細胞膜ドメインの構造および形成機構の解明を指向したガングリオシドプローブの創製

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Title

細胞膜ドメインの構造および形成機構の解明を指向したガ

_{ングリオシドプローブの創製( 本文(Fulltext) )}

Author(s)

河村, 奈緒子

Report No.(Doctoral

Degree)

博士(農学) 乙第151号

Issue Date

2018-09-21

Type

博士論文

Version

ETD

URL

http://hdl.handle.net/20.500.12099/77257

※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。

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⣽

⣽⬊⭷ࢻ࣓࢖ࣥࡢᵓ㐀࠾ࡼࡧᙧᡂᶵᵓࡢゎ᫂ࢆ

ᣦྥࡋࡓ࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢ๰〇

㸰㸮㸯㸶ᖺ

ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ㎰Ꮫ◊✲⛉

㸦ᒱ㜧኱Ꮫ㸧

Ἑᮧዉ⥴Ꮚ

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⣽

⣽⬊⭷ࢻ࣓࢖ࣥࡢᵓ㐀࠾ࡼࡧᙧᡂᶵᵓࡢゎ᫂ࢆ

ᣦྥࡋࡓ࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢ๰〇

Ἑᮧዉ⥴Ꮚ

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04 06 11 11 14 15 15 18 23 24 28 28 31 31 35 35 37 39 40 42

┠

┠ḟ

␎ྕ⾲ ⥴ゝ ➨୍㒊⺯ග࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢ㛤Ⓨ ➨୍❶ ◊✲⫼ᬒ ➨஧❶ ศᏊタィ ➨୕❶ ࢞ࣛࢡࢺ࣮ࢫ6 ఩ᶆ㆑ GM3 ࣉ࣮ࣟࣈࡢྜᡂ ➨୍⠇ ྜᡂᡓ␎ ➨஧⠇ ࢩ࢔ࣜࣝ࢞ࣛࢡࢺ࣮ࢫ౪୚యࡢྜᡂ ➨୕⠇ GM3 㦵᱁ࡢᵓ⠏ ➨ᅄ⠇ ⺯ගⰍ⣲ࡢᑟධ ➨ᅄ❶ ࢩ࢔ࣝ㓟9 ఩ᶆ㆑ GM3 ࣉ࣮ࣟࣈࡢྜᡂ ➨୍⠇ ྜᡂᡓ␎ ➨஧⠇ ࢩ࢔ࣜࣝ࢞ࣛࢡࢺ࣮ࢫ౪୚యࡢྜᡂϨ ➨୍㡯 1,4-ࣛࢡࢺࣥయࡢྜᡂ ➨஧㡯 ౪୚య࡬ࡢኚ᥮ ➨୕⠇ GM3 㦵᱁ࡢྜᡂ᳨ウϨ ➨ᅄ⠇ ࢩ࢔ࣜࣝ࢞ࣛࢡࢺ࣮ࢫ౪୚యࡢྜᡂϩ ➨஬⠇ GM3 㦵᱁ࡢྜᡂ᳨ウϩ ➨භ⠇ ⺯ගⰍ⣲ࡢᑟධ ➨஬❶ GM3 ࣉ࣮ࣟࣈࡢᶵ⬟ホ౯࡜୍ศᏊほᐹᐇ㦂

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42 50 50 53 55 55 57 58 61 62 64 65 70 70 70 72 74 76 76 78 81 82 ➨୍⠇ ࣛࣇࢺศ㓄≉ᛶࡢ᳨ド ➨஧⠇ ୍ศᏊほᐹᐇ㦂 ➨୍㡯 ᣑᩓಀᩘࡢィ⟬ ➨஧㡯 GPI ࢔࣮ࣥ࢝ᆺཷᐜయ࡜ࡢぶ࿴ᛶࡢ᳨ド ➨භ❶ ࢩ࢔ࣝ㓟9 ఩ᶆ㆑ GM1 ཬࡧ GM2 ࣉ࣮ࣟࣈࡢྜᡂ ➨୍⠇ ྜᡂᡓ␎ ➨஧⠇ ࢩ࢔ࣜࣝ࢞ࣛࢡࢺ࣮ࢫཷᐜయࡢྜᡂ ➨୕⠇ GM1 ࢚ࣆࢺ࣮ࣉ㦵᱁ࡢᵓ⠏࡜౪୚య࡬ࡢኚ᥮ ➨ᅄ⠇ GM1 㦵᱁ࡢᵓ⠏࡜⺯ගⰍ⣲ࡢᑟධ ➨஬⠇ GM2 ࢚ࣆࢺ࣮ࣉ㦵᱁ࡢᵓ⠏࡜౪୚య࡬ࡢኚ᥮ ➨භ⠇ GM2 㦵᱁ࡢᵓ⠏࡜⺯ගⰍ⣲ࡢᑟධ ➨୐⠇ ⺯ගGM1 ཬࡧ GM2 ࣉ࣮ࣟࣈྜᡂࡢࡲ࡜ࡵ ➨୐❶ ࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢᶵ⬟ホ౯࡜୍ศᏊほᐹᐇ㦂 ➨୍⠇ ᶵ⬟ホ౯ ➨୍㡯 ࣛࣇࢺศ㓄≉ᛶࡢ᳨ド ➨஧㡯 ᣑᩓಀᩘࡢィ⟬ ➨஧⠇ GPI ࢔࣮ࣥ࢝ᆺཷᐜయ࡜ࡢぶ࿴ᛶࡢ᳨ド ➨୕⠇ ࢞ࣥࢢࣜ࢜ࢩࢻ⤖ྜࢱࣥࣃࢡ㉁࡜ࡢぶ࿴ᛶࡢ᳨ド ➨୍㡯 ⢾㙐ㄆ㆑ࢱࣥࣃࢡ㉁࡟ᑐࡍࡿ⤖ྜ⬟ࡢ᳨ド ➨஧㡯 EGF ཷᐜయ࡜ࡢ஧Ⰽྠ᫬୍ศᏊほᐹ ⥲ᣓ ᐇ㦂ࡢ㒊

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143 143 149 153 155 155 158 160 162 162 166 170 172 175 176 178 179 200 201 ➨ ➨஧㒊 ග཯ᛂᛶ࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢ㛤Ⓨ ➨୍❶ ◊✲⫼ᬒ ➨஧❶ ศᏊタィ ➨୕❶ ྜᡂᡓ␎ ➨ᅄ❶ ⢾㙐ಟ㣭ᆺග཯ᛂᛶࣉ࣮ࣟࣈࡢྜᡂ ➨୍⠇ Trifluoromethylphenyl diazirine (TPD) ࣘࢽࢵࢺࡢྜᡂ ➨஧⠇

ග཯ᛂᛶ

GM3 ࣉ࣮ࣟࣈࡢྜᡂ ➨୕⠇

ග཯ᛂᛶ

GM1 ࣉ࣮ࣟࣈࡢྜᡂ ➨஬❶ ⬡㉁ಟ㣭ᆺග཯ᛂᛶGM3 ࣉ࣮ࣟࣈࡢྜᡂ ➨୍⠇ ග཯ᛂᛶ⬡⫫㓟ࡢྜᡂ ➨஧⠇ Sph ཷᐜయࡢྜᡂ ➨୕⠇ Glc-Sph ཷᐜయࡢྜᡂ ➨ᅄ⠇ ⢾⬡㉁㦵᱁ࡢᵓ⠏࡜⬡⫫㓟ࡢᑟධ ➨஬⠇ ⺯ගⰍ⣲ࡢᑟධ ➨භ⠇ ࣅ࢜ࢳࣥࡢᑟධ ⥲ᣓ ᐇ㦂ࡢ㒊 ⤖ㄒ ㅰ㎡

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␎グ୍ぴ

ᮏㄽᩥ࡟࠾࠸࡚௨ୗ࡟♧ࡍ␎ྕࢆ⏝࠸ࡓࠋ Ac : acetyl Bn : benzyl Bz : benzoyl

CAN : cerium(IV) ammonium nitrate Cer : ceramide Chol : cholesterol DBU : 1,8-diazabicyclo[5.4.0]undec-7-ene DIEA : N,N-diisopropylethylamine DMAP : 4-N,N-dimethylaminopyridine DMF : N,N-dimethylformamide DMTST : dimethyl(methylthio)sulfonium triflate DOPE : 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DRM : detergent-resistant membrane DTBMP : 2,6-di-tert-butyl-4-methylpyridine EDC : 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Gal : galactose Glc : glucose GPI : glycosylphosphatidylinositol HOOBt : hydroxyl-3,4-dihydro-4-oxo-1,2,3-benzotriazine MP : p-methoxyphenyl MS : molecular sieves

MS AW : acid-washed molecular sieves Neu : neuraminic acid

NHS : N-hydroxysuccinimide NIS : N-iodosuccinimide Pfp : pentafluorophenyl PMB : p-methoxybenzyl Pyr : pyridine Sph : sphingosine

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TBAI : tetrabutylammonium iodide TBS : t-butyldimethylsilyl TEA : triethylamine Tf : trifluoromethanesulfonyl TFAc : trifluoroacetyl THF : tetrahydrofuran

TMSOTf : trimethylsilyl trifluoromethanesulfonate Troc : 2,2,2-trichloroethoxycarbonyl Ts : p-toluenesulfonyl

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⥴ゝ

⣽⬊ࡣ⏕యࢆᵓᡂࡍࡿᇶᮏ༢఩࡛࠶ࡾࠊDNA ࡢไᚚࡢࡶ࡜⏕࿨άື࡟ᚲせ࡞ࢱࣥࣃࢡ㉁ ࢆྜᡂࡋࠊቑṪ࣭ศ໬ࢆ㔜ࡡ࡚⏕࿨ࢆႠࡴࠋࡇࢀࡽ୍ࡘ୍ࡘࡢ⣽⬊ࡣࠊ⣽⬊⭷࡟ࡼࡗ࡚ໟࡲ ࢀࡓ⊂❧ࡋࡓᙧ≧ࢆᣢࡗ࡚࠾ࡾࠊ⣽⬊⭷ࡣࠊ⭷እ⏺࠿ࡽ⣽⬊ࢆ༊⏬࣭ಖㆤࡍࡿᙺ๭ࢆᢸ࠺ࠋ ࡋ࠿ࡋ࡞ࡀࡽࠊ⣽⬊⭷ࡣእ⏺࠿ࡽ㝸࡚ࡿᶵ⬟ࡔࡅ࡛࡞ࡃࠊ≀㉁ࡢᤵཷࡸእ⏺࡜ࡢ┦஫స⏝ࡢ ཯ᛂሙ࡜ࡋ࡚ࡢᙺ๭ࢆᢸ࠺ࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡾࠊ⣽⬊⭷⮬యࡢᛶ≧ࡸᶵ⬟ࡀὀ┠ࡉࢀ࡚࠸ ࡿࠋ 1972 ᖺࠊSinger ࡜ Nicolson ࡣࠊ⣽⬊⭷ࡢືⓗᴫᛕ࡜ࡋ࡚ࠊὶືࣔࢨ࢖ࢡࣔࢹࣝࢆᥦၐࡋ ࡓ1_{ࠋࡇࢀ࡟ࡼࡾࠊ⣽⬊⭷ࡣ୧ぶ፹ᛶࡢ⬡㉁ศᏊࡀὶືⓗ࡞⬡㉁஧㔜⭷ࢆᙧᡂࡋ࡚࠾ࡾࠊࡑ} ࡢ୰ࢆ⭷ࢱࣥࣃࢡ㉁ࡀ⮬⏤ᣑᩓࡍࡿ࡜࠸࠺⪃࠼᪉ࡀᗈࡲࡗࡓࠋࡇࡢᇶᮏᴫᛕࡣ⌧ᅾࡶཷࡅ ධࢀࡽࢀ࡚࠸ࡿࡀࠊࡑࡢᚋࡢ◊✲࡛ࠊ⭷ࢱࣥࣃࢡ㉁ࡢࡳ࡞ࡽࡎ⬡㉁ศᏊࡶ୙ᆒ୍࡟Ꮡᅾࡋࠊ ࢻ࣓࢖ࣥᵓ㐀ࢆ࡜ࡿࡇ࡜ࡀศ࠿ࡗ࡚ࡁࡓࠋ1997 ᖺࠊSimons ࡽࡣ⣽⬊⭷ࢻ࣓࢖ࣥࡢ᪂ࡓ࡞ᴫ ᛕ࡜ࡋ࡚ࣛࣇࢺ௬ㄝࢆᥦၐࡋࡓ2_{ࠋᙼࡽࡣࠊ⣽⬊⭷ୖ࡟ࡣࠊࢫࣇ࢕ࣥࢦ⢾⬡㉁ࡸࢥࣞࢫࢸࣟ} ࣮ࣝ࡟ᐩࡴࠊ⬡㉁ࣛࣇࢺ࡜࿧ࡤࢀࡿ≉Ṧ࡞⭷㡿ᇦࡀᏑᅾࡋࠊࡑࡇ࡬ᵝࠎ࡞ࢩࢢࢼࣝศᏊࡀ⃰ ⦰ࡉࢀࡿࡇ࡜࡟ࡼࡗ࡚ࠊ⣽⬊ෆ࡬ࡢࢩࢢࢼࣝఏ㐩ࡀ⾜ࢃࢀࡿ࡜⪃࠼ࡓ (Figure 1)ࠋ⬡㉁ࣛࣇ ࢺࡢᏑྰࠊᐃ⩏࡟ࡘ࠸࡚ࡣ⌧ᅾࡶ㆟ㄽࡀ⥆࠸࡚࠸ࡿࡀࠊ⣽⬊ࡢቑṪࡸ᥋╔࡞࡝ࠊᗈ⠊ᅖ࡟ࢃ ࡓࡿ⏕࿨⌧㇟࡟࠾࠸࡚⬡㉁ࣛࣇࢺࡢ௓ᅾࢆᨭᣢࡍࡿ⤖ᯝࡀ⵳✚ࡋࡘࡘ࠶ࡿࠋ

1_{S. J. Singer, G. L. Nicolson, Science 1972, 175, 720-731.}

2_{a) K. Simons, E. Ikonen, Nature 1997, 387, 569-572. b) D. A. Brown, J. K. Rose, Cell 1992, 68,}

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Figure 1. ⬡㉁ࣛࣇࢺࡢᵓ㐀

⏕యศᏊࡢᣲືࢆ⏬ീ࡜ࡋ࡚ᤊ࠼ࡿ᪂ࡓ࡞◊✲㡿ᇦࠕศᏊ࢖࣓࣮ࢪࣥࢢࠖࡀ㏆ᖺᛴ㏿࡟Ⓨ ᒎࡋ࡚ࡁࡓ⫼ᬒ࡟ࡣࠊࡇࡢࡼ࠺࡟⏕యศᏊࡢಶࠎࡢᛶ㉁ࢆ⌮ゎࡍࡿᚲせᛶࡀ㧗ࡲࡗ࡚ࡁࡓ ஦ᐇࡀᏑᅾࡍࡿࠋศᏊ࢖࣓࣮ࢪࣥࢢࡣ⏕ࡁ࡚࠸ࡿ⣽⬊ࠊ⤌⧊ࠊ࠶ࡿ࠸ࡣಶయ࡛ࡢ⏕యศᏊࡢ ᣲືࡸάᛶࠊ㔞ࢆྍど໬ࡍࡿࡶࡢ࡛࠶ࡾࠊ୺࡟࣏ࢪࢺ᩿ࣟࣥᒙ (PET)ࠊ༢୍ศᏊᨺᑕ᩿ᒙ᧜ ᙳ (SPECT)ࠊ⺯ග࢖࣓࣮ࢪࣥࢢࡢ㡿ᇦ࡟ศࡅࡽࢀࡿࠋ୰࡛ࡶ⺯ග࢖࣓࣮ࢪࣥࢢࡣࠊ⺯ගᶆ㆑ ࡋࡓ⏕యศᏊࡢᣲືࢆ⏕⣽⬊୰࡛┤᥋ほᐹࡍࡿ஦ࢆྍ⬟࡜ࡋࠊ⏕࿨⛉Ꮫ◊✲ࡢ㌍㐍࡟ᚲ㡲 ࡞ᢏ⾡࡜ࡋ࡚ὀ┠ࢆ㞟ࡵ࡚࠸ࡿࠋ ࡇࡢ◊✲㡿ᇦ࡟࠾࠸࡚ࠊᴋぢࠊ㕥ᮌࡽࡣࠊ⏕⣽⬊⭷ୖ࡟࠾ࡅࡿ୍ศᏊほᐹἲ3_{ࢆ☜❧ࡋࠊ} ⬡㉁ࣛࣇࢺᵓᡂᡂศࡢືែࡢヲ⣽࡞ゎᯒࢆ㐍ࡵ࡚࠸ࡿࠋ⬡㉁ࣛࣇࢺࢆ௓ࡋࡓࢩࢢࢼࣝఏ㐩 ࡣࣜ࢞ࣥࢻࡢ୍่⃭࡛᫬ⓗ࡟ᘬࡁ㉳ࡇࡉࢀࡿࡓࡵࠊࡑࢀࡽࢆ㟼ⓗ࡞≧ἣୗ࡛෌⌧ࡍࡿࡇ࡜ ࡣ㞴ࡋ࠸ࠋࡑࢀ࡟ᑐࡋࠊ୍ศᏊほᐹἲ࡛ࡣࠊ」ᩘࡢ⏕యศᏊࡢࣜ࢔ࣝࢱ࢖࣒࡞ほᐹࢆྍ⬟࡜ ࡍࡿࡓࡵࠊ⬡㉁ࣛࣇࢺࡢ኱ࡁࡉࡸᑑ࿨ࠊᵓᡂศᏊࡢᣲື࡞࡝࡟㛵ࡍࡿヲ⣽࡞ゎᯒ⤖ᯝࡀࡇࢀ ࡲ࡛࡟ᚓࡽࢀ࡚࠸ࡿࠋ≉࡟ࠊ⬡㉁ࣛࣇࢺࡢ୺せᡂศ࡛࠶ࡿ GPI ࢔࣮ࣥ࢝ᆺཷᐜయࡢほᐹ⤖ ᯝࡣ⬡㉁ࣛࣇࢺᙧᡂᶵᵓࢆゎࡃୖ࡛㔜せ࡞▱ぢࢆ࠸ࡃࡘ࠿୚࠼࡚࠸ࡿࠋࡑࢀࡣࠊࡲࡎ୍Ⅼ┠

3_{A. Kusumi, T. A. Tsunoyama, K. M. Hirosawa, R. S. Kasai, T. K. Fujiwara, Nat. Chem. Biol. 2014, 10,}

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࡟ࠊGPI ࢔࣮ࣥ࢝ᆺཷᐜయࡀࣜ࢞ࣥࢻ่⃭๓ࠊ༶ࡕࣛࣇࢺᙧᡂ๓࡟ྠ✀ศᏊ㛫࡛ࡢࡳࠊ≉␗ ⓗ࡟࣍ࣔࢲ࢖࣐࣮ࢆᙧᡂࡍࡿ࡜࠸࠺ࡇ࡜࡛࠶ࡿ4_{(Figure 2}5_{)ࠋᴋぢ࣭㕥ᮌࡽࡣࡇࢀࢆ࣍ࣔࢲ} ࢖࣐࣮ࣛࣇࢺ࡜࿧ࡧࠊGPI ࢔࣮ࣥ࢝ᆺཷᐜయࡀ㛵୚ࡍࡿ᭱ᑠᵓ㐀ࡢࣛࣇࢺ࡜ࡋࡓࠋࡉࡽ࡟ࠊ ࣍ࣔࢲ࢖࣐࣮ࣛࣇࢺࡀ఍ྜࡋࡓࢸࢺ࣐࣮ࣛࣛࣇࢺࡀᙧᡂࡉࢀࡿ஦ࡶ᫂ࡽ࠿࡟ࡉࢀࡓࡀࠊ㦫 ࡃ࡭ࡁࡇ࡜࡟ࠊࡇࡢẁ㝵࡛ࡣ␗✀ࡢ࣍ࣔࢲ࢖࣐࣮ࣛࣇࢺࡢ఍ྜࢆチᐜࡋࡓ࣊ࢸࣟࢸࢺ࣐ࣛ ࣮ࡀᏳᐃࡋ࡚Ꮡᅾࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡘࡲࡾࠊࡇࡢẁ㝵ࡢ఍ྜ࡛ࡣࠊࢱࣥࣃࢡ㉁ࡢ ≉␗ⓗ┦஫స⏝࡛ࡣ࡞ࡃࠊ≉␗ᛶࡢ࡞࠸⬡㉁㛫┦஫స⏝ࡀࢸࢺ࣐࣮ࣛࣛࣇࢺᙧᡂ࡟ᐤ୚ࡋ ࡚࠸ࡿ࡜᥎ ࡉࢀࡿࠋࡇࢀࡽࡢ⤖ᯝࡣ GPI ࢔࣮ࣥ࢝ᆺཷᐜయࡢ࣍ࣔࢲ࢖࣐࣮ࣛࣇࢺࡀ⬡㉁ ࣛࣇࢺᙧᡂ᫬ࡢ᭱ᑠ༢఩࡛࠶ࡿࡇ࡜ࢆ♧ࡋࠊࡇࢀࢆ㉳Ⅼ࡟ᵓ㐀୙ᆒ୍࡞⬡㉁ศᏊࡢ఍ྜࡀ ㄏ㉳ࡉࢀࡿࡇ࡜ࢆ♧၀ࡍࡿ㔜せ࡞⤖ᯝ࡛࠶ࡿࠋ

Figure 2. ᐃᖖ≧ែ࡟࠾ࡅࡿ GPI ࢔࣮ࣥ࢝ᆺཷᐜయࡢ࣍ࣔࢲ࢖࣐࣮ࣛࣇࢺ

4_{K. G. N. Suzuki, R. S. Kasai, K. M. Hirosawa, Y. L. Nemoto, M. Ishibashi, Y. Miwa, T. K. Fujiwara, A.}

Kusumi, Nat. Chem. Biol. 2012, 8, 774-783.

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ࡉࡽ࡟ࠊࣜ࢞ࣥࢻ่⃭ᚋࡢ୍ศᏊほᐹ࡟ࡼࡾࠊ఍ྜࡋࡓCD59 ࢡࣛࢫࢱ࣮ࡣ⣽⬊⭷ୖ࡛༢

⣧ࣈࣛ࢘ࣥ㐠ືࢆࡋࠊୗὶࢩࢢࢼࣝศᏊࡢ఍ྜ᫬࡟ STALL (Stimulation-induced Temporary

Arrest of LateraL diffusion) ࡜࿧ࡤࢀࡿ≉␗ⓗ࡞୍᫬೵␃ࢆ♧ࡍࡇ࡜6_{ࡀ᫂ࡽ࠿࡟ࡉࢀࡓ}

(Figure 3)ࠋࡇࡢࡼ࠺࡟⤌ᡂࡸᵓ㐀ࡀ୙ᆒ୍࠿ࡘ୙Ᏻᐃ࡛࠶ࡾࠊࡑࡢୖືⓗ࡞ᛶ㉁ࢆᣢࡘ⬡ ㉁ࣛࣇࢺࡢ◊✲࡟࠾࠸࡚ࡣࠊ୍ศᏊほᐹἲࡀᴟࡵ࡚᭷⏝࡛࠶ࡿࡇ࡜ࡀࠊ௨ୖࡢほᐹ⤖ᯝ࡟ࡼ ࡾᐇドࡉࢀࡓࠋ

Figure 3. GPI ࢔࣮ࣥ࢝ᆺཷᐜయࡢ STALL

⬡㉁ࣛࣇࢺࡣྛࠎࡢ⤌ᡂ࡟ᇶ࡙ࡁࠊ␗࡞ࡿᛶ㉁ࢆⓎ⌧ࡍࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋࡑࡢᛶ㉁ࡢ Ỵᐃ࡟㛵୚ࡍࡿࣛࣇࢺᵓᡂᡂศࡢ୍ࡘࡀ࢞ࣥࢢࣜ࢜ࢩࢻ࡛࠶ࡿࠋ࢞ࣥࢢࣜ࢜ࢩࢻࡣᵓ㐀୰ ࡟୍ṧᇶ௨ୖࡢࢩ࢔ࣝ㓟ࢆྵࡴࢫࣇ࢕ࣥࢦ⢾⬡㉁࡛࠶ࡾࠊࡑࡢ⢾㙐ᵓ㐀࡟ᇶ࡙ࡃ≉␗ⓗ࡞ ᶵ⬟ࡀ⏕⌮ⓗࠊ་Ꮫⓗ࡟ὀ┠ࡉࢀ࡚࠸ࡿࠋ౛ࢆᣲࡆࡿ࡜ࠊ࢔࣑ࣟ࢖ࢻβ ࢱࣥࣃࢡ㉁ࡣ⬡㉁ࣛ ࣇࢺ୰ࡢ࢞ࣥࢢࣜ࢜ࢩࢻGM1 ୖ࡟≉␗ⓗ࡟จ㞟ࡍࡿࠋࡇࡢจ㞟యࡢᙧᡂࡣ࢔ࣝࢶࣁ࢖࣐࣮ ⑓࡟≉ᚩⓗ࡞⑓⌮ኚ໬ࢆࡶࡓࡽࡍࡇ࡜࠿ࡽࠊ࢔ࣝࢶࣁ࢖࣐࣮⑓ࡢⓎ⑕ࡢ୍ᅉ࡛࠶ࡿ࡜⪃࠼ ࡽࢀ࡚࠸ࡿ7_{ࠋࡲࡓ஭ࣀཱྀࡽࡣ࢞ࣥࢢࣜ࢜ࢩࢻ} _{GM3 ࡀ⬡㉁ࣛࣇࢺࢆ௓ࡋ࡚࢖ࣥࢫࣜࣥཷᐜ}

6_{a) K. G. N. Suzuki, T. K. Fujiwara, F. Sanematsu, R. Iino, M. Edidin, A. Kusumi, J. Cell Biol. 2007,} 177, 717-730. b) K. G. N. Suzuki, T. K. Fujiwara, M. M. Edidin, A. Kusumi, J. Cell Biol. 2007, 177,

731-742.

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యࡢᶵ⬟ㄪ⠇࡟㛵ࢃࡗ࡚࠸ࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿ8_{ࠋࡋ࠿ࡋ࡞ࡀࡽࠊ⬡㉁ࣛࣇࢺࢆ௓ࡋࡓ࢞} ࣥࢢࣜ࢜ࢩࢻࡢከࡃࡢᶵ⬟ࡀぢฟࡉࢀ࡚࠸ࡿࡶࡢࡢࠊ࢞ࣥࢢࣜ࢜ࢩࢻࡢᣲືࢆᤊ࠼ࠊᛶ㉁ࢆ ヲ⣽࡟ゎᯒࡍࡿࡇ࡜ࡣᅔ㞴࡛࠶ࡿ࡜ࡉࢀ࡚ࡁࡓࠋࡑࢀࡣࠊኳ↛ࡢ࢞ࣥࢢࣜ࢜ࢩࢻࡢᶵ⬟ࢆಖ ᣢࡋࡓほᐹ⏝ࣉ࣮ࣟࣈࡢ㛤Ⓨࡀ㐜ࢀ࡚࠸ࡿࡇ࡜࡟ࡼࡿࠋࡇࡢࡼ࠺࡞ㄢ㢟ࢆ⫼ᬒ࡜ࡋ࡚ࠊ௒᪥ ࡛ࡣඃࢀࡓほᐹ⏝࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢ๰〇ࡀ୍ᒙᚲせ࡜ࡉࢀࡿࡼ࠺࡟࡞ࡗ࡚ࡁࡓࠋ ࡇࡢせồ࡟ᛂ࠼ࡿࡇ࡜ࢆ┠ᣦࡋࠊᮏ◊✲࡛ࡣ⣽⬊⭷ࢻ࣓࢖ࣥࡢᙧᡂᶵᵓࡸᵓ㐀ࡢゎ᫂ࢆ ᣦྥࡋࡓᶵ⬟ᛶ࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢ໬Ꮫྜᡂࢆ⾜࠺ࡇ࡜࡜ࡋࡓࠋᮏㄽᩥࡢ➨୍㒊࡛ ࡣࠊ୍ศᏊほᐹᐇ㦂ࢆᣦྥࡋࡓ⺯ග࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢ㛤Ⓨ࡟ࡘ࠸࡚㏙࡭ࡿࠋࡇࡇ࡛ ࡣࠊ⺯ගࣉ࣮ࣟࣈࡢ໬Ꮫྜᡂἲࡢ㛤Ⓨࠊ⥆࠸࡚⺯ගࣉ࣮ࣟࣈࡢ⏕⣽⬊⭷ୖ࡟࠾ࡅࡿᶵ⬟ࡢ᳨ ドࠊࡉࡽ࡟ࡣ୍ศᏊほᐹᐇ㦂࡟ࡼࡿࣛࣇࢺ㛵㐃ศᏊ࡜ࡢ┦஫స⏝ࡢほᐹ⤖ᯝ࡟ࡘ࠸࡚ㄽ㏙ ࡍࡿࠋ➨஧㒊࡛ࡣ࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢᶵ⬟ᣑᙇࢆᅗࡾࠊ㏆᥋ศᏊ࡜ࡢᯫᶫᙧᡂࡀྍ⬟ ࡞ග཯ᛂᛶ࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢ㛤Ⓨࢆヨࡳࡓࠋ✀ࠎࡢග཯ᛂᛶ࢞ࣥࢢࣜ࢜ࢩࢻࣉࣟ ࣮ࣈࡢ໬Ꮫྜᡂ࡟ࡘ࠸࡚ㄽ㏙ࡍࡿࠋ

8_{S. Tagami, J. Inokuchi, K. Kabayama, H. Yoshimura, F. Kitamura, S. Uemura, C. Ogawa, A. Ishii, M.}

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➨

➨୍㒊

⺯ග࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢ㛤Ⓨ

➨୍❶ ◊✲⫼ᬒ

࢞ࣥࢢࣜ࢜ࢩࢻࢆ⺯ගᶆ㆑ࡍࡿᡭἲ࡜ࡋ࡚ࠊᚑ᮶࡛ࡣࠊ࢞ࣥࢢࣜ࢜ࢩࢻ⤖ྜࢱࣥࣃࢡ㉁ࡸ ᢠయࡢ⺯ගᶆ㆑యࢆ฼⏝ࡋࡓ㛫᥋ⓗ࡞ᶆ㆑ἲࠊཪࡣኳ↛࠿ࡽࡢ࢞ࣥࢢࣜ࢜ࢩࢻᢳฟ≀ࢆ໬ Ꮫἲཪࡣ㓝⣲ἲ࡟ࡼࡾ⺯ගᶆ㆑య࡬࡜ㄏᑟࡍࡿ┤᥋ⓗ࡞ᶆ㆑ἲࡀ฼⏝ࡉࢀ࡚ࡁࡓࠋ 㛫᥋ⓗ࡞ᶆ㆑ἲ࡜ࡋ࡚ࡣࠊ౛࠼ࡤࠊ⺯ගᶆ㆑ࡋࡓࢥࣞࣛẘ⣲ B ࢧࣈࣘࢽࢵࢺཬࡧࢥ࣒ࢠ ⬇ⱆจ㞟⣲➼ࢆ⏝࠸ࡿࡇ࡜࡛ࠊ⣽⬊⭷ୖࡢGM1 ཬࡧ GM3 ࢆᶆ㆑໬ࡍࡿᡭἲࡀᗈࡃ฼⏝ࡉ ࢀ࡚࠸ࡓࠋࡋ࠿ࡋࠊࡇࢀࡽࡢࢱࣥࣃࢡ㉁ࡣከ౯࡛࠶ࡿࡓࡵࠊ࢞ࣥࢢࣜ࢜ࢩࢻࢆจ㞟ࡋࠊࡑࡢ ᒁᅾࡸᣲືࢆኚ࠼ࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ9,10_{ࠋࡲࡓࠊ࢞ࣥࢢࣜ࢜ࢩࢻᢠయࢆ฼⏝ࡍࡿච␿⺯} ගᰁⰍ࡛ࡣࠊ᭱ึࡢᕤ⛬࡜ࡋ࡚⣽⬊⭷ୖࡢ࢞ࣥࢢࣜ࢜ࢩࢻࢆࣃ࣒ࣛ࣍ࣝ࢔ࣝࢹࣄࢻࡸࢢࣝ ࢱࣝ࢔ࣝࢹࣄࢻ࡟ࡼࡿᅛᐃࢆ⾜࠺ࠋࡋ࠿ࡋ࡞ࡀࡽࠊᴋぢ࣭㕥ᮌࡽࡣࠊࡇࡢࡼ࠺࡞ฎ⌮࡟ࡼࡗ ࡚⬡㉁ศᏊࡢṤ࡝ࡣᅛᐃࡉࢀࡎࠊ⮬⏤ᣑᩓࡍࡿࡇ࡜ࢆᣦ᦬ࡋ࡚࠸ࡿࠋࡘࡲࡾࠊᅛᐃ໬ࡉࢀ࡚ ࠸࡞࠸࢞ࣥࢢࣜ࢜ࢩࢻ࡟ᑐࡋ࡚࢞ࣥࢢࣜ࢜ࢩࢻᢠయࠊ⺯ගᶆ㆑ࡋࡓ஧ḟᢠయࢆస⏝ࡉࡏࡿ ࡇ࡜࡛ࠊ࢞ࣥࢢࣜ࢜ࢩࢻࡢ఍ྜࢆㄏ㉳ࡍࡿྍ⬟ᛶࡀ࠶ࡿࠋࡑࡢⅭࠊච␿⺯ගᰁⰍ࡟࠾࠸࡚ࡶ ⏕⌮᮲௳࡜ࡣ␗࡞ࡿ⤖ᯝࢆ୚࠼ࡿ஦ࡀᠱᛕࡉࢀࡿ11_ࠋ ୍᪉࡛ࠊ┤᥋ⓗ࡞ᶆ㆑ἲ࡛ࡣࠊኳ↛ࡼࡾᢳฟࡋࡓ࢞ࣥࢢࣜ࢜ࢩࢻࢆཎᩱ࡜ࡋࡓᵓ㐀ኚ᥮ࡀ ⾜ࢃࢀ࡚ࡁࡓࠋ༶ࡕࠊGM1 ࢆ౛࡟ᣲࡆࡿ࡜ࠊNeu5 ఩ࠊGalN2 ఩ཬࡧ Sph2 ఩ࡢ࢔࣑ࢻ⤖ྜ

9_{D. J. Chinnapen, W. T. Hsieh, Y. M. te Welscher, D. E. Saslowsky, L. Kaoutzani, E. Brandsma, L. Dಬ}

Auria, H. Park, H., J. S. Wagmer, K. R. Drake, M. Kang, T. Benjamin, M. D. Ullman, C. E. Costello, A. K. Kenworthy, T. Baumgart, R. H. Massoi, W. I. Lencer, Dev. Cell 2012, 23, 573-586.

10_{A. T. Hammond, F. A. Heberte, T. Baumgart, D. Holowka, D. Baird, G. W. Geigenson, Proc. Natl.} Acad. Sci. USA 2005, 102, 6320-6325.

11_{K. A. K. Tanaka, K. G. N. Suzuki, Y. M. Shirai, S. T. Shibutani, M. S. Miyahara, H. Tsuboi, M. Yahara,}

(15)

ࢆ࢔ࣝ࢝ࣜຍỈศゎࡋࠊ㐟㞳࡜࡞ࡗࡓ࢔࣑ࣀᇶࡢ཯ᛂᛶᕪࢆ฼⏝ࡋ࡚┠ⓗ఩⨨࡟⺯ගศᏊ ࢆᑟධࡍࡿᡭἲࡸ12,13_{ࠊࢫࣇ࢕ࣥࢦ⬡㉁ࢭ࣑ࣛࢻ}_{N-ࢹ࢔ࢩ࣮ࣛࢮࢆ฼⏝ࡋ Sph2 ఩ࡢ࢔࣑ࢻ} 㒊ศ࡬⺯ගศᏊࢆᑟධࡍࡿᡭἲࡀ࡜ࡽࢀ࡚࠸ࡓ (Figure 4)ࠋࡉࡽ࡟ࠊChinnapen ࡽࡣࠊኳ↛ ࡢGM1 ࡢࢩ࢔ࣝ㓟ഃ㙐ࢆ㐣ࣚ࢘⣲㓟㓟໬࡟ࡼࡾ㑅ᢥⓗ࡟ C7-࢔ࣝࢹࣄࢻㄏᑟయ࡜ࡋࡓᚋ࡟ 14_{ࠊ⺯ගⰍ⣲ഃࡢ࢔࣑ࣀᇶ࡜཯ᛂࡉࡏࡿࡇ࡜࡟ࡼࡾࠊࢩ࢔ࣝ㓟ഃ㙐ಟ㣭ᆺࡢ} _{GM1 ࣉ࣮ࣟࣈ} ࢆ㛤Ⓨࡋࡓ9_ࠋ_{(ࡇࡢሙྜࠊഃ㙐ࡣኳ↛ᵓ㐀ࡼࡾ஧Ⅳ⣲ศᑡ࡞࠸ࠋ) ࡋ࠿ࡋ࡞ࡀࡽࠊࡇࢀࡽࡢ} ᪉ἲ࡟ࡣ࠸ࡃࡘ࠿ၥ㢟ⅬࡀᏑᅾࡍࡿࠋࡑࢀࡣࠊ௨ୗࡢ୕Ⅼ࡟ࡲ࡜ࡵࡽࢀࡿࠋ(1) ᢳฟ࡛ᚓࡓ ࢞ࣥࢢࣜ࢜ࢩࢻࡣᵓ㐀㢮⦕యࡢΰᅾ࡟ࡼࡾ⣧ရࢆᚓࡿࡇ࡜ࡀ㞴ࡋ࠸ࡇ࡜ࠋ(2) ໬Ꮫⓗ᰿ᣐ࡟ ᇶ࡙࠸ࡓᵓ㐀ࡢド᫂ࡀᅔ㞴࡛࠶ࡿࡇ࡜ࠋ(3) ⬡㉁ࣛࣇࢺᙧᡂ࡟㔜せ࡞␯Ỉ⤖ྜࡸỈ⣲⤖ྜ➼ ࡟ᐤ୚ࡍࡿᐁ⬟ᇶࡢᵓ㐀ኚ᥮ࢆ⾜ࡗ࡚࠸ࡿ࡟ࡶ࠿࠿ࢃࡽࡎࠊࣉ࣮ࣟࣈࡢᛶ㉁ࡣホ౯ࡉࢀ࡚ ࠸࡞࠸ࡇ࡜ࠋ ࡇࢀࡲ࡛࡟࢞ࣥࢢࣜ࢜ࢩࢻࡢືែ࡟㛵ࡋᵝࠎ࡞ゎ㔘ࡀᏑᅾࡋࡓ⫼ᬒ࡟ࡣࠊ⺯ගࣉ࣮ࣟࣈ ໬ἲ࡟ࡼࡗ࡚ࡣ࢞ࣥࢢࣜ࢜ࢩࢻࡢ≀ᛶࡀᮏ᮶࡜ࡣ␗࡞ࡿࡶࡢ࡬ኚ໬ࡋ࡚࠸ࡓⅭ࡜⪃࠼ࢀࡤ ㄝ᫂ࡀࡘࡃ࡛࠶ࢁ࠺ࠋࡑࡢࡓࡵࠊኳ↛ࡢ࢞ࣥࢢࣜ࢜ࢩࢻࡢ┿ࡢືែࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵ࡟ࡣࠊ ኳ↛࡜ᛶ㉁ࡢ㏆ఝࡋࡓ⺯ගࣉ࣮ࣟࣈࡢ㛤Ⓨࡀᚲせ࡜⪃࠼ࡽࢀࡿࠋࡑࡇ࡛ᮏ◊✲࡛ࡣࠊ⺯ගࣉ ࣮ࣟࣈ໬ἲ࡜ࡋ࡚ࠊศᏊタィࡢ⮬ᅾᛶࡢ㧗࠸໬Ꮫྜᡂἲࢆ㑅ᢥࡋࡓࠋከᵝ࡞⺯ගࣉ࣮ࣟࣈࡢ ೃ⿵ࢆసᡂࡋࠊࡑࢀࡽࡢ⣽⬊⭷ୖ࡟࠾ࡅࡿᶵ⬟ࢆホ౯ࡍࡿࡇ࡜࡛ࠊඃࢀࡓ⺯ග࢞ࣥࢢࣜ࢜ࢩ ࢻࡢ㛤Ⓨࢆ┠ᣦࡍࡇ࡜࡜ࡋࡓࠋ

12_{S. M. Polyakova, V. N. Belov, S. F. Yan, C. Eggeling, C. Ringemann, G. Schwarzmann, A. de Meijere,}

S. W. Hell, Eur. J. Org. Chem. 2009, 2009, 5162-5177.

13_{C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B.}

Hein, C. von Middendorff, A. Schönle, S. W. Hell, Nature 2009, 457, 1159-1163.

(16)

(17)

➨஧❶ ศᏊタィ

ᮏ◊✲࡛ࡣࠊᶆⓗ࢞ࣥࢢࣜ࢜ࢩࢻ࡜ࡋ࡚⬡㉁ࣛࣇࢺ࡟㇏ᐩ࡞࢞ࣥࢢࣜ࢜⣔࢞ࣥࢢࣜ࢜ࢩ ࢻa ⣔ิࡢ GM3ࠊGM2ࠊGM1 ࢆ㑅ᢥࡋࡓࠋ⺯ග࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࢆタィࡍࡿୖ࡛ࠊ ⺯ගศᏊࢆᑟධࡍࡿ఩⨨ࡢỴᐃࡀ᭱ࡶ㔜せ࡛࠶ࡿࠋࡑࡢࡓࡵࠊ௨ୗࡢ୕Ⅼࡢࡇ࡜ࢆ⪃៖ࡋ࡚ ࡑࢀࡒࢀࡢᑟධ఩⨨ࡢೃ⿵ࢆᣲࡆࡓࠋ(1) ⺯ගศᏊࡣࣛࣇࢺᙧᡂ࡟㔜せ࡞ࢭ࣑ࣛࢻࢆ❧యⓗ ࡟㞀ᐖࡋ࡞࠸఩⨨࡟ᑟධࡍࡿࡇ࡜ࡀᮃࡲࡋ࠸15_ࠋ_{(2) ࢱࣥࣃࢡ㉁ࡢㄆ㆑㒊఩࡟࡞ࡾᚓࡿࠊࢩ} ࢔ࣝ㓟࢝ࣝ࣎࢟ࢩࣝᇶࠊ࢔ࢭࢺ࢔࣑ࢻᇶࡢᵓ㐀ࡣಖᣢࡍࡿࡇ࡜ࠋ(3) ྜᡂࡢ㒔ྜୖࠊ཯ᛂᛶ ࡢⰋ࠸఩⨨ࡀᮃࡲࡋ࠸ࡇ࡜ࠋ௨ୖࡢⅬࢆ⪃៖ࡋࠊ㠀㑏ඖᮎ➃ഃ⢾ṧᇶࡢ୍⣭Ỉ㓟ᇶ஧⟠ᡤࡎ ࡘࢆࡑࢀࡒࢀࡢᑟධೃ⿵఩⨨࡜ࡋ࡚ᣲࡆࡓ (Figure 5)ࠋGM3 ࡟ࡘ࠸࡚ࡣࠊᮎ➃ࡢ Neu9 ఩ࡢ ௚ࠊෆ㒊ࡢGal6 ఩ࢆೃ⿵఩⨨ࡢ୍ࡘ࡟タᐃࡋࡓࠋࡑࢀࡒࢀᶆ㆑఩⨨ࡢ␗࡞ࡿ⺯ගࣉ࣮ࣟࣈ ࡢ⣽⬊⭷ୖ࡟࠾ࡅࡿᛶ㉁ࢆ᳨ド࣭ẚ㍑ࡍࡿࡇ࡜࡛ࠊኳ↛ࡢᛶ㉁࡟㏆ఝࡋࡓ⺯ග࢞ࣥࢢࣜ࢜ࢩ ࢻࡢ㑅ูࡀྍ⬟࡛࠶ࡿ࡜⪃࠼ࡓࠋ

15_{a) P. S. Niemelä, M. T. Hyvönen, I. Vattulainen, Biophys. J. 2006, 90, 851-863. b) K. Simons, W. L.}

(18)

➨୕❶ ࢞ࣛࢡࢺ࣮ࢫ

6 ఩ᶆ㆑ GM3 ࣉ࣮ࣟࣈࡢྜᡂ

➨୍⠇ ྜᡂᡓ␎

⺯ග࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢྜᡂ◊✲ࢆ㛤ጞࡍࡿ࡟࠶ࡓࡾࠊࡲࡎࡣࣛࣇࢺ࡟㇏ᐩ࡟Ꮡ ᅾࡍࡿ࢞ࣥࢢࣜ࢜ࢩࢻࡢ୰࡛ࡶࠊᵓ㐀ࡀ᭱ࡶ༢⣧࡞GM3 ࢆᶆⓗศᏊ࡜ࡋࠊ⺯ග࢞ࣥࢢࣜ ࢜ࢩࢻࣉ࣮ࣟࣈࡢྜᡂἲࡢ☜❧ࢆᅗࡗࡓࠋᶆ㆑఩⨨ࡢೃ⿵࡜ࡋ࡚Neu9 ఩ࠊGal6 ఩ࡢ஧ࡘ ࡢ୍⣭Ỉ㓟ᇶࢆᣲࡆࡓࡀࠊึࡵ࡟Gal6 ఩ᶆ㆑ᆺࡢྜᡂ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ ⺯ගⰍ⣲ࡣ㧗౯࡛ࠊࡋ࠿ࡶ໬Ꮫⓗ࡟୙Ᏻᐃ࡛࠶ࡿࡓࡵࠊྜᡂࡢ᭱⤊཯ᛂ࡛ᑟධࡍࡿࡢࡀ ᮃࡲࡋ࠸࡜⪃࠼ࡓࠋࡑࡇ࡛GM3 ⢾㙐ࡢᩘ࠶ࡿỈ㓟ᇶࡢ୰࠿ࡽᮃࡴ Gal6 ఩ࡢࡳ࡟⺯ගⰍ⣲

(19)

ࢆᑟධࡍࡿࡓࡵ࡟ࠊỈ㓟ᇶࡼࡾ཯ᛂᛶࡢ㧗࠸࢔࣑ࣀᇶ࡛Gal6 ఩Ỉ㓟ᇶࢆ⨨᥮ࡋࡓ୰㛫యࢆ タィࡋࠊࡇࢀࢆ⺯ගⰍ⣲ᑟධࡢ๓㥑య࡜ࡋࡓ (Figure 6)ࠋࡇࢀ࡟ᑐࡋ࡚⺯ගⰍ⣲ᑟධヨ⸆ ࢆస⏝ࡉࡏࡿࡇ࡜࡛ࢳ࢜࢘ࣞ࢔⤖ྜࡸ࢔࣑ࢻ⤖ྜࢆ௓ࡋ࡚⺯ගⰍ⣲ࢆ఩⨨㑅ᢥⓗ࡟⤖ྜࡉ ࡏࡿࡇ࡜ࡀྍ⬟࡜⪃࠼ࡓࠋ

ྜᡂᡓ␎ࢆ Scheme 1 ࡟♧ࡋࡓࠋGal6 ఩Ỉ㓟ᇶࢆ࢔ࢪࢻᇶ࡛⨨᥮ࡋࡓ Neu-Gal ౪୚య࡜

Glc-Cer ཷᐜయࡢ⦰ྜ཯ᛂ࡟ࡼࡾ GM3 㦵᱁ࢆᵓ⠏ࡋࡓᚋࠊ⬺ಖㆤࠊ࢔ࢪࢻᇶࡢ㑏ඖ࡟ࡼࡾ Gal6 ఩࢔࣑ࣀయ࡜ࡋࠊ᭱ᚋ࡟ྛ✀⺯ගⰍ⣲ࢆᑟධࡍࡿࡇ࡜࡟ࡼࡾ┠ⓗ໬ྜ≀࡬࡜ᑟࡃ⤒㊰ ࢆ❧᱌ࡋࡓࠋ

(20)

ࡇࡢྜᡂ࡟࠾࠸࡚ࠊ࢞ࣥࢢࣜ࢜ࢩࢻ㦵᱁ࢆຠ⋡Ⰻࡃᵓ⠏ࡍࡿ஦ࡀ᭱ࡶ㔜せ࡛࠶ࡿࠋࡑࡇ࡛ࠊ (1) Glc-Cer ࢝ࢭࢵࢺࡢ฼⏝ࠊ(2) Neu-Gal ஧⢾ࣘࢽࢵࢺ࡟ᑐࡍࡿ┤᥋ⓗ࡞ Gal6 ఩࢔ࢪࢻ໬ࠊ ࡢ஧ࡘࡢᡓ␎ࢆ࡜ࡗࡓࠋࡇࢀࡽࡣࠊ࢞ࣥࢢࣜ࢜ࢩࢻྜᡂ࡟࠾ࡅࡿඹ㏻ࡢㄢ㢟࡜ࡋ࡚ᣲࡆࡽࢀ ࡿ஧ࡘࡢㄢ㢟ࠊ(1) Cer ᑟධ᫬ࡢప཰⋡ࠊ(2) ࢩ࢔ࣝ㓟ᑟධ᫬ࡢప཰⋡࣭ప❧య㑅ᢥᛶࠊࢆゎ Ỵࡍࡿᡭẁ࡜ࡋ࡚᥇⏝ࡋࡓࠋ Cer ࡢ 1 ఩Ỉ㓟ᇶࡣ 2 ఩࢔࣑ࢻᇶ࡜ࡢỈ⣲⤖ྜ࡟ຍ࠼ࠊ஧ᮏࡢ㛗㙐࢔ࣝ࢟ࣝ㙐࡟ࡼࡿ❧య 㞀ᐖཬࡧ㧗จ㞟ᛶ࡟ࡼࡾᴟࡵ࡚ప཯ᛂᛶ࡛࠶ࡿࠋࡑࡢࡓࡵࠊᚑ᮶ࡢ࢞ࣥࢢࣜ࢜ࢩࢻྜᡂἲ࡛ ࡣ⢾㙐࡟ᑐࡍࡿCer ᑟධ᫬ࡢప཰⋡ࡀᗘࠎㄢ㢟࡜ࡉࢀࡓࠋᙜ◊✲ᐊ࡛㛤Ⓨࡉࢀࡓ Glc-Cer ࢝ ࢭࢵࢺ࢔ࣉ࣮ࣟࢳࡣࡇࡢㄢ㢟ࢆゎỴࡍࡿ⏬ᮇⓗ࡞⬡㉁ᑟධἲ࡛࠶ࡿࠋ⢾㙐㑏ඖᮎ➃ഃࡢGlc ࡟ணࡵCer ࢆᑟධࡋࡓ Glc-Cer ཷᐜయ࡜⢾㙐౪୚యࢆࢢࣜࢥࢩࢻ໬཯ᛂ࡟౪ࡍࡿࡇ࡜࡛ࠊ㧗 ཰⋡࡟࡚ᵝࠎ࡞࢞ࣥࢢࣜ࢜ࢩࢻ㦵᱁ࡢᵓ⠏ࡀྍ⬟࡛࠶ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ16,17_ࠋᮏ◊✲ ࡛ࡣࠊࡑࡢ୰࡛ࡶ᭱ࡶỗ⏝ࡉࢀ࡚࠸ࡿ3,6 ఩ PMB ᆺ Cer ཷᐜయ 4 ࡜ࠊ2,3,6 ఩ Ac ᆺ Glc-Cer ཷᐜయ 3 ࢆ⏝࠸ࠊຠ⋡ࢆẚ㍑ࡍࡿ஦࡜ࡋࡓࠋ2,3,6 ఩ Ac ᆺ Glc-Glc-Cer ཷᐜయ 3 ࡣࠊ㞄᥋఩ ࡢỈ㓟ᇶࢆᵓ㐀ࡢᑠࡉ࡞Ac ᇶ࡛ಖㆤࡍࡿࡇ࡜࡟ࡼࡾࠊ4 ఩Ỉ㓟ᇶ࡬ࡢ❧య㞀ᐖࢆ㍍ῶࡋࠊ ཷᐜయࡢ཯ᛂᛶࡀྥୖࡍࡿྍ⬟ᛶࢆᮇᚅࡋ࡚㑅ᢥࡋࡓࠋ ஧Ⅼ┠ࡢㄢ㢟࡜ࡋ࡚ᣲࡆࡓࢩ࢔ࣜࣝ໬཯ᛂࡣࠊ᏶඲࡞❧యไᚚἲࡀࡇࢀࡲ࡛࡟☜❧ࡉࢀ ࡚࠸࡞࠸ࡓࡵࠊ㏻ᖖ┠ⓗ࡜ࡍࡿ α-ࢢࣜࢥࢩࢻ࡜ࡑࡢ❧య␗ᛶయ࡛࠶ࡿ β-ࢢࣜࢥࢩࢻࡢΰྜ ≀ࢆ୚࠼ࡿࠋ᭦࡟ࠊ1 ఩࢝ࣝ࣎࢟ࢩࣝᇶ࡟ࡼࡿ࢜࢟ࢯ࢝ࣝ࣋ࢽ࣒࢘࢖࢜ࣥࡢ୙Ᏻᐃ໬ࠊ3 ఩ ࢹ࢜࢟ࢩᵓ㐀࡟ࡼࡿ2,3-ene యࡢ⏕ᡂಁ㐍࡟ࡼࡾࠊ⦰ྜ཰⋡ࡢపୗࡶㄢ㢟࡜࡞ࡿࠋࡇࢀࡲ࡛ ࡟ࠊࢩ࢔ࣜࣝ໬཯ᛂࡢ❧య㑅ᢥᛶࢆ㧗ࡵࡿࡓࡵ࡟ࠊࢽࢺࣜࣝ⁐፹ຠᯝ18_{ࡢ฼⏝ࠊ4,5-࢜࢟ࢧ}

16_{K. Fujikawa, S. Nakashima, M. Konishi, T. Fuse, N. Komura, T. Ando, H. Ando, N. Yuki, H. Ishida, M.}

Kiso, Chem. Eur. J. 2011, 17, 5641-5651.

17_{a) A. Imamura, H. Ando, H. Ishida, M. Kiso, J. Org. Chem. 2009, 74, 3009-3023. b) H. Tamai, H.}

Ando, H. N. Tanaka, R. Hosoda-Yabe, T. Yabe, H. Ishida, M. Kiso, Angew. Chem. Int. Ed. 2011, 50, 2330-2333. c) S. Nakashima, H. Ando, R. Saito, H. Tamai, H. Ishida, M. Kiso, Chem. Asian J. 2012, 7, 1041-1051.

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ࢰࣜࢪࣀࣥ౪୚యࢆ⏝࠸ࡓ⁐፹㠀౫Ꮡⓗ࡞ࢢࣜࢥࢩࢻ໬19_{࡞࡝ࡀሗ࿌ࡉࢀ࡚ࡁࡓࠋࡋ࠿ࡋ࡞} ࡀࡽࡇࡢࡼ࠺࡞ඃࢀࡓຠᯝ࣭౪୚యࢆࡶࡗ࡚ࡶ౫↛῝้࡞ၥ㢟࡛࠶ࡿࡢࡀࠊ࠸ࡎࢀࡶ❧య㑅 ᢥᛶࡀ౪୚య࣭ཷᐜయࡢᵓ㐀࡟౫Ꮡࡋ࡚኱ࡁࡃኚືࡍࡿ࡜࠸࠺Ⅼ࡛࠶ࡿࠋࡇࡢၥ㢟Ⅼࢆཷࡅࠊ ᮏ◊✲࡟࠾࠸࡚ࡣࠊỈ㓟ᇶࢆ❅⣲ᐁ⬟ᇶ࡛⨨᥮ࡋࡓ༢⢾ࣘࢽࢵࢺࢆࢩ࢔ࣜࣝ໬཯ᛂ࡟౪ࡍ ࡿࡇ࡜ࡣᚓ⟇࡛ࡣ࡞࠸࡜࠸࠺⪃࠼࡟⮳ࡗࡓࠋࡑࡇ࡛ࠊᮏ◊✲࡛ࡣࠊ኱㔞౪⤥ࡀྍ⬟࡞N-Troc Neu-Gal ஧⢾ࣘࢽࢵࢺ 120_࠿ࡽࠊ_Gal6-N 3౪୚య2 ࡬ㄏᑟࡍࡿ࢔ࣉ࣮ࣟࢳࢆヨࡳࡿࡇ࡜࡜ࡋࡓࠋ

N-Troc Neu-Gal 1 ࡣ N-Troc Neu ౪୚య21_{ࡢ฼⏝࡟ࡼࡾ㧗཰⋡࣭㧗㑅ᢥᛶ࡛ᵓ⠏ྍ⬟࡛࠶ࡿࡔ} ࡅ࡛࡞ࡃࠊ㧗࠸⤖ᬗᛶ࡟ࡼࡾ❧య␗ᛶయࡢศ㞳ࡀ⡆౽࡛࠶ࡿ฼Ⅼࢆࡶࡘࠋࡇࢀࡽࡢ᳨ウࡢヲ ⣽ࢆḟ⠇࠿ࡽ㏙࡭ࡿࠋ

➨஧⠇ ࢩ࢔ࣜࣝ࢞ࣛࢡࢺ࣮ࢫ౪୚యࡢྜᡂ

ึࡵ࡟N-Troc Neu-Gal (໬ྜ≀ 1) ࡢ Troc ᇶࢆ ZnCu/AcOH ࡟ࡼࡾ㑏ඖⓗ࡟㝖࠸ࡓᚋ࡟࢔

ࢭࢳࣝ໬ࠊ᥋ゐỈ⣲ῧຍἲ࡟ࡼࡿ⬺࣋ࣥࢪࣝ໬ࢆ⾜࠸ࠊGal2,6 ఩ࡢỈ㓟ᇶࡀ㐟㞳ࡢ໬ྜ≀ 5

࡬࡜ᑟ࠸ࡓ (Scheme 2)ࠋ

19_{a) H. Tanaka, Y. Nishiura, T. Takahashi, J. Am. Chem. Soc. 2006, 128, 7124-7125. b) D. Crich, W. Li, J.} Org. Chem. 2007, 72, 2387-2391. c) M. D. Farris, C. De Meo, Tetrahedron Lett. 2007, 48, 1225-1227.

d) T. Aoyagi, S. Ohira, S. Fuse, J. Uzawa, Y. Yamaguchi, H. Tanaka, Chem. Eur. J. 2016, 22, 6968-6973.

20_{T. Fuse, H. Ando, A. Imamura, N. Sawada, H. Ishida, M. Kiso, T. Ando, S. C. Li, Y. T. Li} Glycoconjugate. J. 2006, 23, 329-343.

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⥆࠸࡚໬ྜ≀5 ࡟ᑐࡍࡿ Gal6 ఩࢔ࢪࢻ໬ࡢ᳨ウࢆ⾜ࡗࡓࠋ࢔ࢪࢻᇶࡣ୺࡟ࣁࣟࢤࣥࡸࢫ

ࣝ࣍ࣥ㓟࢚ࢫࢸࣝࢆ⬺㞳ᇶ࡜ࡋࡓồ㟁Ꮚయ࡬ࡢ࢔ࢪࢻ࢔ࢽ࢜ࣥࡢ SN2 ཯ᛂ࡟ࡼࡾᑟධࡉࢀ

ࡿ௚ࠊගᘏ཯ᛂ࡟ࡼࡿᑟධἲࡶỗ⏝ࡉࢀ࡚࠸ࡿࠋከࡃࡢሗ࿌࡛ࠊࢫࣝ࣍ࣥ㓟࢚ࢫࢸࣝࡣప

᮲௳୍࡛⣭Ỉ㓟ᇶ㑅ᢥⓗ࡞ᑟධࡀྍ⬟࡛࠶ࡿࠋErcégovic22_ࡽࡣ_{Neu9 ఩࡟ Ts ᇶࢆᴟࡵ࡚㧗཰}

⋡࡟࡚ᑟධࡋࠊ⥆ࡃ࢔ࢪࢻᇶ࡬ࡢኚ᥮࡟࠾࠸࡚ࡶⰋዲ࡞⤖ᯝࢆ♧ࡋ࡚࠸ࡿࠋࡑࡇ࡛ᙼࡽࡢᡭ ἲࢆཧ⪃࡟ࠊGal6 ఩ࡢ㑅ᢥⓗ Ts ໬ࢆ⾜ࡗࡓ (Scheme 3)ࠋCH2Cl2ࠊPyr ࡢΰྜ⁐፹୰ࠊ0 °C

࡟࡚TsCl ࢆస⏝ࡉࡏࠊ90%࡟࡚ Gal6-Ts యࢆᚓࡓࠋ⥆࠸࡚໬ྜ≀ 6 ࡟ᑐࡋ NaN3

ࠊ18-crown-6 ࢆస⏝ࡉࡏࠊ࢔ࢪࢻᇶ࡬ࡢኚ᥮ࢆヨࡳࡓࠋࡋ࠿ࡋ࡞ࡀࡽࠊ┠ⓗ࡜ࡍࡿ Galࠊ18-crown-6-N3య (໬ྜ≀

7) ࡢ௚࡟ Gal6 ఩ࡀ N3ᇶࡢ1,2-ࣛࢡࢺࣥయ (໬ྜ≀ 8) ࡢ๪⏕ࡀ┠❧ࡘ⤖ᯝ࡜࡞ࡗࡓࠋࡇࢀ ࡣᙉሷᇶᛶ࣭㧗 ᮲௳ୗࠊGal2 ఩Ỉ㓟ᇶࡀ㞄᥋ࡍࡿ Neu1 ఩ࡢ࢝ࣝ࣎ࢽࣝⅣ⣲ࢆồ᰾ᨷᧁ ࡋࠊศᏊෆ࡛࢚ࢫࢸࣝ஺᥮཯ᛂࡀ㉳ࡇࡗࡓ⤖ᯝࠊᏳᐃ࡞භဨ⎔ᙧᡂࡀಁ㐍ࡉࢀࡓ࡜⪃ᐹࡋࡓࠋ

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┠ⓗ࡜ࡍࡿ Gal6-N3యࡢ཰⋡ࡀపࡃࠊࡲࡓ 1,2-ࣛࢡࢺࣥయ࡜ࡢศ㞳ࡀᅔ㞴࡛࠶ࡗࡓࡓࡵࠊ ኚ᥮ࡢ㡰ᗎࢆኚ᭦ࡋࡓࠋࡍ࡞ࢃࡕࠊGal6 ఩࡬ࡢ⬺㞳ᇶᑟධᚋ࡟ Gal2 ఩Ỉ㓟ᇶࢆಖㆤࡋࠊࡑ ࡢᚋ࢔ࢪࢻ໬࡟౪ࡍ⤒㊰ࢆ࡜ࢀࡤࠊࣛࢡࢺࣥᙧᡂࡣᅇ㑊࡛ࡁࡿ࡜⪃࠼ࡓࠋࡑࡇ࡛໬ྜ≀6 ࡢ 2 ఩Ỉ㓟ᇶࢆ࣋ࣥࢰ࢖ࣝ໬ࡋࠊ໬ྜ≀ 9 ࡬࡜ᑟ࠸ࡓᚋࠊ෌ࡧ࢔ࢪࢻ໬ࡢ᳨ウࢆ⾜ࡗࡓ㸦Table 1㸧ࠋ ࡋ࠿ࡋ࡞ࡀࡽࠊࡇࢀ࡟ࡼࡾࣛࢡࢺࣥᙧᡂࡢㄢ㢟ࡣᅇ㑊ࡉࢀࡓࡶࡢࡢࠊTLC ୖ࡛ཎᩱ࡜⏕ ᡂ≀ࡢᴟᛶᕪࡀぢฟࡏࡎࠊ཯ᛂ⤊஢ࡢぢᴟࡵཬࡧ⢭〇ࡀᴟࡵ࡚ᅔ㞴࡜࠸࠺ၥ㢟ࡀ᪂ࡓ࡟ᾋ

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ୖࡋࡓࠋࡑࡢࡓࡵࠊ᮲௳ 1 ࡛ࡣ཯ᛂ᫬㛫ࢆ 19 ᫬㛫࡜ࡾࠊཎᩱࡢ᏶඲࡞ᾘ㈝ࢆᅗࡗࡓ⤖ᯝࠊ ┠ⓗ≀ࡢ༢㞳࡟ᡂຌࡋࠊ62%ࡢ཰⋡ࢆᚓࡓࠋࡇࡢ᫬ࠊNeu1 ఩ࡀ࢝ࣝ࣎ࣥ㓟࡛࠶ࡿ๪⏕ᡂ≀ ࡢ⏕ᡂࢆ☜ㄆࡋࠊࡇࢀ࡟ࡼࡗ࡚཰⋡ࡀ୰⛬ᗘ࡜࡞ࡗࡓࠋࡇࢀࡣࠊCurtius ㌿఩ࡸ Schmidt ㌿఩ ࡜ྠᵝ࡟ࠊ⣔ෆ࡛ Neu ࢝ࣝ࣎ࢽࣝⅣ⣲ࡀ࢔ࢪ໬≀࢖࢜ࣥ࠿ࡽࡢồ᰾ᨷᧁࢆཷࡅࠊ୙Ᏻᐃ࡞ ࢔ࢪࢻ࢚ࢫࢸࣝࢆᙧᡂࡋ࡚࠸ࡿⅭ࡛࠶ࡿ࡜᥎ ࡋࡓࠋ ࡼࡾ ࿴࡞᮲௳࡛཯ᛂࡀ㐍⾜ࡍࡿࡇ࡜ࢆᮇᚅࡋࠊ࢔ࢪࢻ໬๣࡜ࡋ࡚ Trimethylsilyl azide (TMSN3) ࡸ Tetramethylguanidinium azide (TMGN3)23ࢆ⏝࠸ࡓ᳨ウࡶ⾜ࡗࡓ (᮲௳ 2ࠊ᮲௳ 3)ࠋ ⁐ゎᛶࡀప࠸NaN3࡟ᑐࡋ࡚TMSN3ཬࡧTMGN3ࡣ࡯࡜ࢇ࡝ࡢ᭷ᶵ⁐፹࡟ྍ⁐࡛࠶ࡾࠊヨ⸆ ࢆ㐣๫࡟⏝࠸ࡿᚲせࡀ࡞࠸Ⅼ࡛ NaN3ࡼࡾࡶඃࢀࡓ཯ᛂ๣࡛࠶ࡿ࡜⪃࠼ࡓࠋࡋ࠿ࡋ࡞ࡀࡽࠊ TMSN3ࢆ⏝࠸ࡓ⣔࡛ࡣ⏕ᡂ≀ࡀᚓࡽࢀࡎࠊཎᩱࡢࡳࡀṧࡿ⤖ᯝ࡜࡞ࡗࡓࠋ୍᪉ࡢTMGN3࡟ ࠾࠸࡚ࡣ཯ᛂࡢ㐍⾜ࡣⱝᖸಁ㐍ࡉࢀࡓࡀࠊཎᩱࡢ᏶඲࡞ᾘ㈝ࡀᅔ㞴࡛࠶ࡾࠊNaN3ࡢ⣔࡜ྠ ᵝࡢ⤖ᯝࢆ୚࠼ࡓࠋΰᅾࡋ࡚࠸ࡿཎᩱࢆศ㞳ࡍࡿᡭẁ࡜ࡋ࡚ࠊ࢔ࢪ໬≀࢖࢜ࣥࡼࡾࡶồ᰾ᛶ ࡢᙉ࠸ࢳ࣮࢜ࣛࢺ࢔ࢽ࢜ࣥ࡟ࡼࡿࢳ࢚࢜ࢫࢸࣝ໬24_{࡟ࡼࡾ┠ⓗ≀࡜ࡢᴟᛶᕪࢆసࡾฟࡍ᳨} ウࡶ⾜ࡗࡓࡀࠊ཯ᛂࡢ㐍⾜ࡣほᐹࡉࢀ࡞࠿ࡗࡓࠋࡇࡢ⤖ᯝࢆཷࡅࠊTs ᇶࡼࡾࡶ⬺㞳⬟ࡢ㧗 ࠸⬺㞳ᇶࢆ⏝࠸ࢀࡤࠊ཰⋡ࡢྥୖ࡜⏕ᡂຠ⋡ࡢᨵၿࡀྍ⬟࡜ᮇᚅࡋࡓࠋࡑࡇ࡛ࠊTs ᇶࡼࡾ ࡶ⬺㞳⬟ࡀ㧗࠸࡜⪃࠼ࡽࢀࡿࢫࣝ࣍ࣥ㓟࢚ࢫࢸࣝ࡜ࡋ࡚ Methanesulfonyl (Ms) ᇶཬࡧ p-Nitrobenzenesulfonyl (Ns) ᇶࢆ㑅ᢥࡋࠊẚ㍑ࡍࡿࡇ࡜࡜ࡋࡓࠋ ⰾ㤶⎔ࢆ᭷ࡍࡿTs ᇶ࡟ᑐࡋ࡚ Ms ᇶࡣ❧యⓗ࡟ᑠࡉ࡞ᵓ㐀࡛࠶ࡾࠊ࢔ࢪ໬≀࢖࢜ࣥࡢ᥋ ㏆ࡀࡼࡾᐜ᫆࡟࡞ࡿ࡜⪃࠼ࡓࠋྠᵝ࡟ࡋ࡚ᇶ㉁ࢆྜᡂࡋࠊ࢔ࢪࢻ໬࡟౪ࡋࡓࠋࡑࡢ⤖ᯝࠊᮇ ᚅ࡟཯ࡋࠊMs యࡢ཯ᛂᛶࡣຎᝏ࡛࠶ࡾࠊ཯ᛂ᫬㛫ࢆ 66 ᫬㛫࠿ࡅ࡚ࡶཎᩱࡀṧᏑࡍࡿ⤖ᯝ ࡜࡞ࡗࡓ (Scheme 4)ࠋ

23_{a) A. J. Papa, J. Org. Chem. 1966, 31, 1426-1430. b) C. Chu, A. Ramamurthy, A. Makriyannis, M. A.}

Tius, J. Org. Chem. 2003, 68, 55-61.

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Ns ᇶࡣ p ఩ࢽࢺࣟᇶࡢ㟁Ꮚồᘬࡢຠᯝ࡟ࡼࡾࠊTs ᇶࡼࡾࡶ⬺㞳ࡀ㎿㏿࡛࠶ࡿࡇ࡜ࢆᮇᚅ ࡋ࡚㑅ᢥࡋࡓࠋྠᵝ࡟ࡋ࡚࢔ࢪࢻ໬ࢆ᳨ウࡋࡓ⤖ᯝࠊண᝿㏻ࡾ཯ᛂࡣ 3 ᫬㛫࡛⤊஢ࡋࡓ (Scheme 5)ࠋࡇࢀ࡟ࡼࡾ࣓ࢳ࢚ࣝࢫࢸࣝࡢศゎࡣⱝᖸᢚไࡉࢀࡓࡶࡢࡢࠊᵓ㐀୙᫂ࡢ๪⏕ᡂ ≀ࡀ」ᩘ⏕ᡂࡋࡓࡓࡵ཰⋡ࡢྥୖ࡟ࡣ⮳ࡽ࡞࠿ࡗࡓࠋ ௨ୖࡢ᳨ウ࠿ࡽMs ᇶཬࡧ Ns ᇶࡣ Ts ᇶ࡟௦ࢃࡿ⬺㞳ᇶ࡟ࡣ࡞ࡾᚓ࡞࠸࡜ุ᩿ࡋࡓࠋ6 ఩ ࢔ࢪࢻ໬ࡣゎỴࡍ࡭ࡁㄢ㢟ࢆṧࡋࡓࠋ ᳨ウࢆ㏻ࡋ࡚Neu (࣓ࢳ࢚ࣝࢫࢸࣝ) Ꮡᅾୗ࡟࠾ࡅࡿ࢔ࢪࢻ໬཯ᛂࡀᅔ㞴࡛࠶ࡿࡇ࡜ࡀ᫂ ࡽ࠿࡜࡞ࡗࡓࠋ༢⢾ࡢẁ㝵࡛Gal6 ఩࡬࢔ࢪࢻᇶࢆᑟධࡍࡿ᪉ἲࡶ୍ࡘࡢᡭẁ࡛࠶ࡿࡀࠊྜ ᡂᡓ␎࡛㏙࡭ࡓᵝ࡟ࠊNeu-Gal 㦵᱁ᵓ⠏ࡢࢩ࢔ࣜࣝ໬཯ᛂ࡟࠾࠸࡚཰⋡ཬࡧ❧య㑅ᢥᛶྥୖ ࡢࡓࡵࡢ᳨ウࠊࡉࡽ࡟❧య␗ᛶయࡢศ㞳࡞࡝ࡢㄢ㢟ࡀ⏕ࡌࡿ࡜ண᝿ࡉࢀࡿࠋ

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⥆࠸࡚ࠊ໬ྜ≀10 ࢆ౪୚య࡬ㄏᑟࡋࡓ (Scheme 6)ࠋCAN ࢆస⏝ࡉࡏ࡚࢔ࣀ࣐࣮఩ࡢ⬺ ಖㆤࢆ⾜ࡗࡓᚋ25_{ࠊ⬺㞳ᇶ࡜ࡋ࡚᭷⏝࡞ࢺࣜࢡࣟࣟ࢔ࢭࢺ࢖࣑ࢹ࣮ࢺᇶ}26_{ࢆᑟධࡋࡓࠋḟ⠇} ࡛ࡣྜᡂࡋࡓNeu-Gal ౪୚య 2 ࢆ⏝࠸ࡓ GM3 㦵᱁ࡢᵓ⠏࡟ࡘ࠸࡚グࡍࠋ

➨୕⠇

GM3 㦵᱁ࡢᵓ⠏

๓⠇࡟࡚ྜᡂࡋࡓ౪୚య2 ࡜ Glc-Cer ཷᐜయࢆࢢࣜࢥࢩࣝ໬཯ᛂ࡟౪ࡋࡓ (Table 2)ࠋࡲ ࡎࡣGlc ࡢ 3,6 ఩Ỉ㓟ᇶࢆ Ac ᇶ࡛ಖㆤࡋࡓ Glc-Cer ཷᐜయ 3 ࡟ᑐࡍࡿࢢࣜࢥࢩࣝ໬ࢆ᳨ウ ࡋࡓࠋ᮲௳1 ࡛ࡣ౪୚య࡟ᑐࡋ࡚ཷᐜయࢆ 1.5 ᙜ㔞⏝࠸᳨࡚ウࢆ⾜ࡗࡓࡀࠊ౪୚య⏤᮶ࡢ࣊ ࣑࢔ࢭࢱ࣮ࣝయ࡜஧㔞యࡢ⏕ᡂࡀ┠❧ࡕࠊ཰⋡ࡣࢃࡎ࠿ 30%࡟␃ࡲࡗࡓࠋཷᐜయࡢప཯ᛂ ᛶࡀ♧၀ࡉࢀࡓࡓࡵࠊ᮲௳2 ࡢ᳨ウ࡛ࡣࡇࢀࢆ⿵࠺ࡓࡵ࡟ཷᐜయࢆ 2.5 ᙜ㔞⏝࠸ࡓࠋ⤖ᯝ࡜ ࡋ࡚཰⋡ 64%࡬ྥୖࡋࡓࡀࠊ౫↛୰⛬ᗘࡢ⤖ᯝ࡛࠶ࡾࠊ㐣๫㔞ࡢཷᐜయࡢ౑⏝ࡣຠᯝⓗ࡛ ࡞࠸࡜ุ᩿ࡋࡓࠋḟ࡟ࠊGlc ࡢ 3 ఩ཬࡧ 6 ఩Ỉ㓟ᇶࢆ PMB ᇶ࡛ಖㆤࡋࡓ Glc-Cer ཷᐜయ 4 1.5 ᙜ㔞ࢆ⏝࠸᳨࡚ウࡋࡓ (᮲௳ 3)ࠋࡑࡢ⤖ᯝࠊཷᐜయࡢᙜ㔞ࢆᢚ࠼࡞ࡀࡽࡶ཰⋡ࡢ኱ᖜ࡞ ྥୖ࡟ᡂຌࡋࠊ84%࡟࡚┠ⓗ≀ࢆᚓࡓࠋ௨ୖࡢ⤖ᯝࡼࡾࠊཷᐜయ 3 ࡢ 3,6 ఩ࡢ Ac ᇶ࡟ࡼࡿ ❧య㞀ᐖᢚไࡢຠᯝࡣపࡃࠊཷᐜయ4 ࡢ 3,6 ఩ࡢ PMB ᇶࡢ㟁Ꮚ౪୚ࡀཷᐜయࡢỈ㓟ᇶࡢ཯

25_{Y. Matsuzaki, Y. Ito, Y. Nakahara, T. Ogawa, Tetrahedron Lett. 1993, 34, 1061-1064.} 26_{R. R. Schmidt, Angew. Chem. Int. Ed. Engl. 1986, 25, 212-235.}

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ᛂᛶྥୖ࡟኱ࡁࡃᐤ୚ࡍࡿ࡜⤖ㄽ௜ࡅࡓࠋࡼࡗ࡚ࠊ௨㝆ࡢ࢞ࣥࢢࣜ࢜ࢩࢻ㦵᱁ᵓ⠏཯ᛂࡣཷ ᐜయ4 ࡢ౑⏝ࢆඃඛࡍࡿ஦࡜ࡋࡓࠋ

➨ᅄ⠇ ⺯ගⰍ⣲ࡢᑟධ

GM3 ࡬ ࡢ ⺯ ග Ⰽ ⣲ ᑟ ධ ࡢ ᳨ ウ ࢆ ࡋ ࡓ ࠋ ⺯ ග ᑟ ධ ヨ ⸆ ࡜ ࡋ ࡚ ึ ࡵ ࡟ ࠊ Fluorescein isothiocyanate (FITC)27_{ࢆ㑅ᢥࡋࡓ (Figure 7)ࠋFITC ࡜ࡣࠊ≉࡟ච␿ᏛࡸศἪᏛ࡛ỗ⏝ࡉࢀ࡚} ࠸ࡿ⺯ගᶆ㆑๣ࡢࡦ࡜ࡘ࡛࠶ࡾࠊᴟࡵ࡚㧗౯࡞⺯ගᶆ㆑๣ࡢ୰࡛ࡶẚ㍑ⓗᏳ౯࡛࠶ࡿࠋࡇࡢ ⺯ගᑟධヨ⸆ࡣࠊ࢔࣑ࣀᇶ࡜≉␗ⓗ࡟཯ᛂࡋ࡚ࢳ࢜࢘ࣞ࢔⤖ྜࢆᙧᡂࡍࡿࠋ⺯ගศᏊࠊ

(28)

Fluorescein ࡢບ㉳Ἴ㛗 (λex) ࡣ 492 nmࠊ⺯ගἼ㛗 (λem) ࡀ 518 nm ࡛࠶ࡾࠊ୧Ἴ㛗ࡀࢱࣥ ࣃࢡ㉁ࡢ⮬ᕫ⺯ගᇦ࡜㞳ࢀ࡚࠸ࡿࡓࡵࠊ⣽⬊⭷ୖࡢศᏊ࢖࣓࣮ࢪࣥࢢ࡟ࡋࡤࡋࡤ฼⏝ࡉࢀ ࡿࠋ ඛࡎࠊ๓⠇࡟࡚ྜᡂࡋࡓGM3 㦵᱁ (໬ྜ≀ 18) ࡢ⬺ಖㆤࢆ⾜ࡗࡓࠋGM3 ㄏᑟయ 18 ࡟ᑐ ࡋ࡚PMB ᇶࡢ㝖ཤࢆ⾜࠸ࠊ⥆࠸࡚ NaOH Ỉ⁐ᾮ࡟ࡼࡿ࢔ࢩࣝ⣔ಖㆤᇶࡢ㝖ཤࠊཬࡧ࣓ࢳࣝ ࢚ࢫࢸࣝࡢࡅࢇ໬ࢆ⾜࠺ࡇ࡜࡛ࠊGal6-N3 GM3 20 ࡬ࡢኚ᥮ࢆ཰⋡Ⰻࡃ⾜ࡗࡓ (Scheme 7)ࠋ ḟ࠸࡛໬ྜ≀20 ࡢ࢔ࢪࢻᇶࢆ࢔࣑ࣀᇶ࡬㑏ඖࡋࡓࠋࡑࡢ᪉ἲ࡜ࡋ࡚࢜ࣞࣇ࢕ࣥᏑᅾୗ ࡟࠾࠸࡚ࡶ໬Ꮫ㑅ᢥⓗ࡞㑏ඖࡀྍ⬟࡛࠶ࡿStaudinger ཯ᛂ28_{ࢆ᥇⏝ࡋࡓ (Scheme 8)ࠋ}

(29)

Staudinger ཯ᛂ࡟ࡣ Triphenylphosphine ࡀ Phosphine ヨ⸆࡜ࡋ࡚ỗ⏝ࡉࢀࡿࡀࠊ๪⏕ࡍࡿ Triphenylphosphine oxide ࡣᙉຊ࡞Ỉ⣲⤖ྜ⏕ᡂ⬟ࡸ␯Ỉᛶࢆ᭷ࡍࡿࡓࡵ⢭〇᫬ࡢጉࡆ࡜࡞ ࡾࡸࡍ࠸ࠋࡑࡇ࡛ࠊ࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡛ࡢ⢭〇ࡀᐜ᫆࡞Phosphine ヨ⸆࡜ࡋ࡚ Tributylphosphine ࢆ㑅ᢥࡋࡓࠋMeOHࠊTHF ΰྜ⁐፹୰ 50 ˚C ࡟࡚཯ᛂࢆ⾜ࡗࡓ⤖ᯝࠊ2 ᫬ 㛫࡛཯ᛂࡢ⤊⤖ࢆ☜ㄆࡋࡓࠋࡑࡋ࡚⏕ᡂ≀ࢆࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮ཬࡧࢤ ࣝℐ㐣ࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡟ࡼࡾ⢭〇ࡋࠊ໬ྜ≀21 ࢆᚓࡓࠋḟ࡟໬ྜ≀ 21 ࡟ FITC ࡜ TEA ࢆస⏝ࡉࡏࡓࠋࡑࡢ⤖ᯝࠊ཯ᛂ㛤ጞ࠿ࡽ 4 ᫬㛫ᚋ࡟ฟⓎ≀㉁ࡢᾘኻ࡜୺⏕ᡂ≀ࡢ⏕ᡂ ࢆ☜ㄆࡋࡓࠋࡋ࠿ࡋ࡞ࡀࡽ⢭〇࡟࠾࠸࡚๪⏕ᡂ≀࡜ࡢศ㞳ࡣᅔ㞴ࢆᴟࡵࠊࡋ࠿ࡶࡇࡢ๪⏕ ᡂ≀ࡣ᫬㛫⤒㐣࡜࡜ࡶ࡟ᚎࠎ࡟ቑຍࡋ࡚࠸ࡿࡼ࠺࡟ぢཷࡅࡽࢀࡓࠋࡑࡇ࡛-78 °C ୍࡛㐌㛫 ಖᏑࡋࠊಖᏑ๓ᚋࡢTLC ࢆẚ㍑ࡋࡓ࡜ࡇࢁࠊ」ᩘࡢࢫ࣏ࢵࢺࡀ⏕ࡌ࡚࠸ࡓࠋࡇࡢ⤖ᯝࡣ໬ ྜ≀22 ࡢᵓ㐀ࡀ୙Ᏻᐃ࡛ࠊ᫬㛫ࡢ⤒㐣࡜ඹ࡟ᚎࠎ࡟ศゎࡋ࡚࠸ࡿࡇ࡜ࢆ♧ࡍࠋࡑࡇ࡛ࠊ ⺯ගⰍ⣲ࡢ⤖ྜᵝᘧࢆᏳᐃ࡞࢔࣑ࢻ⤖ྜ࡬࡜ኚ᭦ࡍࡿ⪃࠼࡟⮳ࡗࡓࠋ

(30)

Tetramethylrhodamine (TMR) ࡣບ㉳Ἴ㛗 (λex) 543 nmࠊ⺯ගἼ㛗 (λem) 578 nm ࡢ⺯ගⰍ⣲ ࡛࠶ࡿ (Figure 8)ࠋࡲࡓࠊᙉ࠸Ⓨගࢆ࿊ࡋࠊ୍ศᏊほᐹ࡟࠾࠸࡚ Fluorescein ࡼࡾࡶග〣Ⰽࡉ ࢀ࡟ࡃ࠸࡜࠸࠺Ⅼ࡛ඃࢀ࡚࠸ࡿࠋ(ග〣Ⰽ᫬㛫ࡣ Fluorescein: ⣙ 100 msࠊTMR: ⣙ 5 s) ࡉࡽ ࡟ࠊFluorescein 㢮ࡣỈ⁐ᛶࡀ㧗ࡃ⣽⬊⭷ࢆ㏱㐣ࡋ࡞࠸୍᪉࡛ࠊRhodamine 㢮ࡣẚ㍑ⓗ⬡⁐ᛶ ࡀ㧗ࡃ⣽⬊ෆ࡬ࡢᑟධࡀྍ⬟࡛࠶ࡿ≉㛗ࢆࡶࡘ29_ࠋࡇࡢ_{TMR ࢆ࢔࣑ࢻ⤖ྜ࡟ࡼࡾ࢔࣑ࣀ୰} 㛫య࡬⤖ྜࡉࡏࡿ᳨ウࢆ⾜ࡗࡓࠋ໬ྜ≀20 ࡟ᑐࡋ Staudinger ཯ᛂࢆ⾜࠸࢔࣑ࣀయ࡜ࡋࡓᚋࠊ

5-Carboxytetramethylrhodamine N-succinimidyl esterࠊTEA ࢆస⏝ࡉࡏࡓ (Scheme 9)ࠋ⢭〇ࡣ PTLC ཬࡧࢤࣝℐ㐣ࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮ࡀຠᯝⓗ࡛࠶ࡾࠊ┠ⓗ໬ྜ≀ࡢ TMR ᶆ㆑ GM3 ࣉ

࣮ࣟࣈࢆ཰⋡43%࡟࡚ᚓࡓࠋࡉࡽ࡟ྠ᮲௳࡟࡚⺯ගⰍ⣲ ATTO594 ࡢᑟධࡶ⾜ࡗࡓࠋATTO594

ࡣTMR ࡜ẚ㍑ࡋ࡚㛗Ἴ㛗 (λex: 543 nmࠊλem: 578 nm) ࡛ぶỈᛶࡀ㧗࠸Ⰽ⣲࡛࠶ࡿࠋࡑࡋ࡚ࠊ

〣Ⰽ᫬㛫ࡀ㛗࠸ࡓࡓࡵ (> 10 s)ࠊ୍ศᏊほᐹᐇ㦂࡟ࡼࡾ㐺ࡋࡓ≉㛗ࢆᣢࡘࠋ࢔࣑ࣀ୰㛫య࡟

ᑐࡋ࡚ATTO594 N-succinimidyl esterࠊTEA ࢆస⏝ࡉࡏࠊ໬ྜ≀ 24 ࢆ཰⋡ 47%࡟࡚ᚓࡓࠋ࡞

࠾ࠊࡇࢀ௨㝆ࠊ᭱⤊໬ྜ≀ྡࡣ⺯ගⰍ⣲ࡢ✀㢮ࠊ⤖ྜ఩⨨࡟‽ࡌ࡚594-G6-GM3 ࡢࡼ࠺࡟࿧

ࡪࡇ࡜࡜ࡍࡿࠋ஧✀㢮ࡢ⺯ගࣉ࣮ࣟࣈࡢ⣽⬊⭷ୖ࡟࠾ࡅࡿᛶ㉁ࡢ᳨ド࡟ࡘ࠸࡚ࡣᚋ㏙ࡍࡿࠋ

(31)

➨ᅄ❶ ࢩ࢔ࣝ㓟

9 ఩ᶆ㆑ GM3 ࣉ࣮ࣟࣈࡢྜᡂ

➨୍⠇ ྜᡂᡓ␎

ᮏ❶࡛ࡣࠊNeu9 ఩ᶆ㆑ᆺࡢ GM3 ࣉ࣮ࣟࣈࡢྜᡂ࡟ࡘ࠸࡚㏙࡭ࡿࠋ⺯ගⰍ⣲ᑟධἲࡣ Gal6

఩ᶆ㆑ᆺGM3 ࣉ࣮ࣟࣈࡢྜᡂ⤖ᯝ࡟ᇶ࡙ࡁࠊ࢔࣑ࢻ⤖ྜࢆ௓ࡋ࡚⺯ගⰍ⣲ࢆ⤖ྜࡉࡏࡿࡇ

(32)

Neu9 ఩ᶆ㆑ᆺ࡟࠾࠸࡚ࡶ࢞ࣥࢢࣜ࢜ࢩࢻ㦵᱁ࡣ N-Troc Neu-Gal ࠿ࡽㄏᑟࡋࡓ Neu9 ఩ಟ

㣭ᆺࡢ஧⢾౪୚య࡜Glc-Cer ཷᐜయࡢࢢࣜࢥࢩࢻ໬཯ᛂ࡟ࡼࡾᵓ⠏ࡍࡿࡇ࡜࡜ࡋࡓࠋࡑࡇ࡛ࠊ

㦵᱁ᵓ⠏࡟㔜せ࡞Neu9 ఩ಟ㣭ᆺ Neu-Gal ౪୚యࡢྜᡂ⤒㊰ࢆ஧⤒㊰❧᱌ࡋࡓ (Scheme 10)ࠋ

⤒㊰A ࡛ࡣ඲࡚ࡢ a ⣔ิ࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢྜᡂ࡟㐺⏝ࡍࡿ஦ࢆ┠ⓗ࡜ࡋࠊGal4 ఩ ࠿ࡽࡢศᒱᵓ㐀ࡢᵓ⠏ࢆྍ⬟࡜ࡋࡓඹ㏻ࣘࢽࢵࢺࢆྜᡂࡍࡿࠋࡍ࡞ࢃࡕࠊGal4 ఩Ỉ㓟ᇶ࡟ 㑅ᢥⓗ㝖ཤࡀྍ⬟࡞ಖㆤᇶࢆᑟධࡋࡓඹ㏻໬ྜ≀࠿ࡽࠊGM3 ྜᡂ⏝ࡢ஧⢾౪୚య࡬ࡢㄏᑟࠊ ୪ࡧ࡟a ⣔ิ࢞ࣥࢢࣜ࢜ࢩࢻྜᡂ⏝ࡢ Gal4-OH ཷᐜయ࡬ࡢㄏᑟࢆᣦྥࡋࡓࠋ⤒㊰ B ࡣ GM3 ࣉ࣮ࣟࣈࡢྜᡂࡢࡳࢆ᝿ᐃࡋ࡚ᕤ⛬ࡢ⡆␎໬ࢆᅗࡗࡓ⤒㊰࡛࠶ࡿࠋಟኈㄽᩥ࡟࠾࠸࡚⤒㊰A ࢆࡲࡎ᳨ウࡋࡓࡀࠊ❧యⓗ࡟㎸ධࡗࡓGal4 ఩Ỉ㓟ᇶ࡟ᑐࡋ࡚╔⬺ྍ⬟࡞ಖㆤᇶࢆぢฟࡍࡇ ࡜ࡀฟ᮶࡞࠿ࡗࡓࡓࡵࠊ⤒㊰B ࡟ࡼࡾྜᡂ◊✲ࢆ㐍ࡵࡓ30_ࠋ ⤒㊰ B ࢆ᳨ウࡍࡿ࡟࠶ࡓࡾㄢ㢟࡜࡞ࡿࡢࡀ Neu9 ఩࢔ࢪࢻᇶࡢ㑏ඖࡢࢱ࢖࣑ࣥࢢࡢỴᐃ ࡛࠶ࡾࠊ௨ୗࡢⅬ࡟␃ពࡍࡿᚲせࡀ࠶ࡿࠋ(1)࢔ࢪࢻᇶࡢ㑏ඖ཯ᛂࡣ๪཯ᛂ࡜ࡋ࡚ Troc ᇶࡢ 30_{Ἑᮧዉ⥴Ꮚ}_{ᒱ㜧኱Ꮫ኱Ꮫ㝔} _{ᛂ⏝⏕≀⛉Ꮫ◊✲⛉} _ಟኈㄽᩥ_2010.

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ሷ⣲ཎᏊࢆỈ⣲ཎᏊ࡬⨨᥮ࡍࡿᜍࢀࡀ࠶ࡾࠊ㏫࡟άᛶ໬ Zn ࡟ࡼࡿ Troc ᇶ㝖ཤࡣ࢔ࢪࢻᇶ ࢆࡶ㑏ඖࡋ࡚ࡋࡲ࠺ࠋ(2)࢔࣑ࣀᇶࡣ㞄᥋఩࠿ࡽࡢ Ac ᇶࡢ㌿఩ࢆཷࡅࡸࡍ࠸ࠋ(3) Bn ᇶ⬺ಖ ㆤࡢẁ㝵ࡲ࡛࡟ࡣ࢔ࢪࢻᇶࢆ᥋ゐỈ⣲ῧຍἲ࡟⪏ᛶࡢ࠶ࡿᐁ⬟ᇶ࡟ኚ᥮ࡍࡿᚲせࡀ࠶ࡿࠋ ௨ୖࡢⅬࢆ⪃៖ࡋ࡚࢔ࢪࢻᇶࡣ Troc ᇶ㝖ཤᚋ࡟ᑟධࡍࡿࡇ࡜࡜ࡋࠊ㑏ඖࡢࢱ࢖࣑ࣥࢢࡣ Neu9 ఩࢔ࢪࢻ໬ࡢ┤ᚋ࡟タᐃࡋࡓࠋ㐟㞳࡜࡞ࡗࡓ࢔࣑ࣀᇶࡣ TFAc ᇶ࡛ಖㆤࡍࡿࡇ࡜࡛ࠊ ⤊┙ࡢ⬺࢔ࢩࣝ໬཯ᛂ࡛㐟㞳ࡢ࢔࣑ࣥ࡬ኚ᥮ྍ⬟࡜⪃࠼ࡓࠋNeu-Gal ࣘࢽࢵࢺࡣ౪୚య࡬࡜ ㄏᑟࡋࡓᚋࠊGlc-Cer ཷᐜయ࡜ࡢ⦰ྜ཯ᛂࠊ᏶඲⬺ಖㆤࠊ⺯ගⰍ⣲ࡢᑟධࡢᡭ㡰࡛ຠ⋡ⓗ࡟ ┠ⓗ໬ྜ≀࡬ᑟࡃ⤒㊰ࢆ❧࡚ࡓ (Scheme 11)ࠋ

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➨஧⠇ ࢩ࢔ࣜࣝ࢞ࣛࢡࢺ࣮ࢫ౪୚యࡢྜᡂϨ

➨୍㡯

1,4-ࣛࢡࢺࣥయࡢྜᡂ

⤒㊰B ࡢ Neu-Gal ࣘࢽࢵࢺࡢྜᡂ࡛ࡣ Gal4 ఩ࡢỈ㓟ᇶࡢಖㆤࢆ⾜ࢃࡎ࡟౪୚య࡬࡜ኚ᥮ ࡍࡿ (Scheme 12)ࠋ໬ྜ≀ 1 ࡢ 5 ఩ Troc ᇶࢆ Ac2O Ꮡᅾୗ࡛㝖ཤࡍࡿ஦࡟ࡼࡾࠊ5 ఩࢔ࢭࢺ ࢔࣑ࢻయ25 ࢆᚓࡓࠋḟ࡟໬ྜ≀ 25 ࡢ Ac ᇶࡢ㝖ཤࢆ᳨ウࡋࡓ࡜ࡇࢁࠊ࣓ࢳ࢚ࣝࢫࢸࣝయ 26 ࡜1,4-ࣛࢡࢺࣥయ 27 ࡢΰྜ≀ࡀ 6:4 ࡢẚ⋡ࢆಖࡗ࡚⏕ᡂࡋࠊ⣔ෆ࡛୧໬ྜ≀ࡀྍ㏫ⓗ࡞㛵 ಀ࡛࠶ࡿࡇ࡜ࢆ☜ㄆࡋࡓࠋࡇࡢ⤖ᯝࡼࡾࠊGal4 ఩ࡢỈ㓟ᇶࡀ㐟㞳ࡢ஧⢾୰㛫యࡣሷᇶᛶࡲ ࡓࡣ㧗 ᮲௳࡟࠾࠸࡚ᐜ᫆࡟ࣛࢡࢺࣥᙧᡂࡍࡿࡇ࡜ࡀศ࠿ࡗࡓࠋࡑࡇ࡛ࠊࡇࡢ1,4-ࣛࢡࢺࣥ ᵓ㐀ࢆ฼⏝ࡋࡓྜᡂ⤒㊰࡟ࡘ࠸᳨࡚ウࢆ⾜ࡗࡓࠋ໬ྜ≀25 ࡢ⬺࢔ࢭࢳࣝ໬཯ᛂᚋࠊ࣓ࢳࣝ ࢚ࢫࢸࣝࢸࢺ࣮ࣛ࢜ࣝయ26 ࡜ 1,4-ࣛࢡࢺࣥࢺ࣮ࣜ࢜ࣝయ 27 ࡢΰྜ≀࡟ᑐࡋ࡚ DBU ࢆస⏝ ࡉࡏࡿࡇ࡜࡛ࠊ1,4-ࣛࢡࢺࣥయ 27 ࡬཰᮰ࡉࡏࡓࠋࡇࡢኚ᥮࡟ࡘ࠸᳨࡚ウࡋࡓ⤖ᯝࠊ཯ᛂ୰࡟ ࢝ࣝ࣎ࣥ㓟ࡢ๪⏕ࢆᢚไࡍࡿࡓࡵ࡟஝⇱๣ (MS4Å) ࢆ⏝࠸ࠊࡉࡽ࡟཯ᛂ⤊஢᫬࡟ࡣ㓑㓟࡟ ࡚୰࿴ࡍࡿࡇ࡜࡛ࠊ෌⌧ᛶⰋࡃ㧗཰⋡ࡀᚓࡽࢀࡓࠋḟ࡟Neu9 ఩Ỉ㓟ᇶࡢࢺࢩࣝ໬ࠊࡑࡋ࡚ ࢔ࢪࢻᇶ࡬ࡢኚ᥮ࢆ⾜ࡗࡓࠋࣛࢡࢺࣥᵓ㐀ࡣ࢔ࢪࢻ໬཯ᛂ࡟࠾࠸࡚㠀ᖖ࡟Ᏻᐃࡋ࡚࠾ࡾࠊࡲ ࡓ❧య㞀ᐖࢆཷࡅ࡟ࡃ࠸Neu9 ఩ࡢ⨨᥮ࡀ㏿ࡸ࠿࡟㐍⾜ࡋࡓࡓࡵࠊ99%࡜࠸࠺㧗཰⋡࡟࡚ 9-N3య 29 ࢆᚓࡓࠋNaN3࡜ TMGN3ࡢ୧᪉᳨࡛ウࡋࡓࡀ཮᪉࡜ࡶ኱ᕪࡢ࡞࠸⤖ᯝ࡜࡞ࡗࡓࡓ ࡵࠊㄪ〇ࡢᚲせࡢ࡞࠸NaN3ࢆ௨ᚋ౑⏝ࡍࡿ஦࡜ࡋࡓࠋ

(35)

⥆࠸࡚Neu9 ఩ࡢ TFAc ࢔࣑ࢻ໬ࡢ᳨ウࢆ⾜ࡗࡓࠋࡲࡎࡣ Gal6 ఩ᶆ㆑ GM3 ࣉ࣮ࣟࣈྜᡂ ᫬࡜ྠᵝ࡟MeOH ࡜ THF ࡢΰྜ⁐፹୰࡛ Tributylphosphine ࢆస⏝ࡉࡏ࡚໬ྜ≀ 29 ࡢ 9 ఩ ࢔ࢪࢻᇶࡢ㑏ඖࢆ⾜ࡗࡓ (Table 3)ࠋࡋ࠿ࡋ࡞ࡀࡽࠊ┠ⓗ࡜ࡍࡿ໬ྜ≀ 30 ࡢ௚࡟࣓ࢳ࢚ࣝࢫ ࢸࣝయ31 ࡀ㢧ⴭ࡟๪⏕ࡋ࡚࠸ࡿࡇ࡜ࡀุ᫂ࡋࡓ (᮲௳ 1)ࠋࡇࢀࡣ཯ᛂ⁐፹ࡢ MeOH ࡟ࡼࡾ ࣛࢡࢺࣥࡀ࢚ࢫࢸࣝ஺᥮ࢆཷࡅࡓ⤖ᯝ⏕ࡌࡓ๪⏕ᡂ≀࡛࠶ࡿ࡜⪃࠼ࡓࠋࡑࡢࡓࡵࠊ᮲௳2 ࡛ ࡣTHF ࢆ⏝࠸᳨࡚ウࡋࡓࠋ࡜ࡇࢁࡀ TLC ࡟࡚」ᩘࡢ⏕ᡂ≀ࡀ☜ㄆࡉࢀࠊ୺⏕ᡂ≀࡜ࡋ࡚᪂ ࡓ࡟࢖࣑ࣀ࣍ࢫ࣍ࣛࣥࡀ⏕ࡌࡓࠋ࢖࣑ࣀ࣍ࢫ࣍ࣛࣥࡣStaudinger ཯ᛂࡢ୰㛫య࡛࠶ࡾࠊ཯ᛂ ࡀࡇࡇ࡛೵Ṇࡍࡿ࡜࠸࠺ࡇ࡜ࡣ༶ࡕࣉࣟࢺࣥࡢ౪⤥ࡀ⁫ࡗ࡚࠸ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿ (Scheme 13)ࠋTHF ࡜ẚ㍑ࡋ࡚ MeOH ࢆ⏝࠸ࡓ⣔ࡢ཰⋡ࡀⰋዲ࡛࠶ࡗࡓせᅉࡣ㓟ᛶᗘࡢ㧗࠸ MeOH ࡀ࢖࣑ࣀ࣍ࢫ࣍ࣛࣥ࡬ࣉࣟࢺࣥࢆ౪⤥ࡋ࡚࠸ࡓࡓࡵ࡜᥎ᐹࡉࢀࡿࠋ

(36)

ᙜ◊✲ᐊࡢMagesh ࡽ31_{ࡣ㓟ࢡࣟࣛ࢖ࢻᏑᅾୗ}_{Ethylenebis(diphenylphosphine) (DPPE) ࢆ࢔} ࢪࢻ໬ྜ≀࡟స⏝ࡉࡏࡿࡇ࡜࡟ࡼࡗ࡚㧗཰⋡࡟࡚࢔࣑ࢻ⤖ྜࡢᙧᡂࢆ㐩ᡂࡋ࡚࠸ࡿࠋᙼࡽ

ࡢሗ࿌ࢆཧ⪃࡟ࠊ࣏࣡ࣥࢵࢺ࡟ࡼࡿ࢔ࢪࢻᇶࡢ TFAc ࢔࣑ࢻᇶ࡬ࡢኚ᥮ࢆ᳨ウࡋࡓ (Table

4)ࠋ

31_{S. Magesh, S. Moriya, T. Suzuki, T. Miyagi, H. Ishida, M. Kiso, Bioorg. Med. Chem. Lett. 2008, 18,}

(37)

᮲௳1 ࡛ THF ⁐፹୰ TFAcOMe ࢆస⏝ࡉࡏࡓ࡜ࡇࢁ TLC ୖ࡛ 3 ࢫ࣏ࢵࢺ⌧ࢀࠊࡑࡢ࠺ࡕ ஧ࡘࡣ┠ⓗ≀ཬࡧ 9 ఩ࡀ TFAc ࢔࣑ࢻᇶࡢ࣓ࢳ࢚ࣝࢫࢸࣝయ࡛࠶ࡿࡇ࡜ࡀศ࠿ࡗࡓࠋTFAc ໬ヨ⸆ࢆ(TFAc)2O ࡟ኚ࠼ࡓ᳨ウࡶ⾜ࡗࡓࡀࠊ⏕ᡂ≀ࡀ」㞧࡟࡞ࡗࡓ (᮲௳ 2)ࠋࡇࢀ࡟ᑐࡋ ࡚MeOH ࢆ཯ᛂ⁐፹࡜ࡋࡓ᮲௳ 3 ࡛ࡣ┠ⓗ≀࡜࣓ࢳ࢚ࣝࢫࢸࣝయࡀ 4:6 ⛬ᗘࡢẚ⋡࡛⏕ᡂ ࡋࠊ9 ఩ࡢ TFAc ࢔࣑ࢻ໬ࡢ᮲௳࡜ࡋ࡚ࡣ᭱㐺࡛࠶ࡿ࡜⪃࠼ࡓࠋ ࡑࡇ࡛ࠊ᮲௳3 ࡢ᮲௳࡛཯ᛂࢆ⾜ࡗࡓᚋ࡟ࠊ๓㏙ࡢ DBU ࢆ⏝࠸ࡓ᮲௳࡟ࡼࡾ෌ࡧࣛࢡࢺ ࣥయ࡬ㄏᑟࡋࡓࠋࡑࡢ⤖ᯝࠊ஧ẁ㝵85%ࡢ཰⋡࡛┠ⓗ≀ 32 ࢆᚓࡓ (Scheme 14)ࠋ

(38)

➨஧㡯 ౪୚య࡬ࡢኚ᥮

ḟ࡟໬ྜ≀32 ࢆ౪୚య࡬ኚ᥮ࡋࡓࠋࢩ࢔ࣝ㓟ࡢ㐟㞳ࡢỈ㓟ᇶࢆ࢔ࢭࢳࣝ໬ࡋࡓᚋࠊ᥋ゐ Ỉ⣲ῧຍἲ࡟ࡼࡿBn ᇶࡢ㝖ཤཬࡧ Gal2,6 ఩Ỉ㓟ᇶ࡬ࡢ Bz ᇶࡢᑟධࢆ⾜࠸໬ྜ≀ 35 ࡬ㄏ ᑟࡋࡓ (Scheme 15)ࠋ⥆࠸࡚࢔ࣀ࣐࣮఩ࡢ⬺ಖㆤࠊࢺࣜࢡࣟࣟ࢔ࢭࢺ࢖࣑ࢹ࣮ࢺᇶࡢᑟධ࡟ ࡼࡾ౪୚య37 ࢆᚓࡓࠋࡇࡢ཯ᛂ࡟࠾࠸࡚ α యࡀ୺⏕ᡂ≀࡜ࡋ࡚ᚓࡽࢀࠊβ యࡣഹ࠿࡟ࡋ࠿ ⏕ᡂࡋ࡞࠿ࡗࡓࠋ1,4-ࣛࢡࢺࣥᵓ㐀ࢆ᭷ࡋࡓ Neu9 ఩ಟ㣭ᆺ Neu-Gal ࣘࢽࢵࢺࢆᏳᐃⓗ࡟౪ ୚య࡬ㄏᑟࡍࡿࡇ࡜࡟ᡂຌࡋࡓࠋḟ⠇࡛ࡣ౪୚య37 ࡜ Glc-Cer ཷᐜయ࡜ࡢ⦰ྜ཯ᛂ࡟ࡘ࠸ ࡚㏙࡭ࡿࠋ

➨୕⠇

GM3 㦵᱁ࡢྜᡂ᳨ウϨ

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ࡲࡎࡣ౪୚య࡟ᑐࡋ࡚ཷᐜయ3 ࢆ 1.5 ᙜ㔞⏝࠸࡚౪୚యࡢ཯ᛂᛶࢆ᳨ドࡋࡓࠋ㦫࠸ࡓࡇ࡜ ࡟⦰ྜ≀38 ࡣ㉁㔞ศᯒ࡟࡚ഹ࠿࡟Ꮡᅾࡀ☜ㄆࡉࢀࡓ⛬ᗘ࡛࠶ࡗࡓ (᮲௳ 1)ࠋ௚ࡢ๪⏕ᡂ≀ ࡜ࡋ࡚౪୚యࡀຍỈศゎࢆཷࡅࡓ࣑࣊࢔ࢭࢱ࣮ࣝయ࡜౪୚యࡢ஧㔞యࢆ☜ㄆࡋࡓࡀࠊࡑࢀ ࡔࡅ࡛࡞ࡃᵓ㐀୙᫂ࡢ๪⏕ᡂ≀ࡀ」ᩘ⏕ᡂࡍࡿ⤖ᯝ࡜࡞ࡗࡓࠋ᮲௳2 ࡛ࡣཷᐜయࢆ 2.5 ᙜ㔞 ࡟ࡋࠊᐊ ࡟᳨࡚ウࢆ⾜ࡗࡓࡀࠊᨵၿࡢ඙ೃࢆぢฟࡍࡇ࡜ࡣ࡛ࡁ࡞࠿ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝࡣ ໬ྜ≀37 ࡢ౪୚య࡜ࡋ࡚ࡢప཯ᛂᛶࢆ♧၀ࡋ࡚࠸ࡿࠋࡇࢀࡲ࡛࡟ሗ࿌ࡉࢀ࡚ࡁࡓ Neu-Gal ౪୚య࡜ẚ㍑ࡋ࡚≉ᚩⓗ࡛࠶ࡿࡢࡀࠊ1,4-ࣛࢡࢺࣥᵓ㐀࡛࠶ࡿࠋࡇࢀࡀప཰⋡ࡢせᅉ࡛࠶ࡿ ࡜ࡍࡿ࡜ࠊ౪୚యࡢ Gal3,4 ఩Ỉ㓟ᇶ㛫ࡢሀᅛ࡞භဨ⎔ᵓ㐀ࡢᙉ࠸⎔ࡦࡎࡳ࡟ࡼࡾࠊ࢜࢟ࢯ ࢝ࣝ࣋ࢽ࣒࢘࢖࢜ࣥࡀ୙Ᏻᐃ໬ࡉࢀࡓ࡜᥎ ࡛ࡁࡿࠋᶫᮏࡽ32_ࡣ _{Neuα(2Ѝ3)Gal 1,4-ࣛࢡࢺ} ࣥ౪୚య࡜Bn ᇶ࡛ಖㆤࡋࡓ Glc-Cer ཷᐜయࢆࢢࣜࢥࢩࣝ໬཯ᛂ࡟౪ࡍࡿࡇ࡜࡟ࡼࡾኳ↛ᆺ GM3 ࢆ㧗཰⋡࡟࡚ᵓ⠏ࡋ࡚࠸ࡿ (Scheme 16)ࠋࡇࡢ⤖ᯝࢆ㋃ࡲ࠼ࡿ࡜ࠊࡼࡾ㧗཯ᛂᛶࡢཷᐜ యࢆ⏝࠸ࡿࡇ࡜࡛౪୚యࡢప཯ᛂᛶࢆ⿵࠼ࡿྍ⬟ᛶࡶᮇᚅ࡛ࡁࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ౪୚యࡢ

(40)

཯ᛂᛶࡢ┦㐪ࢆ᳨ドࡍࡿࡓࡵࠊ᪂ࡓ࡟࣓ࢳ࢚ࣝࢫࢸࣝᆺࡢ౪୚యࢆྜᡂࡋࠊ஧ࡘࡢࢢࣜࢥࢩ ࢻ໬཯ᛂࡢ⤖ᯝࢆẚ㍑ࡍࡿࡇ࡜࡜ࡋࡓࠋ

➨ᅄ⠇ ࢩ࢔ࣜࣝ࢞ࣛࢡࢺ࣮ࢫ౪୚యࡢྜᡂϩ

࣓ࢳ࢚ࣝࢫࢸࣝ౪୚యࡢྜᡂἲ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ1,4-ࣛࢡࢺࣥᵓ㐀ࡣฟⓎ≀㉁ࡢ N-Troc

Neu-Gal ࠿ࡽ Neu9 ఩࢔ࢪࢻయ࡬ㄏᑟࡍࡿୖ࡛᭷⏝࡛࠶ࡾࠊ≉࡟ Neu9 ఩࢔ࢪࢻ໬࡟࠾࠸࡚

ࡣ࢝ࣝ࣎࢟ࢩࣝᇶࢆಖㆤࡍࡿᙺ๭ࢆᢸࡗ࡚㧗཰⋡ࢆ୚࠼ࡓࠋ୍᪉࡛TFAc ࢔࣑ࢻ໬ࡢ᳨ウ࠿

ࡽࠊ1,4-ࣛࢡࢺࣥࡣ MeOH ୰࡛ᐜ᫆࡟࣓ࢳ࢚ࣝࢫࢸࣝ࡬㛤⎔ࡉࢀࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ

௨ୖࡢࡇ࡜ࢆࡩࡲ࠼࡚ࠊ1,4-ࣛࢡࢺࣥయ 29 ࡢ TFAc ࢔࣑ࢻ໬᫬࡟ᚓࡽࢀࡿ࣓ࢳ࢚ࣝࢫࢸࣝయ

33 ࠿ࡽࡢㄏᑟࡀྍ⬟࡜⪃࠼ࡓࠋ

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᮲௳1 ࡛ࡣ MeOH ⁐፹୰ DPPEࠊTFAcOMe ࢆస⏝ࡉࡏ࡚࣓ࢳ࢚ࣝࢫࢸࣝయ 33 ࢆ 60%࡟ ࡚ᚓࡓࠋ᮲௳2 ࡛ࡣ TFAcOMe Ꮡᅾୗ࡛ Lindlar ゐ፹࡟ࡼࡿ࢔ࢪࢻᇶ㑏ඖ࡜࢔࣑ࢻ໬ࢆ࣡ࣥ ࣏ࢵࢺ࡛⾜࠸ࠊ┠ⓗ≀ࢆ཰⋡82%࡟࡚ᚓࡓࠋ୧཯ᛂ࡜ࡶࣛࢡࢺࣥయ 33 ࡀྠ⛬ᗘࡢ๭ྜ࡛ṧ Ꮡࡋࡓࡀࠊ᮲௳2 ࡛ࡣࡑࡢ௚ࡢ๪⏕ᡂ≀ࡀ࡯࡜ࢇ࡝⏕ᡂࡋ࡞࠿ࡗࡓࡓࡵࠊ཰⋡ࡀ኱ᖜ࡟ྥୖ ࡋࡓࠋ⥆࠸࡚ࠊ࣓ࢳ࢚ࣝࢫࢸࣝయ33 ࢆ࢔ࢭࢳࣝ໬ࡋࠊ㡰ḟಖㆤᇶࡢኚ᥮ࢆ⾜࠺ࡇ࡜࡛ࠊࢺ ࣜࢡࣟࣟ࢔ࢭࢺ࢖࣑ࢹ࣮ࢺయ43 ࢆᚓࡓ (α:β = 4:1) (Scheme 17)ࠋ

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➨஬⠇

GM3 㦵᱁ࡢྜᡂ᳨ウϩ

ྜᡂࡋࡓ࣓ࢳ࢚ࣝࢫࢸࣝ౪୚య43 ࢆ⏝࠸࡚ GM3 㦵᱁ᵓ⠏ࡢ᳨ウࢆ⾜ࡗࡓ (Table 7)ࠋ᮲ ௳1 ࡛ࡣ౪୚య࡟ᑐࡋ࡚ Glc-Cer ཷᐜయ 3 ࢆ 1.5 ᙜ㔞౑⏝ࡋࠊ0 ǀC ࡛ TMSOTf ࢆຍ࠼ࡓᚋ Ỉ ࡟࡚ᨩᢾࡋࡓࠋ཯ᛂࡣ1 ᫬㛫࡛⤊⤖ࡋࠊ୺⏕ᡂ≀ࢆ༢㞳ࡋࡓ⤖ᯝࠊ┠ⓗ࡜ࡍࡿ⦰ྜ≀ 44 ࡛࠶ࡿࡇ࡜ࡀ☜ㄆࡉࢀࡓࠋ᮲௳2 ࡛ࡣཷᐜయ 3 ࢆ 2.5 ᙜ㔞࡟ቑຍࡋ᳨࡚ウࡋࡓ⤖ᯝࠊ61%࡬ ᨵၿࡉࢀࡓࠋ཰⋡ࡣ୰⛬ᗘ࡛࠶ࡿࡀࠊ๓❶ࡢGal6-N3ᆺ౪୚యࡢࢢࣜࢥࢩࢻ໬཯ᛂࡢ཰⋡࡜ ㏆ఝࡋࡓ⤖ᯝࡀᚓࡽࢀࡓࡇ࡜࠿ࡽࠊ୧౪୚యࡢ཯ᛂᛶࡣྠ➼࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋࡲࡓࡇࡢ ⤖ᯝ࠿ࡽࠊ1,4-ࣛࢡࢺࣥ౪୚య 37 ࡜ẚ㍑ࡋ࡚࣓ࢳ࢚ࣝࢫࢸࣝ౪୚య 43 ࡢ᪉ࡀ㧗཯ᛂᛶ࡛࠶ ࡾࠊ1,4-ࣛࢡࢺࣥ౪୚య 37 ࡢప཯ᛂᛶࡣࣛࢡࢺࣥᵓ㐀࡟㉳ᅉࡋ࡚࠸ࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋ ࡉࡽ࡟ࠊ᮲௳3 ࡛ࡣ㧗཯ᛂᛶࡢཷᐜయ 4 ࢆࢢࣜࢥࢩࣝ໬࡟౪ࡋࠊ95%࡜࠸࠺ᴟࡵ࡚㧗཰⋡࡟ ࡚GM3 㦵᱁ 45 ࢆᚓࡓࠋ

(43)

➨භ⠇ ⺯ගⰍ⣲ࡢᑟධ

ḟ࡟ࠊ⺯ගⰍ⣲ࡢᑟධ࡟ྥࡅࠊGM3 㦵᱁ 45 ࡢ⬺ಖㆤࢆ⾜ࡗࡓ (Scheme 18)ࠋGM3 㦵᱁ 45 ࡢ PMB ᇶࢆ TFAcOH ࡟ࡼࡾ㝖ཤࡋࡓᚋࠊ඲࢔ࢩࣝ⣔ಖㆤᇶࢆຍỈศゎࡍࡿࡇ࡜࡟ࡼࡗ࡚ Neu9-NH2య47 ࡬࡜཰⋡Ⰻࡃㄏᑟࡋࡓࠋ ᭱ᚋ࡟ࠊᚓࡽࢀࡓ Neu9 ఩࢔࣑ࣀయ 47 ࡟ྛ✀⺯ගⰍ⣲ࡢάᛶ࢚ࢫࢸࣝࢆ཯ᛂࡉࡏ⺯ග࢞ ࣥࢢࣜ࢜ࢩࢻ࡬ㄏᑟࡋࡓ (Scheme 19)ࠋ࠸ࡎࢀࡶ 67~86%ࡢ㧗཰⋡࡟࡚ᑟධࢆ㐩ᡂࡋࠊ⏕ᡂ ≀ࡣLH-20 ୪ࡧ࡟ PTLC ࡟ࡼࡗ࡚⢭〇ࡋࡓࠋGM3 ࡢ Gal6 ఩࡬ࡢ⺯ගⰍ⣲ࡢᑟධ཰⋡ࡀ 4 ๭ ⛬ᗘ࡛࠶ࡗࡓࡢ࡟ᑐࡋࠊNeu9 ఩࡬ࡢᑟධ཰⋡ࡀୖᅇࡗࡓせᅉࡣࠊNeu9 ఩㏆ഐࡢ❧య㞀ᐖࡀ ᑡ࡞ࡃࠊᔞ㧗࠸⺯ගⰍ⣲ࡢᑟධຠ⋡ࡀⰋ࠸Ⅽ࡛࠶ࡿ⪃࠼ࡽࢀࡿࠋ࡞࠾ࠊࡑࢀࡒࢀࡢ⺯ගⰍ⣲ ࡢᵓ㐀ࢆFigure 10 ࡟♧ࡋࡓࠋ

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(45)

➨஬❶

GM3 ࣉ࣮ࣟࣈࡢᶵ⬟ホ౯࡜୍ศᏊほᐹᐇ㦂

➨୍⠇ ࣛࣇࢺศ㓄≉ᛶࡢ᳨ド

๓㏙ࡋࡓࡼ࠺࡟ࠊࡇࢀࡲ࡛࡟ࠊᮏ◊✲࡜ࡣ␗࡞ࡿศᏊタィࡢ⺯ග࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈ ࡣ໬Ꮫ㓝⣲ἲ࡟ࡼࡾྜᡂࡉࢀ࡚࠸ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊࡇࢀࡽࡢࣉ࣮ࣟࣈࡣࣛࣇࢺᵓᡂศᏊ (௨ᚋࣛࣇࢺ࣐࣮࣮࢝࡜グࡍ) ࡜ࡋ࡚ࡢᛶ㉁ࢆホ౯ࡍࡿࡇ࡜࡞ࡃ฼⏝ࡉࢀ࡚ࡁࡓࠋ⺯ගᶆ㆑ ࡟ࡼࡗ࡚ᑐ㇟ศᏊࡢ≀⌮໬Ꮫⓗᛶ㉁ࡀኚ໬ࡍࡿྍ⬟ᛶࢆ⪃៖ࡍࡿ࡜ࠊ⺯ගࣉ࣮ࣟࣈࡢࣛࣇ ࢺぶ࿴ᛶࢆཝᐦ࡟ホ౯ࡋ࡚ࣉ࣮ࣟࣈࢆ㑅ูࡍࡿࡇ࡜ࡀࠊࡼࡾጇᙜᛶࡢ㧗࠸ほᐹ⤖ᯝࢆᚓࡿ Ⅽ࡟㔜せ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ ࡑࡇ࡛ࠊྜᡂࡋࡓGal6 ఩ᶆ㆑ GM3 ࣉ࣮ࣟࣈ (594-G6-GM3, TMR-G6-GM3) ཬࡧ Neu9 ఩ ᶆ㆑GM3 ࣉ࣮ࣟࣈ (647N-S9-GM3, TMR-S9-GM3, Fl-S9-GM3, 488-S9-GM3, 594-S9-GM3) ࡢ ⏕⣽⬊⭷ୖ࡟࠾ࡅࡿᶵ⬟ࢆࡲࡎࡣ᳨ドࡋࡓࠋྜᡂࡋࡓ⺯ගGM3 ࣉ࣮ࣟࣈࡀࣛࣇࢺᵓᡂศᏊ ࡜ࡋ࡚ࡢᶵ⬟ࢆ♧ࡍ࠿ྰ࠿ࢆㄪ࡭ࡿࡓࡵ࡟ࠊ௦⾲ⓗ࡞ホ౯᪉ἲ࡛࠶ࡿ DRM ศ㓄ᐇ㦂࡜ Lo/Ld ศ㓄ᐇ㦂ࠊࡉࡽ࡟ࡣ GPI ࢔࣮ࣥ࢝ᆺཷᐜయࡢ CD59 ࢡࣛࢫࢱ࣮࡜ࡢぶ࿴ᛶࡢホ౯࡟ࡼ ࡾ᳨ドࡋࡓࠋ ࣛࣇࢺ࡟Ꮡᅾࡍࡿ⭷ศᏊࡣ⬡㉁㛫┦஫స⏝࡟ࡼࡾจ㞟ࡋࡓ⬡㉁⎔ቃ࡟࠶ࡿࡓࡵࠊప ࡛ Triton-X100 ࡞࡝ࡢ㠀࢖࢜ࣥᛶ⏺㠃άᛶ๣࡛ฎ⌮ࢆࡋ࡚ࡶ୙⁐ᛶ⭷⏬ศ (DRM ⏬ศ) ࡟ศ㓄 ࡉࢀࡿᛶ㉁ࡀሗ࿌ࡉࢀ࡚࠸ࡿ33_{ࠋࡇࢀࡼࡾࠊDRM ⏬ศ࡟ศ㓄ࡉࢀࡿ⭷ศᏊࡀࣛࣇࢺ࣐࣮࢝} ࣮34_{࡜࡞ࡾᚓࡿᛶ㉁ࢆ♧ࡍศᏊ࡛࠶ࡾࠊ୍᪉࡛}_{DRM ⏬ศ࡟ศ㓄ࡉࢀ࡞࠸ศᏊࡣ㠀ࣛࣇࢺ࣐} ࣮࣮࡛࢝࠶ࡿ࡜ᐃ⩏ࡉࢀࡿࡼ࠺࡟࡞ࡗࡓࠋFigure 11 ࡟♧ࡍࡼ࠺࡟ࠊDRM ศ㓄ᐇ㦂࡟࠾࠸࡚

33_{a) A. K. Kenworthy, B. J. Nichols, C. L. Remmert, G. M. Hendirix, M. Kumar, J. Zimmerberg, J.}

Lippincott-Schwartz, J. Cell Biol. 2004, 165, 735-746. b) B. J. Nichols, A. K. Kenworthy, R. S. Polishchuk, R. Lodge, T. H. Roberts, K. Hirschberg, R. D. Phair, J. Lippincott-Schwartz, J. Cell Biol.

2001, 153, 529-541.

34_{ཝᐦ࡟ࡣࠊࣛࣇࢺ࣐࣮࣮࡛࢝ࡣ࡞ࡃ DRM ศᏊ࡜࿧ࢇࡔ࡯࠺ࡀⰋ࠸ࠋ}

(46)

T24 ⣽⬊ୖࡢኳ↛ࡢ࢞ࣥࢢࣜ࢜ࢩࢻ GM1 ཬࡧ GM3 ࡣ DRM ⏬ศ (TX-100 ୙⁐ᛶ⭷⏬ศ) ࡬ ศ㓄ࡉࢀࡿࡇ࡜ࢆ☜ㄆࡋࡓ (ᅗࡣ TX-100 ୙⁐ᛶ/ྍ⁐ᛶ⭷⏬ศࡢᡂศࢆ PTLC ୖ࡛ᒎ㛤ࡋࡓ ⤖ᯝ)ࠋ

Figure 11. ኳ↛ᆺ࢞ࣥࢢࣜ࢜ࢩࢻࡢ DRM ศ㓄ᐇ㦂

ࡑࡇ࡛ Kenworthy ࡽࡢᡭἲ࡟ᚑ࠸⺯ග࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢ T24 ⣽⬊ୖ࡟࠾ࡅࡿ DRM ศ㓄ᐇ㦂ࢆ⾜࠺ࡇ࡜࡜ࡋࡓࠋ௨ୗ࡟ᐇ㦂ࡢᡭἲࢆ♧ࡍࠋ ࠙᪉ἲࠚ (1) T24 ⣽⬊࡟ࠊ᭱⤊⃰ᗘ 1.2 μM ࡟࡞ࡿࡼ࠺࡟ࠊྛࣉ࣮ࣟࣈࢆຍ࠼ࠊ25 ศ㛫ࠊ25 °C ࡛࢖ࣥ ࣮࢟ࣗ࣋ࢩࣙࣥࡋࡓࠋ (2) ࢖࣮ࣥ࢟ࣗ࣋ࢩࣙࣥᚋࠊࣂࢵࣇ࢓࣮ (HBSS with PIPES) ࡛ 3 ᅇὙίࡋࡓࠋ (3) 2.8 °C ࡟෭ࡸࡋࡓ 1% TX-100 ࡛ࠊon ice ࡛ 15 ศ㛫ฎ⌮ࡋࡓࠋࡇࡢ࡜ࡁࡢࢩ࣮ࣕࣞෆࡢ ᗘࡣ2.8 °Cࠋ (4) ࢖࣮ࣥ࢟ࣗ࣋ࢩࣙࣥᚋࠊࣂࢵࣇ࢓࣮ (HBSS with PIPES) ࡛ 3 ᅇὙίࡋࠊࣃ࣒ࣛ࣍ࣝ࢔ࣝ ࢹࣄࢻ࡛90 ศ㛫ᅛᐃࡋࡓᚋࠊ⺯ග㢧ᚤ㙾 (Olympus IX70 epifluorescence microscope) ࡛ほᐹࡋࡓࠋ

Figure 12 ࡀ⏺㠃άᛶ๣ฎ⌮๓ᚋࡢ T24 ⣽⬊⭷ࡢ⺯ග㢧ᚤ㙾࡟ࡼࡿほᐹ⤖ᯝ࡛࠶ࡾࠊᅗ୰

࡜Table 8 ࡟ྛࣉ࣮ࣟࣈࡢ DRM ࡬ࡢศ㓄⋡ࢆ♧ࡋࡓ (ฎ⌮ᚋ/ฎ⌮๓ẚ)ࠋ⭷ୖࡢⓑⰍࡢ㒊ศ

ࡀTX-100 ฎ⌮ᚋ࡟ṧᏑࡋࡓࣉ࣮ࣟࣈࢆ♧ࡋ࡚࠾ࡾࠊ⏺㠃άᛶ๣ฎ⌮๓࡟࠾࠸࡚ࠊ࠸ࡎࢀࡢ

(47)

ࡣ␗࡞ࡿศ㓄≉ᛶࢆ♧ࡋࡓࠋࡍ࡞ࢃࡕࠊNeu9 ఩ᶆ㆑ GM3 ࣉ࣮ࣟࣈ (TMR-S9-GM3, Fl-S9-GM3, 488-S9-Fl-S9-GM3, 594-G6-GM3) ࡣ⣽⬊⭷࡟ṧᏑࡋࠊࣛࣇࢺ࣐࣮࣮࢝ࡢᛶ㉁ࢆ♧ࡋࡓࡢ࡟ᑐ ࡋ࡚ࠊGal6 ఩ᶆ㆑ GM3 ࣉ࣮ࣟࣈ (594-G6-GM3, TMR-G6-GM3) ୪ࡧ࡟ ATTO647N ࡀ⤖ྜࡋ ࡓNeu9 ఩ᶆ㆑ GM3 ࣉ࣮ࣟࣈ (647N-S9-GM3) ࡣࠊTX-100 ฎ⌮࡟ࡼࡗ࡚⣽⬊⭷࠿ࡽ⁐ฟࡋ ࡓࠋࡑࡢࡓࡵࠊࡇࢀࡽࡢࣉ࣮ࣟࣈࡣࣛࣇࢺ࡬ࡢศ㓄ࡀⴭࡋࡃపୗࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿࠋࡇ ࡢ⤖ᯝࡣ⺯ගⰍ⣲ࡢᑟධ఩⨨࡟ࡼࡗ࡚࢞ࣥࢢࣜ࢜ࢩࢻࡢࣛࣇࢺ࡬ࡢศ㓄ࡀኚືࡍࡿ࡜࠸࠺ ⯆࿡῝࠸஦ᐇࢆ♧ࡋࡓࠋ ࡉࡽ࡟≉グࡍ࡭ࡁࡇ࡜࡟ࠊࡇࢀࡲ࡛ࣛࣇࢺ◊✲࡟฼⏝ࡉࢀ࡚ࡁࡓBodipyFL-GM1 (ᵓ㐀ࡣ Figure 4 ࡟♧ࡍ) ࡢ DRM ࡬ࡢศ㓄ࢆホ౯ࡋࡓ⤖ᯝࠊ㦫ࡃࡇ࡜࡟ࠊTX-100 ྍ⁐ᛶ⏬ศ࡟ඃඛ ⓗ࡟ศ㓄ࡉࢀࡿࡇ࡜ࢆ☜ㄆࡋࠊࣛࣇࢺ࡬ࡢศ㓄ࡀᴟࡵ࡚ప࠸ࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓࠋ

(48)

Figure 12. DRM ศ㓄ᐇ㦂ࡢ⤖ᯝ

⥆࠸࡚ࣛࣇࢺぶ࿴ᛶࢆホ౯ࡍࡿࡶ࠺୍ࡘࡢᐇ㦂࡜ࡋ࡚ࠊGiant PM vesicle (GPMV) ୖ࡛ࡢ Lo/Ld ศ 㓄 ᐇ 㦂 ࡟ ࡼ ࡿ ホ ౯ ࡶ ⾜ ࡗ ࡓ ࠋ ศ Ꮚ ࣉ ࣟ ࣮ ࣈ ࢆ ᑟ ධ ࡋ ࡓ ⣽ ⬊ (RBL-2H3) ࢆ Formaldehyde ࡜ Dithiothreitol (DTT) ࡟ࡼࡿ⸆๣ฎ⌮࡟౪ࡍࡇ࡜࡛ࠊ⭷㦵᱁ࡀ㝖ཤࡉࢀࡓࠊ ࣈࣞࢵࣈ⭷ (┤ᚄ 5㹼20 μm ࡯࡝ࡢᕧ኱࣋ࢩࢡࣝࡢࡼ࠺࡞⭷㡿ᇦ) ࡀᙧᡂࡉࢀࡿࠋࣈࣞࢵࣈ ⭷ࡣప ୗ (10 ˚C) ࡛ࡣ⭷ୖࡢศᏊࡀࣛࣇࢺᵝࡢ㡿ᇦ (ᾮయ⛛ᗎ┦) ࡜ࣛࣇࢺ࠿ࡽ᤼㝖ࡉ

(49)

ࢀࡓ㡿ᇦ (ᾮయ↓⛛ᗎ┦) ࡟┦ศ㞳ࡉࢀࡿ35_{ࠋࡇࡢࣈࣞࢵࣈ⭷ୖ࡛㠀ࣛࣇࢺ࣐࣮࣮࡛࢝࠶ࡿ} ࣜࣥ⬡㉁ (BodipyFL-PC) ➼࡜࡜ࡶ࡟ࠊ⺯ග࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࢆ┦ศ㞳ࡉࡏࠊศ㓄ഴ ྥࢆ඲཯ᑕ⺯ග㢧ᚤ㙾 (TIRF) ࡟ࡼࡾほᐹࡋࡓ36_{ࠋࡑࡢほᐹ⤖ᯝࡢ୍౛ࢆ}_{Figure 13 ࡟♧ࡍࠋ} ౛࠼ࡤࠊᕥࡢ⤖ᯝࡣ⥳ⰍࡢBodipyFL-PC ࢆ Ld ┦ࡢᶆ㆑ศᏊ࡜ࡋࠊ㉥Ⰽࡢ 594-S9-GM3 ඹᏑ ୗ࡛ࣈࣞࢵࣈ⭷ࡢ┦ศ㞳ࢆ⾜ࡗࡓ⤖ᯝ࡛࠶ࡿࠋ஧ࡘࡢศᏊࡢ␗࡞ࡿศᕸࡀ᫂♧ࡉࢀࠊ 594-S9-GM3 ࡀ Lo ┦࡟ศ㓄ࡉࢀ࡚࠸ࡿࡇ࡜ࡀぢ࡚ྲྀࢀࡿࠋ

Figure 13. Lo/Ld ศ㓄ᐇ㦂

Lo/Ld ศ㓄ᐇ㦂ࡢ⤖ᯝࠊDRM ᐇ㦂࡟࡚ࣛࣇࢺ࣐࣮࣮࢝ࡢᛶ㉁ࢆ♧ࡋࡓ Neu9 ఩ᶆ㆑ GM3 ࣉ࣮ࣟࣈ (Fl-S9-GM3, 488-S9-GM3, 594-G6-GM3) ࡣ Ld ┦࡜ࡣ᏶඲࡞┦ศ㞳ࢆ♧ࡋࡓࠋࡇࡢ ᐇ㦂࠿ࡽࡶࠊࡇࢀࡽࡣࣛࣇࢺぶ࿴ᛶࡢ㧗࠸ศᏊ࡛࠶ࡿࡇ࡜ࡀ☜࠿ࡵࡽࢀࡓ (Table 8)ࠋࡉࡽ ࡟ࠊDRM ศ㓄ᐇ㦂࡛㠀ࣛࣇࢺ࣐࣮࣮࢝ࡢᛶ㉁ࢆ♧ࡋࡓ 647N-S9-GM3 ࡜ BodipyFL-GM1 ࡣ 㠀ࣛࣇࢺ┦ࡢLd ┦࡬ศ㓄ࡋࠊGal6 ఩ᶆ㆑ GM3 ࣉ࣮ࣟࣈ (594-G6-GM3, TMR-G6-GM3) ࡜ TMR ࡛ᶆ㆑ࡋࡓ Neu9 ఩ᶆ㆑ GM3 ࣉ࣮ࣟࣈ (TMR-S9-GM3) ࡶ Lo ┦࡬ࡢศ㓄⋡ቑຍࢆ♧

35_{a) D. Lingwood, J. Ries, P. Schwille, K. Simons, Proc. Natl. Acad. Sci. USA 2008, 105, 10005-10010.}

b) T. Baumgart, A. T. Hammond, P. Sengupta, S. T. Hess, D. A. Holowka, B. A. Baird, W. W. Webb,

Proc. Natl. Acad. Sci. USA 2007, 104, 3165-3170. c) I. Levental, M. Grzybek, K. Simons, Proc. Natl. Acad. Sci. USA 2011, 108, 11411-11416.

36_{H. J. Kaiser, D. Lingwood, I. Levental, J. L. Sampaio, L. Kalvodova, L. Rajendran, K. Simons, Proc.} Natl. Acad. Sci. USA 2009, 106, 16645-16650.

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ࡋࡓ (Table 8)ࠋBodipyFL-GM1 ࡟㛵ࡋ࡚ࡣࠊBurns ࡽ37_ࡀ_{Ld ┦࡬ࡢศ㓄ࢆ♧ࡋ࡚࠾ࡾࠊࡇࡢ} ሗ࿌࡜ᐇ㦂⤖ᯝࡢ୍⮴ࢆ☜ㄆࡋࡓࠋLo/Ld ศ㓄ᐇ㦂ࡢ⤖ᯝࡣ DRM ศ㓄ᐇ㦂ࡢ⤖ᯝ࡜ᴫࡡ୍ ⮴ࡋࡓࠋࡍ࡞ࢃࡕࠊGM3 ࡬ࡢ⺯ගⰍ⣲ࡢᑟධ఩⨨ࡀࣛࣇࢺศ㓄≉ᛶ࡟኱ࡁࡃᙳ㡪ࡋࠊGal6 ఩ᶆ㆑యࡀ㠀ࣛࣇࢺぶ࿴ᛶࠊNeu9 ఩ᶆ㆑యࡀࣛࣇࢺぶ࿴ᛶࢆ♧ࡍࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ ࡉࡽ࡟ࠊ㦫ࡃ࡭ࡁࡇ࡜࡟Lo/Ld ศ㓄ᐇ㦂ࡼࡾࠊ⤖ྜࡉࡏࡓ⺯ගⰍ⣲ࡢぶ␯Ỉᛶࡢ㐪࠸ (Figure 14) ࡟ࡼࡗ࡚ࣛࣇࢺ࡬ࡢศ㓄≉ᛶࡀ኱ࡁࡃ␗࡞ࡿࡇ࡜ࡀศ࠿ࡗࡓࠋࡘࡲࡾࠊNeu9 ఩࡟ぶỈ ᛶࡢⰍ⣲࡛࠶ࡿFluoresceinࠊATTO488ࠊATTO594 ࢆ⤖ྜࡉࡏࡓศᏊࡣࣛࣇࢺ┦࡟ከࡃศ㓄 ࡉࢀࡓࡀࠊࡸࡸ␯ỈᛶࡢTMR ࡀ⤖ྜࡍࡿ࡜㠀ࣛࣇࢺ┦࡬ࡢศ㓄⋡ࡀቑࡋࠊࡉࡽ࡟␯Ỉᛶࡢ 㧗࠸ATTO647N ࡀ⤖ྜࡋࡓ GM3 ࡣࠊ࡯ࡰ᏶඲࡟㠀ࣛࣇࢺ┦࡟ศ㓄ࡉࢀࡓࠋ

Figure 14. ⺯ගⰍ⣲ࡢᛶ㉁

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ࡉࡽ࡟ࠊ⺯ග࢞ࣥࢢࣜ࢜ࢩࢻ࡜ࠊ⬡㉁ࣛࣇࢺࣔࢹࣝ (CD59 ࣃࢵࢳ) ࡢぶ࿴ᛶࢆᐇ㝿࡟ㄪ ࡭ࡓࠋT24 ⣽⬊⭷ୖ୍࡛ḟᢠయ࡜஧ḟᢠయࢆ⏝࠸࡚ GPI ࢔࣮ࣥ࢝ᆺཷᐜయ CD59 ࢆᯫᶫࡋ ࡓࠋࡇࢀ࡟ࡼࡾࠊ1 μm2_௨ୖ38_{ࡢ኱ࡁࡉࡢᕧ኱࡞}_{CD59 ࣃࢵࢳࢆᙧᡂࡉࡏࡓ}39_{ࠋࡑࡢ⣽⬊⭷ୖ} ࡛ࡢ⺯ග GM3 ࣉ࣮ࣟࣈࡢᒁᅾࢆㄪ࡭ࡿࡇ࡜࡟ࡼࡾ CD59 ࣃࢵࢳ࡬ࡢぶ࿴ᛶࢆㄪ࡭ࡓ (Figure 15ࠊTable 8)ࠋ

Figure 15. CD59 ࣃࢵࢳ࡜ࡢぶ࿴ᛶࡢ᳨ド

38_{⬡㉁ࣛࣇࢺࡢࢧ࢖ࢬ࡟ࡘ࠸࡚ࡣㅖㄝ࠶ࡿࡀࠊࣛࣇࢺ௬ㄝ࡛ࡣ┤ᚄ 100 nm㹼ᩘ࣑ࢡࣟࣥࢧ࢖} ࢬࠊ㏆ᖺ࡛ࡣࢼࣀ࣭࣓ࢰࢧ࢖ࢬ࡛࠶ࡿ࡜ሗ࿌ࡉࢀ࡚࠸ࡿࠋ

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ࡑࡢ⤖ᯝࠊኳ↛ᆺࡢGM3 (anti-GM3 antibody ࡜⾲グ)ࠊぶỈᛶⰍ⣲ࡀ⤖ྜࡋࡓ Neu9 ఩ᶆ㆑ GM3 ࣉ࣮ࣟࣈ (TMR-S9-GM3, Fl-S9-GM3, 488-S9-GM3, 594-S9-GM3) ࡢࡳ CD59 ࣃࢵࢳ࡜ඹ ᒁᅾࡍࡿ⤖ᯝࢆᚓࡓࠋ

Table 8. ࣛࣇࢺศ㓄≉ᛶࡢ᳨ド⤖ᯝ

௨ୖࡢDRM ศ㓄ᐇ㦂ࠊLo/Ld ศ㓄ᐇ㦂ࠊCD59 ࣃࢵࢳ࡜ࡢඹᒁᅾࡢ୕ࡘࡢ᳨ドࡣ࡯ࡰ୍ ⮴ࡍࡿホ౯⤖ᯝࢆ୚࠼ࠊ㧗࠸ࣛࣇࢺศ㓄ᛶࡀド᫂ࡉࢀࡓࣉ࣮ࣟࣈࡣࠊぶỈᛶࡢ⺯ගศᏊ࡛ᶆ ㆑ࡋࡓS9-GM3 ࣉ࣮ࣟࣈ (Fl-S9-GM3, 488-S9-GM3, 594-S9-GM3) ࡛࠶ࡗࡓࠋࡑࡋ࡚ࠊᶵ⬟ホ ౯ࡼࡾ஧ࡘࡢ㔜せ࡞▱ぢࢆᚓࡓࠋ ୍Ⅼ┠ࡣࠊᶆ㆑఩⨨ࡀ࢞ࣥࢢࣜ࢜ࢩࢻࡢ⣽⬊⭷ୖ࡟࠾ࡅࡿ≀⌮໬Ꮫⓗᛶ㉁࡟኱ࡁࡃᙳ㡪 ࡋࠊኳ↛ࡢ࢞ࣥࢢࣜ࢜ࢩࢻࡢࣛࣇࢺศ㓄≉ᛶࢆಖᣢࡍࡿⅭ࡟ࡣࠊNeu9 ఩ࡢᶆ㆑ࡀᮃࡲࡋ࠸ ࡜࠸࠺ࡇ࡜ࠋᚑ᮶ࣛࣇࢺ◊✲࡟฼⏝ࡉࢀ࡚ࡁࡓ⬡㉁ಟ㣭ᆺGM1 (BodipyFL-GM1)ࠊ⬡㉁࡟㏆ ࠸ෆ㒊Gal6 ఩ಟ㣭 GM3 ࣉ࣮ࣟࣈࡣ⣽⬊⭷ୖ࡟ᒁᅾࡋࠊ⭷ศᏊ࡜ࡋ࡚ࡢṇᖖ࡞᣺⯙࠸ࡣ♧ ࡋࡓࡀ (ศᏊ㐠ື࡟ࡘ࠸࡚ࡣḟ⠇࡛㏙࡭ࡿ)ࠊࣛࣇࢺ࡬ࡢぶ࿴ᛶࡣᦆ࡞ࡗ࡚࠸ࡓࠋࡇࢀࡽࡢ せᅉࡣࠊࣛࣇࢺ㛵㐃ศᏊ࡜ࡢ⬡㉁㛫␯Ỉ⤖ྜ࡟ᐤ୚ࡍࡿࢭ࣑ࣛࢻࡢᶵ⬟ࡀపୗࠊཪࡣ㜼ᐖࡉ

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ࢀ࡚࠸ࡿࡇ࡜࡟ࡼࡿ࡜⪃࠼ࡽࢀࡿࠋࡘࡲࡾࠊBodipyFL-GM1 ࡛ࡣࢭ࣑ࣛࢻࡀ୍ᮏ㙐࡬ᵓ㐀ᨵ ኚࡉࢀ࡚࠸ࡿࡇ࡜࡟ࡼࡾ␯Ỉ⤖ྜࡀኳ↛ᆺࡼࡾࡶᙅࡲࡾࠊGal6 ఩ಟ㣭 GM3 ࣉ࣮ࣟࣈ࡛ࡣᵓ 㐀ࡀᔞ㧗࠸⺯ගศᏊࡀࢭ࣑ࣛࢻ࡟㏆᥋ࡋࡓ఩⨨࡟࠶ࡿࡇ࡜࡟ࡼࡾࠊ⺯ගศᏊࡀ⬡㉁㛫ࡢ␯ Ỉ⤖ྜࢆ㜼ᐖࡋࡓ࡜⪃࠼ࡓࠋࣛࣇࢺศ㓄ᛶࡣࠊẚ㍑ⓗ␯Ỉᛶࡢ㧗࠸TMR ࡟ࡼࡿᶆ㆑య࡛㢧 ⴭ࡟పࡃࠊᵓ㐀୰࡟㧗ᴟᛶᇶࢆྵࡴ ATTO594 ࡢᶆ㆑య࡛ࡣ࠶ࡿ⛬ᗘྥୖࡋࡓࡇ࡜࠿ࡽࡶࠊ ⺯ගศᏊࡢ␯Ỉᛶࡢᵓ㐀ࡀ⬡㉁㛫ࡢ┦஫స⏝ࢆጉࡆࡓ࡜᥎ ࡋࡓࠋࡇࡢ᥎ ࡀጇᙜ࡛࠶ࡿ ࡜ࡍࡿ࡜ࠊGM3 ࡼࡾࡶ⢾㙐㛗ࡢ㛗࠸ GM1 ࡸ GM2 ࠾࠸࡚ࡣࠊNeu9 ఩ࡢࡳ࡞ࡽࡎࠊศᒱ⢾㙐 ࡢ௚᪉ࡢᮎ➃⢾ṧᇶࡶ⺯ගⰍ⣲ᑟධࡢೃ⿵఩⨨࡟࡞ࡿ࡜⪃࠼ࡽࢀࡿࠋࡇࡢ᳨ド࡟ࡘ࠸࡚ࡣ ➨ᅄ❶ࠊ➨஬❶࡟グࡍࠋ ஧Ⅼ┠࡟ᚓࡓ▱ぢࡣࠊ⺯ගศᏊࡢぶ␯Ỉᛶࡀ࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢࣛࣇࢺぶ࿴ᛶ࡟ ኱ࡁࡃᙳ㡪ࡍࡿ࡜࠸࠺Ⅼ࡛࠶ࡿࠋ⢾㙐ࡢ㧗࠸ぶỈᛶ࡟ᙳ㡪ࡋ࡞࠸Ⅽ࡟ࡣ㧗ぶỈᛶࡢ⺯ගศ Ꮚ (ATTO594, ATTO488, Fluorescein ➼) ࡢ౑⏝ࡀዲࡲࡋ࠸஦ࡀ♧၀ࡉࢀࡓࠋ

௨ୖࡼࡾࠊᮏ◊✲࡛ࡣࣛࣇࢺぶ࿴ᛶࡢᛶ㉁ಖᣢࡀド᫂ࡉࢀࡓ⺯ග࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟ ࣈࢆึࡵ࡚㛤Ⓨࡋࡓࠋࡉࡽ࡟ࡑࡢホ౯ࡢ㐣⛬࡛ࠊᶆ㆑఩⨨ࡸ⺯ගⰍ⣲ࡢᛶ㉁ࡀ࢞ࣥࢢࣜ࢜ࢩ ࢻ࡟ཬࡰࡍᙳ㡪ࢆึࡵ࡚᫂ࡽ࠿࡟ࡋࠊࣉ࣮ࣟࣈࡢᶵ⬟ࡢ☜ㄆཬࡧ㑅ูࡢᚲせᛶࢆ❧ドࡋࡓࠋ

➨஧⠇ ୍ศᏊほᐹᐇ㦂

➨୍㡯 ᣑᩓಀᩘࡢィ⟬

ྜᡂࡋࡓ⺯ග࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈ୍ศᏊࡢ⣽⬊⭷ୖ࡟࠾ࡅࡿᣲືࢆ඲཯ᑕ⺯ග㢧ᚤ 㙾࡟ࡼࡾほᐹࡋࡓ40_{ࠋࡲࡎࡣྛ✀⺯ග࢞ࣥࢢࣜ࢜ࢩࢻࢆ}_{T24 ⣽⬊ࠊPtK2 ⣽⬊⭷࡟ᑟධࡋࠊᐃ}

40_{a) I. Koyama-Honda, K. Ritchie, T. Fujiwara, R. Iino, H. Murakoshi, R. S. Kasai, A. Kusumi, Biophys.} J. 2005, 88, 2126-2136. b) T. Fujiwara, K. Ritchie, H. Murakoshi, K. Jacobson, A. Kusumi, J. Cell Biol. 2002, 157, 1071-1081.

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ᖖ≧ែ࡟࠾ࡅࡿ࢞ࣥࢢࣜ࢜ࢩࢻࡢᣑᩓ㐠ືࢆ᳨ドࡍࡿࡇ࡜࡛ࠊ⭷ศᏊ࡜ࡋ࡚ࡢ㐠ືᛶࢆㄪ ࡭ࡓࠋ௦⾲౛࡜ࡋ࡚594-S9-GM3 ࢆ᫬㛫ศゎ⬟ 0.5 ࣑ࣜ⛊୍࡛ศᏊほᐹࡋࡓ⤖ᯝᚓࡽࢀࡓᣑ ᩓ㌶㊧ࢆFigure 16 ࡟♧ࡍࠋ㐠ື㌶㊧ࡼࡾࠊ594-S9-GM3 ࡀ೵␃ࡍࡿࡇ࡜࡞ࡃᖖ᫬ᣑᩓࡋ࡚ ࠸ࡿᵝᏊࡀศ࠿ࡿࠋࡉࡽ࡟ࠊT24 ⣽⬊ୖ࡛ࠊ100 ࣑ࣜ⛊㛫࡟࠾ࡅࡿྛ⺯ග࢞ࣥࢢࣜ࢜ࢩࢻࣉ ࣮ࣟࣈࡢᣑᩓಀᩘࢆồࡵࡓ (Table 9)ࠋࡇࡢ᳨ドࡣࠊᐃᖖ≧ែࠊࡘࡲࡾࣛࣇࢺᙧᡂࢆㄏ㉳ࡋ ࡞࠸᮲௳࡛⾜ࡗ࡚࠸ࡿࡓࡵࠊ⺯ග࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈࡢᣑᩓ㐠ືࡣ㠀ࣛࣇࢺ࣐࣮࣮࢝ ࡢࣜࣥ⬡㉁ (DOPE) ௨ୖ࡟㏿࠸ࡣࡎ࡛࠶ࡿࠋ(ࡍ࡞ࢃࡕᣑᩓಀᩘࡣ DOPE ࡼࡾ኱ࡁ࠸ࡣࡎ࡛ ࠶ࡿࠋ) ᣑᩓಀᩘ D100msࡢ୰ኸ್ࢆồࡵࡓ⤖ᯝࠊ௒ᅇྜᡂࡋࡓ඲࡚ࡢ⺯ග GM3 ࣉ࣮ࣟࣈ

(0.26~0.33 μm2_{/s)ࠊBodipyFL-GM1 (0.34 μm}2_{/s) ࡜ࡶ࡟ࠊATTO594 ᶆ㆑ DOPE (594-DOPE) (0.20}

μm2_{/s) ࡜ྠ➼ࡶࡋࡃࡣࡑࢀ௨ୖࡢ್ࢆ♧ࡋࡓࠋࡇࢀ࡟ࡼࡾࠊᐃᖖ≧ែࡢ⣽⬊⭷ୖ࡟࠾࠸࡚ࠊ} ࡇࢀࡽࡢ⺯ග࢞ࣥࢢࣜ࢜ࢩࢻࡀ⭷ࢻ࣓࢖ࣥ࡟ྲྀࡾ㎸ࡲࢀࡿࡇ࡜࡞ࡃᖖ᫬༢⣧ࣈࣛ࢘ࣥ㐠ື ࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ୍᪉࡛GM1 ⤖ྜࢱࣥࣃࢡ㉁࡛࠶ࡿࢥࣞࣛẘ⣲ B ࢧࣈࣘࢽ ࢵࢺࢆCy3 ࡛ᶆ㆑ࡋ (Cy3-CTB)ࠊT24 ⣽⬊ୖ࡟Ⓨ⌧ࡋ࡚࠸ࡿෆᅉᛶ GM1 ࡢ㐠ືࢆほᐹࡋࡓ ⤖ᯝࠊ࢖࣮ࣥ࢟ࣗ࣋ࢩࣙࣥ᫬㛫࡟కࡗ࡚ᚎࠎ࡟ᣑᩓ㐠ືࡀపୗࡍࡿࡇ࡜ࡀศ࠿ࡗࡓ (~0.01 μm2_{/s)ࠋࡇࢀࡣࠊCTB ࡀ஬㔞య࡛࠶ࡿࡓࡵࠊ᫬㛫⤒㐣࡜࡜ࡶ࡟ GM1 ࡀࢡࣛࢫࢱ࣮໬ࡋࠊࡇ} ࢀ࡟ࡼࡗ࡚༢ศᏊࡼࡾࡶGM1 ࡢ㐠ືࡀపୗࡍࡿࡇ࡜ࢆ᫂♧ࡋ࡚࠸ࡿࠋ⺯ග CTB ࢆ฼⏝ࡋ ࡓGM1 ࡢほᐹἲࡣ࢞ࣥࢢࣜ࢜ࢩࢻࣉ࣮ࣟࣈㄪ〇ࡢᚲせࡀ࡞ࡃࠊ⡆౽࡛࠶ࡿ฼Ⅼࡀ࠶ࡿࠋࡋ ࠿ࡋ࡞ࡀࡽࠊኳ↛ࡢ࢞ࣥࢢࣜ࢜ࢩࢻ୍ศᏊࡢ᣺⯙࠸ࠊ௚ࡢ⭷ศᏊ࡜ࡢ┦஫స⏝ࢆ᫂ࡽ࠿࡟ࡍ ࡿ࡜࠸࠺ᮏ◊✲ࡢ┠ⓗ࡟ࡣἢࢃࡎࠊࡸࡣࡾ⺯ගศᏊࡀ┤᥋໬Ꮫ⤖ྜࡋࡓ࢞ࣥࢢࣜ࢜ࢩࢻࣉ ࣮ࣟࣈࢆྜᡂࡍࡿᚲせᛶࡀᙉㄪࡉࢀࡓࠋ

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Figure 16. 594-S9-GM3 ࡢᣑᩓ㌶㊧

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➨஧㡯

GPI ࢔࣮ࣥ࢝ᆺཷᐜయ࡜ࡢぶ࿴ᛶࡢ᳨ド

ࣛࣇࢺศ㓄ᛶࡀド᫂ࡉࢀࡓ Neu9 ఩ᶆ㆑ GM3 ࡢ୰࡛ࡶࠊATTO594 ᶆ㆑య (594-S9-GM3) ࡣ⺯ගⰍ⣲ࡢග〣Ⰽ᫬㛫ࡀ᭱ࡶ㛗࠸ࡓࡵࠊ୍ศᏊほᐹ࡟࡜ࡾࢃࡅ㐺ࡋࡓࣉ࣮ࣟࣈ࡛࠶ࡿࠋ GPI-࢔࣮ࣥ࢝ᆺཷᐜయ (CD59) ࡟ᑐࡍࡿ่⃭᮲௳ୗࠊ594-S9-GM3 ࡀ CD59 ࡜ᐇ㝿࡟┦஫ స⏝ࡍࡿᵝᏊࢆ஧Ⰽྠ᫬୍ศᏊほᐹ࡟ࡼࡾᤊ࠼ࡿᐇ㦂ࢆ⾜ࡗࡓࠋึࡵ࡟ࠊ๓⠇ࡢඹᒁᅾᐇ 㦂࡛⏝࠸ࡓ CD59 ࣃࢵࢳ࡜ࡢ஧Ⰽྠ᫬୍ศᏊほᐹࡢ⤖ᯝࢆ♧ࡍ (Figure 17)ࠋᕥࡢ⤖ᯝࡣࠊ ⥳Ⰽࡢ CD59 ࣃࢵࢳ࡟ᑐࡍࡿ㠀ࣛࣇࢺ࣐࣮࣮࢝ᛶࡢ DOPE ࡢ㐠ື㌶㊧࡛࠶ࡿࠋDOPE ࡣ CD59 ࣃࢵࢳ࡬ࡢぶ࿴ᛶࡀప࠸ࡓࡵࠊࣃࢵࢳ࠿ࡽᙎ࠿ࢀࠊᖖ᫬እഃ࡛ᣑᩓࡋ࡚࠸ࡿᵝᏊࡀ ศ࠿ࡿࠋࡇࢀ࡟ᑐࡋ࡚ࠊྑࡢ⤖ᯝ࡛ࡣࠊ594-S9-GM3 ࡀ CD59 ࣃࢵࢳ࡬ࡢฟධࡾࢆ㢖⦾࡟⧞ ࡾ㏉ࡋࠊCD59 ࣃࢵࢳ (⬡㉁ࣛࣇࢺࣔࢹࣝ) ࡟ᑐࡋ࡚㧗࠸ぶ࿴ᛶࢆ♧ࡋࡓࠋࡇࡢࡼ࠺࡞ࠊ⬡ ㉁ࣛࣇࢺࣔࢹࣝ࡜࢞ࣥࢢࣜ࢜ࢩࢻࡢ୍ศᏊ࢖࣓࣮ࢪࣥࢢࡣᮏ◊✲࡟ࡼࡗ࡚ึࡵ࡚㐩ᡂࡋࠊ ⬡㉁ࣛࣇࢺࣔࢹࣝ࡜ࡢືⓗ┦஫స⏝ࢆྍど໬ࡍࡿࡇ࡜࡟ᡂຌࡋࡓࠋ

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Figure 17. CD59 ࣃࢵࢳ࡜ࡢඹᒁᅾ

ḟ࡟ࠊࡼࡾ⏕⌮ⓗ᮲௳࡟㏆࠸఍ྜ≧ែࡢ CD59 ࡜࢞ࣥࢢࣜ࢜ࢩࢻࡢඹᒁᅾࢆほᐹࡍࡿࡓ ࡵࠊT24 ⣽⬊ୖ࡟࠾࠸࡚ࠊᢠ CD59 ᢠయ࡜ yellow-green beads ࡛ᶆ㆑ࡋࡓ CD59 ࢡࣛࢫࢱ࣮࡜ ࡢ஧Ⰽྠ᫬ほᐹࢆ⾜ࡗࡓࠋࡇࡢ᮲௳ୗࠊyellow-green beads ࡟ᑐࡋ࡚ᖹᆒ 3~6 ศᏊࡢ CD59 ࡀ ఍ྜࡍࡿ6_{ࠋᐇ㦂ࡢ⤖ᯝࠊ⺯ග}_{GM3 ࣉ࣮ࣟࣈ (594-S9-GM3) ࡀ CD59 ࢡࣛࢫࢱ࣮࡜┦஫స} ⏝ࡍࡿᵝᏊࢆึࡵ࡚ᤊ࠼ࠊ᭷ព࡞ඹᒁᅾ᫬㛫 (98 ms) ࢆ☜ㄆࡋࡓ (Figure 18)ࠋᚋ࡟グࡍࡀࠊ ᑐ↷ⓗ࡟ࠊ㠀ࣛࣇࢺ࣐࣮࣮࢝ᛶࡢATTO594 ᶆ㆑ DOPE ࡣ 50 ms ⛬ᗘࡢࡳ CD59 ࢡࣛࢫࢱ࣮ ࡜ඹᒁᅾࢆ♧ࡋࡓࠋኳ↛ࡢ࢞ࣥࢢࣜ࢜ࢩࢻࡣ GPI ࢔࣮ࣥ࢝ᆺཷᐜయ࡜ᙉࡃ┦஫స⏝ࡍࡿࡇ ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࡓࡵࠊࡇࡢ▱ぢ࡜ྜ⮴ࡍࡿほᐹ⤖ᯝࢆᚓࡓࠋࡇࡇ࡛ࡣࣜ࢞ࣥࢻ่⃭࡟ࡼࡾ ㄏ㉳ࡉࢀࡿ CD59 ࢡࣛࢫࢱ࣮ࡢࣔࢹࣝ࡟ᑐࡍࡿぶ࿴ᛶࢆㄪ࡭ࡓࡀࠊᐃᖖ≧ែ࡛Ꮡᅾࡍࡿࠊ CD59 ༢ศᏊࡲࡓࡣ஧㔞య࡟ᑐࡍࡿ࢞ࣥࢢࣜ࢜ࢩࢻࡢぶ࿴ᛶ࡟ࡘ࠸࡚ࡶࡉࡽ࡟᳨ドࡋࡓࠋࡑ

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ࡢ⤖ᯝ࡟ࡘ࠸࡚ࡣᚋ࡟㏙࡭ࡿࠋ

Figure 18. GPI ࢔࣮ࣥ࢝ᆺཷᐜయࣉ࣮ࣟࣈ࡜ࡢ୍ศᏊほᐹᐇ㦂⤖ᯝ

➨භ❶ ࢩ࢔ࣝ㓟

9 ఩ᶆ㆑ GM1 ཬࡧ GM2 ࣉ࣮ࣟࣈࡢྜᡂ

➨୍⠇ ྜᡂᡓ␎

⺯ගGM3 ࣉ࣮ࣟࣈࡢ⤖ᯝࢆ㋃ࡲ࠼࡚ࠊ⬡㉁ࣛࣇࢺ࡟Ꮡᅾࡍࡿ௚ࡢ a ⣔ิ࢞ࣥࢢࣜ࢜ࢩ (GM1 ཬࡧ GM2) ࡢྜᡂ࡟╔ᡭࡋࡓࠋ๓❶ࡢ⺯ග GM3 ࣉ࣮ࣟࣈࡢᶵ⬟ホ౯ࡼࡾࠊ⺯ගⰍ⣲ ࡢᑟධ఩⨨ࡀ࢞ࣥࢢࣜ࢜ࢩࢻࡢ≀⌮໬Ꮫⓗᛶ㉁࡟኱ࡁࡃᙳ㡪ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡾࠊ⺯ ගⰍ⣲ࡢᙳ㡪ࢆ᭱ᑠ㝈࡟ࡍࡿࡓࡵ࡟ࡣࠊᶆ㆑఩⨨ࢆ Neu9 ఩࡟タᐃࡍࡿࡢࡀዲࡲࡋ࠸࡜࠸ ࠺♧၀ࢆᚓࡓࠋࡑࡇ࡛ࠊGM1 ཬࡧ GM2 ࡟࠾࠸࡚ࡶࠊᶆ㆑఩⨨ࢆ Neu9 ఩࡜ࡋࡓ⺯ගࣉࣟ ࣮ࣈࢆ᭱ึࡢೃ⿵࡜ࡋࠊGM3 ࡜ྠᵝ࡟ᶵ⬟ࢆ᳨ドࡍࡿࡇ࡜࡜ࡋࡓࠋࡲࡓࠊGM1 ཬࡧ GM2 ࡣศᒱᵓ㐀ࢆ᭷ࡋࠊ௚᪉ࡢᮎ➃⢾ṧᇶࡶᶆ㆑఩⨨ࡢೃ⿵࡟ᡂࡾᚓࡿࠋࡇࢀࡽࡢ⺯ගࣉ࣮ࣟ

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ࣈࡢྜᡂ⤖ᯝ࡟ࡘ࠸࡚ࡣᚋ㏙ࡍࡿࠋ

Neu9 ఩ᶆ㆑ GM2 ཬࡧ GM1 ࣉ࣮ࣟࣈࡢ๓㥑యࢆ Figure 19 ࡢࡼ࠺࡟タィࡋࡓࠋ

GM1 ཬࡧ GM2 ࡢ⺯ගࣉ࣮ࣟࣈࡢྜᡂ࡟࠾࠸࡚ࡶ Neu-Gal ࣘࢽࢵࢺࡢタィ࣭ྜᡂࡀ᭱ࡶ 㔜せ࡛࠶ࡿࠋ➨஧❶࡛㏙࡭ࡓ㏻ࡾࠊNeu-Gal ࡢ Gal4 ఩Ỉ㓟ᇶ࡬╔⬺ྍ⬟࡞ಖㆤᇶࢆᑟධࡍ ࡿ࢔ࣉ࣮ࣟࢳࡣ㐩ᡂ࡟ࡣ⮳ࡽ࡞࠿ࡗࡓ (⤒㊰ A)ࠋ୍᪉࡛ࠊ⤒㊰ B ࡢ GM3 ྜᡂ⤒㊰ࢆ㐙⾜ࡍ

ࡿ㐣⛬࡛᭷⏝࡞ࣘࢽࢵࢺࢆᚓࡿࡇ࡜ࡀ࡛ࡁࡓࠋࡍ࡞ࢃࡕࠊScheme 20 ࡟♧ࡍ Neu9 ఩ TFAc

࢔࣑ࢻㄏᑟయ33 ࡢ㐟㞳ࡢỈ㓟ᇶࢆ㐺ษ࡟ಖㆤࡍࡿࡇ࡜࡛ࠊGal4 ఩Ỉ㓟ᇶࡀ㐟㞳ࡢཷᐜయ࡬