CHEMICAL BIOLOGY OF METALLOPROTEASES AND CYSTEINE PROTEASES HU MINGYU NATIONAL UNIVERSITY OF SINGAPORE 2012 CHEMICAL BIOLOGY OF METALLOPROTEASES AND CYSTEINE PROTEASES HU MINGYU (B.Sc., SOOCHOW UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2012 ACKNOWLDGEMENTS I would like to express my sincere gratitude to my supervisor Professor Yao Shao Qin for his guidance, encouragement and continuous support throughout my Ph.D studies The extensive knowledge, vision, and creative thinking of Prof Yao have been the source of inspiration for me throughout my studies Under his supervision and training, I have learnt a lot about the research field of Chemical biology, and have made great improvement on my mental discipline, leadership skills, and planning ability I would like to take this opportunity to extend my best wishes to him and hope his research group will attain greater success in the future Next, I am very thankful to a number of my senior lab-mates in both Chemistry and DBS labs: Wang Jun, Mahesh, Li Junqi, Rajavel Srinivisan, Sun Hongyan, Resmi C Panicker, Dr Sun Liping, Ng Su Ling, Chattopadhaya Souvik, Huang Xuan, Tan Lay Pheng, Candy Lu, Wei Lin They gave me a lot of help and support during my first 1-2 years study, which was probably the hardest time for me Besides, I also have many thanks to those junior lab-mates such as Kalesh, Wu Hao, Yang Pengyu, Shi Haibin, Liu Kai, Liqian, Ge Jingyan, Dr Li Lin, Zhang Chongjing, Cheng Xiamin, Na Zhenkun, Su Ying, Tan Ching Tian, Lee Jiexun, Zhang Xiaohua, Tan Mei Xuan, Lee Mei Ying, Wendy Leong, Ong Li Bing, David Ng, Derek Sim, Low Joo Leng, Shuyun, Yuhui, Tan Choon Meng, Liang Xian, Kaijia, Wee Liang, Farhana, Kitty, Cindy, Wang Jigang, Dr Li Zhengqiu and Wu Xiaoyuan Working with all of them has been a great experience and happy time for me I I appreciate the support of the service laboratory staff –– Ms Ler Peggy and Mdm Han Yanhui from the NMR lab, Mdm Lai Hui Ngee and Mdm Wong Lai Kwai from the Mass Spectrometry lab, Mdm Tong Yan and Dr Xiao Yong from DBS confocal Lab –– in providing training and technical expertise I would like to express my greatest thanks to my parents for their understanding and supports over these years This thesis will be dedicated to them Last but not least, I am also grateful to the National University of Singapore, for providing me the opportunity and scholarship for my research study I greatly acknowledge the following who had helped in part of the work described in this thesis: In Chapter 2, the PTP probes 2-P5 and 2-P6 were synthesized and characterized by Su Ying The two-photon dye 2-24 was designed and synthesized by Dr Li Lin and he also participated in the two-photon image experiment The bacteria lysates and transfected HeLa cells were provided by Wu Hao Mass Spectrometry analysis was carried out by Wang Jigang in A/P Lin Qingsong’s lab In Chapter and 5, a proportionate amount of work was done by my senior lab-mate Mr Wang Jun My major contribution is the synthesis of eight warheads Therefore, only relavent experimental details were described in this thesis II Table of Contents Page Summary VIII List of Publications IX List of Figures XI List of Tables XIV List of Schemes XV List of Abbreviations and Symbols XVI List of 20 Natural Amino Acids Chapter 1.1 XX Introduction Summary 1.2 Chemical Biology of Metalloproteases 1.2.1 The Nature of Matrix Metalloproteases 1.2.2 Development of MMP Inhibitors 1.2.2.1 1.2.2.2 1.3 Hydroxamic acid-based MMP Inhibitors Non-Hydroxamic acid-based MMP Inhibitors Chemical Biology of Cysteine Proteases 14 1.3.1 14 1.3.2 Activity-based protein profiling 17 1.3.3 1.4 The Nature of Caspases Fluorescent Probes for Bioimaging Applications 18 Project Objective 20 III Chapter Multicolor, One- and Two-Photon Imaging of Enzymatic Activities in Live Cells with Fluorescently Quenched Activity-Based Probes (qABPs) 22 2.1 Summary 22 2.2 Introduction 23 2.3 Results and Discussion 28 2.3.1 Design Principle of Quinone (or Quinolimine) MethideBased qABPs 28 2.3.2 Chemical Synthesis of the Probes 2.3.3 Mechanistic Studies of the Probes with Recombinant Enzymes 32 2.3.4 In-Cell Bioimaging of Apoptosis Using qABPs 36 2.3.5 One- and Two-Photon qABPs for Tyrosine Phosphatases 39 2.3.6 2.4 29 Live Cell Imaging with CPP-Containing Probe (2-P7) 43 Conclusion Chapter 45 In Situ “Click” Assembly of Small Molecule Matrix Metalloprotease Inhibitors Containing Zinc-Chelating Groups 47 3.1 Summary 47 3.2 Introduction 47 3.3 Results and Discussion 49 3.3.1 Design and synthesis of our new small molecule MMPIs 49 3.3.2 Inhibitor Fingerprinting with MMPs 51 IV 3.3.3 3.4 Docking Simulations 54 Conclusion Chapter 55 Rapid Assembly of Matrix Metalloprotease Inhibitors Using Click Chemistry 56 4.1 Summary 56 4.2 Introduction 57 4.3 Results and Discussion 58 4.3.1 Design and synthesis of alkyne and azide building blocks 4.3.2 58 Rapid assembly and in situ screening of metalloprotease inhibitors 4.3.3 4.4 61 Docking Simulations 64 Conclusion Chapter 65 “Click” Synthesis of Small Molecule Probes for Activity-based Fingerprinting of Matrix Metalloproteases 66 5.1 Summary 66 5.2 Introduction 66 5.3 Results and Discussion 68 5.3.1 68 5.3.2 5.4 Chemical design of the activity-based MMP probes Activity based fingerprinting of metalloproteases 69 Conclusion Chapter 6.1 72 Experimental Procedures 73 General Information 73 V 6.2 Solution Phase Synthesis 75 6.2.1 General Procedure for Synthesis of Dabcyl-containing linker 75 6.2.2 General Procedure for Synthesis of probe 2-P1 to 2-P4 77 6.2.3 General Procedure for Synthesis of 2-P5 (1-P PTP) and 2-P6 (2-P PTP) 6.2.4 89 General Procedure for Synthesis of Alkyne building blocks (3-D and 3-E) 97 6.2.5 General Procedure for Synthesis of Azide building blocks (3-C) 99 6.2.6 Synthesis of succinyl hydroxamate-based alkyne-containing warheads (4-8) 6.2.7 105 Synthesis of succinyl hydroxamate-based warheads with P1’ substitution 6.2.8 6.3 123 General Procedure of in situ Click Chemistry 129 Solid Phase Synthesis 131 6.3.1 131 6.3.2 General procedure for Fmoc deprotection 131 6.3.3 General procedure for coupling of Fmoc-amino acids 132 6.3.4 Procedure for coupling of compound 2-17 132 6.3.5 Procedure for coupling of Fluorescein(OAc)2-NHS 132 6.3.6 Cleavage of peptide from resin 133 6.3.7 6.4 Loading of Fmoc-Lys(Biotin)-OH onto Rink Amide AM Resin Purification of the peptides 133 Microplate-Based Enzyme Profiling Procedures VI 134 6.4.1 Caspase activity assay on microplate 134 6.4.2 High-Throughput Inhibitor Screening against MMPs 135 6.4.3 IC50 Measurements against MMPs 137 6.4.4 Ki Measurements against MMPs 138 6.5 Gel-Based Labeling Experiments with 2-P1 and 2-P5 138 6.6 Western Blot 140 6.7 Mass Spectrometry 140 6.8 Cell Culture 141 6.9 Transfection Experiment 141 6.10 Live Cell Imaging 142 6.11 Docking Simulations 143 Chapter Conclusion Remarks 144 7.1 Conclusion 144 Chapter References 145 Chapter Appendix 160 9.1 Supplemental Tables 160 9.2 Supplemental Figures 162 9.3 Supplemental Spectra 190 VII Summary Proteases are known to recognize a larger number of different substrates and are involved in almost every process in the cells Abnormal proteases activities are linked to many serious diseases, such as cancer, inflammation, arteriosclerosis, neurodegeneration and infection Due to their essential roles in physiological and pathological events, proteases are very important targets for drug development Therefore, it will be interesting to establish a chemical biology platform to study and understand the activities, localizations and functions of different enzymes In this thesis, we report a high-throughput strategy for assembling and in-situ screening of small molecule-based MMP inhibitors A “one-pot” click chemistry protocol was developed for this application (Chapter 3) Furthermore, we also successfully developed fluorescent quenched activity-based probes suitable for biomedical applications (Chapter 2) This approach provides real-time imaging of endogenous Caspase-3/7 activities in the apoptotic cells and could potentially target different classes of enzymes including glycosidases, phosphatases and various proteases VIII 1H normal range AC300 Dabcyl-3C-Tri(D)-Gly-COOH NAME : ma20hmy EXPNO : PROCNO 4.5470 4.5207 4.4998 4.4730 4.3404 4.3070 4.2818 4.2681 4.2325 4.2100 4.1865 3.0689 2.7538 2.7341 2.7007 2.6832 2.5835 2.5544 2.5298 1.7069 1.6844 1.6625 1.3601 5.8191 5.7506 6.8562 6.8255 10.1854 8.6810 8.6624 8.0275 7.9990 7.8839 7.8587 7.8313 7.8143 7.8012 7.7864 7.7267 7.7026 7.6900 7.6018 7.5738 7.5459 7.3996 7.3755 7.3152 7.2983 7.2917 7.2753 *** Current Data Parameters *** : *** Acquisition Parameters *** AQ_mod FmocHN CPDPRG O CPDPRGT : O N : CPDPRGB : HN N : 300.1300000 MHz BYTORDA : OH N H O : 300.1300000 MHz BF3 O au_zg : 300.1300000 MHz BF2 O : BF1 OtBu H N dqd AUNM O : DATE_t O 03:21:08 : Mar 20 2009 DBP07 17 : DATE_d HN N : edit DBPNAM0 : DBPNAM1 : DBPNAM2 : DBPNAM3 : DBPNAM4 : DBPNAM5 : DBPNAM6 : DBPNAM7 : DE : 6.0 usec 10.0 9.0 8.0 7.0 6.0 : 32 DECNUC : off DECSTAT : 9.0000 1.8316 0.8952 1.1295 7.4399 3.1147 1.0503 0.9419 1.9319 2.3288 4.0255 3.2452 2.9884 6.6686 1.6031 0.7322 0.8364 11.0 off DECIM Integral DECBNUC : DO 4.0 3.0 2.0 1.0 DIGTYP 0.0 : DP07 5.0 (ppm) : edit DPNAME0 : DPNAME1 : DPNAME2 : DPNAME3 : DPNAME4 : DPNAME5 : DPNAME6 : DPNAME7 : DR : 18 DS : DSLIST 13C AMX500 Dabcyl-3C-Tri(D)-COOH : SSSSSSSSSSSSSSS DSPFVS : 12 38.6922 38.0072 37.4242 29.8162 28.2275 28.1619 47.0800 52.6767 66.2385 74.9032 80.7622 155.4650 154.3938 153.3007 144.2716 144.1915 143.1348 141.1672 139.8846 139.2871 137.8952 135.2645 132.4151 129.3981 128.8078 128.3123 128.1083 127.7657 127.5180 125.7326 125.5431 121.9577 121.8411 120.5513 120.4857 119.6841 112.0469 110.1959 *** Current Data Parameters *** EXP NAME F1LIST : EXPNO : PROCNO : F2LIST F3LIST : my1228 : 111111111111111 : 222222222222222 : 333333333333333 *** Acquisition Parameters *** FL1 LOCNUC FL2 : : : NSFL3 : : NUCLEUS : : FL4 O O1FOV 13204.57 Hz 20.00 cm zgpg30 125000.00 Hz N H OH SOLVENT : : HL1 DMSO1 dB HL2 SW HN O HN 35 dB 238.7675 ppm : : 16 65536 dB TEHL4 O : : TDHL3 O N : : SFO1 : GRDPROG : 125.7709936 MHz O O N 90 dB PULPROG : : FW OtBu H N FmocHN 90 dB 2H 15901 dB 90 off dB 90 : : 17 dB 300.0 K INSTRUM : spect *** Processing Parameters *** N 17 O LBLOCNUC : : SF NBL : : 2H 1.00 Hz 125.7577890 MHz NC : -2 NS : 16 *** 1D NMR Plot Parameters *** NUCLEUS : NUCLEUS : off off O1 : 1853.43 Hz O2 : 1853.43 Hz O3 : 1853.43 Hz PAPS : QP PARMODE : 1D PHP : PH_ref : POWMOD : 0.000 degree low PR 140 120 100 (ppm) 80 60 40 20 high : mm QNP 1H/13C/31P/19F Z3246/0214 PROSOL 160 11 : PROBHD 180 : PRGAIN : no PULPROG : zg30 QNP : RG : 322.5000000 RO : SEOUT : SFO1 : 300.1318534 MHz SFO2 : 300.1318534 MHz SFO3 : 300.1318534 MHz SOLVENT : 190 20 Hz HR DMSO SW : SW_h : 5387.931 Hz TD : 32768 TE : 298.2 K TP07 : edit TPNAME0 : TPNAME1 : TPNAME2 : TPNAME3 : TPNAME4 : 17.9519 ppm 1H AMX500 *** Current Data Parameters *** Dabcyl-Glu-Allyl(cdcl3/minor meod) NAME : suy1111 EXPNO : 37 PROCNO : 7.5298 7.5008 7.4668 7.4617 7.4352 7.3672 7.3495 7.3369 7.3331 7.2600 6.7242 6.7065 6.3006 6.2224 6.1632 6.0358 6.0257 5.8921 5.8833 5.8719 5.8606 5.8492 5.8366 5.8253 5.8139 5.8013 5.7900 5.7799 5.7686 5.3765 5.3311 5.3286 5.2970 5.2680 5.2592 5.2479 5.2264 5.2101 5.1861 5.1647 4.6553 4.6452 4.6427 4.6188 4.6099 4.5999 4.5797 4.5683 4.5545 4.5431 4.3502 4.3338 4.2998 4.1838 4.1687 4.1536 4.1397 4.0893 4.0691 4.0577 4.0439 4.0338 4.0023 3.9771 3.9682 3.4173 3.4060 3.2181 3.2042 3.1891 3.1765 3.0555 2.9054 2.8386 2.7743 2.6016 2.5159 2.3974 2.3848 2.3469 2.3343 2.1112 1.9397 1.9271 1.7317 1.7178 1.7040 1.6914 1.6775 suy1111 37 *** Acquisition Parameters *** AQ_mod : dqd AUNM : au_zg BF1 : 500.1300000 MHz BF2 : 500.1300000 MHz BF3 : 500.1300000 MHz DATE_t : DATE_d : Dec 18 2010 DBP07 : edit INSTRUM : av500 NS : O1 : 3088.51 Hz O2 : 3088.51 Hz 21:42:55 44 PARMODE : SFO1 1D : 500.1330885 MHz SOLVENT : CDCl3 SW : 20.6557 ppm TE : 300.0 K *** Processing Parameters *** AZFW : 0.100 ppm LB : 0.30 Hz MAXI : 4.50 cm MI : 0.00 cm PC : 1.00 WDW : EM *** 1D NMR Plot Parameters *** 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 0.5 (ppm) : 487.29 AQ_time 0.0 : Hz_cm 1.0 : Stop 9.0000 6.1032 1.5026 2.0787 1.9524 6.4929 3.9499 1.4880 1.5046 2.3376 1.8627 6.2365 1.5465 0.6335 1.1011 3.2505 0.6406 1.3236 0.8678 1.2612 3.5207 7.3639 8.4565 3.4589 4.0496 Integral Start : 1.5859710 sec NAME : suy1111 Dabcyl-Glu-Allyl(cdcl3/minor meod) EXPNO : 38 PROCNO : -4.18 ppm *** Current Data Parameters *** suy1111 38 40.5559 40.0822 37.8595 31.3738 27.9050 46.9615 54.6278 80.9133 67.0455 66.7102 65.9232 *** Acquisition Parameters *** 165.6873 156.5417 155.3830 154.9166 154.3992 152.7231 143.5118 143.4973 141.1362 141.1143 131.2691 130.7954 127.6181 127.5962 126.9695 125.2788 124.9072 122.0505 119.8425 119.8206 111.3527 172.7852 170.6209 13C AMX500 16.48 ppm AQ_mod : qsim AUNM : au_zg BF1 : 125.7577890 MHz BF2 : 500.1300000 MHz BF3 : 125.7577890 MHz DATE_t : DATE_d : Dec 18 2010 DBP07 : edit INSTRUM : av500 NS : O1 : 13204.57 Hz O2 : 2000.52 Hz PARMODE : SFO1 21:45:48 824 1D : 125.7709936 MHz SOLVENT : CDCl3 SW : 238.7675 ppm TE : 300.0 K *** Processing Parameters *** AZFW : LB : 0.500 ppm 1.00 Hz MAXI : 10000.00 cm MI : 0.00 cm PC : 1.40 WDW : EM *** 1D NMR Plot Parameters *** Start 170 160 150 140 130 120 110 100 90 80 70 (ppm) 191 60 50 40 30 20 10 : AQ_time 180 : Hz_cm 190 : Stop : 224.26 ppm -14.53 ppm 1416.51 1.0911740 sec 1H AMX500 *** Current Data Parameters *** suy1111 49 NAME : suy1111 Dabcyl-Glu-COOH(repurifiedCDCl3/minor MeOD) EXPNO : 49 PROCNO : 5.2763 4.2853 4.1530 4.1391 4.1252 4.0874 4.0735 3.6915 3.5049 3.4911 3.4041 3.2566 2.9893 2.6124 2.5758 2.5632 2.5506 2.3047 2.1572 2.0955 2.0526 1.9404 1.5899 1.4210 1.3365 1.2533 1.1940 1.1802 1.1663 0.9885 0.9747 0.8751 -0.0074 6.6240 7.7725 7.7145 7.6212 7.4674 7.3930 7.3666 7.2607 7.1535 *** Acquisition Parameters *** INSTRUM : LOCNUC : av500 2H NS : 201 NUCLEUS : O1 off : 3088.51 Hz PULPROG : SFO1 zg30 : 500.1330885 MHz SOLVENT : SW TD CDCl3 : 20.6557 ppm : 32768 *** Processing Parameters *** LB : 0.30 Hz *** 1D NMR Plot Parameters *** 9.0 8.0 7.0 6.0 5.0 (ppm) 4.0 3.0 2.0 off 4.2635 9.6651 6.1212 2.3379 4.2328 2.9039 3.0502 8.3545 0.8209 0.5415 0.8388 6.4583 2.1365 13.377 10.060 Integral NUCLEUS : 1.0 0.0 : suy1111 EXPNO : 50 PROCNO : 80.8368 77.2587 77.0036 76.7485 67.1584 67.1146 66.3130 65.7592 60.4613 54.8719 49.8581 49.6833 49.5156 49.3407 46.9213 40.1076 35.1449 31.8511 31.5085 29.6211 29.2859 22.6107 19.0690 14.9589 14.0917 14.0261 NAME Dabcyl-Glu-COOH(repurifiedCDCl3/minor MeOD) 111.3781 *** Current Data Parameters *** suy1111 50 172.5556 156.5598 156.5234 154.7380 152.6174 143.7924 143.5446 141.0888 128.0152 127.5342 126.9731 125.2897 125.0055 122.0250 119.7586 13C AMX500 *** Acquisition Parameters *** INSTRUM : LOCNUC : 2H NS : 1824 NUCLEUS : O1 : PULPROG : SFO1 av500 off 13204.57 Hz zgpg30 : 125.7709936 MHz SOLVENT : SW : TD : CDCl3 238.7675 ppm 65536 *** Processing Parameters *** LB : 1.00 Hz *** 1D NMR Plot Parameters *** NUCLEUS : 180 160 140 120 100 (ppm) 80 60 192 40 20 off 190 180 170 160 8.0 150 7.0 140 130 6.0 120 110 5.0 100 90 4.0 (ppm) 80 193 70 60 41.6441 40.7787 38.5749 37.2222 30.1673 9.0 67.4997 67.4415 66.5833 10.0 155.7140 155.5758 153.3721 151.0229 150.9356 147.1682 144.0917 134.8405 134.7387 134.0768 129.9167 129.7566 129.0875 128.4911 126.6874 125.9601 125.6547 122.7236 120.7599 120.6945 119.4726 111.9887 170.0709 169.8018 168.3036 1.8873 5.7351 1.9792 2.0511 1.8346 2.0535 2.0000 4.0286 1.0850 0.9813 0.8579 2.3674 2.0400 0.9060 5.0259 3.8587 5.8613 2.4263 Integral 5.8660 5.8468 5.8282 5.8117 5.7893 5.7707 5.7548 5.7356 5.5948 5.5696 5.2644 5.2601 5.2069 5.2025 5.1845 5.1806 5.1494 5.1461 4.5462 4.5270 4.0510 4.0318 4.0132 3.9945 3.4401 3.4198 3.2275 3.2067 3.1864 3.1410 3.0413 1.6886 8.0727 8.0459 8.0223 7.9916 7.8733 7.8448 7.8382 7.8070 7.7873 7.7588 7.6131 7.6021 7.5966 7.5698 7.4591 7.4465 7.4312 7.4180 7.4082 7.3627 7.3435 7.3233 7.1759 7.1485 6.8220 6.7913 6.6987 6.6680 6.2834 6.2637 6.2435 1H normal range AC300 ag04suy before de-allyl in cdcl3 *** Current Data Parameters *** NAME 3.0 2.0 50 40 1.0 30 20 10 : ag04suy EXPNO : PROCNO : *** Acquisition Parameters *** BF1 : 300.1300000 MHz LOCNUC : NS : O1 : SFO1 SW SFO1 SW 2H 1853.43 Hz 32 PULPROG : : 300.1318534 MHz zg30 SOLVENT : : 17.9519 ppm CDCl3 *** Processing Parameters *** LB : 0.30 Hz PHC0 : 320.653 degree PHC1 : 2.436 degree (ppm) 0.0 13C Standard AC300 ag04suy before de-allyl in cdcl3 *** Current Data Parameters *** NAME : ag04suy EXPNO : PROCNO : *** Acquisition Parameters *** BF1 : LOCNUC : 2H NS : 13545 O1 PULPROG : : : SOLVENT : : 75.4677490 MHz 7924.11 Hz 75.4756731 MHz zgpg30 238.2968 ppm CDCl3 *** Processing Parameters *** LB : PHC0 : 40.610 degree PHC1 : 1.021 degree 1.00 Hz 31P AC300 ag04suy -6.4682 before de-allyl in cdcl3 *** Current Data Parameters *** NAME : ag04suy EXPNO : PROCNO : *** Acquisition Parameters *** BF1 : 121.4948510 MHz LOCNUC : NS : O1 : PULPROG : SFO1 16 -6074.78 Hz zgpg30 : 121.4887762 MHz SOLVENT : SW 2H : CDCl3 399.5734 ppm *** Processing Parameters *** LB 80 70 60 50 40 30 20 10 -10 -20 -30 -40 -50 -60 -70 -80 1.00 Hz : -78.535 degree PHC1 90 : PHC0 : 34.595 degree -90 (ppm) 1H normal range AC300 ma15suy 1.7228 1.7009 1.6795 2.5062 2.5002 2.4947 3.6764 3.6189 3.5855 3.5318 3.3061 3.0508 5.5950 5.5703 5.9347 6.8326 6.8019 8.6361 8.1228 8.0976 8.0110 7.9825 7.8105 7.7891 7.7820 7.7541 7.7311 7.6549 7.6330 7.6105 7.5864 7.5295 7.5010 7.2380 7.2101 after deallyl-63mg *** Current Data Parameters *** NAME : ma15suy EXPNO : PROCNO : *** Acquisition Parameters *** BF1 : 300.1300000 MHz LOCNUC : NS : O1 : PULPROG : SFO1 32 1853.43 Hz zg30 : 300.1318534 MHz SOLVENT : SW 2H : DMSO 17.9519 ppm *** Processing Parameters *** 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 (ppm) 194 3.0 2.2783 6.8559 2.3404 6.9757 3.5000 0.5138 0.8469 1.9508 1.9785 2.4178 4.5630 6.0119 2.3785 1.7091 0.5154 9.0 2.5 2.0 1.5 1.0 0.5 0.0 : 58.372 degree PHC1 9.5 : PHC0 Integral LB 0.30 Hz : -10.187 degree 13C Standard AC300 ma15suy 40.3412 40.0648 39.7885 39.5048 39.2284 38.9521 38.6757 38.2902 37.0029 29.3517 66.3785 66.3204 73.8406 111.5439 121.4934 119.9151 119.8569 167.9315 165.6841 165.6041 154.9201 153.9600 152.8109 149.7999 149.7126 146.7961 142.6651 134.7811 134.4466 134.3084 129.6100 129.0573 128.8464 128.3154 125.0789 124.9262 after deallyl-63mg *** Current Data Parameters *** NAME : ma15suy EXPNO : PROCNO : *** Acquisition Parameters *** BF1 : LOCNUC : 2H NS : 2609 O1 : 7924.11 Hz PULPROG : SFO1 : SOLVENT : SW : 75.4677490 MHz zgpg30 75.4756731 MHz DMSO 238.2968 ppm *** Processing Parameters *** LB 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 : 8.017 degree PHC1 190 : PHC0 1.00 Hz : -24.869 degree (ppm) 31P AC300 ma15suy -6.1511 after deallyl *** Current Data Parameters *** NAME : ma15suy EXPNO : PROCNO : *** Acquisition Parameters *** BF1 : 121.4948510 MHz LOCNUC : NS : O1 : PULPROG : SFO1 -6074.78 Hz zgpg30 : 121.4887762 MHz SOLVENT : SW 2H : DMSO 399.5734 ppm *** Processing Parameters *** LB 80 70 60 50 40 30 20 10 -10 -20 (ppm) 195 -30 -40 -50 -60 -70 -80 -90 1.00 Hz : -62.449 degree PHC1 90 : PHC0 : 23.903 degree 1.8173 1.8034 1.7896 1.4101 1.3660 1.3521 3.4852 3.4714 3.4575 3.1751 3.1625 3.1486 4.7699 7.2788 7.2636 6.6976 6.6925 6.5929 6.5753 6.5349 6.5299 6.5173 6.5135 7.9394 8.2092 8.2067 8.1928 8.1903 suying 1-P linker 1H 500MHz *** Current Data Parameters *** NAME : gjy0807 EXPNO : PROCNO : *** Acquisition Parameters *** BF1 : 500.1300000 MHz LOCNUC : NS : O1 : PULPROG : SFO1 29 3088.51 Hz zg30 : 500.1330885 MHz SOLVENT : SW 2H : MeOD 20.6557 ppm *** Processing Parameters *** 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 146.720 degree : -0.006 degree 2.1843 9.0000 1.9673 2.0488 1.9281 3.8647 2.1195 1.8795 1.8956 0.8752 0.7393 0.8705 9.0 0.30 Hz : PHC1 9.5 : PHC0 Integral LB 1.5 1.0 0.5 0.0 (ppm) 13.2398 37.3319 36.7708 33.8485 30.8024 29.0097 25.8543 45.0055 66.5980 77.9517 77.7039 77.4489 112.0931 110.3441 123.7019 121.1586 129.9836 137.6426 149.2368 157.0562 166.0488 165.7719 173.7661 suying 2-P linker *** Current Data Parameters *** NAME : gjy0807 EXPNO : PROCNO : *** Acquisition Parameters *** BF1 : 125.7577890 MHz LOCNUC : 2H NS : 105 O1 : PULPROG : SFO1 zgpg30 : 125.7709936 MHz SOLVENT : SW 13204.57 Hz : CDCl3 238.7675 ppm *** Processing Parameters *** LB 180 170 160 150 140 130 120 110 100 90 80 (ppm) 196 70 60 50 40 30 20 10 1.00 Hz : 169.946 degree PHC1 190 : PHC0 : 16.046 degree 1H AMX500 condensation 1# NAME : my0317 EXPNO : PROCNO 0.0026 0.2585 6.6177 7.2606 *** Current Data Parameters *** : *** Acquisition Parameters *** D[1] : D[33] : DATE_t : 05:05:52 DATE_d : Mar 17 2007 NS : O2 : SFO1 : 500.1330885 MHz SOLVENT : SW : SW_h : 1.0000000 sec 0.0000000 sec 3088.51 Hz CDCl3 20.6557 ppm 10330.579 Hz *** Processing Parameters *** LB : 0.30 Hz *** 1D NMR Plot Parameters *** Start : 9.00 ppm Stop : -1.00 ppm SR : 12.74 Hz O TMS S NH 8.5 8.0 7.5 7.0 9.0957 1.0000 Integral S 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 (ppm) 13C AMX500 condensation 1# NAME : my0317 EXPNO : PROCNO -0.0093 -0.5195 77.2587 77.0036 76.7486 99.8422 115.8526 112.9668 139.3471 165.9387 192.2680 *** Current Data Parameters *** : *** Acquisition Parameters *** D[1] : D[33] : DATE_t : 05:09:58 DATE_d : Mar 17 2007 NS : 300 O2 : 2000.52 Hz SFO1 : 125.7709936 MHz SOLVENT : SW : SW_h : 2.0000000 sec 0.0000000 sec CDCl3 238.7675 ppm 30030.030 Hz *** Processing Parameters *** LB : 1.00 Hz *** 1D NMR Plot Parameters *** Start : 224.40 ppm Stop : -14.39 ppm SR : -0.21 Hz O TMS S NH S 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 (ppm) 197 60 50 40 30 20 10 -10 1H normal range AC300 condensation 2# 0.2582 -0.0032 2.0951 4.8388 6.7228 10.8328 *** Current Data Parameters *** NAME : ma08hmy EXPNO : PROCNO : *** Acquisition Parameters *** AQ_mod : dqd AUNM : au_zg BF1 : 300.1300000 MHz DATE_t : 05:31:52 DATE_d : Mar 08 2007 NS : O2 : 16 1853.43 Hz PARMODE : 1D RG : 143.6999969 SFO1 : 300.1318534 MHz SOLVENT : CDCl3 SW SW_h : TE : YMAX_a : 1220161152.0000000 YMIN_a acetic acid : 17.9519 ppm : -926127360.0000000 5387.931 Hz 300.0 K *** 1D NMR Plot Parameters *** SR : 11.64 Hz 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 TMS 8.9823 1.3328 2.0000 0.9239 Integral O 2.0 1.0 N S COOH S 0.0 (ppm) 20.6693 29.7170 44.3066 77.3624 76.9406 76.5115 *** Current Data Parameters *** 99.8578 115.9457 113.3929 136.8483 164.8785 171.4460 177.8171 192.2177 13C Standard AC300 condensation 2# NAME : ma08hmy EXPNO : PROCNO : *** Acquisition Parameters *** AQ_mod : dqd AUNM : au_zg BF1 : DATE_t : 05:36:09 DATE_d : Mar 08 2007 NS : 212 O2 : 1200.52 Hz 75.4677490 MHz PARMODE : 1D RG :32768.0000000 SFO1 : SOLVENT : 75.4756731 MHz CDCl3 SW : SW_h : 238.2968 ppm TE : YMAX_a : 1221259136.0000000 YMIN_a : -915546560.0000000 17985.612 Hz 300.0 K *** 1D NMR Plot Parameters *** acetic acid acetic acid SR : 7.70 Hz O TMS S N S 220 200 180 160 140 120 100 80 (ppm) 198 60 40 20 COOH 1H normal range AC300 pdt-8 NAME : ag31hmy EXPNO : PROCNO 2.5061 2.5000 2.4946 2.4885 1.9084 3.3092 3.8708 7.1327 7.1058 7.5759 7.5474 7.5216 7.5112 7.4860 9.1992 *** Current Data Parameters *** : *** Acquisition Parameters *** DATE_t : DATE_d : Aug 30 2007 NS : SFO1 : 300.1318534 MHz SOLVENT : 16:48:09 16 DMSO *** Processing Parameters *** SF : 300.1300011 MHz *** 1D NMR Plot Parameters *** Start : 11.00 ppm Stop : -0.50 ppm O N N N O S NH 10.0 9.0 8.0 7.0 6.0 5.0 4.0 0.5823 3.0000 1.0993 3.9876 0.9234 Integral S 3.0 2.0 1.0 0.0 (ppm) 1H normal range AC300 pdt-23 -0.0013 1.3853 1.3623 1.3387 2.0712 1.9074 4.1530 4.1295 4.1065 4.0829 7.1711 7.1410 7.8275 7.7973 7.6050 9.0974 *** Current Data Parameters *** NAME : ag31hmy EXPNO : PROCNO : *** Acquisition Parameters *** DATE_t : DATE_d : Aug 30 2007 NS : SFO1 : 300.1318534 MHz SOLVENT : 16:54:43 16 DMSO *** Processing Parameters *** SF : 300.1300011 MHz *** 1D NMR Plot Parameters *** Start : 11.00 ppm Stop : -0.50 ppm O O N N N S NH 10.0 9.0 8.0 7.0 6.0 5.0 3.0000 2.2058 1.8885 0.7232 1.7109 0.6512 Integral S 4.0 3.0 (ppm) 199 2.0 1.0 0.0 1H normal range AC300 pdt-25 NAME : ag31hmy EXPNO : PROCNO 1.3870 1.3640 1.3360 1.3130 1.2895 4.2292 4.2112 4.1882 4.1651 4.1416 8.9714 7.7738 7.7328 7.7108 7.7059 7.5766 7.5711 7.5476 7.5240 7.5185 7.3547 7.3295 7.1926 7.1668 7.1416 7.1378 9.6234 *** Current Data Parameters *** : *** Acquisition Parameters *** DATE_t : DATE_d : Aug 30 2007 NS : SFO1 : 300.1318534 MHz SOLVENT : 16:59:33 16 DMSO *** Processing Parameters *** SF : 300.1300010 MHz *** 1D NMR Plot Parameters *** Start : 11.00 ppm Stop : -0.50 ppm O O N N N S NH 10.0 9.0 8.0 7.0 6.0 5.0 3.0000 2.0298 0.9954 0.9775 0.9935 1.9225 0.7660 0.1801 Integral S 4.0 3.0 (ppm) 200 2.0 1.0 0.0 201 202 203 204 .. .CHEMICAL BIOLOGY OF METALLOPROTEASES AND CYSTEINE PROTEASES HU MINGYU (B.Sc., SOOCHOW UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY... activities 1.2 Chemical Biology of Metalloproteases Metalloproteases, together with serine, threonine, cysteine, aspartate and glutamic acid proteases, represent the six major classes of proteolytic... Structures of AHA and new ZBGs examined by Cohen’s group Table 1-1 Pyrone-based MMP inhibitors: compounds 1-22 and 1-23 and their IC50 values 13 1.3 Chemical Biology of Cysteine Proteases As one of the