Hydrogen peroxide-triggered gene silencing in mammalian ... · S1 Supplementary Information...

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


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


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


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


S16


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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-


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ﾋﾟーｸ# 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





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



m AU

0 5 10 15 20 25 300

200

400

600

800

1000

検出器A C h1 260nmﾋﾟーｸ# 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



ennnnn1n1111e1en1n11111e1 ]]] ]



] ] ]] ] ] ]] ]0]13]23z] ]z ] ]1nynsen1n111e1n1e11n11ynn11nn1e


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





MALDI-TOF MS

Calcd. 4561.71 [M-H]-

2016/12/16 19:10:27 Page 1 / 2

D :\m orishohei\161204 PO caged1.lcd



m AU

0 5 10 15 20 25 300

100

200

300

検出器A C h1 260nmﾋﾟーｸ# 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



ennnnn1n1111e1en1n11111e1 ]]] ]



] ] ]] ] ] ]] ]0]02]20z] ]z ] ]1nynsen1n111e1n1e11n11ynn11nn1e


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’


HPLC





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



m AU

0 5 10 15 20 25 300

100

200

300

検出器A C h1 260nmﾋﾟーｸ# 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’


HPLC





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



m AU

0 5 10 15 20 25 300

100

200

検出器A C h1 260nmﾋﾟーｸ# 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’


HPLC





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



m AU

0 5 10 15 20 25 300

100

200

300

400

500

検出器A C h1 260nmﾋﾟーｸ# 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’


HPLC





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



m AU

0 5 10 15 20 25 300

100

200

300

検出器A C h1 260nmﾋﾟーｸ# 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



ennnnn1n1111e1en1n11111e1 ]]] ]



] ] ]] ] ] ]] ]0]05]66z] ]] ]1nynsen1n111e1n1e11n11ynn11nn1e


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.

Hydrogen peroxide-triggered gene silencing in mammalian ... · S1 Supplementary Information...

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