SHANGHAI JIAO TONG UNIVERSITY 学士学位论文 THESIS OF BACHELOR 论文题目 : D-NNA 手性转化中转氨酶的鉴定 学生姓名 : 陈忠炜 学生学号 : 专 业 : 药 学 指导教师 : 郝 彬 学院 ( 系 ): 药学院

SHANGHAI JIAO TONG UNIVERSITY 学士学位论文 THESIS OF BACHELOR 论文题目 : D-NNA 手性转化中转氨酶的鉴定 学生姓名 : 陈忠炜 学生学号 : 5061719022 专 业 : 药 学 指导教师 : 郝 彬 学院 ( 系 ): 药学院

上海交通大学 本科生毕业设计 ( 论文 ) 任务书 课题名称 : D-NNA 手性转化中转氨酶的鉴定 执行时间 : 2010 年 3 月至 2010 年 6 月 教师姓名 : 郝彬 职称 : 助研 学生姓名 : 陈忠炜 学号 : 5061719022 专业名称 : 药学 学院 ( 系 ): 药学院

毕业设计 ( 论文 ) 基本内容和要求 : 王教授课题组在国际率先发现 D- 硝基精氨酸 (D-NNA) 的体内手性转化, 并提出 D-NNA 手性转化的两步反应机制 : 即 D-NNA 首先在 D- 氨基酸氧化酶 ( D-amino acid oxidase, DAO) 作用下氧化脱氨生成 α- 酮酸 (nitro-5-gunidino-2-oxopentanoic acid), 后者在转氨酶参与下, 通过立体选择性获取氨基生成 L-NNA(Xin et al.,2005,2007) 本研究在此基础上, 利用体外酶学实验, 确定参与 D-NNA 手性转化的转氨酶特异亚类, 期望完整地阐明 D-NNA 的体内手性转化机制, 为 D- 氨基酸体内代谢揭示新的通路 实验内容 :1 本实验应用 L-nitro-Arginine 与 pyruvic acid( 或 oxaloacetate 或 a-ketoglutarate) 在辅酶磷酸吡哆醛 (PLP) 及磷酸吡哆胺 (PMP) 作用下发生转氨基反应生成 nitro-argketoanalog, 此酮酸可与 2- 硝基苯肼发生反应, 生成黄色固体 借助于此转氨基反应考察与 L-nitro-Arginine 相关的转氨酶 2 D-nitro-Arginine 在 DAO 作用下可生成 L-nitro-Argketoanalog, 向孵育体系中加入不同转氨酶 ( Aminotransferase: AspAT, AlaAT, Aromatic amino acid aminotransferase,branched-chain Amino Acid Aminotransferase), 考察 L-nitro-Arginine 的生成量 pyruvic acid PLP PMP Ala L-nitro-Arginine + oxaloacetate ------------- Asp + nitro-argketoanalog a-ketoglutarate Glu aminotransferase

nitro-argketoanalog + 2-nitrophenylhydrazine 2-nitrophenylhydrazone( yellow )

毕业设计 ( 论文 ) 进度安排 : 序号 毕业设计 ( 论文 ) 各阶段内容 时间安排 备注 1 α- 酮酸的制备 3.1-3.31 2 转氨酶体外孵育 4.1-4.31 3 肾脏中转氨酶亚型鉴定 5.1-6.30 课题信息 : 课题性质 : 设计 论文 课题来源 * : 国家级 省部级 校级 横向 预研 项目编号其他 指导教师签名 : 年 月 日 学院 ( 系 ) 意见 : 院长 ( 系主任 ) 签名 : 年月日 学生签名 : 年月日

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D-NNA 手性转化中转氨酶的鉴定 摘要 手性药物代谢动力学对于药物研究具有重要意义, 手性药物在代谢过程中具有立体选择性, 从而导致异构体在体内的药理活性强度 性质和毒副作用方面都可能不同 研究表明 D- 氨基酸在体内发生手性转化具有一定普遍性, 但机制尚不清楚 王永祥课题组在国际率先发现 D- 硝基精氨酸 (N G -nitro-d-arginine, D-NNA) 的体内手性转化, 并提出 D- 硝基精氨酸手性转化的两步反应机制 : 即 D- 硝基精氨酸首先在 D- 氨基酸氧化酶 (D-amino acid oxidase, DAO) 作用下氧化脱氨生成 α- 酮酸 (N G- nitro-5-gunidino-2-oxopentanoic acid), 后者在转氨酶参与下, 通过立体选择性获取氨基生成 L- 硝基精氨酸 本文在此基础上期望通过体外孵育的方法寻找转氨酶, 以阐明 D- 硝基精氨酸的体内手性转化机制, 为最终揭示这条药物代谢新途径奠定基础 D- 硝基精氨酸的手性转化现象的发现源于 D- 硝基精氨酸所表现出体内和体外的巨大活性差异 硝基精氨酸对一氧化氮合酶 (nitric oxide synthase, NOS) 活性的抑制具有立体选择性, 在体外 L- 硝基精氨酸 (N G -nitro-l-arginine, L-NNA) 可高活性地抑制 NOS, 而其 D- 异构体对 NOS 则几乎无抑制作用 但是研究发现在大鼠体内 D- 硝基精氨酸具有与 L- 硝基精氨酸类似的升高动脉血压的生物活性 根据 D- 硝基精氨酸体内的作用特点, 研究者认为 D- 硝基精氨酸可能在体内发生手性转化, 随后 D- 硝基精氨酸的手性转化假设被证实 本研究首先建立了高效液相色谱 (HPLC) 拆分手性 D- 硝基精氨酸和 L- 硝基精氨酸的方法, 快速并且分辨良好 分离柱为 C18 耐水柱, 前装保护柱, 分离流动相条件为 1% 甲醇和 99% 醋酸铵缓冲液 ( 含 2 mm 阿斯巴甜和 1mM 硫酸铜 )(V/V), 流速 0.8 毫升 / 分钟, 紫外检测波长 280nm 在此基础上制作了 L- 硝基精氨酸的标准曲线, 曲线结果表明 HPLC 的方法结果比较精确, 线性度良好 本研究首先制备 α- 酮酸, 通过 D- 硝基精氨酸和 D 型氨基酸氧化酶反应, 加入过氧化氢酶防止生成的过氧化氢将 α- 酮酸氧化脱去羰基为相应的羧酸, 在反应足够长时间后生成 α- 酮酸 通过截留分子量 3000kDa 的超滤管滤去 D 型氨基酸氧化酶, 合成的 α- 酮酸进行了质谱和 13C-NMR 检验, 证明了生成物 α- 酮酸 本研究随后证实了肾脏匀浆中 D- 硝基精氨酸和 α- 酮酸的转化, 在 ph=8.2 的 TrisHCl 缓冲液体外孵育中,D- 硝基精氨酸和 α- 酮酸均有不同程度的转化, 在 L- 硝基精氨酸的位置上出现了峰,α- 酮酸的转化量和 L- 硝基精氨酸的生成量随着孵育时间的延长而增加 本研究拆分肾匀浆组分, 分为肾匀浆胞浆部分和粗制线粒体部分, 确定了肾匀浆胞浆部分和粗制线粒体中 D- 硝基精氨酸和 α- 酮酸的转化, 两者转化的量均十分强烈 为了探究线粒体中转氨酶转化 α- 酮酸的情况, 本研究又使用不连续蔗糖梯度浓度的方法, 超速离心得到纯化的线粒体, 体外孵育证明了纯线粒体中存在能够将 α- 酮酸转化为 L- 硝基精氨酸的转氨酶, 但是转化速率不高, 而且因为超速离心的原因失去了辅酶 5- 磷酸吡哆醛和大部分的氨基供体 之后的实验考察了线粒体中此类转氨酶对于氨基供体的选择性, 证明丙氨酸和谷氨酸是较好的氨基供体, 相对来说 L- 亮氨酸 L- 缬氨酸 L- 苏氨酸等氨基酸较

差 本研究在粗制肾匀浆胞浆的基础上进行了一定程度的纯化, 得到了 20,000 g 的上清 探索了胞浆中转氨酶对于氨基供体的选择性, 结果表明丙氨酸是现在阶段胞浆转氨酶最有效的氨基供体, 苯丙氨酸 谷氨酸次之, 而亮氨酸较差 本研究同时考察了天冬氨酸转氨酶 (Aspartate Aminotransferase, AST, GOT, EC 2.6.1.1) 对于 α- 酮酸转氨反应的催化能力, 最终结果表明, 无论增加酶浓度或者延长孵育时间, 天冬氨酸转氨酶不具有将此 α- 酮酸转化为 L- 硝基精氨酸的能力, 排除了天冬氨酸转氨酶转化 α- 酮酸的可能性 本研究又考察了丙氨酸转氨酶 (Alanine Aminotransferase, ALT, GPT, EC 2.6.1.2) 催化转化 α- 酮酸的能力, 在长时间的孵育中, 有 L- 硝基精氨酸的生成, 虽然速率不高, 仍然表明了丙氨酸转氨酶在体外孵育中有将此 α- 酮酸转化为 L- 硝基精氨酸的能力 本研究还考察了支链氨基酸转氨酶 (Branched-chain aminotransferase, BCAT, EC 2.6.1.42) 体外催化转化 α- 酮酸的能力 在半个小时的孵育中, 有相当量的 L- 硝基精氨酸生成, 表明支链氨基酸转氨酶对于 α- 酮酸的转化能力十分强大 但由于支链氨基酸转氨酶在肾里的含量未知, 因此不能断定支链氨基酸转氨酶是体内转化 α- 酮酸的主要转氨酶 在支链氨基酸转氨酶体外孵育的基础上, 本研究利用胞浆支链氨基酸转氨酶 (BCATc, cytosolic brached-chain aminotransferase) 专一性竞争性抑制剂加巴喷丁 (Gabapentin), 将加巴喷丁以不同浓度加入已经加入 L- 亮氨酸的胞浆中, 证明了加巴喷丁能够抑制胞浆对于 α- 酮酸的转化和 L- 硝基精氨酸的生成, 随着浓度的升高, 抑制作用加强, 但是不能完全抑制 L- 硝基精氨酸的生成, 表明了很可能有其它的转氨酶参与了 α- 酮酸的转氨反应 在将加巴喷丁加入线粒体后, 对比未加加巴喷丁的线粒体反应, 加巴喷丁不能抑制线粒体对于 α- 酮酸的转化 通过多方面的实验, 本研究证明了 D- 硝基精氨酸手性转化两步机制中的第二步, 证明了丙氨酸转氨酶和支链氨基酸转氨酶对于 α- 酮酸转化的能力, 证明了天冬氨酸转氨酶不能催化转化这个反应, 同时加巴喷丁实验证明了胞浆支链氨基酸转氨酶在体内参与了 α- 酮酸的转氨反应 本研究成果对最终揭示这条药物代谢通路和增加手性药物代谢动力学新内容具有一定的学术意义 关键词 D- 硝基精氨酸, 手性转化, 高效液相色谱,α- 酮酸

STUDY OF AMINOTRANSFERASE IN THE CHIRAL INVERSION OF D-NNA ABSTRACT Enantimoers may differ significantly not only in their bioavailability, rate of metabolism and excretion, but also in pharmacological active strength, properties and toxic effects, so study of enantimoers has great significance to drug studies. The importance of chiral pharmacokinetics and chiral inversion has gained greater attention due to recent development of chiral separation technology. Recent studies have indicated the universality of chiral inversion of D-amino acids, however, the mechanism of chiral inversion is not fully revealed. King's Lab has discovered the two-step metabolic inversion of D-NNA(N G -nitro-d-arginine), which is D-NNA, catalyzed by DAO(D-amino acid oxidase), is oxidized and deaminized to α-keto acid(n G -nitro-5-gunidino-2-oxopentanoic acid), then α-keto acid obtains the amino group with the catalysis of aminotransferases and is converted to L-NNA(N G -nitro-l-arginine), an inhibitor of nitric oxide sythase. Derived from the result, this study aimed to study the mechanism of transaminaiton between α-keto acid and L-NNA and establish a solid foundation of the metabolic pathway of this newly discovered drug. The chiral inversion of D-NNA was first discovered through the enormous difference of activation of D-NNA in vivo and in vitro. NNA inhibits nitric oxide synthase(nos) with structural specification. In vitro, L-NNA is able to inhibit nitric oxide synthase with high activation, while D-NNA does not have that ability. However, further study shows that in vivo, D-NNA gains the ability to inhibit nitric oxide synthase just like L-NNA. Based on the function and properties of D-NNA, researchers hold the opinion that D-NNA has performed a chiral inversion in vivo. Later, the hypothesis of the chiral inversion of D-NNA was confirmed. The chiral seperation of D-NNA, L-NNA and α-keto acid was achieved by chiral ligand exchange method via high performance liquid chromatography with high resolution and efficiency. The column is C18 water-tolerant column with protection column installed before. The mobile phase condition is 1% methanol and 99% ammonium acetate buffer solution with 2 mm aspartame and 1mM copper sulfate, the flow rate is 0.8ml/min and the UV detection wavelength is 280nm. Grounded on the condition, the standard curve of L-NNA was determined, which demonstrated a good linear relation. α-keto acid was prepared firstly. It was by the oxidation reaction catalyzed by D-amino acid oxidase from D-NNA that α-keto acid was produced. And after ultrafiltration at a molecular cut off at 3000kDa, α-keto acid was proved by mass spectrum and 13 C-NMR. The inversion of D-NNA and α-keto acid was observed in kidney homogenate. Incubated in

the TrisHCl buffer(50 mm, ph = 8.2), the peak of generated L-NNA was clearly detected in HPLC chromatogram. As the incubation time extended, the quantity of product increased. Then the kidney homogenate was separated to cytosol and crude mitochondria, using the differential centrifugation. The following incubation experiment confirmed that both in cytosolic part and crude mitochondria were able to provide a good condition for both the chiral inversion of D-NNA and the transamination reaction of α-keto acid. As the incubation time was prolonged, the generation of L-NNA increased significantly. To study the aminotranferases in mitochondria, it is necessary to purify the crude mitochondria for a condition of more simplicity. Purified mitochondria were separated by differential centrifugation and sucrose gradient centrifugation. Further experiment shows that there exists aminotransferases in mitochondria that are able to convert α-keto acid to L-NNA but without good efficiency. And due to the loss of pyridoxal-5 -phosphate, the coenzyme of aminotranferases, and the amino donors, pure mitochondria is unable to carry out the transamination. The amino donor selectivity of mitochondrial aminotranferase has been studied, which shows that L-alanine and L-glutamate are the best amino donors, and L-leucine, L-valine and L-threonine are poor amino donors. And 3,000 g kidney homogenate supernatant, the crude cytosol, was centrifugated at 20,000 g, and incubation experiment manifested that 20,000 g supernatant was equal to 3,000 g supernatant on the transamination ratio. The amino donor selectivity of cytosolic aminotransferase has also been studied, which demonstrated that L-alanine was the best amino donor, while L-leucine and L-phenylalanine was poorer than L-alanine. Our study also proves that Aspartate aminotransferase(ast, GOT, EC 2.6.1.1) is not the right aminotransferase to convert α-keto acid to L-NNA. However the incubation time was extended, and however the amount of enzyme was added, no signal of L-NNA was detected on HPLC. It is certain and reliable that Aspartate aminotransferase does not catalyze the reaction. Also, Alanine Aminotransferase(ALT, GPT, EC 2.6.1.2), a common aminotransferase in vivo, has been proved to participate in the transamination reaction. With a 50 Units of Alanine Aminotransferase and an incubation duration of 3 hours, a clear signal of L-NNA was detected by HPLC. In spite of the low efficiency of Alanine Aminotransferase, it is doubtless that it is Alanine Aminotransferase that is involved in the transamination of α-keto acid. Furthermore, branched-chain aminotransferease(bcat, EC 2.6.1.42), an aminotransferase distributed both in cytosol and mitochondria, has been testified to be engaged in the transamination of α-keto acid. During 30 minutes incubation, a strong signal of L-NNA was detected, which affirm the strong reaction ratio of branched-chain aminotransferease. However, the exterminaattori of the quantity of branched-chain aminotransferease in vivo hinders us from saying that branched-chain aminotransferease is the main aminotransferase catalyzing the transamination of α-keto acid. Additionally, gabapentin, a structural analog of isoleucine and a competitive inhibitor of cytosolic branched-chain aminotransferease(bcatc), was added to both cytosol and mitochondria. The experiment in cytosol displayed evident effect that gabapentin inhibited the aminotransferase and reduce the generation of L-NNA, and the inhibitory action enhanced when the concentration

of gabapentin was raised. Nevertheless, in mitochondria, there was no effect like what was demonstrated in cytosol. This experiment shows that BCATc have been involved in the transamination of α-keto acid in biological homogenate. It has been through many experiments that proves the second step of chiral inversion of D-NNA exists. Alanine Aminotransferase and branched-chain aminotransferease suffice to the transamination from α-keto acid to L-NNA, while Aspartate aminotransferase does not. The experiment involving gabapentin shows that cytosolic branched-chain aminotransferase has taken part in the transamination. The result of the study will contribute to the final reveal of the complete pathway of the chiral inversion of D-NNA. KEY WORDS N G -nitro-d-arginine, Chiral inversion, HPLC, α-keto acid

Contents Chapter 1 Introduction... 5 1. Phenomenon of Chiral Inversion in vivo... 5 2. Chiral Inversion of 2-arylpropionic acids... 5 2.1 The mechanism of the chiral inversion of 2-arylpropionic acid drugs... 6 3. Chiral inversion of D-amino acids... 7 3.1 Phenomenon of chiral inversion of amino acids in vivo... 9 3.2 Mechanism of chiral inversion of amino acids in vivo... 9 3.3 DAO in chiral inversion of amino acid in vivo... 11 3.4 Aminotransferases in chiral inversion of amino acid in vivo... 12 4 Chiral ligand exchange method... 12 4.1 Mechanism of Chiral ligand exchange method... 13 4.2 Influence factor in Chiral ligand exchange method... 13 4.3 Use of HPLC in Chiral ligand exchange method... 14 Chapter 2 Detection of D- and L-NNA in biological samples... 17 1 Instruments and methods... 17 1.1 Reagents... 17 1.2 Instruments... 17 1.3 Chromatographic condition... 17 1.4 Sample treatment... 18 1.5 Standard curve of concentration of L-NNA... 18 2 Result and discussion... 18 2.1 Separation of D- and L-NNA... 18 2.2 Separation of D- and L-NNA in biological samples... 19 2.3 Standard Curve of L-NNA... 19 3 Conclusions... 20 Chapter 3 Incubation of D-NNA with kidney homogenate... 21 1 Reagents and methods... 21 1.1 Reagents... 21 1.2 Preparation of Kidney Homogenate, Crude mitochondria and 3,000 g Supernatant 21 1.3 Incubation Mixture... 21 1.4 Incubation Temperature... 21 1.5 Sample procedures... 21 1.6 Detection Method... 21 2 Result and discussion... 22 2.1 HPLC Chromatograms of Incubation of kidney homogenate with D-NNA... 22 2.2 Discussion... 22 3 Conclusions... 23 Chapter 4 Preparation of N G -nitro-5-gunidino-2-oxopentanoic acid... 24 1 Reagents and methods... 25 1.1 Reagents... 25 1

1.2 Incubation Mixture... 25 1.3 Sample procedures... 25 1.4 Identification Method... 25 2 Result... 26 2.1 Comparison if incubation with and without catalase... 26 2.2 ESI-MS and 13 C-NMR... 27 3 Conclusions... 28 Chapter 5 Incubation of N G -nitro-5-gunidino-2-oxopentanoic acid with kidney homogenate... 29 1 Reagents and methods... 29 1.1 Reagents... 29 1.2 Preparation of Kidney Homogenate, Crude mitochondria and 3,000 g Supernatant 30 1.3 Incubation Mixture... 30 1.4 Incubation Temperature... 30 1.5 Sample procedures... 30 1.6 Detection Method... 30 2 Result and discussion... 30 2.1 Chromatogram... 30 2.2 Discussion... 31 3 Conclusions... 31 Chapter 6 Incubation of N G -nitro-5-gunidino-2-oxopentanoic acid with purified kidney mitochondria... 32 1 Reagents and methods... 32 1.1 Reagents... 32 1.2 Preparation of Purified kidney mitochondria... 32 1.3 Incubation Mixture... 32 1.4 Incubation Temperature... 34 1.5 Sample procedures... 34 1.6 Detection Method... 35 2 Result and discussions... 35 2.1 Incubation of mitochondria with L-alanine and L-glutamate... 35 2.2 Incubation of mitochondria and keto acid with different L-amino acids of 3 hours. 36 3 Conclusions... 38 Chapter 7 Incubation of N G -nitro-5-gunidino-2-oxopentanoic acid with kidney cytosol... 39 1 Reagents and methods... 39 1.1 Reagents... 39 1.2 Preparation of kidney cytosol... 39 1.3 Incubation Mixture... 39 1.4 Incubation Temperature... 40 1.5 Sample procedures... 40 1.6 Detection Method... 40 2 Result and Discussion... 40 2.1 Incubation of 3,000 g supernatant... 40 2.2 HPLC Comparison of 3,000 g supernatant and 20,000 g supernatant... 41 3 Conclusion... 42 2

Chapter 8 Incubation of Alanine Transaminase... 43 1 Reagents and methods... 43 1.1 Reagents... 43 1.2 Icubation Mixture... 43 1.3 Incubation Temperature... 44 1.4 Sample procedures... 44 1.5 Detection Method... 44 2 Result and Discussion... 45 2.1 Incubation of GPT with keto acid and L-alanine and L-glutamate... 45 3 Conclusion... 47 Chapter 9 Incubation of Aspartate Transaminase... 48 1 Reagents and methods... 48 1.1 Reagents... 48 1.2 Icubation Mixture... 48 1.3 Incubation Temperature... 49 1.4 Sample procedures... 49 1.5 Detection Method... 49 2 Result and Discussion... 50 2.1 Incubation of keto acid with L-glutamate... 50 2.2 Incubation of keto acid with L-glutamate... 51 2.3 Incubation of positive reaction of GOT... 52 3 Conclusion... 53 Chapter 10 Incubation of Branched-Chain Aminotransferase... 54 1 Reagents and methods... 55 1.1 Reagents... 55 1.2 Incubation Mixture... 55 1.3 Incubation Temperature... 56 1.4 Sample procedures... 56 1.5 Detection Method... 56 2 Result and Discussion... 56 2.1 HPLC Chromatogram of Incubation of keto acid with L-amino acids... 56 2.2 AUC of HPLC Chromatogram of Incubation of keto acid with L-amino acids... 57 2.3 Discussion... 57 3 Conclusion... 58 Chapter 11 Incubation of kidney cytosol and mitochondria with gabapentin... 59 1 Reagents and methods... 59 1.1 Reagents... 59 1.2 Incubation Mixture... 60 1.3 Incubation Temperature... 61 1.4 Sample procedures... 61 1.5 Detection Method... 61 2 Result and Discussion... 62 2.1 Incubation of 20,000 Kidney homogenate supernatant (30 min)... 62 2.2 Incubation of 20,000 Kidney homogenate supernatant (3 h)... 63 3

2.3 AUC of Repeated Experiments in 20,000 supernatant... 64 2.4 Discussion of Incubation of 20,000 g supernatant with and without gabapentin... 64 2.5 Mitochondrial Incubation of L-alanine with and without gabapentin... 65 2.6 Mitochondrial Incubation of L-leucine with and without gabapentin... 66 2.7 AUC of Repeated Experiments of Incubation... 67 2.8 Discussion of Incubation of Mitochondria with and without gabapentin... 67 3 Conclusion... 67 Chapter 12 Conclusions and prospects... 68 Reference... 69 Appendix I Seperation of cytosol and mitochondria in kidney homogenate... 72 1 Materials... 72 1.1 Animals... 72 1.2 Materials... 72 2 Methods... 73 2.1 Isolate kidney... 73 2.2 Homogenize kidney... 73 2.3 Separation of each part... 73 3 Result... 74 Appendix II Resolution of Rat Kidney Mitochondria using discontinuous sucrose gradient... 75 1 Materials... 75 2 Method... 76 3 Flow Chart... 77 Acknowledgments... 78 4

Chapter 1 Introduction 1. Phenomenon of Chiral Inversion in vivo As linked to four different groups, the carbon atom becomes the asymmetric center, thus forming a chiral molecule. Enantiomers are stereoisomers that mirror-imaged but unable to be overlapped. Despite the difference on systemic arrangement of groups on the chiral carbon atom, most enantimoers owns similar physical and chemical properties, such as melting point and solubility, while the most significant difference lies on the different directions of rotation of plane of polarization of polarized light [1]. Since enantimoers have such similar physical and chemical properties, it is hard to distinguish them; however, in chiral environment such as human body, enantimoers show obviously different characters. Molecular asymmetry plays a key role in life science, for many life processes have stereoselectivity. After drug enters the body, it takes effect only when strictly recognized and matched, which consequently makes difference in pharmacological activities, distribution and metabolism for enantimoers of a chemical compound [2,3]. Data shows about one-fourth of clinical drugs exist in the form of raceme, while only one among the enantimoers performs therapeutical effect, and the others are often activeless or toxic [2]. And chiral pharmacokinetics, an important aspect of chiral drug studies, including absorption, distribution,,metabolitics and excretion of chiral drugs, is still in infant age. Drugs of enantimoers show stereoselectivity rising from the interactions between chiral drugs and endogenous macromolecules in vivo in all process of absorption, distribution, metabolitism and excretion, for the ability of enantimoers to combine enzymes or reacting as the substrate of enzymes is different. Chiral drugs have diversities of stereoselectivity and metabolic priority among different species, even in the same species, just like the ligand-receptor process, which chiral inversion may perform during metabolism [2,4]. Chiral inversion in metabolism has two mechanisms; one is intermediate epimerization after reversible combination to one specific group, while another one is an inversion between two enantimoers [3]. Our study is mainly about the latter one. As natural chiral compounds, amino acids play a key role in life process in nature. Present studies show that the chiral inversion of D-amino acids is unique but also universal. After the chiral inversion of D-phenylalanine (D-Phe) [5] and D-leonine (D-Leu) [6] were discovered, the chiral inversion of N G -nitro-d-arginine(d-nna) has been discovered in rat [7] while D-dopa was proved to have similar bioactivity with L-dopa in vivo [8]. We are going to introduce some studies of chiral inversion of amino acids, as well as chiral separation using chromatography 2. Chiral Inversion of 2-arylpropionic acids 2-arylpropionic acids, a kind of non-steroidal anti-inflammatory drugs(nsaids) developed in recent years, also named as profens. It is commonly considered that the anti-inflammatory 5

mechanism of profens is the inhabitation of prostaglandin (PG) in tissues [9]. Profens, including ibruprofen, ketoprofen and flurbiprofen, have a variety of nearly 20 types [1]. Most 2-arylpropionic acid NSAIDs are sold in racemic form. These enantimoers have different biological properties, including stereoselective protein binding [10], Glucuronidation [11] and metabolism [12] and chiral inversion between R- and S-isomers [13]. Figure 1 Chemical structure of 2-arylpropionic acids Chiral inversion of 2-arylpropionic acid drugs is one-way inversion from inert R-form to anti-inflammatory S-enantimoer. In other words, it is rare that S-enantimoers is converted to R-form. 2.1 The mechanism of the chiral inversion of 2-arylpropionic acid drugs In 2-arylpropionic acid drugs, S-enantimoers are the anti-inflammatory ingredient, but S-isomers are not. R-enantimoers can be converted to S-isomer in vivo, which is the reason for the difference of drug effects of profens in vivo and in vitro [2]. Based on the study of the metabolic pathway of profens in vivo, it is considered to be three steps in the chiral inversion of profens [14]. The mechanism is shown as the figure below. Step 1: R-2-arylpripionic acid is converted stereoseletively to sulfolipidcoa by Dicarboxylate-CoA ligase in microsomes or mitochondria. The unidirectional reaction has decided the monodirection of chiral inversion of profens [14,15]. Step 2: the sulfolipidcoa is converted to its enantimoer through epimerization by CoA racemase, which is reversible [16]. Step 3: S-enantimoer is generated catalyzed by hydrolase. Dicarboxylate-CoA ligase (EC 6.2.1.23) is distributed in microsomes, perixisomes and 6

mitochondria in liver, and the racemases is mainly distributed in mitochondria and cytosol in liver cells [16]. Hutt and Caldwell has proved that the hydrolase in Step3 demonstrates the strongest activation in kidney [17]. Figure 2 Chiral inversion processes of aryl-2-propionic acids (APA). A three-step process which begins with the enantiomer specific enzymatic formation of a thioester between the R-enantiomer of the 2-APA and CoA. This thioester may be hydrolysed to regenerate the R-enantiomer or may undergo epimerization to yield the thioester in which the 2-arylpropionyl moiety has the S configuration. Subsequent hydrolysis of this S(+)profen-CoA completes the inversion process. 3. Chiral inversion of D-amino acids α-amino acids, except Glycine, have enantimoers for their α-carbon atom. Life processes, including protien synthesis, prefer L-amino acids, while microbes, such as bacteria, are able to metabolize and produce D-amino acids. We used to conclude the fact that D-amino acids exist in mammals to food absorption or prodution of bacteria metabolism [18], and D-amino acids do not take part in other vital process. However, recent studies show that there are many residues of D-amino acids in proteins and polypeptides in higher developed animals [19]. With the development of chiral chromatogram seperation technologies, free D-amino acids are detected in higher developed animals [20], which play important roles in bioactivities. For example, D-Aspartate (D-Asp) of high concentration are detected in neuroendocrine and endocrine organs [21], which is considered regulatory factors of several hormones, like testosterone and melatonin [19]. Free D-serine(D-Ser) are detected in brain 7

of mice, rats, cattle and human [22], which is proved to effect the NMDA receptor(n-methyl-d-aspartate receptor, a kind of important excitatory amino acids receptor ) [23] which plays a role not only in development of nervous system but also in the forming process of neuronal circuit. Since some D-amino acids exist in food and water, researchers believe that D-amino acids enter the body through food absorption [19]. On the other hand, reports show that some natural D-amino acids and their derivations have similar biological activities with their L-enantimoer or perform chiral inversion. Table 1 lists several natural amino acids that can possibly be chiral inversed. Table 1 The structure of some amino acids Amino Acid Symbol Structure pi Leucine Leu 5.98 N G -Nitro-Arginine NNA 12.5 Phenylalanine Phe 5.48 Dihydroxyphenylalanine dopa Tryptophan Trp 5.89 Ethionine 8

3.1 Phenomenon of chiral inversion of amino acids in vivo In recent 40 years, some researchers have found that D-amino acids are able to maintain same growth rate of some mammals when take the place of L-amino acids, despite several times higher concentration compared to control group using L-amino acid. So researchers believe D-amino acids can be converted to L-amino acids in vivo [19]. By using gas chromatography(gc), Hasegawa et found that soon after an intravenous injection to rats of 1mg/kg or 10mg/kg D-[ 2 H 7 ]-Leu(D-[ 2 H 7 ]-Leucine), L-[ 2 H 7 ]-Leu was detected in blood plasma. The inversion rate from D-Leu to L-Leu was 29.8 ± 15.3 % (1 mg/kg) and 35.5 ± 13.2% (10 mg/kg). No evidence of dose dependence was found, nor chiral inversion from L-[ 2 H 7 ]-Leu to D-[ 2 H 7 ]-Leu [24,25]. By using isotope labeling and LC-ESI-MS(liquid chromatogram-electrode Spray ionazation-mass Spectrum), one third of D-phenylalanine(D-Phe) was converted to L-Phe after oral administration of 25mg/kg of D-[ 2 H 5 ]-Phe of healthy volunteers, while 27% of D-Phe was excreted through urine, nevertheless, when oral administrated with same dose of L-[ 15 N]-Phe, No D-[ 15 N]-Phe was detected and excretion rate through urine was 0.25%. L-NNA is an inhibitor of nitric oxide synthase (NOS), which inhibits the generation of NO in vivo and raise the arterial blood pressure. D-NNA does not inhibit the NOS in vitro, but raise arterial blood pressure in vivo, and its effect can be antagonized by L-Argine (L-Arg), a substrate of NOS, when D-Arg cannot. It is presumed that there is chiral inversion of D-NNA in vivo, and L-NNA is generated. HPLC Spectrum show that L-NNA is detected in blood plasma after intravenous injection of 50mg/kg dose of D-NNA of rats, and L-NNA content reached its peak at about an hour, and the chiral inversion rate from D-NNA to L-NNA is around 40%. No inversion from L-NNA to D-NNA [7]. As a precursor of dopamine, L-dopa is widely used to treat Parkinson's disease. D-dopa is used to be considered no therapeutic effect. However, Karoum et found that in rotation behavior experiment with Striatonigral degeneration of rats, L-dopa and D-dopa showed same bioactive effect, which indicate that D-dopa can be converted to L-dopa in vivo [8]. Though the mechanism has not been fully revealed, researchers believe that D-dopa can be metabolized to L-dopa through some path, and finally converted to dopamine and takes effect. 3.2 Mechanism of chiral inversion of amino acids in vivo 3.2.1 The discovered pathway of chiral inversion of amino acids in vivo Report shows that amino acids can perform isomeric inversion catalyzed by acids, bases or enzymes. The mechanism of base catalyzed racemization: (a) Proton abstraction-addition mechanism. One OH - ion gain one H + from α-ch on the amino acid, making the carbon atom negatively charged and unstablized, and then that carbon atom gains one proton form solution and becomesa an amino acid again, thus making itself DL-racemized.(b)Elimination-addition mechanism. After deprotonation just like the process in (a), that carbon atom reacts with activated H atom and becomes an amino acid [19]. The mechanism of acid catalyzed isomeric inversion: The amino acid that has got a proton is easy to lose α-ch and becomes unsterilized, and D- or L- amino acid is generated after proton 9

performs nucleophilic reaction from both side [19]. Studies show that some enzymes on amino acid can perform racemiztion mechanism as Figure 4: A: L-amino acid, catalyzed by bacteria racemase(pyridoxal-5'-phosphate as the coenzyme), generates D-amino acid by proton's stereoselective transference. B: D-Lysine (D-Lys) chiral inversion in vivo among plants and animals. D-Lys is cyclized to L-pipecolic acid and decyclized to L-Lys. This inversion exists in some hereditary peroxisome-disordered patients whose concentration of L-pipecolic acid in blood plasma and urine is much higher than normal people. As to other D-amino acid rich diet induced high concentration of L-pipecolic acid, it is normal metabolism. Figure 3 Isomeric inversion in vivo: (A) catalysis of inversion of an L-amino acid by the pyridoxal phosphate (PLP) coenzyme of a microbial racemase involving stereoselective shifts of H atoms; (B) conversion of D- to L-lysine by plant and microbial enzymes via pipecolic acid 3.2.2 New mechanism of chiral inversion of D-amino acids Chiral inversion in vivo can be carried out by amino acid racemase, however, these racemase only exist in simple organisms like bacteria, though recent study has found D-Serine racemase in mammal brains, the high substrate specificity of racemase limits the chiral inversion of so many kinds of amino acids. On the other hand, racemase catalyze in both directions, while D-Leu, D-Phe and D-NNA perform a one-way inversion, which indicates that the chiral inversion of these amino acid is not catalyzed by amino acid racemase. These D-amino acid inversion pathway is also different from the proved R- to S- chiral inversion pathway which mainly performed in liver [26,27]. Wang and Cheng has discovered that D-NNA can be chirally converted to L-NNA effectively in kidney homogenate [28,29]. Research shows that ligation of both kidneys can block the pressure rise effect of D-NNA and block 80% inversion from D-NNA to L-NNA [30]. And some researchers found that a higher concentration of D-Phe and D-Lys was detected in 10

patients suffering from renal failure, compared to normal people [31]. The difference of occuring place of chiral inversion indicates that the chiral inversion pathway of these amino acids is different from aryl-2-propionic acids. Figure 4 Proposed pathway of D-amino acid inversion in vivo. Though the molecular mechanism of chiral inversion of D-amino acid are not clearly revealed yet, the combined action of D-amino acid oxidase (DAO) and aminotransferase through two-step reaction is a preferred truth [24]. DAO catalytically oxidize D-amino acid to α-keto acid, and then α -keto acid gains amino group catalyzed by aminotransferase and generate L-enantimoer. 3.3 DAO in chiral inversion of amino acid in vivo D-amino acid oxidase (DAO, EC1.4.3.3) is the second discovered enzyme coenzymed by flavine-adenine dinucleotide (FAD), and FAD is the prosthetic group of all DAOs which is noncovalent-bonded. Recent studies mainly focus on their Biological method among eucaryotes and the mechanism of DAO catalization [32]. 3.3.1 Mechanism of catalization of DAO The mechanism is shown as Figure 5. Amino acid becomes imino acid after the dehydrogenation with the reduction of FAD, and FAD is oxidased with the existance of oxygen molecule, at the same time H 2 O 2 is generated. The imino acid is hydrolyzed without the catalization effect of enzyme and creates α-keto acid and NH 3. α-keto acid is decarboxylazed at the existance of H 2 O 2 in vivo and generates homologous keto acid. Figure 5 Scheme of catalyzed by D-amino acid oxidase 11

DAOs have strong substrate stereoselectivity, which means they only catalyze D-amino acids. At the same time, DAOs demonstrate wide substrate specificity. DAO is able to catalyze neutral and alkaline D-amino acids, not for acid D-amino acids, like D-Asp and D-Glu, while the best substrate is amino acids with hydrophobic side chain [32]. 3.3.2 Physical function of DAO DAO exists widely in prokaryote and eukaryote, including yeast, epiphyte, insects, Amphibian, birds and mammals. Yeast s ablity to use D-amino acid is closely linked to DAO. In spite of the expression level in higher developed animals' liver and kidney, concentration of D-amino acid is low in vivo, so it is still unclear for the physical function of DAO in higher developed animals. For example, D'Aniello et al(1993)proved that a huge amount of D-amino acid exists in mammals including human. The D-amino acids can be oxidized by DAO and D-aspartate oxidase(daspo, EC1.4.3.1, mainly oxidize D-Asp and D-Glu), raising the amount of NH 3 and H 2 O 2. Further experiment proved that the accumulation of D-amino acids was harmful to organs and tissues. Base on the fact, DAO and DASPO is considered to be enzymes metabolizing D-amino acids absorbed from nature to prevent the toxic accumulation when the animals age.[33,34] Also, DAO has connection with D-Ser in rats' brain. Study shows that D-Ser is able to adjust NMDA nerve conduction, and DAO is able to affect brain function by adjusting the concentration of D-Ser [35,36]. 3.4 Aminotransferases in chiral inversion of amino acid in vivo At present, aminotransferases has a variety of more than 80 kinds, and there are about 13 aminotransferases distribute in kidney. Though a lot or research work has been done, it is still in misery whether aminotransferases take part in chiral inversion of D-amino acids, not to say further information. Since aminotransferases have strong substrate specificity, we believe it different for aminotransferases engaged in chiral inversion. Activation difference influence the inversion a lot, for instance, D-Alanine(D-Ala), best substrates for most DAOs, lacks chiral inversion in vivo(dolle et al., 2000), while DAO's specificity on D-Phe and D-Leu differs much, however, D-Phe and D-Leu demonstrate similar chiral inversion rate in vivo. All these make us believe that aminotransferase is the determinate role in the chiral inversion of D-amino acids. 4 Chiral ligand exchange method To study the pharmacology and pharmacokinetics of chiral drugs, it is necessary to establish a analytic method with high resolution and high sensitivity. Recent years chiral chromatography, especially liquid chromatography and gas chromatography, has provided efficient methods. These analytic methods can be sorted as Chiral Derivatization Chromatography, chiral mobile phase chromatography, chiral stationary phase chromatography, chiral gas chromatography and chiral capillary electrophoresis [4]. Among all, chiral mobile phase addictive chromatography, though without high resolution, has been used widely because of rapidity, strong selectivity and inexpensiveness. 12

chiral mobile phase additive chromatography requires additive of chiral selector or chrial ligand to the mobile phase to separate the chiral compounds. This methods includes complex substance, chiral ligand-exchange chromatography (CLEC) and chiral ion pair. As a separation method mainly on separations of free amino acids and their derivatives, hydroxyl acid, alkyd-amine and polypeptides, chiral ligand-exchange use the properties as reference that in biological systems amino acids and metal ions form ligands. This study also uses this method to carry out a separation of D- and L-NNA with high resolution, high selectivity and high sensitivity. 4.1 Mechanism of Chiral ligand exchange method Chiral ligand-exchange method is a method that add a certain metal ion and a certain chiral ligand to form two pseudo-heterchiral complexes, and since the diastereomers have different stability or affinity to the column, they can be separated through chromatography process. Also, CLEC allows to bond chiral ligand to the immobile phase to become chiral stationary phase chromatography. The mechanism of the formation of pseudo-heterchiral complexes from enantimoers with ligand[4] 2[L-LE]+M2 + [L-LE]-M2 + -[L-LE] 2[L-LE]-M 2 +[L-LE] + [D-AX] [L-AX] [L-LE]-M 2 +-[D-AX] [L-LE]-M 2 +-[L-AX] +2[L-LE] LE:chrial ligand M2+:metal ion AX:pseudo-heterchiral complexes Fig 6 pseudo-homochiral and pseudo-heterchiral complexes Chiral ligand-exchange method was first established by Karge. The more stable complex will stay in the column for a shorter time, so it will be washed out first. Influenced by ph value of mobile phase or the organic content, concrete analysis is required in separation, more than just the elution sequence. 4.2 Influence factor in Chiral ligand exchange method The factors influencing chiral ligand-exchange method includes metal ion, concentration of metal complex compound, ph value of mobile phase, organic modifier, ionic strength of mobile phase and chiral selector [37] 13

4.2.1 Metal Ion Common metal ions used in CLEC include Cu 2+, Ni 2+, and Co 2+ etc. Among all, Cu 2+ is able to form complex with many chiral ligands and enantimoers to separate effectively on chromatography columns, which makes it the most commonly used ion. 4.2.2 ph value of mobile phase Different ph value may change the balance of metal ions. Proper ph value will keep the free amino acid neural and form complex easily. Generally speaking, the efficiency of separation of nonpolar amino acids tends to rise as ph rises, while the efficiency of separation of polar amino acids tends to be best in neural conditions. In a condition with too low ph value, H+ ion will compete for the chelating center with ions, enantimoers and chiral ligands and leads to the decrease of efficiency of separation. And condition of too high ph value does not fit the chromatographic system and will cause precipitation of metal ions. ph5.0~8.0 is preferred in common use. The elution sequence will change as ph varies. 4.2.3 Organic modifier Some organic solutions, such as methanol, acetonitrile and tetrahydrofuran, are able to adjust the elution order and the efficiency of separation. Reduction of content of organic solutions will promote the hydrophobic interaction between solute and adsorbent, thus leading to the increase of efficiency of separation and prolong the retention time. 4.2.4 The ion strength of mobile phase Ion strength will change the retention time of enantimoers, as well as response of peaks. 4.2.5 Chiral selector Different selectors form different complex with enantimoers, which may change the efficiency of separation. L-proline, L-hydroxyproline and aspartame are commonly used. 4.3 Use of HPLC in Chiral ligand exchange method High performance liquid chromatography (HPLC) is an important analytical tool for separating and quantifying components in complex liquid mixtures. By choosing the appropriate equipment (i.e. column and detector), this method is applicable to samples with components ranging from small organic and inorganic molecules and ions to polymers and proteins with high molecular weights. The various types of HPLC and their characteristics are summarized in the table below. 14

Table 2 Various Types and Applications of HPLC TYPE SAMPLE POLARITY MOLECULAR WEIGHT RANGE STATIONARY PHASE MOBILE PHASE Adsorption non-polar to somewhat polar 10 0-10 4 silica or alumina non-polar polar to Partition (reversed-phase) non-polar to somewhat polar non-polar liquid adsorbed 10 0-10 4 or chemically bonded to the packing material relatively polar Partition (normal-phase) somewhat polar to highly polar 10 0-10 4 highly polar liquid adsorbed or chemically bonded to the packing material relatively non-polar Ion Exchange highly polar to ionic 10 0-10 4 ion-exchange resins made of insoluble, high-molecular weight solids functionalized typically with sulfonic acid (cationic exchange) or amine (anionic exchange) groups aqueous buffers with added organic solvents to moderate solvent strength Size-Exclusion non-polar to ionic 10 3 10 6 small, porous, silica or polymeric particles polar non-polar to 4.3.1 Principles of Chiral HPLC Chiral HPLC columns are made by immobilizing single enantimoers onto the stationary phase. Resolution relies on the formation of transient diastereoisomers on the surface of the column packing. The compound which forms the most stable diastereoisomer will be most retained, whereas the opposite enantimoer will form a less stable diastereoisomer and will elute first. To achieve discrimination between enantimoers there needs to be a minimum of three points of interaction to achieve chiral recognition. The forces that lead to this interaction are very weak and require careful optimization by adjustment of the mobile phase and temperature to maximize selectivity. Chromatography is a multi-step method where the separation is a result of the sum of a large number of interactions. Typically a free energy of interaction difference of only 0.03 kj/mol between the enantimoers and the stationary phase will lead to resolution. The intermolecular forces involved with chiral recognition are polar/ionic interactions, pi-pi interactions, hydrophobic effects and hydrogen bonding. These can be augmented by the formation of inclusion complexes and binding to specific sites such as peptide or receptor sites in complex phases. The analyst may manipulate these intermolecular forces by choosing suitable mobile phases, for instance polar interactions may be controlled by the ph. 15

The effect of temperature is important in chiral HPLC. Lower temperature will increase chiral recognition, but as it alters the kinetics of mass transfer, it may actually make the chromatography worse by broadening peaks. There is often an optimum temperature for a separation and knowledge of this gives the analyst another factor to exploit in the method development process. The type of column used for separating a class of enantimoer is often very specific, this combined with the high cost of chiral columns, makes the choice of which column to use seem at first bewildering. Fortunately, taking the time to study the structure of chiral phases and visualizing the potential interactions with the analyte can narrow down the choice significantly. There are still a few occasions where a brute force method of screening through many chiral columns is still necessary but these occasions are becoming rarer with time. 4.3.2 Columns - Chiral Stationary Phases Chiral Stationary Phases (CSP's) may be classified according to their interaction mechanism with the solute. Type I CSP's are those which differentiate enantimoers by the formation of complexes based on attractive interactions. These may be hydrogen bonds, p-p interactions, dipole stacking. Type II CSP's are those which involve a combination of attractive interactions and inclusion complexes to produce a separation. Most type II phases are based on cellulose derivatives. Type III CSP's rely on the solute entering into chiral cavities to form inclusion complexes. The classic inclusion complex column is the cyclodextrin type of column. Other CSP's in this class are crown ethers and helical polymers such as poly (triphenylmethyl methacrylate). Type IV CSP's separate by means of diastereomeric metal complexes. This technique is also known as Chiral Ligand Exchange Chromatography (CLEC). Type V CSP's are proteins where separations rely on a combination of hydrophobic and polar interactions. 16

Chapter 2 Detection of D- and L-NNA in biological samples This study aims to clarify the mechanism of chiral inversion of D-NNA, thus requiring for a method to separate and analyze D- and L-NNA. Chiral separation method at present includes chiral liquid chromatographic separation, chiral gas chromatographic separation, chiral capillary electrophoresis and chiral capillary electrochromatography. High performance liquid chromatography technique with C18 column is based on the mechanism of adsorption and desorption, and the chiral separation technique based on chiral ligand exchange principle is applied to the chiral separation of D- and L-NNA, with an advantage of fast, precise and economical. This study aims at setting up a liquid chromatographic method base on ligand exchange principle, thus laying a foundation for further studies. Chiral ligand exchange method uses a mental ion (such as Cu 2+ ) in mobile phase and chiral selective body(such as L-Proline, L-Hydroxyproline and Aspartame) to combine the enantimoers and becomes ternary complex of different stability. And this ternary complex, with different adsorption strength with stationary phase because of combination strength of hydrogen bond, is eluted separatedly, which performs a chiral separation [37,38]. Xin have developed a separation method using 5%methnol and 95% 50 mm NH 4 Ac buffer (ph 5.0, containing 1mM Cu 2+ and 2 mm Aspartate) which worked well on CEC (capillary electrochromatography). This method forms the base of analysis method of this study. 1.1 Reagents 1 Instruments and methods D-NNA and L-NNA were bought from GL Biochem (Shanghai) LTD. Formic acid was from Sigma (HPLC grade). Methanol (HPLC), Copper sulfate (AR), Ammonium Acetate (AR), Acetic acid were obtained from Sinopharm Chemical Reagent Co., LtdS. Aspartame was purchased from Aladdin Reagent(Shanghai). Purified water was from Ultra Pure Water System-Milli-Q Plus TM (Millipore, US). 1.2 Instruments LC-2010HT liquid chromatography, Shimadzu, Japan Ultimate TM Column, AQ-C18, 5µm, 4.6*150mm Protection column: LBBM-612111 C18 column 1.3 Chromatographic condition 1.3.1 Chromatographic condition of Detection of D- and L-NNA Mobile phase: 1% methanol, 99% 10mM NH4Ac buffer(ph=5.0, containing 2 mm Aspartame, 17

1mM CuSO 4 ) Flow Rate = 0.8 ml/min, Oven Temperature = 35 Detector: LC-2010 UV detector, wavelength=280 nm 1.3.2 Chromatographic condition of Detection of α-ketoglutarate and oxaloacetic acid Mobile phase: 1%methnol, 99% 0.1% HCOOH Flow Rate = 0.8 ml/min, Oven Temperature = 35 Detector: LC-2010 UV detector, wavelength= 233 nm 1.4 Sample treatment Add methanol of triple volume to the sample to precipitate protein, while finally inject into HPLC after 10minutes of centrifugation at 15,000 g 1.5 Standard curve of concentration of L-NNA Concentration Point Flow Rate Sample Volume Mobile Phase Oven Temperature Detector 5 μm 25 μm 50 μm 100 μm 500 μm 1 mm 0.8 ml/min 5 μl 1% methanol, 99% 10mM NH 4 Ac buffer(ph=5.0 containing 2 mm Aspartame, 1mM CuSO 4 ) 35 LC-2010 UV detector, wavelength=280 nm Table 1 Parameters of Standard Curve of L-NNA 2.1 Separation of D- and L-NNA 2 Result and discussion Figure 1 Typical chromatograms of 50 mm D-NNA + 50 mm L-NNA, in 50 mm TrisHCl (ph = 8.2) From Figure 1 we may conclude that D-NNA and L-NNA can be separated by the mobile phase, which consists of 1%methnol and 99%NH 4 Ac buffer(ph = 5.0) with 2 mm Aspartame and 18

1mM CuSO 4. L-NNA was washed out ahead of D-NNA, which between two peaks is about 2 minutes, and the peaks are clear and without any interference. 2.2 Separation of D- and L-NNA in biological samples Figure 2 Typical Chromatograms of D-NNA in Kidney Homogenate: (a) L-NNA in kidney homogenate. (b) D-NNA in Kidney Homogenate (c) L-NNA in 50 mm TrisHCl (ph=8.2) (d) D-NNA in 50 mm TrisHCl (ph=8.2) From Figure 2 may conclude that D-NNA and L-NNA in biological samples such as kidney homogenate can be separated by the mobile phase with a good efficiency of seperation. L-NNA was washed out ahead of D-NNA, and the peaks are clear and without any interference. 2.3 Standard Curve of L-NNA 2.3.1 AUC of each concentration point of L-NNA [L-NNA] AUC 5 μm 17972 25 μm 104971 50 μm 191181 100 μm 407840 500 μm 2.09874E6 1 mm 3.91313E6 Table 2 AUC of each concentration point of L-NNA 2.3.2 Linear Fit of each concentration point of L-NNA 19

Figure 3 Linear Fit of each concentration point of L-NNA Linear Fit was carried out by OriginLab v7.5 AUC = 16757 + 3948*[L-NNA] [L-NNA] = (AUC-16757)/3948 R = 0.99938 SD = 61886 3 Conclusions The mobile phase, which consists of 1% methanol, 99% 10mM NH 4 Ac buffer(ph5.0, containing 2 mm Aspartame and 1mM CuSO 4 ), is able to separate D- and L-NNA in biological samples under the present HPLC conditions. The standard curve of L-NNA shows that the HPLC detection of L-NNA is reliable and precise, thus enabling the standard curve to be applied on crude quantitative determination. Further experiment should be focus on daily divergence. 20

Chapter 3 Incubation of D-NNA with kidney homogenate To find out the aminotransferase, we must first make sure that D-NNA is able to carry out a chiral inversion in kidney homogenate, and it is very important to find out the suitable incubation method, which builds up a solid foundation for further studies. Recent studies show that kidney is the main organism for D-NNA's chiral inversion. The ligation of both kidneys blocks the way for the chiral inversion from D-NNA to L-NNA. And the incubation of D-NNA with heart homogenate, lung homogenate, liver homogenate and kidney homogenate demonstrate the fact that kidney is the main site for the inversion [30]. Based on earlier researches, kidney is chosen as the final research direction. 1 Reagents and methods 1.1 Reagents D-NNA was purchased from GL Biochem (Shanghai) LTD. Methanol(HPLC grade) was obtained from Sinopharm Chemical Reagent Co., LtdS. Purified water was from Ultra Pure Water System-Milli-Q Plus TM (Millipore, US). 1.2 Preparation of Kidney Homogenate, Crude mitochondria and 3,000 g Supernatant The preparation of kidney homogenate, crude mitochondria and 3,000 g Supernatant was performed as Appendix I. 3,000 g supernatant is usually considered as crude cytosolic part of kidney cells. 1.3 Incubation Mixture D-NNA 50 mm 5 µl (in 50 mm TrisHCl, ph = 8.2) Kidney Homogenate or Crude Mitochondria or 3,000 g Supernatant 100 µl Add TrisHCl (50 mm ph=8.2) to final volume of 300 µl 1.4 Incubation Temperature Incubated at 37 C 1.5 Sample procedures Sample at 30 minutes and 5 hours after starting, add methanol of same volume to precipitate protein, while finally inject into HPLC after 10 minutes of centrifugation at 15,000 g. 1.6 Detection Method Detection Method is carried out as is described in Chapter 2. 21

2 Result and discussion 2.1 HPLC Chromatograms of Incubation of kidney homogenate with D-NNA Figure 1 The chromatogram of Incubation of D-NNA with kidney homogenate, crude kidney mitochondria and 3,000 g supernatant along with sampling point time: (a)kidney Homogenate+D-NNA-30 min (b)crude Kidney Mitochondria+D-NNA-30 min (c)kidney 3,000 g Supernatant+D-NNA-30 min (d)kidney Homogenate+D-NNA-5h (e)crude Kidney Mitochondria+D-NNA-5 h (f)kidney 3,000 g Supernatant+D-NNA-5 h (g)kidney Homogenate (Boiled in boiling water)+d-nna 2.2 Discussion In Figure 1, (a) (b) (c) are the chromatograms of samples of 30 min. It is obvious that L-NNA is generated in this short time, in mitochondria and in cytosol, which remind us that the D-NNA is able to react in the incubation mixture, thus assuring us that the mixture is suitable for the reaction. Compared to (g), the control group, it is indubitably that L-NNA has generated. (d) (e) (f) are the chromatograms of sample of 5 hours, and the content of L-NNA increased as the incubation time extended. Compared to the reaction rate demonstrated by (a )(b) (c), we may think that the reaction is near to the end, the amino donors should be completely consumed during incubation. As is shown, (e) D-NNA's incubation with crude kidney mitochondria is the inversion with the highest rate among the three, which gives us a suspect that there are more aminotransferases, more amino donors, or more DAOs or other reasons unknown. 22

3 Conclusions D-NNA can be converted to L-NNA under the condition of 50 mm TrisHCl (ph = 8.2), which means the incubation method is suitable for the experiment. There are aminotransferases in both mitochondria and cytosolic part. In this experiment, we cannot exclude other factors such as DAO, so further experiment is required. To exclude unrelated factors, usage of keto acid is acceptable. 23

Chapter 4 Preparation of N G -nitro-5-gunidino-2-oxopentanoic acid To determine the aminotransferase catalyzing the reaction from N G -nitro-5-gunidino-2-oxopentanoic acid (α-keto acid) to L-NNA, it is important to prepare α-keto acid to exclude unrelated factors of aminotransferases. DAO (EC 1.4.3.3) was applied to biotransformation of some D-amino acids like D-methionine or D-phenylalanine to the corresponding α-keto acid starting with the racemic mixtures as substrates, while FAD (Flavin adenine dinucleotide) is the coenzyme. D-NNA is also able to be biotranformed to N G -nitro-5-gunidino-2-oxopentanoic acid through the oxidation of DAO. Figure 1 Biotransformation of D-NNA However, hydrogen peroxide is formed as by-product in the DAO catalyzed reaction. The reactive species is disadvantage for the racemate resolution process due to the chemical decarboxylation of the α-keto acids as well as the inactivation of DAO. In vivo, hydrogen peroxide is converted to water and molecular oxygen by catalase (EC 1.11.1.6). To immediately remove the generated H 2 O 2 during the biotransformation, catalase should be added to the reaction mixture in vitro. 24

Figure 2 Conversion of D-NNA during Incubation without removing H 2 O 2 1.1 Reagents 1 Reagents and methods D-NNA was purchased from GL Biochem (Shanghai) LTD. DAO was bought from Kayon Biology (Shanghai). Methanol(HPLC grade) was obtained from Sinopharm Chemical Reagent Co., LtdS. Purified water was from Ultra Pure Water System-Milli-Q Plus TM (Millipore, US). 1.2 Incubation Mixture 1.2.1 Incubation with catalase 50 mm D-NNA(solved in 50 mm TrisHCl, ph8.2) 400µl, DAO 25Unit/ml(5 Units) 200µl,Catalase 10µl 1.2.2 Incubation without catalase 50 mm D-NNA(solved in 50 mm TrisHCl, ph8.2) 400 µl DAO 25Unit/ml(5 Units) 200 µl 1.2.3 Incubation Temperature and time 37, 5hours 1.2.4 Process after incubation The final mixture was ultrafiltered with a ultrafiltrator with a cut-off molecular weight of 3,000 1.3 Sample procedures Sample at 2 hours and 5 hours after starting, add methanol of triple volume to precipitate protein, while finally inject into HPLC after 10minutes of centrifugation at 15,000 g. 1.4 Identification Method The identification of the products were carried out on ESI-MS and 13 C-NMR. 25

2 Result 2.1 Comparison if incubation with and without catalase Figure 3 HPLC Chromatogram of incubation with and without catalase (Keto acid = N G -nitro-5-gunidino-2-oxopentanoic acid) (Acid = N G -nitro-l-arginine) (A) Separation of D- and L-NNA. (B) Incubation with catalase. (C) Incubation without catalase. From Figure 3, the chromatogram shows that biotransformation product is obviously different between incubation with and without catalase. ESI-MS and 13 C-NMR is used to identify the products. 26

2.2 ESI-MS and 13 C-NMR 2.2.1 Identification of N G -nitro-5-gunidino-2-oxopentanoic acid Figure 4 ESI-MS and 13 C-NMR Spectrum of N G -nitro-5-gunidino-2-oxopentanoic acid ESI-MS: Negative Scan[M-H] - =217 27

2.2.2 Identification of N G -nitro-l-arginine Figure 5 ESI-MS and 13 C-NMR Spectrum ESI-MS: Negative Scan [M-H] - =189 3 Conclusions N G -nitro-5-gunidino-2-oxopentanoic acid (α-keto acid) is available through incubation of D-NNA with DAO, however, the by-product of this process in vitro is hydrogen peroxide, which is easy to perform a decarbonylation reaction and may lead to product loss and DAO inactivation. The add-in of catalase, which is a commercially available, catalyzes hydrogen peroxides to water and molecular oxygen. 28

Chapter 5 Incubation of N G -nitro-5-gunidino-2-oxopentanoic acid with kidney homogenate To further study the mechanism of aminotransferase, it is necessary to carry out an incubation of α-keto acid with kidney homogenate. Aminotransferases catalyze the reaction between keto acids and the corresponding amino acid. Most aminotransferases accept α-ketoglutarate as amino acceptor. Pyridoxal-5'-Phosphate (PLP) is the coenzyme of all aminotransferases. Aminotransferases, widely distributed in vivo, catalyze specific transamination reactions. Among all aminotransferases, glutamate-pyruvate transaminase (GPT, Alanine Transaminase, EC 2.6.1.2) and glutamate-oxaloacetate transaminase (GOT, Aspartate Transaminase, EC 2.6.1.1). GPT catalyzes the reaction between L-alanine and α-ketoglutarate and generates glutamate and pyruvate. GOT catalyzes the reaction between L-aspartate and α-ketoglutarate to generate glutamate and oxaloacetic acid. PLP, the coenzyme of all aminotransferases, combines to the ε-amino group of lysine (Lys) at the active site of aminotransferases. Figure 1 Common transamination reaction coenzymed by PLP 1 Reagents and methods 1.1 Reagents Methanol(HPLC grade) was obtained from Sinopharm Chemical Reagent Co., LtdS. Purified 29