DEVELOPING HIGH-THROUGHPUT AMENABLE CHEMISTRY FOR CHEMICAL BIOLOGY APPLICATIONS RAJAVEL SRINIVASAN NATIONAL UNIVERSITY OF SINGAPORE 2009 DEVELOPING HIGH-THROUGHPUT AMENABLE CHEMISTRY FOR CHEMICAL BIOLOGY APPLICATIONS RAJAVEL SRINIVASAN (M.Sc., Anna University, Chennai, India) A THESIS SUBMITTED FOR THE DOCTOR OF PHILOSOPHY DEGREE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2009 THESIS DEDICATED TO PROF. E. BALASUBRAMANIAM (Retired Professor of chemistry, VOC College, MS University, India) ACKNOWLDGEMENTS First of all, I would like to express my sincere thanks to my advisor Prof. Yao Shao Qin for giving me an opportunity to work and learn in his laboratory. I had a wonderful chance to improve my mental discipline, leadership skills, and planning ability under his training. I always respect his straight forward approaches. It was only because of his smart ideas, deadlines and my passion towards chemistry that I was able to successfully complete most of the projects assigned to me. I am always grateful for his support and helps. I also wish for my relation/contact with Prof. Yao continues for ever. Next, I am very grateful to Prof. Bahulayan (Calicut University) and Prof. Chang Young-Tae (NUS) for their instant and timely helps. I am also very thankful to Prof. V. Murugesan (Anna University), Dr. N. Somanathan (CLRI), Prof. Zhu Qing (Zhejiang University), Prof. Suresh (NUS) and Prof. S. Kumaresan (MS University) for their kind support. Thanks are due to my colleagues (past and present) for having provided a nice friend circle and making the working place an enjoyable one. Resmi, Souvik, Kalesh, Pengyu Hongyan, Aparna, Mahesh, Derek, Wu Hao, Ching Tian, Jiexun, Xiaohua, Mingyu, Huang Xuan, Lay Pheng, Wang Jun, Haibin, Dawn, Wang Gang, Jingyan, Su Ling and Junqi – Working with all of them have been a great experience. Special thanks to Subbu for the great chemistry discussions. i I am grateful to Prof. Vittal for recruiting me into NUS Chemistry graduate program and I am also thankful to NUS for awarding me the research scholarship. I would like to express my greatest thanks to my parents Srinivasan and Dhanalakshmi, soul mate-cum-wife Resmi, baby Gayathri, baby Srinidhi, brothers, sisters, all in-laws, friends and all family members for their well wishes and ever lasting love. ii TABLE OF CONTENTS Table of Contents iii Abbreviations vii List of Publications xii Summary xiv List of Schemes xvii List of Figures xviii List of Tables xxii Chapter Introduction 1.1 Catalomics 1.2 Protein Tyrosine Phosphatases (PTPs) 1.2.1 Catalytic mechanism of PTPs 1.2.2 Inhibitor development for PTPs 1.3 High-throughput amenable chemistry to study PTPs 1.3.1 Click Chemistry 1.3.1.1 Click-based fragment assembly and in situ screening 1.3.1.2 Amide-bond formation and in situ screening 1.3.2 Solid-phase Chemistry 10 1.3.2.1 Split and mix synthesis 11 1.3.2.2 Parallel Synthesis 12 iii 1.3.2.3 Traceless resins 13 1.3.3 Microwave Assisted Synthesis 14 1.4 Activity-based Protein profiling/fingerprinting 15 1.4.1 Components of activity-based probes 16 1.4.2 Click chemistry-based design concepts 18 1.4.3 Imaging of Enzyme activity 20 1.5 Bioimaging 21 Chapter High-Throughput Assembly of Protein Tyrosine Phosphatases (PTPs) Inhibitors Using “Click Chemistry” 2.1 Summary 24 2.2 Introduction 2.2.1 Fragment-based drug discovery of PTP inhibitors 25 2.2.2 Click Chemistry as a High-throughput tool 27 2.2.3 Bidentate inhibitors against Protein Tyrosine Phosphatases 1B 28 2.2.4 PTP1B bidentate inhibitor design 29 2.3 Results and discussion 2.3.1 Chemical Synthesis of the inhibitor library 30 2.3.2 Biological screening results 33 2.4 High-throughput synthesis of 3250-member PTP inhibitor library 36 2.4.1 Traceless solid-phase synthesis of 325 azide fragments 38 2.4.2 General Procedure for the High-throughput ‘click’ assembly 40 2.5 Conclusion 42 iv Chapter Solid-Phase Assembly and in situ screening of Protein Tyrosine Phosphatases inhibitors 3.1 Summary 44 3.2 Introduction 44 3.2.1 High-throughput amenable chemical reactions 44 3.2.2 Limitations of Wong’s in situ screening approach 45 3.2.3 Introduction to solid-phase combinatorial library 46 3.2.4 Design of the traceless Solid-phase library 47 3.3 Results and Discussion 48 3.3.1 Chemical Synthesis of the inhibitor library 49 3.3.2 Biological screening results 51 3.4 Conclusion 54 Chapter Versatile Microwave-Assisted Strategies for the Synthesis of Azide fragments 4.1 Summary 56 4.2 Introduction 56 4.2.1 Synthesis of azides – a literature review 57 4.2.2 Drawbacks of the existing methods 59 4.2.3 Design of our azide library 60 4.3 Results and Discussion 4.3.1 Chemical synthesis of the azide library 4.3.1.1 Traceless solid-phase synthesis form PS-TsCl resin 61 61 61 v 4.3.1.2 Solution-phase MW-assisted azidation 62 4.3.1.3 Utilization of the azides in click assembly 63 4.4 Conclusion 65 Chapter Activity-based fingerprinting of Enzymes 5.1 Summary 67 5.2 Introduction 67 5.2.1 Activity-based fingerprinting 67 5.2.2 Design of our activity-based protease probes 69 5.3 Results and Discussion 71 5.3.1 Chemical synthesis of the probe 71 5.3.2 Fingerprinting experiments 73 5.4 Activity-based probe for Protein Tyrosine Phosphatases (PTPs) 75 5.4.1 Design of the probe 75 5.4.2 Chemical synthesis of the probe 77 5.5 Conclusion 79 Chapter Bioimaging using small molecule probes 6.1 Summary 80 6.2 Introduction 80 6.2.1 Native chemical ligation 80 6.2.2 Small-molecular probe design 82 6.3 Results and discussions 6.3.1 Chemical synthesis of the probes 83 83 vi 6.3.2 Imaging experiments 84 6.4 Conclusion 86 Chapter Experimental Section 88 Chapter Concluding Remarks 195 Chapter References 196 Chapter 10 Appendix 204 vii 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 2-C-2-A10-5C 2-C-2-E1-6C 2-C-2-E2-6C 2-C-2-E3-6C 2-C-2-E4-6C 2-C-2-E5-6C 2-C-2-E6-6C 2-C-2-A9-5C 2-D-2-A1-5C 2-D-2-A4-5C 2-D-2-A6-5C 2-E-2-A1-2C 2-E-2-A2-2C 2-E-2-A5-2C 2-E-2-E1-4C 2-E-2-A1-5C 2-E-2-A2-5C 2-E-2-A3-5C 2-E-2-A4-5C 2-E-2-A5-5C 2-F-2-A6-2C 2-F-2-A8-2C 2-F-2-A9-2C 2-F-2-A11-2C 2-F-2-A12-2C 2-F-2-B3-2C 2-F-2-B4-2C 2-F-2-B5-2C 2-F-2-A1-5C 2-F-2-A2-5C 2-G-2-A1-2C 2-G-2-A2-2C 2-G-2-A1-5C 2-G-2-A2-5C 2-G-2-A3-5C 2-G-2-A4-5C 2-G-2-A7-5C 548.088 512.172 512.172 530.164 530.162 526.191 526.152 528.148 578.234 596.2 592.212 471.086 471.097 485.114 499.129 513.143 513.139 531.135 531.13 527.155 482.133 484.088 498.107 514.109 494.163 494.158 480.147 540.164 510.178 510.173 452.139 452.141 494.179 494.177 512.168 512.169 544.111 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 2-I-2-A6-2C 2-I-2-A8-2C 2-I-2-A9-2C 2-I-2-A11-2C 2-I-2-A12-2C 2-I-2-B1-2C 2-I-2-B2-2C 2-I-2-B3-2C 2-I-2-B4-2C 2-I-2-B5-2C 2-I-2-B6-2C 2-I-2-B7-2C 2-I-2-B8-2C 2-J-2-A1-2C 2-J-2-A2-2C 2-J-2-A4-2C 2-J-2-A5-2C 2-J-2-A6-2C 2-J-2-A8-2C 2-J-2-A9-2C 2-J-2-A11-2C 2-J-2-A12-2C 2-J-2-E4-4C 2-J-2-E5-4C 2-J-2-E6-4C 2-J-2-A1-5C 2-J-2-A2-5C 2-J-2-A3-5C 2-J-2-A4-5C 2-J-2-A5-5C 2-J-2-A6-5C 2-J-2-A11-5C 2-J-2-A12-5C 2-J-2-E1-6C 2-J-2-E2-6C 2-J-2-E3-6C 2-J2-E4-6C 470.125 472.082 486.098 502.098 482.154 530.15 498.148 482.145 468.129 528.159 496.163 484.104 522.101 438.122 438.125 456.119 452.139 452.145 454.098 468.112 484.153 464.163 484.153 480.176 480.178 480.153 480.153 480.163 498.15 498.153 494.183 526.146 506.2 494.176 494.176 512.163 512.168 222 Table 10.3 Summary of characterization and IC50 of traceless PTP1B inhibitors Summary Of Characterization and IC50 of Traceless PTP inhibitors # Product ID Acid used Amine used Est % Puriy Obs MS NMR (1H) 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 3-A1 3-A2 3-A3 3-A4 3-A5 3-A6 3-A7 3-A8 3-A9 3-A10 3-A11 3-A12 3-A13 3-A14 3-A15 3-A16 3-A17 3-A18 3-A19 3-A20 3-A21 3-A22 3-A23 3-A24 3-A25 3-A26 3-A27 3-A28 3-A29 3-A30 3-A31 3-A32 3-A33 3-A34 3-A35 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-A 3-a1 3-a2 3-a3 3-a4 3-a5 3-a6 3-a7 3-a8 3-a9 3-a10 3-a11 3-a12 3-a13 3-a14 3-a15 3-a16 3-a17 3-a18 3-a19 3-a20 3-a21 3-a22 3-a23 3-a24 3-a25 3-a26 3-a27 3-a28 3-a29 3-a30 3-a31 3-a32 3-a33 3-a34 3-a35 >95 % >95 % >95 % >95 % >95 % >95 % >95 % >95 % >95 % >95 % >95 % >60 % >95 % >95 % >95 % >50 % >80 % >95 % >95 % >95 % >95 % >95 % >90 % >95 % - 463 412 520 438 482 425 484 478 440 439 424 472 494 467 466 424 464 423 466 512 - Yes Yes Yes Yes Yes Yes Yes Yes Yes - IC50 against PTP1B 115.3 167.8 106.8 216.4 111 77.3 177.1 125.9 395.3 66.6 120.9 139.4 60.8 145.9 115.9 >400 182.9 216.5 27.6 148.7 99.4 192.6 8.8 68 157.9 75.5 75 147.2 150.7 20.6 34.2 223 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 3-B1 3-B2 3-B3 3-B4 3-B5 3-B6 3-B7 3-B8 3-B9 3-B10 3-B11 3-B12 3-B13 3-B14 3-B15 3-B16 3-B17 3-B18 3-B19 3-B20 3-B21 3-B22 3-B23 3-B24 3-B25 3-B26 3-B27 3-B28 3-B29 3-B30 3-B31 3-B32 3-B33 3-B34 3-B35 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-B 3-a36 3-a37 3-a38 3-a39 3-a40 3-a41 3-a42 3-a43 3-a44 3-a45 3-a46 3-a47 3-a48 3-a49 3-a50 3-a51 3-a52 3-a53 3-a54 3-a55 3-a56 3-a57 3-a58 3-a59 3-a60 3-a61 3-a62 3-a63 3-a64 3-a65 3-a66 3-a67 3-a68 3-a69 3-a70 >70 % >60 % >90 % >95 % >95 % >95 % >95 % >95 % >95 % >95 % >95 % >95 % >95 % >95 % >95 % >95 % >95 % 454 440 467 509 452 512 481 529 469 467 524 487 500 451 451 493 Yes Yes Yes Yes Yes Yes - >400 >400 >400 236.3 >400 36.5 58.7 >400 >400 183.2 204.2 256.8 >400 174.1 >400 >400 304.7 149.7 105.8 275.2 117.2 365.9 20.5 197.7 378.8 244.5 >400 >400 >400 379.3 >400 >400 209.8 224 10.8 10.2 9.6 9.0 8.4 7.8 7.2 6.6 6.0 5.4 4.8 4.2 3.6 3.0 2.4 1.8 2.1656 1.6201 2.1370 1.4559 2.0884 1.0292 0.3141 4.8216 0.0220 2.2083 0.5563 7.0 1.0020 7.2 1.4932 0.5536 7.4 0.9565 7.6 1.9794 0.7591 7.8 2.0939 2.1143 8.0 1.4570 8.2 0.6269 1.0500 1.0000 Integral 0.9565 1.9794 0.7591 2.0939 2.1143 1.4570 0.6269 1.0500 6.8709 7.3172 7.2996 7.2466 7.2139 7.1962 7.1823 7.5606 7.5442 7.9287 7.9110 7.8909 7.8732 8.2666 2.5391 2.5038 2.5000 2.4962 2.3651 2.3500 2.3361 2.1810 1.8848 1.8696 1.8545 1.8406 1.7587 1.7524 1.7448 1.7385 1.7322 1.5961 1.5797 1.5658 1.5507 3.5943 4.6773 4.4226 4.4087 4.3949 5.2660 5.2345 5.0795 6.8709 8.2666 7.9287 7.9110 7.8909 7.8732 7.5606 7.5442 7.3172 7.2996 7.2466 7.2139 7.1962 7.1823 9.9270 10.2 NMR Spectra for selected compounds Compound 2-A-13 1H AMX500 A13 (ppm) 6.8 (ppm) 1.2 0.6 0.0 225 Compound 2-B8-2C-2-I 1H normal range AC300, B08-MeOD 2.0350 5.4443 5.3972 8.2652 8.0077 7.9863 7.9786 7.9573 7.6932 7.6861 7.6702 7.6625 7.2949 7.2872 7.2588 7.2308 7.0566 6.9531 6.9448 6.9240 6.9163 6.8966 6.8890 *** Current Data Parameters *** NAME : EXPNO : ag06kka PROCNO : 5.3972 5.4443 7.0566 6.9531 6.9448 6.9240 6.9163 6.8966 6.8890 7.2949 7.2872 7.2588 7.2308 7.6932 7.6861 7.6702 7.6625 8.0077 7.9863 7.9786 7.9573 8.2652 *** Acquisition Parameters *** DATE_t : DATE_d : Aug 07 2007 03:58:43 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 32 *** 1D NMR Plot Parameters *** 8.2 8.0 7.8 7.6 7.4 7.2 : 4.70 Hz 4.1048 1.1294 1.0000 3.1590 2.0503 1.0528 0.9276 SR 5.4 7.0 (ppm) 10.0 9.0 8.0 4.1048 1.1294 1.0000 3.1590 2.0503 1.0528 0.9276 Integral (ppm) 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 (ppm) Compound 2-B10-2C-I 1H Water suppression, B10 NAME 2.5124 2.5064 2.5003 2.4949 2.4888 3.7160 3.7083 5.4702 5.3316 8.3295 7.9931 7.9706 7.9641 7.9416 7.5044 7.4967 7.4666 7.4589 7.2984 7.2907 7.0700 7.0617 7.0535 7.0409 7.0333 7.0212 7.0135 6.9927 6.9850 6.9198 6.8908 10.3560 *** Current Data Parameters *** : ag09kka EXPNO : 12 PROCNO : 6.8908 6.9198 7.0535 7.0409 7.0333 7.0212 7.0135 6.9927 6.9850 7.5044 7.4967 7.4666 7.4589 7.2984 7.2907 7.9706 7.9641 7.9416 *** Acquisition Parameters *** DATE_t : DATE_d : Aug 08 2007 19:48:25 NS : O1 : 1002.80 Hz O2 : 1853.43 Hz O3 : 1853.43 Hz SFO1 : 300.1310028 MHz SFO2 : 300.1318534 MHz SFO3 : 300.1318534 MHz 20 7.6 1.1319 3.2326 1.0696 7.4 7.00 (ppm) 9.0 8.0 0.80 Hz 6.90 7.0 6.0 6.6385 2.1873 3.2326 1.0696 1.1263 1.0687 1.0000 Integral 10.0 : (ppm) 2.2571 7.8 SR 1.1319 8.0 1.1263 1.0687 *** 1D NMR Plot Parameters *** 5.0 4.0 3.0 2.0 1.0 0.0 (ppm) 226 Compound 2-C8-2C-2-I 1H Water suppression 1.5843 1.5585 1.5350 1.5103 1.4873 1.3273 1.3054 1.2824 1.2715 1.2605 1.2501 1.2337 1.2222 0.8704 0.8485 0.8250 2.5063 2.4948 5.4702 5.3475 8.3294 7.9925 7.9700 7.9635 7.9410 7.5000 7.4918 7.4803 7.4523 7.1504 7.1225 7.0513 7.0409 7.0190 7.0118 6.9916 6.9839 10.3948 *** Current Data Parameters *** NAME : EXPNO : ag09kka PROCNO : 7.5 7.3 7.2 0.8704 0.8485 0.8250 1.3273 1.3054 1.2824 1.2715 1.2605 1.2501 1.2337 1.2222 1.5843 1.5585 1.5350 1.5103 1.4873 1.9411 7.1504 7.1225 7.0513 7.0409 7.0190 7.0118 6.9916 6.9839 7.4 7.1 2.3928 1.4 : 1002.10 Hz O2 : 1853.43 Hz O3 : 1853.43 Hz SFO1 : 300.1310021 MHz SFO2 : 300.1318534 MHz SFO3 : 300.1318534 MHz 20 : 1.27 Hz (ppm) 6.0 2.3928 2.1240 2.1076 1.9411 2.2555 3.3286 1.0000 Integral 1.0241 7.0 : O1 0.8 1.2 (ppm) 8.0 NS SR 1.6 9.0 Aug 08 2007 7.0 (ppm) 10.0 19:27:52 : 5.0 4.0 3.0 3.5399 7.6 5.1684 7.7 : DATE_d 3.5399 7.8 2.2555 7.5000 7.4918 7.4803 7.4523 7.9 DATE_t *** 1D NMR Plot Parameters *** 5.1684 8.0 3.3286 1.0241 7.9700 7.9635 7.9410 *** Acquisition Parameters *** 2.0 1.0 0.0 (ppm) Compound 2-C7-2C 1H AMX500 SL-C7 Bruker 1.1649 1.1498 1.1346 2.5567 2.5416 2.5328 2.5013 2.4975 2.4937 2.4899 4.0053 7.4862 7.4685 7.1571 7.1407 10.0392 *** Current Data Parameters *** NAME : my0310sl EXPNO : PROCNO : *** Acquisition Parameters *** INSTRUM : NS : O1 : PULPROG : av500 3088.51 Hz zg30 SFO1 : 500.1330885 MHz SW : 20.6557 ppm *** Processing Parameters *** LB : SI : 0.30 Hz 16384 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 Start : 12.00 ppm Stop : -0.48 ppm SR : 7.76 Hz 3.2834 9.5844 2.0000 2.0224 1.9492 0.8778 Integral *** 1D NMR Plot Parameters *** 2.0 1.0 0.0 (ppm) 227 10.8 10.2 9.6 9.0 8.4 7.8 7.2 6.6 6.0 7.5 6.0 7.4 5.5 7.3 5.4 5.0 7.2 4.8 4.5 4.2 4.0 3.5 3.6 3.0 3.0 2.5 2.4 1.8268 1.8129 1.7978 1.7839 1.7700 6.5 3.6 2.1403 7.0 7.0 2.3853 2.3626 2.3475 2.3336 7.2 1.9912 4.0000 0.8341 1.1307 (ppm) 3.2882 7.5 3.3548 3.3409 3.3270 8.0 4.0825 8.5 7.4 7.1798 7.1760 7.1622 9.0 7.6 2.2026 2.1658 7.5190 7.5164 7.5026 9.5 0.9460 0.9640 Integral 7.8 2.2026 10.0 7.5190 7.5164 7.5026 7.1798 7.1760 7.1622 9.8954 10.5 2.1658 1.0000 Integral Integral 4.0000 0.8341 1.1307 0.9460 0.9640 3.6215 3.6041 3.5879 3.5693 3.5542 7.0295 7.2848 7.2605 7.2326 7.2024 7.1746 7.7306 7.7248 7.6969 7.6690 7.6632 7.5878 7.5820 7.5669 7.5599 -0.0000 3.6215 3.6041 3.5879 3.5693 3.5542 7.2848 7.2605 7.2326 7.2024 7.1746 7.0295 7.7306 7.7248 7.6969 7.6690 7.6632 7.5878 7.5820 7.5669 7.5599 Compound 2-G3-3C PyBop-II-3 (ppm) 3.5 (ppm) 2.0 1.8 1.5 1.2 1.0 0.6 0.5 0.0 Compound 2B-2C B7-C4 500MHz (ppm) 7.1 (ppm) 0.0 228 Compound 2-F11-6C 1H normal range AC300, 21, Traceless azide 1.6013 1.5756 1.5509 1.5268 1.5011 1.3416 1.3186 1.1943 2.2637 3.2942 3.2712 7.5789 7.5493 7.3439 7.3187 7.3154 7.2907 7.0491 6.9456 6.9220 6.9160 6.8968 9.8476 *** Current Data Parameters *** NAME : EXPNO : ma14kka PROCNO : 3.2942 3.2712 7.0491 6.9456 6.9220 6.9160 6.8968 7.3439 7.3187 7.3154 7.2907 7.5789 7.5493 *** Acquisition Parameters *** AQ_mod : dqd AUNM : au_zg BF1 : 300.1300000 MHz DATE_t : 03:40:08 DATE_d : Mar 14 2007 NS : O2 : PARMODE : RG SFO1 7.2 2.0000 Integral 4.0183 1.0169 1.9616 2.0209 7.4 3.3 7.0 3.2 (ppm) (ppm) : 300.1318534 MHz SW : SW_h : : YMAX_a : 1216539136.0000000 7.0 5.0 4.0 3.0 YMIN_a : -928285824.0000000 2.0 : 12.52 Hz 2.1012 4.0640 2.0187 2.0000 6.0 300.0 K *** 1D NMR Plot Parameters *** 4.0183 1.0169 1.9616 2.0209 0.9404 8.0 DMSO 17.9519 ppm 5387.931 Hz TE SR 9.0 1D : 287.3999939 SOLVENT : 7.6 16 1853.43 Hz 1.0 0.0 (ppm) Compound 3-31A 229 10.8 10.2 7.40 9.6 7.30 9.0 7.20 8.4 7.8 7.2 6.6 6.0 5.4 4.8 4.2 2.1730 7.50 2.0000 6.2234 Integral 6.2234 Integral 7.4086 7.3865 7.3621 7.3331 7.3088 7.2902 7.2670 2.9641 2.9397 2.9165 3.5630 3.5387 3.5143 7.4086 7.3865 7.3621 7.3331 7.3088 7.2902 7.2670 Compound 3-16B Compound 4-5 2-phenylethyl azide thru OMs (ppm) 7.10 (ppm) 3.6 3.0 2.4 1.8 1.2 0.6 0.0 230 Compound 4-22 8.1 8.0 7.9 7.8 7.6 3.0978 3.0931 3.0873 3.9370 4.3398 4.1982 7.3867 8.0460 8.0205 7.8859 7.8615 7.5771 7.5527 7.5295 7.3867 7.7 2.0066 7.5771 7.5527 7.5295 1.1175 7.8859 7.8615 2.3589 2.7543 Integral 8.0460 8.0205 9.2091 9.1987 A2-Urea 7.5 7.4 10.8 10.2 9.6 9.0 8.4 7.8 7.2 6.6 6.0 5.4 4.8 4.2 5.2933 6.5655 3.2343 5.9928 2.0066 1.1175 2.3589 2.7543 2.0000 Integral (ppm) 3.6 3.0 2.4 1.8 1.2 0.6 0.0 (ppm) 10.3 LC-MS spectra for selected compounds Compound 2-A-2-A3-2C mAU(x100) 254nm,4nm (1.00) 5.0 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 900 m/z Inten.(x1,000,000) 3.0 613.921 2.0 1.0 250.969 401.871 635.900 0.0 200 300 400 500 600 700 800 231 Compound 2-B-2-B5-2C m AU(x100) 1.5 254nm ,4nm (1.00) 1.0 0.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 m in 900 m /z 15.0 900 m /z Inten.(x1,000,000) 3.0 528.159 2.0 1.0 475.336 566.110 287.965362.242 0.0 200 300 400 500 600 700 800 Compound 2-C-2-A10-5C mAU(x100) 7.5 254nm,4nm (1.00) 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 Inten.(x10,000,000) 1.00 548.088 0.75 0.50 0.25 0.00 100 387.101 200 300 400 572.058 500 600 700 800 232 Compound 2-D-2-B4-2C mAU(x1,000) 254nm,4nm (1.00) 1.0 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 900 m/z 15.0 m in 900 m /z Inten.(x10,000,000) 1.5 480.147 1.0 0.5 502.128 0.0 200 300 400 500 600 700 800 Compound 2-E-2-B1-2S m AU(x100) 254nm 4nm (1.00)/sm th 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 Inten.(x1,000,000) 536.048 4.0 3.0 2.0 538.043 1.0 0.0 200 300 400 500 600 700 800 233 Compound 2-F-2-B1-2CAlk-6 mAU(x100) 254nm,4nm (1.00) 5.0 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 900 m/z Inten.(x10,000,000) 1.5 512.131 1.0 0.5 0.0 200 300 400 500 600 700 800 Compound 2-H-2-A7-5C mAU(x100) 254nm ,4nm (1.00) 7.5 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 Inten.(x10,000,000) 1.00 0.75 544.111 0.50 0.25 0.00 100 200 300 400 500 600 700 800 900 m/z 234 Compound 2-J-2-E4-4C mAU(x1,000) 1.0 254nm,4nm (1.00) 0.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 900 m/z Inten.(x10,000,000) 1.0 484.153 0.5 186.224 506.126 0.0 200 300 400 500 600 700 800 Compound 3-A26 mAU(x1,000) 254nm,4nm (1.00) 3.0 2.0 1.0 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 Inten. (x10,000) 1.0 1440 494 0.5 0.0 164256330413 250 623 748 500 750 899 1092 1000 1313 1473 1639 1790 1954 1250 1500 1750 m/z 235 Compound 3-A33 mAU(x1,000) 254nm,4nm (1.00) 3.0 2.0 1.0 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 Inten. (x10,000) 512 1.0 536 268 381 463 0.0119 165 229 303 358 100 200 300 400 500 732 583629 600 744 803835 881 929 975 700 800 900 m/z Compound 3-B15 mAU(x1,000) 254nm,4nm (1.00) 3.0 2.0 1.0 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 Inten. (x10,000) 481 2.5 231 141 182 242 0.0 100 200 420 327 373 422 300 400 630 522 582 667 720 500 600 700 986 787 810 800 879 904943 900 m/z 236 Compound 3-B23 mAU(x1,000) 254nm,4nm (1.00) 3.0 2.0 1.0 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 Inten. (x1,000) 487 5.0 802 154 422 142 213243 291 351 397 0.0 100 200 300 523 400 500 603 661 718 600 700 787 800 856891 948982 900 m/z Compound 4-1 AU 1.00 2487Channel (254.00 nm) 0.50 0.00 0.00 2.00 4.00 6.00 8.00 Minutes 10.00 12.00 14.00 Compound 4-6 1.00 2487Channel (254.00 nm) AU 0.50 0.00 0.00 2.00 4.00 6.00 8.00 Minutes 10.00 12.00 14.00 237 [...]... in a high- throughput manner “Catalomics” is the emerging subfield of Chemical Biology in which one aims at studying enzymes at the organism wide scale by employing high- throughput chemistry and technology3 High- throughput platform offers an efficient and rapid analysis of proteome on a global scale My Ph.D research mainly concentrates on the development or fine-tuning of high- throughput amenable chemical. .. the HT amenable chemistry tools (Fig 1.5), we have explored on Click chemistry, Solid-phase chemistry and Microwave (MW) assisted reactions for synthesis for the enzyme inhibitor libraries and fragments 5 ‘Click’ Chemistry Solid-phase Chemistry High- throughput Chemistry tools Microwave assisted Synthesis Multicomponent Reactions (MCRs) Fig 1.5 High- throughput Chemistry tools 1.3.1 Click Chemistry Sharpless... in a high- throughput manner “Catalomics” is the emerging subfield of Chemical Biology in which one aims at studying enzymes at the organism wide scale by employing high- throughput chemistry and technology High- throughput platform offers an efficient and rapid analysis of proteome on a global scale The major part of my Ph.D research concentrates on the development or fine-tuning of highthroughput amenable. .. biology to medicinal chemistry/ chemical biology It is for the same reasons that click chemistry has emerged as an integral part of the drug discovery pipeline by providing a high- throughput amenable chemical reaction platform for compound synthesis 6 N N N N N N Cu(I) Aq Solvent 1,4-regioisomer Fig 1.6 Cu(I) catalyzed azide-alkyne ligation Other salient features of Click Reaction10 1 Highly exothermic... Graduate Congress, NUS, Singapore, Sep - 2007 2 Srinivasan, R.; “Highthroughput amenable Chemistry in Catalomics” A*STARNoyori Forum Joint Symposium on Organic Chemistry Singapore, May 2007 3 Srinivasan, R.; “Highthroughput amenable Chemistry in Catalomics”, 3rd MPSGC, Kuala lampur, Malaysia, Dec – 2007 xiii AWARDS 1 Best Graduate Researcher in Chemistry, NUS – Sep 2007 2 Top Graduate Researcher in the Faculty... amenable chemistry to study PTPs One of the main challenges in the field of Catalomics is the development of highthroughput (HT) amenable chemical reactions that allow rapid synthesis of diverse chemical libraries for the interrogation of different classes of enzymes High- throughput amenable reactions are mainly characterized by near-perfect, modular and robust and biocompatible nature Among the HT amenable. .. classes of enzymes High- throughput (HT) amenable reactions are mainly characterized by near-perfect, modular and robust and biocompatible nature Among the HT amenable chemistry tools, we have explored on Click chemistry, Solid-phase chemistry and Microwave (MW) assisted reactions xv synthesis for enzyme inhibitor libraries and fragments We have successfully adopted the click chemistry platform to construct... Yang, P-Y.; Yao, S.Q “Highthroughput amenable Chemistry in Catalomics” A*STAR-Noyori Forum Joint Symposium on Organic Chemistry Singapore, May 2007 2 Srinivasan, R.; Uttamchandani, M.; Yao, S.Q ‘Click’-assembly of bidendate PTP1B inhibitors: Presented at the Singapore International chemical conference (SICC-4) Singapore, December 2005 3 Srinivasan, R.; Huang, X.; Yao, S.Q Chemical Biology of Phosphatases:... highthroughput amenable chemical platform to study enzymes especially Protein Tyrosine Phosphatases (PTPs), the last part of my research work is focused on Activity-based profiling and Bioimaging One of the main challenges in the field of Catalomics is the development of highthroughput (HT) amenable chemical reactions that allow rapid synthesis of diverse chemical libraries for the interrogation of... some high- throughput amenable chemistry tools to develop better inhibitors against PTPs, which is discussed in the following section 4 O HO P HO HO COO H O O F HOO C F Difluorophosphonate Cinnamic acid X O O-malonyltyrosine halo ketone O O O HO O O HO O OH HO NH N O O Keto acids Squaric acid Oxamic acid Isoxazole carboxylic acid Fig 1.4 Various Pharmacophores targeting PTPs 1.3 High- throughput amenable . DEVELOPING HIGH-THROUGHPUT AMENABLE CHEMISTRY FOR CHEMICAL BIOLOGY APPLICATIONS RAJAVEL SRINIVASAN . NATIONAL UNIVERSITY OF SINGAPORE 2009 DEVELOPING HIGH-THROUGHPUT AMENABLE CHEMISTRY FOR CHEMICAL BIOLOGY APPLICATIONS RAJAVEL SRINIVASAN (M.Sc.,. development for PTPs 4 1.3 High-throughput amenable chemistry to study PTPs 5 1.3.1 Click Chemistry 6 1.3.1.1 Click-based fragment assembly and in situ screening 7 1.3.1.2 Amide-bond formation