STEREOCHEMICAL ASSIGNMENT AND TOTAL SYNTHESIS OF AN ANTIMALARIAL LIPOPEPTIDE SHIBAJI KUMAR GHOSH NATIONAL UNIVERSITY OF SINGAPORE 2011 STEREOCHEMICAL ASSIGNMENT AND TOTAL SYNTHESIS OF AN ANTIMALARIAL LIPOPEPTIDE SHIBAJI KUMAR GHOSH (M.Sc, UNIVERSITY OF CALCUTTA) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2011 To my wife ACKNOWLEDGEMENTS I would like to express my sincere thanks and appreciation to my research advisor, Assistant Prof. Dr. Martin J. Lear, for his guidance, support, encouragement and patience throughout the completion of this work. I would like to thank our collaborators, Dr. Mark Butler and Dr. Brinda for their suggestions and supply of the natural product. I would also like to thank Dr. Kevin Tan group for helping me to test the antimalarial activity of the lipopeptide. I am grateful to all past and present members of Dr. Lear group for their kind help, useful discussions and friendship. I would especially thank Dr. Patil, Dr. Bastien Reux, Mun Hong, Santosh, Stanley, Eugene, Sandip, Kunal for their timely help, co-operation and constant support. I wish to thank Mdm. Han Yanhui and Mr. Wong Chee Peng for their timely assistance with NMR measurements and Mdm. Wong Lai Kwai with Mass Spectroscopy measurements. I am also grateful to my parents and younger brother for their continuous support and enthusiasm. This thesis is dedicated to my beloved wife Tumpa for her incredible support and sacrifice during my graduate studies. i TABLE OF CONTENTS Acknowledgments i Table of contents ii Summary vi List of Tables vii List of Figures viii List of Schemes x Abbreviations and symbols xiii Publications xx Chapter Introduction 1.1 Malaria background 1 1.1.1 Life cycle of the malaria parasite 1.1.2 Haemoglobin metabolism 1.2 Antimalarial drugs 1.2.1 Casual prophylaxis 1.2.2 Suppressive prophylaxis 1.2.3 Clinical cure 1.2.4 Radical cure 1.2.5 Controlling transmission 1.2.6 Qunoline with secondary alcohols 1.2.7 8-aminoquinolines ii 1.2.8 4-aminoquinolines 1.2.9 Antifolates 1.2.10 Antibiotics 1.2.11 Phenanthrenes 1.2.12 Artimisinins/Sesquiterpene 1.3 Antimalarial drug resistance 1.4 Antimalarial drug targets 10 1.4.1 Protein kinases 10 1.4.2 Choline transporter 11 1.4.3 DOXP reductoisomerase 11 1.4.4 Purine salvage enzyme HGXPRT 12 1.4.5 Lactate dehydrogenase 12 1.4.6 Plasmepsins 13 1.4.7 Falcipains 13 1.5 Antimalarial peptides 14 1.6 Aims of this study 15 Chapter References 16 Hydrolysis of the peptide and identification of amino acids 21 2.1 Hydrolysis of peptide N1708 21 2.2 Stereochemical assignment of the aspartic acid residue 23 2.3 Conclusion 25 Chapter Synthesis of 2-Amino-3, 4-Dimethylpent-4-Enoic Acid 3.1 Direct methylation of aspartic acid 26 26 iii 3.2 Synthetic plan via 1, 3-oxazin-6-ones 29 3.3 Synthetic plan with threonine 31 3.4 Proline catalysed Mannich reaction 33 3.5 Conclusion 38 References 39 Synthesis of 3-Amino-2-Hydroxy-2-Methylsuccinic Acid 41 Chapter 4.1 1st Generation synthesis from L-Tartaric acid 41 4.2 2nd Generation synthesis from hydroxyacetone 43 4.3 Sharpless Asymmetric Aminohydroxylation 45 4.4 Sharpless Asymmetric Dihydroxylation 45 4.5 Changing TBS group to benzyl group 46 4.6 Inserting p-Methoxybenzyl (PMB) group 50 4.7 Conclusion 68 References 69 Chapter Synthesis of 5-Methyl-∆3,4 Decanoic Acid 72 5.1 Asymmetric methylation using Evan’s chiral auxiliary 72 5.2 Model studies of Julia-Kocienski olefination 74 5.3 2nd Generation synthetic plan 75 5.4 Synthesis of other enantiomer of the fatty acid 79 5.5 Conclusion 80 References 81 Chapter Coupling of Fragments & Full Structural Assignment 6.1 Peptide coupling 82 82 iv 6.2 Conclusion 94 References 95 Experimental Section Appendices 96 160 v SUMMARY Malaria is one of the three prime causes (together with tuberculosis and AIDS) responsible for the high mortality in this world. 300-500 Millions people suffer from the disease every year resulting in about one million deaths. In recent years, malaria is considered as a complex multisystem disorder. As more than 40% of the world’s population lives in malaria endemic areas, the challenge is to understand the complexities of this disease and develop potential tools for improving the present scenario. There is also the immediate need for the discovery of cost effective drugs or vaccines to fight mainly chloroquine-resistant strains of P. falciparum. The lipopeptide (N1708) isolated from Streptomyces sp. using bioassay-guided isolation by MerLion Pharmaceuticals exhibits promising activity against Plasmodium falciparum (IC50= 0.8 µM against 3D7 strain). NMR and mass analyses suggest that this peptide contains two non-proteinogenic amino acids, one aspartic acid and a ten carbon long chain fatty acid containing a trans-double bond and a chiral centre. As it is a wellknown problem that the half life of peptide drugs is short because of the enzymatic hydrolysis of the amide bond formed by proteinogenic amino acids, thereby we are interested to find out the configuration of these six chiral centres (one of them is quaternary) present in this lipopeptide. Synthesis and stereochemical assignment of the non-proteinogenic amino acids and rest of the fragments have been performed in this work. The non-proteinogenic amino acids have been synthesised and their absolute configuration assigned to the chiral centres with the help of Marfey’s reagent. The full structure of N1708 has been confirmed by the total synthesis of the targeted lipopeptide. vi LIST OF TABLES Table 3.1: β-Methylation studies of protected aspartic acid. 27 Table 3.2: Further β-methylation studies of protected aspartic acid. 28 Table 3.3: Reaction conditions to manipulate the cyano group. 32 Table 3.4: Wittig reaction for the methenylation. 34 Table 3.5: PMP deprotection. 35 Table 4.1: Oxidation of azido-alcohol. 42 Table 4.2: Oxidation of Boc protected amino-alcohol (4-8). 43 Table 4.3: Benzylation of tri-substituted allylic alcohol. 47 Table 4.4: Debenzylation. 48 Table 4.5: PMB protection of hydroxyacetone. 51 Table 4.6: Oxidation studies of 4-68R. 56 Table 4.7: Oxidation studies of 4-79R. 59 Table 5.1: Methylation using different bases. 73 Table 5.2: Oxidation studies. 79 Table 6.1: Chemical shift values (δppm). 89 vii 8.8 8.4 8.0 7.6 165 160 7.2 155 150 6.8 145 140 6.4 135 130 6.0 125 (S) 5.6 120 115 5.2 110 105 4.8 100 4.4 95 90 85 4.0 80 75 3.6 70 3.2 65 60 2.8 55 2.4 50 45 2.0 40 35 1.6 30 25 1.2 6.1040 170 1.1091 175 7.4302 Ph 1.0480 180 5-4 1.7637 1.6119 1.5933 1.5736 1.5527 1.5319 1.3895 1.3780 1.3615 1.3374 1.3007 1.2624 1.2509 1.2361 1.1068 1.0783 1.0520 1.0257 0.9019 0.8800 0.8663 0.8427 185 N 0.9643 190 1.0127 O 3.5053 3.4861 3.4702 3.4511 3.4061 3.3842 3.3716 3.3497 7.2603 195 1.0000 Integral 20 0.8 14.0057 17.3368 22.5079 26.9080 33.3737 31.8100 37.9121 37.7012 55.3528 65.9932 129.4428 128.9192 127.3191 DEPT135 AC300 O O (ppm) 15 10 OH 5-5 (ppm) 0.4 230 14.0494 16.5513 22.6170 26.6171 33.0901 32.1228 35.7375 68.3932 77.4263 77.0045 76.5827 (S) OH 5-5 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 95 90 85 80 75 70 65 60 55 50 45 40 65 60 55 50 45 40 35 30 25 20 15 10 10 (ppm) (S) 16.5586 14.0494 22.6315 26.6317 35.7375 33.1047 32.1446 68.3278 DEPT135 AC300 OH 5-5 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 35 30 25 20 15 (ppm) 231 195 190 185 180 175 170 165 160 155 150 145 140 (S) S 135 130 5.6 125 120 5.2 115 110 4.8 105 100 4.4 95 90 4.0 85 3.6 80 75 3.2 70 65 2.8 60 55 2.4 50 45 2.0 N N N N Ph 5-14 40 35 1.6 30 25 20 13.9826 6.0 22.5161 21.5396 19.0546 6.4 26.4148 6.8 32.9079 31.8585 7.2 35.8374 7.6 15 3.0904 3.0000 7.3961 2.1206 1.0148 1.0338 1.0387 S 40.5304 8.0 77.2515 77.0037 76.7487 8.4 123.8470 8.8 133.7651 129.9975 129.7133 154.7236 4.8238 Integral (S) N N N N Ph 5-14 (ppm) 1.2 0.8 (ppm) 10 232 3.4698 3.4584 3.4446 3.4332 3.2706 3.2555 3.2454 3.2302 1.9393 1.9267 1.9140 1.9014 1.6922 1.4766 1.4715 1.4665 1.4627 1.4526 1.4425 1.4312 1.4236 1.4110 1.3631 1.3593 1.3543 1.3492 1.3442 1.3366 1.3253 1.3102 1.2988 1.2849 1.2648 1.2585 1.2484 1.2333 1.2295 1.2232 1.0290 1.0164 0.8828 0.8689 0.8550 7.2608 7.5936 7.5886 7.5722 7.5596 7.5432 7.5407 7.5356 7.5306 7.5230 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 5.2 115 110 4.8 105 4.4 100 95 4.0 90 85 3.6 (S) O S O 80 75 3.2 70 65 2.8 60 55 2.4 50 45 2.0 40 35 1.6 30 25 20 13.9541 5.6 19.6780 6.0 22.4635 6.4 28.2237 25.9255 6.8 31.5693 7.2 36.4859 7.6 61.8396 8.0 77.4256 77.0038 76.5819 8.4 125.1293 8.8 133.0714 131.3986 129.6312 154.0831 3.0516 3.0000 7.3225 1.0103 0.9957 0.9999 1.0033 4.8738 Integral (S) O S O N N N N Ph 5-15 (ppm) 1.2 0.8 N N N N Ph 5-15 (ppm) 15 10 233 2.3597 2.3378 2.3159 2.2973 2.2743 1.6037 1.5462 1.5401 1.5275 1.5210 1.5100 1.4980 1.3983 1.3840 1.3588 1.3528 1.3320 1.3144 1.2766 1.1605 1.1380 0.8981 0.8772 0.8537 3.8351 3.8187 3.7869 3.7710 3.6138 3.5869 3.5656 3.5393 7.2603 7.6953 7.6898 7.6816 7.6794 7.6711 7.6640 7.6520 7.6459 7.6235 7.6081 7.5846 7.5736 7.5671 195 190 185 180 175 170 165 160 155 150 145 140 135 130 5.6 125 120 5.2 115 110 4.8 105 100 4.4 95 90 4.0 85 3.6 80 75 3.2 70 65 2.8 (S) ( E) 60 55 2.4 50 45 2.0 40 35 1.6 30 25 20 14.0995 6.0 26.9582 26.8491 22.6307 20.6816 19.2270 6.4 37.0676 36.7258 36.0785 32.0202 6.8 64.1159 7.2 77.4255 77.0037 76.5746 7.6 124.5110 8.0 129.4784 127.5511 8.4 135.5950 134.1332 138.6642 8.8 15 1.2 10 3.6361 3.0000 10.696 10.918 1.1237 2.4021 2.2615 2.1337 7.1225 4.7695 Integral (S) ( E) OTBDPS 5-18 (ppm) 0.8 OTBDPS 5-18 (ppm) 234 1.2948 1.2663 1.2455 1.0543 0.9562 0.9338 0.8954 0.8899 0.8741 1.5446 1.4422 2.2897 2.2809 2.2678 2.2596 2.2492 2.0678 2.0487 2.0284 3.7015 3.6791 3.6566 5.3741 5.3560 5.3478 5.3369 7.6986 7.6932 7.6899 7.6800 7.6740 7.6680 7.4247 7.4132 7.4088 7.4001 7.3842 7.3749 7.3562 7.2604 195 190 185 180 175 170 165 160 155 150 145 140 6.0 135 130 5.6 125 120 5.2 ( S) ( E) 115 110 4.8 105 100 4.4 95 90 4.0 85 80 3.6 75 3.2 70 65 2.8 60 55 2.4 50 45 2.0 40 35 1.6 30 25 20 14.0556 6.4 22.6159 20.3176 6.8 26.8633 7.2 31.9326 29.6853 7.6 37.5474 36.7692 36.6092 8.0 77.4253 76.9961 76.5743 8.4 118.9468 8.8 141.3477 176.6581 1.2 15 10 4.0956 3.0000 11.958 1.4416 1.8623 1.8293 Integral ( S) ( E) OH 5-20 O (ppm) 0.8 OH 5-20 O (ppm) 235 0.9809 0.9670 0.8889 0.8763 0.8611 1.2545 3.0775 3.0674 5.4742 5.4704 5.4666 5.4628 7.2606 195 190 185 180 175 170 165 160 155 150 145 140 135 6.4 BocHN 130 125 6.0 120 115 5.6 110 105 5.2 100 4.8 95 90 4.4 85 80 4.0 75 3.6 70 65 3.2 60 55 2.8 50 2.4 45 40 2.0 35 1.6 30 25 20 13.9900 6.8 20.5705 7.2 28.2368 7.6 42.9354 8.0 55.9944 8.4 2.9677 9.7859 3.0000 1.0626 0.6734 2.2327 0.7301 1.1720 BocHN 80.0719 77.2517 77.0039 76.7488 8.8 112.5225 9.2 145.3084 9.6 155.6711 176.9065 Integral (R) (S) CO2 H 6-1 (ppm) 1.2 15 0.8 (R) (S) 6-1 CO2 H (ppm) 10 236 1.0730 1.0591 1.4247 1.7840 2.6539 2.6426 4.4467 4.9825 4.9661 4.8463 4.7757 7.2606 8.9689 195 190 185 180 175 170 165 160 155 150 145 140 135 130 (S) BocHN H N O 125 120 115 110 4.8 105 100 4.4 95 90 4.0 85 80 3.6 75 3.2 70 65 2.8 60 55 2.4 50 45 2.0 40 35 1.6 30 25 20 13.8805 13.8513 13.8367 5.2 21.0804 5.6 28.1929 6.0 35.9831 6.4 42.3887 6.8 52.6784 52.6639 51.9133 48.6777 7.2 2.9248 9.3960 2.8910 0.9985 1.0223 1.0498 3.0000 2.9911 0.9211 O 2.0138 BocHN H N 57.0363 57.0144 56.9780 7.6 1.6990 0.9534 (S) 80.1300 80.1154 80.0935 77.2515 76.9964 76.7413 8.0 112.3402 8.4 145.9568 8.8 155.6344 171.2075 170.7193 Integral (R) (S) CO2Me CO2Me 6-3 (ppm) 1.2 0.8 (R) (S) CO2Me CO2Me 6-3 (ppm) 15 10 237 1.0329 1.0190 1.4502 1.4363 1.7969 3.0475 3.0387 3.0135 3.0034 2.9177 2.9089 2.8836 2.8748 2.7248 2.7109 2.6971 3.7649 3.6968 4.2515 4.2389 4.2251 4.8907 4.8113 4.7974 7.0151 7.0024 7.2861 195 190 185 180 175 170 O 165 160 155 O N3 HO 150 145 140 N H 135 130 O O 125 120 115 110 H N 105 100 95 90 4.0 85 3.6 80 75 3.2 70 65 2.8 60 2.4 CO2Me CO 2Me 6-15 55 50 45 2.0 40 35 1.6 30 25 20 14.1136 4.4 20.8618 4.8 23.9224 5.2 29.6576 29.6212 5.6 35.8519 6.0 38.5846 6.4 2.9747 3.0451 2.8968 1.0696 1.0511 1.0921 2.9535 3.0000 3.0404 0.9757 0.9074 0.9809 0.9752 0.9763 O 42.1409 6.8 H N 52.8970 52.7586 52.0298 48.7141 7.2 0.9735 HO N H 55.5205 7.6 0.9130 0.9638 N3 65.8394 8.0 O 77.2514 76.9964 76.7486 8.4 O 112.5588 8.8 145.5997 172.6213 171.2440 170.6683 170.4569 169.2618 Integral O CO 2Me CO2Me 6-15 (ppm) 1.2 0.8 (ppm) 15 10 238 1.0995 1.0856 1.4777 1.8029 1.6958 3.0208 3.0120 2.9868 2.9779 2.8998 2.8897 2.8657 2.8557 2.8040 2.8002 2.7901 2.7762 3.8920 3.8567 3.8478 3.7508 3.6852 4.2488 4.9094 4.8451 4.8022 4.7934 4.7871 4.7833 4.7770 4.7682 4.5702 4.5576 4.5526 4.5400 6.9997 6.9845 7.2606 7.2026 7.1850 180 () 170 (S) 160 O 10 150 O 140 5.8 O N H 5.6 130 5.4 N OH H 120 5.2 5.0 O O 110 4.8 H N 4.6 100 4.4 4.2 90 4.0 3.8 80 3.6 3.4 70 3.2 3.0 60 2.8 2.6 2.4 50 2.2 40 2.0 1.8 30 1.6 1.4 20 1.2 3.4209 2.9772 2.9127 1.0515 1.0377 0.9822 0.9683 0.8851 0.8801 0.8713 0.8574 1.2558 1.7601 2.1686 2.1585 2.1446 2.1320 3.0372 3.0284 2.9956 2.9842 2.8922 2.8834 2.8582 2.8481 2.7598 2.7472 2.7334 3.7508 3.7193 3.6827 4.8993 4.8602 4.8413 4.8161 4.7820 4.7732 4.7644 4.7581 4.4480 4.4353 4.4316 4.4240 4.4190 5.5498 5.5360 5.5183 5.5057 5.4981 5.4868 5.4679 5.4553 6.8824 6.8673 7.2606 7.1522 7.1358 7.1182 7.1056 7.1005 7.0854 7.0665 7.0564 1.4613 6-16 9.2993 CO 2Me CO2Me 3.1374 3.0000 0.9966 1.0233 1.0633 2.9653 2.8264 2.8239 2.9323 0.9424 1.0026 4.0038 1.9185 0.8154 O 14.1500 14.0844 6.0 H N 23.9661 22.5961 20.9346 20.4026 6.2 N OH H 36.7700 36.7263 35.8810 31.9749 29.6940 26.9248 6.4 N H O 42.1627 40.1878 6.6 10 O 48.6557 6.8 (S) O 57.5609 55.1852 52.9188 52.8022 52.0444 7.0 O 77.2587 77.0036 76.7485 76.4425 7.2 0.8092 0.8718 Integral 112.6826 7.4 119.9262 7.6 142.4806 7.8 145.5851 175.8568 172.7305 171.7540 171.2876 170.6536 170.0342 () (ppm) 1.0 0.8 0.6 CO 2Me CO2Me 6-16 (ppm) 10 239 2D NMR Experiment (COSY) of compound 6-16. SG0487 ppm Current Data Parameters NAME skg1022 EXPNO PROCNO F2 - Acquisition Parameters Date_ 20101022 Time 13.36 INSTRUM spect PROBHD mm TXI 1H-13 PULPROG cosygs TD 2048 SOLVENT C6D6 NS 16 DS SWH 3822.630 Hz FIDRES 1.866518 Hz AQ 0.2680592 sec RG 181 DW 130.800 usec DE 6.00 usec TE 298.0 K D0 0.00000300 sec D1 1.00000000 sec D13 0.00000300 sec D16 0.00020000 sec IN0 0.00026160 sec ======== CHANNEL f1 ======== NUC1 1H P0 8.20 usec P1 8.20 usec PL1 -2.00 dB SFO1 500.2320434 MHz ====== GRADIENT CHANNEL ===== P16 1500.00 usec F1 - Acquisition parameters ND0 TD 512 SFO1 500.232 MHz FIDRES 7.466074 Hz SW 7.642 ppm FnMODE QF F2 - Processing parameters SI 512 SF 500.2300053 MHz WDW SINE SSB LB 0.00 Hz GB PC 1.40 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 1.0 0.5 ppm F1 - Processing parameters SI 512 MC2 QF SF 500.2300062 MHz WDW SINE SSB LB 0.00 Hz GB 240 2D NMR Experiment (HMQC) of compound 6-16. SG0487 ppm 10 20 30 40 50 60 70 80 90 100 Current Data Parameters NAME skg1022 EXPNO 12 PROCNO F2 - Acquisition Parameters Date_ 20101022 Time 21.19 INSTRUM spect PROBHD mm TXI 1H-13 PULPROG inv4gstp TD 512 SOLVENT MeOH NS 16 DS SWH 3894.081 Hz FIDRES 7.605627 Hz AQ 0.0659192 sec RG 128 DW 128.400 usec DE 6.00 usec TE 298.1 K D0 0.00000300 sec D1 1.00000000 sec D2 0.00345000 sec d4 0.00172500 sec D11 0.03000000 sec D13 0.00000300 sec D16 0.00020000 sec d20 0.00002500 sec d21 0.00174600 sec IN0 0.00000946 sec ======== CHANNEL f1 ======== NUC1 1H P1 8.20 usec P2 16.40 usec PL1 -2.00 dB SFO1 500.2320981 MHz ======== CHANNEL f2 ======== CPDPRG2 garp NUC2 13C P3 13.20 usec P4 26.40 usec PCPD2 70.00 usec PL2 -2.00 dB PL12 12.49 dB SFO2 125.7942545 MHz ====== GRADIENT CHANNEL ===== P16 1500.00 usec 110 120 130 140 150 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm F1 - Acquisition parameters ND0 TD 512 SFO1 125.7943 MHz FIDRES 51.601719 Hz SW 210.026 ppm FnMODE TPPI F2 - Processing parameters SI 512 SF 500.2300083 MHz WDW QSINE SSB LB 0.00 Hz GB PC 1.40 F1 - Processing parameters SI 512 MC2 TPPI SF 125.7829116 MHz WDW QSINE SSB LB 0.00 Hz GB 241 2D NMR Experiment (HMBC) of compound 6-16. SG0487 ppm 20 40 60 80 100 120 140 160 180 200 220 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 1.0 0.5 ppm Current Data Parameters NAME skg1022 EXPNO 13 PROCNO F2 - Acquisition Parameters Date_ 20101022 Time 23.49 INSTRUM spect PROBHD mm TXI 1H-13 PULPROG inv4gplplrnd TD 2048 SOLVENT CDCl3 NS 32 DS SWH 3894.081 Hz FIDRES 1.901407 Hz AQ 0.2631416 sec RG 8192 DW 128.400 usec DE 6.00 usec TE 298.4 K D0 0.00000300 sec D1 2.00000000 sec D2 0.00345000 sec D6 0.10000000 sec D13 0.00000300 sec D16 0.00020000 sec IN0 0.00001728 sec ======== CHANNEL f1 ======== NUC1 1H P1 8.20 usec P2 16.40 usec PL1 -2.00 dB SFO1 500.2320981 MHz ======== CHANNEL f2 ======== NUC2 13C P3 13.20 usec PL2 -2.00 dB SFO2 125.7967701 MHz ====== GRADIENT CHANNEL GPNAM1 sine.100 GPNAM2 sine.100 GPNAM3 sine.100 GPX1 0.00 GPX2 0.00 GPX3 0.00 GPY1 0.00 GPY2 0.00 GPY3 0.00 GPZ1 50.00 GPZ2 30.00 GPZ3 40.00 P16 1500.00 ===== % % % % % % % % % usec F1 - Acquisition parameters ND0 TD 512 SFO1 125.7968 MHz FIDRES 56.530392 Hz SW 230.082 ppm FnMODE QF F2 - Processing parameters SI 2048 SF 500.2300083 MHz WDW QSINE SSB LB 0.00 Hz GB PC 1.40 F1 - Processing parameters SI 512 MC2 QF SF 125.7828890 MHz WDW SINE SSB LB 0.00 Hz GB 242 180 170 ()3 160 10 150 O O N H 140 5.8 O 5.6 130 5.4 N OH H 120 5.2 5.0 O 4.8 H N O 110 4.6 100 4.4 4.2 90 4.0 3.8 80 3.6 3.4 70 3.2 3.0 2.8 60 2.6 2.4 50 2.2 40 2.0 1.8 30 1.6 1.4 20 1.2 14.1209 14.1063 6.0 1.0 4.0842 3.5232 3.1663 10.491 3.3353 3.0000 1.4244 1.2598 1.0218 3.2891 3.3266 2.8712 3.3309 1.1299 0.9996 4.5723 2.3374 0.8562 0.9045 1.0070 Integral O 23.9588 22.6107 20.9491 20.4172 6.2 N OH H H N 36.7627 36.7409 35.8664 31.9749 29.6940 26.9394 6.4 N H O 42.1481 40.1878 6.6 O 48.6339 6.8 10 O 57.5245 55.1415 52.9480 52.8314 52.0662 7.0 O 77.2587 77.0036 76.7485 76.4352 7.2 112.7044 7.4 119.9116 7.6 142.4952 7.8 145.5559 175.8787 172.7597 171.7613 171.3241 170.6609 170.0269 ()3 CO 2Me CO2 Me N1708M (ppm) 0.8 0.6 CO 2Me CO2 Me N1708M (ppm) 10 243 1.0478 1.0339 0.9797 0.9671 0.8839 0.8700 0.8561 1.2520 1.4625 1.5937 1.7575 2.1572 2.1446 2.1320 3.0448 3.0359 2.9968 2.9855 2.8960 2.8872 2.8619 2.8519 2.7598 2.7460 2.7321 3.7520 3.7205 3.6839 5.5460 5.5158 5.5057 5.5032 5.4969 5.4843 5.4666 5.4540 4.9005 4.8804 4.8690 4.8627 4.8501 4.8438 4.8148 4.7808 4.7719 4.7644 4.7568 4.7480 4.4492 4.4366 4.4328 4.4252 4.4202 4.4089 6.8912 6.8761 7.2606 7.1383 7.1219 7.0904 7.0715 180 () 170 (R) 160 10 150 O O 140 N H O 130 5.6 5.4 N OH H 120 5.2 5.0 O 4.8 H N O 110 4.6 100 4.4 4.2 90 4.0 3.8 80 3.6 3.4 70 3.2 3.0 60 2.8 2.6 50 2.4 2.2 40 2.0 1.8 30 1.6 1.4 20 1.2 14.1354 14.0917 5.8 1.0 3.8126 2.8523 3.0112 13.673 3.0640 3.0858 1.1169 1.0723 1.1789 3.4044 3.0030 2.8422 3.0427 0.9997 1.0543 4.0876 1.8796 0.8852 O 23.9661 22.5961 20.9273 20.4099 6.0 H N 26.9321 6.2 N OHH 31.9676 29.6940 6.4 N H O 36.7554 35.8810 6.6 O 42.1700 40.2388 6.8 10 O 57.5682 55.1852 52.9188 52.8095 52.0516 48.6630 7.0 (R) 77.2587 77.0036 76.7485 76.4352 7.2 O 112.6826 7.4 0.8855 1.0000 Integral 119.9772 7.6 142.4588 7.8 145.5851 175.8349 172.6868 171.7759 171.3095 170.6609 170.0561 () CO 2Me CO2Me 6-17 (ppm) 0.8 0.6 CO 2Me CO2 Me 6-17 (ppm) 10 244 1.0503 1.0364 0.9872 0.9734 0.8851 0.8801 0.8713 0.8574 1.2545 1.4625 1.7601 2.1698 2.1496 2.1370 2.1232 3.0384 3.0296 2.8947 2.8859 2.8607 2.8506 2.7598 2.7472 2.7334 3.7520 3.7205 3.6839 4.4542 4.4404 4.4379 4.4240 5.5498 5.5372 5.5196 5.5057 5.4943 5.4817 5.4641 5.4515 4.8980 4.8589 4.8489 4.8400 4.8148 4.7846 4.7745 4.7669 4.7593 4.7505 6.8988 6.8837 7.2606 7.1459 7.1295 7.0829 7.0640 195 190 185 180 175 170 165 160 155 150 145 4.2 O () 140 135 4.0 O 130 3.8 N H 125 3.6 120 3.4 115 110 3.2 (S) OH O N OH H 105 100 3.0 H N O 95 90 2.8 85 2.6 80 2.4 75 2.2 70 65 2.0 60 1.8 55 50 1.6 45 1.4 40 35 1.2 30 H N O 25 3.4585 3.3076 3.1328 N OHH 8.3575 N H 3.1362 3.0000 1.3863 1.2468 (S) 1.0 20 15.5691 14.4396 4.4 1.8699 OH O 24.4524 23.6727 20.9545 20.4517 4.6 2.1916 1.2187 2.5494 2.0378 () O 28.1034 4.8 59.6214 57.1291 50.3372 49.5138 49.3461 49.1785 49.0036 48.8360 48.6684 48.4935 44.3324 40.6304 38.0944 38.0070 36.8337 33.1536 5.0 76.5135 5.2 113.3804 5.4 121.7463 5.6 142.7995 5.8 147.0116 176.3579 174.1353 173.9531 173.5887 173.2827 172.6632 Integral O CO 2H CO2H 6-18 (ppm) 0.8 15 0.6 10 0.4 CO 2H CO2 H 6-18 (ppm) 245 1.0829 1.0691 0.9997 0.9858 0.9064 0.8926 0.8774 1.2960 1.4397 1.7637 2.1319 2.8227 2.8101 2.7975 2.6891 2.6752 2.6563 2.9765 2.9665 3.3144 3.3106 3.3081 4.4428 4.4264 4.7869 4.6167 4.6079 4.5966 4.5840 5.5182 5.5156 5.5106 5.5056 [...]... Ghosh, Martin J Lear; Stereochemical Assignment and Total Synthesis of an Anti-malarial Lipopeptide , The 6th Mathematics and Physical Science Graduate Congress, University of Malaya, Malaysia , 13th - 15th Dec, 2010 (Oral Presentation) 2 Shibaji K Ghosh, Brinda Somanadhan, Mark S Butler, Martin J Lear; “Amino Acid Stereochemical Assignment and Total Synthesis of A Natural Anti-malarial Peptide”,... Synthesis of a Novel Antimalarial Lipopeptide by the de Novo Preparation of Chiral Nonproteinogenic Amino Acids, Shibaji K Ghosh, Brinda Somanadhan, Kevin S.-W Tan, Mark S Butler, and Martin J Lear.- Org Lett 2012, 14, 1560-1563 2 Synthesis of 2-C-Methylerythritols and 2-C-Methylthreitols via Enantiodivergent Sharpless Dihydroxylation of Trisubstituted Olefin, Shibaji K Ghosh, Mark S Butler, and Martin... responsible for membrane transport and signalling (e.g protein kinases and the choline transporter) b) Enzymes involved in macromolecular and metabolite synthesis (e.g DOXP reductoisomerase, parasite HGXPRT and lactate dehydrogenase) c) Targets taking part in the processes occurring in the digestive vacuole (e.g haemoglobin digestion and haem detoxification) Proteases namely plasmepsins and falcipans are the... that PKs of Plasmodium and mammalian are different in their compositions and organisation of signalling pathways.31 PfCPK and PfCPK2, the calcium-dependent protein kinases have been described in P falciparum.32 Previously 10 Chapter 1 this class of enzymes has been isolated only in plants and some protozoan species This makes the target promising as it may be significantly different from mammalian PKs... site of action of the drug It is known that malaria-infected human RBCs have better permeability than normal RBCs and show a new permeation pathway (NPP).33 NPP may consist of single or multiple channels, which prefer anions over cations Choline carrier activity is much (10 fold) higher in infected RBCs The antimalarial activity of choline analogues is due the inhibition of the de novo synthesis of major... inhibitors Sulfadoxine and pyrimethamine are used in combination for drug therapy in some parts of Africa.18 1.2.10 Antibiotics Antibiotics are used along with other antimalarial drugs Tetracycline and doxycycline are the common drugs for this purpose 1.2.11 Phenanthrenes Halofantrine (1-11) is a popular antimalarial drug in this class This was identified during the Second World War This class of drug acts on...LIST OF FIGURES Figure 1.1: Global malaria distribution and endemicity 2 Figure 1.2: Life cycle of Plasmodium falciparum 3 Figure 1.3: General haemoglobin catabolism pathway 4 Figure 1.4: Structurally different antimalarial drugs 6 Figure 1.5: Radical mechanism of artemisinin class of drug 8 Figure 1.6: Example of antimalarial peptides 14 Figure 1.7: Linear structure and fragments of the isolated... Scheme 6.1: Synthesis of dipeptide 6-4 82 Scheme 6.2: Synthesis of tripeptide 84 Scheme 6.3: Boc protection 84 xi Scheme 6.4: Functional group manipulation 85 Scheme 6.5: One step procedure for azide reduction, Boc protection 85 Scheme 6.6: Oxidative cleavage of PMB 86 Scheme 6.7: Synthesis of azido tripeptide and Staudinger reaction 86 Scheme 6.8: Final coupling and synthesis of trimethyl ester of target... ago but huge parasite resistance has forced a reduction in its use This class may also have an important role in the heme poisoning process 1.2.9 Antifolates This class of antimalarials works by inhibiting dihydrofolate reductase (DHFR) and deoxyhypusine synthase (DHPS) Pyrimethamine (1-5) and proguanil (1-6) are common DHFR inhibitors whereas sulfadoxine (1-7, sulfonamides) and dapsone (1-8, 7 Chapter... chloroquine and mefloquine 1.2.3 Clinical cure These type of agents are involved in killing erytrocytic schizogony and prevent clinical attack They are also called blood schizonticides These include the 4aminoquinolines (e.g chloroquine), the phenanthrenes (e.g halofrantrine), the antifolates (e.g pyrimethamine, proguanil, dapsone and sulfadoxine), the artemisinin group (e.g dihydroartemisinin, artesunate and . STEREOCHEMICAL ASSIGNMENT AND TOTAL SYNTHESIS OF AN ANTIMALARIAL LIPOPEPTIDE SHIBAJI KUMAR GHOSH NATIONAL UNIVERSITY OF SINGAPORE 2011 STEREOCHEMICAL. ASSIGNMENT AND TOTAL SYNTHESIS OF AN ANTIMALARIAL LIPOPEPTIDE SHIBAJI KUMAR GHOSH (M.Sc, UNIVERSITY OF CALCUTTA) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF. suggestions and supply of the natural product. I would also like to thank Dr. Kevin Tan group for helping me to test the antimalarial activity of the lipopeptide. I am grateful to all past and present