硼中子捕獲治療(Boron neutron capture therapy BNCT)是一種用於局部侵入性惡性腫瘤的有效試劑，其中Sodium borocaptate (BSH)鈉硼硫醇金屬簇化物，是一個有效的藥物，但是其中卻包含部分的缺點，包括無法有效進入癌細胞之中，因此在此論文，我們選擇Folic acid葉酸與Biotin生物素維生素H做為引金屬試劑，試圖克服BSH本身的缺點，並期望應用在BNCT的治療上。
    在本研究之中，我們合成出BSH非放射性20% 10B之試劑，並利用1-(2-Aminoethyl) maleimide與葉酸和生物素偶合，試圖利用其對硫醇的選擇性做為前驅藥物，利將BSH成功帶入細胞之中，而不需使用100% 10B之試劑。目前已能成功合成此二項前驅化合物，並測試其螢光指示劑之程度，同時也利用U2OS人骨肉瘤細胞測試其生物毒性，皆得到合理的成果，未來將應用在BNCT之中，使其有選擇性而可以避免100% 10B之鈉硼硫醇金屬簇化物。

Boron neutron capture therapy (BNCT) is a potential approach to treat cancer and Sodium borocaptate (BSH) has been used for BNCT application as a boron delivery agent. Even though, BSH were used in clinical trials, this agent till exhibits some disadvantage in the case of selective targeting of cancer cells and cellular uptake ability. Folic acid and biotin were considered as an excellent targeting ligand which can help to overcome the disadvantage of BSH in BNCT application. In this study, we prepared the natural sodium borocaptate (BSH) from natural abounded starting materials and we also successfully conjugated this potential BNCT agent with folic acid/biotin using 1-(2-Aminoethyl)maleimide as the short crosslinker. 
Folic acid/biotin-conjugated BSH exhibited the potential boron delivery agent which decreased the cytotoxicity of BSH treatment effects on U2OS cell line. Interestingly, folate-conjugated products which showed two maleimide groups and it also exhibited the high fluorescence emission. Of these, this complex might provide a new strategy to enhance and also detect the boron accumulation. These observations offered two of new BSH delivery platforms which were described as a small molecule weight and highly targeting of cancer cells which could be used as starting material in further investigation.

Contents
Acknowledgment	I
Abstract	III
Abbreviation	V
Contents	VIII
List of Figures	XI
List of Tables	XV
List of Schemes	XVI
Chapter 1. Introduction	1
1.1. Boron Neutron Capture Therapy (BNCT)	1
1.2. Boron Delivery Agents	2
1.2.1. Boron	2
1.2.2. L- Boronophenylalanine (BPA)	5
1.2.3. Sodium borocaptate (BSH)	6
1.2.4. Improve BSH Uptake in Cancer Cells	7
1.3. Ligands and biomimetic strategies for targeted	11
1.3.1. Folate and folate receptor	11
1.3.2. Folate-based delivery application in BNCT	14
1.3.3. The blood-brain barrier (BBB) and the role of folate-based carrier	16
1.3.4. Biotin and biotin-specific uptake systems (B-SUS)	17
Chapter 2. Technical Approach	19
2.1. Preparation of compounds	19
2.1.1. Synthesis of [NEt3H]2[B12H12]	19
2.1.2. Synthesis of Na2[B12H12]	20
2.1.3. Synthesis of Sodium borocaptate Na2[B12H11SH]	20
2.1.4. Synthesis of Folic acid and Biotin conjugated BSH via Maleoylimide substituted	22
2.2. X-Ray crystallography	24
2.3. Bioactivity assessment	25
Chapter 3. Results and Discussions	27
3.1. Synthesis of BNCT agent: Sodium borocaptate (BSH)	27
3.1.1. Preparation of Na2[B12H12] from Na[BH4] and I2	27
3.1.2. Preparation Na2B12H11SH (BSH) from Na2[B12H12] and N-Methylbenzothiazole-2-thione	31
3.2. Synthesis of folate/biotin-conjugated BSH via carbonyl-reactive and sulfhydryl-reactive crosslinkers	37
3.2.1. Preparation of crosslinker, 1-(2-Aminoethyl)maleimide hydrochloride (C6H8N2O2.HCl)	37
3.2.2. Synthesis of folic acid-conjugated 1-(2-Aminoethyl)maleimide via carbonyl-reactive group	41
3.2.3. Synthesis of biotin-conjugated 1-(2-Aminoethyl)maleimide via carbonyl-reactive group	45
3.2.4. Synthesis of folate/biotin-conjugated BSH via “click” chemistry and its chemical properties	48
3.3. Fluorescence properties of conjugated products	56
3.3.1. Folate-conjugated	56
3.3.2. Biotin-conjugated products	58
3.4. Cytotoxicity	60
3.4.1. Cytotoxicity of BSH on U2OS cells	61
3.4.2. Cytotoxicity of FA-MAL and FA-MAL-BSH on U2OS cells	62
3.4.2. Cytotoxicity of BIO-MAL and BIO-MAL-BSH on U2OS cells	64
Conclusion	67
References	68
Appendix 11B NMR and 1H NMR spectra	80

List of Figures 
Figure 1-1. Two clinical boron delivery agents for BNCT [1] 4 
Figure 1-2. Folic acid structure. Blue and Red represent for pteroate and glutamate moiety, respectively 12 
Figure 1-3. Structure of FRα bound to folic acid.  13 
Figure 1-4. Folic acid affinities of FRa ligand-binding-pocket mutants. 14 
Figure 1-5. The structure of Biotin.17 
Figure 3-1. Crystal structure of Na2[B12H12].6[THF] and Na2[B12H12].4[THF]. Thermal ellipsoids are shown at the 50% probability level 29 
Figure 3-2. 11B NMR and 1H NMR of Na2B12H12 and Na2B12H11SH in comparison 33 
Figure 3-3. Crystal structure of Na2[B12H11SH] in packing complex with 18-crown-6-ether [C12H24O6], THF [C4H8O], and DMSO [C2H6OS]. Thermal ellipsoids are shown at the 50% probability level 36 
Figure 3-4. 1H NMR spectra of 1-(2-Aminoethyl)maleimide (D2O), 300 MHz : 3.25-3.28 [2H, t, CH2], 3.85-3.89 [2H, t, CH2], 6.94 [2H, s, maleimide] 39 
Figure 3-5. The crystal structure of 1-(2-Aminoethyl)maleimide hydrochloride 40 
Figure 3-6. 1H NMR spectrum in the comparison between FA-conjugated product and Folic acid. 300Hz. D2O 43 
Figure 3-7. ESI spectrum of FA-MAL with the main peak at 708.47 m/z which represents for [M+Na]+ ion and another at 686.27 which represents for [M+H]+ 44 

Figure 3-8. 1H NMR spectrum in the comparison between Biotin-conjugated product and Biotin. 300Hz. D2O  47 
Figure 3-9. ESI spectrum of BIO-MAL with the main peak at 389 m/z which represents for [M+Na]+ ion  48 
Figure 3-10. 1H NMR in the comparison between folic acid/biotin-conjugated compounds 50 
Figure 3-11. 11B NMR chemical shifts in the comparison between FA-MAL-BSH, BIO-MAL-BSH, and BSH. 600 Hz 51 
Figure 3-12. ESI spectrum of BIO-MAL-BSH 52 
Figure 3-13. ESI spectra of FA-MAL-BSH 52 
Figure 3-14. Two parallel reactions of a succinimidyl thioether in solution [68] 53 
Figure 3-15. Excitation and Emission spectra in the comparison between Folic acid with its conjugated products at 0.1 μM concentration in NaHCO3 and PBS solution. 57 
Figure 3-16. Excitation and Emission spectra in the comparison between FA-MAL and FA-MAL adding BSH (the ratio is 1:1 molar). All compound was prepared at 0.1 μM concentration in NaHCO3 and PBS solution. 58 
Figure 3-17. Excitation and Emission spectra of Biotin-conjugated products at 1 μM concentration which was prepared in DMSO  59 
Figure 3-18. Excitation and emission spectra of Biotin-conjugated products at 1 μM concentration which was prepared in PBS solution 60 
Figure 3-19. Effects of BSH with varying concentrations on the viability of U2OS cells after 24h. The results are the mean SE of 3 separate measurements. 61

Figure 3-20. Effects of FA-MAL with varying concentrations on the viability of U2OS cells after 24h. The results are the mean SE of 3 separate measurements. 62 
Figure 3-21. Effects of FA-MAL-BSH with varying concentrations on the viability of U2OS cells after 24h. The results are the mean SE of 3 separate measurements.63 
Figure 3-22. Effects of BIO-MAL with varying concentrations on the viability of U2OS cells after 24h. The results are the mean SE of 3 separate measurements.  64 
Figure 3-23. Effects of BIO-MAL-BSH with varying concentrations on the viability of U2OS cells after 24h. The results are the mean SE of 3 separate measurements.  65 
Figure 0-1. 11B NMR spectrum of Na2[B12H12] in D2O solvent, 600 MHz 80 
Figure 0-2. 11B NMR spectrum of Na2[B12H11SH] in D2O solvent, 600 MHz 81 
Figure 0-3. 11B NMR spectrum of FA-MAL-BSH in DMSO d6 solvent, 600 MHz  82 
Figure 0-4. 11B NMR spectrum of BIO-MAL-BSH in DMSO d6 solvent, 600 MHz  83 
Figure 0-5. 1H NMR spectrum of Na2[B12H12] in D2O solvent, 300 MHz  84
Figure 0-6. 1H NMR spectrum of Na2[B12H11SH] in D2O solvent, 300 MHz 85
Figure 0-7. 1H NMR spectrum of FA-MAL-BSH in DMSO d6 solvent, 600 MHz  86 
Figure 0-8. 1H NMR spectrum of BIO-MAL-BSH in DMSO d6 solvent, 600 MHz  87

Figure 0-9. 1H NMR spectrum of Boc-Ethylenediamine in CDCl3 solvent, 300 MHz88
Figure 0-10. 1H NMR spectrum of N-Boc-2-Maleimidoethylamine in CDCl3 solvent, 300 MHz  89
Figure 0-11. 1H NMR spectrum of 1-(2-Aminoethyl)- maleimide hydrochloride in CD3OD d4 solvent, 300 MHz  90
Figure 0-12. 1H NMR spectrum of Folic acid in DMSO d6 solvent, 300 MHz . 91
Figure 0-13. 1H NMR spectrum of FA-MAL in DMSO d6 solvent, 300 MHz . 92 
Figure 0-14. 1H NMR spectrum of FA-MAL in DMSO d6 solvent, 600 MHz . 93 
Figure 0-15. 1H NMR spectrum of Biotin in DMSO d6 solvent, 300 MHz 94
Figure 0-16. 1H NMR spectrum of BIO-MAL in DMSO d6 solvent, 300 MHz 95
Figure 0-17. 1H NMR spectrum of BIO-MAL in DMSO d6 solvent, 600 MHz 96

List of Tables 
Table 1-1. Boron isotopes [8] 3 
Table 3-1. 11B NMR and 1H NMR data of Na2B12H12 in D2O, 600 MHz 28 
Table 3-2. Crystallographic parameters of Na2[B12H12] in two different packing complex with THF 30 
Table 3-3. Crystal structure data of Na2[B12H12] in the comparison between of two complexes  31 
Table 3-4. 11B NMR and 1H NMR data of Na2B12H11SH in D2O, 600 MHz 33 
Table 3-5. Crystallographic parameters of Na2[B12H11SH] in packing complex with 18-crown-6-ether [C12H24O6], THF [C4H8O], and DMSO [C2H6OS] 35 
Table 3-6. Crystal structure data of Na2[B12H11SH] in complex with 18-crown-6-ether [C12H24O6], THF [C4H8O], and DMSO [C2H6OS] 36 
Table 3-7. Crystal structure data of 1-(2-Aminoethyl)maleimide hydrochloride  40 
Table 3-8. Crystallographic parameters of 1-(2-Aminoethyl)maleimide hydrochloride 41 
Table 3-9. Comparison 11B NMR data between Na2B12H11SH, FA-MAL-BSH, BIO-MAL-BSH in D2O, DMSO-d6, 600 MHz 51 
Table 3-10. Some physical and chemical properties of BSH and Folic acid/Biotin-conjugated compounds  54 
Table 3-11. Comparison IC50 of Folic acid-conjugated compounds in effect on U2OS cells viability 63 
Table 3-12. Comparison IC50 of Biotin-conjugated compounds in effect on U2OS cells viability 65 

List of Schemes 
Scheme 3-1. The N-methyl benzothiazole-2-thione routes to synthesize BSH. [54]  32 
Scheme 3-2. Maleimide thiols reaction [64]37 
Scheme 3-3. Synthesis of 1-(2-Aminoethyl)maleimide hydrochloride [58] [59] [60] [61] 38 
Scheme 3-4. Synthesis of folic acid-conjugated 1-(2-Aminoethyl)maleimide via carbonyl-reactive group 43 
Scheme 3-5. Synthesis of Biotin-conjugated 1-(2-Aminoethyl)maleimide using DMSO as the solvent, DMAP and TEA as an organic base, NHS and DCC as the additive reagent in activated step 46 
Scheme 3-6. Synthesis of Biotin-conjugated 1-(2-Aminoethyl)maleimide using DMF as the solvent and NHS, DCC as the additive reagent in activated step 46 
Scheme 3-7. Preparation of Folate-conjugated BSH via “click” chemistry 49 
Scheme 3-8. Preparation of Biotin-conjugated BSH via “click” chemistry 49

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