14ࢸ࣮࣐ 2.

࣍ࢱࣝࡢගࢆసࡗ࡚ࡳࡼ࠺(1)࣍ࢱࣝ⏕≀Ⓨගᇶ㉁ࡢྜᡂ࡜ᶵჾศᯒ
1ࡣࡌࡵ࡟ a, b)࣍ࢱࣝ࡟௦⾲ࡉࢀࡿගࡿ⏕≀࡟ࡣᵝࠎ࡞ࡶࡢࡀ▱ࡽࢀ࡚࠸ࡿࠋࣂࢡࢸࣜ࢔ࠊ࢟ࣀࢥ㢮ࠊ࡯࡜ࢇ࡝ࡢ↓⬨᳝ື≀㛛ࡸ◳㦵㨶㢮࡞࡝ࠊᗈ⠊࡞⏕≀⩌࡛Ⓨගࡍࡿࡶࡢࡀぢฟࡉࢀࡿࠋ
ࡑࡢᩘࡣ 1000 ✀௨ୖ࡟ࡢࡰࡿࡀࠊⓎග⏥⹸ࡸ࢟ࣀࢥ㢮ࢆ㝖ࡅࡤ࡯࡜ࢇ࡝ࡣᾏ᳇࡛࠶ࡿ㸦Ⓨගࡍࡿ᳜≀ࡣ࠸ࡲࡢ࡜ࡇࢁぢฟࡉࢀ࡚࠸࡞࠸㸧ࠋගࡾ᪉ࡶከᙬ࡛࠶ࡿࠋ࣍ࢱࣝ㸦෗┿ 1㸧ࡣ᫂⁛ࡋࠊ࢜࣡ࣥࢡࣛࢤ㸦෗┿ 2㸧ࠊ࣍ࢱࣝ࢖࢝ࠊኪග⹸࡞࡝ࡣᶵᲔⓗ࡟่⃭ࡍࡿ࡜ගࡾࠊࣖࢥ࢘ࢱࢣ㸦෗┿ 5㸧ࡣ᫨ኪගࡾ⥆ࡅࡿࠋ࠸ࡎࢀࡶయෆ࡟࠶ࡿⓎගჾᐁ࡛ࡢⓎග࡛࠶ࡿࡀࠊ࣑࢘࣍ࢱࣝ㸦෗┿ 3㸧ࡸⓎගࢦ࢝࢖㸦෗┿ 4㸧ࡣⓎගᾮࢆయእ࡟ᨺฟࡋ࡚ගࡿࠋ

෗┿ 1 ࣊࢖ࢣ࣎ࢱࣝ

෗┿ 2 ࢜࣡ࣥࢡࣛࢤ

෗┿ 4 Odontosyllis ᒓࡢⓎගࢦ࢝࢖෗┿ 3 ࣑࢘࣍ࢱࣝ෗┿ 5 ࣖࢥ࢘ࢱࢣ

⏕≀Ⓨගࡢ㐍໬ⓗ㉳※ࡣᆅ⌫኱Ẽ⎔ቃࡀ᎘Ẽⓗ࠿ࡽዲẼⓗ࡟ኚ໬ࡋࡓ㝿ࡢ㓟⣲ẘᛶ࡟ᑐࡍࡿ㜵ᚚࢩࢫࢸ࣒࡛࠶ࡗࡓ࡜ゝࢃࢀࡿࡀࠊᐇࡣࡼࡃゎࡗ࡚࠸࡞࠸ࠋ㐍໬ࡢ㐣⛬࡛㓟⣲㓟໬࡟࡜ࡶ࡞࠸ຠ⋡ࡼࡃගࡀసࡾฟࡉࢀࡿࢩࢫࢸ࣒ࡀసࡾฟࡉࢀࠊᵝࠎ࡞┠ⓗ࡟✚ᴟⓗ࡟฼⏝ࡍࡿࡼ࠺࡟࡞ࡗࡓ࡜⪃࠼ࡽࢀࡿࠋ࣍ࢱࣝࡢ௰㛫ࠊ࣍ࢱࣝ࢖࢝ࠊⓎගࢦ࢝࢖ࡣ㞤㞝㛫ࡢྜᅗ࡟ࠊ࣑࢘࣍ࢱࣝࡸⓎග࢖࢝ࡣᩛ࡬ࡢጾᄐ࣭ᩛ࠿ࡽࡢ㏨ஸ࡟ࠊࢳࣙ࢘ࢳࣥ࢔ࣥࢥ࢘ࡸ࢔ࣛ࢟ࣀ࢝ࣥࣃ㸦⺅ࡢ௰㛫㸧ࡢᗂ⹸࡛ࡣᤕ㣗ື≀ࡢㄏᅉ࡞࡝࡟฼⏝ࡋ࡚࠸ࡿࠋࡲࡓ࣑ࢵࢻࢩࢵࣉ࣐ࣥ࡜࠸࠺㨶ࡣ⭡ഃࢆගࡽࡏࡿࡇ࡜࡟ࡼࡾ⫼ᬒගࡢ୰࡟⮬ศࡢጼࢆ㞃ࡋࠊୗ࠿ࡽࡸࡗ࡚ࡃࡿᤕ㣗⪅࡟ࡳࡘ࠿ࡾ࡟ࡃࡃࡋ࡚࠸ࡿ㸦࢝࢘ࣥࢱ࣮࢖࣑ࣝࢿ࣮ࢩࣙࣥ㸧ࠋ⏕≀Ⓨග࡜ࡣ㓝⣲࣭ࢱࣥࣃࢡ㉁ࡀ㛵୚ࡍࡿࠕ໬ᏛⓎග㸸≀㉁ࡢ㓟໬࡟కࡗ࡚ගࡾࡀᨺฟࡉࢀࡿ⌧㇟࡛ࠖ࠶ࡿࠋࡍ࡞ࢃࡕⓎගᇶ㉁ࣝࢩࣇ࢙ࣜࣥࡀⓎග㓝⣲ࣝࢩࣇ࢙࣮ࣛࢮࡢാࡁ࡛㓟⣲㓟໬ࡉࢀ࡚୙Ᏻᐃ㸦㧗࢚ࢿࣝࢠ࣮≧ែ㸧࡞㐣㓟໬≀࡜࡞ࡾࠊࡘ࠸࡛ࡇࡢࡶࡢࡀศゎࡍࠋࡇࡿ㐣⛬࡛㟁Ꮚⓗບ㉳㸯㔜㡯㸦S 1 㸧≧ែࡢศᏊ㸦࢜࢟ࢩࣝࢩࣇ࢙ࣜࣥ㸧ࢆ⏕ࡌࡿ㸦ᅗ 1㸧

15ࡢࡶࡢࡀᇶᗏ㸦S 0 㸧≧ែ࡟ᡠࡿ᫬࡟㐣๫࡞࢚ࢿࣝࢠ࣮ࡀග࡜ࡋ࡚ᨺฟࡉࢀࡿࠋࣝࢩࣇ࢙ࣜࣥࡸࣝࢩࣇ࢙࣮ࣛࢮࡣ⏕≀✀࡟ࡼࡾ␗࡞ࡿࠋᅗ 2 ࡟௦⾲ⓗ࡞ࣝࢩࣇ࢙ࣜࣥࡢᵓ㐀ࢆ♧ࡍࠋ

ᅗ 1 ⏕≀Ⓨග཯ᛂ࡜࢚ࢿࣝࢠ࣮ᴫᛕᅗ
HOONHONNSS

H CO H
2NH
NH

࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧OHN

NH
N

N
H

NH2NHNHO࣑࢘࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ

Ⓨගࢡࣛࢤࡢࣝࢩࣇ࢙ࣜࣥ
㸦ࢭࣞࣥࢸࣛࢪࣥ㸧ᅗ 2 ྛ✀Ⓨග⏕≀ࡢࣝࢩࣇ࢙ࣜࣥࡢ໬Ꮫᵓ㐀࣍ࢱࣝࡢ⏕≀Ⓨගࢩࢫࢸ࣒ࡣࣂ࢖࢜࢖࣓࣮ࢪࣥࢢࢆࡣࡌࡵ࡜ࡋ࡚⏕࿨⛉Ꮫࡸ⎔ቃ⛉Ꮫࠊ⮫ᗋ࣭⾨⏕᳨ᰝ࡞࡝ࠊᵝࠎ࡞ศ㔝࡛ᖜᗈࡃ฼⏝ࡉࢀ࡚࠸ࡿࠋᮏయ㦂ᐇ㦂࡛ࡣ࣍ࢱࣝࡢ⏕≀Ⓨගᇶ㉁࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡢྜᡂᐇ㦂ࢆ⾜࡞࠸ࠊࠕ᭷ᶵྜᡂࠖ࡜ᵓ㐀Ỵᐃࡢ⌧ሙࢆయឤࡍࡿࠋᮏᐇ㦂࡛ྜᡂࡋࡓ࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࢆ⏝࠸࡚࣍ࢱࣝࡢගࢆసࡿ㸦⏕≀Ⓨගࡉࡏࡿ㸧ᐇ㦂ࢆ⾜࠺ࠋ2య㦂ᐇ㦂㸸࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡢ᭷ᶵྜᡂࠕ᭷ᶵྜᡂࠖ࡜ࡣḧࡋ࠸᭷ᶵ໬ྜ≀㸦Ⅳ⣲໬ྜ≀㸧ࢆᏳ౯࡛⡆༢࡟ධᡭࡋᚓࡿࡶࡢ࠿ࡽ໬Ꮫ཯ᛂࢆ⤌ࡳྜࢃࡏ࡚సࡾୖࡆࡿࠊ᭷ᶵ໬Ꮫࡢ୍኱ศ㔝࡛࠶ࡿࠋ⌧௦ࡢ᭷ᶵྜᡂࡢຊ㔞ࡣྜᡂ࡛ࡁ࡞࠸ࡶࡢࡣ↓࠸࡜࠸ࡗ࡚ࡶ㐣ゝ࡛ࡣ࡞࠸ࠋ་⸆࣭㎰⸆ࠊᶵ⬟ᛶ≀㉁࣭ᮦᩱࡢ㛤Ⓨࡸ⏕⏘࡛ࡣࡶࡣࡸࠕ᭷ᶵྜᡂࠖࡣḞ࠿ࡏ࡞࠸ࡶࡢ࡜࡞ࡗ࡚࠸ࡿࠋࠕ᭷ᶵྜᡂࠖࡢ㸲ᮏᰕࡣ16ձ Ⅳ⣲̺Ⅳ⣲⤖ྜࡢᙧᡂղ ⎔ᵓ㐀ࡢᵓ⠏ճ ᐁ⬟ᇶ㸦≀⌮࣭໬Ꮫⓗᛶ㉁ࢆỴࡵࡿࠊ≉ᚩⓗ࡞ཎᏊ࣭ཎᏊᅋࡢࡇ࡜㸧ࡢᑟධ࣭┦஫ኚ᥮մ ❧య໬Ꮫ(ᵓᡂཎᏊࡢ✵㛫㓄⨨)ࡢไᚚ࡛࠶ࡿࠋᶆⓗ໬ྜ≀ࡢྜᡂࡣ୍⯡࡟ࡣከẁ㝵ࢆせࡍࡿࠋࡑࡢ㝿ձ—մࡢࢫࢸࢵࣉࡣࠊಶู࡟ࠊ࠶ࡿ࠸ࡣྠ᫬࡟ࠊࡑࡋ࡚⧞ࡾ㏉ࡋ⾜࡞ࢃࢀࡿࠋᐇ㝿ࡢྜᡂ࡛ࡣᶆⓗ໬ྜ≀ࡢᵓ㐀ࢆ຺᱌ࡋ࡚ࠊ㐺ษ࡞ฟⓎཎᩱ࡜⤒㊰ࢆ⪃᱌ࡍࡿࠋᅗ 3 ࡟࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ(1)ࡢ᭱᪂ࡢྜᡂ⤒㊰ࢆ♧ࡍࠋc) ࡇࡢ᪉ἲ࡛ࡣ 1 ࡢ㸴ဨ⎔㸦࣋ࣥࢮࣥ⎔㸧ᵓ㐀࡟╔┠ࡋࠊp-࢔ࢽࢩࢪࣥࢆฟⓎཎᩱ࡟㑅ࢇ࡛࠸ࡿࠋࡲࡎ࣋ࣥࢮࣥ⎔㒊࡟ࢳ࢔ࢰ࣮ࣝ⎔㸦Sࠊ N ࢆྵࢇࡔ㸳ဨ⎔ᵓ㐀㸧ࡀ⦰⎔ࡋࡓ࣋ࣥࢰࢳ࢔ࢰ࣮ࣝ⎔ᵓ㐀㸦Ⅼ⥺࡛ᅖࡗࡓ㒊ศ㸧ࢆ㸰ẁ㝵࡛ᵓ⠏ࡍࡿࠋ᭱ᚋ࡟ࠊCHBT ࡢࢽࢺࣜࣝᇶ㸦CӇN㸧࡜ D-ࢩࢫࢸ࢖ࣥ࡜ࢆ⦰ྜࡉࡏࠊ୙ᩧⅣ⣲㸦‫ە‬༳Ⅳ⣲㸧ࡢ❧య໬Ꮫࢆไᚚ㸦ಖᣢ㸧ࡋࡘࡘࠊࡶ࠺୍ࡘࡢ㸳ဨ⎔㒊ࢆᵓ⠏ࡋࠊ1 ࡢྜᡂࢆ㐩ᡂࡋ࡚࠸ࡿࠋᮏయ㦂ᤵᴗ࡛ࡣ᭱ᚋࡢẁ㝵㸦㉥࠸ᅄゅ࡛ᅖࡗࡓ཯ᛂ㸧ࢆ⮬ศ࡛⾜࡞ࡗ࡚ࡳࡿࠋ᧯స⮬యࡣ⡆༢࡛࠶ࡿࡀࠊ᭷ᶵྜᡂࡢ㸱ᮏᰕࡀ㸯ẁ㝵࡛㐩ᡂࡉࢀࡿࠊ࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ྜᡂࡢぢ஦࡞᭱⤊ࢫࢸࢵࣉ࡛࠶ࡿࠋຍ⇕ճղ࣋ࣥࢰࢳ࢔ࢰ࣮ࣝ⎔p-࢔ࢽࢩࢪࣥճղճմ࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ (1)D-ࢩࢫࢸ࢖ࣥ

ᅗ 3 ࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡢྜᡂ⤒㊰ c)

2. 1 ౑⏝ࡍࡿヨ⸆
࣭CHBT㸦2-cyano-6-hydroxybenzothiazole㸧㸦ศᏊ㔞 176.2㸧44.0 mg (0.250 mmol)
࣭D-ࢩࢫࢸ࢖ࣥሷ㓟ሷ୍Ỉ࿴≀㸦ศᏊ㔞 175.6㸧44.4 mg (0.253 mmol)
㸦ศᏊ㔞 138.2㸧35.0 mg (0.253 mmol)࣭↓ỈⅣ㓟࣒࢝ࣜ࢘㸦K 2 CO 3 㸧
࣭1 M ሷ㓟 1.0 mL (1.0 mmol)࣭࣓ࢱࣀ࣮ࣝ

30 mL㸦౑⏝┤๓࡟⬺Ẽࡋ࡚࠾ࡃ㸧࣭㓑㓟࢚ࢳࣝ 10 mL࣭⵨␃Ỉ

30 mL㸦౑⏝┤๓࡟⬺Ẽࡋ࡚࠾ࡃ㸧࣭୓⬟ pH ヨ㦂⣬

17

2. 2 ౑⏝ࡍࡿჾල
࣭$15/25 ඹ㏻᧿ࢼࢫࣇࣛࢫࢥ㸹20 mL 2 ಶ
࣭$15/25 ඹ㏻᧿୕᪉ࢥࢵࢡ 㸯ಶ࣭$15/25 ඹ㏻᧿ῶᅽ⏝㐃⤖⟶ 㸯ಶ࣭᱒ᒣ࣮ࣟࢺ V-8 (I 8 mm) or S-21 (I 21 mm) 㸯ಶ࣭ࢩࣜࢥ࣮ࣥࢦ࣒〇࣮ࣟࢺ࢔ࢲࣉࢱ࣮㸦ῶᅽ⏝㐃⤖⟶࡬ࡢ᱒ᒣ࣮ࣟࢺ⿦╔⏝㸧࣭ࢁ⣬㸦V-8 ⏝I 8 mm or S-21 ⏝I 21 mm㸧㸰ࠥ㸱ᯛ࣭࣐ࢢࢿࢳࢵࢡࢫࢱ࣮࣮ࣛ
࣭☢Ẽ᧠ᢾᏊ

1ྎ

1ಶ࣭࢔ࢫࣆ࣮ࣞࢱ࣮(Ỉὶ࣏ࣥࣉ) 1 ྎ࣭୕ゅࣇࣛࢫࢥ㸦30ࠥ50 mL㸧࣭ࣃࢫࢶ࣮ࣝࣆ࣌ࢵࢺ

2ಶ

5 ᮏ ࡲࡓࡣ㥖㎸ࣆ࣌ࢵࢺ㸦2 mL㸧 5 ᮏ࣭ࣆ࣌ࢵࢺ⏝ࢦ࣒࢟ࣕࢵࣉ5ಶ࣭ⷧᒙࢡ࣐ࣟࢺࢢࣛࣇ⏝ ᒎ㛤ᒙ㸦ĳFP[FP ⛬ᗘࡢ⛗㔞ࡧࢇ࡛௦⏝ྍ㸧 㸯ಶ࣭ࢫࣃ࣮ࢸࣝࠊࢧࣥࣉࣝ⟶㸦ࠥ2 mLࠊヨ⸆⛗㔞⏝㸧ࠊ⸆ໟ⣬ࠊࢦ࣒㢼⯪

2. 3 ᐇ㦂᧯స
ձ 20 mL ࢼࢫࣇࣛࢫࢥࢆᐇ㦂ྎ࡟ࢡࣛࣥࣉ࡛ᅛᐃࡋࠊࣇࣛࢫࢥෆ࡟☢Ẽ᧠ᢾᏊࢆධ
ࢀࠊ࢔ࣝࢦࣥ㢼⯪ࢆ⿦╔ࡋࡓ୕᪉ࢥࢵࢡࢆྲྀࡾ௜ࡅࡿࠋࣇࣛࢫࢥࡢୗ࡟ࡣ࣐ࢢࢿ
ࢳࢵࢡࢫࢱ࣮࣮ࣛࢆ⨨ࡃ(ᅗ 4)ࠋ
ղ CHBT 44.0 mg (0.250 mmol)ࠊD-ࢩࢫࢸ࢖ࣥሷ㓟ሷ୍Ỉ࿴≀ 44.4 mg (0.253 mmol)ࠊ
↓ỈⅣ㓟࣒࢝ࣜ࢘ 35.0 mg (0.253 mmol)ࢆࡑࢀࡒࢀࢧࣥࣉࣝ⟶࡟㔞ࡾྲྀࡿ㸦ᅗ 5㸧ࠋճ 㔞ࡾ࡜ࡗࡓ CHBT ࢆ⬺Ẽ࣓ࢱࣀ࣮ࣝ㸯mLࠊD-ࢩࢫࢸ࢖ࣥሷ㓟ሷ୍Ỉ࿴≀ࢆ⬺Ẽ⵨␃Ỉ 0.25 mLࠊ↓ỈⅣ㓟࣒࢝ࣜ࢘ࢆ⬺Ẽࡋࡓ⵨␃Ỉ 0.25 mL ࡟⁐ゎࡍࡿࠋմ CHBT ࡢ࣓ࢱࣀ࣮ࣝ⁐ᾮ࡜ D-ࢩࢫࢸ࢖ࣥሷ㓟ሷỈ⁐ᾮࢆࠊࣃࢫࢶ࣮ࣝࣆ࣌ࢵࢺࢆ⏝࠸࡚ࣇࣛࢫࢥෆ࡟ὀධࡋ㸦ᅗ 6㸧ࠊ࣐ࢢࢿࢳࢵࢡࢫࢱ࣮࣮࡛ࣛ᧠ᢾࡍࡿࠋյ ࡘ࠸࡛Ⅳ㓟࣒࢝ࣜ࢘Ỉ⁐ᾮࢆࠊࣃࢫࢶ࣮ࣝࣆ࣌ࢵࢺࢆ⏝࠸࡚ຍ࠼ࠊࣇࣛࢫࢥෆࢆ࢔ࣝࢦࣥ㞺ᅖẼ㸦࢔ࢫࣆ࣮ࣞࢱ࣮࡛ῶᅽࡋࠊ࢔ࣝࢦࣥ࢞ࢫ࡛࣮ࣜࢡࡍࡿ᧯సࢆ 3ᅇ࡯࡝⧞ࡾ㏉ࡍ㸧࡜ࡋࡓࡢࡕࠊ཯ᛂ⁐ᾮࢆᐊ࡟࡚᧠ᢾࡍࡿ㸦ᅗ 7㸧ࠋ30 ศᚋࠊ཯ᛂࡢ㐍⾜ලྜࢆⷧᒙࢡ࣐ࣟࢺࢢࣛࣇ࢕㸦TLC㸧࡛ࣔࢽࢱ࣮ࡍࡿ㸦ᚋ㏙㸧ࠋն ཯ᛂࡀ⤊஢ࡋࡓࡇ࡜ࢆ☜ㄆࡋࡓࡢࡕࠊ཯ᛂ⁐ᾮࢆị෭Ỉ 5 mL ࡛ᕼ㔘ࡋࠊࣇࣛࢫࢥ
ࡈ࡜ịᾎ࡛෭༷ࡍࡿࠋ
շ 1 M ሷ㓟 1.0 mL (1.0 mmol) ࢆຍ࠼࡚㸦ⓎἻὀព㸧㓟ᛶ㸦pH㸱௨ୗ㸧࡟ࡍࡿࠋ⤖ᬗࡀ⏕ࡌࡓࡇ࡜ࢆ☜ㄆࠋո ᪂ࡓ࡟ 20 mL ࢼࢫࣇࣛࢫࢥࢆࢡࣛࣥࣉ࡛ᅛᐃࡋࠊῶᅽ⏝㐃⤖⟶ࠊ᱒ᒣ࣮ࣟࢺࢆ⿦╔ࡋࠊ᱒ᒣ࣮ࣟࢺ࡟ࢁ⣬ࢆࢭࢵࢺࡍࡿ㸦ᅗ 8㸧ࠋࣇࣛࢫࢥෆᐜ≀ࢆ᱒ᒣ࣮ࣟࢺ࡟ὀ
ࡂ㎸ࡴ㸦ᅗ 9ࠊ㥖㎸ࣆ࣌ࢵࢺࢆ⏝࠸࡚⛣ࡋ࡚ࡶⰋ࠸㸧ࠋῶᅽ⏝㐃⤖⟶ࢆ࢔ࢫࣆ࣮ࣞ
ࢱ࣮࡟᥋⥆ࡋࠊ྾ᘬࡍࡿࠋࣇࣛࢫࢥෆ࡟ṧࡗࡓ⤖ᬗࡣࢁᾮࢆ౑ࡗ࡚Ὑ࠸ฟࡍࠋչ ࢁ⣬ୖ࡟ṧࡗࡓ⤖ᬗࢆᑡ㔞ࡢị෭Ỉ࡛Ὑ࠺ࠋպ ᚓࡽࢀࡓ⤖ᬗࢆࢧࣥࣉࣝ⎼࡟⛣ࡋ᭰࠼ࠊῶᅽ஝⇱ࡋࠊ㔜ࡉࢆ㔞ࡾࠊ

18཰⋡[(ᐇ㝿ࡢ཰㔞㸭⌮ㄽ཰㔞) × 100%]ࢆィ⟬ࡍࡿࠋ࡞࠾⌮ㄽ཰㔞ࡣ 70.1 mg ࡛࠶ࡿ [280.3㸦1 ࡢࣔࣝ㉁㔞㸧× 0.250 mmol (⏝࠸ࡓ CHBT ࡢ≀㉁㔞㸧= 70.1 mg]ࠋᅗ 4 ཯ᛂᐜჾ඲యᅗ

ᅗ 5 ヨ⸆ࡢ⛗㔞

ᅗ 6 ヨ⸆ࢆ⛣ࡋ᭰࠼ࡿᵝᏊᅗ 7 ཯ᛂ୰ࡢᵝᏊᅗ 8 ྾ᘬࢁ㐣⿦⨨ᅗ 9 ྾ᘬࢁ㐣ࡢᵝᏊ[ᐇ㦂᧯సࡢ⿵㊊]࣭ᐇ㦂ჾලࡣࡋࡗ࠿ࡾ࡜ᅛᐃࡉࢀ࡚࠸ࡿ࠿ࠊゐࡗ࡚☜࠿ࡵࡿࡇ࡜ࠋ࣭཯ᛂࡢ㐍⾜ࢆ TLC㸦ᚋ㏙㸧࡛ࢳ࢙ࢵࢡࡍࡿࡓࡵ࡛ CHBT ࢆࡈࡃᑡ㔞ṧࡋ࡚࠾ࡃࡇ࡜ࠋ࣭྾ᘬࢁ㐣⿦⨨ࡢ᱒ᒣ࣮ࣟࢺ࡟ࢁ⣬ࢆࢭࢵࢺࡍࡿ᫬ࡣࠊࡲࡎࢁ⣬ࢆ┠─㒊࡟⨨ࡁࠊỈ࡛⃿ࡽࡋࠊ࢔ࢫࣆ࣮ࣞࢱ࣮࡛྾ᘬࡋࠊࢁ⣬࡜࣮ࣟࢺ┠─㒊ࢆᐦ╔ࡉࡏࡿ஦ࠋ࣭྾ᘬࢁ㐣ࡢᡭ㡰ࡣࠕ࢔ࢫࣆ࣮ࣞࢱ࣮ࡢࢫ࢖ࢵࢳࢆධࢀࡿЍ࢔ࢫࣆ࣮ࣞࢱ࣮࡜㐃⤖⟶ࢆ⧅ࡄࠖࡢ㡰࡛⾜࡞࠺ࡇ࡜ࠋ྾ᘬࢆṆࡵࡿ࡜ࡁࡣࡑࡢ㏫࡛ࠕ㐃⤖⟶࠿ࡽ࢔ࢫࣆ࣮ࣞࢱ࣮ࢆࡣࡎࡍЍ࢔ࢫࣆ࣮ࣞࢱ࣮ࡢࢫ࢖ࢵࢳࢆṆࡵࡿࠖࠋእࡉࡎ࡟࢔ࢫࣆ࣮ࣞࢱ࣮ࡢࢫ࢖ࢵࢳࢆษࡿ࡜Ỉࡀ࢔ࢫࣆ࣮ࣞࢱ࣮࠿ࡽ㏫ὶࡍࡿࠋ

2. 4 ⷧᒙࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮㸦TLC㸧ᅛᐃ┦࡜≀㉁࡜ࡢ྾╔࡞࡝ࡢ┦஫స⏝ࡢ㐪࠸ࢆ฼⏝ࡋ࡚ΰྜ≀ࢆศ㞳ࡍࡿᡭἲࢆࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡜࠸࠺ࠋⷧᒙࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮㸦TLC㸧ࡣࢩࣜ࢝ࢤࣝ࡞࡝ࡢᚤ⢏Ꮚ≧ࡢᅛᐃ┦ࢆⷧ⭷㸦㏻ᖖ 0.25㹫㹫㸧࡟ࡋࡓࣉ࣮ࣞࢺࢆ⏝࠸ࡿࠋ཯ᛂࡢ㐍⾜ලྜࡸ≀㉁ࡢ⣧ᗘ࡞࡝ࢆࠊ▷᫬㛫࡟⡆౽࡟᳨ᐃ࡛ࡁࡿᴟࡵ࡚౽฼࡞ศᯒἲࠋ⛣ື┦㸦ᒎ㛤⁐፹㸧ࡣ᭷ᶵ⁐፹ࠊ᭷ᶵ⁐፹-Ỉΰྜ⁐፹ࠊỈ࡞࡝ࢆ⏝࠸ࡿࠋ19໬ྜ≀ࡀྠࡌ࡛࠶ࢀࡤࠊྠ୍᮲௳ୗ࡛ࡣྠࡌᒎ㛤ᣲືࢆࡋࡵࡍࠋ໬ྜ≀ࡢᒎ㛤㊥㞳࡜⁐፹ࡢඛ➃ࡲ࡛ࡢ㊥㞳ࡢẚࡣ୍ᐃ࡜࡞ࡿࠋࡇࡢẚࢆ Rf ್࡜࠸࠸ࠊRf ್ࡀ኱ࡁࡅࢀࡤࡑࡢ⁐፹࡛ࡣ࠶ࡲࡾ྾╔ࡉࢀࡎࡼࡃᒎ㛤ࡉࢀࡿࡇ࡜ࢆ♧ࡍ(ᅗ 10)ࠋRf ್ࡣ 0.1㹼0.8 ࡢ㛫࡟࡞ࡿࡼ࠺࡟⁐፹ࢆㄪᩚࡍࡿࠋ(Rf㸻a/b)[TLC ࡢ᧯సἲ]ձ ཎᩱ CHBT ࢆࡈࡃᑡ㔞ࢧࣥࣉࣝ⟶࡟࡜ࡾࠊ0.1 mL ⛬ᗘࡢ㓑㓟࢚ࢳࣝ࡟⁐ゎࡍࡿࠋ
ղ 2 cm×5 cm ⛬ᗘ࡟ษࡾฟࡋࡓ TLC ⏝ࢩࣜ࢝ࢤࣝࣉ࣮ࣞࢺࢆ⏝ពࡍࡿࠋཎᩱࠊ཯ᛂ⁐ᾮࠊ࠾ࡼࡧࡑࡢΰྜ≀ࢆࠊࣉ࣮ࣞࢺࡢୗ࠿ࡽ⣙ 1 cm ࡢ఩⨨࡟ẟ⣽⟶࡛ࢫ࣏ࢵࢺࡍࡿࠋճ ᒎ㛤ᒙ࡟㓑㓟࢚ࢳࣝࢆ῝ࡉ 0.5 cm ⛬ᗘධࢀࠊ⵹ࢆࡋࠊᩘศ㛫ᨺ⨨ࡍࡿࠋմ ࢫ࣏ࢵࢺࡋࡓⅬࢆୗ࡟ࡋ࡚ࣉ࣮ࣞࢺࢆᒎ㛤ᒙ࡟ࡑࡗ࡜⨨ࡁࠊ⵹ࢆࡋࠊᒎ㛤ࡍࡿࠋյ ⁐፹ࡢඛ➃ࡀୗ࠿ࡽ 4.5 cm ⛬ᗘ࡟㐩ࡍࡿࡲ࡛ᒎ㛤ࡍࡿࠋն UV ࣛࣥࣉ࡛ࢫ࣏ࢵࢺࡢ఩⨨ࢆ☜ㄆࡍࡿࠋ࠶ࡿ࠸ࡣ㐺ᙜ࡞ⓎⰍ๣࡛ⓎⰍࡉࡏࡿࠋ‫ە‬ᒎ㛤๓‫ە‬ᒎ㛤୰

‫ە‬ᒎ㛤ᚋ

ᅗ 10 ⷧᒙࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮ࡢᵝᏊ

3࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡢᵓ㐀Ỵᐃ㸸ᶵჾศᯒ d)᭷ᶵྜᡂࠊ཯ᛂ㛤Ⓨࠊ⏕యᡂศࡢᢳฟ࣭༢㞳◊✲࡞࡝ࠊ࠸࠿࡞ࡿሙྜ࡛ࡶᡭ࡟ࡋࡓ໬ྜ≀ࡀ┠ⓗ≀࠿࡝࠺࠿ࠊᵓ㐀☜ㄆ࣭Ỵᐃࡍࡿᚲせࡀ࠶ࡿࠋࡑࡢࡓࡵ࡟ྛ✀ࡢᶵჾศᯒࢆ⾜࡞࠺ࠋᶵჾศᯒἲ࡜ࡋ࡚ࡣୗグࡢ᪉ἲࡀ࠶ࡿࠋձ᰾☢Ẽඹ㬆ศගἲ㸦NMR㸧㸸Ⅳ⣲㦵᱁ࡸᐁ⬟ᇶ㸦≀⌮࣭໬Ꮫⓗᛶ㉁ࢆỴࡵࡿࠊ≉ᚩⓗ࡞ཎᏊ࣭ཎᏊᅋ㸧ࡢ୪ࡧ᪉ࢆỴᐃ࡛ࡁࡿࠋղ㉥እศගἲ㸸≉ᐃࡢᐁ⬟ᇶࡢ᭷↓ࡀゎࡿࠋճ㉁㔞ศᯒἲ㸸ศᏊ㔞ࡸศᏊᘧ㸦ཎᏊࡢ⤌ᡂ㸧ࡀỴᐃ࡛ࡁࡿࠋ࠶ࡿ⛬ᗘࠊᵓ㐀᝟ሗ㸦≉ᐃࡢᐁ⬟ᇶࡢ᭷↓ࠊⅣ⣲㦵᱁᝟ሗ㸧➼ࡀᚓࡽࢀࡿࠋմ⣸እ࣭ྍどศගἲ㸸࡝ࡢࡼ࠺࡞ȧ㟁Ꮚ⣔ࢆᣢࡘ࠿Ỵᐃ࡛ࡁࡿࠋյX ⥺ᵓ㐀ゎᯒ㸸ᵓᡂཎᏊࡢ 3 ḟඖⓗ㓄ิ㸦㸻ศᏊᵓ㐀㸧ࡀ┤᥋ồࡲࡿࠋ୍⯡࡟ࡣ༢⤖ᬗࡀᚲせ㸦᭱㏆㸪ᑡ㔞࣭㠀ᬗ㉁࡛༢⤖ᬗࡀᚓࡽࢀ࡞࠸ࡶࡢ࡛ࡶྍ⬟࡜࡞ࡗࡓ㸧ࠋ௒ᅇࠊྜᡂࡋࡓ࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧࡟ࡘ࠸࡚ࠊNMR ࢫ࣌ࢡࢺࣝ࡜㉥እ྾཰ࢫ࣌ࢡ
ࢺࣝࠊ࣐ࢫࢫ࣌ࢡࢺࣝࢆ ᐃࡋࠊࢫ࣌ࢡࢺࣝゎᯒࢆ⾜࡞࠸ࠊᵓ㐀ࡀṇࡋ࠸࠿᳨ウࡍࡿࠋ

20

3. 1 ᰾☢Ẽඹ㬆ศගἲ㸦NMR㸧᭷ᶵ໬ྜ≀ࡢ᭱ࡶ㔜せ࡞ᶵჾศᯒἲ࡛࠶ࡿࠋᚓࡽࢀࡿ᝟ሗࡶከ࠸ࠋ
᰾☢Ẽඹ㬆ศගἲ࡛ࡣ☢ሙࡢ୰࡟࠾࠿ࢀࡓ᭷ᶵศᏊ୰ࡢ 㸯H ᰾㸦ኳ↛Ꮡᅾẚ 99.9%㸧ࡸ 13C᰾㸦ኳ↛Ꮡᅾẚ 1.1%㸧ࡢືࡁࢆほ ࡍࡿࠋ㸯H ࡸ 13C ᰾ࡣ☢ሙࡢ୰࡛ࡣᑠࡉ࡞Წ☢▼ࡢࡼ࠺࡟ࡩࡿࡲ࠺ࠋศᏊ࡟☢ሙࢆ࠿ࡅࡿ࡜㸪ศᏊࢆᵓᡂࡍࡿ 㸯H ࡸ 13C ᰾Წ☢▼ࡣࢥ࣐ࡢᵝ࡟ࡳࡑࡍࡾ㐠ື㸦ṓᕪ㐠ື㸧ࡍࡿࠋࡳࡑࡍࡾ㐠ືࡢ࿘Ἴᩘ(͆ඹ㬆࿘Ἴᩘ͇࡜࠸࠺㸧ࡣࠊศᏊࡢ୰ࡢ 㸯H ࡸ 13C ᰾ࡢ࠾࠿ࢀࡓ⎔ቃ(ࡍ࡞ࢃࡕศᏊᵓ㐀)ࢆ཯ᫎࡋ࡚ᑡࡋࡎࡘ␗࡞ࡿࠋࡇࡢඹ㬆࿘Ἴᩘࡢ㐪࠸ࢆࠊ㟁☢ㄏᑟࢆ฼⏝ࡋࡓ㟁Ẽಙྕ࡜ࡋ᳨࡚ฟࡍࡿࠋᶓ㍈࡟ಶࠎࡢ 㸯H ࡸ 13C ᰾ࡢࡳࡑࡍࡾ㐠ືࡢඹ㬆࿘Ἴᩘࠊ⦪㍈࡟ಙྕᙉᗘ(ࢩࢢࢼࣝ)ࢆࣉࣟࢵࢺࡍࡿ࡜᰾☢Ẽඹ㬆ࢫ࣌ࢡࢺࣝࡀᚓࡽࢀࡿࠋࢩࢢࢼࣝࡢ఩⨨㸦ඹ㬆࿘Ἴᩘ㸧ࡣ⏝࠸ࡿ⿦⨨ࡢ☢ሙࡢᙉࡉ࡟ẚ౛ࡍࡿࠋࡑࡇ࡛ヨᩱ࡜ᇶ‽≀㉁㸦ࢸࢺ࣓ࣛࢳࣝࢩࣛࣥ TMS㸧ࡢࢩࢢࢼࣝࡢඹ㬆࿘Ἴᩘࡢᕪ㸦Hz ༢఩㸧ࢆࡑࡢ⿦⨨ࡀ♧ࡍᏙ❧ࡋࡓ 㸯H ࡸ 13C ᰾࡟ᑐࡍࡿඹ㬆࿘Ἴᩘ㸦MHz ༢఩㸧࡛๭ࡾࠊ106ಸࡋࡓ್ࠊį㸦ppm㸧࡛⾲グࡍࡿࠋࡇࢀࢆ໬Ꮫࢩࣇࢺ࡜࿧ࡪ㸦TMS ࡢࢣ࣑࢝ࣝࢩࣇࢺ್ࡣ į = 0 ࡜࡞ࡿ㸧ࠋࡇࢀ࡟ࡼࡾࠊᵓ㐀ࡀྠࡌ࡛࠶ࢀࡤ࡝ࡢ⿦⨨࡛ ᐃࡋ࡚ࡶࠊ໬Ꮫࢩࣇࢺࡣྠࡌ್ࢆᣢࡘࡇ࡜࡟࡞ࡿࠋ᭷ᶵ໬ྜ≀୰ࡢ 㸯H ࡸ 13C ᰾ࡢ࠾ࡼࡑࡢ໬Ꮫࢩࣇࢺ್ࢆ⾲ 1 ࡜⾲ 2 ࡟♧ࡍࠋᮏᐇ㦂࡛ࡣ᪥ᮏ㟁Ꮚᰴᘧ఍♫〇 ECA500 (1Hࠊ 500 MHz㸹13C, 125 MHz)ࢆ⏝࠸࡚ࠊ ᐃ⁐፹ DMSO-d 6࡛ࢫ࣌ࢡࢺࣝࢆ ᐃࡍࡿࠋᅗ 11 ࡟࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡢ 1H NMRࠊᅗ 13 ࡟ 13C NMRࢫ࣌ࢡࢺࣝࢆ♧ࡍࠋᵓ㐀Ỵᐃ࡛ࡣ㹆ࡸ㹁ࡢᩘࡸ໬Ꮫࢩࣇࢺ್࡜ࠊ㞄᥋㹆᰾࡜ࡢࢫࣆࣥʊࢫࣆࣥ࢝ࢵࣉࣜࣥࢢ㸦┦஫స⏝㸧࡟ࡼࡿࢩࢢࢼࣝࡢศ⿣ࡢ኱ࡁࡉ࣭ࣃࢱ࣮ࣥࢆヲ⣽࡟᳨ウࡋࠊH ࡸ C ࡢࡘ࡞ࡀࡾࢆồࡵࠊᵓ㐀ࢆᑟࡁฟࡍࠋᅗ 12 ࡟࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡢ࢝ࢵࣉࣜࣥࢢࣃࢱ࣮ࣥࢆ♧ࡍࠋࡓ࡜࠼ࡤ࣋ࣥࢮࣥ⎔ୖࡢỈ⣲ Hd ࡣ㞄ࡢỈ⣲ He ࡜ࡢࢫࣆࣥíࢫࣆࣥ࢝ࢵࣉࣜࣥࢢ࡟ࡼࡾ࢝ࢵࣉࣜࣥࢢᐃᩘ J d-e Hz ࡛ࠊ㸰㔜⥺࡟ศ⿣ࡍࡿࠋỈ⣲ Hj ࡣ He ࡜ J e-j Hz࡛࢝ࢵࣉࣜࣥࢢࡋࠊ㸰㔜⥺࡟ศ⿣ࡍࡿࠋỈ⣲ He ࡣ Hd (J d-e Hz)࠾ࡼࡧ Hj㸦J e-j Hz㸧࡜࢝ࢵࣉࣜࣥࢢࡋࠊ㸲㔜⥺࡟ศ⿣ࡍࡿࠋ13C NMR ࡢሙྜࡣ┤⤖ࡍࡿỈ⣲ࡢᩘ࡛ศ⿣ࣃࢱ࣮ࣥ㸦ከ㔜ᗘ㸧ࡀつ๎ⓗ࡟ኚ໬ࡍࡿࠋ┤⤖Ỉ⣲ࢆᣢࡓ࡞࠸Ⅳ⣲ࡣ㸯㔜⥺ࠊ1 ಶࡢሙྜࡣ㸰㔜⥺ࠊ㸰ಶࡢሙྜࡣ㸱㔜⥺ࠊ㸱ಶࡢሙྜࡣ㸲㔜⥺࡜࡞ࡿࠋ⾲ 1 ᭷ᶵ໬ྜ≀୰ࡢỈ⣲ࡢࢣ࣑࢝ࣝࢩࣇࢺࡢᴫ␎್
CHᐁ⬟ᇶ

CO2HCCCHXC໬ᏛࢩࣇࢺG ppm)

㹼10

7-9

5-7

C

3-5CC

H

X: O, N etc.

CH3CH2CCH
C

0.5-3

21

⾲ 2 ᭷ᶵ໬ྜ≀୰ࡢⅣ⣲ࡢࢣ࣑࢝ࣝࢩࣇࢺࡢᴫ␎್
Cᐁ⬟ᇶONC

CX

CCC

C CCCCCetc.

CCHCC

180-160

160-120150-100

100-3090-30

40-10G ppm)

ᅗ 11 ࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡢ 1H-NMR ࢫ࣌ࢡࢺࣝACC໬Ꮫ
ࢩࣇࢺ

CH2C

X=O, N,CO2HCH3B

ᅗ 12 ࢫࣆ࣮ࣥࢫࣆࣥ࢝ࢵࣉࣜࣥࢢ࡟ࡼࡿࢩࢢࢼࣝࡢศ⿣ࣃࢱ࣮ࣥ(1H NMR)C22ࢩࢢࢼࣝࡢከ㔜ᗘs: 㸯㔜⥺ (singlet)d: 㸰㔜⥺ (doublet)t: 㸱㔜⥺ (triplet)⁐፹(DMSO-d6)

ᅗ 13 ࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡢ 13C-NMR ࢫ࣌ࢡࢺࣝ
C1, C4, C5, C6, C9, C11 ࡣỈ⣲ཎᏊ࡜⤖ྜࡋ࡚࠸࡞࠸ࡓࡵ㸯㔜⥺(s)ࠊ
C2, C7, C8, C10 ࡣ 1 ಶࡢỈ⣲ཎᏊࡀ⤖ྜࡍࡿࡓࡵ㸰㔜⥺(d)ࠊ
C3 ࡣ㸰ಶࡢỈ⣲ཎᏊࡀ⤖ྜࡍࡿࡓࡵ㸱㔜⥺(t)࡜࡞ࡿࠋ

3. 2 ㉥እศගἲศᏊࡢඹ᭷⤖ྜࡣࡤࡡࡢࡼ࠺࡟ఙ⦰ࡸኚゅ᣺ືࡋ࡚࠸ࡿࠋࡇࡢࡼ࠺࡞ศᏊ࡟㉥እ⥺㡿ᇦἼᩘ 4000 cm-1㹼400 cm-1㸦Ἴ㛗 2.5 ȝP㹼25 ȝP㸧ࡢ㟁☢Ἴࢆ↷ᑕࡍࡿ࡜ࠊศᏊࡢ≉ᐃࡢᐁ⬟ᇶ(CO 2 H ᇶࠊOH ᇶ࡞࡝)ࡸศᏊᅛ᭷ࡢ᣺ືࢿࢿࣝࢠ࣮ࡀ྾཰ࡉࢀࡿࠋࡇࡢ྾཰ࡢᵝᏊ࠿ࡽ≉ᐃࡢᐁ⬟ᇶࡢᏑᅾ࡞࡝ࠊ᭷⏝࡞ᵓ㐀᝟ሗࡀᚓࡽࢀࡿࠋ⾲ 3 ࡟୺࡞⤖ྜࡢ྾཰㡿ᇦࢆ♧ࡍࠋᮏᐇ㦂࡛ࡣࢧ࣮ࣔࣇ࢕ࢵࢩ࣮ࣕࢧ࢖࢚ࣥࢸ࢕ࣇ࢕ࢵࢡᰴᘧ఍♫〇ࣇ࣮࢚ࣜኚ᥮㉥እศගගᗘィ(Nicolet6700 FTIR)ࢆ⏝࠸ࠊ඲཯ᑕἲ㸦ATR㸧࡛㉥እ྾཰ࢫ࣌ࢡࢺࣝࢆ ᐃࡍࡿࠋᅗ 14࡟࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡢ IR ࢫ࣌ࢡࢺࣝࢆ♧ࡍࠋ

ᅗ 14 ࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡢ IR ࢫ࣌ࢡࢺࣝ

23

⾲ 3 ⤖ྜࡢ✀㢮࡜㉥እ⥺྾཰㡿ᇦ⤖ྜࡢ✀㢮
C㸫H, O㸫H, N㸫H&Ł&&Ł1
C㸻C, C㸻O, C㸻N
C㸫C, C㸫O, C㸫N྾཰㡿ᇦ㸦cm-1㸧
3800 㹼 2700
2300 㹼 2000
1900 㹼 1500
1300 㹼 800

3. 3 ㉁㔞ศᯒἲఱࡽ࠿ࡢ᪉ἲ࡛ࠊ㉁㔞 m ࡢศᏊ ࡟㟁Ⲵ z ࢆᣢࡓࡏ㸦࢖࢜ࣥ໬ࡋ㸧ࠊⲴ㟁ࡋࡓศᏊ࢖࢜ࣥࡸศゎ࡛⏕ࡌࡓⲴ㟁ศᏊ᩿∦㸦ࣇࣛࢢ࣓ࣥࢺ࢖࢜ࣥ㸧ࢆఱࡽ࠿ࡢ᪉ἲ
࡛㉁㔞/㟁Ⲵẚ(m/z) ࡟ᛂࡌ࡚ศ㞳᳨࣭ฟࡋࠊ
ศᏊ㸯ಶࡢ㉁㔞 m㸦ࡍ࡞ࢃࡕศᏊ㔞㸧ࡸศᏊࡢᵓ㐀᝟ሗ㸦≉ᐃࡢᐁ⬟ᇶࡢ᭷↓ࡸ㦵᱁᝟ሗ㸧ࢆᚓࡿࠋ⢭ᐦ࡞ ᐃࢆࡍࡿ࡜ඖ⣲⤌ᡂࡀุู࡛ࡁࡿ㹙౛࠼ࡤ࢚ࢳࣞࣥ
C 2 H 4 (m/z 28.03)ࠊ❅⣲ N 2 (m/z 28.01)ࠊ୍
㓟໬Ⅳ⣲ CO (m/z 27.99) ࡀ༊ู࡛ࡁࡿ㸹ᩚᩘ㉁㔞ࡣ࠸ࡎࢀࡶ 28㹛ࠋ࢖࢜ࣥ໬ἲ࡟ࡣ㟁Ꮚ⾪ᧁἲࠊ࢚ࣞࢡࢺࣟࢫࣉ࣮ࣞ㸦ESI㸧ἲ㸦2002ࠊࣀ࣮࣋ࣝ㈹㸧ࠊMALDI ἲ㸦2002ࠊ⏣୰⪔୍ࠊࣀ࣮࣋ࣝ㈹㸧➼ࡀᣲࡆࡽࢀࡿࠋ᳨ฟἲ࡜ࡋ࡚ࡣ㣕⾜᫬㛫ᆺ㸦Time of Flight=TOF ᆺ㸧ࠊ☢ሙᆺࠊ࢖࢜ࣥࢺࣛࢵࣉᆺࠊ㸲㔜ᴟᆺ㸦Q ࣏࣮ࣝᆺ㸧࡞࡝ࡀ࠶ࡿࠋᮏᐇ㦂࡛ࡣ᪥ᮏ㟁Ꮚᰴᘧ఍♫〇ᅗ 15 ࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡢ
ESI㸫TOF ࣐ࢫࢫ࣌ࢡࢺࣝ-06í7/& ᆺ㉁㔞ศᯒィࢆ⏝࠸ࠊESIἲ࡛ ᐃࡍࡿࠋᅗ 15 ࡟࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡢ ESI㸫TOF ࣐ࢫࢫ࣌ࢡࢺࣝࢆ♧ࡍࠋ

4࣏࣮ࣞࢺㄢ㢟

1. CHBT ࡜ࢩࢫࢸ࢖ࣥ࠿ࡽ࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡀ⏕ᡂࡍࡿ཯ᛂᶵᵓࢆ⪃࠼ࡼ࠺ࠋ
2. ᐇ㦂཰⋡ࡣ 100%࡟࡞ࡽ࡞࠸ࠋࡑࡢ⌮⏤࡜ࡋ࡚࡝ࢇ࡞஦ࡀ⪃࠼ࡽࢀࡿࡔࢁ࠺࠿ࠋ
3. ᅗ 13㸪B ࡟♧ࡋࡓ HbࠊHcࠊHc̓ࡢࢩࢢࢼࣝࡢศ⿣ࣃࢱ࣮ࣥࢆㄝ᫂ࡋ࡚ࡃࡔࡉ࠸ࠋ
ཧ⪃ᩥ⊩a) ௒஭୍ὒࠊ㏆Ụ㇂ඞ⿱⦅㸸ࣂ࢖࣭࢜ࢣ࣑࣑ࣝࢿࢵࢭࣥࢫࣁࣥࢻࣈࢵࢡࠊ୸ၿࠊ2006㸬
b) Shimomura, O㸬Bioluminescence: Chemical Principles and Methods; World Scientific, 2006.
c) McCutcheon, D. C.; Porterfield, W. B.; Prescher, J. A. Org. Biomol. Chem. 2015, 13, 2117-2121.
d) Ⲩᮌ㉅࡯࠿ヂࠊ᭷ᶵ໬ྜ≀ࡢࢫ࣌ࢡࢺࣝ࡟ࡼࡿྠᐃἲ㸦➨㸳∧㸧㸪ᮾி໬Ꮫྠேࠊ2006.

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ࢸ࣮࣐ 2.
2. 1

࣍ࢱࣝࡢගࢆసࡗ࡚ࡳࡼ࠺ (2) ⏕≀Ⓨගᐃ࣍ࢱࣝࡢ⏕≀Ⓨග཯ᛂ࡟ࡘ࠸࡚ a, b)࣍ࢱࣝࡢ⏕≀Ⓨගࡣ㓝⣲཯ᛂ࡛࠶ࡿ(ᅗ 1)ࠋᇶ㉁࡛࠶ࡿ࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡣ Mg2+࢖࢜ࣥඹᏑ໬࡛ 㓝⣲ࣝࢩࣇ࢙࣮ࣛࢮࡢస⏝࡛࢔ࢹࣀࢩࣥ୕ࣜࣥ㓟㸦ATP㸸⏕యෆ࡟ᬑ㐢ⓗ࡟Ꮡᅾࡍࡿࠊ⏕యෆ࢚ࢿࣝࢠ࣮ࡢ㐠ࡧᒇ㸧࡜཯ᛂࡋ࡚ࣝࢩࣇ࢙ࣜࣝ AMP2 ࡜࡞ࡿࠋࡘ࠸

࡛ 2 ࡣࣝࢩࣇ࢙࣮ࣛࢮࡢస⏝࡛㓟⣲ศᏊ࡜཯ᛂࡋ࡚ࠊ࣌ࣝ࢜࢟ࢩࢻ 3 ࢆ୚࠼ࡿࠋࡉࡽ࡟ 3ࡣࢪ࢜࢟ࢭࢱࣀࣥ୰㛫య 4 ࢆ⤒࡚㟁Ꮚⓗບ㉳୍㔜㡯≧ែ㸦S 1 ≧ែ㸧ࡢ࢜࢟ࢩࣝࢩࣇ࢙ࣜࣥ
5㸨 ࡜ CO 2 ࡟ศゎࡍࡿࠋࡑࡋ࡚㓝⣲࡜⤖ྜࡋࡓ 5㸨 ࠿ࡽ㯤⥳Ⰽ㸦560 nm㸧ࡢගࡀฟࡿࠋ࣍ࢱ
ࣝ⏕≀ⓎගࡢⓎගຠ⋡ࡣ⣙ 41%㸦100 ಶࡢࣝࢩࣇ࢙ࣜࣥศᏊ࠿ࡽ 41 ಶࡢࣇ࢛ࢺࣥࡀ⏕ᡂࡍࡿ㸧࡛ࠊ኱ኚຠ⋡ࡀ㧗࠸ࠋⓎගຠ⋡ࡣձࠕ㓟໬཯ᛂ㸦1Ѝ5㸧ࡢ໬Ꮫ཰⋡ࠖղࠕບ㉳≧ែࡢ5㸨 ࡀ⏕ᡂࡍࡿຠ⋡ࠖճࠕ5㸨 ࠿ࡽ⺯ගࢆⓎࡍࡿຠ⋡ࠖࡢ᥃ࡅ⟬࡛࠶ࡿࠋ࣍ࢱࣝࡢ⏕≀Ⓨගࢩࢫࢸ࣒ࡀᴟࡵ࡚ຠ⋡ࡼࡃ໬Ꮫ཯ᛂ࢚ࢿࣝࢠ࣮ࢆගࡾ࢚ࢿࣝࢠ࣮࡟ኚ᥮ࡋ࡚࠸ࡿࡇ࡜ࡀ⌮ゎ࡛ࡁࡿࠋ࣍ࢱࣝࡢගࡀࠕ෭ගࠖ࡜ゝࢃࢀࡿᡤ௨࡛࠶ࡿࠋ࣍ࢱࣝ࡜㏆⦕ࡢࣄ࢝ࣜࢥ࣓ࢶ࢟ࡸ㕲㐨⹸࡞࡝ࡢⓎග⏥⹸ࡶ࣍ࢱࣝ࡜࠾࡞ࡌ 1 ࢆⓎගᇶ㉁࡜ࡋ࡚⏝࠸࡚࠸ࡿࡀࠊࣄ࢝ࣜࢥ࣓ࢶ࢟ࡣಶయ࡟ࡼࡗ࡚⥳ࠊ㯤⥳ࠊ࢜ࣞࣥࢪⰍࠊ࡜ⓎගⰍࡀ␗࡞ࡿࠋ୍᪉ࠊ㕲㐨⹸ࡣ୍ಶయ࡛㢌ࡀ㉥ࡃࠊ⬗యࡀ⥳Ⰽ࡟ගࡿࠋᇶ㉁ 1 ࡣྠࡌ࡞ࡢ࡟Ⰽࡢ㐪࠸ࡀฟࡿ࣓࢝ࢽࢬ࣒࡟ࡘ࠸࡚ࡣㅖ
ㄝࡀ࠶ࡗࡓࡀࠊ᭱㏆ࠊ㟁㏻኱ࠊᖹ㔝ࡽ࡟ࡼࡾゎỴࢆぢ࡚࠸ࡿࠋc)ᅗ12. 2࣍ࢱࣝࡢ⏕≀Ⓨග཯ᛂ࣍ࢱࣝ⏕≀Ⓨග⣔ࡢᛂ⏝ d, e)

25ᅗ 1 ࡟♧ࡋࡓࡼ࠺࡟ࠊ࣍ࢱࣝࡢ⏕≀Ⓨග⣔ࡣ ATP ࢆᚲせ࡜ࡍࡿࠋࡇࢀࢆ㏫࡟฼⏝ࡍࢀࡤᴟࡵ࡚㧗ឤᗘ࡞ ATP ᳨ฟ⣔ࢆᵓ⠏࡛ࡁࡿࠋᐇ㝿࡟㣗ရࠊ⎔ቃỈࠊᡭ⾡ᐊ࡞࡝ࡢࣂࢡࢸࣜ࢔
ࡢࣔࢽࢱࣜࣥࢢ(ATP ࢆ᳨ฟ)࡞࡝ࡢ⾨⏕᳨ᰝࠊATP ⏘⏕࣭ᾘ㈝࡜ࣜࣥࢡࡋࡓྛ✀⏕యᚤ㔞ᡂศࡢ᳨ฟ࣭ྛ✀㓝⣲άᛶࡢᐃ࡞࡝ࡢ⮫ᗋ᳨ᰝࡸ㧗㏿㑇ఏᏊ㸦DNA㸧ゎᯒᢏ⾡࡟฼⏝ࡉࢀ࡚࠸ࡿࠋࡲࡓ࠶ࡿ㓝⣲཯ᛂ࡛࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡀ⏕ᡂࡍࡿࡼ࠺࡟ࡋ࡚࠾ࡅࡤ㧗ឤᗘ࡞㓝⣲άᛶᐃ⣔ࡀᵓ⠏࡛ࡁࡿࠋࡲࡓ⭾኱࡞ᩘࡢ໬Ꮫ≀㉁ࡢ༴㝤ᛶホ౯࡟ࠊ᫬㛫࡜ᡭ㛫࡜㈝⏝ࡢ࠿࠿ࡿື≀ᐇ㦂࡟᥮ࢃࡾࠊపࢥࢫࢺ࡛ࣁ࢖ࢫ࣮ࣝࣉࢵࢺ໬ࡀྍ⬟࡞࣍ࢱࣝࡸࣂࢡࢸࣜ࢔ࡢ⏕≀Ⓨග⣔ࡀ฼⏝ࡉࢀࡘࡘ࠶ࡿࠋᆅ⌫እ⏕࿨᥈ᰝ(㸻ATP ࢆ᳨ฟ)࡟ࡶ࣍ࢱࣝࡢ
Ⓨග⣔ࡀ฼⏝ࡉࢀࡿ࡜࠸࠺ࠋ࣍ࢱࣝⓎග⣔ࡣ⥳Ⰽ⺯ගࢱࣥࣃࢡ㉁ GFP㸦ୗᮧ⬶ࠊ2008 ᖺࣀ࣮࣋ࣝ໬Ꮫ㈹㸧࡜࡜ࡶ࡟⏕࿨⌧㇟ࡢࣂ࢖࢜࢖࣓࣮ࢪࣥࢢࢶ࣮ࣝ࡜ࡋ࡚ᗈࡃ฼⏝ࡉࢀ࡚࠸ࡿࠋ࣍ࢱࣝࡢ㓝⣲ࣝࢩࣇ࢙࣮ࣛࢮࡢ㑇ఏᏊࡣ࣍ࢱࣝ௨እࡢ⏕≀࡛ࡶⓎ⌧ࡉࡏࡿࡇ࡜ࡀ࡛ࡁࡿ㸦㸻ࣝࢩࣇ࢙࣮ࣛࢮࡀ⏕ᡂࡍࡿ㸧ࠋࡑࡇ࡛ࣔࢽࢱ࣮ࡋࡓ࠸㑇ఏᏊࡀⓎ⌧ࡋࡓ࡜ࡁࠊྠ᫬࡟ࣝࢩࣇ࢙࣮ࣛࢮ㑇ఏᏊࡶⓎ⌧ࡍࡿࡼ࠺࡟㑇ఏᏊ᧯సࡋ࡚࠾ࡅࡤࠊATPࠊ㹋g ࢖࢜ࣥࠊ㓟⣲ࡣ⏕యෆ࡟ᖖᅾࡋ࡚࠸ࡿࡢ࡛࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧ࡢᢞ୚࡟ࡼࡾࠊ≉ᐃࡢ㑇ఏᏊࡀⓎ⌧ࡍࡿࢱ࢖࣑ࣥࢢࡸሙᡤࢆ࣍ࢱࣝࡢග࡛ࣔࢽࢱ࣮࡛ࡁࡿ㸦ࣉ࣮ࣟࣔࢱ࣮࢔ࢵࢭ࢖࠶ࡿ࠸ࡣ࣏࣮ࣞࢱ࣮࢔ࢵࢭ࢖㸧ࠋ࢘࢖ࣝࢫࡸ⑓ཎ⳦ឤᰁࡢྍど໬ࠊ㑇ఏᏊ἞⒪ࡢホ౯ࠊᐇ㦂ື≀࡟࠾ࡅࡿᢠࡀࢇ๣ࡢホ౯ࡸࡀࢇ㌿⛣
ࡢ᳨ฟࠊoncolytic ἞⒪(࢘࢖ࣝࢫ࡟ࡼࡿࡀࢇ⣽⬊◚ቯ἞⒪)ࡢホ౯ࠊiPS ⣽⬊ࡢ◊✲࡛ࡶ࣍ࢱࣝࡢ⏕≀ⓎගࡀጾຊࢆⓎ᥹ࡋ࡚࠸ࡿࠋࡋ࠿ࡋࠊ⏕య῝㒊࠿ࡽࡢගࢆ࡜ࡽ࠼ࡿ࡟ࡣࠊ⾑ᾮ࡟ࡼࡿගࡢ྾཰ࡀ࠶ࡿࡓࡵࠊ࣍ࢱࣝࡢග(560ࠥ600 nm)࡛ࡣ୙༑ศ࡛࠶ࡿࠋ⾑ᾮࡀ࠶ࡗ࡚ࡶ㏱
㐣ࡍࡿࠊ㏆㉥እ㡿ᇦࡢ 700ࠥ1300 nm㸦͆⏕యࡢ❆͇࡜࿧ࡪ㸧࡛Ⓨගࡍࡿࡶࡢࡀᮃࡲࡋ࠸ࠋ
᭱㏆ࠊ㟁㏻኱ࠊ∾ࡽࡣ“⏕యࡢ❆͇࡟ࡲ࡛Ⓨග㡿ᇦࢆᣢࡘ࣍ࢱࣝேᕤᇶ㉁࢔࣑࢝ࣝࢿ‫ࢆט‬㛤Ⓨࡋࠊୡ⏺ึࡢᐇ⏝໬࡟ᡂຌࡋ࡚࠸ࡿ(ᅗ 2)ࠋf)࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ(1)ᅗ2࢔࣑࢝ࣝࢿ‫ט‬࣐࢘ࢫయෆ࡛ࡢ࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ(1)࡜࢔࣑࢝ࣝࢿ‫≀⏕ࡢט‬Ⓨගࡢẚ㍑࣍ࢱࣝࣝࢩࣇ࢙࣮ࣛࢮ㑇ఏᏊࡀⓎ⌧ࡋ࡚࠸ࡿ⭘⒆⣽⬊ࢆ࣐࢘ࢫ࡟᳜࠼௜ࡅࠊᙜ
ࣔࣝ㔞ࡢ࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ(1)࡜࢔࣑࢝ࣝࢿ‫ࢆט‬ᢞ୚ࡋ࡚Ⓨගࡉࡏࠊ࣐࢘ࢫయ
⾲࡛ග㔞ࢆィࡋࡓࠋ࢔࣑࢝ࣝࢿ‫ࢆט‬ᢞ୚ࡋࡓ᪉㸦ྑ㸧ࡀࠊ᫂ࡽ࠿࡟య⾲㠃࡟ฟ࡚ࡃࡿග㔞ࡀከ࠸ࠋ㸦෗┿ᥦ౪ࠊᮾᕤ኱ࠊཱྀ୸㧗ᘯຓᩍࠊ㏆⸨⛉Ụᩍᤵ㸧

26ᮏయ㦂ᐇ㦂࡛ࡣࢸ࣮࣐㸯࡛ྜᡂࡋࡓ࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧࡜ᕷ㈍ࡢ㏆㉥እⓎගᛶேᕤ࣍ࢱࣝⓎගᇶ㉁࢔࣑࢝ࣝࢿ‫≀⏕࡚࠸⏝ࢆט‬Ⓨග཯ᛂࢆ⾜࠸ࠊⓎගࢫ࣌ࢡࢺࣝ࡜Ⓨගࡢ⤒᫬ኚ໬ࢆィ

2. 3ࡋࠊ୧⪅ࡢ㐪࠸ࢆẚ㍑᳨࣭ウࡍࡿࠋ࣍ࢱࣝ⏕≀Ⓨගࡢィ

2. 3. 1ᐇ㦂࡟⏝࠸ࡿ⿦⨨ࠊヨ⸆ࠊჾල㸯㸧ᐃ⿦⨨࣭pHᐃ㸸ᇼሙ〇సᡤ〇 F-23 ᆺ࢞ࣛࢫ㟁ᴟᘧỈ⣲࢖࢜ࣥ⃰ᗘᣦ♧ィࢆ౑⏝ࠋ࣭⏕≀Ⓨග㔞ᐃ㸸࢔ࢺ̿〇 Luminescencer-PSN AB-2200 ࢆ౑⏝ࠋ

࣭⏕≀Ⓨගࢫ࣌ࢡࢺࣝᐃ㸸࢔ࢺ̿〇 AB-1850 LumiFL Spectrocaptureࢆ౑⏝ࠋ㸰㸧ヨ⸆࣭࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸸ᐇ㦂ࢸ࣮࣐㸯࡛ྜᡂࡋࡓࡶࡢࠋ࣭࢔࣑࢝ࣝࢿ‫ט‬㸸࿴ග⣧⸆⸆ရᕤᴗ〇ࠊ≉⣭ࠋ࣭㉸⣧Ỉ㸸MILLIPORE 〇 Milli-5;Į ࠿ࡽ᥇Ỉࡋࡓࡶࡢࠋ
࣭ࣝࢩࣇ࢙࣮ࣛࢮ (໭⡿⏘࣍ࢱࣝ Photinus pyralis ⏤᮶)㸸Promega ♫〇ࠊ⤌ࡳ᥮࠼ᆺࣝࢩࣇ࢙࣮ࣛࢮ (QuantiLum‫ ט‬Recombinant Luciferase)
࣭ATP-Mg㸸Sigma ♫〇ࠋ࣭ࣜࣥ㓟Ỉ⣲஧࣒࢝ࣜ࢘࡜ࣜࣥ㓟஧Ỉ⣲࣒࢝ࣜ࢘㸸࿴ග⣧⸆ᕤᴗ〇ࠊ≉⣭ࠋ࣭ࢺࣜࢫ㸦ࣄࢻࣟ࢟ࢩ࣓ࢳࣝ㸧࢔࣑ࣀ࣓ࢱࣥ㸦2-࢔࣑ࣀ-2-ࣄࢻࣟ࢟ࢩ࣓ࢳࣝ-1,3ࣉࣟࣃࣥࢪ࣮࢜ࣝ㸧(㏻⛠ Tris)㸸㛵ᮾ໬Ꮫ〇ࠊ≉⣭ࠋ࣭ࢢࣜࢭ࣮ࣟࣝ㸸࿴ග⣧⸆ᕤᴗ〇ࠊ≉⣭ࠋ࣭㸴M ሷ㓟࠾ࡼࡧ㸯M ሷ㓟ࠋ㸱㸧࣐࢖ࢡࣟࣆ࣌ࢵࢺ (20 PL, 200 PL, 1000 PL)࡜࣐࢖ࢡࣟࣆ࣌ࢵࢺ⏝ࢳࢵࣉ㸲㸧ྛ✀ࣉࣛࢫࢳࢵࢡࢳ࣮ࣗࣈ㸦ᅗ 3㸧
޽ 0.3 mL ࢚ࢵ࣌ࣥࢳ࣮ࣗࣈ㸸㓝⣲ࢫࢺࢵࢡ⏝޾ 1.5 mL ࢚ࢵ࣌ࣥࢳ࣮ࣗࣈ㸸Ⓨග཯ᛂ㓝⣲⁐ᾮ⏝
޿ 2 mL ࢚ࢵ࣌ࣥࢳ࣮ࣗࣈ㸸
ᇶ㉁ࢫࢺࢵࢡ⏝߀ ߁ 15 mL ࡜ 50 mL ࣇ࢓ࣝࢥࣥࢥࢽ࢝ࣝࢳ࣮ࣗࣈ㸸࠾ࡶ࡟⦆⾪ᾮࡢㄪ〇࡟౑⏝ࠋ޽޾޿߀ᅗ32. 3. 2߁ྛ✀ࣉࣛࢫࢳࢵࢡࢳ࣮ࣗࣈ⦆⾪ᾮࡢㄪ〇

㸯㸧pH ࣓࣮ࢱ࣮ࡢ᧯సἲ
⦆⾪ᾮࡢㄪ〇࡛ࡣỈ⣲࢖࢜ࣥ⃰ᗘ (pH) ࢆㄪ⠇ࡍࡿᚲせࡀ࠶ࡿࠋpH ㄪ⠇ࡣᇼሙ〇సᡤ〇 F-23 ᆺ࢞ࣛࢫ㟁ᴟᘧỈ⣲࢖࢜ࣥ⃰ᗘᣦ♧ィ㸦pH ࣓࣮ࢱ࣮㸧࡛⾜࠺ࠋᅗ 4 ࡟pH ࣓࣮ࢱ࣮ࡢ᧯సἲ࡜࢞ࣛࢫ㟁ᴟࢆ౑⏝ࡋࡓ pHᐃࡢᵝᏊࢆ♧ࡍࠋ

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ձ pH ࣓࣮ࢱ࣮ᮏయ㸦ᕥ෗┿㸧ࡢ㟁※ࢆධࢀࡿࠋղ ᰯṇ࣎ࢱࣥࢆᢲࡍࠋճ ࢞ࣛࢫ㟁ᴟࢆ pH 4, 7, 9 ࡢᶆ‽ᾮ࡟ᾐࡋࠊᰯṇࢆ⾜࠺ࠋ
մ ESC ࢆᢲࡋ࡚ᰯṇ⤊஢ࠋյ ヨᩱ⁐ᾮࡢ pH ࢆᐃࠋ⁐ᾮΰྜࢆ⧞ࡾ㏉ࡋ┠ⓗࡢ pH ࡟࠶ࢃࡏࡿࠋ‫ە‬pHᐃࡣ⁐ᾮᨩᢾࡋ࡞ࡀࡽ⾜࠺ࠋᨩᢾᏊࡀ࢞ࣛࢫ㟁ᴟ࡟ࡪࡘ࠿ࡽ࡞࠸ࡼ࠺࡟ࡍࡿࠋ㟁ᴟಖㆤ࢟ࣕࢵࣉ㸸ᐃ᫬࡟࠶ࡅࠊ⤊஢᫬࡟㛢ࡵࡿ࢞ࣛࢫ㟁ᴟ㒊ᐃࡢࡓࡧ࡟⵨␃Ỉ࡛Ὑ⁡ࡍࡿࠋ‫⏝౑ە‬๓ᅗ4‫ە‬㛤ጞ᫬pH ࣓࣮ࢱ࣮᧯సἲ࡜࢞ࣛࢫ㟁ᴟ࡟ࡼࡿ pH‫⏝౑ە‬୰ᐃࡢᵝᏊ

㸰㸧500 mM pH 8.0 ࠾ࡼࡧ pH 6.0 ࣜࣥ㓟⦆⾪ᾮࡢㄪ〇ձ ࣜࣥ㓟Ỉ⣲஧࣒࢝ࣜ࢘㸦K 2 HPO 4 㸸≉⣭ࠊศᏊ㔞 174㸧4.35 gࠊࣜࣥ㓟஧Ỉ⣲࣒࢝ࣜ࢘㸦KH 2 PO 4 㸸≉⣭ࠊศᏊ㔞 136㸧3.40 g ࢆࡑࢀࡒࢀ㉸⣧Ỉ 45 mL ࡟⁐ゎࡋࠊ᭱⤊ⓗ࡟య✚ࢆ 50 mL ࡟࣓ࢫ࢔ࢵࣉࡋࠊ⃰ᗘ 500 mM ࡢ⁐ᾮࢆㄪ〇ࠋK 2 HPO 4Ỉ⁐ᾮࡣ pH 8㹼9ࠊKH 2 PO 4 Ỉ⁐ᾮࡣ pH 4㹼5 ࡜࡞ࡗ࡚࠸ࡿࡇ࡜ࢆ pH ࣓࣮ࢱ࣮࡛☜ㄆࠋ
ղ 500 mM pH 8.0 ࣜࣥ㓟⦆⾪ᾮࡢㄪ〇㸸pH ࣓࣮ࢱ࣮࡛ࣔࢽࢱ࣮ࡋ࡞ࡀࡽ pH 8.0 ࡟࡞ࡿࡼ࠺ 500 mM K 2 HPO 4 (pH 8㹼9) 10 mL ࡟ 500 mM KH 2 PO 4 (pH 4㹼5)ࢆຍ࠼
࡚࠸ࡃࠋճ 500 mM pH 6.0 ࣜࣥ㓟⦆⾪ᾮࡢㄪ〇㸸500 mM KH 2 PO 4 (pH 4㹼5) 10 mL ࡟ 500
mM K 2 HPO 4 (pH 8㹼9)ࢆຍ࠼࡚⾜ࡁࠊpH 6.0 ࡟ㄪᩚࠋ㸱㸧50 mM pH 6.0 ࡢࣜࣥ㓟⦆⾪ᾮࡢㄪ〇

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500 mM pH 6.0 ࣜࣥ㓟⦆⾪ᾮ 5 mL ࢆ㉸⣧Ỉ࡛ 10 ಸᕼ㔘ࡋࠊㄪ〇ࠋ㸲㸧ࢢࣜࢭ࣮ࣟࣝࢆ 35%ྵࡴ 50 mM pH 8.0 ࣜࣥ㓟⦆⾪ᾮࡢㄪ〇
500 mM pH 8.0 ࣜࣥ㓟⦆⾪ᾮ 1 mL ࡜ࢢࣜࢭ࣮ࣟࣝ 3.5 mL ࢆΰྜࡋࠊࡉࡽ࡟㉸⣧Ỉ࡛඲యࡢయ✚ࢆ 10 mL ࡟ࡋ࡚ㄪ〇ࡍࡿࠋ
㸳㸧500 mM pH 8.0 ࠾ࡼࡧ pH 9.0 Tris-HCl ⦆⾪⁐ᾮࡢㄪ〇ձ ࢺࣜࢫ㸦ࣄࢻࣟ࢟ࢩ࣓ࢳࣝ㸧࢔࣑ࣀ࣓ࢱࣥ(2-࢔࣑ࣀ-2-ࣄࢻࣟ࢟ࢩ࣓ࢳࣝ-1,3-ࣉࣟࣃࣥࢪ࣮࢜ࣝ)(㏻⛠ Trisࠊ≉⣭ࠊศᏊ㔞 121)

3.03 g ࢆ⣙ 40 mL ࡢ㉸⣧Ỉ࡟⁐ゎࡋ㸦pH 10㸧ࡋࠊ㸴M ሷ㓟࡛ pH ࢆ 8.0 ࡟ㄪ⠇ࡋࡓᚋࠊᐊ

࡟෭༷㸦pH ࡟ᗘ౫Ꮡᛶࡀ࠶ࡿࡓࡵ㸧ࠋ㸯M ሷ㓟࡛ pH ࢆ pH 8.0 ࡟ㄪ⠇ᚋ ඲యࡢయ✚ࢆ 50 mL
࡟ࡋ࡚ࠊ500 mM pH 8.0 Tris-HCl ⦆⾪⁐ᾮࢆㄪ〇ࠋղ ྠᵝ࡟ࡋ࡚ pH 9.0 ࡢ 500 mM Tris-HCl ⦆⾪⁐ᾮࢆㄪ〇ࠋ㸴㸧ࢢࣜࢭ࣮ࣟࣝࢆ 10%ྵࡴ 50 mM pH 8.0 Tris-HCl ⦆⾪ᾮࡢㄪ〇ձ 500 mM pH 8.0 ࡢ Tris-HCl ⦆⾪ᾮ 20 mL ࢆ㉸⣧Ỉ࡛ 5 ಸᕼ㔘ࡋࠊ100 mM pH 8.0Tris-HCl ⦆⾪ᾮࢆㄪ〇ࡍࡿ
ղ 100 mM pH 8.0 Tris-HCl ⦆⾪ᾮ 20 mL ࡜ࢢࣜࢭ࣮ࣟࣝࢆ 4 mL ࢆΰྜࡋࠊ㉸⣧Ỉ࡛඲యࡢయ✚ࢆ 40 mL ࡟ࡋࠊࢢࣜࢭ࣮ࣟࣝࢆ 10%ྵࡴ 50 mM pH 8.0 Tris-HCl⦆⾪ᾮࢆᚓࡿࠋ

2. 3. 3ヨᩱㄪ〇㸯㸧ᇶ㉁⁐ᾮձ ࢫࢺࢵࢡ⁐ᾮ㸸࣭࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥ㸦1㸧(ศᏊ㔞 280)㸸 1.4 mg㸦5 ȝmol㸧ࢆ㟁Ꮚኳ⛗࡛ 2 mL࢚ࢵ࣌ࣥࢳࣗíࣈ࡟㔞ࡾྲྀࡾࠊ50 mM pH 6.0 ࣜࣥ㓟࣒࢝ࣜ࢘⦆⾪ᾮ 1.0 mL ࡟⁐ゎࡋࠊ5 mM ⁐ᾮࢆㄪ〇ࠋ࣭࢔࣑࢝ࣝࢿ‫ט‬㸦ศᏊ㔞 302.4㸧
㸸1.5 mg㸦5 ȝmol㸧ࢆ㟁Ꮚኳ⛗࡛⛗㔞ࡋࠊ50 mM,pH 6.0 ࣜࣥ㓟࣒࢝ࣜ࢘⦆⾪ᾮ 10 mL ࡟⁐ゎࡋࠊ0.5 mM ⁐ᾮࢆㄪ〇ࠋղ ᐇ㝿࡟౑⏝ࡍࡿᇶ㉁⁐ᾮ㸸࣭ୖグࢫࢺࢵࢡ⁐ᾮࢆࣜࣥ㓟⦆⾪ᾮ㸦50 mM pH 8.0㸧࡛ᕼ㔘ࡋࠊȝ0 ࡢ⃰ᗘ࡟ࡋ࡚⏝࠸ࡿ㸦࣍ࢱࣝࣝࢩࣇ࢙ࣜࣥࡣ 50 ಸᕼ㔘ࠊ࢔࣑࢝ࣝࢿ‫ ࡣט‬5 ಸᕼ㔘ࡍࡿ㸧ࠋ㸰㸧㓝⣲⁐ᾮձ ࣇ࢓ࣥࢺ࣒ィ⏝㸸㉎ධࡋࡓ㓝⣲⁐ᾮࢆᕼ㔘ࡏࡎ࡟⏝࠸ࡿࠋ࡞࠾㉎ධࡋࡓࣝࢩ

ࣇ࢙࣮ࣛࢮࡣࠊ㔜㔞ࡣ 1 mg ࡟⤫୍ࡉࢀ࡚࠸ࡿࡀࠊ⃰ᗘࡣࣟࢵࢺ㛫࡛ࣂࣛࡘࡁࡀ࠶ࡿ㸦ྲྀᢅㄝ᫂᭩࡟⃰ᗘࡣグ㍕ࡉࢀ࡚࠸ࡿ㸧ࠋ㐣ཤ࡟㉎ධࡋࡓࣝࢩࣇ࢙࣮ࣛࢮࡢ
⃰ᗘࡣ 13.2 mg/mLࠊ14.7 mg/mL ࡞࡝࡛࠶ࡿࠋղ ࢫࢺࢵࢡ⁐ᾮ㸸㉎ධࡋࡓࣝࢩࣇ࢙࣮ࣛࢮ 1 mg ࢆ 1 mg/mL ࡟࡞ࡿࡼ࠺࡟ࠊࢢࣜ
ࢭ࣮ࣟࣝࢆ 10%ྵࡴ Tris-HCl ⦆⾪ᾮ (50 mM, pH 8.0) ࡛ᕼ㔘ࡋࠊ20ࠥ30 ȝL ࡎ
ࡘ 0.3 mL ࡢ࢚ࢵ࣌ࣥࢳ࣮ࣗࣈ㸦ᅗ 3ࠊ޽㸧࡟ᑠศࡅࡋࠊ–80 Υࡢ෭෾ᗜ࡟ಖ⟶ࡍࡿࠋճ ᐇ㝿࡟౑⏝ࡍࡿ㓝⣲⁐ᾮ㸸෭෾ಖᏑࡋ࡚࠶ࡗࡓ 1 mg/mL ࢫࢺࢵࢡ⁐ᾮࢆ

29

20ࠥ30 ȝL ࢆゎ෾ࡋࠊࢢࣜࢭ࣮ࣟࣝࢆ 35%ྵࡴࣜࣥ㓟⦆⾪ᾮ㸦50 mM pH 8.0㸧࡛
100 ಸᕼ㔘ࡋࠊȝJmL ࡢ⃰ᗘ࡟ࡋ࡚⏝࠸ࡿࠋ
㸱㸧ATP-M㹥⁐ᾮࡢㄪ〇ձ 10 mM ATP-M㹥ࡢ⁐ᾮࡢㄪ〇㸸
ATP-Mg 㸦2Mg ሷࡢศᏊ㔞㸸551㸧11 mg ࢆ㉸⣧Ỉ 2 mL ࡟⁐ゎࡋࠊㄪ〇ࠋ
ղ 200 PM ATP-Mg ⁐ᾮࡢㄪ〇㸸
10 mM ATP-M㹥 0.1 mL ࢆ 4.9 mL ࡢ㉸⣧Ỉ࡛ 50 ಸᕼ㔘ࡋࠊㄪ〇ࠋ

2. 3. 4⏕≀Ⓨගᐃ㸯㸧Luminescencer-PSN AB-2200 ࡟ࡼࡿ⏕≀Ⓨග㔞ᐃ

Luminescencer-PSN AB-2200 ࡢ᧯సἲࢆᅗ 5 ࡟♧ࡍࠋ
⾲ 1 ࡟♧ࡋࡓᅗ 6 ࡟グ㍕ࡋࡓᡭ㡰࡛Ⓨග㔞ࢆᐃ⁐ᾮࡢయ✚࡛ࠊᐃࡍࡿࠋձ 㟁※ࢆධࢀࡿ࡜ࠊ“Set Conditions”࡜⾲♧ࡉࢀࡿࡢ࡛ Enter ࢆᢲࡍ࡜ᐃ᮲௳ࡢタᐃ⏬㠃࡟࡞ࡿࠋղ ୖ࠿ࡽ Mul, 35 sec, Integral, 0 sec, 35 sec, off ࢆ㑅ࢇ࡛ Enter ࢆᢲࡍࠋࡍࡿ࡜“SetConditions”࡜⾲♧ࡉࢀࡿࠋճ UNK ࢆᢲࡍࠋSample name ࢆධࢀࡿࠋ

ᐃᐃᅇᩘࢆධຊࡍࡿࠋ

մ “UNK Analysis”࡜⾲♧ࡉࢀࡿࡢ࡛ヨᩱࢆࢭࢵࢺࡋࠊStart ࢆᢲࡍ࡜յ

ᐃ㛤ጞࠋᐃ⤊஢᫬ࡣ ESC ࢆᢲࡍࠋն “Save a UNK File”࡜⾲♧ࡉࢀࡿࡢ࡛ Save ࡍࡿሙྜࡣ“Yes”ࠊࡋ࡞࠸ሙྜࡣ
“No”ࢆ㑅ᢥࠋ
ᅗ5
⾲1

Luminescencer-PSN AB-2200 ࡢ᧯సἲLuminescencer-PSN AB-2200 ࡟ࡼࡿ⏕≀Ⓨග㔞ࡢᐃ᮲௳య✚ ȝ/᭱⤊⃰ᗘ

500 mMȝ/

100 mMᇶ㉁ᇶᮏࡣ ȝ0㸨ȝ/ȝ0㓝⣲10 ȝJP/ȝ/ȝJmLMg-ATPȝ0ȝ/ȝ0⃰ᗘpH 8 ࣜࣥ㓟⦆⾪⁐ᾮ㸨Ⓨගᙉᗘࡀ㣬࿴ࡍࡿሙྜࡣ 10 ȝ0 ⃰ᗘࡢᇶ㉁⁐ᾮࢆ⏝࠸ࡿ࢔࣑࢝ࣝࢿ‫ࡢט‬ሙྜࡣ 0.5 mM ⁐ᾮࢆ⏝࠸ࡿ㸦᭱⤊⃰ᗘࡣ 0.1 mM㸧

30Luminescencer-PSN AB-2200(࢝࢘ࣥࢺࡣ 9 ᱆ࡄࡽ࠸࡟࡞ࡿ࡜㣬࿴ࡍࡿࡢ࡛ὀព)ᇶ㉁ࠊ㓝⣲࡞࡝ࡢࢫࢺࢵࢡ
⁐ᾮࡣࡇࡢࡼ࠺࡟ࠊịୖ࡛෭༷ࡋ࡞ࡀࡽ౑⏝ࡍࡿࠋ

ճATP ࢆධࢀࠊⓎගࢆ㛤ጞࡉࡏࡿࠋղ࢟ࣗ࣋ࢵࢺࢆィ ჾ࣍ࣝࢲ࣮࡟⿦ሸࠋձ ィ ⏝࢟ ࣗ࣋ࢵ ࢺ࡟ 㓝⣲ ࡜ᇶ㉁⁐ᾮࠊ500 mM
pH 8 ࣜࣥ㓟⦆⾪ᾮࢆධࢀࡿࠋ

ձ Luminescencer ⏝ࡢィ࢟ࣗ࣋ࢵࢺෆ࡛ࠊࣜࣥ㓟࣒࢝ࣜ࢘⦆⾪⁐ᾮ(500 mM, pH 8.0,20 ȝL)ࠊᇶ㉁⁐ᾮ>ȝ0 Ⓨගᙉᗘࡀ㣬࿴ࡍࡿሙྜࡣ ȝ0 ȝ/@ࠊ㓝⣲⁐ᾮ(10ȝJP/ȝ/ ࢆⰋࡃΰྜࡍࡿࠋղィճ࢟ࣗ࣋ࢵࢺࢆᐃ⿦⨨ෆ࡟ᤄධࠋᐃ⿦⨨ࡢୖ㒊࠿ࡽ࣐࢖ࢡࣟࢩࣜࣥࢪࢆ⏝࠸࡚ᐃ㛤ጞ஬⛊ᚋ࡟ ATP-Mg ⁐ᾮȝ0ȝL)ࢆὀධ㸦஦๓࡟ ATP-Mg ⁐ᾮࡶຍ࠼ࠊ☜ᐇ࡟ΰྜࡉࡏ࡚࠿ࡽィ⏝࢟ࣗ࣋ࢵࢺࢆ
մ 30 ⛊㛫Ⓨගᐃ⿦⨨ෆ࡟ᤄධࡋ࡚ィࡋ࡚ࡶⰋ࠸㸧ࠋᐃࠋ⏕≀Ⓨග㔞ࡣⓎග㛤ጞ࠿ࡽ 30 ⛊㛫ࡢⓎගᙉᗘࡢ⥲࿴࡛࠶ࡿࠋյ ࢔࣑࢝ࣝࢿ‫ࡣט‬᭱኱Ⓨගᙉᗘ࡟㐩ࡍࡿࡢࡀ㐜࠸ࠋኳ↛ᆺᇶ㉁ 1 ࡜࢔࣑࢝ࣝࢿࢆẚ㍑ࡍࡿሙྜࡣ 180 ⛊㛫ࡢⓎග
ᅗ6

ᐃࢆ⾜࠺ࠋ

Luminescencer-PSN AB-2200 ࡟ࡼࡿ⏕≀Ⓨග㔞ᐃࡢᡭ㡰

31

㸰㸧AB-1850 LumiFL Spectrocapture࡟ࡼࡿ⏕≀Ⓨගࢫ࣌ࢡࢺࣝࡢ

ᐃ

Ⓨගࢫ࣌ࢡࢺࣝࡣ AB-1850 LumiFLSpectrocapture㸦ᅗ 7㸧⏝࠸࡚ࡓᐃࡍࡿࠋᏳᐃࡋ

ᐃࢆ⾜࡞࠺࡟ࡣ luminescencer-PSN AB-2200

࡛ 107㹼8ࡢ࢝࢘ࣥࢺᩘࢆ♧ࡍ⁐ᾮࡀᮃࡲࡋ࠸ࠋᴟ኱Ἴ㛗ࡀࢃ࠿ࢀࡤⰋ࠸ࡢ࡛࠶ࢀࡤࠊ࢝࢘ࣥࢺ
ᩘ 5㹼6 ᱆⛬ᗘ࡛ィࡢ࡞ࡽ࡛ࡁࡿࠋ㛗᫬㛫Ⓨගࡍࡿࡶᐃ᫬㛫ࢆ㛗ࡃࡍࢀࡤࡁࢀ࠸࡞Ⓨගࢫ࣌ࢡࢺࣝࡀᚓࡽࢀࡿࠋ

ᐃ᫬㛫࡛ࡢ⥲Ⓨග㔞ࡀ 5

㹼7 ᱆࠶ࢀࡤⓎගࢫ࣌ࢡࢺࣝࢆᐃྍࠋ⤒᫬ኚ໬ᐃࡍࡿᶵ⬟ࡶഛࢃࡗ࡚࠸ࡿࡀࠊ▷᫬㛫ࡢ⤒ᅗ7᫬ኚ໬ࢆぢࡿࡢࡣ㞴ࡋ࠸ࠋAB-2200 ࡛࢝࢘ࣥࢺ⏕≀Ⓨගࢫ࣌ࢡࢺࣝᐃ⿦⨨ AB-1850LumiFL Sectrocaptureࡀ 108⛬ᗘ࡛㣬࿴ࡍࡿᜍࢀࡀ࠶ࡿࠋAB-1850
LumiFL Spectrocapture ࡢ᧯సᡭ㡰ࢆ௨ୗ࡟♧ࡍࠋձࣃࢯࢥࣥࠊ⿦⨨ࡢ㟁※ࢆධࢀࡿࠋ

ղࣃࢯࢥࣥࡢࠕLumiFL Spectrocaptureࠖ࡜࠸࠺࢔ࣉࣜࢣ࣮ࢩࣙࣥࢆ㛤ࡃࠋCCD ࣓࢝ࣛࡢ෭༷࡟ᩘศ࠿࠿ࡿࠋ“ready”ࡢࣛࣥࣉࡀ࢜ࣞࣥࢪ࠿ࡽ⥳࡟࡞ࡗࡓࡽճィ᮲௳タᐃ㸸ᐃ࣮ࣔࢻࠊᇶᮏⓗ࡟ࡣୗグタᐃ࡛ࢩࣥࢢࣝᐃ᫬㛫㐺᫬ᇶᮏࡣ⮬ືࠊヨᩱࡢࣂࢵࢡࢢࣛ࢘ࣥࢻࢆྲྀࡾࡓ࠸᫬ࡣࣂࢵ
ࢡࢢࣛ࢘ࣥࢻ㛤࡟ࡍࡿィ⟬᪉ἲᐃ್̿ࣂࢵࢡࢢࣛࣥࢻ࡟ࡍࡿ
1 mmࢫࣜࢵࢺᖜ
࣭ᐃ᫬㛫ࠊࣂࢵࢡࢢࣛ࢘ࣥࢻࠊィ⟬᪉ἲࢆタᐃࠋᐃࠋᐃ࣮ࣔࢻࣂࢵࢡࢢࣛ࢘ࣥࢻ

ᐃྍࠋᐃ࡟౑࠼ࡿࢳ࣮ࣗࣈ㸸୺࡟ PCR ࢳ࣮ࣗࣈ(ȝL)ࠊ▼ⱥࢭࣝࠊࢹ࢕ࢫ࣏ࢭࣝࠊ࣌ࢺࣜ─ࠊ࣑ࣝࢳ࣮ࣗࣈࠋմィࢳ࣮ࣗࣈ࡟ࣜࣥ㓟࣒࢝ࣜ࢘⦆⾪⁐ᾮ(500 mM, pH 8.0, 5 ȝL)ࠊᇶ㉁⁐ᾮ>ȝ05 ȝ/@ࠊ㓝⣲⁐ᾮ(1 mg/mL, 5 ȝ/ ࠊATP-Mg ⁐ᾮ ȝ010 ȝL)ࢆຍ࠼ࠊⰋࡃΰྜ
ࡍࡿࠋյ ヨᩱࢆࢭࢵࢺࡋ࡚⏬㠃ୖࡢ㟷࠸୸ࢆࢡࣜࢵࢡ(ࣂࢵࢡࢢࣛ࢘ࣥࢻࣛ࢘ࣥࢻᐃࡣᐃ᫬㛫ࢆኚ࠼ࡿࡓࡧ࡟ᚲせ.

ն ㉥୸ࢆࢡࣜࢵࢡࡋ࡚շᐃ)ࠋࣂࢵࢡࢢ

ᐃ㛤ጞࠋᐃࡀ⤊ࢃࡗࡓࡽ⾲♧Ѝ෌ィ⟬ЍගᏊ࡟࠿࠼ࡿࠊ࡛ࣇ࢓࢖ࣝࢆಖᏑࠋո ಖᏑࡋࡓࣇ࢓࢖ࣝࢆ୍ᣓࣇ࢓࢖ࣝኚ᥮࡛ asc ࣇ࢓࢖ࣝ࡟ኚ᥮ࠋ32չ ⤊஢᫬㸸ࣃࢯࢥࣥୖࡢ࢔ࣉࣜࢣ࣮ࢩࣙࣥࢆ⤊஢ࡉࡏࡿࠋࡍࡿ࡜ࠕ෭༷ゎ㝖୰ࠖ࡜࠸࠺⾲♧ࡀฟࡿࡢ࡛ᾘ࠼ࡿࡲ࡛ࡲࡘࠋࡑࢀࡀᾘ࠼ࡓࡽᶵᲔࠊࣃࢯࢥࣥࢆࢩࣕࢵࢺࢲ࢘ࣥࠋ⏕≀ⓎගἼ㛗ᐃჾ࡟ᑐᛂࡋ࡚࠸ࡿᐃᐜჾࡣᵝࠎ࠶ࡿࡀࠊᑠࡉ࠸య✚࡛ᐃྍ⬟࡞ PCR ࢳ࣮ࣗࣈࢆࡼࡃ⏝࠸ࡿ㸦ᅗ 3ࠊ޽㸧ࠋࡑࡢ㝿ࡢࠊ ᐃ⁐ᾮࡢయ✚ࢆ⾲ 2 ࡟♧ࡍࠋࡇࡢ᮲௳ࡢ㓝⣲⃰ᗘ࡛࠶ࢀࡤࠊ㸱ศ㛫✚⟬࡛ኳ↛ᆺᇶ㉁࡛ࡣ⥡㯇࡞ࢫ࣌ࢡࢺࣝࡀᚓࡽࢀࡿࠋ࢔࣑࢝ࣝࢿ‫ࠊࡣ࡛ט‬ᴟ኱ⓎගἼ㛗ࡣศ࠿ࡿࡶࡢࡢࣀ࢖ࢬࡀฟࡿ࡜ᛮࢃࢀࡿࠋ⥡㯇࡞ࢫ࣌ࢡࢺࣝࢆᚓࡓ࠸ሙྜ࡟ࡣࠊ㓝⣲⃰ᗘࢆ⃰ࡃࡍࡿ࠿ࠊᐃ᫬㛫ࢆ㛗ࡃࡍࡿᚲせࡀ࠶ࡿࠋ࠸ࡎࢀࡢሙྜࡶⓎගᙉᗘࡀ㣬࿴ࡋ࡞࠸ࡼ࠺࡟ὀពࡍࡿࠋ
⾲2AB-1850 LumiFL Spectrocapture ࡟ࡼࡿ⏕≀Ⓨගࢫ࣌ࢡࢺࣝᐃ᮲௳⃰ᗘయ✚ ȝ/᭱⤊⃰ᗘ

pH 8 ࣜࣥ㓟⦆⾪⁐ᾮ

500 mMȝ/

100 mMᇶ㉁100 ȝ0㸨

ȝ/

ȝ0

㓝⣲

1 mg/mLȝ/

0.2 mg/mL

Mg-ATPȝ0ȝ/ȝ0㸨࢔࣑࢝ࣝࢿ‫ࡢט‬ሙྜࡣ 0.5 mM ⁐ᾮࢆ౑⏝㸦᭱⤊⃰ᗘࡣ 0.1 mM㸧

2. 5࣏࣮ࣞࢺㄢ㢟

1. ࣍ࢱࣝ⏕≀Ⓨග⣔ࡣ࡝ࡢࡼ࠺࡞ࡇ࡜࡟฼⏝ࡉࢀ࡚࠸ࡿ࠿ࠊㄪ࡭࡚ࡳࡼ࠺ࠋ
2. ࣍ࢱࣝ⏕≀Ⓨග⣔ࡢ᪂ࡋ࠸฼⏝ἲࢆ⪃࠼࡚ࡳࡼ࠺ࠋཧ⪃ᩥ⊩a) ௒஭୍ὒࠊ㏆Ụ㇂ඞ⿱⦅㸸ࣂ࢖࣭࢜ࢣ࣑࣑ࣝࢿࢵࢭࣥࢫࣁࣥࢻࣈࢵࢡࠊ୸ၿࠊ2006㸬b) Shimomura, O㸬Bioluminescence: Chemical Principles and Methods; World Scientific, 2006.
c) Hirano, T.; Hasumi, Y.; Ohtsuka, K.; Maki, S.; Niwa, H.; Yamaji, M.; Hashizume, D. J. Am.
Chem. Soc. 2009, 131, 2385-2396.
d) Adams, S. T. ; Miller, S. C. Curr. Opin. Chem. Biol. 2014, 21, 112–120.
e) Porterfield, W. B. ; Prescher, J. A. Curr. Opin. Chem. Biol. 2015, 22, 121–130.
f) Iwano, S.; Obata, R.; Miura, C.; Kiyama, M.; Hama, K.; Nakamura, M.; Amano, Y.; Kojima, S.;
Hirano, T.; Maki, S.; Niwa, H. Tetrahedron, 2013, 69, 3847-3856.