DEVELOPING HIGH-THROUGHPUT CHEMICAL APPROACHES FOR PROTEOMIC PROFILING OF ASPARTIC PROTEASES AND PROTEIN KINASES SHI HAIBIN (M.Sc., SOOCHOW UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENTS I would like to give special thank to my helpful supervisor, Prof. Yao Shao Qin for his invaluable guidance, patience, encouragement and endless support throughout my four-year study. He has brought me into the fascination world of chemical biology and ignited in me an inexorable passion for science. The supervision and support that he gave truly help me make great progression and improvement in many aspects. His unparalleled passion, indefatigable spirits and professionalism have been deeply engraved upon my mind. My deepest appreciation is for Prof Yao-my teacher and mentor. I sincerely thank all the labmates both in Chemistry and DBS Labs-Liu Kai, Chongjing, Xiamin, Grace, Jigang, Laypheng, Candy, Wu Hao, Pengyu, Mingyu, Li Lin, Jingyan, Liqian, Zhenkun, Su Ying, Zhengqiu, Xiaoyuan, Wendy, Ashley as well as some senior members in Yao lab: Mahesh, Kalesh, Hongyan, Raja, Souvik. I would like to take this opportunity to thank each of you for invaluable assistances, fruitful discussions and happy memories over these years. The cooperation is much appreciated. I would like to give special thanks to my collaborators, Liu Kai helped me to carry out many biological works, and I have learnt more from him. Besides, I am very thankful to Wu Hao‟s help. Thank Kalesh, Chongjing and Xiamin help me to synthesize the kinase probes, Jigang taught me how to carry out the protein identification work. Thanks again for your helps. I also appreciate the support from Mdm Han Yanhui and Ms Peggy Ler from i NMR lab as well as other chemistry support labs. Last but not least, I would like to express my heartful gratitude to my parents, my wife and my son for their love, understanding, encouragement, patience and moral supports over these years. This thesis will be dedicated to them. I also acknowledge kind support from NUS for providing me research scholarship. ii Table of Contents Page Chapter 1. Introduction 1.1 Summary 1.2 Aspartic Proteases as Therapeutic Targets 1.3 1.4 1.2.1 Catalytic Mechanism of Aspartic Proteases 1.2.2 Inhibitor Development for Aspartic Proteases Protein Kinases as Therapeutic Targets 1.3.1 Catalytic Mechanism and Clarification of Protein Kinase 1.3.2 Development of Protein Tyrosine Kinases Inhibitors High-throughput Amenable Chemistry 10 1.4.1 Solid-phase Chemistry 11 1.4.2 Click Chemistry 13 1.4.3 Small Molecule Microarray 16 1.5 Activity-based Protein Profiling (ABPP) 18 1.6 Affinity-based Protein Profiling (AfBP) 19 1.7 Research Objectives 20 Chapter 2. Expedient Solid-phase Synthesis of Both Symmetric and Asymmetric Diol Libraries Targeting Aspartic Proteases 21 2.1 Summary 21 2.2 Introduction 21 iii 2.3 2.4 Results and Discussion 2.3.1 Solution-phase Synthesis of Diamino Diols 2.3.2 Traceless Synthesis of Symmetric and Asymmetric 24 24 Inhibitor Library 26 2.3.3 Inhibitor Fingerprinting 27 2.3.4 IC50 Measurements of Selected Inhibitors 28 2.3.5 Ki Measurements 30 Conclusion Chapter 3. 31 High-throughput Synthesis of Aspartic Protease Inhibitors and Probes for Proteome Profiling of Plasmapsins in Malaria Parasites 33 3.1 Summary 33 3.2 Introduction 34 3.2.1 High-throughput Amenable Chemical Reactions in Inhibitor Discovery 3.2.2 Functional Profiling, Identification and Inhibition of Plasmepsins in Intraerythrocytic Malaria Parasites 3.3 Results and Discussion 3.3.1 34 35 37 Solution-cum-solid Phase Synthesis of N, C-terminal Azide Warheads 37 3.3.2 Traceless Synthesis of Alkyne Building Blocks 49 3.3.3 “Click” Assembly of Aspartic Protease Inhibitor Library 40 3.3.4 Preliminary Screening Experiments with Aspartic Proteases 41 iv 3.3.5 “Click” Synthesis of Affinity-based Probes 42 3.3.6 Labeling of Recombinant Aspartic Proteases 43 3.3.7 Profiling of Aspartic Protease Activity Throughout Different of Blood Stages P. Falciparum 45 3.3.8 Inhibitor Library Screening for Plasmepsins 47 3.3.9 Identification of the 37-kDa Band 48 3.3.10 Molecule Modelling G16 Binding to Plasmepsins 3.4 Conclusion Chapter 4. 51 52 Small Molecule Microarray (SMM)-facilitated Screening of Affinity-based Probes for -secretase 54 4.1 Summary 54 4.2 Introduction 54 4.3 Results and Discussion 57 4.3.1 Synthesis of N, C-terminal Warheads 57 4.3.2 Solid-phase Synthesis of Inhibitor Library 59 4.3.3 Inhibitor Fingerprinting of HAP and Its Mutants 60 4.3.4 Inhibitors Fingerprinting of Cellular Lysates 62 4.3.5 “Click” Synthesis of TER/Biotin Probes 63 4.3.6 In-gel Fluorescence Scanning of Cellular Lysate with Probe and Validation 4.4 Conclusion Chapter 5. 64 65 Applying Small Molecule Microarrays and Resulting Affinity Probe v Cocktails for Sub-Proteomic Profiling of Mammalian Cell Lysates 66 5.1 Summary 66 5.2 Introduction 67 5.3 Results and Discussion 72 5.3.1 5.4 Design and Synthesis of Hydroxyethylamine-containing Inhibitor Library 72 5.3.2 Profiling of Mammalian Cell Lysates on SMMs 74 5.3.3 Click Assembly of Select AfBPs 76 5.3.4 Activity-based Profiling of the AfBPs Library with Recombinant Cathepsin D 77 5.3.5 In vitro Proteome Profiling 78 5.3.6 Pull-Down Experiments and Target Identification 82 Conclusion Chapter 6. 87 Developing Photo-affinity Probes for Proteomic Profiling of Cellular Targets of DasatinibTM 88 6.1 Summary 88 6.2 Introduction 89 6.3 Results and Discussion 91 6.3.1 Design and Synthesis of Dasatinib-like Probes 91 6.3.2 Molecule Modelling and Determination of IC50 Values 93 6.3.3 Effects on Cell Proliferation and Phosphorylation of c-Src 95 6.3.4 Cellular Imaging using DA-2 96 vi 6.4 6.3.5 In vitro Labeling of Purified Kinase 6.3.6 Labeling of c-Src in Bacterial and Mammalian Proteomes 100 6.3.7 Labeling of Endogenous c-Src in Cancer Cell Lysates 101 6.3.8 In situ Labeling in Cultured Cells 102 6.3.9 Target Identification and Validation 104 Conclusion 98 107 Chapter 7. A “Clickable”, Cell Permeable Probe for Proteome Profiling of Potential Cellular Targets of Staurosphorine 108 7.1 Summary 108 7.2 Introduction 109 7.3 Results and Discussion 111 7.4 7.3.1 Design and Synthesis of Staurosphorine-like Probe 111 7.3.2 Biological Characterization of Probe STS-1 112 7.3.3 In vitro Labeling with Purified Kinases 114 7.3.4 Proteome Profiling of Mammalian Cellular Lysates 115 7.3.5 Target Identification and Validation 116 Conclusion Chapter 8. 8.1 8.2 Experimental Section General Information 118 119 119 8.1.1 Materials 119 8.1.2 Instruments 121 Solution-phase Synthesis 122 vii 8.3 8.4 8.5 8.2.1 General Procedure for Synthesis of Diol-amine Warhead 122 8.2.2 Procedure for Synthesis of N-terminal Azides 129 8.2.3 Procedure for Synthesis of C-terminal Azides 136 8.2.4 Synthesis of Rhodamine/biotin Bp Alkyne Compounds 140 8.2.5 Chemical Synthesis of Biotin Acid Linkers 144 8.2.6 Chemical Synthesis of Dasatinib-like Probes 147 8.2.7 Chemical Synthesis of the Staurosphorine-derived Probe 156 Solid-phase Synthesis 8.3.1 Procedure for Synthesis of the Diol Library 159 8.3.2 Synthesis of Eight Hydroxyethylamine Azide Warheads 168 8.3.3 Traceless Synthesis of Alkyne Building Blocks 170 8.3.4 Procedure for Synthesis of the 198-member N-terminal Library 173 8.3.5 Procedure for Synthesis of the 100-member C-terminal Library 176 Click Chemistry Synthesis 178 8.4.1 Construction of the 152-member Inhibitor Library Against PMs 179 8.4.2 Synthesis of Affinity-based Probes Targeting Aspartic Proteases 183 Microplate Assay 8.5.1 8.5.2 184 Inhibition Activity of the Diols Library against Aspartic Proteases 184 Inhibition Activity of Dasatinib/staurosphorine Probes against Protein Kinases 8.6 159 Microarray-based Screening 186 188 viii 8.7 8.8 8.9 8.6.1 Preparation of Avidin Slides 187 8.6.2 Microarray Preparation 189 8.6.3 Protein/proteome Labeling and Screening on SMM 189 8.6.4 Data Extraction and Analysis 190 Fluorescent Profiling of Aspartic Proteases and Protein Kinases 8.7.1 Labeling of Recombinant Aspartic Proteases 191 8.7.2 Labeling and Identification of PM I, PM II and HAP 192 8.7.3 Characterization of Known Inhbitors for FV Plasmepsins 194 8.7.4 Characterization of AfBPs with ɤ-30 Cell Lysates 195 8.7.5 Fluorescent Labeling of Mammalian Cell Lysates 196 8.7.6 Proteome Profiling of Protein Kinases 197 8.7.6.1 Labeling of Kinases Present in Bacterial Proteome 197 8.7.6.2 Labeling of Kinases Present in Mammalian Proteome 198 Pull-down and Mass Spectrometry Identification 199 8.8.1 Pull-down Assay with Aspartic Protease Probes 199 8.8.2 Pull-down Assay with Kinase Probes 201 8.8.3 Mass Spectrometric Analysis 202 Cell Culture and Lysates Preparation 204 8.9.1 Parasite Cultures 204 8.9.2 Mammalian Cell Cultures 204 8.9.2.1 8.10 191 Preparation of Membrane Fractions of ɤ-30 Cells Western Blotting 205 205 ix 1H normal range AC300 hit-3 1.2346 1.2001 1.1765 1.1529 0.9634 0.9415 0.8598 0.8385 2.3418 4.5463 4.5003 4.1908 4.1612 4.1469 4.1163 4.0407 4.0204 3.9273 3.8955 3.8626 3.7843 3.7536 3.7246 3.3980 8.3649 8.3336 8.0537 8.0345 8.0148 7.7984 7.5496 7.5420 7.5343 7.5277 7.5140 7.4067 7.3475 7.3196 7.2911 7.2571 *** Current Data Parameters *** NAME : jl09shb EXPNO : PROCNO : *** Acquisition Parameters *** DATE_t : 01:59:53 DATE_d : Jul 09 2008 DBPNAM7 : O N H F N N N OH Cl O O S N H CH3 DS : NS : O1 : 1853.43 Hz O2 : 1853.43 Hz O3 : 1853.43 Hz SFO1 : 300.1318534 MHz SFO2 : 300.1318534 MHz SFO3 : 300.1318534 MHz SOLVENT : CH3 DMSO *** Processing Parameters *** AZFW : 0.100 ppm *** 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 : 0.42 Hz 3.0000 3.4488 1.4102 1.2486 5.7521 0.9213 1.0597 2.0027 1.3657 0.7815 2.0348 1.8452 2.1980 0.7948 0.9130 0.7602 Integral SR 1.0 0.5 0.0 (ppm) 13C Standard AC300 hit-3 20.7462 20.2880 15.7206 40.0779 39.8015 39.5251 39.2487 38.9651 27.4228 55.9912 53.6929 69.6135 124.0374 123.9574 117.0262 116.6989 114.5461 114.2552 143.3108 140.2344 138.6270 134.1760 133.6668 131.0122 130.8740 130.5831 130.4522 166.1335 *** Current Data Parameters *** NAME : jl09shb EXPNO : 12 PROCNO : *** Acquisition Parameters *** DATE_t : 02:02:04 DATE_d : Jul 09 2008 DBPNAM7 : O N H F Cl N N N OH O N H O S CH3 DS : NS : 358 O1 : 7924.11 Hz O2 : 1200.52 Hz O3 : 7546.30 Hz SFO1 : 75.4756731 MH SFO2 : 300.1312005 MH SFO3 : SOLVENT : CH3 75.4752953 MH DMSO *** Processing Parameters *** AZFW : 0.500 ppm *** 1D NMR Plot Parameters *** SR 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 (ppm) 265 : 34.28 Hz 1H normal range AC300 hit-4 1.2340 1.2115 0.9628 0.9404 0.8587 0.8368 2.2125 2.0706 2.9986 2.9745 2.9510 2.9301 2.7642 2.5023 4.5540 4.5080 4.1913 4.1623 4.1464 4.1162 4.0286 3.9272 3.8955 3.8637 3.8001 3.7738 3.7459 5.3418 5.3177 8.3605 8.3287 8.0641 7.8263 7.7392 7.7151 7.5392 7.5118 7.4614 7.4368 7.3431 7.3152 7.2938 *** Current Data Parameters NAME : jl09shb EXPNO : PROCNO : *** Acquisition Parameters ** DBPOFFS[2]: 0.0000000 DBPOFFS[4]: 0.0000000 SFO1 : 300.1318534 SFO2 : 300.1318534 SFO3 : 300.1318534 SOLVENT : O N N 8.5 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 2.0 1.5 1.0 AZFW : LB : 0.100 0.30 MAXI : 10000.00 MI : 0.00 PC : 1.00 WDW : EM 3.0189 3.5478 1.2108 2.5 6.3305 3.0 1.1232 0.9918 0.8096 1.1993 2.1985 2.3885 2.1595 1.0452 8.0 1.0980 9.0 S 1.7194 9.5 0.9460 0.8622 Integral N H O 1.1656 Cl O 2.3732 OH 1.6511 N 0.8391 N H F DMSO *** Processing Parameters ** 0.5 0.0 (ppm) 13C Standard AC300 hit-4 40.3607 40.0843 39.8079 39.5316 39.2479 38.9715 38.6879 38.2079 37.5388 33.3786 31.8876 29.7857 27.4220 23.4946 20.2872 19.7054 15.7125 14.0761 55.9831 53.7066 69.6054 143.2737 137.8625 134.1606 134.1169 131.0186 130.8732 130.5823 130.4586 127.0403 126.7348 124.0075 117.0108 116.6836 114.5235 114.2398 153.1795 160.3143 166.1036 163.6162 *** Current Data Parameters *** NAME : jl09shb EXPNO : 14 PROCNO : *** Acquisition Parameters *** DATE_t : 02:29:44 DATE_d : Jul 09 2008 DBPNAM7 : O N H F Cl N N N OH O N H O S DS : NS : 374 O1 : 7924.11 Hz O2 : 1200.52 Hz O3 : 7546.30 Hz SFO1 : 75.4756731 MH SFO2 : 300.1312005 MH SFO3 : SOLVENT : 75.4752953 MH DMSO *** Processing Parameters *** AZFW : 0.500 pp *** 1D NMR Plot Parameters *** SR 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 (ppm) 266 : 34.89 Hz 190 180 170 8.0 160 7.5 F 150 7.0 6.5 N H Cl 140 130 6.0 N 120 5.5 5.0 110 4.5 N OH 100 90 4.0 3.5 80 3.0 70 2.5 60 2.0 50 1.5 40 6.0000 1.0369 OH 1.7844 N 1.0165 0.6132 1.4244 1.0304 0.9681 3.9689 0.9482 Cl 0.9762 2.8988 0.9502 1.0304 0.9681 3.9689 8.0 N 53.2917 52.8858 50.5392 50.0891 40.4150 40.3412 40.0608 39.7804 39.5074 39.2269 38.9465 38.8359 38.6661 38.2750 34.5928 30.6745 24.4096 24.2398 23.8561 23.3986 21.5907 21.2660 20.7569 19.2146 14.0861 8.5 1.9343 0.9783 0.9390 N H 59.7706 9.0 0.9482 8.5 1.9343 9.0 0.9783 0.9390 9.5 0.9427 0.9427 F 72.7284 71.8798 168.6208 165.8536 165.6839 163.6399 160.3414 144.5352 134.5143 133.9313 133.8870 131.3191 131.1863 130.9944 130.6549 130.5369 130.4188 129.8211 128.3822 128.0501 127.7697 126.5669 125.2239 124.7811 123.7849 123.6005 117.1215 117.0182 116.7895 116.6862 114.5314 114.4208 114.2437 Integral 7.7605 7.7350 7.5748 7.5550 7.5458 7.5365 7.5272 7.5156 7.5075 7.4831 7.4645 7.4413 7.3983 7.3705 7.3473 7.3391 7.3299 7.3194 7.3101 7.3020 7.2904 7.2823 1.7618 1.6469 1.6004 1.5644 1.5215 1.4739 1.4391 1.4263 1.3927 1.2429 1.2023 1.1791 1.1547 0.9342 0.9237 0.9133 2.0879 1.9928 4.6764 4.6381 4.6195 4.5650 4.5267 4.3514 4.3270 4.2968 4.2817 4.2516 4.1784 4.0705 4.0473 4.0241 3.9997 3.7524 3.7292 3.3752 2.5395 2.3886 5.4053 5.3845 7.7605 7.7350 7.5748 7.5550 7.5458 7.5365 7.5272 7.5156 7.5075 7.4831 7.4645 7.4413 7.3983 7.3705 7.3473 7.3391 7.3299 7.3194 7.3101 7.3020 7.2904 7.2823 hit-6 *** Current Data Parameters *** O N O N H 7.5 F 1.0 30 0.5 O N O N H F (ppm) 20 10 267 NAME : 11jlshb EXPNO : PROCNO : *** Acquisition Parameters *** DATE_t : 05:22:27 DATE_d : Jul 11 2008 NS : SFO1 : 300.1315000 MHz SOLVENT : *** Processing Parameters *** Start : Stop : YScale : SR : Hz_cm : CDCl3 SF : 300.1300014 MHz XDIM *** 1D NMR Plot Parameters *** : 8192 10.00 ppm 100.00 % -0.50 ppm 151.51 1.37 Hz (ppm) (ppm) 0.0 hit-6 *** Current Data Parameters *** NAME : 11jlshb EXPNO : PROCNO : *** Acquisition Parameters *** DATE_t : 05:23:37 DATE_d : Jul 11 2008 NS : SFO1 : SOLVENT : SF : XDIM : 75.4760840 M 245 *** Processing Parameters *** CDCl3 75.4677836 M *** 1D NMR Plot Parameters *** 64 Start : Stop : -5.00 pp YScale : 100.00 % SR : Hz_cm : 200.00 pp 743.80 64.64 H 190 180 8.5 170 8.0 160 7.5 150 7.0 F 140 6.5 6.0 N H Cl 130 5.5 120 110 5.0 OH N N H 100 4.5 O 90 3.8 N H 4.0 80 3.5 3.0 70 2.5 60 2.0 50 1.5 40 30 6.0000 O 3.0178 N 0.9718 0.8483 0.8483 1.3159 1.5246 Cl 1.3159 1.5246 2.9029 4.0 F OH 40.3412 40.0682 39.7878 39.5074 39.2343 38.9539 38.7621 38.6735 38.3341 34.9028 24.3579 24.2030 23.8192 23.3765 21.5686 21.2292 9.0 4.2 2.8723 4.4 1.8739 4.6 2.8460 2.9029 Integral N H 53.2326 52.9006 50.5097 50.0522 9.5 0.9156 0.9173 4.8 72.5882 71.7322 165.8241 165.6544 165.5659 165.0788 163.6251 162.2747 160.3266 144.6754 133.9092 133.8575 131.1199 130.9797 130.6550 130.5369 130.4115 129.9835 129.8654 123.8514 123.6595 117.0994 116.9961 116.7600 116.6641 115.3506 115.0628 114.5020 114.3913 114.2215 114.1035 Integral 4.5545 4.5243 4.5058 4.4512 4.2887 4.2562 4.2237 4.2098 4.1773 4.1262 4.0937 4.0612 4.0368 4.0136 3.9892 3.9707 3.9382 3.9057 3.7339 3.7095 3.6851 4.6009 4.5615 4.5545 4.5243 4.5058 4.4512 4.2887 4.2562 4.2237 4.2098 4.1773 4.1262 4.0937 4.0612 4.0368 4.0136 3.9892 3.9707 3.9382 3.9057 3.7339 3.7095 3.6851 3.1709 2.9956 2.5046 2.4988 2.4942 1.9835 1.7269 1.7153 1.6074 1.5586 1.5470 1.5122 1.4797 1.4669 1.4309 1.3868 1.3427 1.3265 1.2986 1.2801 1.2545 1.2278 1.1930 1.1698 1.1454 0.9144 0.8993 0.8935 0.8796 9.0814 9.0628 9.0443 8.4175 8.3873 8.3234 8.2933 7.9775 7.9590 7.9508 7.9300 7.9218 7.5214 7.5121 7.5051 7.4900 7.4819 7.4761 7.3217 7.2915 7.2625 hit-7 *** Current Data Parameters *** O N N O S (ppm) 1.0 0.5 O N N O S (ppm) 20 10 268 NAME : 11jlshb EXPNO : PROCNO : *** Acquisition Parameters *** DATE_t : 05:37:23 DATE_d : Jul 11 2008 NS : SFO1 : 300.1315000 MHz SOLVENT : *** Processing Parameters *** Start : Stop : YScale : SR : Hz_cm : CDCl3 SF : 300.1300017 MHz XDIM *** 1D NMR Plot Parameters *** : 8192 10.00 ppm 100.00 % -0.50 ppm 151.51 1.72 Hz (ppm) 0.0 hit-7 *** Current Data Parameters *** NAME : 11jlshb EXPNO : PROCNO : *** Acquisition Parameters *** DATE_t : 05:38:31 DATE_d : Jul 11 2008 NS : SFO1 : SOLVENT : SF : XDIM : Start : Stop : YScale : SR : Hz_cm : 75.4760840 MHz 155 *** Processing Parameters *** CDCl3 75.4677831 MHz *** 1D NMR Plot Parameters *** 64 200.00 ppm 100.00 % -5.00 ppm 743.80 64.08 Hz 190 180 8.5 170 8.0 160 7.5 150 7.0 140 6.5 130 6.0 120 5.5 F 110 5.0 N H Cl 100 4.5 90 4.0 N 80 3.5 70 3.0 OH N 2.5 60 2.0 50 1.5 40 30 6.0000 OH 0.7991 N 2.1066 1.0076 0.7419 0.9859 1.9274 Cl 1.0735 2.1619 1.3363 0.9761 0.8809 0.9859 7.6 1.9274 7.8 1.9386 F N 40.3412 40.0608 39.7878 39.5074 39.2270 38.9466 38.8137 38.0980 24.2177 23.8340 23.3839 21.5760 21.2439 9.0 8.0 1.9386 2.9177 1.2490 N H 53.2031 52.8489 50.5024 50.0301 9.5 8.2 2.9177 1.2490 8.4 72.5956 71.7322 165.8389 165.6765 163.6399 160.3340 142.9192 142.8601 139.3772 137.2446 133.8870 131.1346 130.9944 130.6402 130.5221 130.3967 129.2382 128.5371 124.1687 123.9473 117.1216 117.0183 116.7821 116.6862 114.5167 114.4134 114.2363 114.1256 Integral 8.3409 8.3107 7.8626 7.8394 7.7988 7.7709 7.6514 7.6224 7.5492 7.5260 7.4993 7.4784 7.3357 7.3275 7.3066 7.2997 7.2788 7.2718 1.5679 1.5226 1.4867 1.4379 1.3926 1.3509 1.3195 1.2267 1.1930 1.1698 1.1466 0.9237 0.9109 0.8900 1.7327 2.5000 2.9945 3.1697 4.2341 4.2051 4.1749 4.1599 4.1285 4.0693 3.6700 4.5673 4.5267 4.4570 4.4187 5.3299 8.4302 8.4001 8.3409 8.3107 7.8626 7.8394 7.7988 7.7709 7.6514 7.6224 7.5492 7.5260 7.4993 7.4784 7.3357 7.3275 7.3066 7.2997 7.2788 7.2718 hit-8 *** Current Data Parameters *** O N O N H F 7.4 1.0 0.5 O N O N H F (ppm) 20 10 269 NAME : 11jlshb EXPNO : PROCNO : *** Acquisition Parameters *** DATE_t : 05:48:08 DATE_d : Jul 11 2008 NS : SFO1 : 300.1315000 MHz SOLVENT : *** Processing Parameters *** Start : Stop : YScale : SR : Hz_cm : CDCl3 SF : 300.1300017 MHz XDIM *** 1D NMR Plot Parameters *** : 8192 10.00 ppm 100.00 % -0.50 ppm 151.51 1.72 Hz (ppm) (ppm) 0.0 hit-8 *** Current Data Parameters *** NAME : 11jlshb EXPNO : PROCNO : *** Acquisition Parameters *** DATE_t : 05:49:05 DATE_d : Jul 11 2008 NS : SFO1 : SOLVENT : SF : XDIM : Start : Stop : YScale : SR : Hz_cm : 75.4760840 MHz 151 *** Processing Parameters *** CDCl3 75.4677831 MHz *** 1D NMR Plot Parameters *** 64 200.00 ppm 200.00 % -5.00 ppm 743.80 64.08 Hz 180 170 160 150 140 130 6.0 F 120 5.5 N H Cl 110 5.0 N OH 100 90 4.5 N H O 80 4.0 3.5 70 3.0 60 2.1637 7.4781 7.4567 7.4485 7.4337 7.4096 7.3296 7.3028 7.2754 7.2672 7.2469 7.2387 7.2129 7.1921 7.1845 7.1636 7.0721 7.0601 7.0442 7.0316 6.6969 6.6695 N Cl OH N H 2.5 50 2.0 40 1.5 30 1.5627 3.2490 1.5039 1.1539 0.7232 3.3326 1.4575 0.4083 2.2016 2.3307 4.0249 2.4407 4.7752 0.9149 0.9820 N H 34.6330 6.5 0.6811 1.0404 2.1637 2.2016 2.3307 4.0249 2.4407 F 54.0010 53.8119 53.2882 7.0 71.9871 7.5 80.3074 8.0 7.5 86.8604 8.5 4.7752 0.9149 0.9820 8.0 94.8243 9.0 117.0870 116.7524 114.9633 114.8833 114.3887 9.5 155.6048 143.9826 143.1607 139.1824 138.6078 133.7494 131.2621 131.1312 130.1784 130.0111 129.1311 128.8547 128.7020 128.4765 127.3856 127.2765 127.1092 126.4473 124.1491 165.6779 165.3797 1.0000 Integral 5.5267 5.5031 5.4806 5.4604 4.6211 4.5811 4.4233 4.2907 4.2661 4.2365 4.2200 4.1899 4.1488 4.1066 4.0864 4.0371 4.0129 3.9664 3.7944 3.7713 3.7450 3.3665 3.0553 3.0159 2.9972 2.9655 2.8592 2.8411 2.8126 2.7967 2.7195 2.6877 2.6400 2.6039 2.5694 2.5579 2.5392 2.5058 2.4998 2.4943 1.3696 1.3466 1.2956 1.2786 1.2512 1.2255 1.1937 1.1696 1.1460 0.8951 0.8710 0.8568 0.8458 0.7762 6.6969 6.6695 7.7285 7.7000 7.5345 7.5110 7.4858 7.4781 7.4567 7.4485 7.4337 7.4096 7.3296 7.3028 7.2754 7.2672 7.2469 7.2387 7.2129 7.1921 7.1845 7.1636 7.0721 7.0601 7.0442 7.0316 1H normal range AC300 hit-10 *** Current Data Parameters *** OH O N N O O S (ppm) 7.0 1.0 0.5 OH O N N O S (ppm) 20 10 270 NAME : jl16shb EXPNO : PROCNO : *** Acquisition Parameters *** DATE_t : 23:59:56 DATE_d : Jul 15 2008 DBPNAM7 : DS : NS : O1 : 1853.43 Hz O2 : 1853.43 Hz O3 : 1853.43 Hz SFO1 : 300.1318534 MHz SFO2 : 300.1318534 MHz SFO3 : 300.1318534 MHz AZFW SR SOLVENT : *** Processing Parameters *** DMSO *** 1D NMR Plot Parameters *** : 0.100 ppm : 1.34 Hz (ppm) 0.0 13C Standard AC300 hit-10 *** Current Data Parameters *** NAME : jl16shb EXPNO : 12 PROCNO : *** Acquisition Parameters *** DATE_t : 00:01:22 DATE_d : Jul 16 2008 NS : SFO1 : SOLVENT : SF : XDIM : Start : Stop : YScale : SR : Hz_cm : 75.4756731 MHz 391 *** Processing Parameters *** DMSO 75.4677831 MHz *** 1D NMR Plot Parameters *** 64 190.01 ppm 100.00 % -5.00 ppm 707.52 34.08 Hz N3-phe-warhead-Br 3.7678 3.7469 3.7330 3.7260 3.7167 3.6865 3.6772 3.6505 3.6424 3.6366 3.6146 3.6006 3.5797 3.5101 3.4892 3.4718 3.1700 3.1595 3.1236 3.1120 3.0609 3.0423 3.0168 2.8624 2.8345 2.8160 2.7869 2.4492 2.3760 1.6517 1.5740 1.5531 7.3661 7.3603 7.3394 7.3185 7.2918 7.2860 7.2721 7.2616 7.2570 7.2512 *** Current Data Parameters *** NAME : ju20shb EXPNO : PROCNO : 3.1700 3.1595 3.1236 3.1120 3.0609 3.0423 3.0168 2.8624 2.8345 2.8160 2.7869 2.4492 2.3760 3.6006 3.5797 3.5101 3.4892 3.4718 *** Acquisition Parameters *** DATE_t : 22:47:46 DATE_d : Jun 19 2007 NS : SFO1 : 300.1315000 MH SOLVENT : CDCl3 *** 1D NMR Plot Parameters *** 3.5 N3 0.8181 0.4629 0.9465 0.4906 0.9471 0.8369 2.0007 OH 3.0 Br Start : 10.00 pp Stop : -0.50 pp 2.5 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 0.8181 0.4629 0.9465 0.4906 0.9471 0.8369 2.0007 5.0000 Integral (ppm) 2.5 2.0 1.5 1.0 0.5 0.0 (ppm) N3-phe-warhead-Br 37.3369 37.0122 36.8794 35.1821 65.9755 64.7800 72.1961 71.9379 77.4206 77.0000 76.5720 136.8747 136.6312 129.4069 129.2446 128.8019 128.6764 127.0825 127.0013 *** Current Data Parameters ** NAME : ju20shb EXPNO : PROCNO : *** Acquisition Parameters *** DATE_t : 22:49:00 DATE_d : Jun 19 2007 NS : SFO1 : SOLVENT : 207 75.4760840 M CDCl3 *** 1D NMR Plot Parameters ** OH N3 170 160 150 140 130 120 Br 110 100 90 80 70 60 50 40 30 20 10 (ppm) 271 Start : 180.01 p Stop : -5.00 p 170 160 7.5 150 7.0 140 6.5 N3 130 6.0 120 5.5 110 13C AMX500n3-leu-war-Br 5.0 100 4.5 90 4.0 80 3.5 70 3.0 60 2.5 50 2.0 1.5 40 30 6.0000 1.5 1.1939 1.6 0.8292 1.7 1.2636 1.8 0.6258 3.5 1.1939 0.8292 1.2636 2.0252 1.0302 N3 25.9050 25.3220 25.0159 24.9212 24.8920 24.6588 23.3617 22.9026 22.6621 21.7949 21.7657 21.4669 21.4378 21.3649 8.0 3.6 44.8157 44.7574 39.8311 39.8093 39.3647 39.1242 36.6538 35.3348 8.5 3.7 2.0252 1.0302 Integral 3.8 77.2591 77.0040 76.7489 73.6445 73.4478 71.9539 65.5337 62.9540 61.9264 60.3305 60.2503 58.4722 54.6536 53.7209 52.4893 171.5358 171.1131 1.6352 1.6238 1.6163 1.6049 1.5129 1.5028 1.4927 1.4839 1.4751 1.4637 1.4536 1.4461 1.4335 1.4272 1.4158 1.4133 1.4057 1.3994 1.3893 3.5351 3.5313 3.5225 3.5187 3.5149 3.5111 3.5086 3.5036 3.4973 3.4872 3.4733 3.4658 3.4532 3.4078 3.3952 3.3826 3.3195 3.3094 3.5528 3.5465 3.5427 3.5389 3.5351 3.5313 3.5225 3.5187 3.5149 3.5111 3.5086 3.5036 3.4973 3.4872 3.4733 3.4658 3.4532 3.4078 3.3952 3.3826 3.3195 3.3094 2.5883 2.5782 2.5328 2.5215 2.0260 1.8407 1.8356 1.8319 1.8281 1.8230 1.8180 1.8092 1.8054 1.7991 1.7966 1.7865 1.7827 1.7726 1.7688 1.7562 1.7512 1.7449 1.7423 1.7386 1.6869 1.6806 1.6768 1.6692 1.6629 1.6579 1.6516 1.6440 1.6352 1.6238 1.6163 1.6049 1.5129 1.5028 1.4927 1.4839 1.4751 1.4637 1.4536 1.4461 1.4335 1.4272 1.4158 1.4133 1.4057 1.3994 1.3893 1.2960 1.2406 0.9758 0.9708 0.9670 0.9569 0.9544 0.9493 0.9355 -0.0164 1H AMX500n3-leu-war-Br *** Current Data Parameters *** OH Br 1.0 0.5 OH Br (ppm) 20 10 272 NAME : shb0616 EXPNO : 17 PROCNO : *** Acquisition Parameters *** NS : O1 : 3088.51 Hz O2 : 3088.51 Hz O3 : 3088.51 Hz SFO1 : 500.1330885 MH SFO2 : 500.1330885 MH SFO3 : 500.1330885 MH SR SR : : *** 1D NMR Plot Parameters *** 12.15 Hz 3.4 (ppm) 1.4 (ppm) (ppm) 0.0 *** Current Data Parameters *** NAME : shb0616 EXPNO : 18 PROCNO : *** Acquisition Parameters *** NS : O1 : O2 : 2000.52 Hz O3 : 12575.30 Hz SFO1 : 125.7709936 MH SFO2 : 500.1320005 MH SFO3 : 125.7703643 MH 13204.57 Hz 88 *** 1D NMR Plot Parameters *** 7.98 Hz 1H AMX500n3-ala-war-Br 0.9121 0.8983 0.8895 0.8768 0.8630 1.3584 4.9503 4.0198 4.0072 3.8496 3.8358 3.7173 3.6895 3.6757 3.6668 3.6542 3.6404 3.6290 3.6189 3.6114 3.6063 3.5950 3.5824 3.5698 3.5559 3.5433 3.5193 3.5130 3.4992 3.4916 3.4727 3.4588 3.4513 3.4475 3.4361 3.4260 3.4134 3.4084 3.3945 3.3819 3.3743 3.3617 3.3454 3.3365 3.3138 3.2647 3.2621 2.7932 2.1968 6.9006 7.2297 *** Current Data Parameters *** NAME : shb0616 EXPNO : 15 PROCNO : 3.4916 3.4727 3.4588 3.4513 3.4475 3.4361 3.4260 3.4134 3.4084 3.3945 3.3819 3.3743 3.3617 3.3454 3.3365 3.3138 3.2647 3.2621 *** Acquisition Parameters *** OH N3 Br NS : O1 : 3088.51 Hz O2 : 3088.51 Hz O3 : 3088.51 Hz SFO1 : 500.1330885 MHz SFO2 : 500.1330885 MHz SFO3 : 500.1330885 MHz *** 1D NMR Plot Parameters *** : 27.60 Hz 2.1331 SR 1.8369 3.6 3.5 3.4 3.3 7.5 7.0 6.5 6.0 5.5 5.0 4.5 13C AMX500n3-ala-war-Br 4.0 3.5 3.0 2.5 2.0 1.5 0.4986 3.0000 0.9009 2.1331 1.8369 Integral (ppm) 1.0 0.5 0.0 (ppm) 0.7999 15.4621 14.5876 18.5009 36.1509 35.3566 34.8465 34.0157 30.1170 27.5883 27.4425 59.2738 59.1717 77.2590 77.0039 76.7489 73.8995 73.4331 125.2973 135.6527 151.3060 151.2622 *** Current Data Parameters *** NAME : shb0616 EXPNO : 16 PROCNO : *** Acquisition Parameters *** OH N3 170 160 150 140 130 110 : O1 : 38 O2 : 2000.52 Hz O3 : 12575.30 Hz SFO1 : 125.7709936 MHz SFO2 : 500.1320005 MHz SFO3 : 125.7703643 MHz 13204.57 Hz *** 1D NMR Plot Parameters *** Br 120 NS SR 100 90 80 70 60 50 40 30 20 10 (ppm) 273 : 21.74 Hz C-Tyr-NH-isobutyl 3.6320 3.6174 3.5911 3.5784 3.5697 3.4372 3.0418 3.0213 2.9970 2.9756 2.9512 2.9220 2.9045 2.8762 2.8373 2.7973 2.7866 2.7759 2.7477 2.7360 2.7165 2.7067 2.6912 2.6775 2.6629 2.6512 2.6376 2.4321 2.4116 2.4019 2.3902 2.3795 2.3405 1.7708 1.7484 1.7269 1.7075 1.6860 1.6636 1.3159 0.9166 0.9010 0.8952 0.8796 7.2600 7.1577 7.1480 7.1295 7.1198 6.9396 6.9113 *** Current Data Parame NAME : EXPNO : PROCNO : 1703 2.4321 2.4116 2.4019 2.3902 2.3795 2.3405 2.9045 2.8762 2.8373 2.7973 2.7866 2.7759 2.7477 2.7360 2.7165 2.7067 2.6912 2.6775 2.6629 2.6512 2.6376 *** Acquisition Paramete BF1 : 300.1300 BF2 : 300.1300 BF3 : 300.1300 DATE_d : Mar 16 *** Processing Paramete SF OH H N N3 : 300.1298 *** 1D NMR Plot Parame Start : Stop : - SR : -18 3.0 2.9 2.8 4.4207 2.6247 1.4895 O 2.7 2.6 2.5 2.4 2.3 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 5.6038 9.0000 0.9936 4.4207 2.6247 1.4895 4.6884 1.4736 1.9381 1.9526 Integral (ppm) 1.0 0.5 0.0 (ppm) C-Tyr-NH-isobutyl -0.0901 20.4228 28.7398 36.4761 36.2681 52.2784 50.8373 50.5218 57.5913 78.3982 77.4231 77.0000 76.5770 70.4970 70.1959 67.1989 65.9155 124.3282 132.5448 132.3943 129.6626 154.0400 *** Current Data Parameters *** NAME : 1703shb EXPNO : PROCNO : *** Acquisition Parameters *** BF1 : BF2 : 300.1300000 MHz 75.4677190 MHz BF3 : DATE_d : 75.4677190 MHz Mar 16 2009 *** Processing Parameters *** SF H N N3 O 200 190 180 170 160 150 140 130 120 110 100 : 75.4677061 MHz *** 1D NMR Plot Parameters *** OH 90 80 70 60 50 40 30 20 10 -10 -20 (ppm) 274 Start : 207.64 ppm Stop : -27.30 ppm SR : -12.91 Hz 170 160 150 140 N3 130 N O 120 110 100 90 80 4.0 3.5 70 3.0 60 2.5 50 2.0 40 1.5 30 1.0 20 -0.0257 4.5 20.0714 19.8205 19.7129 15.2604 5.0 22.9680 5.5 30.3530 29.6790 26.9114 6.0 6.2169 5.2950 0.0540 11.371 4.7793 1.9892 1.0773 1.0874 1.0727 1.0837 0.6638 1.0773 1.0874 1.0727 O 52.2425 6.5 N 1.5513 0.6048 1.3716 1.0837 0.6638 1.5513 0.6048 N3 60.0361 59.0610 58.5663 58.3584 7.0 77.4158 76.9928 76.5698 72.8845 72.6407 7.5 122.1772 8.0 125.5900 8.5 1.3653 1.0083 3.6 128.7878 9.0 1.0183 4.0 137.1335 134.3086 134.0504 151.2652 9.5 1.0029 1.0000 Integral 3.7986 3.7781 3.7499 3.5366 3.5151 3.4937 3.4246 3.3749 3.3437 3.3038 3.1596 3.1314 3.1129 3.0856 3.0272 2.9950 2.9736 2.9483 2.9269 4.2329 4.2144 4.2018 3.9904 3.9583 3.9174 3.9008 3.8687 3.7986 3.7781 3.7499 3.5366 3.5151 3.4937 3.4246 3.3749 3.3437 3.3038 3.1596 3.1314 3.1129 3.0856 3.0272 2.9950 2.9736 2.9483 2.9269 2.3376 2.3123 2.1681 1.8370 1.8156 1.7932 1.6841 1.4270 1.3977 1.3763 1.3315 1.3218 1.2994 1.2828 1.2546 1.1085 0.9439 0.9195 0.8942 0.8777 0.8543 0.7140 0.7033 0.6916 0.6819 0.6702 0.6566 0.6342 7.2600 9.0794 9.0735 9.0657 8.5787 8.5534 8.2963 8.2681 8.0966 8.0694 7.7197 7.6905 7.6642 7.6389 7.5834 7.5687 7.5561 7.5415 C-Ala-1wh *** Current Data Parameters *** OH S O N 3.2 0.5 OH S O N (ppm) 10 275 NAME : 1803shb EXPNO : PROCNO : *** Acquisition Parameters *** DATE_t : 23:51:11 DATE_d : Mar 17 2009 NS : SFO1 : 300.1312500 MH SOLVENT : *** Processing Parameters *** SOLVENT : SF : XDIM : CDCl3 SF : 300.1298200 MH XDIM *** 1D NMR Plot Parameters *** : 8192 Start : Stop : -0.50 ppm YScale : 12.50 % SR : -180.04 Hz Hz_cm : 10.00 ppm (ppm) 151.51 (ppm) 0.0 C-Ala-1wh *** Current Data Parameters *** NAME : 1803shb EXPNO : PROCNO : *** Acquisition Parameters *** DATE_t : 23:53:02 DATE_d : Mar 17 2009 NS : SFO1 : 75.4745111 MH 74 *** Processing Parameters *** CDCl3 75.4677034 MH *** 1D NMR Plot Parameters *** 64 Start : Stop : -4.99 pp YScale : 200.00 % SR : -15.61 Hz Hz_cm : 671.21 180.00 pp 40 210 200 35 190 30 180 25 170 6.5 20 160 6.0 15 150 5.5 140 5.0 130 4.5 120 4.0 O 110 100 3.5 N O 90 80 3.0 N O 70 2.5 60 2.0 H N 50 1.5 O 40 16.494 34.109 3.3038 1.1884 1.0655 1.5882 N H 1.8131 O 0.7723 4.1975 O 4.6547 0.9672 N 4.5197 0.6080 N H N 80.3763 72.3330 69.1525 55.3608 51.0514 49.8486 49.5682 49.2804 49.0000 48.7122 48.4318 48.1440 46.8969 42.8236 40.1966 39.0381 36.6398 33.0388 31.6294 30.7070 30.1240 29.9395 29.5115 29.0983 27.2978 26.3901 24.9659 23.9919 23.6967 14.3547 12.8272 11.3882 7.0 1.0000 1.0539 O 97.3706 7.5 1.1128 1.1084 O 115.4348 114.8888 8.0 1.6827 2.7461 5.7015 O 14.3547 12.8272 11.3882 175.8704 174.4979 172.9187 172.8302 169.6276 169.3324 159.2820 157.2527 156.9944 143.9701 138.9523 137.2772 136.5614 133.7426 133.6024 133.1818 132.3774 131.7354 131.3296 131.2189 130.9090 130.5474 129.8538 129.5069 128.9387 8.5 40.1966 39.0381 36.6398 33.0388 31.6294 30.7070 30.1240 29.9395 29.5115 29.0983 27.2978 26.3901 24.9659 23.9919 23.6967 42.8236 Integral 4.6472 4.6287 4.6182 4.5985 4.1655 4.1469 3.8811 3.6269 3.6037 3.5805 3.4180 3.3193 3.1917 3.1719 3.1452 3.1255 3.0442 3.0210 2.9978 2.9491 2.9177 2.9038 2.8713 2.5231 2.3884 2.3675 2.1876 2.1342 2.1098 2.0855 1.6455 1.6258 1.6061 1.3971 1.3716 1.3472 1.2938 1.2787 1.2602 1.2358 1.2137 0.9108 0.8864 0.8597 0.8377 7.6211 7.5874 7.5735 7.5630 7.5549 7.5444 7.4736 7.4481 7.4284 7.3587 7.3320 7.2310 7.1997 7.0186 6.9908 6.8886 6.8817 TER-alkyne *** Current Data Parameters O N+ H N O 1.0 N H 30 0.5 O N+ O H N O N (ppm) 10 (ppm) 20 10 276 NAME : oc12shb EXPNO : PROCNO : *** Acquisition Parameters * DATE_t : 03:36:14 DATE_d : Oct 12 2007 NS : SFO1 : 300.1315000 SOLVENT *** Processing Parameters ** : MeOD SF : 300.1300227 XDIM : *** 1D NMR Plot Parameters 8192 Start : 9.00 Stop : -0.50 YScale : 50.00 SR : 22.66 Hz_cm : 147.73 N (ppm) 0.0 TER-alkyne *** Current Data Parameters NAME : oc12shb EXPNO : PROCNO : *** Acquisition Parameters ** DATE_t : 03:38:58 DATE_d : Oct 12 2007 NS : 5871 SFO1 : 75.4760840 SOLVENT *** Processing Parameters ** : CDCl3 SF : 75.4676436 XDIM : *** 1D NMR Plot Parameters 64 Start : Stop : -4.99 YScale : 25.00 SR : -75.36 Hz_cm : 879.80 220.01 190 180 O 170 8.5 H HN 160 8.0 7.5 S N H H 150 140 7.0 6.5 130 120 6.0 H N O 110 5.5 5.0 O N H 100 90 4.5 80 4.0 70 3.5 60 3.0 50 40 2.5 2.0 30 20 12.570 O 9.1241 N H 2.3972 4.9478 O 1.0376 H N 1.5774 1.5568 N H H 6.0364 S 4.3047 O 2.4791 1.5774 HN 1.9297 3.2 1.7007 1.4107 3.6 1.5568 6.0364 4.3047 2.4791 1.9297 1.7007 H 72.7198 61.1703 59.8393 59.3448 55.4828 53.3518 40.3695 40.0931 39.8895 39.8095 39.5331 39.2567 38.9804 38.7040 38.3622 35.2784 35.0820 33.7220 28.9582 28.2745 28.1000 27.7799 26.1362 26.0344 25.3944 24.9362 24.2962 20.8124 14.1358 9.0 1.3869 1.0000 4.0 81.4183 9.5 162.8978 174.5128 172.0545 Integral 4.0475 4.0240 3.9998 3.9763 3.8240 3.8158 3.8059 3.7977 3.6552 3.0477 3.0395 3.0312 2.9934 2.9743 2.9184 2.8811 2.8400 2.8236 2.7990 2.7820 2.7190 2.6762 2.5984 2.5574 2.5004 6.4350 4.3280 4.3028 4.2869 4.1467 4.1319 4.1209 4.1061 4.0475 4.0240 3.9998 3.9763 3.8240 3.8158 3.8059 3.7977 3.6552 3.0477 3.0395 3.0312 2.9934 2.9743 2.9184 2.8811 2.8400 2.8236 2.7990 2.7820 2.7190 2.6762 2.5984 2.5574 2.5004 2.2012 2.1766 2.1525 2.0857 2.0615 2.0374 2.0144 1.9717 1.9005 1.6205 1.5953 1.5740 1.5542 1.5088 1.4852 1.4627 1.4414 1.4058 1.3784 1.3559 1.3313 1.3083 1.2836 1.2754 1.2480 1.2179 1.1948 1.1817 1.1576 1.1340 7.8507 7.8332 7.8151 8.3005 1H normal range AC300 biotin-alkyne *** Current Data Parameters *** O H N O (ppm) 2.8 1.5 O H N O (ppm) 10 277 NAME : ag18shb EXPNO : PROCNO : *** Acquisition Parameters *** DATE_t : DATE_d : Aug 18 2008 SOLVENT : AZFW SR AZFW SR : : SOLVENT : : : 04:29:50 DBPNAM7 : DS : NS : O1 : 1853.43 Hz O2 : 1853.43 Hz O3 : 1853.43 Hz SFO1 : 300.1318534 MH SFO2 : 300.1318534 MH SFO3 : 300.1318534 MH *** Processing Parameters *** DMSO *** 1D NMR Plot Parameters *** 0.100 ppm 1.00 Hz (ppm) 1.0 13C Standard AC300 biotin-alkyne *** Current Data Parameters *** NAME : ag18shb EXPNO : 12 PROCNO : *** Acquisition Parameters *** DATE_t : DATE_d : Aug 18 2008 04:32:43 DBPNAM7 : DS : NS : 605 O1 : 7924.11 Hz O2 : 1200.52 Hz O3 : 7546.30 Hz SFO1 : 75.4756731 MH SFO2 : 300.1312005 MH SFO3 : 75.4752953 MH *** Processing Parameters *** DMSO *** 1D NMR Plot Parameters *** 0.500 ppm 29.28 Hz O Cl N N H N N N H S O H N N H HN O O DA-1 O Cl N N H N S O N H N H N N H HN O O DA-1 278 O H N Cl N N S O N N H H N N H HN N N O DA-2 O H N Cl N N S O N N H N H H N HN N N O DA-2 279 H N N O N O N N H O O N N H O H N O STS-1 H N N O N O N N H O O N N H O H N O STS-1 280 [...]... enzymology.4 This chapter will provide a brief overview of applications of high- throughput technologies in chemical proteomics Much attention will be focused on studies of high- throughput strategies for proteomic profiling of aspartic proteases as well as protein kinases 1 1.2 Aspartic Proteases as Therapeutic Targets Proteases belong to one of the largest enzyme family encoded by the human genome... Fluorescent Labeling of different recombinant proteins 98 6.5 Fluorescent labeling of recombinantly purified protein kinases 99 6.6 Labeling profiles of bacterial and mammalian proteomes 6.7 In vitro (A) and in situ (B) labeling profilings of K562 and HepG2 cells using 100 DA-2 103 7.1 Overall strategy of affinity-based proteome profiling 110 7.2 Biological activities of STS-1 in vitro and in situ 113 7.3... falciparum aspartic proteases 44 3.5 Profiling, identification and proteome characterization of P falciparum aspartic proteases 46 3.6 Inhibition of P.falciparum aspartic proteases 49 3.7 Molecular docking of G16 in the active site of PM-II, HAP and PM-IV 52 4.1 Overall strategy of the small molecule microarray (SMM)-facilitated platform for high- throughput identification of AfBPs 58 4.2 Microarray screening... capables of studing enzyme activities in a high- throughput manner.3 High- throughput amenable reactions are mainly characterized by near-perfect, modular and robust and biocompatible nature and offer an efficient and rapid analysis of proteomes on a global scale To date, many high- throughput methods have been applied to drug synthesis and proteome analysis, especially in the field of high- throughput. .. I, xiv and (c) Plasmepsin II 30 2.4 Characterization of inhibitor hit 31 3.1 High- throughput amenable chemical reactions 35 3.2 Assembly of affinity-based probes (AfBPs) and the 152-membered library of potential inhibitors against all four FV plasmepsins in P falciparum 37 3.3 Inhibition profiles of the 152-member library against 5 aspartic proteases 42 3.4 Profiling of P falciparum aspartic proteases. .. inhibitors Chapter 3 presents a high throughput synthetic strategy for rapid assembly of 475-member aspartic protease inhibitors library using “click” reaction With this platform, new inhibitors against malaria were discovered Chapter 4 and 5 present a small molecule microarray-facilated platform for proteome profiling of aspartic proteases in cellular lysates Chapter 6 and 7 describes the cell permeable,... studies of a significant number of enzymes present in any particular organism, development of high- throughput amenable chemistry and enzyme-screening tools are becoming very urgent This thesis examines and addresses these challenges by combination of various synthetic approaches and introducing high throughput screening platforms Chapter 2 describes a solution-cum-solid phase strategy for rapid assembly of. .. Thus far, one of the main challenges in the field of Catalomics is the development of high- throughput (HT) amenable chemical reactions that allow rapid synthesis of diverse chemical libraries for the interrogation of different classes of enzymes Herein, we have rapidly and efficiently synthesized the enzyme inhibitor libraries by using high- throughput amenable reactions (Click chemistry and solid-phase)... images of recombinantly purified cathepsin D and mammalian cell lysates labeled with different AfBPs 79 5.6 Proteome profiling of recombinant cathepsin D and T47D cell lysates 80 5.7 Pull-down results using the biotinylated probe cocktail and target validation 83 6.1 Activity-based proteome profiling 92 6.2 Determination of biological activities of DA-1 and 2 95 6.3 Biological activities of DA-1 and 2... drug-like probes for proteome profiling of cellular on/off-targets of drug dasatinib and staurosphorine xi List of Publication (2006-2012) 1 Sun, H.; Lu, C H S.; Shi, H.; Gao, L.; Yao, S Q.* Peptide Microarrays for High- throughput Studies of Ser/Thr Phosphatases Nat Protocols (2008), 3, 1485-1493 2 Shi, H.; Liu, K.; Leong, W Y W.; Yao, S Q.* Expedient Solid-phase Synthesis of Both Symmetric and Asymmetric . DEVELOPING HIGH- THROUGHPUT CHEMICAL APPROACHES FOR PROTEOMIC PROFILING OF ASPARTIC PROTEASES AND PROTEIN KINASES SHI HAIBIN (M.Sc.,. Extraction and Analysis 190 8.7 Fluorescent Profiling of Aspartic Proteases and Protein Kinases 191 8.7.1 Labeling of Recombinant Aspartic Proteases 191 8.7.2 Labeling and Identification of PM. Inhibition profiles of the 152-member library against 5 aspartic proteases 42 3.4 Profiling of P. falciparum aspartic proteases 44 3.5 Profiling, identification and proteome characterization of P.