【作者】 李睿；

【导师】 冯大诚；

【作者基本信息】 山东大学 ， 物理化学， 2009， 博士

【摘要】 β-内酰胺类抗生素在对抗细菌感染方面发挥着重要作用。但是随着它们的广泛使用,细菌的抗药性也大大增加了。而产生β-内酰胺酶是耐药性菌株对β-内酰胺类抗生素产生抗药性的主要途径,它们可以催化水解β-内酰胺类抗生素中的酰胺键,使其在到达靶点之前就被开环失活。β-内酰胺酶可根据其活性点的不同而分为A、B、C、D四类。其中除B类为金属酶外,A、C、D类β-内酰胺酶在活性位点上都有丝氨酸,故又称活性位点丝氨酸酶。临床上最常见的是A和C类酶。为了对抗β-内酰胺酶,人们将β-内酰胺酶抑制剂与β-内酰胺类抗生素组成联合复方使用。β-内酰胺酶抑制剂的作用是通过抑制β-内酰胺酶以保护β-内酰胺类抗生素。它们的抗菌活性较低,但可以与β-内酰胺酶不可逆的结合,从而使其失去催化β-内酰胺类抗生素水解反应的能力,这是一种最有效的对抗β-内酰胺酶的方法。因此,了解β-内酰胺酶抑制剂的抑酶机理非常重要,它对于将来开发新的抑制剂或者新的对抗β-内酰胺酶的方法具有指导意义。目前,实验工作者已经针对β-内酰胺酶抑制剂的抑酶机理做了很多有意义的研究。但是,其中还存在着不少争议。与实验相比,理论工作显得有点落后。它们大多只是集中在抑制剂的酰化反应上,而β-内酰胺开环以后的反应,理论研究少之又少。基于这种研究现状,本论文把研究重点放在了抑制剂β-内酰胺环开环后的后续反应上。本论文取得的创新性成果主要包括以下几个方面:1.用量子化学密度泛函方法利用模型化合物详细研究了盘尼西林(penicillin)与酶作用的机理,并与抑制剂舒巴坦(sulbactam)中间体的反应进行了比较。结果显示,与先前研究的舒巴坦相比,进行的反应有很多相似之处,即可发生β-内酰胺环开环和噻唑开环反应,从而得到烯胺和亚胺的产物,这两种产物可发生相互转化。水催化对反应产生影响,水的参与使得能垒大大降低。但又不完全相同。其中,开环反应的势垒都有不同程度的提高,这使得反应进行的很困难,这可能是盘尼西林不能抑制β-内酰胺酶的主要原因。2.用量子化学结合分子反应动力学的方法研究了抑制剂克拉维酸(clavulanic acid)在抑制β-内酰胺酶过程中开环反应的机理,结果显示由于N4或C6上的氢迁移到01上而导致oxazolidine环的断裂开环,这样的开环有三种方式。这三种方式得到的产物不同,这些产物的形式分别是亚胺,顺式烯胺和反式烯胺。我们的分子动力学模拟证实了水分子可能参与这些反应,水催化大大降低了反应的势垒。3.Blanchard等人发现在抑制A类β-内酰胺酶过程中,克拉维酸酰化酶中间体能很快地脱去羧基,而舒巴坦酰化酶中间体不能够发生脱羧反应。为了解释这一现象,我们分别研究了在抑制A类β-内酰胺酶过程中,克拉维酸和舒巴坦酰化酶中间体脱羧反应的机理。结果显示,克拉维酸和舒巴坦酰化酶中间体均可发生直接的09-C3氢迁移而脱羧,但是这种反应的势垒非常高,以致其很难自发进行。除此之外,克拉维酸酰化酶中间体还可能采取分步氢迁移的反应路径,即羰基氧可以作为氢迁移的中继,它的存在使得克拉维酸中间体脱羧反应的势垒被大大降低了。4.一些新的抑制剂已经被开发出来用来对抗C类β-内酰胺酶,如6-methylidene penem和,但是它们的抑制机理还不是很明确。(a)我们用量子化学的方法研究了一种新的methylidene penem抑制剂在抑制β-内酰胺酶GC1过程中,七元环中间体形成的反应机理,以及各中间体间相互转化的反应。结果显示,七元环中间体能通过两种不同的机理,即分步机理和协同机理而形成。这两种机理分别产生了两种不同的产物。通过比较势垒,我们发现分步反应机理更容易发生,在这个机理中,得到了一种从未报道过的三角硫醇中间体。(b)对抑制剂DVR-Ⅱ-41S形成稳定的双环中间体的机理进行了研究。结果表明,侧链吡啶上的N进攻C6原子,离去的亚磺酸去进攻C7原子,得到一个三环相连的不稳定中间体,然后C6上的氢迁移到砜基氧上得到最终的烯胺式双环产物。烯胺反应物也可先通过噻唑环内的氢迁移得到亚胺式反应物,从而得到亚胺式双环产物。更多还原

【Abstract】 β-lactams are widely used in medicine as potent antibacterials.In response to their extensive use and misuse,resistance to these drugs has become widespread.β-lactamases are bacterial enzymes responsible for most resistance againstβ-lactam antibiotics.They present a serious and growing threat to the efficacy of antibacterial chemotherapy and thus pose a major challenge to human health.These defensive enzymes,prevalent in nearly every pathogenic bacterial strain,hydrolyze theβ-lactam ring and release the cleaved,inactive antibiotics.The enzyme family ofβ-lactamases is divided into four classes,according to their sequence relationships.Those of class B are zinc-dependent proteins,whereas classes A,C,D and penicillin-binding-proteins(PBPs) are active site serine enzymes(β-lactamases are believed to have evolved in bacteria from PBPs,the natural targets of(?)-lactam antibiotics).The most studied of these enzymes,clinically,biochemically,and structurally,are the serine-reactiveβ-lactamases in classes A and C,which can inactivateβ-lactam antibiotics through an acylation-deacylation mechanism.An effective approach to combat resistance is to co-administer the enzyme-susceptibleβ-lactam with aβ-lactamase inhibitor.β-lactamase inhibitor can bind toβ-lactamases in an irreversible(so-called "suicidal substrates"),consequently protecting the antibiotics.So understanding the inhibition mechanism ofβ-lactamase by inhibitors is very important,and it is potentially useful for the design of new inhibitors and new methods againstβ-lactamase.The inhibition mechanism ofβ-lactamase by inhibitors have been extensively studied experimentally.However,there is certainly no consensus as to how they proceed.Moreover,most theoretical studies were concentrated in enzymatic acylation reaction,viz.β-lactam ring opening.That is to say,the inhibition mechanisms ofβ-lactamase are not yet fully understood.Based on the above research background,our research was focused on reactions after enzymatic acylation.The following works have been done by us. 1.The density functional theory(DFT) methods were used on the model molecules of penicillin to determine the possible reactions after their acylation onβ-lactamase,and the results were compared with sulbactam we have studied.The results show that,the acylated-enzyme tetrahedral intermediate can evolves with opening ofβ-lactam ring as well as the thiazole ring;the thiazole ring-open products may be formed viaβ-lactam ring-open product or from tetrahedral intermediate directly.Those products,in imine or enamine form,can tautomerize via hydrogen migration.In virtue of the water-assisted,their energy barriers are obviously reduced. Compared with sulbactam,the energy barrier has great increase in penicillin ring-open reaction,which makes the reaction difficult to process.2.The reaction mechanism of formation of acyclic clavulanate intermediates in inhibition of class Aβ-lactamase was investigated.The oxazolidine ring undergoes cleavage as a result of proton transfer via three paths,and three different products (cis-enamine,trans-enamine,imine) are obtained.MD simulation provided evidence that water molecule can catalyze the proton transfer,and QC calculation shows water assistance can decrease the energy barrier greatly.3.Blanchard et al.(J.-E.Hugonnet,J.S.Blanchard,Biochemistry.2007,46, 11998) found clavulanate can be rapidly decarboxylated,while sulbactam can not undergo decarboxylation in the class Aβ-lactamase BlaC’s inhibition in a mass spectrometry study.In order to investigate the mechanism of decarboxylation of lactam inhibtor and explain why sulbactarn can not undergo decarboxylation,we performed a quantum chemistry computation on clavulanate and sulbactm respectively.The results show clavulanate can undergo the indirect proton transfer and the ketone oxygen atom can be as a proton relay which can decrease the energy barrier.While sulbactam can only undergo direct proton transfer and the energy barrier are too large for the mechanism to be operative.4.Some newβ-lactamase inhibitors have been explored to inactivate both class A and class Cβ-lactamases,for example,the 6-methylidene penem and the sulfone DVR-Ⅱ-41S.However,the inhibition mechanisms of these componds are still under discussion.(a) We studied the reaction mechanism of 6-methylidene penem based on a quantum chemical modeling.The results indicated the seven-membered ring intermediate which can stabilize the acyl ester bond to hydrolysis can be obtained via two possible mechanisms.Besides,a new thiirane intermediate which has never been reported was found.(b) The reaction mechanism of DVR-Ⅱ-41S was also been studied,and a new mechanism has been suggested.In the acyl-enzyme intermediate, the side chain pyridine nitrogen atom attacks the C6 atom,which is followed by cleavage of the C6-S1 bond.The unsaturated C7 is attacked by the leaving sulfinate, and a tricyclic structure can be obtained.This structure is unstable,and the proton is transferred from C6 to the sulfone to yield the bicyclic end product.更多还原