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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://repository.lib.gifu-u.ac.jp/handle/123456789/50375
※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
┠ ḟ
⌮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
ᐇ㦂䛾㒊
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㎡ .................................................................................................................................73
➨୍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Ᏻ඲ᛶ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
1
Fig. 1-2. ᪂⸆㛤Ⓨ䛾ᡂຌ⋡䠄⣼✚ᡂຌ⋡䠅
་⸆ရ⏘ᴗ䝡䝆䝵䞁 㻞㻜㻝㻟㻌
ೃ⿵໬ྜ≀䛜⮫ᗋヨ㦂䛷୙㐺᱁䛸䛺䜛୺䛺⌮⏤䛿䚸䐟ࣄࢺ࡟࠾࠸࡚྾཰ࡀ୙༑
ศࠊ࠶ࡿ࠸ࡣ⾑୰࠿ࡽࡢᾘኻࡀᴟࡵ࡚᪩࠸࡞࡝ࡢዲࡲࡋࡃ࡞࠸ືែ≉ᛶࢆ♧ࡍ
ࡇ࡜ࠊղ἞⒪࡟᭷ຠ࡞⃰ᗘࡢ⸆๣ࡀᶆⓗ㒊఩࡟฿㐩ࡋ࡞࠸ࡓࡵࠊ᭷ຠᛶࡀಖド
ฟ᮶࡞࠸ࡇ࡜ࠊճᶆⓗ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
2
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
⛉Ꮫᢏ⾡ືྥ 㻞㻜㻝㻝 ᖺ 㻝 ᭶ྕ䛛䜙୍㒊ኚ䛘䛶ᘬ⏝㻌
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
3
➨஧⠇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ᨺᑕᛶྠ఩ඖ⣲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ᮍኚ໬య䜔௦ㅰ≀䛾⸆≀ືែᏛⓗ᝟ሗ䜢
ᚓ䜛䚹
Ϲ䠅ᨺᑕᛶྠ఩ඖ⣲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ᮍኚ໬య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
ᒙ᧜ᙳἲ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
Single-Photon Emission Computed Tomography 䠄SPECT䠅䜢⏝䛔䛶ྠᵝ䛻 ᐃ䛩䜛䚹
4
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
5
➨୕⠇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ṇ཯ᑐ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
6
ᅗ䜢୍㒊ᨵኚ䛧䛶ᘬ⏝
http://www.riken.go.jp/r-world/info/release/news/2008/feb/index.htm
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
13
N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
7
18
F ᶆ㆑໬ἲ
18
F 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
18
F ᶆ ㆑ ศ Ꮚ ᅋ ([18F]FBA䚸 [18 F]SFB 䛺 䛹 䠅 䜢 ௓ 䛧 䛯 㛫 ᥋ ⓗ 䛺
18
F- 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
཯ᛂ䛜䛒䜛䚹䜰䝆䝗䛸䜰䝹䜻䞁䛾௜ຍ⎔໬཯ᛂ䛿䚸1893 ᖺ䛻 Michael
8
8)
䛻䜘䛳䛶ึ䜑
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⎔໬௜ຍ཯ᛂ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⎔໬௜ຍ཯ᛂ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Ϻ཯ᛂ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࠸ࡿࠋ࡜ࡇࢁ࡛ࠊ㏆ᖺࠊ᰾㓟་⸆ࡣࠊ
ᢠయ་⸆࡟ḟࡄḟୡ௦་⸆ရ࡜ࡋ࡚ὀ┠ࡉࢀࡿ࡟ࡘࢀ࡚ࠊࢡࣜࢵࢡ཯ᛂࢆ⏝࠸
ࡓ᰾㓟࢜ࣜࢦ࣐࣮ࡢ PET ᶆ㆑໬ࡀ┒ࢇ࡟◊✲ࡉࢀ࡚࠸ࡿࠋ
9
➨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)
10
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
䛭䛾ᚋ䚸pri-miRNA 䛜䚸Drosha 䛻䜘䛳䛶ษ᩿䛥䜜䚸䝦䜰䝢䞁ᵓ㐀䜢ᣢ䛴 pre-miRNA 䛸
11
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
䜙䚸⒴䜔 HIV 䜈䛾ᛂ⏝䛜ᮇᚅ䛥䜜䛶䛔䜛䚹
12
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Ᏻ඲ᛶ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
13
➨஬⠇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
18
F 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ᨺᑕ໬Ꮫ䜽䝸䝑䜽཯ᛂ䛷⏕䛨䜛
๪⏕ᡂ≀䛾⏕ᡂ䜢ᢚไ䛧䛶䛔䜛䚹㻌 㻌
Scheme 1-6. [18F]FPy5yne 䛾ྜᡂἲ
14
TBTA
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,
18
35)
F 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
15
㻌
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఩Ꮚ TBTA 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
16
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)
Scheme 1-12. BE 䜢ᑟධ䛧䛯᰾㓟䜸䝸䝂䝬䞊䛾䜽䝸䝑䜽཯ᛂ
17
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
18
➨஧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⟬
19
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 ᵓ㐀㻌 㻔ᕥ㻕㻌 䛸஧ᮏ㙐ᙧᡂ䝰䝕䝹㻌 㻔ྑ㻕㻠㻝㻕
㻌
㻌
20
➨஧⠇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
Scheme 2-3. Reagents and conditions; (a) I2, AgOTf, CH2Cl2, rt, 12 h, 93%.
21
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య 3 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.
Scheme 2-7. Reagents and conditions; (a) I2, AgOTf, CH2Cl2, rt, 24h.
22
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ᮏ໬ྜ≀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཯ᛂ 49) 䛻䜘䜚䚸ᑐ
ᛂ䛩䜛䜰䝹䜻䝙䝹య 6 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,
piperidine, rt, 4 h, 85%; (b) K2CO3, MeOH, H2O, rt, 12 h, quant.
23
➨୕⠇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).
24
➨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
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
ே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ᮍ཯ᛂ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
26
➨஧⠇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ㄏᑟయ 8 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 ME ࡜ BE 䛾ᵓ㐀䜢 Fig. 3-2 ࡟♧ࡋࡓࠋ
Fig. 3-2. ࢚ࢳࢽࣝ䝧䞁䝊䞁⨨᥮ᆺேᕤ᰾㓟 ME ࡜ BE 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
E
E
ON 13 (dT7-M -dT7)
5'-d(TTTTTTTM TTTTTTT)-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
E
E
ON 16 (dT7-B -dT7)
5'-d(TTTTTTTB TTTTTTT)-3'
4417.7
4417.4
ON 17 (dT14-BE)
5'-d(TTTTTTTTTTTTTTBE)-3'
4417.7
4418.4
27
౛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
ON 12 (ME-dT14)
ON 13 (dT7-ME-dT7)
ON 14 (dT14-ME)
Fig. 3-3. ME 䜢ᑟධ䛧䛯 DNA䠄ON 12䚸ON 13䚸ON 14䠅䛾㓄ิ
28
➨୕⠇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
ಖᣢ᫬㛫 9.5 ศ䛾఩⨨䛻ཎᩱ ON 13䠄dT7-ME-dT7䠅⏤᮶䛾䝢䞊䜽䛜ほᐹ䛥䜜䛯䚹䜽䝸
29
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 ࡢࢡࣜࢵࢡ཯ᛂຠ⋡
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 BE ྵ᭷᰾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
TTTTTTTTTTTTTT)-3'
4568.8
4569.3
5'-d(TTTTTTTMFBTTTTTTTT)-3'
4568.8
4570.2
)-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
4568.8
4569.6
FBT
ON 18 (M
-dT14)
ON 19 (dT7-MFBT-dT7)
FBT
ON 20 (dT14-M
)
FBT
ON 23 (dT14-B
)
FBT
5'-d(M
FBT
5'-d(TTTTTTTTTTTTTTM
FBT
5'-d(TTTTTTTTTTTTTTB
30
)-3'
➨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ME ཬ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Tm ್䜢 ᐃ䛧䛯䠄Fig. 3-6䠅䚹
Fig. 3-6. ஧ᮏ㙐 DNA 䛾⇕ⓗᏳᐃᛶ
䛭䛾⤖ᯝ䚸ኳ↛ᆺ ON 11䠄dT15䠅/ON 10䠄dA15䠅䛸ẚ㍑䛧䛶䚸ME ཬ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 Tm ್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 Tm ್䛿䚸ኳ↛ᆺ ON 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 Tm ್䛿䚸BE 䜢ෆ㒊䛻᭷䛩䜛 ON 16䠄dT7-BE-dT7䠅/ON
10䠄dA15䠅䛸ẚ㍑䛧䛶⣙ 5 ºC 㧗䛔್䜢♧䛧䛯䚹
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
୰ኸ㒊䛻 ME 䜢᭷䛩䜛஧ᮏ㙐 DNA [ON 13䠄dT7-ME-dT7䠅/ON 10䠄dA15䠅]䛾 Tm ್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
32
⏤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
Marker
DNA step ladder
(10~300 bp)
ON 13/ON 10
Fig. 3-8. ஧ᮏ㙐 DNA 䛾㟁ẼὋື⏬ീ
33
ON 19/ON 10
➨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ㄏᑟయ 8 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
No.of ON
Sequence
ON 24 (A15)
ON 25 (U15)
E
Calculated
Observed
5'-r(AAAAAAAAAAAAAAA)-3'
4872.9
4872.8
5'-r(UUUUUUUUUUUUUUU)-3'
4527.4
4527.8
E
ON 26 (M -U14)
5'-r(M UUUUUUUUUUUUUU)-3'
4445.4
4445.8
ON 27 (U7-ME-U7)
5'-r(UUUUUUUMEUUUUUUU)-3'
4445.4
4445.1
4445.4
4445.6
E
ON 28 (U14-M )
E
E
5'-r(UUUUUUUUUUUUUUM )-3'
E
ON 29 (B -U14)
5'-r(B UUUUUUUUUUUUUU)-3'
4445.4
4447.2
ON 30 (U7-BE-U7)
5'-r(UUUUUUUBEUUUUUUU)-3'
4445.4
4446.8
4445.4
4445.9
E
ON 31 (U14-B )
E
5'-r(UUUUUUUUUUUUUUB )-3'
౛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
34
ON 26 (ME-rU14)
ON 27 (rU7-ME-rU7)
ON 28 (rU14-ME)
Fig. 4-1. ME 䜢ᑟධ䛧䛯 DNA䠄ON 26䚸ON 27䚸ON 28䠅䛾㓄ิ
35
➨஧⠇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
No.of ON
Sequence
FBT
ON 32 (M
-rU14)
ON 33 (rU7-MFBT-rU7)
FBT
ON 34 (rU14-M
)
FBT
ON 35 (B
-rU14)
ON 36 (rU7-BFBT-rU7)
FBT
ON 37 (rU14-B
)
Calculated
Observed
UUUUUUUUUUUUUU)-3'
4596.5
4595.2
5'-r(UUUUUUUMFBTUUUUUUU)-3'
4596.5
4593.9
)-3'
4596.5
4594.1
UUUUUUUUUUUUUU)-3'
4596.5
4596.2
5'-r(UUUUUUUBFBTUUUUUUU)-3'
4596.5
4595.3
4596.5
4593.1
FBT
5'-r(M
FBT
5'-r(UUUUUUUUUUUUUUM
FBT
5'-r(B
FBT
5'-r(UUUUUUUUUUUUUUB
)-3'
୍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 BE ྵ᭷᰾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⋡
36
୍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
ON 12
ON 13
ON 14
Fig. 4-3. ME 䜢᭷䛩䜛୍ᮏ㙐 DNA 䛾ศᏊ䝰䝕䝹
37
➨୕⠇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ME ཬ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Tm ್䜢 ᐃ䛧䛯䠄Fig. 4-4䠅䚹
Fig. 4-4. ஧ᮏ㙐 RNA 䛾⇕ⓗᏳᐃᛶ
䛭䛾⤖ᯝ䚸ኳ↛ᆺ ON 25䠄rU15䠅/ON 24䠄rA15䠅䛸ẚ㍑䛧䛶䚸ME ཬ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 Tm ್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 Tm ್䛿䚸ኳ↛ᆺ ON 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 Tm ್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ME ᡈ䛔䛿 BE 䜢஧ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛾ᮎ
➃䛻ᑟධ䛧䛯ሙྜ䚸஧ᮏ㙐䛾⇕ⓗᏳᐃᛶ䛻䜋䛸䜣䛹ᙳ㡪䜢୚䛘䛺䛔䛸䛔䛖⤖ᯝ䜢ᚓ䛯䚹
38
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
39
➨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 Tm ್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
40
➨஧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Ᏻᐃ໬స⏝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Tm ್ ᐃ⏝ 0.01 M 䝸䞁㓟⦆⾪ᾮ䛻 5 mM 䛾
MgCl2 䜢ῧຍ䛧䚸Tm ್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
Tm ್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 Tm ್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
ຠ⋡䜢䛸ẚ㍑䛧䛯䠄Fig. 4-7䠅䚹
41
Scheme 4-2. Mg2+ῧຍᚋ୰ኸ㒊䛻 ME ࢆ᭷ࡍࡿ஧ᮏ㙐 RNA ࡢࢡࣜࢵࢡ཯ᛂ
dsRNA
2+
dsRNA (mg )
Substrate Consumption (%)
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⏬ീ
42
➨஬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Ᏻ඲ᛶ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་⸆ရ࡜ࡋ࡚㛤Ⓨࡍࡿ࡟ࡣࠊPET ࡟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᰾㓟࢜ࣜࢦ࣐࣮ࡢᮎ➃ࢆࢱ࣮ࢤࢵࢺ࡟ࡋ࡚࠸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ࡋ࠿ࡋࠊsiRNA ࡞࡝ࡢ஧ᮏ㙐᰾㓟࢜ࣜࢦ࣐࣮࡛ࡣࠊ3’ᮎ➃ࡢࢲࣥ
ࢢࣜࣥࢢ࢚ࣥࢻ㒊఩ࡣࠊ᰾㓟࢜ࣜࢦ࣐࣮ࡀ RISC ࡟ྲྀࡾ㎸ࡲࢀࠊRISC ࡢ PAZ ࢻ
࣓࢖ࣥࡢ␯Ỉᛶ࣏ࢣࢵࢺࢆㄆ㆑ࡋࠊ⤖ྜࡍࡿࡢ࡟㔜せ࡞ᙺ๭ࢆᯝࡓࡍࠋࡲࡓࠊ5’
ᮎ➃ࡢࣜࣥ㓟ᇶࡣࠊ᰾㓟࢜ࣜࢦ࣐࣮ࡀ RISC ࡢ Mid ࢻ࣓࢖ࣥ࡟ㄆ㆑ࡉࢀࠊᅛᐃࡉ
ࢀࡿࡢ࡟㔜せ࡞ᙺ๭ࢆᯝࡓࡍࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡑࡇ࡛ࠊⴭ⪅ࡣ᰾㓟࢜ࣜ
ࢦ࣐࣮ࡢᮎ➃ࡢᵓ㐀ࢆኚ࠼ࡎࠊෆ㒊࡛ࡢ PET ࣛ࣋ࣝ໬ࡸ໬Ꮫಟ㣭ࢆ┠ᣦࡋࠊศ
Ꮚࣔࢹࣝ࡟ࡼࡿຊሙィ⟬ࢆ⾜࠸ࠊ஧ᮏ㙐ࡢ⇕ⓗᏳᐃᛶ࡟ᙳ㡪ࡀᑡ࡞࠸࡜⪃࠼ࡽ
ࢀࡿ(S)-4-Ethynylmandelol䠄ME䠅ࢆタィࡋࡓ㸦➨஧❶➨୍⠇ࠊ➨஧⠇㸧ࠋࡲࡓࠊࡇ
43
ࢀࡲ࡛ࡢ▱ぢࢆᇶ࡟ࠊ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ME ཬ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
44
䜜䛯䚹䜎䛯䚸ME 䛿䚸᰾㓟࢜ࣜࢦ࣐࣮ࡢ௵ពࡢ఩⨨࡟ᑟධ࡛ࡁࡿࡇ࡜࠿ࡽࠊ⮬⏤࡞
ศᏊタィࡀ⾜࠼ࡿࠋࡉࡽ࡟ࠊ✀ࠎࡢ࢔ࢪࢻ໬ྜ≀ࢆ௜ຍࡉࡏࡿࡇ࡜࡛ࠊ᪂つࡢ
ࢻࣛࢵࢢࢹࢨ࢖ࣥࡸ⺯ගࣉ࣮ࣟࣈ໬࡞࡝ PET ᶆ㆑໬௨እࡢ⏝㏵ࡶᮇᚅ࡛ࡁࡿࠋ
ᚑࡗ࡚ࠊᮏ◊✲ࡣࠊsiRNAࠊmiRNAࠊ࢔ࣥࢳࢭࣥࢫࠊ࢔ࣉࢱ࣐࣮࡞࡝ࠊᵝࠎ࡞
᰾㓟་⸆ࢆ㛤Ⓨࡍࡿୖ࡛ࠊᚲ㡰࡛࠶ࡿ⸆≀ືែゎᯒ࡟࠾࠸࡚㔜せ࡞᰾㓟ศᏊࡢ
PET ࣛ࣋ࣝ໬ࡸ⺯ගࣛ࣋ࣝ໬࡞࡝࡟㐺⏝ྍ⬟࡞ࣘࢽࣂ࣮ࢧࣝ࡞ᶆ㆑໬ἲࢆᥦ౪
ࡍࡿࡶࡢ࡜⪃࠼ࡿࠋ
45
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
SHIMADZU
Tm ᐃᶵ
UV-2450
UV-VISIBLE
SPECTROPHOTOMETER
UV 䝷䞁䝥
AS ONE Handy UV Lamp SLUV-6 (254/365 nm)
pH 䝯䞊䝍
HORIBA F-52
46
*1
1
H 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ᮾ໬Ꮫ; 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
Bis(triphenylphosphine)palladium( ϩ )
䛆Aldrich䛇
Silver(Ϩ) Oxide
䛆㛵ᮾ໬Ꮫ䛇
THF (Dehydrated)䚸Copper(I) Iodide
47
Dichloride 䚸
䛆䜻䝅䝎໬Ꮫ䛇
CH2Cl2 (୍ᩯ⨁)䚸Pyridine*1
䛆ᮾி໬ᡂ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 (SiMe4 ྵ᭷)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
48
dA-CE
Phosphoramidite 䚸 dT-CE
Phosphoramidite 䚸
䛆Glen research䛇
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
䛆䛭䛾௚䛇
EtOH (୍ᩯ⨁)
49
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
䜚䜣㓟஧Ỉ⣲䝘䝖䝸䜴䝮
50
2. ᐇ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
51
(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) 䛾ྜ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⁐
ゎ䛧䚸㣬࿴㣗ሷỈ䛷 2 ᅇ䚸5%䛾ሷ㓟Ỉ⁐ᾮ䛷 1 ᅇ䚸㣬࿴Ⅳ㓟Ỉ⣲໬䝘䝖䝸䜴䝮Ỉ⁐ᾮ
52
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) 䛾ྜ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ῶᅽ␃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,
J = 7.0 Hz and 4.9 Hz, 1H), 3.61 (d, J = 4.9 Hz, 1H), 3.60 (d, J = 7.0 Hz, 1H), 3.43 (s,
53
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,
6.21%.
54
2-2. ➨஧❶➨୕⠇䛻㛵䛩䜛ᐇ㦂
Scheme 2-10 䛻㛵䛩䜛ᐇ㦂
(S)-2-(4,4'-Dimethoxytrityloxy)-1-(4-ethynylphenyl)ethanol 䛾ྜᡂ(7)
50 mL 䝘䝇䝣䝷䝇䝁䛻(S)-4-ethynylmandelol (1) (301 mg, 1.86 mmol) 䜢ຍ䛘䚸⬺Ẽᚋ䚸
䜰䝹䝂䞁⨨᥮䛧䛶䚸pyridine (6.0 mL) 䛻⁐ゎ䛧䚸4,4'-dimethoxytrityl chloride (818 mg,
2.42 mmol, 1.3 eq.) 䛾 pyridine (4.0 mL) ⁐ᾮ䜢䜖䛳䛟䜚⁲ୗ䛧䛶䚸ᐊ ୗ 2 ᫬㛫ᨩᢾ
䛧䛯䚹TLC 䛷཯ᛂ䛾㐍⾜䜢☜ㄆ䛧䛯ᚋ䚸཯ᛂᾮ䜢䝆䜽䝻䝻䝯䝍䞁 (80 mL) 䛸Ỉ (80
mL)䚸㣬࿴Ⅳ㓟Ỉ⣲䝘䝖䝸䜴䝮Ỉ⁐ᾮ (80 mL × 2) 䛷ᢳฟ䛧䛯䚹䛭䛧䛶䚸䝆䜽䝻䝻䝯䝍䞁
ᒙ䜢㣬࿴㣗ሷỈ (80 mL) 䛷Ὑί䛧䚸↓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 = 20 : 1 →
0 : 1) 䛻䛶⢭〇䛧 䚸 (S)-2-(4,4'- dimethoxytrityloxy)-1-(4-ethynylphenyl)ethanol (7)
(264 mg, 568 μmol, 31%) 䜢↓Ⰽ⤖ᬗ䛸䛧䛶ᚓ䛯䚹1H NMR (400 MHz, CD3OD) δ 7.41
(d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.0 Hz, 2H), 7.29 (d, J = 7.9 Hz, 2H), 7.23-7.19 (m,
7H), 6.79-6.76 (m, 4H), 4.73 (dd, J = 5.8 Hz, 1H), 3.74 (s, 2H), 3.73 (s, 6H), 3.43 (s,
1H);
13
C NMR (100 MHz, CD3OD) δ 160.0, 146.4, 144.8, 137.3, 132.7, 131.3, 130.4,
129.3, 128.6, 127.7, 122.8, 114.0, 87.5, 84.4, 78.5, 74.0, 70.0, 55.7
(S)-1-O-[(2-Cyanoethyloxy)(N,N-diisopropylamino)phosphanyl]-2-(4,4'-dimethoxyt
rityloxy)-1-(4-ethynylphenyl)ethane 䛾ྜᡂ (8)
䜾䝻䞊䝤䝞䝑䜾 (䜰䝹䝂䞁⨨᥮䚸᏶඲↓Ỉ) ୰䛷཯ᛂ䜢⾜䛳䛯䚹
30 mL 䝘䝇䝣䝷䝇䝁䛻(S)-2-(4,4'-dimethoxytrityloxy)-1-(4-ethynylphenyl)ethanol (7)
(238 mg, 512 μmol) 䜢ຍ䛘䚸୍ᬌ┿✵䝫䞁䝥䛻䛶஝⇱䛧䛯䚹⩣᪥䚸THF (3.0 mL) 䛻
55
⁐ ゎ 䛧 䚸 N,N-di isopropylethylamine (360 μL, 2.06 mmol, 4.0 eq.) 䜢 ຍ 䛘 䚸
2-cyanoethyl diisopropylchloro phosphoramidite (300 μL, 1.34 mmol, 2.6 eq.) 䜢᧠ᢾ
䛧䛺䛜䜙ᑡ㔞䛪䛴ຍ䛘䛯ᚋ䚸䜾䝻䞊䝤䝞䝑䜾䛛䜙ྲྀ䜚ฟ䛧䚸ᐊ ୗ 1.5 ᫬㛫᧠ᢾ䛧䛯䚹
TLC 䛷཯ᛂ䛾᏶⤖䜢☜ㄆ䛧䛯ᚋ䚸཯ᛂᾮ䜢䜽䝻䝻䝩䝹䝮 (40 mL) 䛸㣬࿴Ⅳ㓟Ỉ⣲䝘
䝖䝸䜴䝮Ỉ⁐ᾮ (40 mL) 䛷ᢳฟ䛧䛯䚹䛭䛧䛶䚸䜽䝻䝻䝩䝹䝮ᒙ䜢㣬࿴㣗ሷỈ (40 mL)
䛷Ὑί䛧䚸↓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 = 1 : 1) 䛻 䛶 ⢭ 〇 䛧 䚸
(S)-1-O-[(2-cyanoethyloxy)(N,N-diisopropylamino)phosphanyl]-2-(4,4'-dimethoxytrityl
oxy)-1-(4-ethynylphenyl)ethane (8) (268 mg, 403 μmol, 79%) 䜢↓Ⰽ⤖ᬗ䛸䛧䛶ᚓ䛯䚹
31
P NMR (160 MHz, CDCl3) δ 149.5, 149.4
ᅛ┦ᢸయ (9 ) 䛾ྜᡂ.
7 (674.9 mg, 1.45 mmol), Succinic anhydride (580.8 mg, 5.80 mmol), DMAP (1.91
mg, 0.017 mmol) 䜢 Pyridine (3.9 mL) 䛻⁐䛛䛧 24 ᫬㛫ᐊ 䛷ᨩᢾ䛧䛯䚹㣬࿴
NaHCO3 Ỉ⁐ᾮ䛷䜽䜶䞁䝏䛧 CHCl3 䛻䛶ᢳฟᚋ䚸᭷ᶵᒙ䜢 brine 䛷Ὑί䛧 Na2SO4 䛻
䛶஝⇱ᚋ䚸ῶᅽ␃ཤ䛧䚸┿✵䝫䞁䝥䛷஝⇱䛥䛫ᑐᛂ䛩䜛䝇䜽䝅䝙䝹య䜢ᚓ䛯䚹䝇䜽䝅䝙
䝹య䛸 Aminopropyl controlled pore glass (CPG) (838 mg, 77 μmol),
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) (720 mg, 3.76
mmol) 䜢 DMF (15 mL) 䛻⁐䛛䛧䚸24 ᫬㛫ᐊ 䛷᣺┞䛥䛫䛯䚹䜾䝷䝇䝣䜱䝹䝍䞊䛷ℐ
㐣䛧䚸䝢䝸䝆䞁䛷Ὑίᚋ䚸䜻䝱䝑䝢䞁䜾䝋䝸䝳䞊䝅䝵䞁 (8 mL, 0.1 M DMAP in
pyridine/Ac2O, 9:1, v/v) ୰䛷 16 ᫬㛫ᐊ ᣺┞䛥䛫䛯䚹ᚓ䜙䜜䛯 CPG ᶞ⬡䜢 Pyridine,
EtOH, 䜰䝉䝖䝙䝖䝸䝹䛾㡰䛷Ὑί䛧┿✵䝫䞁䝥䛷஝⇱䛥䛫 7 䜢ᢸయ䛧䛯 CPG ᶞ⬡ 9 䜢
26.5 μmol/g 䛾άᛶ䛷ᚓ䛯䚹
56
2-3. ➨஧❶➨ᅄ⠇䛻㛵䛩䜛ᐇ㦂
Table 2-1 䛻㛵䛩䜛ᐇ㦂
䜶䝏䝙䝹䝧䞁䝊䞁㦵᱁䜢᭷䛩䜛ேᕤ᰾㓟 BE ཬ䜃 ME 䜢⁛⳦Ỉ䛻⁐ゎ䛧䚸260 nm 䛻䛚
䛡䜛྾ගᗘ䜢 ᐃ䛧䛶䚸䛭䛾䝰䝹྾ගィᩘ ε (M-1cm-1) 䜢ồ䜑䛯*1䚹BE 䛿ⴭ⪅䛾◊✲
Absorbance at 260 nm of BE
ᐊ䛷ྜᡂ䛥䜜䛯䜒䛾䜢⏝䛔䛯䚹௨ୗ䛻䚸BE ཬ䜃 ME 䛾 ε ್⟬ฟ⤖ᯝ䜢♧䛩䚹
1
0.8
0.6
y = 627.33x
0.4
0.2
⃰ᗘ C (mmol/L)
0.752
1.2100
0.576
0.9075
0.383
0.6050
0.191
0.3025
0
0
0
0.0005
0.001
௨ୖࡼࡾࠊB ࡢ ε ್ࢆ⟬ฟࡍࡿ࡜ࠊ
0.0015
ε = 627 (M-1cm-1)
1
0.8
y = 672.01x
0.6
0
E
Concentration (mol/L)
Absorbance at 260 nm of ME
྾ගᗘ Abs260
0.4
0.2
྾ගᗘ Abs260
⃰ᗘ C (mmol/L)
0.867
1.2875
0.691
1.0300
0.343
0.5150
0.175
0.2575
0
0
0
E
0
0.0005
0.001
௨ୖࡼࡾࠊM ࡢ ε ್ࢆ⟬ฟࡍࡿ࡜ࠊ
0.0015
ε = 672 (M-1cm-1)
Concentration (mol/L)
*1 ேᕤ᰾㓟䛿Ỉ⁐ᾮ䛸䛧䚸Ἴ㛗 260 nm 䛷䛾྾ගᗘ (Abs260) 䛜䚸྾ගᗘィ䛾᭷ຠ
⠊ᅖ䛻䛺䜛䜘䛖䛻ᕼ㔘䛧䛯䚹ග㊰㛗 (l) 1 cm 䛾྾ගᗘ ᐃ⏝▼ⱥ䝉䝹䜢⏝䛔䚸ᐊ 䛻
䛶 Abs260 䜢 ᐃ䛧䛯䚹OD260 ್䛾ィ⟬䛻䛿௨ୗ䛾ᘧ䜢⏝䛔䛯䚹䛣䛣䛷䛾⁐ᾮ㔞 (V) 䛿䚸
ᕼ㔘⁐ᾮ䛾䛖䛱 ᐃ䛻౑⏝䛧䛯 1 mL 䜢♧䛩䚹
OD260 (Mε-1 䞉 mL-1 䞉 cm-1) = Abs260 (Mε-1) 䞉 V-1 (mL) 䞉 l-1 (cm)
䛭䛧䛶䚸ε ್䛿 Beer 䛾ἲ๎䛻䜘䜚ḟᘧ䜢⏝䛔䛶ồ䜑䛯䚹 ε260 = Abs260 䞉 C-1 䡡 l-1
57
2-4. ➨୕❶➨஧⠇䚸➨ᅄ❶➨୍⠇䛻㛵䛩䜛ᐇ㦂
Table 3-1䚸4-1 䛻㛵䛩䜛ᐇ㦂
᰾㓟䜸䝸䝂䝬䞊䛾ྜᡂ䛿䚸᰾㓟⮬ືྜᡂᶵ䛻䜘䜛䝩䝇䝩䝻䜰䝭䝎䜲䝖ἲ䛻ᚑ䛔 1 μmol
䝇䜿䞊䝹䛷⾜䛳䛯䚹ኳ↛䛾䝩䝇䝩䝻䜰䝭䝎䜲䝖䛿 0.1 M䚸ྜᡂ䛧䛯䝩䝇䝩䝻䜰䝭䝎䜲䝖䛿
0.15 M 䛻䛺䜛䜘䛖䛻䜰䝉䝖䝙䝖䝸䝹䛷ㄪ〇䛧䚸ྛ CPG ᶞ⬡䛿ྛ䚻䛾άᛶ䛻ᇶ䛵䛝 1
μmol ศ䜢䜹䝷䝮䛻㔞䜚ྲྀ䜚䚸᰾㓟⮬ືྜᡂᶵ䛻ྲྀ䜚௜䛡䛯䚹rA-CPG ᶞ⬡䚸rU-CPG ᶞ
⬡ཬ䜃 BE-CPG 䛿䚸ⴭ⪅䛾◊✲ᐊ䛷ྜᡂ䛥䜜䛯䜒䛾䜢⏝䛔䛯䚹
䠘DNA 䛾ሙྜ䠚
ྜᡂ⤊஢ᚋ䚸CPG ᶞ⬡䜢䝃䞁䝥䝹䝇䝖䝑䜽䝏䝳䞊䝤䛻⛣䛧䚸䜰䞁䝰䝙䜰Ỉ (1.2 mL) 䜢
ຍ䛘䚸55 ºC 䛷 12 ᫬㛫䜲䞁䜻䝳䝧䞊䝖䛩䜛䛣䛸䛻䜘䜚䚸᰾㓟䜸䝸䝂䝬䞊䛾 CPG ᶞ⬡䛛䜙
䛾ษ䜚ฟ䛧ཬ䜃⬺ಖㆤ䜢⾜䛳䛯䚹཯ᛂᚋ䚸⁐ᾮ䜢ᐊ 䛻ᡠ䛧䚸ୖΎ䜢䜶䝑䝨䞁䝗䝹䝣
䝏䝳䞊䝤䛻⛣䛧䛯ᚋ䚸CPG ᶞ⬡䜢⁛⳦Ỉ (1 mL) 䛷 3 ᅇὙί䛧䚸ྠᵝ䛻ୖΎ䜢䜶䝑䝨
䞁䝗䝹䝣䝏䝳䞊䝤䛻㞟䜑䚸⁐፹䜢㐲ᚰῶᅽ␃ཤ䛧䛯䚹ṧ´䜢⁛⳦Ỉ (1 mL) 䛻⁐ゎ䛧䚸
Millex®-HV 䝣䜱䝹䝍䞊䛻㏻䛧䛯ᚋ䚸HPLC*1 䛻䛶┠ⓗ䛾᰾㓟䜸䝸䝂䝬䞊䜢⢭〇䛧䛯䚹
⁐ฟᾮ䛿䜶䝑䝨䞁䝗䝹䝣䝏䝳䞊䝤䛻㞟䜑䚸⁐፹䜢㐲ᚰῶᅽ␃ཤ䛧䛯䚹
䠘RNA 䛾ሙྜ䠚
ྜᡂ⤊஢ᚋ䚸CPG ᶞ⬡䜢䝃䞁䝥䝹䝇䝖䝑䜽䝏䝳䞊䝤䛻⛣䛧䚸䜰䞁䝰䝙䜰Ỉ : 䜶䝍䝜䞊
䝹 = 3 : 1 ⁐ᾮ (1.2 mL) 䜢ຍ䛘䚸ᐊ 䛷 12 ᫬㛫᣺䛸䛖䛩䜛䛣䛸䛻䜘䜚䚸᰾㓟䜸䝸䝂䝬䞊
䛾 CPG ᶞ⬡䛛䜙䛾ษ䜚ฟ䛧ཬ䜃⬺ಖㆤ䜢⾜䛳䛯䚹཯ᛂᚋ䛾ୖΎ䜢䜶䝑䝨䞁䝗䝹䝣䝏䝳
䞊䝤䛻⛣䛧䛯ᚋ䚸CPG ᶞ⬡䜢⁛⳦Ỉ : 䜶䝍䝜䞊䝹 = 3 : 1 ⁐ᾮ (1 mL) 䛷 3 ᅇὙί䛧䚸
ྠᵝ䛻ୖΎ䜢䜶䝑䝨䞁䝗䝹䝣䝏䝳䞊䝤䛻㞟䜑䚸⁐፹䜢㐲ᚰῶᅽ␃ཤ䛧䛯䚹ḟ䛻䚸ṧ´
䛻 1 M TBAF in THF ⁐ᾮ (1 mL) 䜢ຍ䛘䚸ᐊ 䛷 12 ᫬㛫᣺䛸䛖䛩䜛䛣䛸䛻䜘䜚䚸᰾㓟
䜸䝸䝂䝬䞊䛾⬺ TBDMS ಖㆤ䜢⾜䛳䛯䚹䛣䛾཯ᛂᾮ䜢 0.1 M TEAA buffer*2 䛻䛶 30 mL
58
䛻ᕼ㔘䛧䚸ᖹ⾮໬䛧䛯 Sep-Pak tC18 ㏫┦䜹䝷䝮䜽䝻䝬䝖䜾䝷䝣䜱䞊䛻㏻䛧䚸䜹䝷䝮䛻᰾㓟
䜸䝸䝂䝬䞊䜢྾╔䛥䛫䛯䚹䜹䝷䝮䜢⁛⳦Ỉ䛷Ὑί䛧䛶ሷ䜢ྲྀ䜚㝖䛔䛯ᚋ䚸50%䜰䝉䝖䝙䝖
䝸䝹 in ⁛⳦Ỉ (3 mL) 䛷᰾㓟䜸䝸䝂䝬䞊䜢⁐ฟ䛧䚸䛣䛾⁐ฟᾮ䜢䜶䝑䝨䞁䝗䝹䝣䝏䝳
䞊䝤䛻㞟䜑䚸⁐፹䜢㐲ᚰῶᅽ␃ཤ䛧䛯䚹ṧ´䜢 loading solution*3 (180 μL) 䛻⁐ゎ䛧䚸
20%ኚᛶ PAGE*4 (500 V, 20 mA) 䛻䜘䜚┠ⓗ䛾᰾㓟䜸䝸䝂䝬䞊䜢ศ㞳䛧䛯䚹㟁ẼὋື
ᚋ䚸┠ⓗ䛾᰾㓟䜸䝸䝂䝬䞊䛾䝞䞁䝗䜢ษ䜚ฟ䛧䚸䝀䝹⁐ฟ⁐ᾮ*5 (20 mL) 䛻ᾐ䛧䚸୍
ᬌ᣺䛸䛖䛧䛯䚹䛣䛾䝀䝹⁐ฟᾮ䜢ᖹ⾮໬䛧䛯 Sep-Pak tC18 ㏫┦䜹䝷䝮䛻෌ᗘ㏻䛧䚸䜹䝷
䝮䛻᰾㓟䜸䝸䝂䝬䞊䜢྾╔䛥䛫䛯䚹䜹䝷䝮䜢⁛⳦Ỉ䛷Ὑί䛧䛶ሷ䜢ྲྀ䜚㝖䛔䛯ᚋ䚸50%
䜰䝉䝖䝙䝖䝸䝹 in ⁛⳦Ỉ (3 mL) 䛷᰾㓟䜸䝸䝂䝬䞊䜢⁐ฟ䛧䚸䛣䛾⁐ฟᾮ䜢䜶䝑䝨䞁
䝗䝹䝣䝏䝳䞊䝤䛻㞟䜑䚸⁐፹䜢㐲ᚰῶᅽ␃ཤ䛧䛯䚹ṧ´䜢⁛⳦Ỉ (1 mL) 䛻⁐ゎ䛧䚸
Millex®-HV 䝣䜱䝹䝍䞊䛻㏻䛧䛯ᚋ䚸HPLC*1 䛻䛶┠ⓗ䛾᰾㓟䜸䝸䝂䝬䞊䜢⢭〇䛧䛯䚹
⁐ฟᾮ䛿䜶䝑䝨䞁䝗䝹䝣䝏䝳䞊䝤䛻㞟䜑䚸⁐፹䜢㐲ᚰῶᅽ␃ཤ䛧䛯䚹
⢭〇䛧䛯᰾㓟䜸䝸䝂䝬䞊䜢⁛⳦Ỉ (1 mL) 䛻⁐ゎ䛧䚸Abs260 䜢 ᐃ䛧䛶཰㔞䜢ồ䜑䛯
*8䚹䛭䛧䛶䚸30 pmol ศ䛾᰾㓟䜸䝸䝂䝬䞊䜢஝ᅛ䛥䛫䚸⁛⳦Ỉ (2 μL) ཬ䜃䝬䝖䝸䝑䜽䝇
⁐ᾮ*9 (2 μL) 䛸䜘䛟ΰ࿴䛥䛫䚸䝥䝺䞊䝖ୖ䛷஝ᅛ䛧䛯ᚋ䚸MALDI-TOF MASS 䛷ᵓ㐀
☜ㄆ䜢⾜䛳䛯䚹
*1
HPLC 䛻䜘䜛᰾㓟䜸䝸䝂䝬䞊䛾⢭〇䛻䛿㏫┦䜹䝷䝮䜢౑⏝䛧䛯䚹䜎䛯䚸㏫┦䜹䝷䝮
⏝ buffer 䛻䛿䚸A ᾮ䛻 5%䜰䝉䝖䝙䝖䝸䝹 in 0.1 M TEAA buffer*2 (2 M TEAA buffer*2
(50 mL)䚸䜰䝉䝖䝙䝖䝸䝹 (50 mL)䚸Milli Q (900 mL) 䜢ΰྜ䛧 1000 mL 䛻䛧䛯䜒䛾) 䜢䚸
B ᾮ䛻 50% 䜰䝉䝖䝙䝖䝸䝹 in 0.1 M TEAA buffer*2 (2 M TEAA buffer*2 (50 mL)䚸䜰
䝉䝖䝙䝖䝸䝹 (500 mL)䚸Milli Q (450 mL) 䜢ΰྜ䛧 1000 mL 䛻䛧䛯䜒䛾) 䜢౑⏝䛧䛯䚹
*2 0.1 M TEAA buffer 䛿䚸2 M TEAA buffer (㓑㓟 (114.38 mL) 䛻䝖䝸䜶䝏䝹䜰䝭䞁
(277.61 mL) 䜢ຍ䛘䚸Milli Q 䛷 1000 mL 䛻 mess up 䛧䚸pH 7.0 䛻ㄪ〇䛧䛯䜒䛾) 䜢⁛
59
⳦Ỉ䛷 20 ಸᕼ㔘䛧౑⏝䛧䛯䚹
*3
Loading solution 䛿䚸䝩䝹䝮䜰䝭䝗 (9 mL) 䛸 10 × TBE buffer*8 (1 mL) 䜢ΰྜ䛧
౑⏝䛧䛯䚹
*4 20%ኚᛶ PAGE 䜢⾜䛖䛻䛒䛯䜚䚸䝀䝹 1 ᯛ䛻䛴䛝 40%䜰䜽䝸䝹䜰䝭䝗 (19 : 1) ⁐ᾮ
*9 (40 mL)䚸ᒀ⣲ (33.6 g)䚸10 × TBE buffer*8 (8 mL) 䜢ຍ䛘䛶⁐ゎ䛧䚸Milli Q 䛷 80
mL 䛻 mess up 䛧䛯䚹⥆䛔䛶 APS (55 mg) 䜢ຍ䛘䛶᏶඲䛻⁐ゎ䛥䛫䚸᭱ᚋ䛻 TEMED
(40 μL) 䜢ຍ䛘䛶᣺䜚ΰ䛬䚸1.5 mm 䝇䝨䞁䝃䞊䜢ᣳ䜣䛷ᅛᐃ䛧䛶䛚䛔䛯 2 ᯛ䛾䜺䝷䝇
ᯈ䛾㛫䛻㟼䛛䛻ὶ䛧㎸䜏䚸6 ᫬㛫௨ୖ㟼⨨䛧䛶ᅛ໬䛥䛫䛯䚹䜎䛯䚸1 × TBE buffer*8 䜢
Ὃື⏝⦆⾪ᾮ䛸䛧䛶⏝䛔䛯䚹
*5 䝀䝹⁐ฟ⁐ᾮ䛿䚸2 M TEAA buffer (1 mL) 䛸 0.1 M EDTA Ỉ⁐ᾮ (EDTA·4Na
(1.809 g) 䜢⁛⳦Ỉ䛷 40 mL 䛻 mess up 䛧䛯䜒䛾) (0.2 mL) 䜢ΰྜ䛧䚸⁛⳦Ỉ䛷 20 mL
䛻 mess up 䛧䛯䜒䛾䜢౑⏝䛧䛯䚹
*6
᰾㓟䜸䝸䝂䝬䞊䛿Ỉ⁐ᾮ䛸䛧䚸Abs260 䛜྾ගᗘィ䛾᭷ຠ⠊ᅖ䛻䛺䜛䜘䛖䛻ᕼ㔘䛧
䛯䚹ග㊰㛗 (l) 1 cm 䛾྾ගᗘ ᐃ⏝▼ⱥ䝉䝹䜢⏝䛔䚸ᐊ 䛻䛶 Abs260 䜢 ᐃ䛧䛯䚹
OD260 ್䛾ィ⟬䛻䛿௨ୗ䛾ᘧ䜢⏝䛔䛯䚹䛣䛣䛷 V 䛿⁐ᾮ䛾඲㔞䜢♧䛩䚹
OD260 (Mε-1 䞉 mL-1 䞉 cm-1) = Abs260 (Mε-1) 䞉 V-1 (mL) 䞉 l-1 (cm)
䜎䛯䚸N1pN2pN3p 䡡䡡䡡 Nn-1pNn 䛷⾲䛥䜜䜛୍ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛾䝰䝹྾ගಀᩘ ε260
䛾⟬ฟ䛻䛿䚸ḟᘧ䜢⏝䛔䛯䚹
ε = 2 {ε (N1pN2) + ε (N2pN3) + 䡡䡡䡡 + ε (Nn-1pNn)} – {ε (N2) + ε (N3) + 䡡䡡䡡 + ε (Nn-1)}
䛣䛣䛷䚸ε (Nn) 䛿䛒䜛᰾㓟 Nn 䛾 ε260 䜢♧䛧䚸ε (Nn-1pNn) 䛿䛒䜛᰾㓟஧㔞య Nn-1pNn 䛾
ε260 䜢♧䛩䚹
䛺䛚䚸ேᕤ᰾㓟䛾 ε260 䛿⟬ฟ䛧䛯୍ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛾 ε260 䛻䛭䛾䜎䜎ຍ⟬䛧䛯䚹
䛭䛧䛶䚸᰾㓟䜸䝸䝂䝬䞊䛾⃰ᗘ C (mol/L) 䛿䚸ḟᘧ䜢⏝䛔䛶ồ䜑䛯䚹
C = Abs260 䞉 ε260-1 䡡 l-1
60
*7
䝬䝖䝸䝑䜽䝇⁐ᾮ䛿䚸3-HPA (4.85 mg) 䛸䛟䛘䜣㓟Ỉ⣲஧䜰䞁䝰䝙䜴䝮 (0.80 mg)
䜢 50%䜰䝉䝖䝙䝖䝸䝹 in Milli Q (50 μL) 䛻⁐ゎ䛥䛫ㄪ〇䛧䛯䚹䛺䛚䚸䛟䛘䜣㓟Ỉ⣲஧
䜰䞁䝰䝙䜴䝮䛿䚸᰾㓟䜸䝸䝂䝬䞊䛻 Na+䜔 K+䛜௜╔䛩䜛䛾䜢㜼ᐖ䛩䜛䛯䜑䛻ຍ䛘䛶䛔
䜛䚹
*8 10 × TBE buffer 䛿䚸䝖䝸䝇(䝠䝗䝻䜻䝅䝯䝏䝹)䜰䝭䝜䝯䝍䞁 (108 g)䚸䜋䛖㓟 (55 g)䚸
EDTA·2Na (7.43 g) 䜢⵨␃Ỉ䛻⁐ゎ䛥䛫 1000 mL 䛻䛧䛯䜒䛾䜢౑⏝䛧䛯䚹䜎䛯䚸1 ×
TBE buffer 䛿䚸10 × TBE buffer 䜢⵨␃Ỉ䛷 10 ಸᕼ㔘䛧౑⏝䛧䛯䚹
*9 40%䜰䜽䝸䝹䜰䝭䝗 (19 : 1) ⁐ᾮ䛿䚸䜰䜽䝸䝹䜰䝭䝗 (190 g)䚸N,N'-䝯䝏䝺䞁䝡䝇䜰䜽
䝸䝹䜰䝭䝗 (10 g) 䜢 Milli Q 䛻⁐ゎ䛥䛫 500 mL 䛻䛧䛯䜒䛾䜢౑⏝䛧䛯䚹
61
2-5. ➨୕❶➨୕⠇䚸➨ᅄ❶➨஧⠇䛻㛵䛩䜛ᐇ㦂
Figure 3-5䚸4-2 䛻㛵䛩䜛ᐇ㦂
䜶䝑䝨䞁䝗䝹䝣䝏䝳䞊䝤䛻䚸䜶䝏䝙䝹䝧䞁䝊䞁ᆺேᕤ᰾㓟 BE 䜎䛯䛿 ME 䜢ྵ䜐୍ᮏ㙐
DNA (100 μM solution in H2O, 20 μL, 2 nmol)䚸4-fluorobenzylazide (1 mM solution in
DMSO, 20 μL, 20 nmol)䚸DMSO䠄17 μL䠅, MeCN (22 μL) 䜢⁛⳦䛧䛯 0.1 M phosphate
buffer (pH 7.0)*1 (156 μL) 䛻ຍ䛘䛯䚹䛭䛾ᚋ䚸(+)-sodium
2
L-ascorbate*
(100 mM
solution in H2O, 2 μL, 200 nmol)䚸copper (ϩ) sulfate pentahydrate (100 mM solution
in H2O, 2 μL, 200 nmol) 䜢㡰ḟຍ䛘䚸1 ⛊䜋䛹䝪䝹䝔䝑䜽䝇䛧䚸ᐊ ୗ 15 ศ㛫㟼⨨䛧
䛯䚹䜰䝹䝂䞁㞺ᅖẼୗ➼≉ู䛺᧯స䛿⾜䜟䛺䛔䚹15 ศᚋ䚸Millex®-HV 䝣䜱䝹䝍䞊䛷
ℐ㐣䛧䚸⁛⳦Ỉ䛷Ὑ䛔㎸䜏䚸┤䛱䛻 HPLC 䛻䛶 ᐃ䛧䛯䚹䛺䛚䚸H2O ཬ䜃 DMSO 䛿
⁛⳦䛧䛯䜒䛾䜢౑⏝䛧䛯䚹⏕ᡂ䛧䛯䝖䝸䜰䝌䞊䝹య䛿 MALDI-TOF MASS 䛻䛶ᵓ㐀☜
ㄆ䜢⾜䛳䛯䚹
*1 0.1 M phosphate buffer (pH 7.0) 䛿䚸䜚䜣㓟Ỉ⣲஧䝘䝖䝸䜴䝮䛸䜚䜣㓟஧Ỉ⣲䝘䝖䝸
䜴䝮䛸䜢䚸䛭䜜䛮䜜 0.1 M 䛻䛺䜛䜘䛖䛻⁛⳦Ỉ䛻⁐ゎ䛧䚸pH 䝯䞊䝍䛻䜘䜚 pH 䜢ㄪ〇䛧
䛯䚹
*2 (+)-Sodium L-ascorbate (100 mM solution in H2O) 䛿䚸䛩䛠䛻㓟໬ຊ䛜పୗ䛧䛶
䛧䜎䛖䛣䛸䛛䜙⏝஦ㄪ〇䛜ᚲせ䛷䛒䜛䚹
62
2-6. ➨୕❶➨ᅄ⠇䚸➨ᅄ❶➨୕⠇䛻㛵䛩䜛ᐇ㦂
Figure 3-6䚸4-4 䛻㛵䛩䜛ᐇ㦂
䜶䝑䝨䞁䝗䝹䝣䝏䝳䞊䝤䛻䚸ྜᡂ䛧䛯᰾㓟䜸䝸䝂䝬䞊䛾┦⿵㙐ྠኈ䜢 600 pmol ศຍ䛘
䛶஝ᅛ䛥䛫䚸 ᐃ⏝⦆⾪ᾮ*1 (200 μL) 䛻⁐ゎ䛧䛶 3 μM 䛻ㄪ〇䛧䛯䚹䛭䛧䛶䚸90 ºC
䛷 5 ศ㛫ຍ⇕䛧䛯ᚋ䚸ᐊ ୗ 1 ᫬㛫௨ୖ㟼⨨䛩䜛䛣䛸䛷䚸᰾㓟䜸䝸䝂䝬䞊䛾┦⿵㙐ྠ
ኈ䜢䝝䜲䝤䝸䝎䜲䝈䛥䛫䛯䚹䛭䛾ᚋ䚸15 ศ㛫⬺Ẽ䜢⾜䛔䚸ᚓ䜙䜜䛯䝃䞁䝥䝹䛾䛖䛱 180
μL 䜢ᑓ⏝䛾 10 mm 䝉䝹䛻ධ䜜䚸Tm ᐃᶵ䛷 A260 䛾 ᗘኚ໬䜢 ᐃ䛧䛯䚹Tm ᐃ⤊
஢ᚋ䚸ᚓ䜙䜜䛯⼥ゎ᭤⥺䛛䜙䚸Tm 䜢୰⥺ἲ䛻䜘䜚⟬ฟ䛧䛯䚹
*1 Tm ᐃ⏝⦆⾪ᾮ䛿䚸10 mM phosphate buffer (pH 7.0)/100 mM NaCl 䛷䛒䜚䚸0.1
M phosphate buffer (pH 7.0) (1 mL)䚸1 M NaCl (1 mL)䚸⁛⳦Ỉ (8 mL) 䜢ΰྜ䛧 10
mL 䛻䛧䛯䜒䛾䜢౑⏝䛧䛯䚹
Figure 4-7 䛻㛵䛩䜛ᐇ㦂
ୖグ䛸ྠᵝ䛻䚸Tm ᐃ⏝⦆⾪ᾮ୰䚸஧ᮏ㙐 RNA 䝃䞁䝥䝹䛾䝝䜲䝤䝸䝎䜲䝈ཬ䜃⬺Ẽ
䜢⾜䛔䚸50 mM MgCl2 Ỉ⁐ᾮ䜢㐺㔞ຍ䛘䛶䝃䞁䝥䝹⃰ᗘ䜢 3 μM 䛻ㄪ〇䛧䚸㟼䛛䛻䝢
䝨䝑䝔䜱䞁䜾䛧䛯䚹䛭䛾ᚋ䛾᧯స䛿ୖグ䛸ኚ䜟䜙䛺䛔䚹
5 mM 䛾 Mg2+䜢ῧຍ䛩䜛ሙྜ䚸䛿䛨䜑䛻 Tm ᐃ⏝⦆⾪ᾮ (180 mL) 䛻⁐ゎ䛧䚸䛭䛾
ᚋ MgCl2 ⁐ᾮ䜢 20 μL ῧຍ䛩䜛䛣䛸䛷䚸䝃䞁䝥䝹⃰ᗘ䜢 3 μM䚸Mg2+⃰ᗘ䜢 5 mM 䛻
ㄪ〇䛧䛯䚹
63
2-7. ➨୕❶➨஬⠇䚸➨ᅄ❶➨ᅄ⠇䛻㛵䛩䜛ᐇ㦂
஧ᮏ㙐᰾㓟䜸䝸䝂䝬䞊䛷䛾䜽䝸䝑䜽཯ᛂ
䜎䛪䚸Ᏻᐃ䛺஧ᮏ㙐䜢⤌䜎䛫䜛䛯䜑䛻䚸䜶䝏䝙䝹䝧䞁䝊䞁ᆺேᕤ᰾㓟 BE 䜎䛯䛿 ME 䜢
ྵ䜐୍ᮏ㙐᰾㓟䜸䝸䝂䝬䞊(100 μM solution in H2O, 20 μL, 2 nmol)䛸┦⿵㙐(100 μM
solution in H2O, 20 μL, 2 nmol)䜢䜶䝑䝨䞁䝗䝹䝣䝏䝳䞊䝤䛻ධ䜜䚸஝ᅛ䛥䛫䚸0.1 M
phosphate buffer (pH 7.0)/1 M NaCl (66 μL) 䛻⁐ゎ䛧䛯䚹䛭䛧䛶䚸90 ºC 䛷 5 ศ㛫ຍ⇕
䛧䛯ᚋ䚸ᐊ ୗ 1 ᫬㛫௨ୖ㟼⨨䛩䜛䛣䛸䛷䚸᰾㓟䜸䝸䝂䝬䞊䛾┦⿵㙐ྠኈ䜢䝝䜲䝤䝸䝎
䜲䝈䛥䛫䛯䚹䛭䛾ᚋ䚸4-fluorobenzylazide (1 mM solution in DMSO, 20 μL, 20 nmol)䚸
DMSO䠄17 μL䠅, MeCN (22 μL) 䜢⁛⳦䛧䛯 0.1 M phosphate buffer (pH 7.0) (90 μL)
䛻ຍ䛘䛯䚹䛭䛾ᚋ䚸(+)-sodium L-ascorbate (100 mM solution in H2O, 2 μL, 200 nmol)䚸
copper (ϩ) sulfate pentahydrate (100 mM solution in H2O, 2 μL, 200 nmol) 䜢㡰ḟຍ
䛘䚸1 ⛊䜋䛹䝪䝹䝔䝑䜽䝇䛧䚸ᐊ ୗ 15 ศ㛫㟼⨨䛧䛯䚹䜰䝹䝂䞁㞺ᅖẼୗ➼≉ู䛺᧯
స䛿⾜䜟䛺䛔䚹15 ศᚋ䚸Millex®-HV 䝣䜱䝹䝍䞊䛷ℐ㐣䛧䚸⁛⳦Ỉ䛷Ὑ䛔㎸䜏䚸┤䛱
䛻 HPLC 䛻䛶 ᐃ䛧䛯䚹䛺䛚䚸H2O ཬ䜃 DMSO 䛿⁛⳦䛧䛯䜒䛾䜢౑⏝䛧䛯䚹⏕ᡂ䛧䛯
䝖䝸䜰䝌䞊䝹య䛿 MALDI-TOF MASS 䛻䛶ᵓ㐀☜ㄆ䜢⾜䛳䛯䚹
64
␎ㄒ㞟
Abs
Absorbance
APS
Ammonium Peroxodisulfate
BPB
Bromophenol Blue
CE
Cyanoethyl
CPG
Controlled Pore Glass
DMF
N,N-dimethylformamide
DMSO
Dimethyl Sulfoxide
DMTrCl
4,4'-Dimethoxytrityl Chloride
DNA
Deoxyribonucleic Acid
DNase
Deoxyribonuclease
dsRNA
double-strand RNA
EDTA
Ethylenediamine-N,N,N',N'-tetraacetic Acid
GNA
Glycol Nucleic Acid
HIV
Human Immunodeficiency Virus
3-HPA
3-Hydroxypicolinic Acid
HPLC
High Performance Liquid Chromatography
MALDI-TOF
Matrix Assisted Laser Desorption Ionization-Time of Flight
mRNA
messenger RNA
miRNA
micro RNA
MS
Mass Spectrometry
NMR
Nuclear Magnetic Resonance
65
ON
Oligonucleotide
OD
Optical Density
PAGE
Polyacrylamide Gel Electrophoresis
PBS
Phosphate Buffered Saline
pH
potential Hydrogen, power of Hydrogen
RCY
Radiochemical Yield
RISC
RNA-induced silencing complex
RNA
Ribonucleic Acid
RNase
Ribonuclease
rRNA
ribosomal RNA
rt
room temperature
shRNA
small hairpin RNA, short hairpin RNA
siRNA
small interfering RNA
snRNA
small nuclear RNA
TBAF
Tetrabutylammonium Fluoride
TBDMS
tert-Butyldimethylsilyl
TBE
Tris-Borate-EDTA
TBTA
Tris[(1-Benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
TEAA
Triethylammonium Acetate
TEMED
N,N,N',N'-Tetramethylenediamine
THF
Tetrahydrofuran
TLC
Thin Layer Chromatography
Tm
Melting Temperature
66
TMS
Trimethylsilyl
tRNA
transfer RNA
UV
Ultraviolet
VIS
Visible
XC
Xylene Cyanol FF
67
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72
ㅰ㎡
ⴭ⪅䜢ᮏ◊✲䜈䛸ᑟ䛝䚸㐙⾜䛩䜛䛻䛒䛯䜚⤊ጞᚚ᠓⠜䛺䜛ᚚᣦᑟ䠈ᚚ㠴᧡䜢㈷䜚䜎
䛧䛯ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ๰⸆་⒪᝟ሗ◊✲⛉ᩍᤵ㻌 ໭ฟᖾኵඛ⏕䛻⾺ᚰ䜘䜚ឤㅰ
䛔䛯䛧䜎䛩䚹
ㄽᩥ䜢ᇳ➹䛩䜛䛻䛒䛯䜚䚸ከ኱䛺䜛ᚚຓゝ䛺䜙䜃䛻ᚚᨭ᥼䜢㈷䜚䜎䛧䛯ᒱ㜧኱Ꮫ኱
Ꮫ㝔㐃ྜ๰⸆་⒪᝟ሗ◊✲⛉෸ᩍᤵ ụ⏣ᑗඛ⏕䛻῝䛟ឤㅰ⮴䛧䜎䛩䚹
ᮏ◊✲䛾ィ⏬䚸ᐇ㦂䚸⪃ᐹ䛾⣽㒊䚸ཎⴭㄽᩥ䛾సᡂ䛻⮳䜚ከ኱䛺䜛䛤ᣦᑟ䜢㈷䜚䜎
䛧䛯ᒱ㜧኱ᏛᕤᏛ㒊⏕࿨ᕤᏛ⛉ຓᩍ㻌 ႐ከᮧᚨ᫛ඛ⏕䛻῝䛟ឤㅰ⮴䛧䜎䛩䚹
䛥䜙䛻䚸ᮏ◊✲䜢⾜䛖㝿䛻ᚚຓゝ䚸ᚚ༠ຊῧ䛘䜢㈷䜚䜎䛧䛯ᒱ㜧⸆⛉኱Ꮫྡ㄃ᩍᤵ
䠄ඖᒱ㜧኱ᏛᕤᏛ㒊⏕࿨ᕤᏛ⛉≉ู༠ຊ◊✲ဨ䠅 ∦ᒸ㈆ඛ⏕䛻῝䛟ឤㅰ⮴䛧䜎䛩䚹
䜎䛯䚸ከ኱䛺ᚚ༠ຊ䛸ᚚຓゝ䜢㡬䛝䜎䛧䛯ᒱ㜧኱ᏛᕤᏛ㒊≉௵ຓᩍ㻌 ᰘ⏣⥤ඛ⏕䚸
ⓑ▼Ꮥ㞝༤ኈ䚸୰ᓥ♩⨾ಟኈ䚸⥘ሙెዉಟኈ䛻῝䛟ឤㅰ⮴䛧䜎䛩䚹
䛭䛧䛶䚸ᮏ◊✲䜢㐙⾜䛩䜛䛻䛒䛯䜚䚸◊✲άື䜢ඹ䛻䛧䛶䛝䛯䚸ᒱ㜧኱ᏛᕤᏛ㒊⏕
࿨ᕤᏛ⛉⏕య཯ᛂᕤᏛㅮᗙ໭ฟ◊✲ᐊ䛾Ꮫ⏕ⓙ䛥䜣䛻῝䛟ឤㅰ⮴䛧䜎䛩䚹
㻌 ᮏㄽᩥ䛾ᑂᰝ䛻䛒䛯䜚᭷┈䛺ᚚຓゝ䜢㈷䜚䜎䛧䛯ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ๰⸆་⒪᝟
ሗ◊✲⛉ᩍᤵ㻌 ㉥ᑿᖾ༤༤ኈ䚸ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ๰⸆་⒪᝟ሗ◊✲⛉ᩍᤵ ୹
⩚㞞அ༤ኈ䚸ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ๰⸆་⒪᝟ሗ◊✲⛉‽ᩍᤵ ụ⏣ᑗ༤ኈ䛻ཌ䛟
73
ᚚ♩⏦䛧ୖ䛢䜎䛩䚹
◊✲άື䛻㛵䛩䜛஦ົᡭ⥆䛝䜢䛧䛶㡬䛝䜎䛧䛯ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ๰⸆་⒪᝟ሗ
◊✲⛉㐃ྜ๰⸆ಀ䛾᪉䚻䛻ឤㅰ⮴䛧䜎䛩䚹
᭱ᚋ䛻䚸㛗ᖺ䛻䜟䛯䜚䛣䛾䜘䛖䛺ຮᏛ䛾ᶵ఍䜢୚䛘䛶䛟䛰䛥䛳䛯∗䚸ẕ䛻ᚰ䜘䜚ឤㅰ
⮴䛧䜎䛩䚹䜎䛯䚸ᖖ䛻ᚰ䛾ᨭ䛘䛸䛺䜚䚸ບ䜎䛧䛶䛟䜜䛯ጔ䛻ឤㅰ䛔䛯䛧䜎䛩䚹
74