)3Fe(3+) (from M?ssbauer isomer shifts). Spin-polarized broken-symmetry (BS) density functional theory (DFT) calculations have been undertaken to determine which oxidation level best represents the M(N) state and to provide a framework for understanding its energetics and spectroscopy. For the Mo(4+)6Fe(2+)Fe(3+) oxidation state, the spin coupling pattern for several spin state alignments compatible with S = (3)/(2) were generated and assessed by energy and geometric criteria. The most likely BS spin state is composed of a Mo3Fe cluster with spin S(a) = 2 antiferromagnetically coupled to a 4Fe' cluster with spin S(b) = (7)/(2). This state has a low DFT energy for the isolated FeMoco cluster and the lowest energy when the interaction with the protein and solvent environment is included. This spin state also displays calculated metal hyperfine and M?ssbauer isomer shifts compatible with experiment, and optimized geometries that are in excellent agreement with the protein X-ray data. Our best model for the actual spin-coupled state within FeMoco alters this BS state by a slight canting of spins and is analogous in several respects to that found in the 8Fe P-cluster in the same protein. The spin-up and spin-down components of the LUMO contain atomic contributions from Mo(4+) and the homocitrate and from the central prismane Fe sites and muS(2) atoms, respectively. This qualitative picture of the accepting orbitals for M(N) is consistent with observations from M?ssbauer spectra of the one-electron reduced states. Similar calculations for the Mo(4+)4Fe(2+)3Fe(3+) oxidation state yield results that are in poorer agreement with experiment. Using the Mo(4+)6Fe(2+)Fe(3+) oxidation level as the most plausible resting state, the geometric, electronic and energetic properties of the one-electron redox transition to the oxidized state, M(OX), catalytically observed M(R) and radiolytically reduced M(I) states have also been explored.