Title エチニルベンゼン置換型グリコールを導入した核酸オリゴマーの合成とその性質( 本文(Fulltext) )
Author(s) REN, QIN
Report No.(Doctoral Degree) 博士(薬科学) 連創博甲第26号 Issue Date 2014-09-30 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/50375 ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。
Synthesis and properties of nucleic acid oligomers
containing the ethynylbenzene-substituted glycol
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⌮ㄽ䛾㒊
➨୍❶㻌 ᗎㄽ ➨୍⠇㻌 ⸆䝥䝻䝉䝇 ... 1 ➨⠇㻌 䝬䜲䜽䝻䝗䞊䝈⮫ᗋヨ㦂 ... 3 ➨୕⠇㻌 㝧㟁Ꮚᨺฟ᩿ᒙ⏬ീᙳἲ䠄PET䠅 ... 6 ➨ᅄ⠇㻌 ᰾㓟་⸆... 10 ➨⠇㻌 䜽䝸䝑䜽ᛂ䜢⏝䛧䛯᰾㓟䜸䝸䝂䝬䞊䛾PET 䝷䝧䝹 ... 14 ➨❶㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺ䜾䝸䝁䞊䝹䛾ྜᡂཬ䜃᰾㓟䜸䝸䝂䝬䞊ᑟධ⏝ 䝴䝙䝑䝖䛾ྜᡂ ➨୍⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺ䜾䝸䝁䞊䝹ME䛾ศᏊタィ ... 19 ➨⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺ䜾䝸䝁䞊䝹ME䛾ྜᡂ ... 21 ➨୕⠇㻌 ᰾㓟䜸䝸䝂䝬䞊ᑟධ⏝䝴䝙䝑䝖䛾ྜᡂ ... 24 ➨ᅄ⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䛾 ε ್䛾⟬ฟ ... 25 ➨୕❶㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛᰾DNA 䛾ྜᡂ䛸 䛭䛾ᛶ㉁ ➨୍⠇㻌 ᰾㓟⮬ືྜᡂᶵ䛾୍⯡ⓗ䛺ྜᡂ䝃䜲䜽䝹 ... 26 ➨⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛DNA 䛾ྜᡂ ... 27➨୕⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛 ୍ᮏ㙐DNA 䛷䛾䜽䝸䝑䜽ᛂ ... 29 ➨ᅄ⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛 ᮏ㙐DNA 䛾⇕ⓗᏳᐃᛶ ... 31 ➨⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛 ᮏ㙐DNA 䛷䛾䜽䝸䝑䜽ᛂ ... 32 ➨ ➨ᅄ❶㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛᰾RNA 䛾ྜᡂ䛸 䛭䛾ᛶ㉁ ➨୍⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛RNA 䛾ྜᡂ ... 34 ➨⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛 ୍ᮏ㙐RNA 䛷䛾䜽䝸䝑䜽ᛂ ... 36 ➨୕⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛 ᮏ㙐RNA 䛾⇕ⓗᏳᐃᛶ ... 38 ➨ᅄ⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛 ᮏ㙐RNA 䛷䛾䜽䝸䝑䜽ᛂ ➨୍㡯㻌 ᮏ㙐RNA 䛷䛾䜽䝸䝑䜽ᛂ ... 40 ➨㡯㻌 䝬䜾䝛䝅䜴䝮䜲䜸䞁ῧຍ䛻䜘䜛ຠᯝ ... 41 ➨❶㻌 ⤖ㄽ ... 43
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1. ⏝ᶵჾཬ䜃⏝ヨ⸆ 1-1. ᐃᶵჾ ... 46 1-2. ྛ✀䜽䝻䝬䝖䜾䝷䝣䜱䞊ᢸయ ... 47 1-3. ྛ✀䝣䜱䝹䝍䞊 ... 47 1-4. ⏝ヨ⸆ ... 47 2. ᐇ㦂䛾ヲ⣽ 2-1. ➨❶➨⠇䛻㛵䛩䜛ᐇ㦂 ... 51 2-2. ➨❶➨୕⠇䛻㛵䛩䜛ᐇ㦂 ... 55 2-3. ➨❶➨ᅄ⠇䛻㛵䛩䜛ᐇ㦂 ... 57 2-4. ➨୕❶➨⠇䚸➨ᅄ❶➨୍⠇䛻㛵䛩䜛ᐇ㦂 ... 58 2-5. ➨୕❶➨୕⠇䚸➨ᅄ❶➨⠇䛻㛵䛩䜛ᐇ㦂 ... 62 2-6. ➨୕❶➨ᅄ⠇䚸➨ᅄ❶➨୕⠇䛻㛵䛩䜛ᐇ㦂 ... 63 2-7. ➨୕❶➨⠇䚸➨ᅄ❶➨ᅄ⠇䛻㛵䛩䜛ᐇ㦂 ... 64 ␎ㄒ㞟 ... 65 ᘬ⏝ᩥ⊩ ... 68 ㅰ㎡ ... 731
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➨୍❶㻌 ᗎㄽ
➨୍⠇㻌 ⸆䝥䝻䝉䝇 ᪂⸆䛾㛤Ⓨ䛻䛿䚸୍⯡䛻⣙1000 ൨䛾㈝⏝䛸 10 ᖺ௨ୖ䛾ᮇ㛫䛜ᚲせ䛸ゝ䜟䜜䛶 䛔䜛䚹᪂⸆ೃ⿵ྜ≀䛾᥈⣴䛻ጞ䜎䜚䚸ᐇ㦂ື≀䜢䛳䛯๓⮫ᗋヨ㦂䚸䝠䝖䛻ᑐ䛧䛶 ⾜䛖⮫ᗋヨ㦂䜢⤒䛶ᢎㄆ䚸㈍䛥䜜䜛䠄Fig. 1-1䠅䚹䛭䛾୰䛷䜒⮫ᗋヨ㦂䛿䚸Ᏻᛶ䜢 ☜ㄆ䛩䜛䛯䜑䛾➨୍┦⮫ᗋヨ㦂䚸⸆ຠ⏝㔞䜢Ỵ䜑䜛䛯䜑䛾➨┦⮫ᗋヨ㦂䚸⸆ຠ䜢 ホ౯䛩䜛䛯䜑䛾➨୕┦⮫ᗋヨ㦂䛛䜙ᵓᡂ䛥䜜䚸3 ᖺ䛛䜙 7 ᖺ䛸㠀ᖖ䛻㛗䛔ᮇ㛫䜢せ䛧䚸 ᪂⸆㛤Ⓨ䛾㈝⏝䛾㒊ศ䜢༨䜑䜛䚹䛣䛾⮫ᗋヨ㦂䜢✺◚䛧䚸ୖᕷ䛥䜜䛶᪂⸆䛸䛺䜛ᡂ ຌ⋡䛿㠀ᖖ䛻ప䛔☜⋡䜢♧䛧䛶䛔䜛䚹1) Fig. 1-1. ⸆䝥䝻䝉䝇㻌 㻞㻜㻜㻜 ᖺ䛛䜙 㻞㻜㻝㻝 ᖺ䜎䛷䛾᪂⸆㛤Ⓨ䛾ᡂຌ⋡䠄⣼✚ᡂຌ⋡䠅䜢 㻠 ᖺ䝇䝟䞁䛷ẚ㍑䛧 䛶ぢ䜛䛸䚸䜋䛸䜣䛹䛾ೃ⿵ྜ≀䛜⮫ᗋヨ㦂䛷㐺ྜ䛸䛺䛳䛶䛔䜛䛣䛸䜔䛣䛣ᩘᖺ䚸ᡂ ຌ☜⋡䛜పୗ䛧䚸⸆䛾㞴䛧䛥䛜୍ẁ䛸ቑ䛩ഴྥ䛜䛒䜛䛣䛸䛜ศ䛛䜛䠄Fig. 1-2䠅䚹㻌2 Fig. 1-2. ᪂᪂⸆㛤Ⓨ䛾ᡂຌ⋡䠄⣼✚ᡂຌ⋡䠅 ೃ⿵ྜ≀䛜⮫ᗋヨ㦂䛷㐺᱁䛸䛺䜛䛺⌮⏤䛿䚸䐟ࣄࢺ࠾࠸࡚྾ࡀ༑ ศࠊ࠶ࡿ࠸ࡣ⾑୰ࡽࡢᾘኻࡀᴟࡵ࡚᪩࠸࡞ࡢዲࡲࡋࡃ࡞࠸ືែ≉ᛶࢆ♧ࡍ ࡇࠊղ⒪᭷ຠ࡞⃰ᗘࡢ⸆ࡀᶆⓗ㒊฿㐩ࡋ࡞࠸ࡓࡵࠊ᭷ຠᛶࡀಖド ฟ᮶࡞࠸ࡇࠊճᶆⓗ㒊௨እࡢṇᖖ⤌⧊㐣⛣⾜ࡋࠊ᭷ᐖ࡞ᛂ㸦ẘᛶ㸧 ࢆᘬࡁ㉳ࡇࡍࡇ࡞ࡀᣲࡆࡽࢀࡲࡍࠋࡇࢀࡽࡣ࠸ࢃࡺࡿ⸆≀ືែ㛵ಀࡍࡿ ၥ㢟࡛࠶ࡿࠋ➨Ϩ┦⮫ᗋヨ㦂䛻ධ䛳䛯ೃ⿵ྜ≀䛾䛖䛱䚸ᐇ㝿䛻ᕷ㈍䛥䜜䜛ྜ≀ 䛿 11䠂⛬ᗘ䛷䛒䜛䛸ゝ䜟䜜䛶䛔䜛䡝2)㻌 ་⸆ရ㛤Ⓨ䛾ᡂຌ☜⋡䜢ୖ䛢䜛䛯䜑䛻䛿䚸᪩ 䛔ẁ㝵䛷䝠䝖䛷䛾⸆≀ືែ䜢ண 䛩䜛䛣䛸䛜ᚲせ䛷䛒䜚䚸䛭䛾䛯䜑䛾᭷ຊ䛺ᡭἲ䛸䛧䛶 ๓⮫ᗋヨ㦂䛾ẁ㝵䛷⾜䛖䝬䜲䜽䝻䝗䞊䝈⮫ᗋヨ㦂䠄MD ヨ㦂䠅䛜ὀ┠䛥䜜䛶䛔䜛䚹 ㏻ᖖ䛾⮫ᗋ㛤Ⓨ䛸䝬䜲䜽䝻䝗䞊䝈⮫ᗋヨ㦂䜢ά⏝䛧䛯⮫ᗋ㛤Ⓨ䜢ẚ㍑䛧䛶ぢ䜛䛸䚸 ㏻ᖖ䛾⮫ᗋ㛤Ⓨ䛷䛿䚸ೃ⿵ྜ≀䛾๓⮫ᗋヨ㦂䛷䛾ඃඛ㡰䛻ᚑ䛔⮫ᗋヨ㦂䜢⾜ 䛳䛶䛔䛟䚹ೃ⿵A䚸B䚸C 䛜⮫ᗋヨ㦂䛾ẁ㝵䛷㐺ྜ䛸䛺䜚䚸ೃ⿵ D 䛜⮫ᗋヨ㦂䜢✺◚ 䛧᭱⤊ⓗ䛻ᕷ㈍䛻⮳䛳䛯ሙྜ䛻䛿䚸ከ䛟䛾㛫䛸䝁䝇䝖䜢↓㥏䛻䛩䜛䛣䛸䛻䛺䜛䚹 ୍᪉䚸䝬䜲䜽䝻䝗䞊䝈⮫ᗋヨ㦂䜢ά⏝䛧䛯ሙྜ䚸ೃ⿵ྜ≀䛾๓⮫ᗋヨ㦂䛷䛾ඃ ඛ㡰䜢䚸䝬䜲䜽䝻䝗䞊䝈⮫ᗋヨ㦂䛾⤖ᯝ䛛䜙ኚ᭦䛧䚸᭱䜒ྍ⬟ᛶ䛾㧗䛔ೃ⿵ D 䛾䜏 ་⸆ရ⏘ᴗ䝡䝆䝵䞁 㻞㻜㻝㻟㻌
3 䛻⮫ᗋヨ㦂䜢⾜䛔䚸ᕷ㈍䛻⮳䜛䛣䛸䛜ྍ⬟䛷䛒䜛䚹䛣䛾䜘䛖䛺䜿䞊䝇䛾ሙྜ䛻䛿䚸ೃ⿵ ྜ≀ C䚸B䚸 A 䛻㛵䛧䛶䚸⮫ᗋヨ㦂䜢ᐇ䛩䜛ᚲせ䛜䛺䛔䚹䛩䛺䜟䛱䚸䝬䜲䜽䝻䝗䞊 䝈⮫ᗋヨ㦂䛿䚸་⸆ရ䛾㛤Ⓨ䝁䝇䝖䜢ୗ䛢䚸㛤Ⓨᮇ㛫䜢▷⦰䛩䜛䛣䛸䛜䛷䛝䜛䠄Fig. 1-3䠅䚹 Fig. 1-3. ㏻㏻ᖖ䛾⮫ᗋ㛤Ⓨ䛸䝬䜲䜽䝻䝗䞊䝈⮫ᗋヨ㦂䜢ά⏝䛧䛯⮫ᗋ㛤Ⓨ䛾ẚ㍑㻌 㻌 㻌 ⛉Ꮫᢏ⾡ືྥ 㻞㻜㻝㻝 ᖺ 㻝 ᭶ྕ䛛䜙୍㒊ኚ䛘䛶ᘬ⏝㻌
4 ➨ ➨⠇㻌 䝬䜲䜽䝻䝗䞊䝈⮫ᗋヨ㦂 䝬䜲䜽䝻䝗䞊䝈⮫ᗋヨ㦂䛸䛿䚸䝠䝖䛻䛚䛔䛶⸆⌮స⏝䜢Ⓨ⌧䛩䜛䛸᥎ᐃ䛥䜜䜛ᢞ㔞 䛾1/100䜢㉸䛘䛺䛔⏝㔞ཪ䛿100μg 䛾䛔䛪䜜䛛ᑡ䛺䛔⏝㔞䛾⿕㦂≀㉁䜢䚸ᗣ䛺⿕ 㦂⪅䛻༢ᅇᢞ䛩䜛䛣䛸䛻䜘䜚⾜䜟䜜䜛⮫ᗋヨ㦂䛾䛣䛸䛷䛒䜛䡝3)㻌 䝬䜲䜽䝻䝗䞊䝈⮫ᗋ ヨ㦂ᐇ䛾┠ⓗ䛿䚸⿕㦂≀㉁䛾䝠䝖䛻䛚䛡䜛⸆≀ືែ䛻㛵䛩䜛ሗ䜢་⸆ရ䛾⮫ᗋ 㛤Ⓨ䛾ึᮇẁ㝵䛻ᚓ䜛䛣䛸䛻䛒䜛䚹ලయⓗ䛻䛿䚸⿕㦂≀㉁䛾྾䜔⾑୰ືែ䚸ἥ ≉ᛶ䚸䝠䝖䛻䛚䛡䜛௦ㅰ≀䝥䝻䝣䜯䜲䝹➼䜢᫂䜙䛛䛻䛩䜛䛣䛸䚸ศᏊ䜲䝯䞊䝆䞁䜾ᢏ⾡䜢 ⏝䛔䛶⿕㦂≀㉁䛾యෆ䛻䛚䛡䜛ᒁᅾ䛻㛵䛩䜛ሗ䜢ᚓ䜛䛣䛸䛺䛹䛷䛒䜛䚹䝬䜲䜽䝻䝗䞊 䝈⮫ᗋヨ㦂䛾ᴫᛕ䛜᭱ึ䛻2003ᖺ䛻Ḣᕞ་⸆ရᗇ䛻䜘䜚♧䛥䜜䛯䚹᪥ᮏ䛷䛿䚸2008 ᖺ䛻䝬䜲䜽䝻䝗䞊䝈⮫ᗋヨ㦂䛾ᣦ㔪䛜ཌ⏕ປാ┬䛻䜘䜚Ⓨ⾲䛥䜜䛯䚹 䝬䜲䜽䝻䝗䞊䝈⮫ᗋヨ㦂䛻䛚䛡䜛䛺⿕㦂≀㉁ ᐃἲ䛻䛿䚸௨ୗ䛻♧䛩୕䛴䛾᪉ ἲ䛜䛒䜛䚹 ϸ䠅14C➼䛾ᨺᑕᛶྠඖ⣲䛷ᶆ㆑䛧䛯⿕㦂≀㉁䜢⿕㦂⪅䛻ᢞ䛧䚸䛘䜀ຍ㏿
ჾ㉁㔞ศᯒィ䠄AMS:㻌 Accelerator Mass Spectrometry䠅䜢⏝䛔䛶⾑ᾮ୰䠄ཪ䛿ᒀ୰ⱝ 䛧䛟䛿⣅୰䠅䛾⃰ᗘ䜢 ᐃ䛧䚸⿕㦂≀㉁䛾ᮍኚయ䜔௦ㅰ≀䛾⸆≀ືែᏛⓗሗ䜢 ᚓ䜛䚹
Ϲ䠅ᨺᑕᛶྠඖ⣲䛷ᶆ㆑䛧䛺䛔⿕㦂≀㉁䜢⿕㦂⪅䛻ᢞ䛧䚸㧗ឤᗘ䛾ᾮయ䜽䝻 䝬䝖䜾䝷䝣㉁㔞ศᯒィ 䠄LC/MS/MS: Liquid Chromatograph / Mass Spectrometry/ Mass Spectrometry䠅➼䛻䜘䜚ᮍኚయ䜔௦ㅰ≀䛾⸆≀ືែᏛⓗሗ䜢ᚓ䜛䚹
Ϻ䠅⿕㦂≀㉁䜢11C䚸13N䚸15O䚸18F➼䛾䝫䝆䝖䝻䞁ᨺฟ᰾✀䛷ᶆ㆑䛧䚸㝧㟁Ꮚᨺᑕ᩿
ᒙᙳἲ䠄PET:㻌 Positron Emission Tomography䠅4) 䜢⏝䛔䛶䚸⿕㦂≀㉁䛾⮚ჾ䞉⤌⧊
䛷䛾ศᕸ⏬ീ䜢⤒ⓗ䛻 ᐃ䛩䜛䚹ཪ䛿⿕㦂≀㉁䜢123I䚸99mTc䚸111In➼䛷ᶆ㆑䛧䚸
5 䝬䜲䜽䝻䝗䞊䝈⮫ᗋヨ㦂䛾୰䛷䜒䚸AMS䜔LC/MS/MS䜢⏝䛧䛯 ᐃἲ䛿䚸⿕㦂 ྜ≀䜢ᢞ䛧䛶ᅇ䛩䜛䜎䛷㛫䜔⃰ᗘ䛺䛹䛿 ᐃ䛷䛝䜛䛜䚸䛭䛾ྜ≀䛜䛹䛾䜘䛖 䛺⤒㊰䜢䛯䛹䛳䛶䛝䛯䛛䛿䜟䛛䜙䛺䛔䚹୍᪉䚸PET䜢⏝䛧䛯 ᐃἲ䛿䚸⿕㦂ྜ≀ 䜢䝫䝆䝖䝻䞁ᨺฟ᰾✀䛷ᶆ㆑ྜᡂ䛧䚸ᨺᑕ⸆䛸䛧䛶⏝䛔䜛䛣䛸䛻䜘䜚䚸⿕㦂ྜ≀䛾 యෆ䛾ศᕸ䜔⃰ᗘ䛜⤒㛫ⓗ䛻⏬ീ䝕䞊䝍䛸䛧䛶᳨ฟ䛥䜜䜛䚹䛥䜙䛻䚸⒪ຠᯝ䜔 స⏝䛻䛝䛟㛵䛩䜛ཷᐜయ䛾༨᭷⋡䠄ྜ≀䛜≉ᐃ䛾ཷᐜయ䛻䛹䛾⛬ᗘ⤖ྜ䛧䛶 䛔䜛䛛䠅䛺䛹䜒ィ ྍ⬟䛷䛒䜛䛣䛸䛛䜙䚸PET䛿䝬䜲䜽䝻䝗䞊䝈⮫ᗋヨ㦂䛻ྍḞ䛺ᢏ ⾡䛷䛒䜛䚹
6 ➨ ➨୕⠇㻌 㝧㟁Ꮚᨺฟ᩿ᒙ⏬ീᙳἲ䠄PET䠅 㻌 㝧㟁Ꮚᨺฟ᩿ᒙ⏬ീᙳἲ䠄PET䠅䛸䛿䚸㝧㟁Ꮚ᳨ฟ䜢⏝䛧䛯䝁䞁䝢䝳䞊䝍᩿ᒙ ᙳᢏ⾡䛷䛒䜛䚹CT 䜔 MRI 䛜䛻⤌⧊䛾ᙧែ䜢ほᐹ䛩䜛䛯䜑䛾᳨ᰝἲ䛷䛒䜛䛾䛻 ᑐ䛧䚸PET 䛿 SPECT 䛺䛹䛾᰾་Ꮫ᳨ᰝ䛸ྠᵝ䛻䚸⏕య䛾ᶵ⬟䜢ほᐹ䛩䜛䛣䛸䛻≉ 䛧䛯᳨ᰝἲ䛷䛒䜛䚹䛻୰ᯡ⚄⤒⣔䛾௦ㅰ䝺䝧䝹䜢ほᐹ䛩䜛䛾䛻⏝䛔䜙䜜䛶䛝䛯 䛜䚸㏆ᖺ䚸⭘⒆⤌⧊䛻䛚䛡䜛⢾௦ㅰ䝺䝧䝹䛾ୖ᪼䜢᳨ฟ䛩䜛䛣䛸䛻䜘䜚⒴䛾デ᩿䛻 ⏝䛥䜜䜛䜘䛖䛻䛺䛳䛯䚹䜎䛯䚸་⸆ရ㛤Ⓨ䛻䜒ᛂ⏝䛥䜜䚸๓⮫ᗋヨ㦂䛾ẁ㝵䛷䝬䜲䜽䝻 䝗䞊䝈⮫ᗋヨ㦂䛻⏝䛔䜙䜜䛶䛔䜛䚹 PET 䜲䝯䞊䝆䞁䜾䛾ཎ⌮ 䝍䞊䝀䝑䝖䛸䛺䜛⸆䜢11C 䜔18F 䛺䛹䛾㝧㟁Ꮚᨺฟ᰾✀䛷ᶆ㆑䛧యෆ䛻ᢞ䛩䜛䚹 యෆ䛷⸆୰䛾㝧㟁Ꮚᨺฟ᰾✀䛜β ᔂቯ䛧䛶 1 ಶ䛾㝧㟁Ꮚ䜢ᨺฟ䛩䜛䚹ᨺฟ䛥䜜䛯 㝧㟁Ꮚ䛿䚸㏆ഐ䛻Ꮡᅾ䛩䜛㟁Ꮚ䛸ᑐᾘ⁛䛧䚸㟁Ꮚ䛾㟼Ṇ㉁㔞䛻➼䛧䛔䜶䝛䝹䜼䞊 (511 keV) 䛾 γ ⥺䛜䚸2 ಶᨺฟ䛥䜜䜛 䠄ᾘ⁛ᨺᑕ⥺䠅䚹䛣䜜䜙䛾ගᏊ䛿䚸ඖ䛾㟁Ꮚ䛸㝧 㟁Ꮚ䛾㐠ື㔞䜢ಖᏑ䛩䜛䛯䜑䛻䚸ṇᑐ䛾㐠ື㔞䜢ᣢ䛱䚸ᑐ᪉ྥ䛻ᑐ䛷ᨺฟ䛥䜜 䜛䚹⿕᳨య䛾࿘ᅖ䜢ྲྀ䜚ᕳ䛟䜘䛖䛻㓄ิ䛥䜜䛯 PET 䜹䝯䝷 (γ ⥺᳨ฟჾ)䛾䛔䛪䜜䛛 2 䛴䛜ྠ䛻γ ⥺䜢᳨ฟ䛧䛯䛸䛝䛻䚸䛭䛾 2 䛴䛾᳨ฟჾ䜢⤖䜆┤⥺ୖ䛾䛹䛣䛛䛷ᑐᾘ⁛ 䛜㉳䛝䛯䛸⪃䛘䜙䜜䜛䚹䛣䜜䜙䛾ሗ䜢㞟䜑䛶 CT 䛸ྠᵝ䛻䝁䞁䝢䝳䞊䝍⏬ീฎ⌮䜢 䛩䛣䛸䛻䜘䜚䚸⸆䛾ศᕸ䜢♧䛩⏬ീ䜢సᡂ䛩䜛(Fig. 1-4)䚹
7 Fig. 1-4.㻌㻌 PET 䛾ཎ⌮ PET 䛿䚸⿕䜀䛟䛜ᑡ䛺䛟⏕య䜈䛾㈇ᢸ䛜ᑡ䛺䛔䛣䛸䜔䚸䝍䞊䝀䝑䝖䜢㧗ឤᗘ䛛䛴ᐃ㔞 ⓗ䛻ほᐹ䛷䛝䜛䛸䛔䛖Ⅼ䛜䛒䜛䚹䛧䛛䛧䚸㝧㟁Ꮚᨺฟ᰾✀䛾ᑑ䛜▷䛔䛣䛸䛛䜙䚸⸆ 䛾PET ᶆ㆑䛿⢭〇䜒ྵ䜑䛶⣲᪩䛟⾜䛖ᚲせ䛜䛒䜛䚹 ⌧ᅾ䚸⮫ᗋ䛷୍⯡ⓗ䛻⏝䛥䜜䛶䛔䜛PET デ᩿⏝㝧㟁Ꮚᨺฟ᰾✀䛻䛿䚸䛻11C䚸 13N䚸15O䚸18F 䛾 4 ✀㢮䛜䛒䜛(Table 1-1)䚹䛣䜜䜙䛾㝧㟁Ꮚᨺฟ᰾✀䛿༙ῶᮇ䛜㠀ᖖ 䛻▷䛔䛣䛸䛛䜙䚸䝅䞁䜾䝹䝣䜷䝖䞁ᨺฟ᰾✀䛷ᶆ㆑䛥䜜䛯ᨺᑕᛶ་⸆ရ䛾䜘䛖䛻ၟᴗⓗ 䛻౪⤥䛩䜛䛣䛸䛿ᅔ㞴䛷䛒䜛䚹18F 䛷ᶆ㆑䛥䜜䛯 ⸆䛾ሙྜ䚸༙ῶᮇ䛜䜔䜔㛗䛔䛣䛸䛛䜙䚸ၟᴗ ⓗ䛺౪⤥䛜ྍ⬟䛷䛒䜚䚸PET ᶆ㆑䛻䜘䛟⏝䛔䜙 䜜䛶䛔䜛䚹 Table 1-1. 䝫䝆䝖䝻䞁ᨺฟ᰾✀䛾༙ῶᮇ䛸〇㐀ἲ ᅗ䜢୍㒊ᨵኚ䛧䛶ᘬ⏝ http://www.riken.go.jp/r-world/info/release/news/2008/feb/index.htm
8 18F ᶆᶆ㆑ἲ 18F ᶆ㆑ἲ䛻䛿௨ୗ䛻♧䛩୕䛴䛾᪉ἲ䛜䛒䜛䚹 ϸ) ồ᰾ヨ⸆([18F]FK 䛺䛹)䜢⏝䛔䛶䚸⮯⣲䚸䝇䝹䝩䞁㓟䜶䝇䝔䝹䚸䝖䝸䜰䝹䜻䝹䜰䞁䝰 䝙䜴䝮䛺䛹䜢┤᥋⨨䛧䛯18F-ᶆ㆑(Scheme 1-1) 5) Scheme 1-1. [18F]FK 䜢⏝䛔䛶⮯⣲䜢┤᥋⨨䛧䛯18F-ᶆ㆑
Ϲ)㻌 ồ㟁Ꮚヨ⸆([18F]FCIO3䚸[18F]F2䚸[18F]AcOF)䜢⏝䛔䛯18F-ᶆ㆑(Scheme 1-2) 6)
Scheme 1-2. [18F]FCIO3䜢⏝䛔䛯18F-ᶆ㆑ Ϻ) 㻌 18F ᶆ ㆑ ศ Ꮚ ᅋ ([18F]FBA䚸 [18F]SFB 䛺 䛹 䠅 䜢 䛧 䛯㛫 ᥋ ⓗ 䛺 18F-ᶆ ㆑ (Scheme 1-3) 7) Scheme 1-3. ᶆ㆑ศᏊᅋ [18F]FBA 䜢䛧䛯㛫᥋ⓗ䛺18F-ᶆ㆑ 䛣䜜䜙䛾ᶆ㆑ἲ䛿䚸ᛂຠ⋡䛜ᝏ䛔䚸䛒䜛䛔䛿ᇶ㉁㐺⏝⠊ᅖ䛜⊃䛟୍⯡ᛶ䛻Ḟ 䛡䜛䛺䛹䛾ၥ㢟Ⅼ䜢᭷䛧䛶䛔䜛䚹䛭䛣䛷᭱㏆䚸୍⯡ᛶ䛾㧗䛔㧗㏿ᛂ䛸䛧䛶䚸䜽䝸䝑䜽 ᛂ䛜ὀ┠䛥䜜䛶䛔䜛䚹
䜽䝸䝑䜽ᛂ䛾䜂䛸䛴䛸䛧䛶䚸copper-catalyzed azide–alkyne cycloaddition䠄CuAAC䠅
9 䛶ሗ࿌䛥䜜䛯䛜䚸1963 ᖺ䚸Huisgen 䜙 9)䛻䜘䛳䛶ヲ䛧䛟ㄪ䜉䜙䜜䚸䜰䝆䝗䛸䜰䝹䜻䞁䛸䜢 100Υ⛬ᗘ䛻䜎䛷ຍ⇕䛩䜛䛸䚸1,3-ᴟᏊ⎔ຍᛂ䛜㐍⾜䛧䚸⨨␗ᛶయ䛾ΰྜ ≀䛺䛜䜙䝖䝸䜰䝌䞊䝹⎔䛜ᙧᡂ䛥䜜䜛䛣䛸䛜ሗ࿌䛥䜜䚸Huisgen ຍ⎔ᛂ䛸䜀䜜 䛯䚹Huisgen ຍ⎔ᛂ䛿䚸༠ዌⓗ䛻ᛂ䛜㐍⾜䛩䜛䛯䜑䚸1,4-⨨య䛸 1,5- ⨨య䛾䝖䝸䜰䝌䞊䝹య䜢⏕ᡂ䛩䜛䚹䛣䛾ᛂ⮬య䛿䚸䝖䝸䜰䝌䞊䝹ㄏᑟయ䛾ྜᡂ䛻䛚 䛔䛶᭷⏝䛷䛒䜛䜒䛾䛾䚸ᐊ 䛷䛿㐍⾜䛧䛺䛔䛯䜑䚸⏕యศᏊ䛾Ꮫಟ㣭ἲ䛸䛧䛶⏝ 䛷䛝䛺䛛䛳䛯(Scheme 1-4)䚹䛭䜜䛜䚸2002 ᖺ䛻䛺䛳䛶䚸Sharpless 10) 䜙䛸Meldal 11) 䜙䛜 䛭䜜䛮䜜⊂❧䛻䚸㖡ሷ䛻䜘䛳䛶䜰䝆䝗䛸䜰䝹䜻䞁䛸䛾⎔ຍᛂ䛜ⴭ䛧䛟ຍ㏿䛥䜜䚸 ⨨㑅ᢥⓗ䛻 1,4-⨨య䛾䝖䝸䜰䝌䞊䝹య䜢⏕ᡂ䛩䜛䛣䛸䜢ぢฟ䛧䛯䛣䛸䛷䚸᪂䛯䛺 ศᏊ㐃⤖ἲ䛸䛧䛶⏝䛷䛝䜛䜘䛖䛻䛺䛳䛯䚹(Scheme 1-5)䚹 Scheme 1-4. ↓ゐ፹䛾䜰䝆䝗䛸䜰䝹䜻䞁䛾ຍ⎔ᛂ Scheme1-5. 㖡ゐ፹䛻䜘䜛ゐ፹䛾䜰䝆䝗䛸䜰䝹䜻䞁䛾ຍ⎔ᛂ 䛣䛾ᛂ䛾Ⅼ䛿䚸ϸ㧗⋡䛛䛴㧗䛔ᐁ⬟ᇶ㑅ᢥᛶ䛷㐍⾜䛩䜛䚸Ϲ⁐፹䜢㑅䜀 䛪䚸Ỉ୰䛷䜒ᛂ䛿㐍⾜䛩䜛䚸Ϻᛂ䛜ྍ㏫ⓗ䛷䛒䜛䚸ϻ㐺ษ䛺㖡(I)ゐ፹䛾ῧຍ 䛻䜘䛳䛶䚸ᮎ➃䜰䝹䜻䞁䛾ሙྜ䛻䛿100 ಸ㏆䛟ᛂ䛜ຍ㏿䛥䜜䜛䚹ᚑࡗ࡚ࠊࢡࣜࢵࢡ ᛂࡣࠊPET ᶆ㆑᭷ຠ࡛࠶ࡿ⪃࠼ࡽࢀ࡚࠸ࡿࠋࡇࢁ࡛ࠊ㏆ᖺࠊ᰾㓟་⸆ࡣࠊ ᢠయ་⸆ḟࡄḟୡ௦་⸆ရࡋ࡚ὀ┠ࡉࢀࡿࡘࢀ࡚ࠊࢡࣜࢵࢡᛂࢆ⏝࠸ ࡓ᰾㓟࢜ࣜࢦ࣐࣮ࡢPET ᶆ㆑ࡀ┒ࢇ◊✲ࡉࢀ࡚࠸ࡿࠋ
10 ➨ ➨ᅄ⠇㻌 ᰾㓟་⸆ ᰾㓟་⸆䛸䛿䚸᰾㓟 䠄DNA, RNA䠅䛭䛾䜒䛾䛜ᶵ⬟䜢ᣢ䛴་⸆ရ䛾⥲⛠䛷䛒䜛䚹㑇 ఏᏊ䜢Ⓨ⌧䛥䛫䛶స⏝䛥䛫䜛㑇ఏᏊ⒪⸆䛸䛿␗䛺䜚䚸㑇ఏᏊ䛾ᶵ⬟䜢ไᚚ䛩䜛䛣䛸 䛻䜘䛳䛶⏕య䛻స⏝䛩䜛䚹᰾㓟་⸆䛾✀㢮䛸䛧䛶䛿䚸䜰䞁䝏䝆䞊䞁᰾㓟䚸䜰䞁䝏䝉䞁䝇 ᰾㓟䚸䝕䝁䜲᰾㓟䚸siRNA䚸miRNA䚸䝸䝪䝄䜲䝮䚸䜰䝥䝍䝬䞊➼䛜ᣲ䛢䜙䜜䜛䠄Fig.㻌 1-5䠅䚹䜎䛷䛻ୖᕷ䛥䜜䛯᰾㓟་⸆䛿䚸Vitravene (䜰䞁䝏䝉䞁䝇᰾㓟)(᪤䛻㈍⤊)䚸 Kynamro 12) (䜰䞁䝏䝉䞁䝇᰾㓟)䛸 Macugen 13) (䜰䝥䝍䝬䞊) 䛾䜟䛪䛛3 ရ┠䛻㐣䛞䛪䚸 ᰾㓟་⸆䛾㛤Ⓨ䛿ᮍ䛰Ⓨᒎ㏵ୖ䛻䛒䜛䚹 Fig. 1-5. ᵝ䚻䛺᰾㓟་⸆ရ䛸䛭䛾స⏝Ⅼ㻌 䜰䞁䝏䝆䞊䞁᰾㓟䛿1 ᮏ㙐 DNA 䛷䛒䜚䚸ᮏ㙐 DNA 䛾ᵓ䛻⤖ྜ䛩䜛䛣䛸䛷䚸୕ ᮏ㙐᰾㓟䜢ᙧᡂ䛧䚸㌿㐣⛬䜢㜼ᐖ䛩䜛䚹䛭䛾⤖ྜᵝᘧ䛿 2 ㏻䜚䛒䜛䚹2 䛴䛸䜒䚸3 ᮏ ┠᰾㓟䛾ሷᇶ䛜䝥䝸䞁䞊䝢䝸䝭䝆䞁ሷᇶᑐ䛾䝥䝸䞁ሷᇶ䛸Ỉ⣲⤖ྜ䛩䜛䛣䛸䛷䚸ᖹ㠃ෆ 䛻3 䛴䛾ሷᇶ䛜㐃⥆䛧䛶୪䜆ᵓ㐀䜢䛸䜛䛣䛸䛻䜘䛳䛶୕㔜㙐ᙧᡂ䛜ྍ⬟䛸䛺䜛䚹14, 15)
11 䝕䝁䜲᰾㓟䛿䚸㌿ᅉᏊ䛾DNA ⤖ྜ㒊䛻┦⿵ⓗ䛺㓄ิ䜢᭷䛩䜛᰾㓟䜸䝸䝂䝬䞊 䛷䛒䜛䚹㌿ᅉᏊ䛻⤖ྜ䛩䜛䛣䛸䛻䜘䜚䚸䛭䛾㌿ᅉᏊ䛜ไᚚ䛧䛶䛔䜛㑇ఏᏊ䛾Ⓨ⌧ 䜢ᢚไ䛩䜛䚹16) ⾑୰䛷䛾ศゎ⪏ᛶ䜔⣽⬊⭷㏱㐣ᛶ䛜ప䛔䛯䜑䚸ᚑ᮶䛾ᮏ㙐ᆺᵓ 㐀䛛䜙䝸䝪䞁ᆺᵓ㐀䛻ᨵⰋ䛩䜛➼䛾ᕤኵ䛜㐍䜑䜙䜜䛶䛔䜛䚹 䜰䞁䝏䝉䞁䝇᰾㓟䛿䚸ᶆⓗ㑇ఏᏊ䛾 mRNA 䛻┦⿵ⓗ䛺㓄ิ䜢᭷䛩䜛୍ᮏ㙐 DNA 䜎䛯䛿 RNA 䛷䛒䜛䚹ᶆⓗ㑇ఏᏊ䛾 mRNA 䛻┦⿵ⓗ䛻⤖ྜ䛩䜛䛣䛸䛻䜘䜚䚸⩻ヂ䛾㜼 ᐖ䜔䚸RNase 䛻䜘䜛ศゎ䜢ಁ䛧䚸ᶆⓗ㑇ఏᏊ䛾Ⓨ⌧䜢ᢚไ䛩䜛䚹17)
siRNA (small interfering RNA) 䛿䚸RNA ᖸ΅䜢ᘬ䛝㉳䛣䛩▷䛔ᮏ㙐 RNA 䛷䛒䜛䚹
ᶆⓗ㑇ఏᏊ䛸┦⿵ⓗ䛺㓄ิ䜢᭷䛩䜛 dsRNA 䜔 shRNA ➼䛾㛗㙐ᮏ㙐 RNA 䛜䚸
Dicer 䛻䜘䛳䛶 siRNA 䛸䛧䛶ษ䜚ฟ䛥䜜䚸18, 19) 䝦䝸䜹䞊䝊䛾ാ䛝䛷୍ᮏ㙐䛻ゎ㞳䛧䚸
RISC䠄RNA-inducedsilencing complex䠅䛻ྲྀ䜚㎸䜏䚸RISC 」ྜయ䜢ᙧᡂ䛩䜛䚹䛣䛾 RISC 」ྜయ䛜ᶆⓗ mRNA 䛻⤖ྜ䛧䚸䝇䝷䜲䝃䞊άᛶ䛻䜘䛳䛶ᶆⓗ mRNA 䜢ษ᩿䛩 䜛䛣䛸䛻䜘䜚䚸ᶆⓗ㑇ఏᏊ䛾Ⓨ⌧䜢ᢚไ䛩䜛(Fig. 1-6)䚹RISC 䛾せ䛺ᵓᡂᅉᏊ䛿䚸 Argonaute 䝍䞁䝟䜽㉁20-22)㻌䛷䛒䜚䚸
N ᮎ➃ (NTD), PAZ, middle (MID), PIWI 䛾 4 䛴䛾
䝗䝯䜲䞁䛛䜙ᵓᡂ䛥䜜䜛䚹23) 䛣䜜䛿䚸ྂ⣽⳦ P.furiosus 䛾 Argonaute 䝍䞁䝟䜽㉁
(PfAGO) 䛾 X ⥺⤖ᬗᵓ㐀ゎᯒ䛻䜘䜚᫂䜙䛛䛻䛺䛳䛯䚹Argonaute 䝍䞁䝟䜽㉁䛜 RISC
䛾ษ᩿άᛶ䜢ᢸ䛳䛶䛔䜛䛸⪃䛘䜙䜜䛶䛚䜚䚸䜺䜲䝗㙐䛻┦⿵䛺䝍䞊䝀䝑䝖RNA 䛜ㄆ㆑䛥 䜜୰ኸ㒊䛷ゐ፹ⓗ䛻ษ᩿䛥䜜䚸㑇ఏᏊⓎ⌧䛜ᢚไ䛥䜜䜛䚹䝠䝖䛻䛚䛔䛶䛿䚸Argonaute 䝍䞁䝟䜽㉁䛾䝃䝤䝣䜯䝭䝸䞊 (AGO) 䛷䛒䜛 Ago2 䛾䜏䛜䝇䝷䜲䝃䞊άᛶ䜢ᣢ䛴䛸䛥䜜䛶 䛔䜛24, 25)䚹 miRNA(microRNA)䛿䚸18ࠥ25 ሷᇶ㛗䛾୍ᮏ㙐 RNA 䛷䛒䜛䚹᰾ෆ䛷䝀䝜䝮䛛䜙 ㌿䛥䜜䛯㛗䛔༢㙐RNA 䛜䚸≉␗ⓗ㓝⣲䠄RNA ྜᡂ㓝⣲ϩ䠅 26) 䛻䜘䛳䛶䝇䝔䝮䝹 䞊䝥ᵓ㐀䜢䜒䛴ᩘⓒࠥᩘ༓ሷᇶ㛗䛾 primary miRNA䠄pri-miRNA䠅䛻ษ䜚ฟ䛥䜜䜛䚹
12 䛺䜚䚸27, 28) Exportin–5 䛸䛔䛖᰾እ㍺㏦䝍䞁䝟䜽㉁䛻䜘䛳䛶᰾ෆ䛛䜙⣽⬊㉁䛻㍺㏦䛥䜜 䜛䚹29) 䛭䛾ᚋ䚸pre-miRNA 䛿 Dicer 䛸䛔䛖㓝⣲䛸⿵ᅉᏊ䠄TRBP䠅䛛䜙䛺䜛」ྜయ䛻䜘 䛳䛶୧➃䛜ษ᩿䛥䜜䚸22 ሷᇶᑐ⛬ᗘ䛾▷䛔ᮏ㙐 miRNA 䛻䛺䜛䚹ᮏ㙐 miRNA 䛿䛭䛾ᚋゎ㞳䛧䛶䚸䜺䜲䝗㙐䛸䜘䜀䜜䜛ഃ䛰䛡䛜㑅ᢥⓗ䛻ṧ䛥䜜䛶ᡂ⇍䛧䚸䜒䛖୍ᮏ 䠄䝟䝑䝉䞁䝆䝱䞊㙐䠅䛿ศゎ䛥䜜䜛䚹ᡂ䛧䛯 miRNA 䛿 RISC 䛻ྲྀ䜚㎸䜎䜜䚸ᶵ⬟ⓗ
miRNA-RISC 」ྜయ䠄miRISC䠅䛸䛺䜛䚹miRNA 䜢ྲྀ䜚㎸䜣䛰 miRISC 䛜䚸ᶆⓗ䛸䛩䜛 mRNA 䛻᥋㏆䛧䛶䚸3’㠀⩻ヂ㡿ᇦ䠄3’UTR䠅䛻䛒䜛┦⿵ⓗ㓄ิ㒊䛻 miRNA 䜢⤖
ྜ䛥䛫䚸㑇ఏᏊ⩻ヂ䜢୰᩿䛥䛫䜛䛛䚸䛭䛾㑇ఏᏊ䜢ศゎ䛧䛶Ⓨ⌧䜢㜼ᐖ䛩䜛䚹miRNA
䛜mRNA 䛾䛺┦⿵ⓗ㓄ิ㒊䛻⤖ྜ䛧䛯ሙྜ䚸siRNA 䛸ྠᵝ䛻䚸mRNA 䛿ศ
ゎ䛥䜜䜛䠄Fig. 1-6䠅䚹
Fig. 1-6. siRNA䚸䚸miRNA 䛻䜘䜛 RNA ᖸ΅ᶵᵓ
䝸䝪䝄䜲䝮䛿䚸ゐ፹䛸䛧䛶䛿䛯䜙䛟୍ᮏ㙐 RNA 䛷䛒䜚䚸RNA ⮬㌟䜢ษ᩿䛧䛯䜚䚸㈞䜚
䛡䛯䜚䚸ᤄධ䛧䛯䜚䚸⛣ື䛧䛯䜚䛩䜛άᛶ䞉⬟ຊ(⮬ᕫ䝇䝥䝷䜲䝅䞁䜾ᶵ⬟)䜢ᣢ䛳䛶䛔
䜛䛯䜑䚸mRNA 䜢ษ᩿䛧䚸䝍䞁䝞䜽㉁䜈䛾⩻ヂ䜢ᢚไ䛩䜛䚹mRNA 䜢ᶆⓗ䛸䛩䜛䛣䛸䛛
13 䜰䝥䝍䝬䞊䛿䚸᰾㓟ᢠయ䛸䜒䜀䜜䜛ᶵ⬟ᛶ᰾㓟䛷䛒䜛䚹ᢠయ䛾䜘䛖䛻ᶆⓗ䝍䞁䝟䜽 䛸≉␗ⓗ䛻⤖ྜ䛩䜛⬟ຊ䜢᭷䛩䜛᰾㓟䛷䛒䜚䚸ᶆⓗ䝍䞁䝟䜽䛾❧యᵓ㐀䛾䛟䜌䜏䛻ධ 䜚䚸Ᏻᐃⓗ䛺୕ḟඖᵓ㐀䜢ᙧᡂ䛧䛶䝍䞁䝟䜽㉁䛾ᶵ⬟䜢㜼ᐖ䛩䜛䚹 䛣䜜䜙䛾᰾㓟་⸆ရ䛿䚸ሷᇶ㓄ิ䜔ᵓ㐀䛜᪤▱䛷䛒䜛᰾㓟䜔䝍䞁䝟䜽㉁䜢ᶆⓗ䛸 䛩䜛䛯䜑䚸≉␗ᛶ䛜㧗䛟䚸స⏝䜒ᑡ䛺䛔䛸⪃䛘䜙䜜䛶䛝䛯䚹䛧䛛䛧䚸䜸䝣䝍䞊䝀䝑䝖ຠ ᯝ䛺䛹䛾㠀≉␗ⓗ䛺స⏝䚸䝚䜽䝺䜰䞊䝊䛻䜘䜛ศゎ䚸⣽⬊㏱㐣ᛶ䛾ᝏ䛥䚸䛸䛔䛳䛯 ၥ㢟Ⅼ䛜䛒䜛䚹᰾㓟་⸆䛾᭷ຠᛶ䞉Ᏻᛶ䜢ྥୖ䛥䛫䜛᪉ἲ䛸䛧䛶䚸᰾㓟䛭䛾䜒䛾䜢 Ꮫಟ㣭䛧䛯ேᕤ᰾㓟䜔䚸་⸆ရ䜢㏦㐩䛩䜛䛯䜑䛾䝗䝷䝑䜾䝕䝸䝞䝸䞊䝅䝇䝔䝮䛜䛒䜛䚹 䛣䜜䜙䜢ά䛛䛧䛯᰾㓟་⸆䛾ᐇ⏝䛻ྥ䛡䛶䚸㛤Ⓨ䛧䛯⸆䛾᭷ຠᛶ䜔యෆືែ䛺䛹 䜢ホ౯䛩䜛ᢏ⾡䠄䠬䠡䠰ᶆ㆑ᢏ⾡䛺䛹䠅䛾㛤Ⓨ䛜ồ䜑䜙䜜䛶䛔䜛䚹䛣䜜䜎䛷䛻᰾㓟䜸䝸 䝂䝬䞊䛾䠬䠡䠰ᶆ㆑䛾◊✲䛜ከ䛟ሗ࿌䛥䜜䛶䛔䜎䛩䛜䚸㝧㟁Ꮚᨺฟ᰾✀䛾༙ῶᮇ䛜 ▷䛔䛯䜑䚸䜽䝸䝑䜽ᛂ䜢⏝䛔䛯䠬䠡䠰ᶆ㆑䛜ὶ䛻䛺䛳䛶䛔䜛䚹
14 ➨
➨⠇㻌 䜽䝸䝑䜽ᛂ䜢⏝䛧䛯᰾㓟䜸䝸䝂䝬䞊䛾PET ᶆ㆑
Inkster 䜙䛿䚸DNA 䜸䝸䝂䝬䞊䛾ᮎ➃䛾䜰䝭䝜䝸䞁䜹䞊䛻䜰䝆䝗ᇶ䜢ᑟධ䛧䚸㐣ཤ䛻ሗ
࿌䛧䛯 18F 䜢᭷䛩䜛䜰䝉䝏䝺䞁 [18F]FPy5yne䠄Scheme 1-6䠅䛸䛾䜽䝸䝑䜽ᛂ䛻䜘䛳䛶
PET ᶆ㆑䛧䛯䠄Scheme 1-7䠅䡝30, 31) 䛧䛛䛧䚸᰾㓟䜸䝸䝂䝬䞊䛸[18F]FPy5yne 䛸䛾㐃⤖
⋡䛿 18%䛸ప䛔䚹㖡ゐ፹䛻⣧ᗘ 99.9999%䛾 CuBr 䜢⏝䛩䜛䛣䛸䛷ᨺᑕᛶ⋡
(RCY; radiochemical yield) 䜢ྥୖ䛥䛫䛶䛔䜛䚹䜎䛯䚸1 ౯㖡䛻䜘䜚⏕䛨䛯䝠䝗䝻䜻䝅䝷䝆
䜹䝹䛻䜘䜛᰾㓟䜸䝸䝂䝬䞊䛾㓟ⓗษ᩿ 32, 33) 䜢㜵䛠䛯䜑䛻䚸Tris[(1-benzyl-1H-1, 2,
3-triazol-4-yl)methyl]amine䠄TBTA䠅➼䛾㓄Ꮚ䜢ῧຍ䛧䛶 1 ౯㖡䛻㓄䛥䛫䚸1 ౯㖡
䜢Ᏻᐃ䛥䛫䛶䛔䜛䚹2,6-Lutidine 䛿 DIPEA 䛸ྠᵝ䛻䚸ᨺᑕᏛ䜽䝸䝑䜽ᛂ䛷⏕䛨䜛
⏕ᡂ≀䛾⏕ᡂ䜢ᢚไ䛧䛶䛔䜛䚹㻌 㻌
15 Scheme 1-7. 䜽䝸䝑䜽ᛂ䜢⏝䛧䛯䜰䝭䞁ྵ᭷ 㻰㻺㻭 䜸䝸䝂䝬䞊䛾 㻼㻱㼀 䝷䝧䝹㻌 ᭱㏆䚸ᶆ㆑ຠ⋡䜢ᨵၿ䛧䛯䛸䛧䛶䚸Kuboyama 䜙䛿䚸㐣ཤ䛻ሗ࿌䛧䛯䜶䝏䝙䝹 䝕䜸䜻䝅䝸䝪䞊䝇䛸18F 䜢᭷䛩䜛䝣䜵䝙䝹䜰䝆䝗䠄Scheme 1-8䠅䜢⏝䛧䛶 DNA 䜸䝸䝂䝬 䞊䛾 PET 䝷䝧䝹ἲ䛸䝷䝑䝖䛾యෆືែ䛻䛴䛔䛶ሗ࿌䛧䛶䛔䜛䠄Scheme 1-9䠅䚹34, 35)㻌 18F 䜢᭷䛩䜛 p-య䛾䝣䜵䝙䝹䜰䝆䝗䛻䜘䜚 PET 䝷䝧䝹䛧䛯 DNA 䜸䝸䝂䝬䞊䜢䝷䝑䝖䛻 ᢞ䛧䛯䛸䛣䜝䚸⭈⮚䚸⫢⮚䚸⭤⬔䚸⭠➼䜈䛾ศᕸ䛿ほᐹ䛥䜜䛯䚹䜎䛯䚸⬨᳝䚸⫘㦵䚸 ⭣㦵➼䛾㦵䛾㒊ศ䛻㞟✚䛜ほᐹ䛥䜜䛯䚹䛣䜜䛿䚸䝣䝑⣲䛜䝧䞁䝆䝹䛾㧗䛔ᛂ ᛶ䛻䜘䜚⬺㞳䛧䚸䝣䝑≀䜲䜸䞁䛸䛧䛶㦵䛻㞟✚䛧䛶䛔䜛䛯䜑䛷䛒䜛䛸⪃䛘䜙䜜䛶䛔䜛䚹 䜎䛯䚸18F 䜢᭷䛩䜛 m-య䛾䝣䜵䝙䝹䜰䝆䝗䛻䜘䜚 PET 䝷䝧䝹䛩䜛䛸䚸p-య䛸䛿␗䛺䜛ศ ᕸ䜢♧䛧䛯䚹䛣䛾ᛂ䜒㖡㓄Ꮚ䛸ຍ⇕䜢ᚲせ䛸䛩䜛䚹 TBTA
16 Scheme 1-8. 18F 䜢᭷䛩䜛䝣䜵䝙䝹䜰䝆䝗䛾ྜᡂἲ Scheme 1-9.㻌 䜽䝸䝑䜽ᛂ䜢⏝䛧䛯䜶䝏䝙䝹䝕䜸䜻䝅䝸䝪䞊䝇ྵ᭷ 㻰㻺㻭 䜸䝸䝂䝬䞊 䛾 㻼㻱㼀 䝷䝧䝹㻌 ᭦䛻ྠᮇ䛻䚸Mercier 䜙䛾䜾䝹䞊䝥䛜18F ᶆ㆑䛧䛯 4-䝣䝹䜸䝻䝧䞁䝆䝹䜰䝆䝗䜢ྜ ᡂ䛧(Scheme 1-10) 36)䚸ᮎ➃䛻䜶䝏䝙䝹ᇶ䜢ຍ䛧䛯ᮏ㙐siRNA 䛻స⏝䛥䛫䜛䛣䛸 䛻䜘䜚PET ᶆ㆑䜢⾜䛳䛯䛜ሗ࿌䛥䜜䛶䛔䜛(Scheme 1-11)䡝37) 䛣䛱䜙䛾ᛂ䛿䚸ᐊ 䛷㐍⾜䛩䜛䜒䛾䛾䚸䜔䛿䜚㖡㓄Ꮚ䜢ᚲせ䛸䛧䜎䛩䚹䛣䛾ᡭἲ䛿䚸ᮏ㙐≧ែ䛷䜒 Ⰻዲ䛻ᛂ䛜㐍⾜䛧䛯䛜䚸䜔䛿䜚㖡㓄ᏊTBTA 䜢⏝䛧䛺䛡䜜䜀䛺䜙䛺䛔ไ㝈䛜䛒 䜛䚹 Scheme 1-10. 4-[18F]䝣䝹䜸䝻䝧䞁䝆䝹䜰䝆䝗䛾ྜᡂἲ
17
Scheme 1-11. 䜽䝸䝑䜽ᛂ䜢⏝䛧䛯ᮏ㙐 siRNA 䛾 PET 䝷䝧䝹
䛣䜜䜙䛾 PET ᶆ㆑ἲ䛻ᑐ䛧䚸ⴭ⪅䛜ᡤᒓ䛩䜛◊✲ᐊ䛷䛿䚸䜶䝏䝙䝹䝧䞁䝊䞁㦵
᱁䛛䜙䛺䜛ேᕤ᰾㓟 (BE)䜢㛤Ⓨ䛧䚸BE 䜢ᮎ➃䛻ᑟධ䛧䛯᰾㓟䜸䝸䝂䝬䞊䜢⏝䛔䛶䚸䜰
䝆䝗ྜ≀䛸䛾 CuAAC ᛂ䜢᳨ド䛧䛯⤖ᯝ䚸㖡㓄Ꮚ㠀Ꮡᅾୗ䚸ᐊ 䛷ຠ⋡ⓗ䛻
㐍⾜䛧䚸PET ᶆ㆑䛻㐺⏝ྍ⬟䛷䛒䜛䛣䛸䜢ぢฟ䛧䛶䛔䜛(Scheme 1-12) 䡝38)
18 䛧䛛䛧䚸ศᏊ䝰䝕䝹䛻䜘䜛ຊሙィ⟬䜢⾜䛳䛯⤖ᯝ䚸BE䛾ẕ᰾䛷䛒䜛䝧䞁䝊䞁䝆䝯䝍䝜 䞊䝹䛾 2 䛴䛾㓟⣲ཎᏊ㛫㊥㞳䛿䚸䝕䜸䜻䝅䝸䝪䞊䝇㢮䛸ẚ㍑䛧䛶⣙ 2 ಸ䛷䛒䜛䛣䛸䛜 ศ䛛䛳䛯䚹䛭䛾䛯䜑䚸BE䜢ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛾ෆ㒊䛻ᑟධ䛧䛯ሙྜ䚸ᮏ㙐䛾⇕ ⓗᏳᐃᛶ䜢పୗ䛩䜛䛣䛸䛜ᠱᛕ䛥䜜䚸ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛾௵ព䛾⨨䛻ᑟධ䛩 䜛䛣䛸䛜ᅔ㞴䛷䛒䜛䛸⪃䛘䜙䜜䛯䚹䛭䛣䛷䚸ᮏ◊✲䛷䛿䚸䜶䝏䝙䝹䝧䞁䝊䞁㦵᱁䜢᭷䛩 䜛᪂䛯䛺ேᕤ᰾㓟(S)-4-Ethynylmandelol䠄ME䠅䜢㛤Ⓨ䛧䠄➨❶➨⠇䠅䚸䛣䜜䜢୰ኸ 㒊䜔ᮎ➃䛻ᑟධ䛧䛯୍ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛜 4-䝣䝹䜸䝻䝧䞁䝆䝹䜰䝆䝗䛸䜽䝸䝑䜽ᛂ 䛩䜛䛣䛸䜢ᐇド䛧䛯䠄➨୕❶➨⠇䚸➨ᅄ❶➨୍⠇䠅䚹䛭䛾ᚋ䚸ᮏ㙐᰾㓟䜸䝸䝂䝬䞊 䛾⇕ⓗᏳᐃᛶ䜢᳨ド䠄➨୕❶➨ᅄ⠇䚸➨ᅄ❶➨୕⠇䠅䛧䚸ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛷䛾 䜽䝸䝑䜽ᛂ䜢᳨ウ䛧䛯䠄➨୕❶➨⠇䚸➨ᅄ❶➨ᅄ⠇䠅䚹
19
➨
➨❶㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺ䜾䝸䝁䞊䝹ཬ䜃㻌
䛭䛾᰾㓟䜸䝸䝂䝬䞊ᑟධ⏝䝴䝙䝑䝖䛾ྜᡂ
㻌 ➨୍⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺ䜾䝸䝁䞊䝹 㻹㻱䛾ศᏊタィ㻌 ⴭ⪅䛾ᡤᒓ䛩䜛◊✲ᐊ䛷䚸㛤Ⓨ䛧䛯ேᕤ᰾㓟BE䛾ሙྜ䚸䜽䝸䝑䜽ᛂ䛻䜘䜛᰾㓟䜸 䝸䝂䝬䞊䛾 PET ᶆ㆑䛻㐺⏝ྍ⬟䛷䛒䜛䛜䚸ศᏊ䝰䝕䝹䛻䜘䜛ຊሙィ⟬䛾⤖ᯝ(Fig. 2-2)䚸BE䛾ẕ᰾䛷䛒䜛䝧䞁䝊䞁䝆䝯䝍䝜䞊䝹䛾2 䛴䛾㓟⣲ཎᏊ㛫㊥㞳䛿䚸䝕䜸䜻䝅䝸䝪 䞊䝇㢮䛸ẚ㍑䛧䛶⣙2 ಸ䛷䛒䜛䛣䛸䛜ศ䛛䛳䛯䚹䛭䛾䛯䜑䚸BE䜢ᮏ㙐᰾㓟䜸䝸䝂䝬䞊 䛾ෆ㒊䛻ᑟධ䛧䛯ሙྜ䚸ᮏ㙐䛾⇕ⓗᏳᐃᛶ䜢పୗ䛩䜛䛣䛸䛜ᠱᛕ䛥䜜䡠39, 40)㻌 ᮏ 㙐᰾㓟䜸䝸䝂䝬䞊䛾 PET ᶆ㆑䜔Ꮫಟ㣭䛺䛹䜢┠ᣦ䛧䚸௵ព䛾⨨䛻ᑟධ䛩䜛䛣䛸 䛜ᅔ㞴䛷䛒䜛䛸⪃䛘䜙䜜䜛䚹䛭䛣䛷䚸ᮏ◊✲䛷䛿䚸᪂䛯䛺ேᕤ᰾㓟䛸䛧䛶䚸䜶䝏䝙䝹䝧 䞁䝊䞁⨨ᆺ䜾䝸䝁䞊䝹(S )-4-Ethynylmandelol䠄ME䠅䜢タィ䛧䛯䠄Fig. 2-1䠅䚹 Fig.2-1.㻌 ศᏊ䝰䝕䝹䛻䜘䜛ศᏊຊሙィ⟬20 ME 䛾⢾㒊䛸䛺䜛䜾䝸䝁䞊䝹䛾㓟⣲ཎᏊ㛫㊥㞳䛜䝕䜸䜻䝅䝸䝪䞊䝇㢮䛻㏆䛔䛣䛸䛛䜙䚸 ME 䜢ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛾ෆ㒊䛻ᑟධ䛧䛯ሙྜ䚸ᮏ㙐䛾Ᏻᐃ䛜ᑠ䛥䛔䛸ぢ ㎸䜎䜜䜛䚹䜎䛯䚸ME 䛜䚸䃟㟁Ꮚ䝸䝑䝏䛺䜶䝏䝙䝹䝧䞁䝊䞁㦵᱁䜢᭷䛧䛶䛔䜛䛣䛸䛛䜙䚸 㧗㏿䜽䝸䝑䜽ᛂ䛻㐺䛧䛶䛚䜚䚸ᵝ䚻䛺䜰䝆䝗ྜ≀䛸䛾䜽䝸䝑䜽ᛂ䛻䜘䜚䚸᰾㓟䜸䝸䝂 䝬䞊䛾PET ᶆ㆑䜔Ꮫಟ㣭䚸⺯ගᶆ㆑䛺䛹䛻㐺⏝ྍ⬟䛷䛒䜛䛸⪃䛘䜙䜜䜛䚹 ⢾㒊䛜䜾䝸䝁䞊䝹㦵᱁䛛䜙䛺䜛᰾㓟䜸䝸䝂䝬䞊䠄GNA䠅䛿䚸䝥䝻䝢䝺䞁䜾䝸䝁䞊䝹䝚䜽 䝺䜸䝅䝗䛸䝩䝇䝩䝆䜶䝇䝔䝹⤖ྜ䛾⧞䜚 ㏉䛧ᵓ㐀䜢䛸䛳䛶䛔䜛 䠄Fig. 2-2䠅䚹䜎䛯䚸 (S )-GNA 䛿䝂䞊䝅䝳ຠᯝ䛻䜘䛳䛶ᮏ㙐䜢Ᏻᐃ䛩䜛䛣䛸䛜▱䜙䜜䛶䛔䜛䡝41-46) Fig. 2-2. (S)-GNA ᵓᵓ㐀㻌 㻔ᕥ㻕㻌 䛸ᮏ㙐ᙧᡂ䝰䝕䝹㻌 㻔ྑ㻕㻠㻝㻕 㻌 㻌
21 ➨ ➨⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺ䜾䝸䝁䞊䝹 㻹㻱 䛾ྜᡂ㻌 ྜᡂᡓ␎䛸䛧䛶䚸䜎䛪䚸ᕷ㈍䛾L-䝬䞁䝕䝹㓟䜢ཎᩱ䛸䛧䛯 Scheme 2-1 䛻♧䛩⤒㊰䜢 ⪃䛘䛯䚹 Scheme 2-1. 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺ䜾䝸䝁䞊䝹 ME䛾ྜᡂᡓ␎ձ 䜎䛪䚸L-䝬䞁䝕䝹㓟䛾 4 㑅ᢥⓗ䝶䜴⣲䜢᳨ウ䛧䛯䚹Auge 47) 䜙䛾᪉ἲ䜢ཧ⪃䛻 䛧䛶◲㓟䛸㓑㓟䛾ΰྜ⁐፹୰䛷L-䝬䞁䝕䝹㓟䚸䝶䜴⣲䚸䝶䜴⣲㓟䝘䝖䝸䜴䝮䛸㐣䝶䜴⣲㓟 䝘䝖䝸䜴䝮䜢70 ºC 䛷᧠ᢾ䛧䛯䚹ᛂ⏕ᡂ≀䛾ᴟᛶ䛜㧗䛟䚸4-䝶䞊䝗య䠄2䠅䛾༢㞳⢭〇䛜 ᅔ㞴䛷䛒䛳䛯䠄Scheme 2-2䠅䚹
Scheme 2-2. Reagents and conditions; (a) I2, NaIO3, NaIO4, H2SO4, AcOH, 70 ºC.
Zheng 48) 䜙䛿䚸䜶䝇䝔䝹ྜ≀䛷䛾䝖䝸䝣䝹䜸䝻䝯䝍䞁䝇䝹䝩䞁㓟㖟䠄AgOTf䠅䜢ゐ፹
䛸䛧䛶⏝䛔䜛4 㑅ᢥⓗ䝶䜴⣲ἲ䜢ሗ࿌䛧䛶䛔䜛䠄Scheme 2-3䠅䚹
22
䛭䛣䛷䚸䜶䝇䝔䝹య䜢⤒⏤䛧䛶ME䜢ྜᡂ䛩䜛䛣䛸䛻䛧䛯䠄Scheme 2-4䠅䚹
Scheme 2-4. 䜶䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺ䜾䝸䝁䞊䝹ME䛾ྜᡂᡓ␎ղ
䜎䛪䚸䜶䝍䝜䞊䝹୰䚸ゐ፹㔞䛾◲㓟䜢⏝䛔䛶䚸䜶䝇䝔䝹䜢⾜䛔䚸97%䛾⋡䛷䜶 䝇䝔䝹య3 䜢ᚓ䛯䠄Scheme 2-5䠅䚹
Scheme 2-5. Reagents and conditions; (a) H2SO4, EtOH, 70 °C, 23 h, 97%.
ḟ䛻䚸㓟㖟䛸䝖䝸䝣䝹䜸䝻䝯䝍䞁䝇䝹䝩䞁㓟䛛䜙ㄪ〇䛧䛯䝖䝸䝣䝹䜸䝻䝯䝍䞁䝇䝹䝩䞁 㓟㖟䠄Scheme 2-6䠅ඹᏑୗ䚸4 㑅ᢥⓗ䛻䝶䜴⣲䜢⾜䛳䛯䠄Scheme 2-7䠅䚹48) 䝆䜽䝻䝻
䝯䝍䞁୰䛷ᐊ ୗ 24 㛫᧠ᢾ䛧䛯䛸䛣䜝┠ⓗ䛾 4-䝶䞊䝗య䠄4䠅䛸ᛮ䜟䜜䜛ྜ≀䛜ᚓ
䜙䜜䛯䚹
Scheme 2-6. Reagents and conditions; (a) Ag2O, H2O, 90 ºC, 3 h.
23 䛧䛛䛧䚸MS ᐃ䜢⾜䛳䛯䛸䛣䜝䚸4 䛷䛿䛺䛟䝧䞁䝆䝹䛷⮬ᕫ⦰ྜ䛧䛯ྜ≀ 4'䛷 䛒䜛䛣䛸䛜♧၀䛥䜜䛯䠄35%䠅䚹ᛂ㛫䜢᳨ウ䛧䛯⤖ᯝ䚸12 㛫䛷䛿ྜ≀ 4'䛾⏕ᡂ 䛿ᢚไ䛥䜜䛯䠄25%䠅䚹3 㛫䛻▷⦰䛧䛯䛸䛣䜝䚸ྜ≀ 4'䛸䛿␗䛺䜛᪂䛯䛺ྜ≀䛜 ⏕ᡂ≀䛸䛧䛶ᚓ䜙䜜䚸MS ᐃ䛾⤖ᯝ䛛䜙䚸┠ⓗ䛾 4-䝶䞊䝗య䠄4䠅䛷䛒䜛䛣䛸䛜ᙉ䛟♧၀ 䛥䜜䛯䚹ᮏྜ≀䛾1H-NMR 䜢ゎᯒ䛧䛯䛸䛣䜝䚸4'䛸㠀ᖖ䛻㓞ఝ䛧䛶䛔䛯䚹䛭䛣䛷䚸䜶䝇 䝔䝹㑏ඖ⏕ᡂ≀䛷䛒䜛䝆䜸䞊䝹య䠄5䠅䛾䜰䝉䝏䝹䜢⾜䛔䚸䜰䝉䝏䝹య䛾 1H-NMR ゎᯒ䜘䜚䜰䝉䝏䝹ᇶ⏤᮶䛾䝯䝏䝹ᇶ䛾䝢䞊䜽䛜2 ᮏほ 䛥䜜䛯䚹䛣䛾⤖ᯝ䛛䜙䚸ୖグ䛾 ྜ≀䛜4-䝶䞊䝗య䠄4䠅䛷䛒䜛䛣䛸䜢ド᫂䛧䛯䠄Scheme 2-8䠅䚹
Scheme 2-8. Reagents and conditions; (a) NaBH4, EtOH, rt, 1 h, 86%;
b) Ac2O, pyridine, rt, 3 h.
⥆䛔䛶䚸䝢䝨䝸䝆䞁୰䚸䝆䜸䞊䝹య䠄5䠅䛸 TMS 䜰䝉䝏䝺䞁䛸䛾ⷵ㢌ᛂ49) 䛻䜘䜚䚸ᑐ
ᛂ䛩䜛䜰䝹䜻䝙䝹య6 䜢⋡ 85%䛷ᚓ䛯ᚋ䚸䝯䝍䝜䞊䝹୰䚸Ⅳ㓟䜹䝸䜴䝮䜢స⏝䛥䛫䛶
TMS ᇶ䜢㝖ཤ䛧䚸䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺ䜾䝸䝁䞊䝹 1䠄ME䠅䜢ᐃ㔞ⓗ䛻ᚓ䛯
䠄Scheme 2-9䠅䚹
Scheme 2-9. Reagents and conditions; (a) trimethylsilylacetylene, PdCl2(PPh3)2, CuI,
24 ➨ ➨୕⠇㻌 ᰾㓟䜸䝸䝂䝬䞊ᑟධ⏝䝴䝙䝑䝖䛾ྜᡂ ᐃἲ䛻ᚑ䛳䛶䚸䝢䝸䝆䞁୰䚸4,4’-䝆䝯䝖䜻䝅䝖䝸䝏䝹䜽䝻䝸䝗䜢⏝䛔䛶䚸1 䛾୍⣭Ỉ㓟ᇶ 䛾 DMTr 䜢⾜䛔䚸䝰䝜䝖䝸䝏䝹య 7 䜢⋡ 31%䛷ᚓ䛯䚹䛭䛾ᚋ䚸ྜ≀ 7 䛾⣭Ỉ 㓟ᇶ䜢ள䝸䞁㓟䛧䚸ME䛾䝩䝇䝩䝻䜰䝭䝎䜲䝖య 8 䜢⋡ 79%䛷ᚓ䛯䚹䜎䛯䚸ྜ≀ 7 䜢䝇䜽䝅䝙䝹䛧䛯ᚋ䚸CPG ᶞ⬡䛸⤖ྜ䛥䛫䚸ME 㐃⤖ᅛ┦ᢸయ 9 䜢ᚓ䛯䠄Scheme 2-10)䚹ᅛ┦ᢸయ 9 䛾άᛶ(26.5 μmol/g) 䛿௨ୗ䛾ᘧ䜘䜚ồ䜑䛯䚹
Scheme 2-10. Reagents and conditions; (a) DMTrCl, pyridine, rt, 2 h, 31%;
(b) (i-Pr)2NP(Cl)O(CH2)2CN, i-Pr2NEt, THF, rt, 1.5 h, 79%; (c) (i) succinic anhydride,
DMAP, pyridine, rt; (ii) CPG, EDCl, DMF, rt, (loading amount was evaluated to be 26.5 μmol/g).
25 ➨ ➨ᅄ⠇ 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䛾 ε ್䛾⟬ฟ 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢ᑟධ䛧䛯᰾㓟䜸䝸䝂䝬䞊䛾ྜᡂ䛻䛒䛯䜚䚸⃰ᗘ ィ⟬䜢䛩䜛䛯䜑䛻䝰䝹྾ගィᩘ ε䠄M-1cm-1䠅 䛜ᚲせ䛸䛺䜛䚹ε ್䛿 Beer 䛾ἲ๎䛻䜘䜚 ௨ୗ䛾ᘧ䛷ồ䜑䜛䛣䛸䛜䛷䛝䜛䚹 ε260 = Abs260 䞉 C-1 䡡 l-1 䛣䛣䛷䚸Abs260䛿Ἴ㛗260 nm 䛷䛾྾ගᗘ䚸C 䛿⃰ᗘ䠄mol/L䠅䚸l 䛿䝉䝹䛾ග㊰㛗䠄cm䠅 䜢♧䛩䚹ⴭ⪅䛾◊✲ᐊ䛷⏝䛔䛶䛔䜛྾ගᗘ ᐃ⏝▼ⱥ䝉䝹䛿䚸l = 1䠄cm䠅䛷䛒䜛䚹 䛭䛣䛷䚸䜶䝏䝙䝹䝧䞁䝊䞁㦵᱁䜢᭷䛩䜛ேᕤ᰾㓟䜢⁛⳦Ỉ䛷⁐ゎ䛧䛶ᗄ䛴䛛䛾㐺 ᙜ䛺⃰ᗘ䜢ㄪ〇䛧䛯ᚋ䚸྾ගᗘ䜢 ᐃ䛧䚸ྛ䚻䛾ε ್䜢⟬ฟ䛧䛯䠄Table 2-1䠅䚹 Table 2-1. ྾ගᗘ ᐃ䛸 ε ್ ேᕤ᰾㓟 ε ್ (M-1cm-1) ME 672 BE 627
26
➨
➨୕❶㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛
DNA 䛾ྜᡂ䛸
䛭䛾ᛶ㉁
➨୍⠇㻌 ᰾㓟⮬ືྜᡂᶵ䛾୍⯡ⓗ䛺ྜᡂ䝃䜲䜽䝹 ேᕤ᰾㓟䜢ᑟධ䛧䛯᰾㓟䜸䝸䝂䝬䞊䛾ྜᡂ䛿䚸ᅛ┦䝩䝇䝩䝻䜰䝭䝎䜲䝖ἲ 50)㻌 䜢⏝ 䛧䛯᰾㓟⮬ືྜᡂᶵ䛻䛶⾜䛖䚹Fig. 3-1 䛻᰾㓟⮬ືྜᡂᶵ䛾୍⯡ⓗ䛺ྜᡂ䝃䜲䜽䝹䜢 ♧䛧䛯䚹 CPG 䛻ᢸᣢ䛧䛯ྜ≀䛾 DMTr ᇶ䜢ᙅ㓟䛷⬺ಖㆤ䛧䛯ᚋ䚸䝔䝖䝷䝌䞊䝹䛷 P-N ⤖ ྜ䜢άᛶ䛧䚸⦰ྜᛂ䜢⾜䛖䚹ᮍᛂ䛾Ỉ㓟ᇶ䜢↓Ỉ㓑㓟䛻䜘䛳䛶䜰䝉䝏䝹䛧䚸 ௨㝆䛾⦰ྜᛂ䛻㛵䛧䛺䛔䜘䛖䜻䝱䝑䝢䞁䜾䛧䛯ᚋ䚸䝶䜴⣲䛷䝸䞁䜢୕౯䛛䜙౯䜈 䛸㓟䛧䚸䝩䝇䝩䝆䜶䝇䝔䝹⤖ྜ䜢ᙧᡂ䛧䚸୍䛴䛾䝃䜲䜽䝹䛜⤊䜟䜛䚹䛭䛾ᚋ䚸䜎䛯䚸⬺ 䝖䝸䝏䝹䛛䜙ጞ䜎䜚䚸䝃䜲䜽䝹䜢⧞䜚㏉䛩䚹 Fig. 3-1. ᅛ┦䝩䝇䝩䝻䜰䝭䝎䜲䝖ἲ㻌27 ➨
➨⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛DNA 䛾ྜᡂ
➨❶➨୕⠇䛷ྜᡂ䛧䛯䝩䝇䝩䝻䜰䝭䝎䜲䝖ㄏᑟయ8 䛒䜛䛔䛿ᅛ┦ᢸయ 9 䜢⏝䛔䛶䚸
᰾㓟⮬ືྜᡂᶵ䛻䛶 ME䜢 5’-ᮎ➃䚸୰ኸ㒊䚸䜎䛯䛿 3’-ᮎ➃䛻 1 䛴ᑟධ䛧䛯 15mer
䛾DNA䠄ON 12䚸ON 13䚸ON 14䠅䜢ྜᡂ䛧䛯䚹ẚ㍑ᑐ㇟䛸䛧䛶䚸BE䜢᭷䛩䜛DNA䠄ON
15䚸ON 16䚸ON 17䠅䜢ྜᡂ䛧䛯䚹䜎䛯䚸┦⿵㙐䠄ON 10䠅䛸ኳ↛㙐䠄ON 11䠅䜢ྜᡂ䛧䛯䚹
MALDI-TOF/MS ᐃ䛻䜘䜚ᵓ㐀䜢ྠᐃ䛧䛯䠄Table 3-1䠅䚹DNA 䛻ᑟධ䛧䛯࢚ࢳࢽࣝ
䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟MEBE䛾ᵓ㐀䜢Fig. 3-2 ♧ࡋࡓࠋ
Fig. 3-2. ࢚ࢳࢽࣝ䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟 MEBE䛾ᵓ㐀
Table 3-1. ྜᡂ䛧䛯 DNA ࡢ㓄ิ MALDI-TOF/MS
No.of ON Sequence Calculated Observed
ON 10 (dA15) 5'-d(AAAAAAAAAAAAAAA)-3' 4632.9 4633.3 ON 11 (dT15) 5'-d(TTTTTTTTTTTTTTT)-3' 4497.7 4496.8 ON 12 (ME-dT14) 5'-d(METTTTTTTTTTTTTT)-3' 4417.7 4418.7 ON 13 (dT7-ME-dT7) 5'-d(TTTTTTTMETTTTTTT)-3' 4417.7 4418.7 ON 14 (dT14-ME) 5'-d(TTTTTTTTTTTTTTME)-3' 4417.7 4418.2 ON 15 (BE-dT14) 5'-d(BETTTTTTTTTTTTTT)-3' 4417.7 4419.2 ON 16 (dT7-BE-dT7) 5'-d(TTTTTTTBETTTTTTT)-3' 4417.7 4417.4 ON 17 (dT14-BE) 5'-d(TTTTTTTTTTTTTTBE)-3' 4417.7 4418.4
28
䛸䛧䛶䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺ䜾䝸䝁䞊䝹ME䜢5’-ᮎ➃䚸୰ኸ㒊䚸3’-ᮎ➃䛻ᑟධ
䛧䛯DNA ᰾㓟䜸䝸䝂䝬䞊䠄ON 12䚸䚸ON 13䚸ON 14䠅䜢 Fig. 3-3 䛻♧䛧䛯䚹
Fig. 3-3. ME䜢ᑟධ䛧䛯DNA䠄ON 12䚸ON 13䚸ON 14䠅䛾㓄ิ
ON 13 (dT7-ME-dT7)
ON 12 (ME-dT14)
29 ➨ ➨୕⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛୍ᮏ㙐DNA 䛷䛾䜽䝸䝑䜽ᛂ ⴭ⪅䛾◊✲ᐊ䛷䛿䚸BE䜢ᮎ➃䛻ᑟධ䛧䛯᰾㓟䜸䝸䝂䝬䞊䜢⏝䛔䛶4-䝣䝹䜸䝻䝧䞁䝆 䝹䜰䝆䝗䛸䛾CuAAC ᛂ䜢ヲ⣽䛻᳨ウ䛧䚸0.1 M 䝸䞁㓟⦆⾪ᾮ䚸MeCN䚸DMSO 䛸䛾 ΰྜ⁐፹୰䚸୍ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛻ᑐ䛧䚸100 ᙜ㔞䛾◲㓟㖡Ỉ≀䚸100 ᙜ㔞䛾 䜰䝇䝁䝹䝡䞁㓟䝘䝖䝸䜴䝮䜢⏝䛔䜛䛣䛸䛻䜘䜚䚸㓄Ꮚ㠀Ꮡᅾୗ䚸ᐊ 䛷ຠ⋡ⓗ䛻 CuAAC ᛂ䛜㐍⾜䛩䜛䛣䛸䜢ぢฟ䛧䛯䡝38)㻌 䛭䛣䛷䚸ྠᛂ᮲௳ୗ䚸➨୍⠇䛷ྜᡂ䛧 䛯ME䛒䜛䛔䛿BE䜢᭷䛩䜛୍ᮏ㙐DNA䠄ON 12-17䠅䛸 4-䝣䝹䜸䝻䝧䞁䝆䝹䜰䝆䝗䛸䛾䜽 䝸䝑䜽ᛂ䜢⾜䛳䛯䚹୰ኸ㒊䛻 ME䜢᭷䛩䜛୍ᮏ㙐 DNA䠄ON13䠅䜢⏝䛔䛯ሙྜ䛾ᛂ ᘧ䜢Scheme 3-1䚸HPLC 䛾䜽䝻䝬䝖䜾䝷䝮䜢 Fig. 3-4 䛻♧䛧䛯䚹䜽䝸䝑䜽ᛂ䛻䜘䜚ᚓ䜙䜜
䛯DNA 䛾ᵓ㐀䛿 MALDI-TOF/MS ᐃ䛻䜘䜚ྠᐃ䛧䛯䠄Table 3-2䠅䚹
Scheme 3-1. DNA䠄ON13䠅䛸 4-䝣䝹䜸䝻䝧䞁䝆䝹䜰䝆䝗䛸䛾䜽䝸䝑䜽ᛂ
Fig. 3-4. DNA䠄ON 13䠅䛸 4-䝣䝹䜸䝻䝧䞁䝆䝹䜰䝆䝗䛸䛾䜽䝸䝑䜽ᛂ䛾䜽䝻䝬䝖䜾䝷䝮
30 䝑䜽ᛂᚋ䠄15 ศᚋ䠅䚸ಖᣢ㛫 9.5 ศ䛾⨨䛾ཎᩱ ON 13䠄dT7-ME-dT7䠅⏤᮶䛾䝢䞊 䜽䛜ῶᑡ䛧䚸12.7 ศ䛾⨨䛻 CuAAC ᛂᡂ⦼య ON 19䠄dT7-MFBT-dT7䠅⏤᮶䛾䝢䞊 䜽䛜ほᐹ䛥䜜䛯䚹 HPLC 䛾䜽䝻䝬䝖䜾䝷䝮䛻䛚䛔䛶ཎᩱ䛸⏕ᡂ≀௨እ䛻䛰䛳䛯䝢䞊䜽䛜᳨ฟ䛥䜜䛺䛛 䛳䛯䛯䜑䚸ཎᩱ䛾ᾘ㈝⋡䜢ᛂຠ⋡䛸䛧䛯䚹 Fig. 3-5. ୍୍ᮏ㙐DNA ࡢࢡࣜࢵࢡᛂຠ⋡ 䛭䛾⤖ᯝ䚸ME 䜢୰ኸ㒊䛻ᑟධ䛧䛯 ON 13䠄dT7-ME-dT7䠅䜢⏝䛔䛯ሙྜ䛜᭱䜒ᛂ ຠ⋡䛜㧗䛛䛳䛯䚹䜎䛯䚸ME䜢᭷䛩䜛᰾㓟䜸䝸䝂䝬䞊䛾 CuAAC ᛂ䛿ྠ㓄ิ䛛䜙䛺 䜛BEྵ᭷᰾㓟䜸䝸䝂䝬䞊䛸ẚ㍑䛧䛶ຠ⋡ⓗ䛻㐍⾜䛧䛯䚹⯆῝䛔䛣䛸䛻ෆ㒊䛻ᑟධ 䛧䛯䜸䝸䝂䝬䞊䛿ᮎ➃䛻ᑟධ䛧䛯䜸䝸䝂䝬䞊䜘䜚䜒ຠ⋡ⓗ䛻4-䝣䝹䜸䝻䝧䞁䝆䝹䜰䝆䝗 䛸ᛂ䛧䛯䠄Fig. 3-5䠅䚹
Table 3-2. CuAAC ᛂ䛻䜘䜚ᚓ䜙䜜䛯 DNA 䛾㓄ิ MALDI-TOF/MS
No.of ON Sequence Calculated Observed
ON 18 (MFBT-dT14) 5'-d(MFBTTTTTTTTTTTTTTT)-3' 4568.8 4569.3 ON 19 (dT7-MFBT-dT7) 5'-d(TTTTTTTMFBTTTTTTTT)-3' 4568.8 4570.2 ON 20 (dT14-MFBT) 5'-d(TTTTTTTTTTTTTTMFBT)-3' 4568.8 4570.6 ON 21 (BFBT-dT14) 5'-d(BFBTTTTTTTTTTTTTTT)-3' 4568.8 4569.9 ON 22 (dT7-BFBT-dT7) 5'-d(TTTTTTTBFBTTTTTTTT)-3' 4568.8 4570.4 ON 23 (dT14-BFBT) 5'-d(TTTTTTTTTTTTTTBFBT)-3' 4568.8 4569.6
31 ➨
➨ᅄ⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛ᮏ㙐DNA 䛾⇕ⓗᏳᐃᛶ
ᮏ㙐≧ែ䛷䜽䝸䝑䜽ᛂ䜈䛾ᒎ㛤䜢┠ᣦ䛧䚸MEཬ䜃 BE䜢᭷䛩䜛ᮏ㙐 DNA 䛾
⇕ⓗᏳᐃᛶ䜢ホ౯䛩䜛䛯䜑䚸10 mM phosphate㻌 buffer䠄pH 7.0䠅/100 mM NaCl ୰䚸ᑐ
ᛂ䛩䜛┦⿵㙐ON10(dA15)䛸ᮏ㙐䜢ᙧᡂ䛧䚸Tm್䜢 ᐃ䛧䛯䠄Fig. 3-6䠅䚹
Fig. 3-6. ᮏ㙐 DNA 䛾⇕ⓗᏳᐃᛶ
䛭䛾⤖ᯝ䚸ኳ↛ᆺON 11䠄dT15䠅/ON 10䠄dA15䠅䛸ẚ㍑䛧䛶䚸MEཬ䜃BE䜢ᮎ➃䛻᭷
䛩䜛 ON 12䠄ME-dT14䠅/ON 10䠄dA15䠅 䚸ON 15䠄BE-dT14䠅/ON 10䠄dA15䠅䚸ON 14
䠄dT14-ME䠅/ON 10䠄dA15䠅䚸ON 17䠄dT14-BE䠅/ ON 10䠄dA15䠅䛾Tm್䛻䛿䚸䜋䛸䜣䛹ᕪ䛜
ぢ䜙䜜䛺䛛䛳䛯䚹୍᪉䚸䛣䜜䜙ேᕤ᰾㓟䜢ෆ㒊䛻᭷䛩䜛 ON 13䠄dT7-ME-dT7䠅/ON
10(dA15)䚸ON 16䠄dT7-BE-dT7䠅/ON 10䠄dA15䠅䛾Tm್䛿䚸ኳ↛ᆺON 11䠄dT15䠅/ON 10
䠄dA15䠅䛸ẚ㍑䛧䛶䛝䛟పୗ䛧䛯䚹䛧䛛䛧䚸ᅇタィ䛧䛯 ME 䜢ෆ㒊䛻᭷䛩䜛 ON 13
䠄dT7-ME-dT7䠅/ON 10䠄dA15䠅䛾Tm್䛿䚸BE䜢ෆ㒊䛻᭷䛩䜛ON 16䠄dT7-BE-dT7䠅/ON
32 ➨
➨⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛ᮏ㙐DNA 䛷䛾䜽䝸䝑䜽ᛂ
୰ኸ㒊䛻ME䜢᭷䛩䜛ᮏ㙐DNA [ON 13䠄dT7-ME-dT7䠅/ON 10䠄dA15䠅]䛾 Tm್䛜
ᐊ 䜘䜚㧗䛛䛳䛯䛯䜑䚸➨⠇䛷⾜䛳䛯୍ᮏ㙐DNA 䛷䛾䜽䝸䝑䜽ᛂ䛸ྠ᮲௳ୗ䚸ON 13䠄dT7-ME-dT7䠅/ON 10䠄dA15䠅䛸4-䝣䝹䜸䝻䝧䞁䝆䝹䜰䝆䝗䛸䛾䜽䝸䝑䜽ᛂ䜢᳨ウ䛧䛯 䠄Scheme 3-2䠅䚹ᛂ䛿 HPLC 䛻䜘䜚㏣㊧䛧䛯䠄Fig. 3-7䠅䚹 Scheme 3-2. ୰ኸ㒊䛻 ME䜢᭷䛩䜛ᮏ㙐DNA 䛾䜽䝸䝑䜽ᛂ Fig. 3-7. ME䜢୰ኸ㒊䛻᭷䛩䜛ᮏ㙐DNA 䛷䛾䜽䝸䝑䜽ᛂ䛾䜽䝻䝬䝖䜾䝷䝮 䜽䝸䝑䜽ᛂ๓䛿䚸ཎᩱ ON 13䠄dT7-ME-dT7䠅/ON 10䠄dA15䠅 ⏤᮶䛾䝢䞊䜽䛜䚸䛭䜜 䛮䜜ON 10䠄dA15䠅䛜ಖᣢ㛫5 ศ䛾⨨䛻䚸ON 13䠄dT7-ME-dT7䠅䛜ಖᣢ㛫11 ศ 䛾⨨䛻ほᐹ䛥䜜䛯䚹䜽䝸䝑䜽ᛂᚋ䛿䚸ಖᣢ㛫11 ศ䛾⨨䛾 ON 13䠄dT7-ME-dT7䠅
33
⏤᮶䛾䝢䞊䜽䛜ῶᑡ䛧䚸14.5 ศ䛾⨨䛻 CuAAC ᛂᡂ⦼య ON 19 䠄dT7-MFBT-dT7䠅
⏤᮶䛾䝢䞊䜽䛜ほᐹ䛥䜜䛯䚹MALDI-TOF/MS ᐃ䛻䜘䜚 CuAAC ᛂᡂ⦼య ON 19 䠄dT7-MFBT-dT7䠅ᵓ㐀䜢ྠᐃ䛧䛯䚹
䜎䛯䚸䜽䝸䝑䜽ᛂ୰䚸ᮏ㙐DNA 䛜ศゎ䛧䛶䛔䛺䛔䛛䜢ᮏ㙐䛾䝬䞊䜹 DNA step
ladder䠄10~300 bp䠅䜢⏝䛔䛯㟁ẼὋື䛻䜘䜚ほᐹ䛧䛯䛸䛣䜝䚸ON 13䠄dT7-ME-dT7䠅/ON
10䠄dA15䠅䚸CuAAC ᛂᡂ⦼య ON 19 䠄dT7-MFBT-dT7䠅/ON 10䠄dA15䠅䛾䝞䞁䝗䛜䚸䝬
䞊䜹䛾15 mer 䛾䝞䞁䝗䛸ྠ䛨⨨䛻ほᐹ䛥䜜䛯䠄Fig. 3-8䠅䚹 㻌 ௨ୖ䛾䛣䛸䛛䜙䚸ᮏ㙐 DNA 䛜ศゎ䛫䛪䚸4-䝣䝹䜸䝻䝧䞁䝆䝹䜰䝆䝗䛸䛾䜽䝸䝑䜽 ᛂ䛜ຠ⋡ⓗ䛻㐍⾜䛧䛯䛣䛸䛜♧၀䛥䜜䛯䚹 Fig. 3-8. ᮏ㙐DNA 䛾㟁ẼὋື⏬ീ ON 13/ON 10 ON 19/ON 10 Marker
DNA step ladder (10~300 bp)
34
➨
➨ᅄ❶㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛
RNA 䛾ྜᡂ䛸
䛭䛾ᛶ㉁
➨୍⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛RNA 䛾ྜᡂ ➨❶➨୕⠇䛷ྜᡂ䛧䛯䝩䝇䝩䝻䜰䝭䝎䜲䝖ㄏᑟయ8 䛒䜛䛔䛿ᅛ┦ᢸయ 9 䜢⏝䛔䛶䚸 ᰾㓟⮬ືྜᡂᶵ䛻䛶 ME䜢 5’-ᮎ➃䚸୰ኸ㒊䚸䜎䛯䛿 3’-ᮎ➃䛻 1 䛴ᑟධ䛧䛯 15mer䛾RNA䠄ON 26䚸ON 27䚸ON 28䠅䜢ྜᡂ䛧䛯䚹ẚ㍑ᑐ㇟䛸䛧䛶䚸BE䜢᭷䛩䜛RNA䠄ON
29䚸ON 30䚸ON 31䠅䜢ྜᡂ䛧䛯䚹䜎䛯䚸┦⿵㙐䠄ON 24䠅䛸ኳ↛㙐䠄ON 25䠅䜢ྜᡂ䛧䛯䚹
MALDI-TOF/MS ᐃ䛻䜘䜚ᵓ㐀䜢ྠᐃ䛧䛯䠄Table 4-1䠅䚹
Table 4-1. ྜᡂ䛧䛯 RNA ࡢ㓄ิ MALDI-TOF/MS
䛸䛧䛶䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺ䜾䝸䝁䞊䝹ME䜢5’-ᮎ➃䚸୰ኸ㒊䚸3’-ᮎ➃䛻ᑟධ
䛧䛯RNA㻌 䠄ON 26䚸ON 27䚸ON 28䠅䜢 Fig. 4-1 䛻♧䛧䛯䚹
No.of ON Sequence Calculated Observed
ON 24 (A15) 5'-r(AAAAAAAAAAAAAAA)-3' 4872.9 4872.8 ON 25 (U15) 5'-r(UUUUUUUUUUUUUUU)-3' 4527.4 4527.8 ON 26 (ME-U14) 5'-r(MEUUUUUUUUUUUUUU)-3' 4445.4 4445.8
ON 27 (U7-ME-U7) 5'-r(UUUUUUUMEUUUUUUU)-3' 4445.4 4445.1
ON 28 (U14-ME) 5'-r(UUUUUUUUUUUUUUME)-3' 4445.4 4445.6
ON 29 (BE-U14) 5'-r(BEUUUUUUUUUUUUUU)-3' 4445.4 4447.2 ON 30 (U7-BE-U7) 5'-r(UUUUUUUBEUUUUUUU)-3' 4445.4 4446.8 ON 31 (U14-BE) 5'-r(UUUUUUUUUUUUUUBE)-3' 4445.4 4445.9
35
Fig. 4-1. ME䜢䜢ᑟධ䛧䛯DNA䠄ON 26䚸ON 27䚸ON 28䠅䛾㓄ิ
ON 27 (rU7-ME-rU7)
ON 26 (ME-rU14)
36 ➨ ➨⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛୍ᮏ㙐RNA 䛷䛾䜽䝸䝑䜽ᛂ ➨⠇䛷⾜䛳䛯୍ᮏ㙐 DNA 䛷䛾䜽䝸䝑䜽ᛂ䛸ྠ᮲௳ୗ䚸୍ᮏ㙐 RNA䠄ON 26-31䠅 䛸4-䝣䝹䜸䝻䝧䞁䝆䝹䜰䝆䝗䛸䛾䜽䝸䝑䜽ᛂ䜢⾜䛳䛯䚹䜽䝸䝑䜽ᛂ䛻䜘䜚ᚓ䜙䜜䛯 RNA 䛾ᵓ㐀䛿MALDI-TOF/MS ᐃ䛻䜘䜚ྠᐃ䛧䛯䠄Table 4-2䠅䚹
Table 4-2. CuAAC ᛂ䛻䜘䜚ᚓ䜙䜜䛯 RNA 䛾㓄ิ MALDI-TOF/MS
୍ᮏ㙐 RNA 䛷䛾䜽䝸䝑䜽ᛂ䛾ຠ⋡䛿䚸୍ᮏ㙐 DNA 䛾ሙྜ䛸ྠᵝ䛻䚸ME䜢ෆ㒊
䛻ᑟධ䛧䛯୍ᮏ㙐RNA ON 27䠄rU7-ME-rU7䠅䛾ሙྜ䛻᭱䜒㧗䛛䛳䛯䚹䜎䛯䚸ME䜢᭷䛩
䜛᰾㓟䜸䝸䝂䝬䞊䛾CuAAC ᛂ䛿ྠ㓄ิ䛛䜙䛺䜛 BEྵ᭷᰾㓟䜸䝸䝂䝬䞊䛸ẚ㍑䛧
䛶ຠ⋡ⓗ䛻㐍⾜䛧䛯䚹䠄Fig. 4-2䠅䚹
Fig. 4-2. ୍ᮏ㙐 RNA 䛾䜽䝸䝑䜽ᛂຠ⋡
No.of ON Sequence Calculated Observed
ON 32 (MFBT-rU14) 5'-r(MFBTUUUUUUUUUUUUUU)-3' 4596.5 4595.2 ON 33 (rU7-MFBT-rU7) 5'-r(UUUUUUUMFBTUUUUUUU)-3' 4596.5 4593.9
ON 34 (rU14-MFBT) 5'-r(UUUUUUUUUUUUUUMFBT)-3' 4596.5 4594.1 ON 35 (BFBT-rU14) 5'-r(BFBTUUUUUUUUUUUUUU)-3' 4596.5 4596.2
ON 36 (rU7-BFBT-rU7) 5'-r(UUUUUUUBFBTUUUUUUU)-3' 4596.5 4595.3
37 ୍ᮏ㙐DNA䚸RNA 䛷䛾ᛂᛶ䛾ẚ㍑䛛䜙䚸୍ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛻䛚䛡䜛䜽䝸䝑䜽 ᛂ䛷䛿䚸ேᕤ᰾㓟䛾ẕ᰾䛾ᵓ㐀ཬ䜃᰾㓟䜸䝸䝂䝬䞊䛻ᑟධ䛩䜛⨨䛜ᛂຠ⋡ 䛻䛝䛟ᙳ㡪䛩䜛䛣䛸䛜♧၀䛥䜜䛯䚹䛣䜜䛿䚸ேᕤ᰾㓟䛾࿘ᅖ䛻Ꮡᅾ䛩䜛᰾㓟ሷᇶ䛸 䜰䝆䝗䛾ⰾ㤶⎔䛸䛾 π-π 䝇䝍䝑䜻䞁䜾┦స⏝䜔䚸ME⢾㒊䛾ᰂ㌾ᛶ䛻䜘䜛䜰䝉䝏䝺䞁 㒊䛾✺ฟ➼ 38) 䛜䜽䝸䝑䜽ᛂ䛾ຠ⋡䛻ᙳ㡪䛧䛯䛸⪃䛘䜙䜜䜛䠄Fig. 4-3䠅䛜䚸⌧ẁ㝵 䛷䛿䚸䛭䛾せᅉ䛾ヲ⣽䛿᫂䛷䛒䜛䚹 Fig. 4-3. ME䜢䜢᭷䛩䜛୍ᮏ㙐DNA 䛾ศᏊ䝰䝕䝹 ON 12 ON 13 ON 14
38 ➨ ➨୕⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛ᮏ㙐RNA 䛾⇕ⓗᏳᐃᛶ ᮏ㙐DNA ᰾㓟䜸䝸䝂䝬䞊䛸ྠ䛨䜘䛖䛻䚸MEཬ䜃BE䜢᭷䛩䜛ᮏ㙐RNA ᰾㓟䜸 䝸䝂䝬䞊䛾⇕ⓗᏳᐃᛶ䜢ホ౯䛩䜛䛯䜑䚸ᑐᛂ䛩䜛┦⿵㙐ON 24䠄rA15䠅䛸ᮏ㙐䜢ᙧᡂ 䛧䚸Tm್䜢 ᐃ䛧䛯䠄Fig. 4-4䠅䚹 Fig. 4-4. ᮏ㙐 RNA 䛾⇕ⓗᏳᐃᛶ
䛭䛾⤖ᯝ䚸ኳ↛ᆺON 25䠄rU15䠅/ON 24䠄rA15䠅䛸ẚ㍑䛧䛶䚸MEཬ䜃BE䜢ᮎ➃䛻᭷䛩
䜛ON 26䠄ME-rU14䠅/ON 24䠄rA15䠅䚸ON 29䠄BE-rU14䠅/ON 24䠄rA15䠅䚸ON 28䠄rU14-ME䠅/
ON 24䠄rA15䠅䚸ON 31䠄rU14-BE䠅/ON 24䠄rA15䠅䛾Tm್䛻䛿䚸䜋䛸䜣䛹ᕪ䛜ぢ䜙䜜䛺䛛䛳
䛯䚹୍᪉䚸䛣䜜䜙ேᕤ᰾㓟䜢ෆ㒊䛻᭷䛩䜛ON 27䠄rU7-ME-rU7䠅/ON 24䠄rA15䠅䚸
ON 30(rU7-BE-rU7)/ON 24(rA15)䛾 Tm್䛿䚸ኳ↛ᆺON 25䠄rU15䠅/ON 24䠄rA15䠅䛸ẚ㍑
䛧䛶ᖜ䛻పୗ䛧䛯䚹 ᅇタィ䛧䛯 ME䜢ෆ㒊䛻᭷䛩䜛ON 27䠄rU7-ME-rU7䠅/ON 24
䠄rA15䠅䛾Tm್䛿䚸BE䜢ෆ㒊䛻᭷䛩䜛ON 30䠄rU7-BE-rU7䠅/ON 24䠄rA15䠅䛸ẚ㍑䛧䛶ᑡ
䛧㧗䛔್䜢♧䛧䛯䚹
௨ୖ䛾 DNA ᆺ䚸RNA ᆺ䛾ẚ㍑䛛䜙䚸MEᡈ䛔䛿 BE䜢ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛾ᮎ
39
䛧䛛䛧䚸ME䛒䜛䛔䛿 BE䜢ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛾୰ኸ㒊䛻ᑟධ䛧䛯ሙྜ䚸ᮏ㙐䛾
⇕ⓗᏳᐃᛶ䜢ୗ䛢䜛䛜䚸ᅇタィ䛧䛯ME䛿BE䛸ẚ㍑䛧䛶ᮏ㙐ᙧᡂ䛾Ᏻᐃ
ຠᯝ䛜ᑠ䛥䛔䛣䛸䛜♧၀䛥䜜䚸➨❶➨୍⠇䛾ศᏊ䝰䝕䝹䛾ຊሙィ⟬䛻䜘䜛ண 䛸 ୍⮴䛧䛯䚹
40 ➨
➨ᅄ⠇㻌 䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺேᕤ᰾㓟䜢᭷䛩䜛ᮏ㙐RNA 䛷䛾䜽䝸䝑䜽ᛂ
➨୍㡯 ᮏ㙐RNA 䛷䛾䜽䝸䝑䜽ᛂ
㻌 ᮏ㙐 RNA 䛾䜽䝸䝑䜽ᛂ䛜ຠ⋡ⓗ䛻㐍⾜䛧 䛯䛣䛸䛛䜙䚸ྠ᮲௳ୗ䚸ON 27
䠄rU7-ME-rU7䠅/ON 24䠄rA15䠅䛸4-䝣䝹䜸䝻䝧䞁䝆䝹䜰䝆䝗䛸䛾䜽䝸䝑䜽ᛂ䠄Scheme 4-1䠅
䜢⾜䛔䚸➨୕❶➨⠇䛻᳨ウ䛧䛯ᮏ㙐DNA 䛾䜽䝸䝑䜽ᛂ䛸ᛂຠ⋡䜢䛸ẚ㍑䛧䛯 䠄Fig. 4-5䠅䚹 Scheme 4-1. ୰ኸ㒊䛻 ME䜢᭷䛩䜛ᮏ㙐RNA 䛾䜽䝸䝑䜽ᛂ Fig. 4-5. ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛸䛾 4-䝣䝹䜸䝻䝧䞁䝆䝹䜰䝆䝗䛸䛾䜽䝸䝑䜽ᛂຠ⋡䛾 ẚ㍑ 㻌 䛭䛾⤖ᯝ䚸ᮏ㙐 RNA 䛷䛿䚸ᛂຠ⋡䛜ᖜ䛻పୗ䛧䛯䚹䛣䜜䛿䚸➨භ⠇䛾᳨ ウ⤖ᯝ䛷䚸୰ኸ㒊䛻ME䜢᭷䛩䜛ᮏ㙐RNA 䛾 Tm್䛜ప䛛䛳䛯䛯䜑䛷䛒䜛䛸⪃䛘䜙 䜜䜛䚹䜽䝸䝑䜽ᛂ๓䚸䜽䝸䝑䜽ᛂᚋ䛾ᮏ㙐䛜㟁ẼὋື䛻䜘䜚☜ㄆ䛥䜜䛯䠄Fig. 4-6䠅䚹
41 ➨
➨㡯 䝬䜾䝛䝅䜴䝮䜲䜸䞁ῧຍ䛻䜘䜛ຠᯝ
ᮏ㙐 RNA 䛾Ᏻᐃ䜢ᅗ䜚䚸㈇㟁Ⲵ䛾୰䛻䜘䜛ᮏ㙐Ᏻᐃస⏝䛜▱䜙䜜䛶
䛔䜛Mg2+䜢ῧຍ䛧䚸ᮏ㙐RNA 䛾⇕ⓗᏳᐃᛶ䜢ホ౯䛧䛯䡝51-54) ᮏ㙐 RNA ᰾㓟䜸
䝸䝂䝬䞊ON 25䠄rU15䠅/ON 24䠄rA15䠅䚸ON 27䠄rU7-ME-rU7䠅/ON 24䠄rA15䠅䚸ON 30
䠄rU7-BE-rU7䠅/ON 24䠄rA15䠅䜢⏝䛔䛶䚸Tm್ ᐃ⏝0.01 M 䝸䞁㓟⦆⾪ᾮ䛻 5 mM 䛾
MgCl2䜢ῧຍ䛧䚸Tm್䜢 ᐃ䛧䛯䠄Fig. 4-6䠅䚹䛭䛾⤖ᯝ䚸Mg2+䜢ῧຍ䛻䜘䜚䚸䛭䜜䛮䜜
Tm್䛜4.1 ºC [ON 25䠄rU15䠅/ON 24䠄rA15䠅]ࠊ4.1 ºC [ON 27䠄rU7-ME-rU7䠅/ON 24
䠄rA15䠅]䚸2.5 ºC [ON 30䠄rU7-BE-rU7䠅/ON 24䠄rA15䠅] ୖ᪼䛧䛯䚹
Fig. 4-6. ᮏ㙐᰾㓟䜸䝸䝂䝬䞊ᑐ䛩䜛䝬䜾䝛䝅䜴䝮䜲䜸䞁䛾ຠᯝ
Mg2+䛾ῧຍ䛻䜘䜚ME䜢୰ኸ㒊䛻᭷䛩䜛ᮏ㙐RNA 䛾 Tm್䛜ᐊ ⛬ᗘ䛻ୖ᪼䛧
䛯䛣䛸䛛䜙䚸䜽䝸䝑䜽ᛂ䛾᭱㐺᮲௳ୗ䛷5 mM 䛾 MgCl2䜢ῧຍ䛧䚸ᮏ㙐RNA [ON
27䠄rU7-ME-rU7䠅/ON 24䠄rA15䠅] 䛸 4-䝣䝹䜸䝻䝧䞁䝆䝹䜰䝆䝗䛸䛾䜽䝸䝑䜽ᛂ䜢᳨ド䛧
䛯䠄Scheme 4-2䠅䚹䛭䛧䛶䚸➨ᅄ❶➨୍㡯䛻᳨ウ䛧䛯ᮏ㙐 RNA 䛾䜽䝸䝑䜽ᛂ䛸ᛂ
42 Scheme 4-2. Mg2+ῧῧຍᚋ୰ኸ㒊䛻MEࢆ᭷ࡍࡿᮏ㙐RNA ࡢࢡࣜࢵࢡᛂ Fig. 4-7. ᮏ㙐 RNA 䛾䜽䝸䝑䜽ᛂຠ⋡䛾ẚ㍑ Mg2+䛾ῧຍ䛻䜘䜚䚸ᮏ㙐RNA 䛜ᑡ䛧Ᏻᐃ䛧䛯䜒䛾䛾䚸ᮏ㙐 RNA 䛸 4-䝣䝹䜸 䝻䝧䞁䝆䝹䜰䝆䝗䛸䛾䜽䝸䝑䜽ᛂ䜢ຠ⋡ⓗ䛻㐍⾜䛥䛫䜛䛣䛸䛜䛷䛝䛺䛛䛳䛯䚹䜽䝸䝑䜽 ᛂ๓䚸䜽䝸䝑䜽ᛂᚋ䛾ᮏ㙐䛜㟁ẼὋື䛻䜘䜚☜ㄆ䛥䜜䛯䠄Fig. 4-8䠅䚹 Fig. 4-8. ᮏ㙐 RNA 䛾㟁ẼὋື⏬ീ dsRNA dsRNA (mg2+) Substrate Consumption (%)
43
➨
➨❶㻌 ⤖ㄽ
㏆ᖺ䚸་Ꮫ䚸ศᏊ⏕≀Ꮫ䛾┠ぬ䜎䛧䛔Ⓨᒎ䛻䜘䜚䚸㑇ఏᏊ⒪䜔⏕་⒪䛾ᐇ⏝ 䛻䜒㐨䛜㛤䛛䜜䛴䛴䛒䜛䚹་⸆ရ䛾㛤Ⓨ䛻┠䜢ྥ䛡䜛䛸䚸పศᏊ་⸆䜔ᢠయ་⸆ 䛻⥆䛟ḟୡ௦䛾་⸆䛸䛧䛶䚸᰾㓟་⸆䛻䛝䛺ᮇᚅ䛜ᐤ䛫䜙䜜䛶䛔䜛䚹䛧䛛䛧䚸᰾㓟 ་⸆䛻䛿䚸㠀≉␗ⓗ䛺స⏝䛾ㄏⓎ䚸䝚䜽䝺䜰䞊䝊䛻䜘䜛ศゎ䚸ప䛔⣽⬊⭷㏱㐣ᛶ ➼䛾ḞⅬ䛜䛒䜛䛯䜑䚸⸆◊✲䛜㞴⯟䛧䛶䛔䜛䚹᰾㓟་⸆ရ䛾᭷ຠᛶ䞉Ᏻᛶ䜢ྥ ୖ䛥䛫䜛䛯䜑䛻䛿䚸㐺ษ䛺Ꮫಟ㣭䜔䝗䝷䝑䜾䝕䝸䝞䝸䞊䝅䝇䝔䝮䛾㛤Ⓨ䛜ᚲせ䛷䛒䜛䚹 ࡲࡓࠊ᰾㓟࢜ࣜࢦ࣐࣮ࢆ་⸆ရࡋ࡚㛤ⓎࡍࡿࡣࠊPET ࡼࡿ⸆≀ືែゎᯒ ࡀ㔜せ࡛࠶ࡿࠋ ᰾㓟་⸆ရ䛾 PET 䝷䝧䝹䜢┠ᣦ䛧䛯◊✲䛜ከᩘ⾜䜟䜜䛶䛔䜛䛜䚸䛭䛾䜋䛸䜣䛹 䛜᰾㓟࢜ࣜࢦ࣐࣮ࡢᮎ➃ࢆࢱ࣮ࢤࢵࢺࡋ࡚࠸ࡿࠋⴭ⪅ࡀᡤᒓࡍࡿ◊✲ᐊ࡛ࡣࠊ 䜶䝏䝙䝹䝧䞁䝊䞁㦵᱁䛛䜙䛺䜛ேᕤ᰾㓟 5-Ethynyl-1,3-benzenedimethanol䠄BE䠅䜢㛤 Ⓨ䛧䛯䚹BE䜢ᮎ➃䛻ᑟධ䛧䛯᰾㓟䜸䝸䝂䝬䞊䜢⏝䛔䛶䜰䝆䝗ྜ≀䛸䛾 CuAAC ᛂ ᮲௳䜢ヲ⣽䛻᳨ウ䛧䚸0.1 M 䝸䞁㓟⦆⾪ᾮ୰䚸㓄Ꮚ㠀Ꮡᅾୗ䚸ᐊ 䛷ຠ⋡ⓗ䛻㐍 ⾜䛩䜛䛣䛸䜢ぢฟ䛧䚸᰾㓟䜸䝸䝂䝬䞊䛾 PET 䝷䝧䝹䛻ᛂ⏝䛷䛝䜛ྍ⬟ᛶ䜢♧䛧䛯䠄➨ ୍❶➨ᅄ⠇䠅䚹ࡋࡋࠊsiRNA ࡞ࡢᮏ㙐᰾㓟࢜ࣜࢦ࣐࣮࡛ࡣࠊ3’ᮎ➃ࡢࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ㒊ࡣࠊ᰾㓟࢜ࣜࢦ࣐࣮ࡀRISC ྲྀࡾ㎸ࡲࢀࠊRISC ࡢ PAZ ࢻ
࣓ࣥࡢỈᛶ࣏ࢣࢵࢺࢆㄆ㆑ࡋࠊ⤖ྜࡍࡿࡢ㔜せ࡞ᙺࢆᯝࡓࡍࠋࡲࡓࠊ5’ ᮎ➃ࡢࣜࣥ㓟ᇶࡣࠊ᰾㓟࢜ࣜࢦ࣐࣮ࡀRISC ࡢ Mid ࢻ࣓ࣥㄆ㆑ࡉࢀࠊᅛᐃࡉ ࢀࡿࡢ㔜せ࡞ᙺࢆᯝࡓࡍࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡑࡇ࡛ࠊⴭ⪅ࡣ᰾㓟࢜ࣜ ࢦ࣐࣮ࡢᮎ➃ࡢᵓ㐀ࢆኚ࠼ࡎࠊෆ㒊࡛ࡢ PET ࣛ࣋ࣝࡸᏛಟ㣭ࢆ┠ᣦࡋࠊศ Ꮚࣔࢹࣝࡼࡿຊሙィ⟬ࢆ⾜࠸ࠊᮏ㙐ࡢ⇕ⓗᏳᐃᛶᙳ㡪ࡀᑡ࡞࠸⪃࠼ࡽ ࢀࡿ(S)-4-Ethynylmandelol䠄ME䠅ࢆタィࡋࡓ㸦➨❶➨୍⠇ࠊ➨⠇㸧ࠋࡲࡓࠊࡇ
44 ࢀࡲ࡛ࡢ▱ぢࢆᇶࠊ䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺ䜾䝸䝁䞊䝹ME䜢᰾㓟䜸䝸䝂䝬䞊䛾௵ ព䛾⨨䛻ᑟධ䛷䛝䜛ヨ⸆䛸䛧䛶䜰䝭䝎䜲䝖య 8 䛸 CPG ᶞ⬡ 9 䜢㛤Ⓨ䛧䛯 (➨❶➨ ୕⠇)䚹䛥䜙䛻䚸BE 䜢ẚ㍑ᑐ㇟䛻䛧䚸15 mer 䛾▷㙐᰾㓟䜸䝸䝂䝬䞊䜢⏝䛔䛶䚸4-䝣䝹 䜸䝻䝧䞁䝆䝹䜰䝆䝗䛸䜽䝸䝑䜽ᛂ䜢⾜䛔䚸䛭䛾ᛶ㉁䜢᳨ウ䛧䛯䠄➨୕❶䚸➨ᅄ❶䠅䚹 ᙜ◊✲ᐊ䛷ぢฟ䛧䛯᰾㓟䜸䝸䝂䝬䞊䛾䜽䝸䝑䜽ᛂ䛾᮲௳ୗ䚸MEཬ䜃BE䜢ᑟධ䛧 䛯୍ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛷䛾䜽䝸䝑䜽ᛂ䜢᳨ウ䛧䛯⤖ᯝ䚸DNA ᆺ䚸RNA ᆺ䛻㛵ಀ 䛺䛟䚸ேᕤ᰾㓟䛜ᮎ➃䜘䜚䜒୰ኸ㒊䛻Ꮡᅾ䛧䚸ୟ䛴BE䜘䜚䜒ME䛾䜽䝸䝑䜽ᛂ䛜㐍⾜䛧 䜔䛩䛔䛸䛔䛖⤖ᯝ䜢ᚓ䛯䚹䛣䜜䜙䛾▱ぢ䛛䜙䚸୍ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛻ᑟධ䛧䛯ேᕤ ᰾㓟䛾ẕ᰾䛾ᵓ㐀ཬ䜃⨨䛜ᛂຠ⋡䛻䛝䛟ᙳ㡪䛩䜛䛣䛸䛜♧၀䛥䜜䛯䠄➨୕❶ ➨୕⠇䚸➨ᅄ❶➨⠇䠅䚹 ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛾 50%⼥ゎ ᗘ䠄Tm䠅䛾 ᐃ䛛䜙䚸ME 䜢୰ኸ㒊䛻ᑟධ䛧䛯 ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛿䚸ኳ↛ᆺ䛸ẚ㍑䛧䛶పୗ䛧䛯䜒䛾䠄DNA ᆺ䛜⣙ 10 ºC䚸RNA ᆺ䛜⣙ 12 ºC䠅䛾䚸BE 䛾ሙྜ䛸ẚ㍑䛧䛶㧗䛔್䜢♧䛧䛯䠄➨୕❶➨ᅄ⠇䚸➨ᅄ❶➨୕ ⠇䠅䚹䛣䛾⤖ᯝ䛛䜙䚸ᅇྜᡂ䛧䛯ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛻䛚䛔䛶䚸ேᕤ᰾㓟䛾ẕ᰾ 䛾ᵓ㐀䛜㔜䜙䛫䜣ᵓ㐀䛾Ᏻᐃᛶ䛻䜒䛝䛟ᙳ㡪䛩䜛䛸䛔䛖䛣䛸䛜♧၀䛥䜜䛯䚹䜎䛯䛣 䛾⤖ᯝ䛿䚸ศᏊ䝰䝕䝹䛻䜘䜛ຊሙィ⟬䛛䜙ኳ↛ᆺ䛸ᕪ䛺䛔㓟⣲ཎᏊ㛫㊥㞳䜢ಖ䛴 䛣䛸䛜ᮇᚅ䛥䜜䛯 ME 䛜䚸BE 䛸ẚ㍑䛧䛶䚸䜘䜚Ᏻᐃ䛺ᮏ㙐䜢ᙧᡂ䛩䜛䛸䛔䛖ศᏊタィ 䛾ண䜢ᨭᣢ䛩䜛䜒䛾䛸䛺䛳䛯䚹 ୍ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛷䛾䜽䝸䝑䜽ᛂ䛸ྠ᮲௳ୗ䚸䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺ䜾䝸 䝁䞊䝹 ME 䜢᭷䛩䜛ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛾䜽䝸䝑䜽ᛂ䜢᳨ド䛧䛯䛸䛣䜝䚸ᮏ㙐䛾 ≧ែ䛷䜒䜽䝸䝑䜽ᛂ䛜㐍⾜䛩䜛䛣䛸䛜♧၀䛥䜜䛯䠄➨୕❶➨⠇䚸➨ᅄ❶➨ᅄ⠇䠅䚹 ௨ୖ䛾䛣䛸䛛䜙䚸ᅇ㛤Ⓨ䛧䛯䜶䝏䝙䝹䝧䞁䝊䞁⨨ᆺ䜾䝸䝁䞊䝹ME䛿䚸ᙜ◊✲ᐊ 䛷㛤Ⓨ䛧䛯BE䜢ᨵၿ䛩䜛䛣䛸䛜䛷䛝䚸䜽䝸䝑䜽ᛂ䜢⏝䛔䛯᰾㓟䜸䝸䝂䝬䞊䛾PET 䝷䝧 䝹䚸≉䛻ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛾ෆ㒊䛷䛾PET 䝷䝧䝹䛻ᛂ⏝䛷䛝䜛䛣䛸䛜♧၀䛥
45 䜜䛯䚹䜎䛯䚸ME 䛿䚸᰾㓟࢜ࣜࢦ࣐࣮ࡢ௵ពࡢ⨨ᑟධ࡛ࡁࡿࡇࡽࠊ⮬⏤࡞ ศᏊタィࡀ⾜࠼ࡿࠋࡉࡽࠊ✀ࠎࡢࢪࢻྜ≀ࢆຍࡉࡏࡿࡇ࡛ࠊ᪂つࡢ ࢻࣛࢵࢢࢹࢨࣥࡸ⺯ගࣉ࣮ࣟࣈ࡞ PET ᶆ㆑௨እࡢ⏝㏵ࡶᮇᚅ࡛ࡁࡿࠋ ᚑࡗ࡚ࠊᮏ◊✲ࡣࠊsiRNAࠊmiRNAࠊࣥࢳࢭࣥࢫࠊࣉࢱ࣐࣮࡞ࠊᵝࠎ࡞ ᰾㓟་⸆ࢆ㛤Ⓨࡍࡿୖ࡛ࠊᚲ㡰࡛࠶ࡿ⸆≀ືែゎᯒ࠾࠸࡚㔜せ࡞᰾㓟ศᏊࡢ PET ࣛ࣋ࣝࡸ⺯ගࣛ࣋ࣝ࡞㐺⏝ྍ⬟࡞ࣘࢽࣂ࣮ࢧࣝ࡞ᶆ㆑ἲࢆᥦ౪ ࡍࡿࡶࡢ⪃࠼ࡿࠋ
46
ᐇ
ᐇ㦂䛾㒊
1. ⏝ᶵჾཬ䜃⏝ヨ⸆ 1-1. ᐃᶵჾ NMR 䝇䝨䜽䝖䝹*1 JEOL JNM-ECS400᰾㓟⮬ືྜᡂᶵ Applied Biosystems Model 3400
HPLC SHIMADZU
DGU-20A3R prominence® DEGASSING UNIT
LC-20AD prominence® LIQUID CHROMATOGRAPH CTO-20A prominence® COLUMN OVEN
CBM-20A prominence® COMMUNICATIONS BUS MODULE
SPD-20A prominence® UV/VIS DETECTOR FRC-10A FRACTION COLLECTOR
྾ගᗘィ HITACHI U-2001 Spectrophotometer
᪕ගィ HORIBA HIGH SENSITIVE POLARIMETER SEPA-300
MALDI-TOF MS SHIMADZU AXIMA-CFR plus
Tm ᐃᶵ
SHIMADZU UV-2450 UV-VISIBLE SPECTROPHOTOMETER
UV 䝷䞁䝥 AS ONE Handy UV Lamp SLUV-6 (254/365 nm)
47 *1 1H NMR 䛾Ꮫ䝅䝣䝖್䛿 CDCl3䛷䛿 SiMe4䜢ෆ㒊ᶆ‽≀㉁䛸䛧䚸CD3OD 䛷䛿 ⁐፹୰䛻ṧᏑ䛩䜛䝥䝻䝖䞁䛻䜘䜛྾ (δ 3.31) 䜢ෆ㒊ᶆ‽䛸䛧䛶 ppm ༢䛷⾲♧䛧 䛯䚹13C NMR 䛾Ꮫ䝅䝣䝖್䛿⁐፹䛾྾ (CDCl3: δ 77.0; CD3OD: δ 49.0) 䜢ෆ㒊 ᶆ‽䛸䛧䛶ppm ༢䛷⾲♧䛧䛯䚹31P NMR 䛾Ꮫ䝅䝣䝖್䛿 H3PO4/CDCl3䜢ෆ㒊ᶆ ‽≀㉁䛸䛧䛶ppm ༢䛷⾲♧䛧䛯䚹 1-2. ྛྛ✀䜽䝻䝬䝖䜾䝷䝣䜱䞊ᢸయ
TLC Merck Silica gel 60 F254 plates
䝅䝸䜹䝀䝹 Silica Gel 60 (spherical) (㛵ᮾᏛ; 63-210 μm)
Sep-Pak® tC18 Waters Corporation
HPLC ⏝㏫┦䜹䝷䝮 Inertsil® ODS-3 (GL Sciences Inc.; 5 μm, 4.6 × 150 mm)
1-3. ྛ✀䝣䜱䝹䝍䞊
Ỉ⣔ MILLIPORE Millex®-HV
Ỉ䞉᭷ᶵ⣔ MILLIPORE Millex®-LG䚸Millicup®-LH
1-4. ⏝ヨ⸆
᭷ᶵྜᡂ⏝ヨ⸆ཬ䜃⁐፹
䛆Aldrich䛇 Bis(triphenylphosphine)palladium( ϩ ) Dichloride 䚸 Silver(Ϩ) Oxide
48
䛆䜻䝅䝎Ꮫ䛇 CH2Cl2 (୍ᩯ⨁)䚸Pyridine*1
䛆ᮾிᡂ䛇 L-(+)-Mandelic Acid䚸Trifluoromethanesulfonic Acid䚸
Trimethylsilylacetylene
䛆䝖䜽䝲䝬䛇 䜽䝻䝻䝩䝹䝮 (୍ᩯ⨁)
䛆䝘䜹䝷䜲䝔䝇䜽䛇 Potassium Carbonate䚸Sulfuric Acid䚸ள◲㓟䝘䝖䝸䜴䝮䚸
䜰䞁䝰䝙䜰Ỉ (28%)䚸Ⅳ㓟Ỉ⣲䝘䝖䝸䜴䝮䚸 ↓Ỉ◲㓟䝘䝖䝸䜴䝮
䛆ග⣧⸆䛇 2-Cyanoethyl Diisopropylchlorophosphoramidite䚸
N,N-Diisopropylethylamine䚸4,4'-Dimethoxytrityl Chloride䚸
EtOH (Dehydrated)䚸Iodine䚸MeOH (୍ᩯ⨁)䚸Piperidine䚸 Sodium Tetrahydroborate䚸㓑㓟䜶䝏䝹 (୍ᩯ⨁)䚸 䝦䜻䝃䞁 (୍ᩯ⨁) 䛆䛭䛾䛇 EtOH (୍ᩯ⨁) *1 Pyridine 䛿Ỉ⣲䜹䝹䝅䜴䝮䛻䜘䜚␃䜢⾜䛔䚸䝰䝺䜻䝳䝷䞊䝅䞊䝤 4A ඹᏑୗಖ Ꮡ䛧䛯䜒䛾䜢⏝䛔䛯䚹 NMR ⏝㔜⁐፹
䛆Cambridge Isotope Laboratories, Inc.䛇CDCl3 (SiMe4ྵ᭷)䚸CD3OD
䜸䝸䝂䝚䜽䝺䜸䝏䝗䛾ྜᡂཬ䜃⢭〇
䛆Aldrich䛇 DMT-2'O-TBDMS-rA(tac) Phosphoramidite䚸
DMT-2'O-TBDMS-rU Phosphoramidite䚸 3-HPA䚸1.0 M TBAF in THF
49
䛆Glen research䛇 dA-CE Phosphoramidite 䚸 dT-CE Phosphoramidite 䚸 dA-CPG䚸
dT-CPG䚸Cap Mix A䚸Cap Mix B䚸Oxidizing Solution
䛆䜻䝅䝎Ꮫ䛇 䛟䛘䜣㓟Ỉ⣲䜰䞁䝰䝙䜴䝮䚸䝖䝸䜽䝻䝻㓑㓟 䛆ྠோ䛇 EDTA·2Na䚸䜰䝉䝖䝙䝖䝸䝹 (ྜᡂ⏝⬺Ỉ⁐፹)䚸 1H-䝔䝖䝷䝌䞊䝹 (᰾㓟ྜᡂ⏝) 䛆䝘䜹䝷䜲䝔䝇䜽䛇 EDTA·4Na䚸N,N'-䝯䝏䝺䞁䝡䝇䜰䜽䝸䝹䜰䝭䝗䚸XC䚸 䜰䜽䝸䝹䜰䝭䝗䚸䜰䞁䝰䝙䜰Ỉ (28%)䚸䜶䝍䝜䞊䝹 (99.5%)䚸 㓑㓟䚸䝖䝸䜶䝏䝹䜰䝭䞁䚸ᒀ⣲ 䛆ග⣧⸆䛇 APS䚸BPB䚸TEMED䚸䜰䝉䝖䝙䝖䝸䝹 (᰾㓟ྜᡂ⏝)䚸 䝆䜽䝻䝻䝯䝍䞁 (䝨䝥䝏䝗ྜᡂ⏝)䚸 䝖䝸䝇(䝠䝗䝻䜻䝅䝯䝏䝹)䜰䝭䝜䝯䝍䞁䚸䜋䛖㓟䚸 䝩䝹䝮䜰䝭䝗 㧗㏿䜽䝸䝑䜽ᛂ⏝ヨ⸆ཬ䜃⁐፹
䛆Aldrich䛇 (+)-Sodium L-ascorbate
䛆䝖䜽䝲䝬䛇 䜽䝻䝻䝩䝹䝮 (୍ᩯ⨁)
䛆䝘䜹䝷䜲䝔䝇䜽䛇 Copper(ϩ) Sulfate Pentahydrate
䛆ග⣧⸆䛇 DMSO (Dehydrated)䚸MeCN (᰾㓟ྜᡂ⏝)䚸
䝯䝍䝜䞊䝹 (୍ᩯ⨁)䚸䜚䜣㓟Ỉ⣲䝘䝖䝸䜴䝮䚸 䜚䜣㓟Ỉ⣲䝘䝖䝸䜴䝮
50
Tm ᐃ⏝ヨ⸆ཬ䜃⁐፹
䛆䝘䜹䝷䜲䝔䝇䜽䛇 ሷ䝬䜾䝛䝅䜴䝮භỈ≀
䛆ග⣧⸆䛇 ሷ䝘䝖䝸䜴䝮䚸䜚䜣㓟Ỉ⣲䝘䝖䝸䜴䝮䚸
51
2. ᐇᐇ㦂䛾ヲ⣽
2-1. ➨❶➨⠇䛻㛵䛩䜛ᐇ㦂
Scheme 2-5 䛻㛵䛩䜛ᐇ㦂
Ethyl (S)-mandelate (3) 䛾ྜᡂ
500 mL 䝘䝇䝣䝷䝇䝁䛻L-(+)-mandelic acid (15.0 g, 98.6 mmol) 䜢ຍ䛘䚸EtOH (150
mL) 䛻⁐ゎ䛧䚸sulfuric acid (1.5 mL) 䜢ୗᚋ䚸70 ºC 䛷 23 㛫᧠ᢾ䛧䛯䚹TLC 䛷
ᛂ䛾㐍⾜䜢☜ㄆ䛧䛯ᚋ䚸⁐፹䜢ῶᅽ␃ཤ䛧䚸ṧ´䜢䝆䜽䝻䝻䝯䝍䞁 (100 mL × 3) 䛸Ỉ
(100 mL) 䛷ᢳฟ䛧䛯䚹䛭䛾ᚋ䚸䝆䜽䝻䝻䝯䝍䞁ᒙ䜢ྜ䜟䛫䚸㣬Ⅳ㓟Ỉ⣲䝘䝖䝸䜴䝮Ỉ
⁐ᾮ (100 mL) 䛷Ὑί䛧䛯䚹䛥䜙䛻䚸㣬Ⅳ㓟Ỉ⣲䝘䝖䝸䜴䝮Ỉ⁐ᾮᒙ䜢䝆䜽䝻䝻䝯䝍
䞁 (100 mL × 2) 䛷ᢳฟ䛧䚸䝆䜽䝻䝻䝯䝍䞁ᒙ䜢ྜ䜟䛫䚸↓Ỉ◲㓟䝘䝖䝸䜴䝮䛷⇱ᚋ䚸
⁐፹䜢ῶᅽ␃ཤ䛧䛯䚹䛭䛧䛶䚸ethyl (S)-mandelate (3) (17.3 g, 95.7 mmol, 97%) 䜢↓ Ⰽ䜸䜲䝹䛸䛧䛶ᚓ䛯䚹1H NMR (400 MHz, CDCl3) δ 7.42-7.27 (m, 5H), 5.15 (d, J = 4.0
Hz, 1H), 4.27-4.10 (m, 2H), 3.68 (d, J = 4.0 Hz, 1H), 1.20 (t, J = 8.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 173.5, 138.4, 128.4, 128.2, 126.4, 72.8, 62.0, 13.9; MS (DRAT)
m/z 180 [M+H]+, HRMS (DART) Calcd for C10H12O3 [M+H]+: 180.2005. Found:
180.2017.
Scheme 2-7 䛻㛵䛩䜛ᐇ㦂
Ethyl (S)-4-iodomandelate (4) 䛾ྜᡂ
200 mL 䝘䝇䝣䝷䝇䝁䛻 ethyl (S)-mandelate (3) (3.20 g, 17.8 mmol)䚸iodine (4.50 g, 17.7 mmol, 1.0 eq.) ཬ䜃 silver trifluoromethanesulfonate (4.56 g, 17.7 mmol, 1.0 eq.)
䜢ຍ䛘䚸CH2Cl2 (80 mL) 䛻ᠱ⃮䛧䚸㐽ග䛧䛶ᐊ ୗ 3 㛫᧠ᢾ䛧䛯䚹TLC 䛷ᛂ䛾 㐍⾜䜢☜ㄆ䛧䛯ᚋ䚸᱒ᒣ䜝㐣䛻䜘䜚䚸ሷ䜢㝖ཤ䛧䚸䜝ᾮ䜢5%䜰䞁䝰䝙䜰Ỉ (100 mL × 2)䚸5%ள◲㓟䝘䝖䝸䜴䝮Ỉ⁐ᾮ (100 mL × 2) ཬ䜃Ỉ (100 mL × 2) 䛷ᢳฟ䛧䛯䚹䝆䜽 䝻䝻䝯䝍䞁ᒙ䜢ྜ䜟䛫䚸↓Ỉ◲㓟䝘䝖䝸䜴䝮䛷⇱ᚋ䚸⁐፹䜢ῶᅽ␃ཤ䛧䛯䚹ṧ´䜢䝅䝸 䜹䝀䝹䜹䝷䝮䜽䝻䝬䝖䜾䝷䝣䜱䞊 (䝦䜻䝃䞁 : 㓑㓟䜶䝏䝹 = 8 : 1) 䛻䛶⢭〇䛧䚸ethyl (S)-4-iodomandelate (4) (3.71 g, 12.1 mmol, 68%) 䜢〓Ⰽ䜸䜲䝹䛸䛧䛶ᚓ䛯䚹1H NMR
52
(400 MHz, CDCl3) δ 7.67 (d, J = 8.5 Hz, 2H), 7.16 (d, J = 8.2 Hz, 2H), 5.09 (s, 1H),
4.25-4.12 (m, 2H), 3.77 (s, 1H), 1.20 (t, J = 8.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
173.0, 138.0, 137.4, 128.3, 94.0, 72.2, 62.2, 13.9; MS (DRAT) m/z 306 [M+H]+, HRMS (DART) Calcd for C10H11IO3 [M+H]+: 306.0907. Found: 306.0955; Anal. Calcd for
C10H11IO3: C, 39.24; H, 3.62. Found: C, 39.37; H, 3.53%.
Scheme 2-8 䛻㛵䛩䜛ᐇ㦂
(S)-4-Iodomandelol (5) 䛾䛾ྜᡂ
100 mL 䝘䝇䝣䝷䝇䝁䛻 ethyl (S)-4-iodomandelate (4) (3.00 g, 9.80 mmol) 䜢ຍ䛘䚸 EtOH (20 mL) 䛻⁐ゎ䛧䛯䚹ị෭ୗ sodium tetrahydroborate (740 mg, 19.6 mmol, 2.0 eq.) 䜢䜖䛳䛟䜚ຍ䛘䚸ᐊ ୗ 1 㛫ᨩᢾ䛧䛯䚹TLC 䛷ᛂ䛾⤖䜢☜ㄆ䛧䛯ᚋ䚸2N H2SO4䛷୰䛧䚸䛧䜀䜙䛟᧠ᢾ䛧䛯䚹ᛂᾮ䜢㓑㓟䜶䝏䝹 (100 mL) 䛸Ỉ (50 mL × 2) 䛷ᢳฟ䛧䚸㓑㓟䜶䝏䝹ᒙ䜢ྜ䜟䛫䚸↓Ỉ◲㓟䝘䝖䝸䜴䝮䛷⇱ᚋ䚸⁐፹䜢ῶᅽ␃ཤ䛧 䛯䚹ṧ´䜢⤖ᬗ (䜽䝻䝻䝩䝹䝮) 䛻䛶⢭〇䛧䚸(S)-4-iodomandelol (5) (2.22 g, 8.41 mmol, 86%) 䜢↓Ⰽ⤖ᬗ䛸䛧䛶ᚓ䛯䚹1H NMR (400 MHz, CD3OD) δ 7.68 (d, J = 8.2 Hz, 2H), 7.17 (d, J = 8.2 Hz, 2H), 4.65-4.62 (m, 1H), 3.63-3.55 (m, 2H); 13C NMR (100 MHz, CD3OD) δ 143.2, 138.3, 129.5, 93.3, 75.2, 68.4; MS (DRAT) m/z 264 [M+H]+,
HRMS (DART) Calcd for C8H9IO2 [M+H]+: 264.9725. Found: 264.9735; Anal. Calcd
for C8H9IO2: C, 36.39; H, 3.44. Found: C, 36.50; H, 3.40%.
䜰䝉䝏䝹య(5')䛾ྜᡂ
50 mL 䝘䝇䝣䝷䝇䝁䛻(S)-4-iodomandelol (5) (1.7 g, 6.44 mmol,䜢ຍ䛘䚸dry pyridine (10 mL) 䛻⁐ゎ䛧䛯䚹ị෭ୗ↓Ỉ㓑㓟 (10 mL) 䜢䜖䛳䛟䜚ຍ䛘䚸3 㛫ᨩᢾ䛧䛯䚹 TLC 䛷ᛂ䛾⤖䜢☜ㄆ䛧䛯ᚋ䚸ᛂᾮ䜢ῶᅽ⃰⦰䛧䚸䝆䜽䝻䝻䝯䝍䞁䠄50 mL䠅䛻⁐
53 䛷2 ᅇᢳฟ䛧䛯䚹᭷ᶵᒙ䜢↓Ỉ◲㓟䝘䝖䝸䜴䝮䛷⇱ᚋ䚸⁐፹䜢ῶᅽ␃ཤ䛧䛯䚹ṧ´䜢 ⤖ᬗ (䜽䝻䝻䝩䝹䝮) 䛻䛶⢭〇䛧䚸䜰䝉䝏䝹య (5')䜢ᐃ㔞ⓗ䛻ᚓ䛯䚹1H NMR (400 MHz, CD3OD) δ 7.68 (d, J = 8.2 Hz, 2H), 7.17 (d, J = 8.2 Hz, 2H), 4.65-4.62 (m, 1H), 3.63-3.55 (m, 2H), 2.11(s, 3H), 2.02(s, 3H) Scheme 2-9 䛻㛵䛩䜛ᐇ㦂 (S)-4-(Trimethylsilylethynyl)mandelol (6) 䛾䛾ྜᡂ 200 mL 䝘䝇䝣䝷䝇䝁䛻(S)-4-iodomandelol (5) (2.16 g, 8.18 mmol) 䜢ຍ䛘䚸⬺Ẽᚋ䚸 䜰䝹䝂䞁⨨䛧䚸piperidine (40 mL) 䛻⁐ゎ䛧䛯䚹ḟ䛻䚸trimethylsilylacetylene (1.92 mL, 13.6 mmol, 1.7 eq.) 䜢 ୗ䛧 䚸䛥 䜙 䛻 bis(triphenylphosphine)palladium( ϩ ) dichloride (25.0 mg, 35.6 μmol, 0.0044 eq.) ཬ䜃 copper(I) iodide (8.90 mg, 46.7 μmol, 0.0057 eq.) 䜢ຍ䛘䚸ᐊ ୗ 4 㛫ᨩᢾ䛧䛯䚹TLC 䛷ᛂ䛾⤖䜢☜ㄆ䛧䛯ᚋ䚸䝆䜽䝻 䝻䝯䝍䞁 (100 mL) 䛸Ỉ (100 mL × 2)䚸5%䜰䞁䝰䝙䜰Ỉ (100 mL × 2) 䛷ᢳฟ䛧䛯䚹 䝆䜽䝻䝻䝯䝍䞁ᒙ䜢ྜ䜟䛫䚸↓Ỉ◲㓟䝘䝖䝸䜴䝮䛷⇱ᚋ䚸⁐፹䜢ῶᅽ␃ཤ䛧䛯䚹ṧ´ 䛿䝅䝸䜹䝀䝹䜹䝷䝮䜽䝻䝬䝖䜾䝷䝣䜱䞊 (䝦䜻䝃䞁 : 㓑㓟䜶䝏䝹 = 5 : 2) 䛻䛶⢭〇䛧䚸 (S)-4-(trimethylsilylethynyl)mandelol (6) (1.63 g, 6.95 mmol, 85%) 䜢↓Ⰽ⤖ᬗ䛸䛧䛶 ᚓ䛯䚹1H NMR (400 MHz, CD3OD) δ 7.40 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.2 Hz, 2H), 4.69-4.66 (m, 1H), 3.64-3.56 (m, 2H), 0.23 (s, 9H); 13C NMR (100 MHz, CD3OD) δ 144.1, 132.7, 127.5, 123.4, 106.3, 94.1, 75.5, 68.5 ; MS (DRAT) m/z 235 [M+H]+, HRMS (DART) Calcd for C13H18O2Si [M+H]+: 235.1154. Found: 235.1184; Anal.
Calcd for C13H18O2Si: C, 66.62; H, 7.44. Found: C, 66.63; H, 7.74%.
(S)-4-Ethynylmandelol (1) 䛾ྜᡂ 100 mL 䝘䝇䝣䝷䝇䝁䛻(S)-4-(trimethylsilylethynyl)mandelol (6) (850 mg, 3.63 mmol) 䜢ຍ䛘䚸MeOH (30 mL) 䛻⁐ゎ䛧䚸㣬Ⅳ㓟䜹䝸䜴䝮Ỉ⁐ᾮ (150 μL) 䜢ୗ 䛧䛶䚸ᐊ ୗ12 㛫᧠ᢾ䛧䛯䚹TLC 䛷ᛂ䛾⤖䜢☜ㄆ䛧䛯ᚋ䚸⁐፹䜢ῶᅽ␃ཤ䛧䚸 ṧ´䜢䝆䜽䝻䝻䝯䝍䞁 (80 mL) 䛸Ỉ (80 mL × 2) 䛷ᢳฟ䛧䛯䚹䝆䜽䝻䝻䝯䝍䞁ᒙ䜢ྜ䜟 䛫 䚸 ↓ Ỉ ◲ 㓟 䝘 䝖 䝸 䜴 䝮 䛷 ⇱ ᚋ 䚸 ⁐ ፹ 䜢 ῶ ᅽ ␃ ཤ 䛧 䛯 䚹 䛭 䛧 䛶 䚸 (S)-4-ethynylmandelol (1) (325 mg, 3.63 mmol, quant) 䜢↓Ⰽ⤖ᬗ䛸䛧䛶ᚓ䛯䚹1H NMR (400 MHz, CD3OD) δ 7.43 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 4.68 (dd,
54
1H); 13C NMR (100 MHz, CD3OD) δ 144.3, 132.9, 127.5, 122.8, 84.3, 78.4, 75.5, 68.5;
MS (DRAT) m/z 163 [M+H]+, HRMS (DART) Calcd for C10H10O2 [M+H]+: 163.0760.
Found: 163.0730; Anal. Calcd for C10H10O2: C, 73.94; H, 6.31. Found: C, 73.94; H,
![Fig. 4-6. ᮏ㙐᰾㓟䜸䝸䝂䝬䞊ᑐ䛩䜛䝬䜾䝛䝅䜴䝮䜲䜸䞁䛾ຠᯝ Mg 2+ 䛾ῧຍ䛻䜘䜚 M E 䜢୰ኸ㒊䛻᭷䛩䜛ᮏ㙐 RNA 䛾 T m ್䛜ᐊ ⛬ᗘ䛻ୖ᪼䛧 䛯䛣䛸䛛䜙䚸䜽䝸䝑䜽ᛂ䛾᭱㐺᮲௳ୗ䛷 5 mM 䛾 MgCl 2 䜢ῧຍ䛧䚸ᮏ㙐 RNA [ON 27 䠄 rU 7 -M E -rU 7 䠅 /ON 24 䠄 rA 15 䠅 ] 䛸 4- 䝣䝹䜸䝻䝧䞁䝆䝹䜰䝆䝗䛸䛾䜽䝸䝑䜽ᛂ䜢᳨ド䛧 䛯䠄Scheme 4-2䠅䚹䛭䛧䛶䚸➨ᅄ❶➨୍㡯䛻᳨ウ䛧䛯ᮏ㙐 RNA 䛾䜽䝸䝑䜽ᛂ](https://thumb-ap.123doks.com/thumbv2/123deta/10093862.1494385/46.892.135.654.525.817/Figᮏ㙐㓟䜸䝸䝂䝬䛩䜛䝬䜾䝛䝅䜴䝮䜲䜸䞁䛾ຠᯝMg䛻䜘䜚⛬ᗘ䛻.webp)
