1、1,The professional English of pharmaceutical engineering,Wang Xinliang East China university of Science and Technology,2,內(nèi)容提要,1. 引言 2. 研究背景及理論基礎(chǔ) 3. 催化抗體的制備方法 4. 催化抗體在有機(jī)合成中的應(yīng)用 5. 催化抗體發(fā)展中存在的問(wèn)題 6. 展望,3,1.引言,1.1 催化抗體（Catalytic Antibody） 又稱異酶、抗體酶(Abzmye)是一類以過(guò)渡態(tài)類似物為半抗原通過(guò)誘導(dǎo)免疫系統(tǒng)產(chǎn)生的具有類似天然酶催化活性的免疫球蛋白（抗體）。 是化學(xué)

2、、生物學(xué)、免疫學(xué)和酶學(xué)交叉滲透融合發(fā)展起來(lái)的新生邊緣學(xué)科。 催化抗體已經(jīng)在酶學(xué)、醫(yī)學(xué)、生物工程、化工和飼料等許多領(lǐng)域顯示出潛在應(yīng)用價(jià)值。,4,1.2 國(guó)內(nèi)外的研究團(tuán)隊(duì),國(guó)外：Scripps Res Inst -Lerner group Univ Calif Berkeley -Schultz group,國(guó)內(nèi)：吉林大學(xué)分子超分子催化與材料國(guó)家實(shí)驗(yàn)室 中科院長(zhǎng)春應(yīng)用化學(xué)研究所國(guó)家重點(diǎn)實(shí)驗(yàn)室 北京大學(xué)生命科學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室,5,2.研究背景及理論基礎(chǔ),2.1 酶與抗體,相同點(diǎn)：都是蛋白質(zhì) 都能特異性地和靶分子結(jié)合 結(jié)合能相同 不同點(diǎn)：酶與化學(xué)反應(yīng)的高能過(guò)渡態(tài)結(jié)合 抗體與基態(tài)的抗原結(jié)合,L. Pau

3、ling, Am.Sci., 36, 51(1948),6,2. 研究背景及理論基礎(chǔ) （二）,2.2 猜想抗體催化的過(guò)渡態(tài)理論,如果抗體也能和過(guò)渡態(tài)特異性的結(jié)合，那么就有可能具有催化性能。 這意味著：抗體一旦能與過(guò)渡態(tài)結(jié)合，它就具有酶的性質(zhì)在溫和條件下，高效高選擇性的催化反應(yīng)。 推理：抗體若能與過(guò)渡態(tài)類似物結(jié)合，則它也就會(huì)與化學(xué)反應(yīng)過(guò)程中的過(guò)渡態(tài)結(jié)合，這樣的抗體亦就具有催化性能。,W.P.Jencks, Catalysis in Chemistry and Enzymology: McGraw-Hill, New York (1969),7,2. 研究背景及理論基礎(chǔ) （三）,Pollack S

4、 J, Jacobs J W, Schultz P G. Science, 1986, 234:1570 Tramontano A, Janda K D, Lerner R A. Science, 1986, 234：1566,2.3 抗體催化的實(shí)現(xiàn),8,3. 催化抗體的制備方法,3.1 免疫誘導(dǎo)法,3.2 免疫拷貝法,3.4 化學(xué)修飾法 把人工合成的，或天然存在的催化基團(tuán)引入到抗體的抗原結(jié)合部位，直接獲得催化抗體。 Kolesnikov V A, Kozyr A V, Alexandrova E S, Proc Natl Acad Sci USA , 2000, 97 (25) : 1352

5、6-135311.,3.3 人工構(gòu)建法 用基因工程和蛋白質(zhì)工程技術(shù)進(jìn)行人工定向構(gòu)建。 基礎(chǔ)：充分了解抗體酶分子立體構(gòu)象和基因組結(jié)構(gòu) 手段：采用定點(diǎn)突變和插入突變技術(shù)，把催化基團(tuán)引入抗體分子的抗原結(jié)合部 位，然后把DNA重組體轉(zhuǎn)化入大腸桿茵中進(jìn)行表達(dá)，獲得高效率的催化抗體。 此外：也可從生成催化抗體的細(xì)胞中提取mRNA，反轉(zhuǎn)錄后，再利用基因工程進(jìn)行高效表達(dá)。 （Baldwin E and Schultz P G. Science,1989,245,1104）,9,4. 催化抗體在有機(jī)合成中的應(yīng)用,迄今為止,科學(xué)家們已成功開發(fā)出能催化所有6 種類型的酶促反應(yīng)(歧化、水解、裂解、異構(gòu)、烷基化和氧化還

6、原)以及幾十種類型的化學(xué)反應(yīng)的抗體酶。 包括：水解、消除、縮合、氧化還原、重排、光分解和聚合、周環(huán)反應(yīng)、異構(gòu)化、環(huán)氧化、 Diels-Alder、能量不利的反應(yīng)等。,張玉彬. 生物催化的手性合成M. 北京:化學(xué)工業(yè)出版社, 2002, 50.,10,5. 催化抗體發(fā)展中存在的問(wèn)題,5.1 催化效率有待提高,一般 K酶/K非109； 現(xiàn)在 K催抗/ K非103 104，個(gè)別106 107，比酶低23個(gè)數(shù)量級(jí),5.2 抗體篩選方法需要革新,現(xiàn)有技術(shù)：根據(jù)對(duì)半抗原結(jié)合力的大小而不是活性的 高低進(jìn)行篩選，而催化活性和結(jié)合力并不一致；現(xiàn)有篩選技術(shù)篩選速度太慢。,Yang Xu, Noboru Y., K

7、im D. Janda, Bioorg. Med. Chem., 2004, 12, 52475268,11,6. 展望,a. 多克隆技術(shù) b. 催化抗體結(jié)合位點(diǎn)的化學(xué)修飾 通過(guò)對(duì)催化結(jié)合位點(diǎn)的有選擇的化學(xué)衍生，把天然或者人工合成的催化基團(tuán)引入抗體，或者利用位點(diǎn)專一性突變，引起抗體結(jié)合部位氨基酸的改變，從而提高抗體的催化性能。 c. 計(jì)算機(jī)輔助設(shè)計(jì)半抗原,Serigo Marti, vicent Miliner and Inaki Tunon Angew.Chem.Int.Ed., 2005, 44, 904,12,Words,biocatalyst antibody antigen hapt

8、en (a. haptenic) abzyme macromolecules viable covalently electrostatically coulombic interaction helix-dipole effect nucleophile (a. nucleophilic) electrophile (a. electrophilic) augment co-factors affinity lactone enantiospecific lipase -ase esterase isomerase,n. 生物催化劑 n. 抗體 n. 抗原 n. 半抗原 (a. 半抗原的)

9、n. 異酶，抗體酶 n. 大分子；高分子 a. 可行的； adv. 共價(jià)地 adv. 靜電地 庫(kù)侖(電荷)相互作用 螺旋偶極子效應(yīng) n. 親核性, 親核試劑 (a.親核性的, 親核試劑的) n. 親電性, 親電試劑 (a.親電性的, 親電試劑的) n. 增加；增大 vt. & vi. 增加；增大 n. 輔助因素；輔酶 n. 親和力；吸引力 n. 內(nèi)酯 對(duì)映特異性脂肪酶 -酶 n. 酯酶 n. 異構(gòu)化酶,13,“酶如何作用”的一個(gè)最有說(shuō)服力的理論就是：相對(duì)于反應(yīng)物和產(chǎn)物的基態(tài)的能量，它們可以降低反應(yīng)的過(guò)渡態(tài)的自由能。這一概念被用來(lái)描述催化抗體作為酶的潛在能力。,One of the most c

10、ogent theories of how enzymes work is that they lower the free energy of the transition state for a reaction relative to 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.,14,可以通過(guò)抗體的方式合成酶也許是可能的：一種方法是將

11、抗體制備到半抗原基團(tuán)上，這類似于一個(gè)給定反應(yīng)的過(guò)渡態(tài)。半抗原是能夠攜帶可刺激產(chǎn)生抗體的小分子化學(xué)半抗原基團(tuán)的大分子。,It might be possible to synthesize a enzyme by means of 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

12、haptenic groups which stimulate the production of antibodies.,15,1986年，位于加利福尼亞的兩個(gè)研究小組率先報(bào)道了制備出具有酶類似活性的抗體，即為人熟知的能催化水解酯和碳酸酯的異酶。盡管這種征求抗體的過(guò)渡態(tài)類似物的程序是第一個(gè)成功產(chǎn)生催化抗體的方法，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 abzyme

13、s which catalyze the hydrolysis of esters and carbonates. While this procedure of eliciting antibodies against transition state analogues was the first successful approach to generating catalytic antibodies, it has some limitations.,16,首先，這種方法制造的異酶的反應(yīng)速度的增強(qiáng)率僅限于103 104倍。其次，它要求必須詳細(xì)清楚地理解反應(yīng)的過(guò)渡態(tài)。再次，適當(dāng)合成穩(wěn)定

14、的過(guò)渡態(tài)類似物必須是可行的。因此，科學(xué)家們嘗試其他方法，以搜尋更有效的催化抗體。,First, abzymes produced in this way show rate enhance-ments 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 ana

15、logue must be viable. As a result, scientists have explored other methods in the search for more efficient catalytic antibodies.,17,氨基酸殘基既可以共價(jià)鍵價(jià)參與酶催化，例如在酶的活性位點(diǎn)提供親核的和一般酸堿催化作用；也通過(guò)庫(kù)侖相互作用或通過(guò)螺旋偶極子效應(yīng)，以靜電價(jià)作用參與酶催化。電荷互補(bǔ)作用也有助于免疫球蛋白所顯示的精妙的結(jié)合特異性。因此，最新的一項(xiàng)半抗原設(shè)計(jì)的策略是，采用這種靜電互補(bǔ)作用將半抗原發(fā)展成催化抗體。,Amino acid residues are i

16、nvolved in enzyme catalysis both covalently, for example contributing nucleophilic and general 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 exquis

17、ite binding specificity exhibited by immunoglobulins. Thus a recent strategy in hapten design uses such electrostatic complementarity to charged haptens to develop catalytic antibodies.,18,This approach has been called the bait and switch strategy. With this method an antibody was produced which cat

18、alyzed a -elimination of ketone. Another component of enzyme catalysis is the ordering 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.,這種做法被

19、稱為“誘導(dǎo)轉(zhuǎn)換”策略。 采用這種方法產(chǎn)生的一種抗體可以催化酮類的消除(反應(yīng))。另一個(gè)酶催化反應(yīng)的組成部分是的反應(yīng)物排序到高能量的構(gòu)象。這種催化特別是涉及到單分子過(guò)程中的熵控制，并體現(xiàn)在酶的分支酸變位酶中。,19,正如酶可通過(guò)獲取輔助因素來(lái)擴(kuò)大其催化能力，現(xiàn)在抗體也可進(jìn)行化學(xué)修飾，以增加20個(gè)天然氨基酸的功能。兩種方法已用于在抗體結(jié)合位點(diǎn)直接引入催化性官能團(tuán)：化學(xué)修飾法和位點(diǎn)特異性突變法。,Just as enzymes have expanded their catalytic repertoire through the acquisition of cofactors, antibodie

20、s have now been modified chemically in order to augment the functionality of the 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.,20,化學(xué)修飾法 抗體已被插入天然或合成的催化劑到其結(jié)合位

21、點(diǎn)，在抗體的領(lǐng)域里半抗原抗體被結(jié)合。這些催化劑包括過(guò)渡金屬配合物、輔助因素、堿或親核試劑。親核試劑通過(guò)在現(xiàn)有側(cè)鏈上的選擇性化學(xué)修飾被插入到抗體的結(jié)合位點(diǎn)。,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 m

22、etal complexes, co-factors, and bases or nucleophiles. And nucleophoiles have been incorporated into antibody combining sites by the selective chemical modification of existing side chains.,21,Site-specific mutagenesis Once important binding site residues have been pin-pointed using X-ray crystallog

23、raphy, they can be replaced by other amino acids to allow comparisons 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.,位點(diǎn)特異性突變法 一旦重要的結(jié)合位點(diǎn)的殘基已利用X射線晶體學(xué)方法準(zhǔn)確

24、找到，它們就可以被其他氨基酸所取代并比較催化效率和結(jié)合的親和力。這種方法已用于在結(jié)構(gòu)明確的膽堿脂酶的活性位點(diǎn)上進(jìn)行修飾。該酶可水解膽堿的2,4-二硝基苯酯。,22,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

25、pharmaceutical field. Since the advent of catalytic antibodies in the mid-1980s, over 70 different chemical reactions have been catalyzed by antibodies.,鑒于生物催化的巨大利益，這并不奇怪，在工業(yè)中已經(jīng)快速見(jiàn)證（或發(fā)現(xiàn)）了具有“可調(diào)變的”催化活性的蛋白質(zhì)在相關(guān)的化學(xué)工業(yè)和制藥領(lǐng)域的（應(yīng)用）潛力。自1980年代中期出現(xiàn)催化抗體，催化抗體已可以催化70多個(gè)不同的化學(xué)反應(yīng)。,23,These reactions range from ester or

26、 carbonate hydrolysis to carbon-carbon bond forming reactions, and bimolecular amide formation, so their application to general synthetic organic chemistry seems to be very promising.,這些反應(yīng)從酯或碳酸酯的水解反應(yīng)到碳-碳鍵形成反應(yīng)，以及雙分子酰胺形成反應(yīng)；所以，其應(yīng)用于一般有機(jī)合成化學(xué)似乎是非常有前途的。,24,Catalytic antibodies have a number of advantages f

27、or the organic chemist over enzymes. Enzymes 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, a

28、bzymes can be of benefit to the synthetic chemist because they are programmable.,對(duì)有機(jī)化學(xué)家而言，催化抗體比酶有很多優(yōu)勢(shì)。酶在合成中有很大的局限性。首先，他們只能對(duì)類似于天然底物的化合物起作用，然后相當(dāng)?shù)偷挠H合性。其次，有許多化學(xué)反應(yīng)并沒(méi)有已知的酶催化劑。在這兩種情況下，對(duì)合成化學(xué)家而言，異酶更有用，因?yàn)樗鼈兪恰翱删幊袒摹薄?25,The range of reactions that can be catalyzed by abzymes appears to be limited only by suffi

29、cient knowledge of the transition state 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 u

30、seful carbon-carbon bond-forming processes in organic chemistry, for which there are no known enzyme catalyst.,可被異酶催化的反應(yīng)范圍看起來(lái)很有限；只有對(duì)那些給定的反應(yīng)過(guò)渡態(tài)足夠了解而且可以合成出適當(dāng)穩(wěn)定的反應(yīng)過(guò)渡態(tài)類似物的有限的反應(yīng)。也許這方面迄今最好的例子就是制造“DielsAlderase”催化抗體。DielsAlder反應(yīng)是有機(jī)化學(xué)中最有用的碳碳鍵成形過(guò)程之一，其中并沒(méi)有任何已知的酶催化劑。,26,Therefore, the acceleration of this reac

31、tion by an abzyme has been an 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 a

32、lso resolve a racemic alcolol mixture using enantiospecific lipase activity.,因此，用異酶加快這一反應(yīng)在催化抗體領(lǐng)域是具有重要里程碑意義的，并在化學(xué)合成中具有相當(dāng)?shù)臐摿?。催化抗體的另一種用途是在手性合成。異酶已經(jīng)顯示可從內(nèi)消旋底物中制造同手性的產(chǎn)品，它們也可以利用對(duì)映體特異性脂酶的活性來(lái)拆分醇類消旋體混合物。,27,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 a 2-benzylcyclopen