基因治療為近年來所發展出最具潛力的醫療技術，可被定義為利用遺傳技術將基因送入病人體內以治療病人的醫療行為。基因治療有兩種系統，分別為病毒載體系統及非病毒載體系統兩種，病毒載體雖然有較好的轉染傳遞效果，但是其安全性及免疫性皆為重要考量。由於非病毒載體擁有較佳的安全性且在製備上較容易，所以目前被廣為開發研究。
本研究選用對基因傳遞可提升效果之輔助性脂質磷脂基乙醇胺(DOPE) 與組胺酸 (Histidine) 進行化學接枝，其進行接枝的方法是先採用固相胜肽合成法，將經Fmoc保護基保護之組胺酸(Fmoc-His(Trt)-OH) 接合至固相載體 (2-Chlorotrityl chloride resin) 上。同時，我們採用Succinic anhydride將DOPE之胺端改為羧端，形成一新化合物DOPE acid。我們採取相同的固相胜肽合成法，將DOPE acid接合上含組胺酸之固相載體；由於2-Chlorotrityl chloride resin可在酸性條件下被移除，因此採用1% 之Trifluoroacetic acid溶液將產物DOPE-Histidine從載體上切除，切除後使用高壓液相層析法 (HPLC) 來進行分離純化。
由HPLC分離純化後，取產物進行電灑游離法 (MS-ESI) 進行質量分析；由實驗結果可得知，Mass圖譜在分子量為980m/z位置有波峰出現，但其純度並不高。因此，我們得知由固相胜肽合成方法可取得我們所要的產物DOPE acid，只是其分離方法或許須朝向傳統有機合成的分離方法，如萃取或是管柱層析法，以期能使產物得到更高的純度。

Gene therapy has been developed rapidly in recent years because of its potential in medical treatments. It can be defined as the use of genetic technology to treat patients by gene delivery. Research in gene therapy has been focused on the development of suitable carriers. Gene therapy has two types of delivery systems, viral vectors and non-viral vectors. Although viral vectors result in better efficiency, but its safety and immunity might present safety concerns. Non-viral vectors have been regarded a safer approach and easier to be prepared.
In this study, we attempted to graft histidine onto 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) . We used Fmoc solid phase peptide synthesis to synthesize the peptide. We chose 2-chlorotrityl chloride resin for the grafting of the protected amino acid (Fmoc-His(Trt)-OH). On the other hand, we synthesized DOPE acid from DOPE by using succinic anhydride to change the amino group to carboxyl group of DOPE. Then we used the solid phase method to graft DOPE acid onto the peptidyl resin. Because the resin contained highly acid-sensitive linkers, we prepared a cleavage solution which contains 1% trifluoroacetic acid to cleavage DOPE-Histidine from peptidyl resin. The obtained product, DOPE-Histidine, was purified by high pressure liquid chromatography. The results of mass spectrometry showed successful synthesis of DOPE-Histidine, but with less desirable purity.
The results suggested that we might use different methods to purify DOPE-Histidine, such as extraction or column chromatography, in order to obtain better purity.

1.McTaggart, S. and M. Al-Rubeai, “Retroviral vectors for human gene delivery,” Biotechnology Advances, Vol. 20, No. 1, p.p. 1-31 (2002)
2.Godbey, W.T., K.K. Wu, and A.G. Mikos, “Poly(ethylenimine) and its role in gene delivery,” Journal of Controlled Release, Vol. 60, No. 2 p.p. 149-160 (1999)
3.Merrifield, R.B., “Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide,” Journal of the American Chemical Society, Vol. 85, No. 14, p.p. 2149-2154 (2002)
4.Nishiuchi, Y., et al., “Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence,” Proceedings of the National Academy of Science U S A, Vol. 95, No. 23, p.p. 13549-13554 (1998)
5.Mourtas, S., et al., Resin-bound aminothiols: synthesis and application. Tetrahedron Letters, Vol. 44, No. 1, p.p. 179-182 (2003)
6.Kirin, S.I., et al., “Manual solid-phase peptide synthesis of metallocene-peptide bioconjugates,” Journal of Chemical Education, Vol. 84, No. 1, p.p. 108-111 (2007)
7.Knorr, R., et al., “New coupling reagents in peptide chemistry,” Tetrahedron Letters, Vol. 30, No. 15, p.p. 1927-1930 (1989)
8.Canne, L.E., et al., “Chemical Protein Synthesis by Solid Phase Ligation of Unprotected Peptide Segments,” Journal of the American Chemical Society, Vol. 121,No. 38: p.p. 8720-8727 (1999)
9.Mergler, M., et al., “Synthesis and application of Fmoc-His(3-Bum)-OH,” Journal of Peptide Science, Vol. 7, No. 9, p.p. 502-510 (2001)
10.Miranda, L.P. and P.F. Alewood, “Accelerated chemical synthesis of peptides and small proteins,” Proceedings of the National Academy of Science U S A, Vol. 96, No. 4, p.p. 1181-1186 (1999)
11.Tam, J.P., W.F. Heath, and R.B. Merrifield, “An SN2 deprotection of synthetic peptides with a low concentration of hydrofluoric acid in dimethyl sulfide: evidence and application in peptide synthesis,” Journal of the American Chemical Society, Vol. 105, No. 21, p.p. 6442- 6455 (2002)
12.Chiva, C., et al., “An HPLC-ESMS study on the solid-phase assembly of C-terminal proline peptides,” Journal of Peptide Science, Vol. 5, No. 3, p.p. 131-140 (1999)
13.胡育誠，「基因治療的過去與展望」，科學發展，372，42- 45頁，2003。
14.Simoes, S., et al., “Cationic liposomes as gene transfer vectors: Barriers to successful application in gene therapy,” Current Opinion in Molecular Therapeutics, Vol. 1, No. 2, p.p. 147-157 (1999)
15.Lee, H., J.H. Jeong, and T.G. Park, “PEG grafted polylysine with fusogenic peptide for gene delivery: high transfection efficiency with low cytotoxicity,” Journal of Controlled Release, Vol. 79, No. 1, p.p. 283-291(2002)
16.Engelhardt, J.F., et al., “Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver,” Proceedings of the National Academy of Science U S A, Vol. 91, No. 13, p.p. 6196-6200 (1994)
17.Lieber, A., et al., “Recombinant adenoviruses with large deletions generated by Cre-mediated excision exhibit different biological properties compared with first-generation vectors in vitro and in vivo,” The Journal of Virology, Vol. 70, No. 12, p.p. 8944-8960 (1996)
18.Weitzman, M.D., et al., “Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA,” Proceedings of the National Academy of Science U S A, Vol. 91, No. 13, p.p. 5808-5812 (1994)
19.Behr, J.P., “Gene transfer with synthetic cationic amphiphiles: prospects for gene therapy,” Bioconjugate Chemistry, Vol. 5, No. 5, p.p. 382-389 (1994)
20.Roth, C.M. and S. Sundaram, “Engineering synthetic vectors for improved DNA delivery: insights from intracellular pathways,” Annual Review of Biomedical Engineering, Vol. 6, p.p. 397-426 (2004)
21.De Smedt, S.C., J. Demeester, and W.E. Hennink, “Cationic polymer based gene delivery systems,” Pharmaceutical Research, Vol. 17, No. 2, p.p. 113-126 (2000)
22.Wu, G.Y. and C.H. Wu, “Receptor-mediated in vitro gene transformation by a soluble DNA carrier system,” Journal of Biologicl Chemistry, Vol. 262, No. 10, p.p. 4429-4432 (1987)
23.Bangham, A.D., M.M. Standish, and J.C. Watkins, “Diffusion of univalent ions across the lamellae of swollen phospholipids,” Journal of Molecular Biology, Vol. 13, No. 1, p.p. 238-252 (1965)
24.Pinto-Alphandary, H., A. Andremont, and P. Couvreur, “Targeted delivery of antibiotics using liposomes and nanoparticles: research and applications,” International Journal of Antimicrobial Agents, Vol. 13, No. 3, p.p. 155-168 (2000)
25.Weiner, N., F. Martin, and M. Riaz, “Liposomes as a Drug Delivery System,”Drug Development and Industrial Pharmacy, Vol. 15, No. 10, p.p. 1523-1554 (1989)
26.R.R.C.New, Liposome: a practical approach Oxford University Press. p.p. 221-225 (2000)
27.Gao, X. and L. Huang, “Cationic Liposomes and Polymers for Gene Transfer,” Journal of Liposome Research, Vol. 3, No. 1, p.p. 17-30 (1993)
28.El-Aneed, A., “An overview of current delivery systems in cancer gene therapy,” Journal of Controlled Release, Vol. 94, No. 1, p.p. 1-14 (2004)
29.Ewert, K., et al., “Efficient synthesis and cell-transfection properties of a new multivalent cationic lipid for nonviral gene delivery,” Journal of Medical Chemistry, Vol. 45, No. 23, p.p. 5023-5029 (2002)
30.Godbey, W.T., K.K. Wu, and A.G. Mikos, “Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 96, No. 9, p.p. 5177-5181 (1999)
31.Zuhorn, I.S., R. Kalicharan, and D. Hoekstra, “Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis,” Journal of Biological Chemistry., Vol.227, No. 20, p.p. 18021-18028 (2002)
32.Pedroso de Lima, M.C., et al., “Cationic lipid-DNA complexes in gene delivery: from biophysics to biological applications,” Advanced Drug Delivery Reviews, Vol. 47, No.2, p.p. 277-294 (2001)
33.May, S. and A. Ben-Shaul, “DNA-lipid complexes: stability of honeycomb-like and spaghetti-like structures,” Biophysical Journal, Vol. 73, No. 5, p.p. 2427-2440 (1997)
34.Dan, N., “The structure of DNA complexes with cationic liposomes-cylindrical or flat bilayers?,” Biochimica and Biophysica Acta, Vol. 1369, No. 1, p.p. 34-38 (1998)
35.Farhood, H., N. Serbina, and L. Huang, “The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer,” Biochimica and Biophysica Acta, Vol. 1235, No. 2, p.p. 289- 295 (1995)
36.Schmitt, L., C. Dietrich, and R. Tampe, “Synthesis and Characterization of Chelator-Lipids for Reversible Immobilization of Engineered Proteins at Self-Assembled Lipid Interfaces,” Journal of the American Chemical Society, Vol. 116, No. 19, p.p. 8485-8491 (2002)
37.Lee, C.L. and M.M. Sim, “Solid-phase combinatorial synthesis of 5-arylalkylidene rhodanine,” Tetrahedron Letters, .Vol.41, No. 30, p.p. 5729-5732 (2000)
38.Batra, S., et al., “A novel isoxazole-based scaffold for combinatorial chemistry,” Tetrahedron Letters, Vol. 41, No. 31, p.p. 5971-5974 (2000)
39.Nash, I.A., B.W. Bycroft, and W.C. Chan, “Dde -- A selective primary amine protecting group: A facile solid phase synthetic approach to polyamine conjugates,” Tetrahedron Letters, Vol. 37, No. 15, p.p. 2625-2628 (1996)
40.Palasek, S.A., Z.J. Cox, and J.M. Collins, “Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis,” Journal of Peptide Science, Vol. 13, No. 3, p.p. 143- 148 (2007)
41.Gelens, E., et al., “Solid-Phase synthesis of 4-substituted imidazoles using a scaffold approach,” Bioorganic & Medicinal Chemistry Letters, Vol. 10, No. 17, p.p. 1935-1938 (2000)
42.Eleftheriou, S., et al., “Attachment of histidine, histamine and urocanic acid to resins of the trityl-type,” Tetrahedron Letters, Vol. 40, No. 14, p.p. 2825-2828 (1999)
43.Di Gioia, M.L., et al., “Solid-Phase Synthesis of N-Nosyl- and N-Fmoc-N-Methyl-α-amino Acids,” The Journal of Organic Chemistry, Vol. 72, No. 10, p.p. 3723-3728 (2007)
44.Friedman, M. and L. David Williams, “Stoichiometry of formation of Ruhemann's purple in the ninhydrin reaction,” Bioorganic Chemistry, Vol. 3, No. 3, p.p. 267-280 (1974)
45.Carpino, L.A. and G.Y. Han, “9-Fluorenylmethoxycarbonyl amino-protecting group. The Journal of Organic Chemistry, 2002. 37(22): p.p. 3404-3409.
46.Barlos, K., et al.,“Darstellung geschützter peptid-fragmente unter einsatz substituierter triphenylmethyl-harze,“ Tetrahedron Letters, Vol. 30, No. 30, p.p. 3943-3946 (1989)