㑇ఏᏊ᳨ฟ䛻ᛂ⏝ྍ⬟䛺⎔ቃឤᛂᆺ⺯ග᰾㓟㻌
Design of Environmentally Sensitive Fluorescent (ESF) purine nucleosides for DNA detectionᕤᏛ㒊㻌 㻌 ⏕࿨ᛂ⏝໬Ꮫ⛉㻌 㻌 ෸ᩍᤵ㻌 㻌 㰻⸨㻌 ⩏㞝Yoshio㻌 Saito, Nihon University, College of Engineering㻌 㻌⤖ᯝ䞉䜎䛸䜑䠄Summary䠅

5NN

NH2OOHOApaGb)

THF

1NH

DMF
2-propanol
MethanolNormalized intensities

0.8Water

ǻȞ

0.6

0.4

0.20㻠㻜㻜

㻠㻡㻜

㻡㻜㻜

㻡㻡㻜

㻢㻜㻜

㻢㻡㻜

㻣㻜㻜

Wavelength (nm)

ᅗ3 a) Normalized UV and fluorescence spectra of ApaG in different solvents.
Spectra are normalized with respect to peak intensities. b) Lippert plot of corrected
Stokes shift in wavenumbers (ǻȞ) against the orientational polarizability of solvents(ǻI). Filled circles: aprotic solvents, open circles: protic solvents. 1, ethyl acetate; 2,tetrahydrofuran; 3, N,N-dimethylformamide; 4, acetonitrile; 5, dimethylsulfoxide; 6,2-propanol; 7, ethanol; 8, methanol; 9, glycerol; 10, water.

ORN
N
HOO

NHN

NH2

ABG : R = Ac

CBG : R = CNOH

DABG : R = NMe
2
MBG : R = OMec)

Dioxane
DMF
EtOH
THF
DMSO
MeOH
AcOEt
CH3CN

ᅗ5 Fluorescence color of ABG in different solvents. Thesample solutions were illuminated with a 365 nm
transilluminator.

ⰾ㤶᪘ⓎⰍᅋ䜢ྵ䜐䝥䝸䞁ሷᇶㄏᑟయG5'-GTATCCA AGATTGAA-3'3'-CATAGGT TCTAACTT-5'ᶆⓗ㻰㻺㻭120
1,4-Dioxane

100

Intensity

AV䝥䝻䞊䝤a)
140c)

THF

80

Acetonitrile

60

DMSOd)
䝥䝻䞊䝤䛾䜏
䝥䝻䞊䝤䠇ᶆⓗDNA

MethanolIntensity

40200㻟㻡㻜 㻠㻜㻜 㻠㻡㻜 㻡㻜㻜 㻡㻡㻜 㻢㻜㻜 㻢㻡㻜 㻣㻜㻜

Wavelength (nm)Emission Maximumb) 600

PVG

550

1

Wavelength (nm)

AVG2
AVA
3500

450400㻟㻡㻠㻡㻡㻡㻢㻡䝥䝻䞊䝤䛾䜏䝥䝻䞊䝤䠇ᶆⓗDNAᅗ6 a) Fluorescence spectra of AVG (10 ȝ0) in various solvents. b) Plots ofemission maximum of PVG , AVG and AVA as a function of ET(30). c)
Fluorescence spectra of single-stranded DNA probe and duplex formed byhybrization with target DNA. Excitation wavelength was 380 nm. d)
Fluorescence image of DNA probe and the duplexes formed with target
DNA under illumination with a transilluminator (365 nm).

NHON
NH

NN

NH2

1,6 HG : R = H
1,6 CNG : R = CN
1,6 AcG : R = Ac
2,7 CNG : R = CN
2,7 AcG : R = Ac

NH200:0OHPetG

౫NNH
NNH2HOO
OH- 0:10:03.
Pyrene labeled f luorescence guanosine derivatives
1,6 HGPetGa)NO
VPyGONNHHOOOHROO
N

1,6 CNG

2,7 CNGb)
1

1

1,6 AcG

0.8
0.60.40.2

1,6 CNG

0.8
0.6
0.4
0.2
00
400

500

600

700

Wavelength (nm)

350

450

550

650

Wavelength (nm)

ᅗ4 a) Normalized fluorescence spectra of PetG, 1,6-HG, 1,6-CNG and 1,6-AcG in THF.
b) Normalized fluorescence spectra of 1,6-CNG and 2,7-CNG in CHCl3.

㻌 䜘䜚㛗Ἴ㛗㡿ᇦ䛷Ⓨග䛧䚸䝋䝹䝞䝖䝣䝹䜸䝻䜽䝻䝭䝑䜽䛺ᛶ㉁䜢᭷䛩䜛໬ྜ≀䛸䛧䛶᪂つ䛾䝢䝺䞁㦵᱁䜢᭷䛩䜛䜾䜰䝜䝅䞁ㄏᑟయ䜢㛤Ⓨ䛧䛯䚹䠎⨨᥮䝢䝺䞁䜢䝁䜰䛸䛧䛶䚸୍᪉䛻ኳ↛䛾᰾㓟ሷᇶ䛾୰䛷᭱䜒㓟໬㟁఩䛾ప䛔䜾䜰䝙䞁ሷᇶ䠄donor䠅䜢䚸䜒䛖୍᪉䛻㟁Ꮚ྾ᘬᇶ䜢ᣢ䛴䝧䞁䝊䞁⎔䠄acceptor䠅䜢䛭䜜䛮䜜୕㔜⤖ྜ䜢௓䛧䛶㐃⤖䛥䛫䜛䛣䛸䛷ʌඹᙺ⣔䛜ᣑ኱䛧䚸䛛䛴ศᏊෆ䝗䝘䞊•䜰䜽䝉䝥䝍䞊ᵓ㐀䜢᭷䛩䜛PushPullᆺ䛾䜾䜰䝜䝅䞁ㄏᑟయ䜢䝕䝄䜲䞁䛧䛯䚹㻌 ᚓ䜙䜜䛯⺯ග᰾㓟ሷᇶ䛻㛵䛧䛶ග໬Ꮫⓗ≉ᛶ䜢ㄪ䜉䛯⤖ᯝ䚸ʌඹᙺ⣔ᣑ኱䛻క䛔⺯ගἼ㛗䛜㛗Ἴ㛗䝅䝣䝖䛧䛶䛔䜛䛣䛸䜢☜ㄆ䛧䛯䚹䜎䛯䚸2,7-⨨᥮䝢䝺䞁㦵᱁䜢᭷䛩䜛㓄ྥᛶ䛾␗䛺䜛䜾䜰䝜䝅䞁ㄏᑟయ䜒ྠᵝ䛻ྜᡂ䜢⾜䛔䚸୧⪅䛾ගᏛ≉ᛶ䛾㐪䛔䛻䛴䛔䛶ẚ㍑᳨ウ䜢⾜䛳䛯⤖
ᯝ1,6-CNG䛿⁐፹ᴟᛶ䛾ኚ໬䛻క䛔⺯ගⓎගᙉᗘ䚸Ἴ㛗䛜኱䛝䛟ኚ໬䛧䝋䝹䝞䝖䝣䝹䜸䝻䜽䝻䝭䝑䜽䛺ᛶ㉁䜢᭷䛩䜛䛣䛸䛜ศ䛛䛳䛯䚹୍᪉䚸
2,7- CNG䛷䛿㢧ⴭ䛺⺯ග䛾ኚ໬䛿ほᐹ䛥䜜䛪㓄ྥᛶ䛾㐪䛔䛻䜘䜚ගᏛ≉ᛶ䛜኱䛝䛟␗䛺䜛䛣䛸䛜☜ㄆ䛥䜜䛯䚹㻌 ᮎ➃䛻㟁Ꮚ྾ᘬᛶ⨨᥮ᇶ䛷䛒䜛䝅䜰䝜ᇶ䜔䜰䝉䝏䝹ᇶ䜢᭷䛩䜛 CBG䜔 ABG䛿䝢䝺䞁ㄏ ᑟ య 䛸 ྠ ᵝ 䛻 ⣙ 500 ࠥ550nm䛸㛗Ἴ㛗㡿ᇦ䛷㢧ⴭ䛺䝋䝹䝞䝖䝣䝹䜸䝻䜽䝻䝭䝑䜽䛺ᛶ㉁䜢♧䛧䛯䚹୍᪉䚸ẚ㍑䛸䛧䛶ྜᡂ䛧䛯㟁Ꮚ౪୚ᛶ⨨᥮ᇶ䜢᭷䛩䜛MBG䜔DABG䛿䚸⺯ගⓎගᙉᗘ䛿኱䛝䛔䜒䛾䛾䝋䝹䝞䝖䝣䝹䜸䝻䜽䝻䝭䝑䜽䛺ᛶ㉁䜢♧䛥䛺䛛䛳䛯䚹䛣䛾
䛣䛸䛛䜙Push-Pullᆺ䛾ㄏᑟయ䛾䜏䛜䝋䝹䝞䝖䝣䝹䜸䝻䜽䝻䝭䝑䜽䛺ᛶ㉁䜢♧䛩䛣䛸䛜ศ䛛䛳䛯䚹䝆䜶䞁ᵓ㐀䜢᭷䛩䜛Push-Pullᆺ䜾䜰䝜䝅䞁ㄏᑟయ䝢䝺䞁㦵᱁䜢᭷䛩䜛Push-Pullᆺ䜾䜰䝜䝅䞁ㄏᑟయ

Normalized IntensityNNHHOa)㻌 ⺯ගⰍ⣲Apa䛸䛭䜜䜢ᑟධ䛧䛯 ApaG䜢ྜᡂ䛧䚸䛭䛾ග໬Ꮫⓗ䛺≉ᛶ䜢ㄪ䜉䛯䚹䛭䛾⤖ᯝ䚸⁐፹ᴟᛶ䛾ኚ໬䛻క䛖㢧ⴭ䛺Stokes shiftኚ໬䛜ほᐹ䛥䜜䛯䚹䜎䛯䚸䛭䛾㝿䛾⺯ග
ⓎගἼ㛗䛿PRODAN䜔䛭䛾ㄏᑟయ䛷䛒䜛Anap䛻ẚ䜉40
ࠥ80nm㛗Ἴ㛗ഃ䛻䝅䝣䝖䛧䛶䛔䜛䛣䛸䛜ศ䛛䛳䛯 䚹ApaG䛿࿘㎶䛾⎔ቃኚ໬䛻క䛔㢧ⴭ䛺⺯ගኚ໬䜢♧䛩䛣䛸䛛䜙䚸⎔ቃឤᛂᆺ䛾⺯ග᰾㓟䛸䛧䛶฼⏝䛷䛝䜛䛸⪃䛘䜙䜜䜛䚹OOHNNormalized Intensity䝢䝺䞁ㄏᑟయApa 䜢䜾䜰䝙䞁ሷᇶ䛻ᑟධ䛧䛯⺯ග᰾㓟㻌 ๓㏙䛾䛔䛟䛴䛛䛾䜰䝥䝻䞊䝏ἲ䛷⎔ቃឤᛂᆺ䛾⺯ග᰾㓟ሷᇶ䜢๰ฟ䛧䛯䛜䚸䛭䛾୰䛷᭱䜒䝅䞁䝥䝹䛺ᵓ㐀䛷䚸䛛䛴㢧ⴭ䛻䝋䝹䝞䝖䝣䝹䜸䝻䜽䝻䝭䝑䜽䛺ᛶ㉁䜢
♧䛩⺯ග᰾㓟ሷᇶ䛷䛒䜛AVG䜢⺯ගDNA䝥䝻䞊䝤䛻ᑟධ䛧䚸⺯ගⰍ䛾㐪䛔䛷ᶆⓗDNA䜢᳨ฟ䛷䛝䜛䛛᳨ウ䜢⾜䛳䛯䚹 AVG䜢ྵ䜐16mer䛛䜙䛺䜛DNA䝥䝻䞊䝤䜢ᐇ㝿䛻ྜᡂ䛧䚸ᶆⓗDNA䛻┦ᙜ䛩䜛DNA㙐䛾᭷↓䛻䜘䜛⺯ග䝇䝨䜽䝖䝹䛾ኚ໬䜢 ᐃ䛧䛯䚹䛭䛾㝿䝥䝻䞊䝤DNA䛿䚸ᶆⓗDNA䛸䛾䝝䜲䝤䝸䝎䜲䝈䛻䜘䜚ಟ㣭᰾㓟㒊఩䛜䝞䝹䝆ᵓ㐀䜢ᙧᡂ䛩䜛䚸ᴟᛶ⎔ቃኚ໬䛜䜘䜚኱䛝䛟䛺䜛䜘䛖䛻䝕䝄䜲䞁䛧䛯䜒䛾䜢⏝䛔䛯䚹㻌 ⺯ග䝇䝨䜽䝖䝹䜢 ᐃ䛧䛯⤖ᯝ䚸ᶆⓗDNA䜢ຍ䛘䜛䛣䛸䛻䜘䜚⺯ගᙉᗘ䛜኱䛝䛟䛺䜚䚸Ἴ㛗䜒⣙25nm䝤䝹䞊䝅䝣䝖䛧䛯䚹䛣䛾ኚ໬䛿ᶳⰍ䛛䜙㯤Ⰽ䜈䛾ኚ໬䛸䛧䛶⫗║䛷䜒ᐜ᫆䛻ほᐹ䛷䛝䚸ᶆⓗDNA䛾᭷↓䜢Ⰽ䛾ኚ໬䛷㆑ูྍ⬟䛷䛒䜛䛣䛸䛜ศ䛛䛳䛯䚹ET (30)

ICT/LE⺯ග䜢฼⏝䛧䛯⎔ቃឤᛂᆺ⺯ග᰾㓟ሷᇶ㻌 ᪂䛯䛻䚸ᚑ᮶䛾ᴟᛶ⎔ቃឤᛂᆺศᏊ䛻䛽䛨䜜䛾せ⣲䜢㏣ຍ䛧䛯䠎✀㢮䛾Ⓨග䝰䞊䝗䜢♧䛩⎔ቃឤᛂᆺ⺯ග᰾㓟ሷᇶ䛾㛤Ⓨ䜢⾜䛺䛳䛯䚹㻌 㻌㻌 ᡃ䚻䛿᭱㏆䚸ศᏊ䛾ᅇ㌿䛻䜘䜛ᖹ㠃ᛶ䛾ኚ໬䛻䜘䜚䚸LE⺯ග䛸ICT⺯ග䛾2✀㢮䛾Ⓨග䝰䞊䝗䜢ษ䜚᭰䛘䜛᪂䛧䛔⺯ග᰾㓟ሷᇶ䛾㛤Ⓨ䛻ᡂຌ䛧䛶䛚䜚䚸䛣䜜䜢DNA㙐䛻ᑟධ䛩䜛䛣䛸䛷䚸ᚑ᮶䜘䜚኱䛝䛺Ἴ㛗ኚ໬䛷ᑐ㠃ሷᇶ䛾✀㢮䜢㆑ู䛩䜛䛣䛸䛜ྍ⬟䛸䛺䛳䛯䚹㻌 ᕥᅗ䛻♧䛩䜘䛖䛻䚸᪂䛯䛻㛤Ⓨ䛧䛯⺯ගDNA䝥䝻䞊䝤䜢⏝䛔䜛䛣䛸䛷⺯ගἼ㛗䛾㐪䛔䛷䝣䝹䝬䝑䝏㓄ิ䠄䝏䝭䞁ሷᇶ䠅䛾㆑ู䛜ྍ⬟䛷䛒䜛䚹NC

major groove
RNN

H
N H
NLE emission

Omajorgroove

H N

twistedNNONA

ICT emission
NH2O

minorgroove

H NN

R

N

cnaO

major groove

NNH2

NO

7-substituted 8-aza-7-deazaA : T pair

HN HNO

minor groove

N㽢NNO
OO

minor groove

O3nzA

3-substituted 3-deazaA : T pair

NN(1)coplanarA G C T ssᅗ 䠓 Discrimination ofThymine by fluorescenceemission.

ᛂ⏝ศ㔝䞉⏝㏵䠄Applications䠅䖃㻌 ᪂䛧䛔䝁䞁䝉䝥䝖䛻ᇶ䛵䛔䛯 SNPs᳨ฟ⏝DNA䝏䝑䝥᪥ᮏ኱Ꮫ⏘ᐁᏛ㐃ᦠ▱㈈䝉䞁䝍䞊㻌䠄㻺㼁㻮㻵㻯㻕㻌
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