鑭系稀土離子之多氨基酸配位化學及生物醫學之應用(II)

行政院國家科學委員會專題研究計畫成果報告

鑭系稀土離子之多氨基酸配位化學及生物醫學之應用

計畫類別 :

個別計畫

整合計畫

計畫編號 : NSC 89-2113-M-009-010 (2

nd

Year)

執行期間 : 89 年 08 月 01 日 至 90 年 12 月 31 日

個別型計畫:

計畫主持人 : 張 正

共同主持人 : N/A

整合型計畫：

總計畫主持人 : N/A

子計畫主持人 : N/A

註 :

整合型計畫總報告與子計畫報告請分開編印各成一冊,

彙整一起繳送國科會。

處理方式 :

可立即對外提供參考

一年候可對外提供參考

兩年候可對外提供參考

(必要時, 本會得展延發表時限)

執行單位 : 交通大學生物科技研究所

中華民國 91 年 03 月 30 日

This is a research program originally started seven years ago (since 1995). Because

the Institute of Biological Science and Technology of National Chiao Tung University was

a new research institute, we spent most of the first two years to establish our laboratories.

This includes renovating laboratory, recruiting postdoctoral research fellows (Dr. Liu,

Yuh-Liang, 劉育良-目前任職永光化學公司; Dr. Chen, Chang-Yuh, 陳成裕-目前任職

永勞工委員會勞工安全衛生研究所), research assistants and graduate students (陳煥

源，陳玉衡，陳桂添，謝發坤，郭永斌，萬磊，潘美蓉，李亮緯，許呈安，林孟嘉，

管佈雲，鄭昇沛，謝明宏，許地利，張志杰，陳家翎，宋婉貞，謝瑞偉，曾繼鋒，

羅千婷，吳柏宏，林志誠，王文宏，管燕芸，藍佩菁，林俊傑，戴金華，康名慰，

黃淑敏，李盈慧), and undergraduate students (陳彥璋，黃崇道，陳伯翰，王文卿)

purchasing chemicals and research equipment (automatic titroprocessor, stopped-flow

uv-vis spectrophotometer, diode-array uv-vis spectrophotometer, PCR, capillary and gel

electrophoresis apparatus), and setting up softwares and hardwares. Over the years, we

have made considerable research progress as proposed and described previously. Recent

progress shall be briefly discussed as follows:

A. Ligand Pr eor ganization in Metal Ion Complexation: Molecular

Mechanics/Dynamics, Kinetics and Laser -Excited Luminescence Studies of

Tr ivalent Lanthanide Complex For mation with Macr ocyclic Ligands TETA

and DOTA

The molecular mechanics and dynamics calculations, kinetics, and laser-excited

luminescence studies were carried out for trivalent lanthanide (Ln

3+

) complexes of

macrocyclic polyaminopolycarboxylate ligands TETA and DOTA (where TETA is

1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid and DOTA is

1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to further understand the

observed thermodynamic, kinetic and structural properties and to examine how ligand

preorganization affects metal ion complexation. Excitation spectroscopy (emission

monitored at 614.0 nm) of the

7

F

0

→

5

D

0

transition of Eu

3+

was used to study the aqueous

properties of the Eu

3+

-TETA system. A stopped-flow spectrophotometric method was used

to study the formation kinetics of the aqueous Ce

3+

-TETA/DOTA systems in the pH range

6.1 – 6.7. Molecular mechanics calculation results are consistent with the proposed

mechanism of Ln(DOTA)

-

formation, i.e. formation of a carboxylate O-bonded precursor,

followed by metal ion moving into the pre-formed macrocyclic cavity. For Ln(TETA)

-formation, at least two carboxylate O-bonded intermediates have been predicted and Ln

3+

ion assisted reorganization of the TETA ligand is present. The calculated bond-distances

and overall structures of Ln(DOTA)

-

and Ln(TETA)

-

were in agreement with the single

crystal and solution NMR structural data. The origin of the difference in thermodynamic

stability of Ln(DOTA)

-

and Ln(TETA)

-

complexes and the corresponding formation

intermediates is mainly due to the differences in water-occupancy energy (i.e. whether

there is an apical coordinated water molecule), the ligand strain energy and the

cation-ligand interaction energy. Kinetic studies revealed that the formation rates of the

Ce(TETA)

-

complex are smaller at lower pH and temperature but become greater at higher

pH and temperature, as compared to those of the Ce(DOTA)

-

complex. This is attributed to

the lanthanide ion and both mono- and di-hydroxide ion assisted TETA conformational

reorganization and higher kinetic activation parameters. The presence of di-hydroxide ion

assisted intermediate rearrangement pathway could make Ce(TETA)

-

complex formation

rate to be faster at higher pH and the higher activation barrier makes Ce(TETA)

-

complex

formation rate slower at lower pH, as compared to those of the Ce(DOTA)

-

complex.

B. Dissociation Kinetics of Lanthanide(III) Complexes of Macr ocyclic Polyaza

Polycar boxylate Ligand DO2A

The acid-catalyzed dissociation rate constants of several lanthanide(III) complexes of

1,4,7,10-tetraazacyclododecane- 1,7-diacetic acid (LnDO2A

+

, Ln = La, Ce, Pr, Nd, Sm, Eu,

Gd, and Lu) have been determined at four different temperatures (i.e. 25.0ºC, 31.0ºC,

37.0ºC, 45.0ºC) in aqueous media (µ = 0.10 M, HC1/LiClO

4

). In the presence of 0.005 –

0.046 M HCl, the dissociation reactions follow the simple rate law: -d[ML]T/dt =

k

H

[H

+

][ML]

T

, where k

H

is the dissociation rate constant for the pathway involving

monoprotonated species. The absence of an acid-independent pathway indicates that

LnDO2A

+

complexes are kinetically rather inert. The obtained k

H

values follow the order:

LaDO2A

+

＞CeDO2A

+

_＞PrDO2A

+

_＞NdDO2A

+

_＞SmDO2A

+

_＞EuDO2A

+

_＞GdDO2A

+

_＞

LuDO2A

+

, consistent with their thermodynamic stability order, i.e. the more

thermodynamically stable the more kinetically inert. The rate activation parameters,

∆

H

‡

,

∆

S

‡

and

∆

G

‡

, for the acid- catalyzed dissociation pathway have also been obtained. In

particular, an isokinetic behavior is found, i.e. a linear relationship between

∆

H

‡

and

∆

S

‡

values, which supports a common reaction mechanism. The data are compared with those

obtained previously for other lanthanide(III) complexes and the dissociation rate order is:

LnK21DA

+

＞LnK22DA

+

_＞LnDO2A

+

_{＞LnDO3A＞LnDOTA}

-, where K21DA is

1,7-diaza-4,10,13- trioxacyclo- pentadecane-N,N’-diacetic acid, K22DA is

1,10-diaza-4,7,13,16- tetraoxacyclooctadecane-N,N’ -diacetic acid, DO3A is

1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, and DOTA is

1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid. This is also consistent with the

order of thermodynamic stability.

C. Macr ocyclic Lanthanide Complexes as Ar tificial Nucleases：Hydr olysis of

Phosphodiester Bonds by LnDO2A and LnK21DA

The phosphodiester bond in DNA is very stable with an estimated

t

1/2

to be 130,000

years and nature has evolved a number of enzymes such as restriction endonucleases and

topoisomerases to efficiently catalyze DNA hydrolysis. Recently considerable effort has

been expended to develop small molecules as artificial nucleases to hydrolyze DNA, and

some trivalent lanthanide complexes are known to promote phosphodiester bond quite

efficiently. We have studied the hydrolysis of phosphodiester bond of a model compound

BNPP（sodium bis(4-nitrophenyl) -phosphate）, a plasmid DNA（pUC19）, and a model

of 5’-cap of mRNA (GpppG) by the use of LnDO2A

+

（Ln = Ce and Eu）and LnK21DA

+

（Ln = La and Eu）complexes, where DO2A is

1,7-diaza-4,10,13-trioxacyclopentadecane-N,N’-diacetic acid. In general, LnDO2A

+

is a better cleavage

agent than LnK21DA

+

, presumably due to the fact that LnDO2A

+

has at least 3

inner-sphere coordinated water molecules, one more than that of LnK21DA

+

. The pH-rate

profile for the hydrolysis of BNPP by both LnDO2A

+

and LnK21DA

+

shows interesting

titration-like curves which is tentatively explained by the stepwise formation of

coordinated hydroxide species, perhaps through forming hydroxo-bridged di- or more

complex multinuclear clusters, and which act as the active catalysts. The rates for the

hydrolysis of plasmid DNA and mRNA model have also been measured which indicates

that our lanthanide(III) complexes are roughly 10-times faster than those of other transition

or lanthanide complexes reported previously.

D. Synthesis and Char acter ization of Macr ocyclic Ligands to be Used for

Lanthanide Ar tificial Nucleases and MRI Contr ast Agents

Lanthanide complexes as artificial nucleases and magnetic resonance imaging（MRI）

contrast agents require high thermodynamic stability and low kinetic lability. We have

designed a number of macrocyclic ligands for such purposes and the synthetic details and

characterizations are reported in this paper.

N N N (CH₂)_n (CH₂)_n (CH₂)_n N N N (CH₂)_n (CH₂)_n (CH₂)_n N N N (CH₂)_n (CH₂)_n (CH₂)_n CHO N N (CH₂)_n (CH₂)_n (CH₂)_n N COOR CHO ROOC N N N (CH₂)_n (CH₂)_n (CH₂)_n COOH HOOC H (1) ₍₂₎ (3) (4) n=2 or 3,

(1) Me2NCH(OMe)2, benzene, reflux, 2-4 h (~100%) (2)EtOH-H2O, room temp., 2 h (70~85%)

(3)BrCH2COOR', iPr2NEt, MeCN (~90%) (4)H+

H

H H

H H

E. Publications (r efer eed, 1996-pr esent) Total publications = 97

1. C.A. Chang. "Selectivity of Macrocyclic Aminocarboxylates for Alkaline Earth Metal Ions

and Stability of Their Complexes", J. C. S. Dalton Trans., 1996, 2347-2350.

2. C.A. Chang. “Dissociation Kinetics of Nickel(II), Zinc(II), and Cadmium(II) Complexes of

1,7-Diaza-4,10,13-trioxacyclopentadecane-N,N'-diacetic Acid and 1,10-Diaza-4,7,13,16-tetraoxacyclooctadecane-N,N'-diacetic Acid”, J. Chin. Chem. Soc. (Taipei), 1996, 43, 419-426.

3. L. Wan, M.-Y. Luo, C.A. Chang, Y.-L. Lin, and E.-R. Chan. “Helicobacter pylori Induced Genes Expression in Human Gastric Cells Identified by mRNA differential Display”, Biochem. Biophys. Res. Commun. 1996, 228, 484-488.

4. C.A. Chang. "Macrocyclic Lanthanide Coordination Chemistry", Proc. Natl. Sci. Counc. ROC(A), 1997, 21, 1-13.

5. J.L. Laing, R.W. Taylor, and C.A. Chang. "The Acid-Catalyzed Dissociation of the Copper(II) and Lead(II) Complexes of Macrocyclic Diazapolyoxa-N,N‘- diacetic Acid", J. C. S. Dalton Trans., 1997, 1195-1200.

6. C.A. Chang, F.-K. Shieh, Y.-L. Liu, Y.-H. Chen, H.-Y. Chen, and C.-Y. Chen. “Capillary

Electrophoresis, Potentiometric and Luminescence Studies of Lanthanide(III) Complexes of 1,7-Dicarboxymethyl-1,4,7,10-tetraaza -cyclododecane (DO2A)”, J. C. S. Dalton Trans., 1998, 3243-3248.

7. C.A. Chang, F.-K. Shieh, Y.-L. Liu, and C.-S. Chung. “Effects of Chain Length and

Terminal N-alkylation on the Protonation Constants and Stability Constants of Some Transition Metal Complexes of Linear Tetraaza and Pentaaza Ligands”, J. Chin. Chem. Soc. (Taipei), 1998, 45, 753-759.

8. E.-R. Chan, C.A. Chang, T.-Z. Wu, and Y.-L. Lin, “Effects of Recombinant Lysostaphin on

Cytotoxicity and Interleukin-8 Level in Normal Human Epidermal Keratinocites Cell Lines”, Biotech. Lett., 1998, 20, 1053-1056.

9. E.-R. Chan, M.-R. Pan, C.A. Chang, T.-Z. Wu, and Y.-B. Kuo, “A Synthetic Complement

C1q-like Peptide Selectively Interacts with Immune Complexes”, Biotech. Lett., 1998, 20, 1119-1123.

10. C.A. Chang, H.-Y. Chen, and C.-Y. Chen. “Determination of Stability Constants of Metal

Ion Complexes by Capillary Electrophoresis.” J. Chin. Chem. Soc. (Taipei), 1999, 46, 519-528.

11. E.R. Chan, C.C. Chang, and C.A. Chang. “Purification and Characterization of Neutral Sphingomyelinase from Helicobacter pylori”, Biochemistry, 2000, 39, 4838-4845.

12. C.A. Chang and Y.-L. Liu. “Dissociation Kinetics of Ce(TETA)- and Ce(DOTA)-”, J. Chin. Chem. Soc. (Taipei), 2000, 47, 1001-1006.

13. K.-T. Chen, J.-D. Lin, T.-C. Chao, C.A. Chang, H.-F. Weng, and E.-C. Chan. “Quantitative Monitoring of Gene Expression Patterns in Metastatic and Follicolar Human Thyroid Carcinoma Using a Complementary DNA Array”, Thyroid, 2001, 11, 41-46.

14. C.A. Chang, Y.-L. Liu, C.-Y. Chen, X.-M. Chou, and J.-S. Ho. “Ligand Preorganization in

Metal Ion Complexation: Molecular Mechanics/Dynamics, Kinetics and the Laser-Excited Luminescence Studies of Trivalent Lanthanide Complex Formation with Macrocyclic

Ligands DOTA and TETA”, Inorg. Chem, 2001, 40, 3448-3455.

15. D.-L. Sheu, H.-A. Fan, K.-C. Hsu, C.A. Chang, Y.-S. Li, C.-C. Chiou, and E.-C. Chan. “Down-Regulation of Matrix Gla Protein Messenger RNA in Human Colorectal Adenocarcinomas”, 2002, Disease of the Colon and Rectum, submitted.

16. C.A. Chang and P.-Y. Kuan. “Effects of pH on the Rates of Phosphate Diester Hydrolysis

by Macrocyclic lanthanide Complexes as Artificial Nucleases”. 2002, to be submitted. 17. C.A. Chang, M.C. Lin, and C.F. Tseng. “Expression and Optimization of recombinant

Human Placental Lactogen in E. coli.” 2002, to be submitted.

18. C.A. Chang and C.-L. Chen. “Dissociation Kinetics of Ln(DO2A)+.” 2002, to be submitted. 19. C.A. Chang and C.-L. Chen. “Kinetics of DNA/RNA Hydrolytic Scission by Macrocyclic

Lanthanide Complexes Ln(DO2A)+.” 2002, to be submitted.

Other Ar ticles 1. 張正，"生化醫藥造福人群-生物科技簡介"、交大友聲雜誌，347期，48-49頁，民國83 年12月31日出刊。 2. 張正，"生物科技蓄勢待發-交大生物科技研究所簡介"、交大友聲雜誌，348期，51-52 頁，民國84年2月28日出刊。 3. 張正，"交大生物科技學系增設計畫"、交大友聲雜誌，356期，8-11頁，民國85年6月1 日出刊。 4. 張正，"從恐龍基因及基因專利談起：淺論生物科技與交大"、交大世紀之慶學術研討論 文集，交大理學院百年校慶特刊，167-174頁，民國85年6月31日出刊。 5. 張正、張永立、“生物科技產業之人力資源培育”、中華亞太經濟與管理學會「國家競 爭力與人力資源再造策略」研討會論文，中華民國 86 年 12 月 5 日，台灣高雄，中山

大學。

6. 李秀眉、沈燕士、張正*，“生物技術產業”，勞委會職訓局「行職業展望」第十三輯，

中華民國 89 年 11 月。

F. Recent Abstr acts and Paper s Pr esented at Scientific Meetings (1996 - 2002)

1. C.A. Chang. "Macrocyclic Lanthanide Complexes as Magnetic Resonance Imaging

(MRI) Contrast Agents." 1996 Symposium on Isotope and Radiation Application, Lungtan, Taiwan, April 26-27, 1996.

2. C.A. Chang. "Metal Complexes as Magnetic Resonance Imaging (MRI) Contrast

Agents." Second APSB International Symposium & Workshop, Hong Kong, July 8-11, 1996.

3. C.A. Chang. "Metal Chelate Nuclear Medicine and Magnetic Resonance Imaging

Contrast Agents“, 1996 Research Conference, Atomic Energy Council, R.O.C., Lung-Tan, Taiwan, November 20-21, 1996.

4. C.A. Chang. “Capillary Electrophoresis, Potentiometric, Luminescence, and Molecular

Mechanics Studies of Lanthanide(III) Complexes of Some Macrocyclic

Tetraazapolycarboxylate Ligands”, 1996 Annual Meeting of the Chinese Chemical Society (Taipei), Kaohsiung, Taiwan, December 6-8, 1996. [Invited Lectur e].

5. Y.-H. Chen and C.A. Chang. “Aqueous Solution Properties of Eu3+-DO2A System: A

Laser-Excited Luminescence Study.” 1996 Annual Meeting of the Chinese Chemical Society (Taipei), Kaohsiung, Taiwan, December 6-8, 1996.

6. F.-K. Shieh, Y.-L. Liu and C.A. Chang. “Effects of Chain Length and Terminal N-alkylation on the Protonation Constants and Stability Constants of Some Transition Metal Complexes of Linear Tetraaza and Pentaaza Ligands.” 1996 Annual Meeting of the Chinese Chemical Society (Taipei), Kaohsiung, Taiwan, December 6-8, 1996.

7. Huan-Yuan Chen, Chang-Yuh Chen and C.A. Chang. “Determination of Stability

Constants of Metal Ion Complexes by Capillary Electrophoresis.” 1996 Annual Meeting of the Chinese Chemical Society (Taipei), Kaohsiung, Taiwan, December 6-8, 1996.

8. Xiu-Mei Chou, Jyh-Shyong Ho and C.A. Chang. “Preorganization in Metal Ion

Complexation: A Molecular Mechanics Study of Lanthanide Complex Formation with

Macrocyclic Ligands DOTA and TETA.” 1996 Annual Meeting of the Chinese

Chemical Society (Taipei), Kaohsiung, Taiwan, December 6-8, 1996.

9. C.A. Chang and Y.-H. Chen. “Laser Luminescence Study of Aqueous Solution

Properties of Eu3+-DO2A System.” 213th ACS National Meeting at San Francisco, April 13-17, 1997.

10. M.-R. Pan, C.-H. Lin, C.A. Chang, and E.-C. Chan. “Development of C1q Like Peptide

for Using as an Universal Tracer in Immunoassay.” The Twelfth Joint Annual Conference of Biomedical Sciences, Taipei, Taiwan, April 19-20, 1997.

11. L. Wan, M.-Y. Lue, C.A. Chang, Y.-L. Lin, and E.-C. Chan. “Helicobacter Pylori Induced Genes Expression in Human Gastric Cells Identified by mRNA Differential Display.” The Twelfth Joint Annual Conference of Biomedical Sciences, Taipei, Taiwan, April 19-20, 1997.

12. C.A. Chang. “Capillary Electrophoresis and Laser-Excitation Fluorescence Studies of

Lanthanide Complex Equilibria”. 6th International Seminars on Electroanalytical Chemistry (6th ISEC), Changchun, China, Oct. 10-12, 1997 (invited speaker).

13. C.A. Chang.“ The Physicoanalytical Chemistry of Lanthanide and Transition Metal

Magnetic Resonance Imaging Contrast Agents”. 7th International Beijing Conference and Exhibition on Instrumental Analysis (7th BCEIA), Shanghai, China, Oct. 14-17, 1997 (invited speaker).

14. Y.M. Wang and C.A. Chang. "Metal Chelate Nuclear Medicine and Magnetic Resonance Imaging Contrast Agents“, 1997 Research Conference, Atomic Energy Council, R.O.C., Lung-Tan, Taiwan, November 26-27, 1997.

15. S.-P. Cheng, E.-C. Chan and C.A. Chang. “The Use of Piezoelectric Crystal Sensor for the Diagnosis of Human Helicobacter Pylori”. 1997 Annual Meeting of the Chinese Chemical Society (Taipei), Hsinchu, Taiwan, December 5-7, 1997.

16. M.-R. Pan, Y.-B. Kuo, C.-H. Lin, E.-C. Chan and C.A. Chang. “Isolation of Porcine Complement C1q and the Development of a C1q-like Peptide for Immunoassay“.. 1997 Annual Meeting of the Chinese Chemical Society (Taipei), Hsinchu, Taiwan, December 5-7, 1997

17. Chang-Yuh Chen and C.A. Chang. “Determination of Stability Constants of Metal Ion

Complexes by Capillary Electrophoresis. II. The Method of Metal-Ligand Double Exchange.” 1997 Annual Meeting of the Chinese Chemical Society (Taipei), Hsinchu, Taiwan, December 5-7, 1997.

18. S.-P. Cheng, E.-C. Chan and C.A. Chang. “Development of a Piezoelectric

Immunosensor for the Detection of Anti-Helicobacter Pylori Antibodies”. 1998 The Thirteenth Joint Annual Conference of Biomedical Sciences, Taipei, Taiwan, April 18-19, 1998.

19. C.A. Chang, Y.-L. Liu and C.-Y. Chen. “Kinetics and the Laser-Excited Luminescence

Study of Solution Properties of the Ce3+/Eu3+-TETA/DOTA Systems”. 1998 Annual Meeting of the Chinese Chemical Society (Taipei), Chia-Yi, Taiwan, November 27-29, 1998.

20. D.-L. Shi, E.-C. Chan and C.A. Chang. “Expression of the Matrix Gla Protein in Colorectal Tumor Cells”. 1999 The Fourteenth Joint Annual Conference of Biomedical Sciences, Taipei, Taiwan, April 10-11, 1999.

21. Y.-L. Yen, C.-C. Chiou, C.A. Chang, and E.-C. Chan. “The Study of Melanoma

Antigen Gene Expression in Colorectal Cancer by RT-PCR”. 1999 The Fourteenth Joint Annual Conference of Biomedical Sciences, Taipei, Taiwan, April 10-11, 1999. 22. C.A. Chang. “Biotechnology in Taiwan”. Taiwan-Australia Collaboration Research

Symposium, Taipei, Taiwan, April 8-9, 1999.

23. C.A. Chang. “Recent Advances of Biotechnology R&D in National Chiao Tung

University”. 高科技產業發展趨勢暨技術合作與投資研討會, Taipei, Taiwan, May 1, 1999.

24. C.A. Chang. “Strategies for Biotechnology Industry and Academic Institutions in

Taiwan.” Annual Conference of Midwest Chinese American Science & Technology Association, St. Louis, MO, U.S.A. June 11-13, 1999.

25. C.A. Chang. “Metal Complexes as Magnetic Resonance Imaging Contrast Agents and

DNA/RNA Cleavage Agents”, Annual Conference of Midwest Chinese American Science & Technology Association, St. Louis, MO, U.S.A. June 11-13, 1999.

26. D.L. Sheu, C.A. Chang, and E.-C. Chan. “Expression of Cell Cycle Regulator Genes

CDC25, Wee1HU, and Proto-Oncogen C-MYC in Human Colonrectal Cancer”. 2000 The

Fifthteenth Joint Annual Conference of Biomedical Sciences, Taipei, Taiwan, March 25-26, 2000.

27. K.-T. Chen, C.A. Chang, and E.-C. Chan. “Quantitative Monitoring of Gene Expression

Tissues”. 2000 The Fifthteenth Joint Annual Conference of Biomedical Sciences, Taipei, Taiwan, March 25-26, 2000.

28. K.-T. Chen, J.D. Lin, M.J. Liou, C.A. Chang, C.C. Chiou, and E.-C. Chan. “Detection of Distinctive Gene Expression in Thyroid Follicular Carcinoma Cell Line by Using cDNA Array Technology”. The Sixteenth Joint Annual Conference of Biomedical Sciences, Taipei, Taiwan, March 24-25, 2001.

29. J.W. Hsieh, J.D. Lin, M.J. Liou, C.A. Chang, and E.-C. Chan. “A Rapid Modified Method

of Polymerase Chain Reaction for Detection of Point Mutated-Iodide Transport Gene”. The Siteenth Joint Annual Conference of Biomedical Sciences, Taipei, Taiwan, March 24-25, 2001.

30. Chih-Huai Chen, Wen-Joan Chiang, Pei-Lin Kang, Lie-Fen Shyur, Ning-Sun Yang, C.A.

Chang, Chi-Meng Tzeng. “Establishment of Platform Technology of Pharmacogenomics

for Herbal Medicine Validation. (Molecular pharmaco-mechanism of Chinese Herbal Medicine (CHM))”. IBC’s Annual International Microtechnology Event, San Diego, CA, U.S.A., Oct. 28-Nov. 1, 2001.

31. Chih-Huai Chen, Wen-Joan Chiang, Pei-Lin Kang, Lie-Fen Shyur, Ning-Sun Yang, C.A.

Chang, Chi-Meng Tzeng. “Establishment of Platform Technology of Pharmacogenomics

for Herbal Medicine Validation. (Molecular pharmaco-mechanism of Chinese Herbal Medicine (CHM))”. IBC’s Annual International Microtechnology Event, San Diego, CA, U.S.A., Oct. 28-Nov. 1, 2001.

32. C.A. Chang; Chia-Ling Chen. “Dissociation Kinetics of Lanthanide(III) Complexes of

Macrocyclic Polyaza Polycarboxylate Ligand DO2A”. 2001 Annual Meeting of the Chinese Chemical Society (Taipei), Tainan, Taiwan, December 29-30.

33. C.A. Chang, Pu-Yun Kuan, Chia-Ling Chen, Po-Hong Wu. “Macrocyclic Lanthanide

Complexes as Artificial Nucleases：Hydrolysis of Phosphodiester Bonds by LnDO2A and LnK21DA”. 2001 Annual Meeting of the Chinese Chemical Society (Taipei), Tainan, Taiwan, December 29-30.

34. C.A. Chang, Wen-Hung Wang, Chun-Chieh Lin, Bo-Hung Wu, Yen-Yun Kwan, and

Chih-Cheng Lin. “Synthesis and Characterization of Macrocyclic Ligands to be Used for Lanthanide Artificial Nucleases and MRI Contrast Agents”. 2001 Annual Meeting of the Chinese Chemical Society (Taipei), Tainan, Taiwan, December 29-30.

35. P.C. Lan and C.A.Chang. “Cloning of Recombinant Porcine Placental Lactogen in E. coli.” The Seventh Joint Annual Conference of Biomedical Sciences, Taipei, Taiwan, March 23-24, 2002.

36. C.F. Teng, M.C. Lin, and C.A. Chang. “Expression and Renaturation of Recombinant Human Placental Lactogen in E. coli.”The Seventh Joint Annual Conference of Biomedical Sciences, Taipei, Taiwan, March 23-24, 2002.

37. K.T. Chen, J.D. Lin, M.J. Liou, C.A. Chang, and E.C. Chan. “Characterization of Distinctive Gene Expression in Thyroid Follicular Carcinoma Cells.” The Seventh Joint Annual Conference of Biomedical Sciences, Taipei, Taiwan, March 23-24, 2002.

38. J.W. Hsieh, F.S. Lo, C.A. Chang, and E.C. Chan. “Development of a Method for a High Throughput Screening Gene Variation.” The Seventh Joint Annual Conference of Biomedical Sciences, Taipei, Taiwan, March 23-24, 2002.

Confer ence/Symposium Or ganized

1. The 3rd International Symposium of Worldwide Chinese Scholar on Analytical Chemistry (第三屆國際華裔學者分析化學研討會). Hong Kong, December 16-19, 1998.

Bio-Medical Technologies Session, Taipei, Taiwan, April 8-9, 1999.

3. 高科技產業發展趨勢暨技術合作與投資研討會,生化組. 台北, 台灣, May 1, 1999.

4. 二十一世紀知識經濟研討會,生物科技發展趨勢及策略分析,交大思源金會,台北,台灣,

May 12, 2001.

Thesis Super vised (Since 1980: total 45+ Ph.D. and M.S. students, 10+ postdoctor al fellows)

學生姓名 論文題目 1. M.S. 謝發坤 F.K. Shieh 多胺與多乙酸基配位子與金屬離子錯合物穩定常數研究

The Stability Constants Studies of Metal Ion Complexes of Polyamines & Polyamino Polycarboxylates 2. M.S. 郭永斌 Y.B. Kuo 豬源補體蛋白 C1q 的純化及定性與定量之研究

The Purification and Characterization of Complement C1q From Swine 3.

M.S.

萬磊 L. Wan

幽門螺旋桿菌感染為組織細胞所引發基因差異表現之研究

Study of the Differential Gene Expression in Human Gastric Cells Infected with Helicobacter pylori 4. M.S. 潘美蓉 M.R. Pan 豬源補體蛋白 C1q 之製備與其類 peptide 應用之研究

The Preparation and Applications of Porcine C1q and The C1q-like Peptide 5.

M.S.

李亮緯 L.W. Lee

結腸直腸癌基因差別表現之研究

Study of Genes Expression in Colorectal Adenocarcinoma by DDRT-PCR Method 6. M.S. 許呈安 T.A. Hsu 甲狀腺腫瘤標記之研究

The Search of Tumor Markers for Thyroid Carcinoma 7.

M.S.

鄭昇沛 S.P. Cheng

壓電晶體免疫感測器於醫療檢驗之應用

The Application of Piezoelectric Crystal Immunosensor in Clinical Diagnosis 8.

M.S.

謝明宏 M.H. Shieh

固定細胞生產和回收溶葡萄球菌素之研究

Study of the in situ Recovery of Lysostaphin by Immobilized Recombinant Cells 9. M.S. 管佈雲 P.Y. Kuan 鑭系金屬與大環配位子錯合物切割磷酸二酯與去氧核醣核酸動力學及催化 效力之研究

Kinetic & Catalytic Activity Study of Lanthanide Complex of DO2A & K21DA in Phosphate Diester & DNA Hydrolysis

10. M.S.

張永立 Y.L. Chang

台灣醫療儀器產業環境與市場競爭策略

The Environment of Medical Instrument Industry and Its Competitive Strategies in Taiwan 11. M.S. 林孟嘉 M.G. Lin 以大腸桿菌表現人類胎盤促乳激素之研究

The Expression of Human Placental Lactogen by E. coli Expression System 12.

M.S.

許地利 D.L. Hsu

大腸直腸分子標幟之研究

Study of the Bio-molecular Markers in Colorectal Cancers 13.

M.S.

張至杰 C.G. Chang

幽門螺旋桿菌鞘磷脂脢的純化及其特性之研究

Purification and Characterization of the Sphingomyelinase of Helicobacter pylori 14. M.S. 陳家翎 G.L. Chen 鑭系金屬與大環多胺多酸基配位錯合物的動力學研究及其在 DNA/RNA 水解 上的應用

Kinetics Studies of Lanthanide Polyaza Polycarboxylate Macrocyclic Complexes Ln(DO2A) and Their Applications in DNA/RNA Hydrolytic Scission

15. M.S.

宋婉貞 W.C. Song

以大腸桿菌表現人類表皮生長因子接受體-2 之研究

Expression of Recombinant Human Epidermal Growth Factor Receptor-2 in E. coli

coli 16. Ph.D. 陳桂添 K.T. Chen 甲狀腺腫瘤分子標螺旋桿菌之膜蛋白 硝磷酸酵素之定性定量研究 Studies of Potential Protein Markers of Thyroid Carcinoma

17. M.S.

曾繼鋒 G.F. Tseng

Optimization of the expression of human placental lactogen by E. coli expression system 18. Ph.D. 羅千婷 C.T. Lo 多醣真菌菌絲體之篩選與基因比對

Polysarcharide and DNA Ringer Printing of Microorganisms 19.

M.S.

管燕芸 Y.Y. Kuan

稀土配位化合物之 DNA/RNA 切割劑 Artificial Lanthanide DNA/RNA Nucleases 20.

M.S.

謝瑞偉 A Rapid Modified Method of Polymerase Chain Reaction for the Dection of

Point Mutation in Sodium Iodide Symporter Gene 21.

M.S.

王文宏 W.H. Wang

稀土配位化合物之 DNA/RNA 切割劑 Artificial Lanthanide DNA/RNA Nucleases 22.

Ph.D.

林志誠 C.C. Lin

雙體磁振造影劑

Dimeric MRI Contrast Enhancement Agents 23.

Ph.D.

藍佩菁 P.G. Lan

The Functional Study of Human Placental Lactogen 24

M.S.

吳柏宏 B.H. Wu

稀土配位化合物之 DNA/RNA 切割劑 Artificial Lanthanide DNA/RNA Nucleases 25

Ph.D.

陳治淮 Fast Screening and Pharmacological Studies of Chinese Herbal Medicines Using

Gene Chips 26

M.S.

林俊傑 稀土配位化合物之 DNA/RNA 切割劑

Artificial Lanthanide DNA/RNA Nucleases 27

Ph.D.

戴金華 Microorganisms Functional Genenomics, Proteomics, Glycomics, and

Polysaccharides Functional Studies

In addition to the above-mentioned publications, abstracts and meetings, the PI has been invited by various institutions to present more than 60 seminars for the last 7 years.

C.A. Chang; Chia-Ling Chen. “Dissociation Kinetics of Lanthanide(III) Complexes of

Macrocyclic Polyaza Polycarboxylate Ligand DO2A”. 2001 Annual Meeting of the Chinese Chemical Society (Taipei), Tainan, Taiwan, December 29-30.

Dissociation Kinetics of Lanthanide(III)

Complexes of Macrocyclic Polyaza

Polycarboxylate Ligand DO2A

Department of Biological Science and Technology, National

Chiao Tung University, Hsinchu, Taiwan 30039, R.O.C.

The acid-catalyzed dissociation rate constants of several

lanthanide(III) complexes of 1,4,7,10-tetraazacyclododecane- 1,7-diacetic

acid (LnDO2A

+

, Ln = La, Ce, Pr, Nd, Sm, Eu, Gd, and Lu) have been

determined at four different temperatures (i.e. 25.0ºC, 31.0ºC, 37.0ºC,

45.0ºC) in aqueous media (µ = 0.10 M, HC1/LiClO

4

). In the presence of

0.005 – 0.046 M HCl, the dissociation reactions follow the simple rate law:

-d[ML]T/dt = k

H

[H

+

][ML]

T

, where k

H

is the dissociation rate constant for

the pathway involving monoprotonated species. The absence of an

acid-independent pathway indicates that LnDO2A

+

complexes are

kinetically rather inert. The obtained k

H

values follow the order: LaDO2A

+

＞CeDO2A

+

_＞PrDO2A

+

_＞NdDO2A

+

_＞SmDO2A

+

_＞EuDO2A

+

_＞

GdDO2A

+

＞LuDO2A

+

, consistent with their thermodynamic stability

order, i.e. the more thermodynamically stable the more kinetically inert.

The rate activation parameters,

∆

H

‡

,

∆

S

‡

and

∆

G

‡

, for the acid- catalyzed

dissociation pathway have also been obtained. In particular, an isokinetic

behavior is found, i.e. a linear relationship between

∆

H

‡

and

∆

S

‡

values,

which supports a common reaction mechanism. The data are compared

with those obtained previously for other lanthanide(III) complexes and the

dissociation rate order is: LnK21DA

+

＞LnK22DA

+

＞LnDO2A

+

＞

LnDO3A＞LnDOTA

-

, where K21DA is 1,7-diaza-4,10,13-

trioxacyclo-pentadecane-N,N’-diacetic acid, K22DA is 1,10-diaza-

4,7,13,16-tetraoxacyclooctadecane-N,N’ -diacetic acid, DO3A is

1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, and DOTA is

1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid. This is also

consistent with the order of thermodynamic stability.

Exper imental

l All reagents are of analytical reagent grade.

l Solutions were prepared and standardized by described methods.

l A HP8453 UV-Visible spectrophotometer equipped with a constant

temperature circulating bath (FIRSTEK SCIENTIFIC B403) was used

l for the kinetic rate measurements using dye indicators（25℃、31℃、37

Scheme 1 Possible Reaction Mechanisms

k

obs

=

k

d

+

k

H

[H

+

]

k

d

= acid-independent rate constant (~0, for the

present cases)

k

H

= acid-catalyzed rate constant。

+ − + +

_

_→

_

₊

+

2 (n 4) n k

L

H

M

nH

ML

obs

(

A

A

) (

k

t

)

A

A

_t

=

_∞

+

₀

−

_∞

exp

−

_obs

[

]

[

]

L

H

Ln

k

HL

Ln

LnHL

L

LnH

HL

Ln

LnHL

L

Ln

H fast k H k k H H d 2 2 * 2 lim 2 1

+

↓

+



→



+



 →





 →





←

+

↓

↓

+ + + +

LnL

Activated Par ameter Calculations and Plots

(Eyr ing equation)

R = 1.98716 cal‧mol-1K-1，kB = Boltzmann’s constant (1.381×10-23 JK-1)，h= Planck’s constant

(6.626×10-34 J‧s)。

Plots of ln(

k

H

/T) vs.1/T give slopes (-ΔH

*

/R) and intercepts

(ΔS

*

/R + ln(

k

B

/h)) and activation enthalpy (ΔH

*

) and

activation entropy (ΔS

*

) could be obtained。ΔG

*

could

then be obtained by

( )

(

)

(

S

_R

)

RT

H

h

k

T

k

_ln

B * *

ln

=









−

∆

+

∆

*

*

*

S

T

H

G

=

∆

−

∆

∆

Results and Discussion

1. 由每一個鑭系金屬錯合物(Ln(DO2A)

+

)觀察到的解離反應速率常數

（k

obs

）與酸濃度作圖之後，所得到的圖形呈現一個線性的關係，即為

一級反應(first-order)，這表示了 Ln(DO2A)

+

_{與酸作用了之後所形成的中}

間體(LnHDO2A)並不是很穩定，因此反應進行中當一形成反應中間體

會很快速的繼續進一步的解離反應。

2. 而以我們所做的八種金屬和 DO2A 所形成的金屬錯合物之解離常數

(

k

obs

) 值 來 比 較 ， 會 發 現 其 解 離 速 率 快 慢 的 順 序 為 ： La(DO2A)

+

＞

Ce(DO2A)

+

＞ Pr(DO2A)

+

_{＞ Nd(DO2A)}

+

_{＞ Sm(DO2A)}

+

_{＞ Eu(DO2A)}

+

_＞

Gd(DO2A)

+

＞Lu(DO2A)

+

，而此順序和金屬錯合物的穩定常數成反比，

亦和金屬的離子半徑(ionic size)的減小及電荷密度的(charge density)的

增加有絕對的關係。

3. 由所求得的自行解離常數(

k

d

)與酸催化解離常數(

k

H

)來看，我們發現所

有 Ln(DO2A)

+

_{的自行解離常數值非常的低，幾乎趨近於零因此可推知}

Ln(DO2A)

+

的解離反應可能極少經由自行解離途徑的，必須一定要在

加入酸時才會進行解離的反應，由此，我們也可印證 Ln(DO2A)

+

_的穩

定性是相當高的。

4. 由所求得的活化參數(activated parameter)來看，Ln(DO2A)

+

在酸解離反

應進行時所需的焓(activation enthalpy、ΔH

*

)其順序為：Lu(DO2A)

+

＞

Gd(DO2A)

+

＞ Eu(DO2A)

+

＞ Sm(DO2A)

+

＞ Nd(DO2A)

+

＞ Pr(DO2A)

+

＞

Ce(DO2A)

+

＞La(DO2A)

+

_{，此順序也是與金屬錯合物的穩定性呈正比的}

對地要使其金屬和配位子之間的離子鍵結斷裂所需要的能量也要越

多，因此，由此可再次證實：擁有高穩定常數的鑭系金屬錯合物，相

對地其會擁有較緩慢的解離速率常數。

5. 由於熱力學穩定常數高的金屬錯合物，其解離的速度會相對地變慢，

而根據以往的文獻整理所得到的鑭系金屬與大環配位子之穩定常數來

看 ， 可 得 知 鑭 系 金 屬 錯 合 物 的 穩 定 常 數 之 順 序 為 Ln(DOTA)

-

_＞

Ln(DO3A)＞Ln(DO2A)

+

，且 12、15、18 圓環的配位子與金屬形成錯合

物後，其穩定常數的順序為 Ln(DO2A)

+

_＞Ln(K22DA)

+

_＞Ln(K21DA)

+

_，

這是因為 K22DA 與 K21DA 比起 12 圓環的 DO2A 來有著較高的結構

柔軟性（more flexible），並且 DO2A 的鹼性較這兩個配位子高，因此

我們可推測 Ln(DO2A)

+

_{之酸解離常數應符合以下的順序 Ln(DOTA)}

-

_＞

Ln(DO3A)＞Ln(DO2A)

+

＞Ln(K22DA)

+

_＞Ln(K21DA)

+

_{，而最後從結果來}

Table1. Kinetic Data (

k

obs

, s

-1

) for the H

+

-catalyzed

Dissociation of Lanthanide DO2A Complexes.

102[H+] (M) Metal T (℃) 0.5769 1.1538 2.3077 3.4615 La 25 0.00503 0.00840 0.00802 0.01370 31 0.00694 0.01100 0.01760 0.02650 37 0.00802 0.01740 0.02990 0.04480 45 0.01370 0.02480 0.04810 0.06830 Ce 25 0.00448 0.00800 0.01370 0.02200 31 0.00692 0.01400 0.02330 0.03860 37 0.01070 0.02230 0.03400 0.05510 45 0.01340 0.03020 0.04530 0.07060 Pr 25 0.00672 0.00970 0.01670 31 0.00840 0.01440 0.02550 37 0.01360 0.02420 0.03980 45 0.02400 0.04350 0.06800 Nd 25 0.004566 0.00553 0.00664 31 0.006588 0.00811 0.01010 37 0.007394 0.01030 0.01380 45 0.008040 0.01410 0.01840 Sm 25 0.000394 0.00044 0.00055 31 0.000485 0.00088 0.00209 37 0.000751 0.00144 0.00283 45 0.00151 0.00348 0.00608 Eu 25 0.00004 0.00017 0.00044 0.00060 31 0.00038 0.00104 0.00197 0.00288 37 0.00072 0.00146 0.00375 0.00570 45 0.00106 0.00436 0.00748 0.01030 Gd 25 0.00002 0.00033 0.00047 0.00061 31 0.00032 0.00057 0.00096 0.00156 37 0.00041 0.00076 0.00174 0.00254 45 0.00115 0.00236 0.00479 0.00713 Lu 25 0.00008 0.00015 0.00021 0.00027 31 0.00010 0.00034 0.00053 0.00088 37 0.00024 0.00044 0.00079 0.00120

45 0.00039 0.00109 0.00232 0.00378 [LaDO2A]=[CeDO2A]=[EuDO2A]=[GdDO2A]=0.538462 × 10-3_M， [PrDO2A]=[NdDO2A]=[SmDO2A]=1.07692 × 10-3_{M，µ=0.10M(LiClO} 4)

Table.2

T(℃) kH LaDO2A 25 0.5872 (

±

0.0271) 31 0.7520 (

±

0.0160) 37 1.3735 (

±

0.0388) 45 1.9738 (

±

0.0741) CeDO2A 25 0.6133 (

±

0.0543) 31 1.0641 (

±

0.0730) 37 1.5488 (

±

0.1080) 45 2.0517 (

±

0.1078) PrDO2A 25 0.4975 (

±

0.0534) 31 0.7068 (

±

0.0579) 37 1.2042 (

±

0.0620) 45 1.9650 (

±

0.0645) NdDO2A 25 0.4566 (

±

0.0023) 31 0.7744 (

±

0.0104) 37 1.3049 (

±

0.0081) 45 1.8220 (

±

0.0124) SmDO2A 25 0.0181 (

±

0.0035) 31 0.0572 (

±

0.0092) 37 0.0854 (

±

0.0120) 45 0.1823 (

±

0.0145) EuDO2A 25 0.0176 (

±

0.0014) 31 0.0840 (

±

0.0042) 37 0.1606 (

±

0.0069) 45 0.3694 (

±

0.0137)

GdDO2A 25 0.0128 (

±

0.0005) 31 0.0446 (

±

0.0028) 37 0.0734 (

±

0.0027) 45 0.2662 (

±

0.0012) LuDO2A 25 0.0062 (

±

0.0009) 31 0.0249 (

±

0.0012) 37 0.0348 (

±

0.0008) 45 0.1165 (

±

0.0013)

Table 3.

ΔH

*

(kcal/mol)

ΔS

*

(e.u.)

ΔG

*

(kcal/mol)

LaDO2A

10.2340 (±0.129)

-21.3270 (±0.388)

16.5890 (±0.302)

CeDO2A

11.5722 (±0.151)

-20.5909 (±0.392)

17.7080 (±0.338)

PrDO2A

13.2942 (±0.642)

-15.4699 (±0.108)

17.9032 (±0.178)

NdDO2A

15.4574 (±0.373)

-8.2263 (±0.321)

17.9088 (±0.699)

SmDO2A

20.1876 (±0.306)

1.6122 (±0.033)

19.0718 (±0.391)

EuDO2A

27.0747 (±0.455)

24.8598 (±0.511)

19.6666 (±0.404)

GdDO2A

28.0271 (±0.338)

26.8541 (±0.453)

20.0246 (±0.297)

LuDO2A

28.1734 (±0.555)

25.7963 (±0.637)

20.4861 (±0.506)

Dissociation Kinetics of EuDO2A

Plots of

k

obs

vs [H+]

Eyr ing plot

Dissociation of EuDO2A [H+_{] (M)} 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040 kobs 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 0.018 0.020 25℃ 31℃ 37℃ 45℃ 1/T 0.00310 0.00315 0.00320 0.00325 0.00330 0.00335 0.00340 ln ( kob s /T ) -10 -9 -8 -7 -6