Supporting Information
DNA photo-cross-linking using pyranocarbazole modified oligodeoxynucleotide with D-threoninol linker Kenzo Fujimoto*a, Tsubasa Yamaguchia, Takahiro Inatsugib, Masahiko Takamurab, Isao Ishimarub, Ayako Kotob, Shigetaka Nakamuraa
Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2019
Compound 2
Figure S1. 1H-NMR spectra of Compound 2
Figure S2. 13C-NMR spectra of Compound 2
9 8 7 6 5 4 3 2 1 ppm
0.760
0.764
0.775
0.781
0.809
1.179
1.197
1.215
1.263
1.354
1.496
2.813
2.814
2.885
3.421
4.142
4.160
4.178
4.196
4.916
6.253
6.277
7.137
7.190
7.221
7.238
7.240
7.248
7.258
7.275
7.294
7.425
7.428
7.443
7.446
7.448
7.464
7.466
7.797
7.820
8.004
8.023
8.053
8.064
3.37
2.04
2.04
1.00
0.94
2.03
1.01
0.97
0.96
0.91
Current Data ParametersNAME 20190303 TY pyrano bromocoupling cdcl3-2EXPNO 10PROCNO 1
F2 - Acquisition ParametersDate_ 20190303Time 17.12 hINSTRUM spectPROBHD Z122623_0030 (PULPROG zg30TD 65536SOLVENT CDCl3NS 32DS 2SWH 8012.820 HzFIDRES 0.244532 HzAQ 4.0894465 secRG 203DW 62.400 usecDE 10.00 usecTE 298.0 KD1 1.00000000 secTD0 1SFO1 400.2324714 MHzNUC1 1HP1 12.00 usecPLW1 9.19999981 W
F2 - Processing parametersSI 65536SF 400.2300377 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.00
180 160 140 120 100 80 60 40 20 ppm
14.17
44.91
62.08
76.71
77.03
77.23
77.35
96.01
108.77
112.63
112.85
119.64
120.56
120.89
121.06
122.61
126.85
141.63
142.98
144.54
153.47
161.50
167.68
Current Data ParametersNAME 20190303 TY pyrano bromocoupling cdcl3 13CEXPNO 10PROCNO 1
F2 - Acquisition ParametersDate_ 20190303Time 18.36 hINSTRUM spectPROBHD Z122623_0030 (PULPROG zgpg30TD 65536SOLVENT CDCl3NS 1024DS 4SWH 24038.461 HzFIDRES 0.733596 HzAQ 1.3631488 secRG 64DW 20.800 usecDE 18.00 usecTE 298.0 KD1 2.00000000 secD11 0.03000000 secTD0 1SFO1 100.6479773 MHzNUC1 13CP1 10.00 usecPLW1 40.59999847 WSFO2 400.2316009 MHzNUC2 1HCPDPRG[2 waltz16PCPD2 90.00 usecPLW2 9.67000008 WPLW12 0.17460790 WPLW13 0.08751346 W
F2 - Processing parametersSI 32768SF 100.6379140 MHzWDW EMSSB 0LB 1.00 HzGB 0PC 1.40
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
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Figure S3. ESI analysis of Compound 2
Compound 3
Figure S4. 1H-NMR spectra of Compound 3
186.221591+
242.284131+
282.279131+
322.107391+
344.089291+
409.162141+
445.313601+
489.339531+
563.551191+
643.207351+
665.189351+
736.543001+
764.574291+
848.667851+
944.728931+
pcxD_000008.d:4+MS
0 100 200 300 400 500 600 700 800 900 m/z0.0
0.5
1.0
1.5
2.0
8x10Intens.
Meas.&m/z Ion&Formula m/z err&[ppm]
322.10739 C19H16NO4 322.107384 0
186.221591+
242.284131+
282.279131+
322.107391+
344.089291+
409.162141+
445.313601+
489.339531+
563.551191+
643.207351+
665.189351+
736.543001+
764.574291+
848.667851+
944.728931+
pcxD_000008.d:4+MS
0 100 200 300 400 500 600 700 800 900 m/z0.0
0.5
1.0
1.5
2.0
8x10Intens.
13 12 11 10 9 8 7 6 5 4 3 2 1 ppm
0.767
0.817
0.840
0.858
0.874
1.155
1.183
1.236
1.255
1.264
1.305
1.343
1.764
1.773
1.917
2.092
2.505
2.509
2.513
2.678
3.176
3.343
3.607
3.613
3.623
3.700
5.309
6.347
6.371
7.287
7.306
7.324
7.481
7.483
7.501
7.519
7.521
7.584
7.605
7.657
8.186
8.209
8.500
13.171
2.28
1.00
1.07
1.06
1.07
1.05
2.15
1.04
0.90
Current Data ParametersNAME 20190310 TY pyrano coupling hydrolysis DMSOEXPNO 10PROCNO 1
F2 - Acquisition ParametersDate_ 20190310Time 20.18 hINSTRUM spectPROBHD Z122623_0030 (PULPROG zg30TD 65536SOLVENT DMSONS 32DS 2SWH 8012.820 HzFIDRES 0.244532 HzAQ 4.0894465 secRG 203DW 62.400 usecDE 10.00 usecTE 298.0 KD1 1.00000000 secTD0 1SFO1 400.2324714 MHzNUC1 1HP1 12.00 usecPLW1 9.19999981 W
F2 - Processing parametersSI 65536SF 400.2300000 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.00
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
Figure S5. 13C-NMR spectra of Compound 3
Figure S6. ESI analysis of Compound 3
180 160 140 120 100 80 60 40 20 ppm
14.43
22.57
25.59
29.18
29.50
30.29
30.82
31.17
31.71
31.76
39.35
39.55
39.76
39.97
40.18
40.39
40.60
44.75
96.90
110.20
112.42
112.52
120.51
120.60
120.77
120.88
122.36
126.97
142.11
143.45
145.90
153.34
161.07
170.31
Current Data ParametersNAME 20190310 TY pyrano coupling hydrolysis DMSO 13CEXPNO 10PROCNO 1
F2 - Acquisition ParametersDate_ 20190311Time 4.54 hINSTRUM spectPROBHD Z122623_0030 (PULPROG zgpg30TD 65536SOLVENT DMSONS 8192DS 4SWH 24038.461 HzFIDRES 0.733596 HzAQ 1.3631488 secRG 101DW 20.800 usecDE 18.00 usecTE 298.0 KD1 2.00000000 secD11 0.03000000 secTD0 1SFO1 100.6479773 MHzNUC1 13CP1 10.00 usecPLW1 40.59999847 WSFO2 400.2316009 MHzNUC2 1HCPDPRG[2 waltz16PCPD2 90.00 usecPLW2 9.67000008 WPLW12 0.17460790 WPLW13 0.08751346 W
F2 - Processing parametersSI 32768SF 100.6379140 MHzWDW EMSSB 0LB 1.00 HzGB 0PC 1.40
164.07516
206.10394
216.54700
244.09285
256.26359 282.27925
294.07620
304.26119
316.05812
328.15032
338.34179
352.24599
360.32386
412.20797
434.19004
459.58427
496.26581
537.53590
563.55155
587.14547
597.33049
609.12747
625.10099
638.30374
693.48064
705.27769
902.19732
pcxD%coupling_000003.d:2+MS
0
2
4
6
8
7x10Intens.
200 300 400 500 600 700 800 900 m/z
Meas.&m/z� Ion&Formula� m/z� err&[mDa]�294.076204� C17H12NO4� 294.076084� B0.1�316.058122� C17H11NNaO4� 316.058029� B0.1�
164.07516
206.10394
216.54700
244.09285
256.26359 282.27925
294.07620
304.26119
316.05812
328.15032
338.34179
352.24599
360.32386
412.20797
434.19004
459.58427
496.26581
537.53590
563.55155
587.14547
597.33049
609.12747
625.10099
638.30374
693.48064
705.27769
902.19732
pcxD%coupling_000003.d:2+MS
0
2
4
6
8
7x10Intens.
200 300 400 500 600 700 800 900 m/z
164.07516
206.10394
216.54700
244.09285
256.26359 282.27925
294.07620
304.26119
316.05812
328.15032
338.34179
352.24599
360.32386
412.20797
434.19004
459.58427
496.26581
537.53590
563.55155
587.14547
597.33049
609.12747
625.10099
638.30374
693.48064
705.27769
902.19732
pcxD%coupling_000003.d:2+MS
0
2
4
6
8
7x10Intens.
200 300 400 500 600 700 800 900 m/z
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
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10
合成スキーム
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10
合成スキーム
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Compound 4
Figure S7. 1H-NMR spectra of Compound 4
Figure S8. 13C-NMR spectra of Compound 4
9 8 7 6 5 4 3 2 1 ppm
1.017
1.033
1.240
1.255
1.264
1.341
2.092
2.499
2.503
2.508
2.512
2.517
3.337
3.368
3.381
3.394
3.494
3.633
4.679
4.762
4.770
5.170
5.179
6.340
6.364
7.275
7.277
7.295
7.313
7.478
7.480
7.498
7.501
7.516
7.519
7.586
7.607
8.035
8.058
8.182
8.189
8.201
8.212
8.498
4.79
0.65
1.36
1.61
1.65
1.63
3.02
2.63
1.00
1.53
1.55
2.96
1.39
3.01
1.45
Current Data ParametersNAME 20190308 pcxD DMSO-d6EXPNO 10PROCNO 1
F2 - Acquisition ParametersDate_ 20190308Time 18.19 hINSTRUM spectPROBHD Z122623_0030 (PULPROG zg30TD 65536SOLVENT DMSONS 32DS 2SWH 8012.820 HzFIDRES 0.244532 HzAQ 4.0894465 secRG 203DW 62.400 usecDE 10.00 usecTE 298.0 KD1 1.00000000 secTD0 1SFO1 400.2324714 MHzNUC1 1HP1 12.00 usecPLW1 9.19999981 W
F2 - Processing parametersSI 65536SF 400.2300000 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.00
180 160 140 120 100 80 60 40 20 ppm
20.77
31.17
39.35
39.56
39.76
39.97
40.18
40.39
40.60
46.14
56.39
61.03
64.42
79.65
96.85
110.31
112.28
112.37
120.45
120.58
120.61
120.84
122.29
126.86
142.28
143.58
145.95
153.30
161.11
167.52
Current Data ParametersNAME 20190309 TY pcxD 13CEXPNO 10PROCNO 1
F2 - Acquisition ParametersDate_ 20190309Time 18.27 hINSTRUM spectPROBHD Z122623_0030 (PULPROG zgpg30TD 65536SOLVENT DMSONS 2048DS 4SWH 24038.461 HzFIDRES 0.733596 HzAQ 1.3631488 secRG 71.8DW 20.800 usecDE 18.00 usecTE 298.0 KD1 2.00000000 secD11 0.03000000 secTD0 1SFO1 100.6479773 MHzNUC1 13CP1 10.00 usecPLW1 40.59999847 WSFO2 400.2316009 MHzNUC2 1HCPDPRG[2 waltz16PCPD2 90.00 usecPLW2 9.67000008 WPLW12 0.17460790 WPLW13 0.08751346 W
F2 - Processing parametersSI 32768SF 100.6379140 MHzWDW EMSSB 0LB 1.00 HzGB 0PC 1.40
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
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Figure S9. ESI analysis of Compound 4
Compound 5
Figure S10. 1H-NMR spectra of Compound 5
235.00021
256.26355
282.27922
335.00432
362.07819
381.14452
403.12667
761.28271
783.26524
PCXD%dioH_000001.d:/+MS
0.0
0.5
1.0
1.5
2.0
2.5
3.0
8x10Intens.
100 200 300 400 500 600 700 800 900 m/z
Meas.&m/z� Ion&Formula� m/z� err&[mDa]�381.144518� C21H21N2O5� 381.144498� 0�
235.00021
256.26355
282.27922
335.00432
362.07819
381.14452
403.12667
761.28271
783.26524
PCXD%dioH_000001.d:/+MS
0.0
0.5
1.0
1.5
2.0
2.5
3.0
8x10Intens.
100 200 300 400 500 600 700 800 900 m/z
9 8 7 6 5 4 3 2 1 ppm
0.991
1.006
2.503
2.508
2.512
2.944
2.948
3.135
3.346
3.716
3.721
3.731
3.930
3.946
4.773
4.784
5.178
5.183
6.319
6.343
6.775
6.797
6.818
7.178
7.196
7.218
7.236
7.253
7.273
7.292
7.311
7.328
7.346
7.426
7.428
7.447
7.592
7.598
7.619
8.165
8.189
8.211
8.326
8.491
3.16
1.05
1.06
6.19
2.17
1.03
1.84
1.00
4.28
11.3
51.
072.
11
3.12
1.01
Current Data ParametersNAME 20190314 TY pcxD DMTrEXPNO 10PROCNO 1
F2 - Acquisition ParametersDate_ 20190314Time 9.56 hINSTRUM spectPROBHD Z122623_0030 (PULPROG zg30TD 65536SOLVENT DMSONS 32DS 2SWH 8012.820 HzFIDRES 0.244532 HzAQ 4.0894465 secRG 101DW 62.400 usecDE 10.00 usecTE 298.0 KD1 1.00000000 secTD0 1SFO1 400.2324714 MHzNUC1 1HP1 12.00 usecPLW1 9.19999981 W
F2 - Processing parametersSI 65536SF 400.2300000 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.00
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
Figure S11. 13C-NMR spectra of Compound 5
Figure S12. ESI analysis of Compound 5
180 160 140 120 100 80 60 40 20 ppm
20.79
39.34
39.55
39.76
39.97
40.18
40.39
40.60
46.21
54.59
55.43
63.36
65.28
79.65
85.66
96.90
110.38
112.24
112.43
113.22
113.44
113.48
120.46
120.54
120.60
120.82
122.32
126.82
126.95
127.88
128.15
129.38
130.06
130.15
136.15
136.28
142.31
143.49
145.43
145.87
153.30
158.33
158.37
161.03
167.55
Current Data ParametersNAME 20190314 TY pcxD DMTr 13CEXPNO 10PROCNO 1
F2 - Acquisition ParametersDate_ 20190314Time 17.42 hINSTRUM spectPROBHD Z122623_0030 (PULPROG zgpg30TD 65536SOLVENT DMSONS 1024DS 4SWH 24038.461 HzFIDRES 0.733596 HzAQ 1.3631488 secRG 114DW 20.800 usecDE 18.00 usecTE 298.0 KD1 2.00000000 secD11 0.03000000 secTD0 1SFO1 100.6479773 MHzNUC1 13CP1 10.00 usecPLW1 40.59999847 WSFO2 400.2316009 MHzNUC2 1HCPDPRG[2 waltz16PCPD2 90.00 usecPLW2 9.67000008 WPLW12 0.17460790 WPLW13 0.08751346 W
F2 - Processing parametersSI 32768SF 100.6379140 MHzWDW EMSSB 0LB 1.00 HzGB 0PC 1.40
303.137801+
357.260971+
386.211261+ 437.19325
1+
489.339531+
515.412591+
537.394541+
577.391971+
651.159511+
705.256831+
951.598771+
pcxD1DMTr_000001.d:8+MS
200 300 400 500 600 700 800 900 m/z0.0
0.5
1.0
1.5
8x10Intens.
Meas.&m/z Ion&Formula m/z err&[ppm] 705.256833 C42H38N2NaO7 705.257122 0.4
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
合成経路
10
合成スキーム
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Figure S13 Photo-cross-linking using PCXD. 10 µM ODNs in 50 mM Cacodylate buffer
containing 100 mM NaCl was annealed and photoirradaition at 400 nm on ice.
A7�
5’-TGC G/A PCXDCCGT-3’�
3’-ACG C/T ----GGGCA-5’�400 nm� 5’-TGC G/A PCXDCCGT-3’�
3’-ACG C/T ----GGGCA-5’�
Photo.cross.link7to7T�Photo.cross.link7to7C�
0 s�
1 s�
5 s�
10 s�
30 s�07 17 27 37 47 57 67 77 87 97 107
dU�
*�
07 17 27 37 47 57 67 77 87 97 107
30 s�
60 s�
600 s�
dU�
0 s�
10 s�
*�
cODN(C)7/7cODN(T)�
PCXD.ODN(C)7/7PCXD.ODN(T)�
RetenWon7Wme7[min]� RetenWon7Wme7[min]�
cODN(C)� cODN(T)� PCXD.ODN(T)�PCXD.ODN(C)�
Figure S14 The photo-splitting of PCXD. The 10 µM photoadduct in 50 mM Cacodylate
buffer containing 100 mM NaCl was photoirradaited at 312 nm under the 60°C condition.
ODN(GGT)� ODN(PCXD)�
RetenWon7Time(min)�
07min�
57min�
307min�
ODN(PCXD�T)�
ODN(PCXD�T)� ODN(APCXDC)�
ODN(GGT)�
Figure S15 Meitng Curve of duplex cnontaining. Sample solution is duplex in 50 mM
Cacodylate buffer containing 100 mM NaCl was measured absorbance at 260 nm from 5°C
to 85°C
17
1.057
1.17
1.157
1.27
07 207 407 607 807
Hype
rchrom
icity�
Temperature(�)�
PCXD�
PCX�
X7=PCXD,7PCX�
5’-TGCGXCCGT-3’�3’-ACGCGGGCA-5’�
Figure S16 The HPLC analysis of (a) pyranocarbazole (b) 3-cyanovinylcarbazole, and (c)
Both
(a)�
(c)�
(b)�
pyranocarbazole�
Mix1soluKon�
3.cyanovinylcarbazole�
01 51 101 151 201 251 301
RetenKon1Kme1[min]�
Retention
time [min]
LogP
PCXD 14.1 1.12 CNVD 15.2 1.35
Figure. S17 Effect of surrounding base of PCXD on its photoreactivity in dsDNA. 10 µM
DNA in 50 mM cacodylate buffer (pH 7.4) containing 100 mM NaCl was 400 nm-irradiated
for 10 s at 4 °C.
Figure S18 UV spectra of PCXD
!0.1%
0%
0.1%
0.2%
0.3%
0.4%
0.5%
0.6%
300% 350% 400% 450% 500%
Wavelength%(nm)�
Absorbance�
Figure S19 Photo-cross-linking using PCXD. 10 µM ODNs in 50 mM Cacodylate buffer
containing 100 mM NaCl was annealed and photoirradaition at 450 nm on ice.
ODN%(GT)�
ODN%(APCXD)�
0%s�
0% 1% 2% 3% 4% 5%
dU�
*�
Reten%on'%me'(min)�
10%s�
30%s�
60%s�
120%s�
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