dna photo-cross-linking using 3-cyanovinylcarbazole

S-1

Supporting Information

DNA Photo-cross-linking using 3-Cyanovinylcarbazole Modified Oligonucleotide with Threoninol Linker Takashi Sakamoto,† Yuya Tanaka,† and Kenzo Fujimoto＊,†,‡

†School of Materials Science, ‡Research Center for Bio-architecture, Japan Advanced Institute of Science and

Technology, 1-1 Asahi-dai, Nomi, Ishikawa 923-1292, Japan

Table of Contents

1. Experimental Procedures S-1

2. Synthetic Procedures S-3

3. Supporting Figures S-6

4. 1H and 13C NMR spectra S-13

5. UPLC chromatograms and MALDI-TOF-MS spectra of ODNs S-19

S-2

1. Experimental procedures

Oligonucleotide synthesis and preparation. All oligonucleotides having CNVD, CNVL or CNVK were synthesized by a

3400 DNA synthesizer (Applied Biosystems) and purified by a reversed-phase HPLC (JASCO PU-980, HG-980-31,

DG-980-50, UV-970 system equipped with an InertSustainTM C18 column (GL Science, 5 µm, 10 × 150 mm)).

Preparation of oligonucleotides was confirmed by MALDI-TOF-MS. Phosphoramidites of CNVD and CNVL were prepared

by the procedure as follows. Phosphoramidite of CNVK was synthesized according to a method described in the literature.1

Other oligonucleotides were purchased from Fasmac (Japan) and used without farther purification.

Photoirradiation and UPLC analysis. Photoirradiation was performed with an LED lamp (ZUV, 366 nm, 1280

mW/cm2, Omron) and a transilluminator (312 nm, Funakoshi) on an aluminum block incubator or water bath. The

photoirradiated samples were analyzed with a UPLC system (Aquity, Waters) equipped with BEH Shield RP18 column

(1.7 µm, 2.1 × 50 mm, elution was with 0.05 M ammonium formate containing 1-10% CH3CN, linear gradient (10 min) at

a flow rate of 0.4 mL/min, 60 ºC).

Enzymatic digestion, HPLC and MALDI-TOF-MS analysis. The enzymatic digestion was carried out with the

treatment of snake venom phosphodiesterase (0.2 U), P1 nuclease (1 U) and calf intestine alkaline phosphatase (20 U) in

50 mM Tris-HCl buffer (pH 9.0) containing 1 mM MgCl2, 0.1 mM ZnCl2 and 1 mM Spermidin at 37ºC for 5 h. After the

purification of the photoadducts by reversed phase HPLC with InertSustainTM C18 (GL Science, 5 µm, 10 × 150 mm,

elution was with 0.05 M ammonium formate containing 1–50% CH3CN, linear gradient (50 min) at a flow rate of 3

mL/min), the molecular masses were analyzed with a MALDI-TOF-Mass spectrometer (Voyager DE-Pro-SF, Applied

Biosystems).

Thermodynamic analysis of the hybridization. Thermodynamic parameters were obtained by the following equations

according to a method in the literature:

where TM is the melting temperature of duplex, ∆Sº is entropy and ∆H is the entropy of duplex formation, respectively. R

is the gas constant and CT is the total strand concentration. TM was measured at various concentrations of duplex in 50 mM

Na-cacodylate buffer (pH 7.4) containing 100 mM NaCl by a spectrophotometer (V–630bio, Jasco) equipped with a

temperature controller.

!!–! = ! ln !!4 + ∆!! /∆!!

S-3

2. Synthetic Procedures

Synthesis of compound 2. 3-Cyanovinylcarbazole (1)1 (1.1 g, 5 mmol) and NaH (60% oil suspension, 0.24 g, 6 mmol)

were solved in dry DMF (20 mL) and stirred for 1 h at ambient temperature under N2 atmosphere. Ethyl bromoacetate (1.1

mL, 9.9 mmol) was drop-wisely added over 30 min and stirred for 2 h at ambient temperature. Reaction mixture was

poured onto water (300 mL) and extracted with chloroform and then dried over sodium sulfate. After removal of the

solvent the residue was subjected to silica gel column chromatography (0–1% MeOH/CHCl3) to afford 2 (white solid, 1.4

g, 4.6 mmol, 92%). 1H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H), 8.09 (d, 1H, J = 7.6 Hz), 7.61-7.47 (m, 3H), 7.40-7.29 (m,

3H), 5.87 (d, 1H, J = 16.4 Hz), 5.00 (s, 2H), 4.22 (q, 2H, J = 7.2 Hz), 1.24 (t, 3H, J = 7.2 Hz). 13C NMR (100 MHz,

CDCl3) δ 168.0, 151.5, 142.3, 141.2, 127.0, 125.7, 125.3, 123.9, 123.0, 120.8, 120.6, 119.2, 109.2, 109.0, 93.0, 62.1, 45.0,

14.3. MS (MALDI): 305.12 calcd. for C19H16N2O2 [(M+H)+], found 304.67.

Synthesis of compound 3. 2 (1.4 g, 4.6 mmol) and NaOH (0.18 g, 23 mmol) were solved in THF/MeOH/H2O (3:2:1,

30 mL) and stirred for 2 h at ambient temperature. After the addition of 1N HCl (250 mL), reaction mixture was extracted

with EtOAc (300 mL) and washed with 1N HCl. Organic layer was dried over MgSO4 and evaporated to afford 3 (white

solid, 1.2 g, 4.4 mmol, 96%). 1H NMR (400 MHz, CDCl3) δ 7.99 (m, 2H), 7.49-7.17 (m, 6H), 5.75 (d, 1H, J = 16.8 Hz),

4.91 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 168.6, 151.4, 142.1, 141.0, 127.0, 125.6, 125.2, 123.7, 122.8, 120.7, 120.5,

119.1, 109.0, 108.9, 92.8, 52.8, 44.6. MS (MALDI): 277.09 calcd. for C17H12N2O2 [(M+H)+], found 277.62.

Synthesis of compound 4 (CNVD). 3 (1.3 g, 4.5 mmol) and D-threoninol (0.47 g, 4.5 mmol) were added to dry DMF (20

mL) containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC; 0.86 g, 4.5 mmol) and

1-hydroxybenzotriazole (HOBt; 0.69 g, 4.5 mmol), and stirred for 5 h at ambient temperature. Sat. NaCl aq. soln. was

added and obtained precipitate was collected and dried under vacuo to afford 4 (white solid, 1.5 g, 4.1 mmol, 91%). 1H

NMR (400 MHz, DMSO-d6) δ8.48 (s, 1H), 8.15 (d, 1H, 7.6 Hz), 7.97 (d, 1H, 8.8 Hz), 7.81-7.73 (m, 2H), 7.65-7.56 (m,

2H), 7.48 (t, 1H, 7.2 Hz), 7.27 (t, 1H, 7.2 Hz), 6.41 (d, 1H, J = 16.8 Hz), 5.17 (s, 2H), 4.72 (d, 1H, 4.4 Hz), 4.65 (t, 1H,

5.6 Hz), 3.90 (m, 1H), 3.63 (m, 1H), 3.50 (m, 1H), 3.38 (t, 1H, 5.5 Hz), 1.01 (d, 3H, 6.4 Hz). 13C NMR (100 MHz,

DMSO-d6) δ 167.2, 151.6, 142.3, 141.2, 126.4, 125.4, 125.2, 122.5, 122.1, 120.7, 120.3, 120.0, 119.6, 110.0, 92.7, 64.0,

60.6, 55.9, 45.6, 20.3. MS (MALDI): 364.16 calcd. for C21H21N3O3 [(M+H)+], found 364.73.

Synthesis of compound 5. 4 (0.40 g, 1.1 mmol), 4,4'-dimethoxytritylchloride (0.41 g, 1.2 mmol) and 4-dimethyl-

aminopyridine (DMAP; 30 mg, 0.25 mmol) in dry pyridine (2 mL) was stirred at ambient temperature for 20 h. The

reaction mixture was diluted with CHCl3, washed with H2O and organic layer was dried over NaSO4. After the removal of

the solvent, residue was subjected to silica gel column chromatography (CHCl3 with 0.2% TEA) to afford 5 (0.63 g, 86%).

S-4

1H NMR (400 MHz, CDCl3) δ 8.13-8.04 (m, 2H), 7.53-7.29 (m, 6H), 7.18-7.11 (m, 3H), 7.07 (m, 2H), 6.96 (d, 4H, 8.8

Hz), 6.67 (m, 4H), 6.35 (d, 1H, 8.7 Hz), 5.82 (d, 1H, J = 16.4 Hz), 4.93 (s, 2H), 3.90 (m, 1H), 3.82 (m, 1H), 3.74 (s, 6H),

3.23 (dd, 1H, 9.6 Hz), 3.03 (m, 1H), 0.91 (d, 3H, 6.3 Hz). 13C NMR (100 MHz, CDCl3) δ167.8, 158.5, 151.1, 144.1,

141.8, 140.8, 135.2, 135.0, 129.7, 127.9, 127.6, 127.4, 126.8, 126.0, 125.5, 123.8, 122.9, 121.1, 120.9, 120.6, 118.9, 113.2,

109.2, 109.0, 93.3, 86.4, 68.2, 64.0, 55.2, 53.9, 45.9, 20.0. MS (MALDI): 666.29 calcd. for C42H39N3O5 [(M+H)+], found

665.09.

Synthesis of compound 6. The residual trivial amount of water in 5 was removed by azeotropic distillation with dry

acetonitrile (twice). Then, 5 (0.38 g, 0.57 mmol) was solved with 0.25 M solution of 5-benzylthio-1H-tetrazole (BTT) in

dry MeCN (2.3 mL, 0.57 mmol) and a solution of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (0.18 mL,

0.57 mmol) in dry acetonitrile (5 mL) was added and stirred under N2 atmosphere for 2 h. The crude mixture was

dissolved in ethyl acetate. The solution containing 6 was washed with water, sat. NaHCO3 aq. soln. and brine. The organic

layer was dried over MgSO4, and then filtered and the ethyl acetate was removed. The yellow foam (0.48 g, 0.55 mmol,

96%) was obtained and immediately used for DNA synthesis without further purification.

Synthesis of compound 4' (CNVL). 3 (0.31 g, 1.1 mmol), L-threoninol (0.14 g, 1.3 mmol) were added dry DMF (4 mL)

containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC; 0.26 g, 1.3 mmol) and

1-hydroxybenzotriazole (HOBt; 0.20 g, 1.3 mmol) and stirred for 18 h at ambient temperature. Sat. NaCl aq. soln. was

added and obtained precipitate was collected and dried under vacuo to afford 4’ (white solid, 0.12 g, 0.34 mmol, 31%). 1H

NMR (400 MHz, DMSO-d6) δ 8.48 (s, 1H), 8.15 (d, 1H, J = 7.6 Hz), 7.97 (d, 1H, 8.8 Hz), 7.81-7.45 (m, 5H), 7.27 (t, 1H,

7.2 Hz), 6.42 (d, 1H, J = 16.6 Hz), 5.15 (s, 2H), 4.72 (d, 1H, 4.4 Hz), 4.65 (t, 1H, 5.2 Hz), 3.90 (m, 1H), 3.64 (m, 1H),

3.49 (m, 1H), 3.38 (t, 1H, 5.4 Hz), 1.01 (d, 3H, 6.4 Hz). 13C NMR (100 MHz, DMSO-d6) δ 167.2, 151.6, 142.3, 141.2,

126.4, 125.4, 125.2, 122.5, 122.1, 120.8, 120.4, 119.9, 119.7, 110.0, 92.7, 64.0, 60.6, 55.9, 45.6, 20.3. MS (MALDI):

364.16 calcd. for C21H21N3O3 [(M+H)+], found 364.74.

Synthesis of compound 5'. 4’ (0.12 g, 0.34 mmol), 4,4'-dimethoxytritylchloride (0.13 g, 0.37 mmol) and 4-dimethyl-

aminopyridine (DMAP; 10 mg, 0.08 mmol) in dry pyridine (1 mL) was stirred sat ambient temperature for 20 h. The

reaction mixture was diluted with CHCl3, washed with H2O and organic layer was dried over NaSO4. After the removal of

the solvent, residue was subjected to silica gel column chromatography (CHCl3 with 0.2% TEA) to afford 5’ (0.18 g, 0.27

mmol, 80%). 1H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H), 8.14 (d, 1H, 7.7 Hz), 7.61-7.48 (m, 3H), 7.47-7.34 (m, 3H),

7.20-7.09 (m, 3H), 7.00 (d, 2H, 7.0 Hz), 6.91 (m, 4H), 6.66 (m, 4H), 6.24 (d, 1H, J = 8.4 Hz), 5.88 (d, 1H, J = 16.6 Hz),

5.02 (s, 2H), 3.87, (br, 1H), 3.77 (s, 6H), 3.25 (m, 1H), 3.02 (m, 1H), 0.89 (d, 3H, J = 6.2 Hz). 13C NMR (100 MHz,

S-5

CDCl3) δ167.9, 158.6, 151.2, 150.0, 144.0, 141.9, 140.9, 135.3, 134.9, 129.8, 128.0, 127.7, 127.0, 126.3, 125.8, 124.1,

123.2, 121.5, 121.2, 120.9, 119.0, 113.3, 109.3, 109.1, 93.7, 86.6, 68.9, 64.5, 55.4, 53.8, 47.3, 20.1. MS (MALDI): 666.29

calcd. for C42H39N3O5 [(M+H)+], found 667.10.

Synthesis of compound 6'. The residual trivial amount of water in 5' was removed by azeotropic distillation with dry

acetonitrile (twice). Then, 5' (0.20 g, 0.30 mmol), 0.25 M solution of 5-benzylthio-1H-tetrazole (BTT) in dry MeCN (1.2

mL, 0.30 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (90 mg, 0.30 mmol) were mixed under

nitrogen for 2 h. The crude mixture was dissolved in ethyl acetate. The solution containing 6’ was washed with water, sat.

NaHCO3 aq. soln. and brine. The organic layer was dried over MgSO4, and then filtered and the ethyl acetate was

removed. The yellow foam (0.21 g, 0.24 mmol, 80%) was obtained and immediately used for DNA synthesis without

further purification.

References

1) Yoshimura, Y.; Fujimoto, K. Org. Lett. 2008, 10, 3227–3230.

S-6

3. Supporting Figures

Figure S1. HPLC analysis of the digested product of photodimer consisting ODN(AD) and cODN

(GT). HPLC was performed with MeCN/50 mM ammonium formate gradient: 1–50% in 50 min.

S-7

Figure S2. UPLC analysis of the photo-cross-linking reaction of CNVL in oligonucleotide duplexes.

(a) UPLC chromatograms of ODN(AL)/cODN(GT) duplexe after the indicated time period of 366

nm photoirradiation. (b) Time course of the photo-cross-linking reaction of ODN(AL)/cODN(GT)

(red) and ODN(AK)/cODN(GT) (blue) duplexes. [duplex] = 15 µM in 50 mM Na-Cacodylate

buffer (pH 7.4) containing 100 mM NaCl and 50 µM dU (as an internal standard). Photoirradiation

(366 nm, 1280 mW/cm2) was performed at 0 ºC.

S-8

Figure S3. UPLC analysis of the photo-cross-linking reaction of CNVD in heteroduplex consisting of

ODN(AD) and cORN(GU). (a) UPLC chromatograms of ODN(AD)/cORN(GU) heteroduplexe

after the indicated time period of 366 nm photoirradiation. (b) Time course of the

photo-cross-linking reaction of ODN(AD)/cORN(GU) (red) and ODN(AK)/cORN(GU) (blue)

duplexes. [duplex] = 15 µM in 50 mM Na-Cacodylate buffer (pH 7.4) containing 100 mM NaCl

and 50 µM dU (as an internal standard). Photoirradiation (366 nm, 1280 mW/cm2) was performed

at 0 ºC.

S-9

Figure S4. UPLC analysis of the photo-splitting reaction of CNVD in oligonucleotide duplexes. (a)

UPLC chromatograms of ODN(AD)/cODN(GT) photodimer after the indicated time period of 312

nm photoirradiation. (b) Time course of the photo-splitting reaction of ODN(AD)/cODN(GT) (red)

and ODN(AK)/cODN(GT) (blue) duplexes. [duplex] = 15 µM in 50 mM Na-Cacodylate buffer (pH

7.4) containing 100 mM NaCl and 50 µM dU (as an internal standard). Photoirradiation (312 nm)

was performed at 60 ºC.

S-10

Figure S5. Energy minimized structures of the duplexes consisting of ODN(GX) and cODN(GC)

estimated by MacroModel (ver. 8.1). Green-colored atoms indicate the vinyl moiety on the

cyanovinyl group and the C5 and C6 carbons on the cytosines possessed at the –1 position at the

complementary strands.

S-11

Figure S6. Absorbance spectra of ODN(AD) and ODN(AK). [ODN] = 8 µM in 50 mM

Na-cacodylate buffer (pH 7.4) containing 100 mM NaCl.

S-12

Figure S7. (a) UV melting curves of the duplexes consisting of ODN(AX) and cODN(GT) at 345

nm. (b) Circular dichroism spectra of the duplexes consisting of ODN(AX) and cODN(GT).

[duplex] = 15 µM in 50 mM Na-Cacodylate buffer (pH 7.4) containing 100 mM NaCl. Circular

dichroism spectra were measured at 10 ºC

S-13

4. 1H and 13C NMR spectra

1H NMR spectrum of Compound 2

13C NMR spectrum of Compound 2

10 9 8 7 6 5 4 3 2 1 ppm

1.224

1.241

1.259

4.193

4.211

4.228

4.246

4.998

5.337

5.368

5.847

5.888

7.302

7.313

7.320

7.334

7.338

7.340

7.346

7.367

7.496

7.499

7.514

7.517

7.519

7.538

7.562

7.565

7.578

8.082

8.101

8.141

3.00

2.01

1.87

0.13

0.85

2.94

2.79

1.85

Current Data ParametersNAME Aug23-2012 carbazolyl esterEXPNO 10PROCNO 1

F2 - Acquisition ParametersDate_ 20120823Time 19.13INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zg30TD 65536SOLVENT CDCl3NS 16DS 2SWH 8223.685 HzFIDRES 0.125483 HzAQ 3.9845889 secRG 128DW 60.800 usecDE 6.50 usecTE 297.5 KD1 1.00000000 secTD0 1

======== CHANNEL f1 ========NUC1 1HP1 13.00 usecPL1 -2.50 dBPL1W 15.18724251 WSFO1 400.1324710 MHz

F2 - Processing parametersSI 32768SF 400.1300096 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.00

180 160 140 120 100 80 60 40 20 ppm

14.258

44.952

62.050

93.014

108.996

109.172

119.154

120.570

120.780

120.810

122.978

123.890

125.272

125.701

127.051

141.226

142.327

151.499

168.039

Current Data ParametersNAME Aug23-2012 carbazolyl esterEXPNO 11PROCNO 1

F2 - Acquisition ParametersDate_ 20120823Time 20.18INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zgig30TD 65536SOLVENT CDCl3NS 1024DS 4SWH 24038.461 HzFIDRES 0.366798 HzAQ 1.3631488 secRG 71.8DW 20.800 usecDE 6.50 usecTE 298.3 KD1 2.00000000 secD11 0.03000000 secTD0 1

======== CHANNEL f1 ========NUC1 13CP1 9.80 usecPL1 -2.00 dBPL1W 57.32743073 WSFO1 100.6228298 MHz

======== CHANNEL f2 ========CPDPRG[2 waltz16NUC2 1HPCPD2 75.00 usecPL2 -2.50 dBPL12 12.72 dBPL2W 15.18724251 WPL12W 0.45654005 WSFO2 400.1316005 MHz


N

O

O

CN

N

O

O

CN

S-14



10 9 8 7 6 5 4 3 2 1 ppm

3.673

3.685

4.889

4.908

4.929

5.250

5.280

5.726

5.768

7.132

7.162

7.192

7.206

7.214

7.228

7.241

7.250

7.264

7.272

7.279

7.285

7.359

7.363

7.406

7.412

7.416

7.424

7.426

7.438

7.447

7.457

7.476

7.913

7.917

7.935

7.938

7.980

7.990

0.87

2.00

0.19

0.69

2.24

0.96

2.75

2.39

0.17

Current Data ParametersNAME Jan21-2014_Cz-OHEXPNO 10PROCNO 1

F2 - Acquisition ParametersDate_ 20140121Time 20.00INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zg30TD 65536SOLVENT CDCl3NS 16DS 2SWH 8012.820 HzFIDRES 0.122266 HzAQ 4.0894465 secRG 32DW 62.400 usecDE 6.50 usecTE 294.9 KD1 1.00000000 secTD0 1

======== CHANNEL f1 ========SFO1 400.1324710 MHzNUC1 1HP1 13.00 usecPLW1 14.50399971 W


180 160 140 120 100 80 60 40 20 ppm

44.568

52.775

92.805

108.896

109.013

119.122

120.464

120.724

122.845

123.703

125.178

125.570

126.993

141.026

142.108

151.385

168.563

Current Data ParametersNAME Jan21-2014_Cz-OHEXPNO 12PROCNO 1

F2 - Acquisition ParametersDate_ 20140121Time 21.07INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zgpg30TD 65536SOLVENT CDCl3NS 1024DS 4SWH 24038.461 HzFIDRES 0.366798 HzAQ 1.3631488 secRG 161DW 20.800 usecDE 6.50 usecTE 295.4 KD1 2.00000000 secD11 0.03000000 secTD0 1

======== CHANNEL f1 ========SFO1 100.6228293 MHzNUC1 13CP1 9.80 usecPLW1 57.32699966 W

======== CHANNEL f2 ========SFO2 400.1316005 MHzNUC2 1HCPDPRG[2 waltz16PCPD2 90.00 usecPLW2 14.50399971 WPLW12 0.30261001 WPLW13 0.24511001 W


N

OH

O

CN

N

OH

O

CN

S-15



10 9 8 7 6 5 4 3 2 1 ppm

0.999

1.015

3.371

3.384

3.398

3.476

3.492

3.627

3.644

3.899

4.639

4.653

4.667

4.712

4.723

5.142

5.150

6.393

6.435

7.251

7.252

7.270

7.288

7.289

7.459

7.462

7.480

7.483

7.498

7.501

7.578

7.599

7.605

7.627

7.752

7.759

7.777

7.781

7.793

7.955

7.977

8.137

8.156

8.479

8.482

3.00

0.96

1.04

0.98

0.97

1.02

0.98

1.97

0.04

0.94

1.02

1.07

1.97

1.91

0.95

0.96

0.93

Current Data ParametersNAME Oct17-2012 carbazolyl threoninolEXPNO 10PROCNO 1

F2 - Acquisition ParametersDate_ 20121017Time 11.47INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zg30TD 65536SOLVENT DMSONS 16DS 2SWH 8223.685 HzFIDRES 0.125483 HzAQ 3.9845889 secRG 101DW 60.800 usecDE 6.50 usecTE 295.1 KD1 1.00000000 secTD0 1



180 160 140 120 100 80 60 40 20 ppm

20.305

45.577

55.892

60.588

63.982

92.659

109.959

110.022

119.610

119.865

120.330

120.714

122.090

122.495

125.157

125.397

126.379

141.223

142.301

151.610

167.213

Current Data ParametersNAME Oct17-2012 carbazolyl threoninol 13CEXPNO 10PROCNO 1

F2 - Acquisition ParametersDate_ 20121017Time 19.58INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zgpg30TD 65536SOLVENT DMSONS 1024DS 4SWH 24038.461 HzFIDRES 0.366798 HzAQ 1.3631488 secRG 71.8DW 20.800 usecDE 6.50 usecTE 297.2 KD1 2.00000000 secD11 0.03000000 secTD0 1


======== CHANNEL f2 ========CPDPRG[2 waltz16NUC2 1HPCPD2 75.00 usecPL2 -2.50 dBPL12 12.72 dBPL13 15.70 dBPL2W 15.18724251 WPL12W 0.45654005 WPL13W 0.22986822 WSFO2 400.1316005 MHz


N

NH

O

OH

HO

CN

N

NH

O

OH

HO

CN

S-16


13 C NMR spectrum of Compound 5

10 9 8 7 6 5 4 3 2 1 ppm

0.904

0.919

3.015

3.024

3.036

3.047

3.215

3.225

3.239

3.249

3.740

3.803

3.811

3.818

3.827

3.894

3.906

4.933

5.795

5.836

6.337

6.655

6.660

6.677

6.682

6.953

6.975

7.073

7.131

7.146

7.150

7.309

7.327

7.345

7.366

7.393

7.413

7.454

7.461

7.470

7.487

7.502

8.061

8.082

8.085

3.00

1.13

1.03

6.11

0.99

1.02

1.93

0.92

0.96

4.04

4.05

2.08

3.05

6.29

1.95

Current Data ParametersNAME Oct19-2012 DMTrcarbazolylamideEXPNO 10PROCNO 1

F2 - Acquisition ParametersDate_ 20121019Time 18.46INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zg30TD 65536SOLVENT CDCl3NS 16DS 2SWH 8223.685 HzFIDRES 0.125483 HzAQ 3.9845889 secRG 22.6DW 60.800 usecDE 6.50 usecTE 296.6 KD1 1.00000000 secTD0 1



180 160 140 120 100 80 60 40 20 ppm

19.970

45.855

53.938

55.189

64.020

68.249

86.386

93.293

109.012

109.165

113.172

118.873

120.601

120.919

121.138

122.942

123.833

125.493

125.965

126.842

127.383

127.644

127.875

129.661

134.981

135.232

140.785

141.811

144.087

151.066

158.458

158.473

167.771

Current Data ParametersNAME Oct19-2012 DMTrcarbazolylamideEXPNO 12PROCNO 1

F2 - Acquisition ParametersDate_ 20121019Time 19.51INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zgpg30TD 65536SOLVENT CDCl3NS 1024DS 4SWH 24038.461 HzFIDRES 0.366798 HzAQ 1.3631488 secRG 161DW 20.800 usecDE 6.50 usecTE 297.5 KD1 2.00000000 secD11 0.03000000 secTD0 1


======== CHANNEL f2 ========CPDPRG[2 waltz16NUC2 1HPCPD2 75.00 usecPL2 -2.50 dBPL12 12.72 dBPL13 15.70 dBPL2W 15.18724251 WPL12W 0.45654005 WPL13W 0.22986822 WSFO2 400.1316005 MHz


N

NH

O

OH

O

CN

DMTr

N

NH

O

OH

O

CN

DMTr

S-17

1H NMR spectrum of Compound 4’

13C NMR spectrum of Compound 4’

10 9 8 7 6 5 4 3 2 1 ppm

0.999

1.015

3.371

3.384

3.476

3.492

3.900

4.639

4.654

4.667

4.712

4.723

5.142

5.150

5.161

5.170

5.715

5.745

6.394

6.436

7.251

7.253

7.270

7.288

7.290

7.478

7.480

7.483

7.498

7.501

7.578

7.599

7.606

7.627

7.753

7.759

7.777

7.781

7.794

7.955

7.977

8.137

8.156

8.479

8.483

2.99

0.83

1.06

1.02

1.00

1.01

1.00

1.99

0.19

0.78

1.00

1.26

1.82

0.22

1.63

1.22

1.00

0.79

0.20

Current Data ParametersNAME TS_140623EXPNO 20PROCNO 1

F2 - Acquisition ParametersDate_ 20140623Time 13.40INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG zg30TD 65536SOLVENT DMSONS 16DS 2SWH 8012.820 HzFIDRES 0.122266 HzAQ 4.0894465 secRG 101DW 62.400 usecDE 10.00 usecTE 298.0 KD1 1.00000000 secTD0 1



cnvL_frac_2

180 160 140 120 100 80 60 40 20 ppm

20.337

45.587

55.903

60.593

63.970

92.674

109.987

110.047

119.652

119.891

120.358

120.755

122.105

122.505

125.174

125.422

126.407

141.240

142.320

151.645

167.244

Current Data ParametersNAME TS_140623EXPNO 30PROCNO 1

F2 - Acquisition ParametersDate_ 20140623Time 14.14INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG zgpg30TD 65536SOLVENT DMSONS 512DS 4SWH 24038.461 HzFIDRES 0.366798 HzAQ 1.3631488 secRG 64DW 20.800 usecDE 18.00 usecTE 298.0 KD1 2.00000000 secD11 0.03000000 secTD0 1




cnvL_frac_2

N

NH

O

OH

HO

CN

N

NH

O

OH

HO

CN

S-18

1H NMR spectrum of Compound 5’

13C NMR spectrum of Compound 5’

10 9 8 7 6 5 4 3 2 1 ppm

0.868

0.884

2.993

3.017

3.229

3.237

3.252

3.764

3.850

3.861

5.010

5.856

5.898

6.218

6.239

6.644

6.663

6.892

6.912

6.982

7.000

7.117

7.136

7.275

7.344

7.362

7.382

7.388

7.410

7.421

7.441

7.505

7.525

7.539

7.579

7.650

7.670

7.688

7.952

7.973

8.002

8.116

3.45

1.01

1.05

7.46

0.99

2.04

0.96

1.00

4.25

4.38

2.13

3.41

3.39

3.10

1.98

0.80

Current Data ParametersNAME TS_140719_cnvLEXPNO 10PROCNO 1

F2 - Acquisition ParametersDate_ 20140719Time 12.31INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG zg30TD 65536SOLVENT CDCl3NS 16DS 2SWH 8012.820 HzFIDRES 0.122266 HzAQ 4.0894465 secRG 203DW 62.400 usecDE 10.00 usecTE 298.0 KD1 1.00000000 secTD0 1



180 160 140 120 100 80 60 40 20 ppm

20.077

47.251

53.798

55.379

64.480

68.856

86.612

93.710

109.088

109.272

113.339

118.972

120.873

121.203

121.456

123.182

124.136

125.756

126.302

127.040

127.666

127.708

128.066

129.757

134.941

135.260

140.889

141.922

144.050

149.981

151.212

158.643

167.892

Current Data ParametersNAME TS_140719_cnvLEXPNO 12PROCNO 1

F2 - Acquisition ParametersDate_ 20140719Time 13.37INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG zgpg30TD 65536SOLVENT CDCl3NS 1024DS 4SWH 24038.461 HzFIDRES 0.366798 HzAQ 1.3631488 secRG 90.5DW 20.800 usecDE 18.00 usecTE 298.0 KD1 2.00000000 secD11 0.03000000 secTD0 1




N

NH

O

OH

O

CN

DMTr

N

NH

O

OH

O

CN

DMTr

S-19

5. UPLC chromatograms and MALDI-TOF-MS spectra of ODNs

Table S1. MALDI-TOF-MS analysis of the synthetic ODNs containing CNVD, CNVK or CNVL

Sequences (5’–3’)a Calcd. [(M+H)+] Found

ODN(AD) TGCACNVDCCGT 2810.56 2809.26

ODN(AK) TGCACNVKCCGT 2781.53 2781.93

ODN(AL) TGCACNVLCCGT 2810.56 2809.26

ODN(GD) TGCGCNVDCCGT 2826.55 2826.23

ODN(GK) TGCGCNVKCCGT 2797.53 2797.70

ODN(XDY)

X=A, Y=A TGCACNVDACGT 2834.57 2833.87

X=A, Y=G TGCACNVDGCGT 2850.57 2849.43

X=A, Y=T TGCACNVDTCGT 2825.56 2824.70

X=G, Y=A TGCGCNVDACGT 2850.57 2849.71

X=G, Y=G TGCGCNVDGCGT 2866.56 2866.58

X=G, Y=T TGCGCNVDTCGT 2841.56 2841.14

X=C, Y=A TGCCCNVDACGT 2810.56 2809.57

X=C, Y=G TGCCCNVDGCGT 2826.56 2826.53

X=C, Y=C TGCCCNVDCCGT 2786.55 2785.50

X=C, Y=T TGCCCNVDTCGT 2801.55 2801.81

X=T, Y=A TGCTCNVDACGT 2825.56 2824.60

X=T, Y=G TGCTCNVDGCGT 2841.56 2840.53

X=T, Y=C TGCTCNVDCCGT 2801.55 2800.98

X=T, Y=T TGCTCNVDTCGT 2816.55 2816.34

S-20

Figure S8. UPLC chromatograms of purified ODNs having CNVD, CNVL or CNVK

S-21

MALDI-TOD-MS spectrum of ODN(AD)

S-22

MALDI-TOD-MS spectrum of ODN(AK)

S-23

MALDI-TOD-MS spectrum of ODN(AL)

S-24

MALDI-TOD-MS spectrum of ODN(GD)

S-25

MALDI-TOD-MS spectrum of ODN(GK)

S-26

MALDI-TOD-MS spectrum of ODN(ADA)

S-27

MALDI-TOD-MS spectrum of ODN(ADG)

S-28

MALDI-TOD-MS spectrum of ODN(ADT)

S-29

MALDI-TOD-MS spectrum of ODN(GDA)

S-30

MALDI-TOD-MS spectrum of ODN(GDG)

S-31

MALDI-TOD-MS spectrum of ODN(GDC)

S-32

MALDI-TOD-MS spectrum of ODN(GDT)

S-33

MALDI-TOD-MS spectrum of ODN(CDA)

S-34

MALDI-TOD-MS spectrum of ODN(CDG)

S-35

MALDI-TOD-MS spectrum of ODN(CDC)

S-36

MALDI-TOD-MS spectrum of ODN(CDT)

S-37

MALDI-TOD-MS spectrum of ODN(TDA)

S-38

MALDI-TOD-MS spectrum of ODN(TDG)

S-39

MALDI-TOD-MS spectrum of ODN(TDC)

S-40

MALDI-TOD-MS spectrum of ODN(TDT)

dna photo-cross-linking using 3-cyanovinylcarbazole

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