制药工程专业英语

1、制药工程专业英语制药工程专业英语第一页，共29页。内容提要v1. 引言引言v2. 研究背景及理论基础研究背景及理论基础v3. 催化抗体的制备方法催化抗体的制备方法v4. 催化抗体在有机合成中的应用催化抗体在有机合成中的应用v5. 催化抗体发展中存在的问题催化抗体发展中存在的问题v6. 展望展望第二页，共29页。1.引言1.1 催化抗体（Catalytic Antibody） 又称异酶、抗体酶(Abzmye)是一类以过渡态类似物为半抗原通过诱导免疫系统产生的具有类似天然酶催化活性的免疫球蛋白（抗体）。 是化学、生物学、免疫学和酶学交叉渗透融合发展起来的新生边缘学科。 催化抗体已经在酶学、医学、生

2、物工程、化工和饲料等许多领域显示出潜在应用价值。第三页，共29页。1.2 国内外的研究团队国外：Scripps Res Inst -Lerner group Univ Calif Berkeley -Schultz group国内：吉林大学分子超分子催化与材料国家实验室 中科院长春应用化学研究所国家重点实验室 北京大学生命科学国家重点实验室第四页，共29页。2.研究背景及理论基础2.1 酶与抗体 相同点：都是蛋白质 都能特异性地和靶分子结合 结合能相同不同点：酶与化学反应的高能过渡态结合 抗体与基态的抗原结合L. Pauling, Am.Sci., 36, 51(1948) 第五页，共29页。

3、2. 研究背景及理论基础 （二）2.2 猜想抗体催化的过渡态理论 如果抗体也能和过渡态特异性的结合，那么就有可能具有催化性能。 这意味着：抗体一旦能与过渡态结合，它就具有酶的性质在温和条件下，高效高选择性的催化反应。 推理：抗体若能与过渡态类似物结合，则它也就会与化学反应过程中的过渡态结合，这样的抗体亦就具有催化性能。W.P.Jencks, Catalysis in Chemistry and Enzymology: McGraw-Hill, New York (1969)第六页，共29页。2. 研究背景及理论基础 （三）Pollack S J, Jacobs J W, Schultz P G

4、. Science, 1986, 234:1570Tramontano A, Janda K D, Lerner R A. Science, 1986, 234：1566 2.3 抗体催化的实现O2NOOON底 物O2NOOOHO过 渡 态O2NPOOON过 渡 态 类 似 物aNF3COOONHO底 物NOHNONHPHNF3COOOHOOOCOO过 渡 态 类 似 物ba: Schultz group, University of California, Berkeeyb: Lerner group, Research Institue of Scripps clinic12Kcat/K

5、= 770Kcat/K = 960第七页，共29页。3. 催化抗体的制备方法3.1 免疫诱导法3.2 免疫拷贝法 3.4 化学修饰法把人工合成的，或天然存在的催化基团引入到抗体的抗原结合部位，直接获得催化抗体。Kolesnikov V A, Kozyr A V, Alexandrova E S, Proc Natl Acad Sci USA , 2000, 97 (25) : 13526-135311. 3.3 人工构建法 用基因工程和蛋白质工程技术进行人工定向构建。基础：充分了解抗体酶分子立体构象和基因组结构手段：采用定点突变和插入突变技术，把催化基团引入抗体分子的抗原结合部 位，然后把DN

6、A重组体转化入大肠杆茵中进行表达，获得高效率的催化抗体。此外：也可从生成催化抗体的细胞中提取mRNA，反转录后，再利用基因工程进行高效表达。 （Baldwin E and Schultz P G. Science,1989,245,1104）第八页，共29页。4. 催化抗体在有机合成中的应用 迄今为止,科学家们已成功开发出能催化所有6 种类型的酶促反应(歧化、水解、裂解、异构、烷基化和氧化还原)以及几十种类型的化学反应的抗体酶。 包括：水解、消除、缩合、氧化还原、重排、光分解和聚合、周环反应、异构化、环氧化、 Diels-Alder、能量不利的反应等。张玉彬. 生物催化的手性合成M. 北京:化

7、学工业出版社, 2002, 50.第九页，共29页。5. 催化抗体发展中存在的问题5.1 催化效率有待提高一般 K酶/K非109；现在 K催抗/ K非103 104，个别106 107，比酶低23个数量级5.2 抗体筛选方法需要革新现有技术：根据对半抗原结合力的大小而不是活性的 高低进行筛选，而催化活性和结合力并不一致；现有筛选技术筛选速度太慢。Yang Xu, Noboru Y., Kim D. Janda, Bioorg. Med. Chem., 2004, 12, 52475268第十页，共29页。6. 展望 a. 多克隆技术b. 催化抗体结合位点的化学修饰 通过对催化结合位点的有选择的

8、化学衍生，把天然或者人工合成的催化基团引入抗体，或者利用位点专一性突变，引起抗体结合部位氨基酸的改变，从而提高抗体的催化性能。c. 计算机辅助设计半抗原Serigo Marti, vicent Miliner and Inaki Tunon Angew.Chem.Int.Ed., 2005, 44, 904第十一页，共29页。Wordsbiocatalystantibodyantigenhapten (a. haptenic)abzyme macromoleculesviablecovalentlyelectrostaticallycoulombic interactionhelix-dipo

9、le effectnucleophile (a. nucleophilic)electrophile (a. electrophilic)augmentco-factorsaffinitylactoneenantiospecific lipase -aseesteraseisomerasen. 生物催化剂n. 抗体n. 抗原n. 半抗原 (a. 半抗原的)n. 异酶，抗体酶n. 大分子；高分子a. 可行的；adv. 共价地adv. 静电地 库仑(电荷)相互作用 螺旋偶极子效应n. 亲核性, 亲核试剂 (a.亲核性的, 亲核试剂的)n. 亲电性, 亲电试剂 (a.亲电性的, 亲电试剂的)n. 增

10、加；增大 vt. & vi. 增加；增大 n. 辅助因素；辅酶n. 亲和力；吸引力n. 内酯对映特异性脂肪酶 -酶 n. 酯酶n. 异构化酶第十二页，共29页。 “酶如何作用”的一个最有说服力的理论就是：相对于反应物和产物的基态的能量，它们可以降低反应的过渡态的自由能。这一概念被用来描述催化抗体作为酶的潜在能力。 One of the most cogent theories of how enzymes work is that they lower the free energy of the transition state for a reaction relative to

11、the energy of the ground states for the reactants and products. This concept was used to describe the potential capacity antibodies to act as enzymes. 第十三页，共29页。 可以通过抗体的方式合成酶也许是可能的：一种方法是将抗体制备到半抗原基团上，这类似于一个给定反应的过渡态。半抗原是能够携带可刺激产生抗体的小分子化学半抗原基团的大分子。 It might be possible to synthesize a enzyme by means o

12、f antibodies: One way to do this to prepare an antibody to a haptenic group which resembles the transition state of a given reaction. Haptens are macromolecules that carry small chemical haptenic groups which stimulate the production of antibodies.第十四页，共29页。 1986年，位于加利福尼亚的两个研究小组率先报道了制备出具有酶类似活性的抗体，即为

13、人熟知的能催化水解酯和碳酸酯的异酶。尽管这种征求抗体的过渡态类似物的程序是第一个成功产生催化抗体的方法，但它仍有一些局限性。 In 1986, two research groups in California first reported the production of antibodies with enzyme-like activities, known as abzymes which catalyze the hydrolysis of esters and carbonates. While this procedure of eliciting antibodies aga

14、inst transition state analogues was the first successful approach to generating catalytic antibodies, it has some limitations.第十五页，共29页。 首先，这种方法制造的异酶的反应速度的增强率仅限于103 104倍。其次，它要求必须详细清楚地理解反应的过渡态。再次，适当合成稳定的过渡态类似物必须是可行的。因此，科学家们尝试其他方法，以搜寻更有效的催化抗体。 First, abzymes produced in this way show rate enhance-ment

15、s usually limited to between 103and 104. Second, it requires that the transition state for the reaction needs to be understood in some detail. Third, synthesis of a suitably stable transition state analogue must be viable. As a result, scientists have explored other methods in the search for more ef

16、ficient catalytic antibodies.第十六页，共29页。 氨基酸残基既可以共价键价参与酶催化，例如在酶的活性位点提供亲核的和一般酸碱催化作用；也通过库仑相互作用或通过螺旋偶极子效应，以静电价作用参与酶催化。电荷互补作用也有助于免疫球蛋白所显示的精妙的结合特异性。因此，最新的一项半抗原设计的策略是，采用这种静电互补作用将半抗原发展成催化抗体。 Amino acid residues are involved in enzyme catalysis both covalently, for example contributing nucleophilic and gener

17、al acid-base catalysis at the active site of the enzymes, and electrostatically, either through coulombic interactions or via helix-dipole effects. Charge-charge complementarity also contributes to the exquisite binding specificity exhibited by immunoglobulins. Thus a recent strategy in hapten desig

18、n uses such electrostatic complementarity to charged haptens to develop catalytic antibodies.第十七页，共29页。 This approach has been called the bait and switch strategy. With this method an antibody was produced which catalyzed a -elimination of ketone. Another component of enzyme catalysis is the orderin

19、g of reactants into high energy conformations. Such catalysis particularly involves control of the entropy in the transition state for unimolecular processes and is exemplified by the enzyme chorismate mutase. 这种做法被称为“诱导转换”策略。 采用这种方法产生的一种抗体可以催化酮类的消除(反应)。另一个酶催化反应的组成部分是的反应物排序到高能量的构象。这种催化特别是涉及到单分子过程中的熵

20、控制，并体现在酶的分支酸变位酶中。第十八页，共29页。 正如酶可通过获取辅助因素来扩大其催化能力，现在抗体也可进行化学修饰，以增加20个天然氨基酸的功能。两种方法已用于在抗体结合位点直接引入催化性官能团：化学修饰法和位点特异性突变法。 Just as enzymes have expanded their catalytic repertoire through the acquisition of cofactors, antibodies have now been modified chemically in order to augment the functionality of t

21、he 20 natural amino acids. Two methods have been used for the direct introduction of catalytic groups into the antibody combining site: chemical modification and site specific and mutagenesis.第十九页，共29页。化学修饰法 抗体已被插入天然或合成的催化剂到其结合位点，在抗体的领域里半抗原抗体被结合。这些催化剂包括过渡金属配合物、辅助因素、碱或亲核试剂。亲核试剂通过在现有侧链上的选择性化学修饰被插入到抗体的

22、结合位点。Chemical modification Antibodies have been modified by the incorporation of natural or synthetic catalysts into their combining sites, the areas in the antibody where the haptens are bound. These have included transition metal complexes, co-factors, and bases or nucleophiles. And nucleophoiles

23、have been incorporated into antibody combining sites by the selective chemical modification of existing side chains.第二十页，共29页。Site-specific mutagenesis Once important binding site residues have been pin-pointed using X-ray crystallography, they can be replaced by other amino acids to allow compariso

24、ns of catalytic rates and binding affinities. This method was used to modify the active site of a structurally resolved choline esterase. This enzyme hydrolyses 2, 4-dinitrophenyl esters of choline.位点特异性突变法 一旦重要的结合位点的残基已利用X射线晶体学方法准确找到，它们就可以被其他氨基酸所取代并比较催化效率和结合的亲和力。这种方法已用于在结构明确的胆碱脂酶的活性位点上进行修饰。该酶可水解胆碱的

25、2,4-二硝基苯酯。第二十一页，共29页。 In view of the tremendous interest in biocatalysis, it is not surprising that industry has been quick to see the potential of proteins with tuneable catalytic activity in relation to both the chemical industry and the pharmaceutical field. Since the advent of catalytic antibodi

26、es in the mid-1980s, over 70 different chemical reactions have been catalyzed by antibodies. 鉴于生物催化的巨大利益，这并不奇怪，在工业中已经快速见证（或发现）了具有“可调变的”催化活性的蛋白质在相关的化学工业和制药领域的（应用）潜力。自1980年代中期出现催化抗体，催化抗体已可以催化70多个不同的化学反应。第二十二页，共29页。These reactions range from ester or carbonate hydrolysis to carbon-carbon bond forming r

27、eactions, and bimolecular amide formation, so their application to general synthetic organic chemistry seems to be very promising. 这些反应从酯或碳酸酯的水解反应到碳-碳键形成反应，以及双分子酰胺形成反应；所以，其应用于一般有机合成化学似乎是非常有前途的。第二十三页，共29页。 Catalytic antibodies have a number of advantages for the organic chemist over enzymes. Enzymes

28、have significant limitations in synthesis. First, they only work on compounds similar to the natural substrate and then with rather low affinities. Second, there are many chemical reactions for which there is no known enzyme catalyst. In both situation, abzymes can be of benefit to the synthetic che

29、mist because they are programmable. 对有机化学家而言，催化抗体比酶有很多优势。酶在合成中有很大的局限性。首先，他们只能对类似于天然底物的化合物起作用，然后相当低的亲合性。其次，有许多化学反应并没有已知的酶催化剂。在这两种情况下，对合成化学家而言，异酶更有用，因为它们是“可编程化的”。第二十四页，共29页。 The range of reactions that can be catalyzed by abzymes appears to be limited only by sufficient knowledge of the transition sta

30、te for any given reaction and the possibility of synthesis of an appropriately stable transition state analogue. Perhaps the best example of this to date has been the production of a Diels-Alderase catalytic antibody. The Diels-Alder reaction is one of the most useful carbon-carbon bond-forming proc

31、esses in organic chemistry, for which there are no known enzyme catalyst. 可被异酶催化的反应范围看起来很有限；只有对那些给定的反应过渡态足够了解而且可以合成出适当稳定的反应过渡态类似物的有限的反应。也许这方面迄今最好的例子就是制造“DielsAlderase”催化抗体。DielsAlder反应是有机化学中最有用的碳碳键成形过程之一，其中并没有任何已知的酶催化剂。第二十五页，共29页。 Therefore, the acceleration of this reaction by an abzyme has been an

32、 important landmark in the field or catalytic antibodies and of considerable potential for chemical synthesis. Another use for catalytic antibodies is in chiral synthesis. Already abzymes have been shown to produce homochiral products from a meso substrate and they can also resolve a racemic alcolol

33、 mixture using enantiospecific lipase activity. 因此，用异酶加快这一反应在催化抗体领域是具有重要里程碑意义的，并在化学合成中具有相当的潜力。催化抗体的另一种用途是在手性合成。异酶已经显示可从内消旋底物中制造同手性的产品，它们也可以利用对映体特异性脂酶的活性来拆分醇类消旋体混合物。第二十六页，共29页。 Although most abzyme catalyzed reactions have been carried out on a small scale, a publication has recently shown that enantioselective reactions can be performed on a gram scale, as in the enantioselective hydrolysis of