hydrogen peroxide-triggered gene silencing in mammalian ... · s1 supplementary information...
TRANSCRIPT
S1
Supplementary Information
Hydrogen peroxide-triggered gene silencing in mammalian cells
through boronated antisense oligonucleotides
Shohei Mori,a Kunihiko Morihiro,*a,b† Takumi Okuda,a Yuuya Kasahara,a,b and Satoshi Obika*a,b
a Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
b National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan
* Corresponding authors. E-mails: [email protected] (K.M.); [email protected] (S.O.)
† Present address: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo
Contents
1. Synthesis of boronated nucleoside analogues and dTB phosphoramidite S2
2. 1H-, 13C- and 31P-NMR spectra of new compounds S10
3. H2O2-decaging of boronated nucleosides S31
4. Peroxynitrite (ONOO-)-decaging of dTBpin S32
5. ESI and MALDI-TOF MS analysis of dTB-modified ODNs S33
6. UV melting experiments of duplexes containing dTB without or with H2O2 S43
7. Reference S44
Electronic Supplementary Material (ESI) for Chemical Science.This journal is © The Royal Society of Chemistry 2017
S2
1. Synthesis of boronated nucleoside analogues and dTB phosphoramidite
1-1. 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)1H-imidazole-1-carboxylate (17)
Na2CO3 (3.5 g, 33.0 mmol) was placed in a flame dried round-bottom flask and triphosgene (2.2 g, 7.4 mmol) in
toluene (15 mL) was added at 0 ˚C. After stirring for 1 h at 0 °C, benzyl alcohol 1 (0.87 g, 3.7 mmol) in toluene (5 mL)
was added and stirred for 6 h at room temperature. The insoluble residues were filtered off through a Celite pad. After
the solvent was removed in vacuo, the resulting chloroformate 16S1 was used without further purification.
Chloroformate 16 (1.09 g, 3.70 mmol) was dissolved in dry toluene (20 mL) and 1H-imidazole (1.00 g, 14.8 mmol) was
added at room temperature. The reaction mixture was stirred for 4 h at room temperature and partitioned between
AcOEt and H2O. The separated organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo.
The residue was purified by a silica gel column chromatography, eluted with hexane/AcOEt (2:1) to give compound 17
(1.06 g, 87% over two steps) as a white foam. IR (KBr): ν 3130 (Ar C-H), 1762 (C=O), 1615 (C=N) cm–1; 1H-NMR (300
MHz, CDCl3): δ 8.15 (1H, s), 7.87 (2H, d, J = 6.0 Hz), 7.48-7.41 (3H, m), 7.05 (1H, s), 5.04 (2H, s), 1.34(12H, s); 13C-
NMR (100 MHz, CDCl3): δ 148.2, 136.8, 136.5, 134.9, 130.4, 127.4, 116.8, 83.6, 69.3, 24.6; FAB-LRMS m/z = 329
(MH+); FAB-HRMS calcd for C17H22N2O4B = 329.1676, found 329.1668.
1-2. 3-Ethyl-1-(((4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)carbonyl-1H-imidazol-3-ium
tetrafluoroborate (2)
Compound 17 (1.21 g, 3.70 mmol) was dissolved in dry DCM (40 mL) and Et3OBF4 (669 mg, 3.52 mmol) was added
at room temperature. The reaction mixture was stirred for 16 h at room temperature and the resulting imidazolium salt
2 was used without further purification.
1 16
ON
B O
O
O
NO
B O
O
Cl
O
1H-imidazole
toluene, rt 87% over 2 steps
17
HO
B O
Otriphosgene
Na2CO3
toluene, rt
Et3OBF4
DCM, rt 17
ON
B O
O
O
N
2
ON
B O
O
O
NEt
BF4-
S3
1-3. (4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl) -3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3,diyl)-2’-
deoxy thymidine (18)
Compound 3 (160 mg, 0.297 mmol) was dissolved in dry MeCN (5 mL) and 2-(4-hydroxymethylphenyl)-4,4,5,5-
tetramethyl-1,3,2-dioxaborolane (139 mg, 0.594 mmol) and DBU (89 µL, 0.594 mmol) were added at room
temperature. The reaction mixture was stirred for 4 h at room temperature and the solvent was removed in vacuo. The
residue was purified by a silica gel column chromatography, eluted with hexane/AcOEt (7:3) to give compound 18
(204 mg, 98%) as a white foam. IR (KBr): ν 2943 (Ar C-H), 1670 (C=O), 1532 (C=N) cm–1; [α]D24 25.0 (c 1.00, CHCl3);
1H-NMR (300 MHz, CDCl3): δ 7.86-7.75 (3H, m), 7.39 (2H, dd, J = 12.0, 7.5 Hz), 6.05 (H, d, J = 6.5 Hz), 5.43 (2H, dd,
J = 13.0, 16.0 Hz), 4.46-4.38 (1H, m), 4.19 (1H, d, J = 13.0 Hz), 4.03-4.00 (1H, m), 3.80-3.78 (1H, m), 2.62-2.52 (1H,
m), 2.38-2.31 (1H, m), 1.99 (3H, s), 1.34 (12H, s), 1.14-0.95 (28H, m) ; 13C-NMR (75 MHz, CDCl3): δ 169.9, 155.4,
144.5, 139.4, 138.7, 134.7, 134.6, 126.9, 125.7, 103.8, 84.9, 84.8, 83.5, 83.4, 83.5, 83.4, 77.2, 68.3, 66.3, 64.4, 59.5,
39.6, 24.6, 17.2, 17.1, 17.0, 16.8, 16.7, 16.6, 13.2, 12.7, 12.4, 12.2, 12,1; MS (FAB) m/z 723 [M+Na]+; HRMS (FAB):
Calcd for C35H57N2O8BSi2Na [M+Na]+: 723.3644. Found: 723.33651.
1-4. (4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-2’-deoxy thymidine (4)
To a solution of 18 (160 mg, 0.227 mmol) in dry THF (2.5 mL) was added 1 M TBAF solution in THF (478 µL, 0.478
mmol) was added dropwise at 0 °C, and the reaction mixture was stirred for 10 min. and then concentrated in vacuo.
O
O
O N
N
O
N
NN
Si
Si
ODBU
MeCN, rt
98% 3
HO
B O
O
O
O
O N
N
OSi
Si
O
O
BO
O
18
O
OH
HO N
N
O
O
BO
O
4
O
O
O N
N
OSi
Si
O
O
BO
O
18
TBAF
THF, 0 °C 72%
S4
The residue was purified by a silica gel column chromatography, eluted with AcOEt/MeOH (97:3) to give 4 (78 mg,
72%) as a white foam. IR (KBr): ν 3335 (-OH), 2977 (Ar C-H), 1752 (C=O), 1660 (C=N) cm–1; [α]D24 33.5 (c 1.00,
MeOH); 1H-NMR (300 MHz, CD3OD): δ 8.08 (1H, s), 7.64 (2H, d, J = 8.0 Hz), 7.32 (2H, d, J = 8.0 Hz), 6.14 (1H, dd, J
= 6.0, 6.5 Hz), 5.31 (2H, s), 4.30-4.26 (1H, m), 3.88-3.84 (1H, m), 3.74 (1H, dd, J = 3.0, 12.0 Hz), 3.64 (1H, dd, J = 3.0,
12.0 Hz), 2.95-2.90 (1H, m), 2.34-2.28 (1H, m), 2.10-2.03 (1H, m), 1.89 (3H, s), 1.22 (12H, s); 13C-NMR (75 MHz,
CD3OD): δ 171.7, 158.0, 142.4, 140.4, 135.9, 128.1, 106.5, 89.1, 87.9, 85.1, 79.5, 71.6, 69.6, 62.4, 54.0, 42.2, 27.1,
25.2, 21.0, 14.0, 12.3; MS (FAB) m/z 481 [M+Na]+; HRMS (FAB): Calcd for C23H31N2O7BNa [M+Na]+: 481.2122.
Found: 481.2126.
1-5. (6-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxycarbonyl-2’-deoxy guanosine (6)
Compound 5 (300 mg, 0.585 mmol) was dissolved in dry 1,4-dioxane (6 mL) and Ph3P (184 mg, 0.702 mmol), DIAD
(138 µL, 0.702 mmol) and 2-(4-hydroxymethylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (144 mg, 0.614 mmol)
were added at room temperature. The reaction mixture was stirred for 4 h at room temperature and cooled in an ice
bath. TBAF solution in THF (1 M, 1.23 mL, 1.23 mmol) was added dropwise at 0 ˚C and the resulting mixture was
stirred for 10 min. The solvent was removed in vacuo and the residue was purified by a silica gel column
chromatography, eluted with AcOEt/MeOH (19:1) to give compound 6 (28 mg, 10%) as a white foam. IR (KBr): ν 3329
(-OH), 1585 (C=N) cm–1; [α]D24 −3.6 (c 1.00, MeOH); 1H-NMR (300 MHz, CD3OD): δ 8.03 (H, s), 7.91 (2H, s), 7.60 (2H,
d, J = 8.0 Hz), 7.40 (2H, d, J = 8.0 Hz), 6.30 (1H, dd, J = 6.5, 9.0 Hz), 5.50 (2H, s), 4.58-4.51 (1H, m), 4.06-4.00 (1H,
m), 3.86-3.67 (2H, m), 2.85-2.70 (1H, m), 2.38-2.27 (1H, m), 1.27-1.20 (12H, m); 13C-NMR (75 MHz, CD3OD): δ 180.1,
162.2, 161.4, 154.2, 140.0, 134.6, 128.3, 115.9, 89.7, 86.8, 79.5, 73.1, 69.4, 63.7, 47.4, 41.2, 24.1, 9.4, 9.3; MS (FAB)
m/z 506 [M+Na]+; HRMS (FAB): Calcd for C23H30N5O6BNa [M+Na]+: 506.3219. Found: 506.3224.
O
OH
HO
N
NN
N
O
NH2
BO
O
O
O
OSi
Si
O
NH
NN
N
O
NH2 PPh3, DIAD then TBAF
1,4-dioxane, rt
10% 5 6
HO
B O
O
S5
1-6. (6-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxycarbonyl-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3,diyl)-2’-deoxy adenosine (19)
Compound 7 (124 mg, 0.25 mmol) was dissolved in dry DCM (10 mL) and compound 2 (444 mg, 1.00 mmol) in DCM
(10 mL) was added at 0 °C. The reaction mixture was stirred for 24 h at room temperature and quenched by addition
of saturated aqueous NaHCO3. The resulting mixture was partitioned between DCM and H2O. The separated organic
layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by a silica gel
column chromatography, eluted with hexane/AcOEt (4:1) to give compound 19 (172 mg, 91%) as a white foam. IR
(KBr): ν1758 (C=O), 1615 (C=N) cm–1; [α]D24 −23.4 (c 1.00, CHCl3); 1H-NMR (300 MHz, CDCl3): δ 10.24 (1H, s), 8.74
(1H, s), 8.04 (1H, s), 7.81 (2H, d, J = 8.0 Hz), 7.39 (2H, d, J = 8.0 Hz), 6.00 (1H, d, J = 6.5 Hz), 5.36-5.23 (2H, m),
5.16-5.08 (1H, m), 4.03-3.98 (2H, m), 3.92-3.84 (1H, m), 2.81-2.56 (2H, m), 1.36 (12H, s), 1.13-1.02 (28H, m); 13C-
NMR (75 MHz, CDCl3): δ 152.3, 151.1, 149.9, 149.5, 144.1, 141.9, 138.1, 134.8, 134.6, 127.6, 125.7, 122.4, 84.9,
83.6, 83.4, 83.3, 77.2, 70.0, 67.2, 64.4, 61.7, 39.4, 24.6(2), 24.5(9), 17.3, 17.0(9), 17.0(7), 17.0(6), 16.9, 16.8, 16.7,
16.6, 13.0, 12.8, 12.5, 12.2; MS (FAB) m/z 754 [M+H]+; HRMS (FAB): Calcd for C36H57N5O8BSi2 [M+H]+: 754.3839.
Found: 754.3846.
1-7. (6-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxycarbonyl-2’-deoxy adenosine (8)
O
O
OSi
Si
O
N
NN
N
NH2
7
O
O
OSi
Si
O
N
NN
NHN O
O
BO
O
19
2
DCM, 0 °C to rt 91%
O
OH
HO
N
NN
N
HN O
O
BO
O
8 O
O
OSi
Si
O
N
NN
NHN O
O
BO
O
19
TASF
DMF, 0 °C 62%
S6
Compound 19 (1.19 g, 1.59 mmol) was dissolved in dry DMF (20 mL) and TASF (1.00 g, 3.63 mmol) was added at
0 °C. The reaction mixture was stirred for 1 h at 0 °C and partitioned between CHCl3/2-propanol (3:1) and H2O. The
separated organic layer was concentrated in vacuo. The residue was purified by a silica gel column chromatography,
eluted with CHCl3/MeOH (19:1) to give compound 8 (520 mg, 62%) as a white foam. IR (KBr): ν 3293 (-OH), 2977 (Ar
C-H), 1757 (C=O), 1619 (C=N) cm–1; [α]D24 1.2 (c 1.00, DMSO); 1H-NMR (300 MHz, DMSO-d6): δ 10.80 (1H, brs),
8.66 (1H, s), 8.63 (1H, s), 7.68 (2H, d, J = 8.0 Hz), 7.46 (2H, d, J = 8.0 Hz), 6.44 (1H, dd, J = 6.5, 7.0 Hz), 5.23 (2H, s),
4.47-4.39 (1H, m), 3.91-3.84 (1H, m), 3.66-3.46 (2H, m), 2.82-2.70 (1H, m), 2.39-2.25 (1H, m), 1.29 (12H, s); 13C-
NMR (75 MHz, DMSO-d6): δ 152.1, 151.6, 151.5, 149.7, 142.8, 139.8, 134.5, 126.9, 123.8, 88.0, 83.7(5), 83.7(2),
70.7, 66.0, 61.6, 48.6, 25.5, 24.7; MS (FAB) m/z 512 [M+H]+; HRMS (FAB): Calcd for C24H31N5O7B [M+H]+: 512.2317.
Found: 512.2321.
1-8. (4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxycarbonyl-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3,diyl)-2’-deoxy cytidine (20)
Compound 9 (416 mg, 0.88 mmol) was dissolved in dry DCM (10 mL) and compound 2 (1.56 g, 3.50 mmol) in DCM
(10 mL) was added at 0 °C. The reaction mixture was stirred for 24 h at room temperature. and quenched by addition
of saturated aqueous NaHCO3. The resulting mixture was partitioned between DCM and H2O. The separated organic
layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by a silica gel
column chromatography, eluted with hexane/AcOEt (4:1) to give compound 20 (593 mg, 92%) as a white foam. IR
(KBr): ν 3151 (Ar C-H), 1747 (C=O), 1622 (C=N) cm–1; [α]D24 27.7 (c 1.00, CHCl3); 1H-NMR (300 MHz, CDCl3): δ 8.80
(1H, drs), 8.22 (1H, d, J = 7.5 Hz), 7.81 (2H, d, J = 8.0 Hz), 7.37 (2H, d, J = 8.0 Hz), 7.23 (1H, d, J = 7.5 Hz), 6.03 (1H,
d, J = 7.0 Hz), 5.21 (2H, s), 4.43-4.30 (1H, m), 4.21 (1H, d, J = 13.0 Hz), 4.02 (1H, dd, J = 3.0, 13.0 Hz), 3.81 (1H, d, J
= 8.0 Hz), 2.63-2.49 (1H, m), 2.41-2.28 (1H, m), 1.34 (12H, s), 1.13-0.92 (28H, m); 13C-NMR (75 MHz, CDCl3): δ
162.4, 137.9, 135.0, 134.8, 134.7, 134.6, 126.9, 125.7, 85.3, 84.9, 83.5(4), 83.5(1), 83.4, 77.2, 67.3, 66.1, 59.5, 39.3,
24.6, 17.2, 17.1(6), 17.0(8), 17.0(1), 16.7(5), 16.6(9), 16.6(5), 16.6, 13.1, 12.7, 12.6, 12.1; FAB-LRMS m/z = 752
O
O
OSi
Si
O
N
N
NH2
O
9
2
DCM, 0 °C to rt 92%
20
O
O
O
N
N
HN
O
O
O
BO
O
Si
Si
O
S7
(MNa+); FAB-HRMS calcd for C35H56O9N3BSi2Na= 752.3546, found 752.3553; MS (FAB) m/z 752 [M+Na]+; HRMS
(FAB): Calcd for C35H56N3O9BSi2Na [M+Na]+: 752.3546. Found: 752.3553.
1-9. (4-(4,4,5, 5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxycarbonyl-2’-deoxy cytidine (10)
Compound 20 (1.99 g, 2.70 mmol) was dissolved in dry pyridine (20 mL) and HF-pyridine (ca 65% HF w/w. 604 µL,
10.8 mmol) was added at room temperature. The reaction mixture was stirred for 12 h at 60 °C and cooled to room
temperature. The reaction was quenched by addition of solid NaHCO3 and the insoluble residues were filtered off
through a Celite pad. After the solvent was removed in vacuo, the residue was purified by a silica gel column
chromatography, eluted with CHCl3/MeOH (9:1) to give compound 10 (684 mg, 52%) as white foam. IR (KBr): ν 3331
(-OH), 2979 (Ar C-H), 1750 (C=O), 1651 (C=N) cm–1; [α]D24 55.7 (c 1.00, MeOH); 1H-NMR (300 MHz, CD3OD): δ 8.37
(1H, d, J = 7.5 Hz), 7.68 (2H, d, J = 8.0 Hz), 7.32 (2H, d, J = 8.0 Hz), 7.22 (H, d, J = 7.5 Hz), 6.15 (1H, t, J = 6.0 Hz),
5.14 (2H, s), 4.37-4.29 (1H, m), 4.00-3.92 (1H, m), 3.79 (1H, dd, J = 3.0, 12.0 Hz), 3.69 (1H, dd, J = 3.0, 12.0 Hz),
2.49-2.38 (1H, m), 2.17-2.06 (1H, m), 1.26 (12H, s); 13C-NMR (75 MHz, CDCl3): δ 163.7, 156.6, 156.5, 153.4, 144.9,
139.2, 134.9, 128.5, 128.4, 128.2, 127.2(3), 127.1(6), 95.6, 88.3, 87.5, 84.1, 70.5, 67.3, 61.4, 41.4, 24.2, 24.0, 17.4;
FAB-LRMS m/z = 488 (MH+); FAB-HRMS calcd for C23H31N3O8B= 488.2119, found 488.2216; MS (FAB) m/z 488
[M+H]+; HRMS (FAB): Calcd for C23H31N3O8B [M+H]+: 488.2119. Found: 488.2119.
1-10. 5’-O-(4,4’-Dimethoxytrityl)-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-2’-deoxy thymidine (21)
10 O
OH
HO
N
N
HN
O
O
O
BO
O
HF-pyridine
pyridine, 60 °C 52%
20 O
O
O
N
N
HN
O
O
O
BO
O
Si
Si
O
O
OH
HO N
N
O
O
BO
O
4
DMTrCl
pyridine, rt 96% 21
O
OH
DMTrO N
N
O
O
BO
O
S8
Compound 4 (450 mg, 0.945 mmol) was dissolved in dry pyridine (10 mL) and DMTrCl (384 mg, 1.13 mmol) was
addedat room temperature. The reaction mixture was stirred for 3 h at room temperature and quenched by addition of
MeOH at 0 °C with 10 min. stirring. After the solvent was removed in vacuo, the residue was purified by a silica gel
column chromatography, eluted with CHCl3/MeOH (19:1 with 0.5% Et3N) to give compound 21 (705 mg, 96%) as a
white foam. IR (KBr): ν 3455 (-OH), 2978 (Ar C-H), 1700 (C=O), 1642 (C=N) cm–1 [α]D24 8.8 (c 1.00, CHCl3); 1H-NMR
(300 MHz, CDCl3): δ 8.54-8.50 (2H, m), 7.79-7.74 (2H, m), 7.69-7.63 (2H, m), 7.51-7.38 (4H, m), 7.32-7.18 (6H, m),
6.85-6.78 (4H, m), 6.50-6.42 (1H, m), 5.11(2H, s), 4.63-4.57 (1H, m), 4.12-4.07 (1H, m), 3.74 (6H, s), 3.51-3.45 (1H,
m), 3.38-3.31 (1H, m), 2.48-2.24 (2H, m), 1.55 (3H, s), 1.31 (12H, s); 13C-NMR (75 MHz, CDCl3): δ 163.3, 163.2,
158.4, 150.7, 150.6, 149.1, 144.2, 139.8, 136.7, 136.2, 135.2, 135.1, 134.7, 133.8, 129.9, 128.9, 128.2, 128.1, 127.9,
127.7, 127.3, 126.9, 123.8, 113.0, 110.1, 110.0, 86.6, 86.1, 85.3, 83.5, 77.2, 74.7, 71.4, 63.3, 55.0, 44.3, 41.0, 24.6,
12.4; MS (FAB) m/z 783 [M+Na]+; HRMS (FAB): Calcd for C44H49N2O9BNa [M+Na]+: 783.3429. Found: 783.3436.
1-11. 3-O-{2-Cyanoethyl(diisopropylamino)phosphino}-5’-O-(4,4’-dimethoxytrityl)- (4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzyl)-2’-deoxy thymidine (11)
Compound 21 (663 mg, 0.850 mmol) was dissolved in dry DCM (10 mL) and N,N-diisopropylamine (440 µL, 2.55
mmol) and 2-cyanoethyl-N,N’-diisopropylchlorophosphoramidite (230 µL, 1.02 mmol) were added at room temperature.
The reaction mixture was stirred for 2 h and partitioned between AcOEt and H2O. The separated organic layer was
washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by a silica gel column
chromatography, eluted with hexane/AcOEt (6:4) to give compound 11 (400 mg, 48%) as a white foam. IR (KBr): ν
2244 (C≡N), 1671 (C=N) cm–1 1H-NMR (300 MHz, CDCl3): δ 8.03-7.93 (1H, m), 7.83 (2H, d, J= 7.5 Hz), 7.48-7.39 (4H,
m), 7.35-7.24 (7H, m), 6.89-6.79 (4H, m), 6.49-6.36 (1H, m), 5.43 (2H, s), 4.78-4.59 (1H, m), 4.30-4.17 (1H, m), 3.77
(3H, s), 3.76 (3H, s), 3.66-3.45 (4H, m), 3.45-3.27 (2H, m), 2.79-2.26 (4H, m), 1.49 (3H, s), 1.34 (12H, s), 1.26-1.03
(12H, m); 13C-NMR (75 MHz, CDCl3): δ 169.8, 169.7, 158.3, 155.5, 155.4, 144.0, 139.6, 138.6, 135.0, 134.9, 134.7,
129.9, 129.8, 127.9, 127.8, 127.6, 126.8, 117.4, 117.2, 112.9, 104.6, 104.5, 86.5, 86.4, 85.9, 83.5, 77.2, 74.5, 68.2,
21
O
OH
DMTrO N
N
O
O
BO
O
11
O
O
DMTrO N
N
O
O
BO
O
Pi-Pr2N O
CN
DIPEA
MeCN, rt 48%
Pi-Pr2N O
CNCl
S9
58.0, 57.8, 54.9, 54.8, 42.9, 42.8 (d, J (C, P) = 5.0 Hz)), 42.7, 24.5, 24.4, 24.3, 24.2, 24.1 (d, J (C, P) = 7.0 Hz), 20.1,
19.9, 19.8, 19.7, 11.3, 44.9 (d, J (C, P) = 5.0 Hz), 24.4, 24.3, 22.8, 22.7, 20.2; 31P-NMR (120 MHz, CDCl3): δ 149.57,
148.96; FAB-LRMS m/z = 961 (MH+); FAB-HRMS calcd for C53H67BN4O10P= 961.4688, found 961.4697.
S10
PPM
12.5
10.0
7.5
5.0
2.5
0.0
single_pulse
0.93
2.04
3.01
1.00
2.00
12.18
8.157.887.86
7.447.42
7.05
5.41
1.34
2. 1H-, 13C- and 31P-NMR spectra of new compounds
2-1. 1H spectrum of compound 17
S11
PPM
200
150
100
50
0
singl
e puls
e dec
ouple
d g
ated
NO
E
148.20
136.81136.51134.95130.37127.44
116.84
83.64 77.42 77.00 76.57
69.33
24.55
2-2. 13C spectrum of compound 17
S12
PPM
12.5
10.0
7.5
5.0
2.5
0.0
single_pulse
4.30
3.30
1.00
2.03
1.28
1.02
1.031.011.02
0.62
1.151.02
3.10
20.04
26.93
7.857.827.797.767.447.417.387.357.326.076.055.485.445.435.394.734.714.464.434.404.374.214.174.034.003.993.803.783.523.502.582.542.522.382.362.342.311.991.341.331.101.091.071.051.031.031.010.99
2-3. 1H spectrum of compound 18
S13
PPM
200
150
100
50
0
singl
e puls
e dec
ouple
d g
ated
NO
E
169.95
155.39
144.49139.37138.67134.72134.59
126.87125.65
103.81
84.93 84.79 83.55 83.39 77.43 77.20 77.00 76.58 68.33 66.31 64.44
59.50
39.55
24.57 17.24 17.15 17.05 16.81 16.73 16.62 13.17 12.69 12.44 12.15 12.10
2-4. 13C spectrum of compound 18
S14
PPM
10.0
7.5
5.0
2.5
0.0
-2.5
single_pulse
1.00
2.02
2.03
1.00
1.99
2.56
0.98
0.99
2.00
2.052.95
12.36
8.08
7.667.637.337.30
6.176.146.12
5.31
4.814.283.873.853.733.723.673.662.362.342.342.322.312.302.292.282.102.082.052.032.011.891.22
2-5. 1H spectrum of compound 4
S15
PPM
200
150
100
50
0
singl
e puls
e dec
ouple
d g
ated
NO
E
171.67
157.99
142.36140.41
135.89
128.16
106.49
89.08 87.94 85.06
79.46
71.58 69.56
62.41 53.99 49.85 49.56 49.28 49.00 48.71 48.43 48.15 42.24
27.11 25.20 20.99
14.01 12.26
2-6. 13C spectrum of compound 4
S16
2-7. 1H spectrum of compound 6
PPM
12.5
10.0
7.5
5.0
2.5
0.0
single_pulse
1.011.37
1.982.01
1.00
1.99
4.08 1.36
0.99
1.001.000.99
0.88
8.42
1.01
0.96
2.56
12.66
8.02607.89897.61517.59227.41137.38506.32636.30576.27945.50564.95164.92764.55454.54534.53624.03254.02453.84943.84023.80823.79903.74753.73723.70633.69603.30463.30003.10773.09743.08373.07453.05963.03562.81582.79522.77012.75062.72432.35922.35232.33972.33282.31572.30762.29511.89331.26951.25691.24551.23291.2088
S17
2-8. 13C spectrum of compound 6
PPM
200
150
100
50
0
singl
e puls
e dec
ouple
d g
ated
NO
E
180.1072
162.2381161.4254
154.2261
139.9901
134.6265
128.3068
115.8873
89.6525 86.8034
79.4798
73.1219
69.4027
63.7236
49.8509 49.5641 49.2868 49.0000 48.7132 48.4359 48.1491 47.4416 41.1219
24.1228
9.3036
S18
2-9. 1H spectrum of compound 19
PPM
12.5
10.0
7.5
5.0
2.5
0.0
singl
e_puls
e
0.99
1.00
1.03
2.85
3.43
1.00
2.101.13
0.46
2.071.32
0.981.00
16.82
30.40
10.2417
8.7366
8.0384 7.8255 7.7992 7.4078 7.3814 7.3288
6.0159 5.9930 5.3578 5.3166 5.2742 5.2330 5.1552 5.1289 5.1014 5.0762 4.7363 4.7203 3.9980 3.9042 3.8904 3.8790 3.8676 2.9473 2.7939 2.7527 2.7287 2.6829 2.6554 2.6268 2.6108
1.3563 1.3426 1.1022 1.0610
S19
2-10. 13C spectrum of compound 19
PPM
200
150
100
50
0
singl
e puls
e dec
ouple
d g
ated
NO
E
152.3103151.1152149.9583149.5281144.1932141.9273138.1412134.9766134.8140134.6324127.5670127.4714125.6931122.3659
84.9068 83.6256 83.4248 83.2623 77.4207 77.2008 77.0000 76.5698 69.9824 67.2098 64.4467 61.7028
39.4453
24.6165 17.2547 17.0826 17.0539 16.9105 16.7766 16.7097 16.6332 13.0288 12.7994 12.4743 12.2066
S20
2-11. 1H spectrum of compound 8
PPM
12.5
10.0
7.5
5.0
2.5
0.0
singl
e_puls
e
0.96
1.95
2.191.97
1.00
1.002.17
1.01
1.00
1.05
2.110.94
1.50
0.99
1.001.02
13.392.53
10.7940
8.6605 8.6284
7.6933 7.6670 7.4712 7.4461
6.4594 6.4365 6.4148 5.3641 5.2301 5.0321 4.4289 4.1142 3.8830 3.8738 3.6346 3.5968 3.5327 3.4938 3.3542 3.1550 2.8036 2.7830 2.7601 2.7372 2.7166 2.4900 2.3630 2.3515 2.3423 2.3320 2.3195 2.3080 2.2989 2.2886 1.8719 1.2767 1.0535 1.0318 1.0112 0.9334 0.9082
S21
2-12. 13C spectrum of compound 8
PPM
200
150
100
50
0
singl
e puls
e dec
ouple
d g
ated
NO
E
152.0591151.6193151.4567149.6593
142.7564139.7639
134.5246
126.9429123.7687
88.0018
83.7186
70.7064
66.0120
61.6236
48.6305 40.3318 40.0545 39.7773 39.5000 39.2227 38.9455 38.6682
25.5221 24.9867 24.6999
S22
2-13. 1H spectrum of compound 20
PPM
12.5
10.0
7.5
5.0
2.5
0.0
single_pulse
0.94
1.12
2.71
3.090.95
1.00
2.09
1.051.001.031.02
1.010.97
15.23
28.68
8.8076
8.23768.21357.82327.79817.37917.35407.32427.23957.2189
6.04236.0205
5.21365.17814.41474.38494.35634.32884.22814.18464.03923.99573.82183.79432.62002.57652.55812.51692.38302.36132.34072.31782.03281.33691.10111.07251.02551.01870.98660.9729
0.0000
S23
2-14. 13C spectrum of compound 20
PPM
200
150
100
50
0
singl
e puls
e dec
ouple
d g
ated
NO
E
162.3873
154.4519152.2529
143.6769137.8735134.9766134.7854134.5750127.4714126.9360125.6644
94.1712 85.2701 84.8685 83.5491 83.3579 77.4207 77.2008 77.0000 76.5698 74.7819 69.2271 67.2576 66.1103 60.0774 59.4655
39.3210
24.5591 17.2451 17.1591 17.0826 17.0157 16.7480 16.6906 16.6524 16.5663 13.9180 13.0766 12.7229 12.6464 12.0727
S24
2-15. 1H spectrum of compound 10
PPM
10.0
7.5
5.0
2.5
0.0
-2.5
single_pulse
1.02
1.67
2.081.25
1.00
2.01
4.79
1.00
0.981.011.02
4.84
0.96
0.98
9.87 2.11
8.38548.36027.69297.66777.33127.30607.23397.20996.17066.15126.13065.13824.85094.34164.32334.31073.96163.95013.81393.80363.77273.76473.72013.70863.68003.66863.58963.56783.54383.51863.30002.47702.45762.44162.43132.41642.39692.15542.13482.11192.08902.06841.26151.14471.12411.1001
S25
2-16. 13C spectrum of compound 10
PPM
200
150
100
50
0
singl
e puls
e dec
ouple
d g
ated
NO
E
164.6474
157.5246154.3600
145.8317
140.1622
135.8790129.5210129.3489129.2246128.2112
96.6032
89.2605 88.4574 85.0538
75.7989
71.5061 68.2459
62.3469 58.2740 49.8414 49.5641 49.2773 49.0000 48.7132 48.4264 48.1491 42.3648
25.2032 25.0024
18.3672
S26
PPM
12.5
10.0
7.5
5.0
2.5
0.0
single_pulse
1.10
1.391.821.94 1.96
9.81
3.94
1.00
2.07
1.12
1.02
6.05
2.07
1.94
3.06
11.24
8.538.517.787.757.697.677.637.497.467.427.397.317.307.287.277.247.206.836.806.486.466.44
5.11
4.60
4.09
3.743.503.463.363.33
2.422.32
1.451.311.261.22
2-17. 1H spectrum of compound 21
S27
PPM
200
150
100
50
0
singl
e puls
e dec
ouple
d g
ated
NO
E
163.34163.28158.41150.74150.68149.08144.15139.80136.67136.23135.22135.11134.66133.85129.88128.90128.20128.13127.91127.75127.34126.88123.76113.01110.06110.01
86.58 86.12 85.33 83.50 77.43 77.20 77.00 76.58 74.70 71.42
63.28
54.98
44.28 41.02
24.59
12.35
2-18. 13C spectrum of compound 21
S28
PPM
12.5
10.0
7.5
5.0
2.5
0.0
single_pulse
0.54 0.472.31
4.58
8.78
4.13
1.00
2.12
0.94
0.99
7.12
5.74
4.34
3.1812.19
5.1311.84
8.017.957.857.827.447.417.347.347.337.327.317.317.306.866.856.836.826.476.456.436.416.395.515.475.455.414.684.214.203.773.763.623.613.603.583.573.553.523.443.393.363.332.772.752.722.702.682.662.642.622.602.432.412.392.382.362.332.312.291.531.511.341.231.191.171.071.04
2-19. 1H spectrum of compound 11
S29
PPM
200
150
100
50
0
singl
e puls
e dec
ouple
d g
ated
NO
E
169.80169.78
158.34155.53155.49143.99139.61138.65135.04134.99134.65129.88129.79127.91127.82127.64126.84117.36117.18112.89
104.58104.50
86.47 86.45 85.96 83.50 77.43 77.20 77.00 76.58 74.52 68.20
58.01 57.76 54.91 54.88
42.98 42.90 42.82 42.73 24.52 24.36 24.28 24.23 24.19 24.13 20.08 19.98 19.88 19.79 11.31
2-20. 13C spectrum of compound 11
S30
PPM
300
200
100
0-100
-200
-300
singl
e puls
e dec
ouple
d g
ated
NO
E
149.57148.96
2-21. 31P spectrum of compound 11
S31
3. H2O2-decaging of boronated nucleosides
3-1. HPLC chromatograms of dABpin after H2O2 addition at different time points.
3-2. HPLC chromatograms of dCBpin after H2O2 addition at different time points.
3-3. HPLC chromatograms of dGBpin after H2O2 addition at different time points.
0Retention time/min.
Reacti
on tim
e/min.
H2O2
5 10 15 20 0
15
20
dABpin Deoxyadenosine
O
OH
HO
N
NN
N
NH2
O
OH
HO
N
NN
N
HN O
O
BO
O
Deoxycytidine dCBpin
O
OH
HO
N
N
NH2
OO
OH
HO
N
N
HN
O
O
O
BO
O
0Retention time/min.
Reacti
on tim
e/min.
H2O2
5 10 15 20 0
15
20
dGBpin Deoxyguanosine
O
OH
HO
NH
NN
N
O
NH2O
OH
HO
N
NN
N
O
NH2
BO
O
0Retention time/min.
Reacti
on tim
e/min.
H2O2
5 10 15 20 0
15
20
S32
4. Peroxynitrite (ONOO-)-decaging of dTBpin
4-1. HPLC chromatograms of dTBpin after peroxynitrite (ONOO-) addition at different time points.
ONOO-
0Retention time/min.
5 10 15 20
01
12
Reacti
on tim
e / h
S33
4. ESI and MALDI-TOF MS analysis of dTB-modified ODNs
4-1. ON 14 5’-d(GCGTTTBTTTCGT)-3’
HPLC
Column: Waters XBridgeTM OST C18 2.5 µm, 4.6 x 50 mm
Gradient: 8-16%MeCN (over 30 min) in triethylammonium acetate butter (pH 7.0, 0.1 M)
Flow rate: 1.0 mL/min
Column temperature: 50 °C
MALDI-TOF MS
Calcd. 3766.30 [M-H]- / citric acid adduct 3922.39 [M-H]-
2016/12/16 19:10:27 Page 1 / 2
D :\m orishohei\161204 PO caged1.lcd
<クロマトグラム>
<ピークレポート>m in
m AU
0 5 10 15 20 25 300
100
200
300
検出器A C h1 260nmピー ク# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
保持時間 0.067 0.417 0.575 0.678 0.817 0.900 1.060 1.254 1.529 1.650 1.724 1.976 4.924 7.676 8.566 11.176 15.068 20.308 21.442 24.300 24.558
面積 9148 8556 5028 6072 4582 4946 5371 5358 6559 1627 2380 81144 1306 12533
4070164 2117 1393 4881 2441 1339 2105
高さ 701 625 590 1281 834 624 616 498 466 409 405 9868 71 465
232341 131 141 269 132 105 153
濃度 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
単位
マーク V V V V V V V V V V SV V SV T V V V V V
化合物名
111111111111111112
11111111
15551155
15111155
511
514
515
518
115
111
5015
]]]]]]1]]]1]1]
1555 1555 1555 1555 4555 4555 5555 5555 5555 5555 5555zzz
rrrrrrrrrrrrrrrrrrrrr
1515 51 51 18 55 151155 55 55]nn1111n1nnnnnnn1n11ennnnnn1n111111e1e11n11 ]] ] ]0 ]] ]] ] 1 15 ]1 ] ]
ennnnn1n1111e1en1n11111e1 ]]] ]
]y1y1ny11e1ynen1ysn1s1e11n1ne11n 5555]
sn1111n1ne1n1en11nn1e1n1e1nnynsen1n11 ]
] ] ]] ] ] ]] ]5]15]11z] ]] ]1nynsen1n111e1n1e11n11ynn11nn1e
orrrrrrrrrrororE E E rn1e
t1n1en1111 C C C
t1n1en111111ye1 ] ] ] ]0 Cz C]
]]] 11z1z1515 1 15 58]E E ] ] ] ] ] ] ]] ]0E]] s] ]
S34
ESI MS
Calcd. 3731.30 [M - 2 x H2O]
m/z250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 3250 3500 3750 4000 4250 4500 4750
%
0
100 160331 CAGED-OLIGO 8 (0.152) TOF MS ES-
3.38e5745.1398338077
744.9386233594
362.7376156055
620.788956481
745.3411274911
931.6680173509
931.9182141734
936.1751133891
936.4258109415
936.676668215
1242.571765560
936.927442484
941.422127049
1242.903856486
1249.238030124
z = 3 [(M- 2 x H2O) - 3H]-3�
z = 2 [(M- 2 x H2O) - 2H]-2
Found: 1864.3360�
z = 4 [(M- 2 x H2O) - 4H]-4�
z = 5 [(M- 2 x H2O) - 5H]-5�
S35
4-2. ON 14 5’-d(GCGTTTBTTTCGT)-3’ + H2O2
ON 14 was dissolved in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl to give a final strand
concentrateon of 4.0 µM. To the ON 14 solutions was added H2O2 (1 mM) and the resulting sample mixture was
incubated for 30 min at room temperature in advance to the HPLC analysis and mass measurement.
HPLC
Column: Waters XBridgeTM OST C18 2.5 µm, 4.6 x 50 mm
Gradient: 8-16%MeCN (over 30 min) in triethylammonium acetate butter (pH 7.0, 0.1 M)
Flow rate: 1.0 mL/min
Column temperature: 50 °C
MALDI-TOF MS
Calcd. 3632.37 [M-H]-
2016/12/16 19:11:38 Page 1 / 1
D :\m orishohei\161204 natural PO M eC N 8-16 50C 1.lcd
<クロマトグラム>
<ピークレポート>m in
m AU
0 5 10 15 20 25 300
200
400
600
800
1000
検出器A C h1 260nmピー ク# 1 2 3 4 5 6 7 8 9 10 11 12 13 合計
保持時間 0.051 0.292 0.524 0.669 0.807 1.057 1.222 1.561 1.706 1.958 2.459 5.146 8.410
面積 1278 1021 2447 5834 10763 3370 3730 20537 37128
5107685 1878 1913 4712
5202296
高さ 72 143 227 1129 1653 452 412 1935 5740
856931 431 214 540
869881
濃度 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
単位
マーク V V V V V V V SV T V S
化合物名
111111111111111112
333333
33
0
1
2
3
4010
]]]]
]]3]]]
3]3]
2000 3000 4000 3000 3000 0000zzz
rrrrrrrrrrrrrrrrrrrrr
2013 12 03 02 3 133233 00 00]nn1111n1nnnnnnn1n11ennnnnn1n111111e1e11n11 ]] ] ]0 ]] ]] ] 1 4 ]3 ] ]
ennnnn1n1111e1en1n11111e1 ]]] ]
]y1y1ny11e1ynen1ysn1s1e11n1ne11n 2300]
sn1111n1ne1n1en11nn1e1n1e1nnynsen1n11 ]
] ] ]] ] ] ]] ]0]13]23z] ]z ] ]1nynsen1n111e1n1e11n11ynn11nn1e
orrrrrrrrrrororE E E rn1e
t1n1en1111 C C C
t1n1en111111ye1 ] ] ] ]0 Cz C]
]]] 12z3z2013 3 01 4 ]E E ] ] ] ] ] ] ]] ]0E]] s] ]
S36
4-3. ASO S0 5’-TCmagtcatgactTCm-3’
n = DNA N = LNA, all internucleosidic linkages are phosphorothioated
HPLC
Column: Waters XBridgeTM OST C18 2.5 µm, 4.6 x 50 mm
Gradient: 10-40%MeCN (over 30 min) in triethylammonium acetate butter (pH 7.0, 0.1 M)
Flow rate: 1.0 mL/min
Column temperature: 80 °C
MALDI-TOF MS
Calcd. 4561.71 [M-H]-
2016/12/16 19:10:27 Page 1 / 2
D :\m orishohei\161204 PO caged1.lcd
<クロマトグラム>
<ピークレポート>m in
m AU
0 5 10 15 20 25 300
100
200
300
検出器A C h1 260nmピー ク# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
保持時間 0.067 0.417 0.575 0.678 0.817 0.900 1.060 1.254 1.529 1.650 1.724 1.976 4.924 7.676 8.566 11.176 15.068 20.308 21.442 24.300 24.558
面積 9148 8556 5028 6072 4582 4946 5371 5358 6559 1627 2380 81144 1306 12533
4070164 2117 1393 4881 2441 1339 2105
高さ 701 625 590 1281 834 624 616 498 466 409 405 9868 71 465
232341 131 141 269 132 105 153
濃度 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
単位
マーク V V V V V V V V V V SV V SV T V V V V V
化合物名
111111111111111112
222222
22
222
222
022
022
2002
]]]]
]]2]]]
2]2]
2222 0222 2222 2222 2222 0222zzz
rrrrrrrrrrrrrrrrrrrrr
2202 02 2 22 2 222000 22 22]nn1111n1nnnnnnn1n11ennnnnn1n111111e1e11n11 ]] ] ]0 ]] ]] ] 0 2 ]2 ] ]
ennnnn1n1111e1en1n11111e1 ]]] ]
]y1y1ny11e1ynen1ysn1s1e11n1ne11n 2222]
sn1111n1ne1n1en11nn1e1n1e1nnynsen1n11 ]
] ] ]] ] ] ]] ]0]02]20z] ]z ] ]1nynsen1n111e1n1e11n11ynn11nn1e
orrrrrrrrrrororE E E rn1e
t1n1en1111 C C C
t1n1en111111ye1 ] ] ] ]0 Cz C]
]]] 02z z2202 0 22 22]E E ] ] ] ] ] ] ]] ]0E]] s] ]
S37
ESI
Calcd. 4562.71 [M]
m/z200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
%
0
100 161206_SCARB1 OLIGO 21 (0.372) TOF MS ES-
2.71e4650.599127090
650.457921019
355.509417303
304.00008353
616.083213854
445.008512441 511.0148
11508
650.745426380
650.886421986
759.367619921
759.198318924
759.034715661
651.027513593
656.01819313
911.245814930759.5370
14773
792.045613289
911.047910496
910.83775583
795.55464826
796.05743320
911.443712107 1139.5543
113041139.3054
10743
1139.05658259
911.83956872
912.03754824
1138.80054654
936.09181762
1139.80328468
1140.05225269
1149.04043956 1519.3802
27781149.29042705 1519.0449
21441154.78991457
1520.04282111
z = 3 [(M - 3H]-3�
z = 4 [(M - 4H]-4�
z = 5 [(M - 5H]-5�
z = 6 [(M - 6H]-6�
z = 7 [(M - 7H]-7�
S38
4-4. ASO S1 5’-TCmagtBcatgactTCm-3’
n = DNA N = LNA, all internucleosidic linkages are phosphorothioated
HPLC
Column: Waters XBridgeTM OST C18 2.5 µm, 4.6 x 50 mm
Gradient: 10-40%MeCN (over 30 min) in triethylammonium acetate butter (pH 7.0, 0.1 M)
Flow rate: 1.0 mL/min
Column temperature: 80 °C
ESI MS
Calcd. 4660.62 [M - 2 x H2O]
2016/12/16 19:07:29 Page 1 / 3
D :\m orishohei\161204scarb1 1caged M EC N 10-40 80C 1.lcd
<クロマトグラム>
<ピークレポート>m in
m AU
0 5 10 15 20 25 300
100
200
300
検出器A C h1 260nmピー ク# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
保持時間 0.664 0.782 0.918 1.062 1.227 1.414 1.691 1.825 2.006 5.000 5.182 5.856 6.617 7.729 8.372 8.631 8.987 9.237 9.449 9.914 10.052
面積 30409 15486 16305 16985 7638 4768 4816 3242 3433 2721 2988 88898 4082
3457658 25866 2965 8192 2386 1140 2558 1282
高さ 5163 3016 2693 1871 920 514 488 457 299 201 284 3420 402
195382 4276 524 1156 445 108 339 266
濃度 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
単位
マーク V V V V V V V V V V V V SV T T T TV T T TV
化合物名
zzz000 000 000 000 000 000 000 000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000
%
0
000 000000)0))))))))))))0 ))))) 00 )0)000) ) ) ))
0)0040000)000000000
000)000000000
000)00000000
000)000000000
000)000000000
000)00000000
000)00000000
000)00000000
000)00000000
000)000000000
000)000000000
000)000000000
000)00000000
000)00000000
000)000000000
000)00000000 000)0000
0000000)0000
0000
000)00000000
000)00000000
000)00000000
000)00000000
0000)00000000
0000)00000000
000)00000000
0000)00000000
0000)00000000
z = 3 [(M- 2 x H2O) - 3H]-3
Found: 1552.0785�
z = 4 [(M- 2 x H2O) - 4H]-4�
z = 5 [(M- 2 x H2O) - 5H]-5�
z = 6 [(M- 2 x H2O) - 6H]-6�
z = 7 [(M- 2 x H2O) - 7H]-7�
S39
4-5. ASO S2 5’-TCmagtBcatBgactTCm-3’
n = DNA N = LNA, all internucleosidic linkages are phosphorothioated
HPLC
Column: Waters XBridgeTM OST C18 2.5 µm, 4.6 x 50 mm
Gradient: 10-40%MeCN (over 30 min) in triethylammonium acetate butter (pH 7.0, 0.1 M)
Flow rate: 1.0 mL/min
Column temperature: 80 °C
ESI MS
Calcd. 4758.52 [M - 4 x H2O]
2016/12/16 19:06:58 Page 1 / 2
D :\m orishohei\161204scarb1 2caged M EC N 10-40 80C 3.lcd
<クロマトグラム>
<ピークレポート>m in
m AU
0 5 10 15 20 25 300
100
200
検出器A C h1 260nmピー ク# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
保持時間 0.657 0.768 0.906 1.049 1.298 1.560 1.664 2.001 3.680 5.766 6.483 7.576 8.959 10.229 10.532 12.498 12.889 13.683 13.897 15.135 16.363
面積 6625 15617 8106 7439 2584 1706 2795 2425 1110 1479 1036
123352 3500761 1002 1344 1490 2286 1530 4328 1672 1518
高さ 1832 3652 1527 647 383 281 363 287 147 101 114 3965
109328 277 269 210 340 184 270 240 184
濃度 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
単位
マーク V V V V V V V V V V V SV T T TV TV T TV TV TV
化合物名
m/z200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
%
0
100 161206_2_CAGED_SCARB1 OLIGO 21 (0.372) TOF MS ES-
1.53e4355.505515334
304.000012667
360.695911962
362.69839952
616.08329069
445.00858223
600.90424222
600.65313790
792.04568684
686.74797923
686.88755871
844.57238603
845.07247408
845.56093740
927.08863030
950.65702711
963.03852081 1188.5641
1486
z = 4 [(M- 4 x H2O) - 4H]-4�
z = 5 [(M- 4 x H2O) - 5H]-5�
z = 6 [(M- 4 x H2O) - 6H]-6�
z = 3 [(M- 4 x H2O) - 3H]-3
Found: 1585.0855 �
S40
4-6. ASO S3 5’-TCmagtBcatBgactBTCm-3’
n = DNA N = LNA, all internucleosidic linkages are phosphorothioated
HPLC
Column: Waters XBridgeTM OST C18 2.5 µm, 4.6 x 50 mm
Gradient: 10-40%MeCN (over 30 min) in triethylammonium acetate butter (pH 7.0, 0.1 M)
Flow rate: 1.0 mL/min
Column temperature: 80 °C
ESI MS
Calcd. 4892.45 [M - 4 x H2O]
2016/12/16 19:10:04 Page 1 / 2
D :\m orishohei\161204 PS Bn M eC N 10-40 80C 1.lcd
<クロマトグラム>
<ピークレポート>m in
m AU
0 5 10 15 20 25 300
100
200
300
400
500
検出器A C h1 260nmピー ク# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
保持時間 0.367 0.656 0.773 1.042 1.208 1.320 1.663 1.817 1.998 2.285 4.924 8.118 8.326 8.642 9.870 12.268 12.518 12.908 13.907 15.153 16.079
面積 3070 8380 14987 4930 2712 5070 6365 2314 2414 1434 23526 1724 3300 13660
5969634 1755 1456 6723 7416 1273 1001
高さ 174 1965 1998 564 457 465 391 257 227 119 1044 171 348 545
433344 171 168 475 315 157 140
濃度 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
単位
マーク V V V V V V V V V V V V V SV V V V V V V
化合物名
zzz000 000 000 000 000 000 000 0000 0000 0000 0000
%
0
000 000000)0))))) )))))0 00 )0)000) ) ))
0)0030000)00000000 000)0000
0000
000)00000000
000)00000000
000)00000000
000)00000000
000)00000000
000)00000000
000)00000000
000)00000000
000)00000000
000)00000000 000)0000
0000
000)00000000
000)00000000 000)0000
0000000)0000
0000
000)00000000
000)00000000
000)00000000
000)00000000
000)00000000
000)00000000 000)0000
0000
000)00000000
000)00000000
000)00000000
z = 5 [(M- 4 x H2O) - 5H]-5�
z = 6 [(M- 4 x H2O) - 6H]-6�
z = 7 [(M- 4 x H2O) - 7H]-7�z = 8
[(M- 4 x H2O) - 8H]-8�
z = 4 [(M- 4 x H2O) - 4H]-4
Found: 1222.30�
S41
4-7. ASO SA 5’-GCmattggtatTCmA-3’
n = DNA N = LNA, all internucleosidic linkages are phosphorothioated
HPLC
Column: Waters XBridgeTM OST C18 2.5 µm, 4.6 x 50 mm
Gradient: 10-40%MeCN (over 30 min) in triethylammonium acetate butter (pH 7.0, 0.1 M)
Flow rate: 1.0 mL/min
Column temperature: 80 °C
MALDI-TOF MS
Calcd. 4324.49 [M-H]-
2016/12/16 19:09:26 Page 1 / 2
D :\m orishohei\161204 PS natural M eC N 10-40 80C 1.lcd
<クロマトグラム>
<ピークレポート>m in
m AU
0 5 10 15 20 25 300
100
200
300
検出器A C h1 260nmピー ク# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
保持時間 0.656 0.774 0.884 1.047 1.183 1.324 1.664 1.883 2.021 4.092 4.299 4.886 8.329 8.918 10.132 10.482 11.907 12.101 12.319 12.475 12.718
面積 3957 4745 5794 3467 2824 3959 6315 1579 1262 44152 25325
4509852 1706 1547 1697 1496 3399 4721 6364 3413 3064
高さ 1128 1120 645 484 406 393 338 170 136 1647 1730
218243 199 102 147 97 264 453 464 394 384
濃度 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
単位
マーク V V V V V V V V SV V V V V V V V V
化合物名
111111111111111112
66666666
060
065
060
065
5000
]]]]]]6]]]6]6]
6000 6500 6000 6500 6000 6500 5000 5500 6000 6500 0000zzz
rrrrrrrrrrrrrrrrrrrrr
6006 00 66 06 0 06666 00 00]nn1111n1nnnnnnn1n11ennnnnn1n111111e1e11n11 ]] ] ]0 ]] ]] ] 6 00 ]6 ] ]
ennnnn1n1111e1en1n11111e1 ]]] ]
]y1y1ny11e1ynen1ysn1s1e11n1ne11n 6500]
sn1111n1ne1n1en11nn1e1n1e1nnynsen1n11 ]
] ] ]] ] ] ]] ]0]05]66z] ]] ]1nynsen1n111e1n1e11n11ynn11nn1e
orrrrrrrrrrororE E E rn1e
t1n1en1111 C C C
t1n1en111111ye1 ] ] ] ]0 Cz C]
]]] 06z z6006 0 6 6 ]E E ] ] ] ] ] ] ]] ]0E]] s] ]
S42
ESI MS
Calcd. 4325.49 [M]
m/z200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
%
0
100 161206_APOB OLIGO 23 (0.406) TOF MS ES-
2.04e4462.342220390
319.002710990
265.13424221
360.69599581
362.69837608
414.94013425
462.677012328
486.044310155
616.88177244
599.36363819
720.03247073
617.02925570
817.04686597
725.85962853
955.08345753
863.83824190
956.09012913
1080.05382840
973.10221706
1200.05332695
1080.29611917
1200.72741582 1440.3827
11761207.72911057
z = 3 [(M - 3H]-3�
z = 4 [(M - 4H]-4�
z = 5 [(M - 5H]-5�
z = 6 [(M - 6H]-6�
z = 7 [(M - 7H]-7�
S43
5. UV melting experiments of duplexes containing dTB without or with H2O2
Equimolecular amounts of the target DNA/RNA and oligonucleotides were dissolved in 10 mM sodium
phosphate buffer (pH 7.2) containing 100 mM NaCl to give a final strand concentration of 4.0 µM. Under the
H2O2 presence condition, to the duplex solution was added H2O2 (1 mM) and the resulting sample mixture was
incubated for 30 min at room temperature in advance to the UV melting experiments.
ODN X�Y�
A� G� C� U�
14 32 39 30 31
14� 46� 39� 30� 32�
15� 47� 37� 29� 30�
N
N
O
O
B OH
OH
N
N
O
O
B OH
OH
NH
N
O
O
ODN cRNA�
5’–d(GCGTTXTTTCGT)–3’�3’–r(CGCAAYAAAGCA)-5’�
Tm/#°C�
H2O2$–$
H2O2$+�
ODN X�Y�
A� G� C� U�
14 31 35 33 34
14� 52� 41� 36� 38�
15� 52� 41� 37� 40�
N
N
O
O
B OH
OH
N
N
O
O
B OH
OH
NH
N
O
O
ODN cDNA�
5’–d(GCGTTXTTTCGT)–3’�3’–d(CGCAAYAAAGCA)-5’�
Tm/#°C�
H2O2$–$
H2O2$+�
ODN14 and RNA target in the presence or absence of H2O2. ODN14 and DNA target in the presence or absence of H2O2.
S44
6. Reference
S1 C. Chung, D. Srikun, C. S. Lim, C. J. Chang and B. R.Cho, Chem. Commun., 2011, 47, 9618–9620.