Organic & Biomolecular Chemistry · 2012-07-04 · S-1 Organic & Biomolecular Chemistry...
Transcript of Organic & Biomolecular Chemistry · 2012-07-04 · S-1 Organic & Biomolecular Chemistry...
S-1
Organic & Biomolecular Chemistry
Supplementary Information
O4-Alkyl-2'-deoxythymidine Cross-linked DNA to Probe
Recognition and Repair by O6-Alkylguanine-DNA Alkyltransferases
Francis P. McManus, Derek K. O'Flaherty, Anne M. Noronha and Christopher J. Wilds* Contents Page Supplementary Figure 1 - 500 MHz
1H NMR spectrum of compound (1a) (in CDCl3) S-2
Supplementary Figure 2 - 500 MHz 1H NMR spectrum of compound (1b) (in CDCl3) S-3
Supplementary Figure 3 - 500 MHz 1H NMR spectrum of compound (2a) (in CDCl3) S-4
Supplementary Figure 4 - 500 MHz 1H NMR spectrum of compound (2b) (in CDCl3) S-5
Supplementary Figure 5 - 500 MHz 1H NMR spectrum of compound (3a) (in CDCl3) S-6
Supplementary Figure 6 - 500 MHz 1H NMR spectrum of compound (3b) (in CDCl3) S-7
Supplementary Figure 7 - 500 MHz 1H NMR spectrum of compound (4a) (in d6-acetone) S-8
Supplementary Figure 8 - 500 MHz 1H NMR spectrum of compound (4b) (in d6-acetone) S-9
Supplementary Figure 9 - 500 MHz 1H NMR spectrum of compound (5a) (in CDCl3) S-10
Supplementary Figure 10 - 500 MHz 1H NMR spectrum of compound (5b) (in CDCl3) S-11
Supplementary Figure 11 - 500 MHz 1H NMR spectrum of compound (6a) (in CDCl3) S-12
Supplementary Figure 12 - 500 MHz 1H NMR spectrum of compound (6b) (in CDCl3) S-13
Supplementary Figure 13 - 500 MHz 1H NMR spectrum of compound (7a) (in d6-acetone) S-14
Supplementary Figure 14 - 500 MHz 1H NMR spectrum of compound (7b) (in d6-acetone) S-15
Supplementary Figure 15 - 202.3 MHz 31
P NMR spectrum of compound (4a) (in d6-acetone) S-16 Supplementary Figure 16 - 202.3 MHz
31P NMR spectrum of compounds (4b) (in d6-acetone) S-17
Supplementary Figure 17 - 202.3 MHz 31
P NMR spectrum of compounds (7a) (in d6-acetone) S-18 Supplementary Figure 18 - 202.3 MHz
31P NMR spectrum of compounds (7b) (in d6-acetone) S-19
Supplementary Figure 19 - ESI MS of oligonucleotide XLTT4 S-20 Supplementary Figure 20 - ESI MS of oligonucleotide XLTT7 S-21 Supplementary Figure 21 - C-18 RP HPLC profile of digested cross-linked duplex XLTT4 S-22 Supplementary Figure 22 - C-18 RP HPLC profile of digested cross-linked duplex XLTT7 S-23 Supplementary Figure 23 - ESI MS of oligonucleotide S4 S-24 Supplementary Figure 24 - ESI MS of oligonucleotide S7 S-25 Supplementary Figure 25 - C-18 RP HPLC profile of digested oligonucleotide S4 S-26 Supplementary Figure 26 - C-18 RP HPLC profile of digested oligonucleotide S7 S-27 Supplementary Figure 27 - Thermodynamic data of Control, XLTT4 and XLTT7 DNA S-28 Supplementary Figure 28 - Tm curves of the various O
4-alkyl-dT mono-adducts in DNA duplexes S-29
Supplementary Figure 29 - CD curves of the various O4-alkyl-dT mono-adducts in DNA duplexes S-30
Supplementary Figure 30 - Repair gel of O4MeT, S4 and S7 DNA by hAGT, Ada-C and OGT S-31
Supplementary Figure 31 - Repair of O4MeT, S4 and S7 by hAGT (red), OGT (green) and
Ada-C (blue) S-31 Supplementary Table 1 - Nucleoside Ratio of the Various Modified Oligonucleotides S-32 Supplementary Table 2 - Kd and stoichiometry of hAGT binding with XLTT4, XLTT7
and control duplex S-32 Supplementary Table 3 - Approximate Kd of hAGT binding to 5' CGAAAXTTTCG S-32 Supplementary Table 4 - Kd of hAGT binding to duplexes containing various
O4-Alkyl-dT mono-adducts and O
6MeG S-33
Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2012
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Supplementary Figure 1 - 500 MHz 1H NMR spectrum of compound (1a) (in CDCl3)
O
OTBDMS
DMTO N
N
O
OOH
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Supplementary Figure 2 - 500 MHz 1H NMR spectrum of compound (1b) (in CDCl3)
O
OTBDMS
DMTO N
N
O
O OH
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Supplementary Figure 3 - 500 MHz 1H NMR spectrum of compound (2a) (in CDCl3)
OTBDMSO
DMTO
O
ODMT
OTBDMSNN
O
O
NN
O
O
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Supplementary Figure 4 - 500 MHz 1H NMR spectrum of compound (2b) (in CDCl3)
OTBDMSO
DMTO
O
ODMT
OTBDMSNN
O
O
NN
O
O
4
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Supplementary Figure 5 - 500 MHz 1H NMR spectrum of compound (3a) (in CDCl3)
OHO
DMTO
O
ODMT
OHNN
O
O
NN
O
O
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Supplementary Figure 6 - 500 MHz 1H NMR spectrum of compound (3b) (in CDCl3)
OHO
DMTO
O
ODMT
OHNN
O
O
NN
O
O
4
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Supplementary Figure 7 - 500 MHz 1H NMR spectrum of compound (4a) (in d6-acetone)
OO
DMTO
O
ODMT
O
P(iPr2)N
OCEt
P N(iPr2)
OCEt
NN
O
O
NN
O
O
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Supplementary Figure 8 - 500 MHz 1H NMR spectrum of compound (4b) (in d6-acetone)
OO
DMTO
O
ODMT
O
P(iPr2)N
OCEt
P N(iPr2)
OCEt
NN
O
O
NN
O
O
4
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Supplementary Figure 9 - 500 MHz 1H NMR spectrum of compound (5a) (in CDCl3)
O
OTBDMS
DMTO N
N
O
OOPac
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Supplementary Figure 10 - 500 MHz 1H NMR spectrum of compound (5b) (in CDCl3)
O
OTBDMS
DMTO N
N
O
O OPac
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Supplementary Figure 11 - 500 MHz 1H NMR spectrum of compound (6a) (in CDCl3)
O
OH
DMTO N
N
O
OOPac
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Supplementary Figure 12 - 500 MHz 1H NMR spectrum of compound (6b) (in CDCl3)
O
OH
DMTO N
N
O
O OPac
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Supplementary Figure 13 - 500 MHz 1H NMR spectrum of compound (7a) (in d6-acetone)
O
O
DMTO N
N
O
O
PN(iPr2)
OCEt
OPac
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Supplementary Figure 14 - 500 MHz 1H NMR spectrum of compound (7b) (in d6-acetone)
O
O
DMTO N
N
O
O
PN(iPr2)
OCEt
OPac
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Supplementary Figure 15 - 202.3 MHz 31P NMR spectrum of compound (4a) (in d6-acetone)
OO
DMTO
O
ODMT
O
P(iPr2)N
OCEt
P N(iPr2)
OCEt
NN
O
O
NN
O
O
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Supplementary Figure 16 - 202.3 MHz 31P NMR spectrum of compound (4b) (in d6-acetone)
OO
DMTO
O
ODMT
O
P(iPr2)N
OCEt
P N(iPr2)
OCEt
NN
O
O
NN
O
O
4
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Supplementary Figure 17 - 202.3 MHz 31P NMR spectrum of compound (7a) (in d6-acetone)
O
O
DMTO N
N
O
O
PN(iPr2)
OCEt
OPac
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Supplementary Figure 18 - 202.3 MHz 31P NMR spectrum of compound (7b) (in d6-acetone)
O
O
DMTO N
N
O
O
PN(iPr2)
OCEt
OPac
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CJW 214
mass6500 6550 6600 6650 6700 6750 6800 6850 6900 6950
%
0
1006715.6
Supplementary Figure 19 - ESI MS of oligonucleotide XLTT4
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CJW 207
mass6500 6550 6600 6650 6700 6750 6800 6850 6900 6950
%
0
1006758.3
Supplementary Figure 20 - ESI MS of oligonucleotide XLTT7
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Supplementary Figure 21 - C-18 HPLC profile of digested cross-linked duplex XLTT4. The column was eluted with a linear gradient of 0-60% buffer B over 30 min (buffer A: 2% ACN, 50 mM sodium phosphate, pH 5.8 and buffer B: 50 mM sodium phosphate, pH 5.8, 50% acetonitrile).
dC
dG
dT
dA
OHO
HO
O
OH
OHNN
O
O
NN
O
O
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Supplementary Figure 22 - C-18 HPLC profile of digested cross-linked duplex XLTT7. The column was eluted with a linear gradient of 0-60% buffer B over 30 min (buffer A: 2% ACN, 50 mM sodium phosphate, pH 5.8 and buffer B: 50 mM sodium phosphate, pH 5.8, 50% acetonitrile).
dC
dG
dT
dA
OHO
HO
O
OH
OHNN
O
O
NN
O
O
4
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mass4000 4100 4200 4300 4400 4500 4600 4700 4800 4900
%
0
1004336.2
Supplementary Figure 23 - ESI MS of oligonucleotide S4 containing mono-adduct O4-butyl-4-ol-dT.
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CJW 200
mass4000 4100 4200 4300 4400 4500 4600 4700 4800
%
0
1004378.3
Supplementary Figure 24 - ESI MS of oligonucleotide S7 containing mono-adduct O4-heptyl-7-ol-dT.
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Supplementary Figure 25 - C-18 RP HPLC profile of digested oligonucleotide S4 containing mono-adduct O4-butyl-4-ol-dT. The column was eluted with a linear gradient of 0-60% buffer B over 30 min (buffer A: 2% ACN, 50 mM sodium phosphate, pH 5.8 and buffer B: 50 mM sodium phosphate, pH 5.8, 50% acetonitrile).
dC
dG
dT
dA
O
OH
HO N
N
O
OOH
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Supplementary Figure 26 - C-18 RP HPLC profile of digested oligonucleotide S7 containing mono-adduct O4-heptyl-7-ol-dT. The column was eluted with a linear gradient of 0-60% buffer B over 30 min (buffer A: 2% ACN, 50 mM sodium phosphate, pH 5.8 and buffer B: 50 mM sodium phosphate, pH 5.8, 50% acetonitrile).
dC
dG
dT
dA
O
OH
HO N
N
O
O OH
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Supplementary Figure 27 - Thermodynamic data for the non-crosslinked control, XLTT4 and XLTT7 DNA
Control (T-A) XLTT4 XLTT7
Trial 0.585 uM 2.719 uM 12.57 uM 58.46 uM 0.585 uM 2.95 uM 0.585 uM 2.95 uM
1 38.0˚C 42.0˚C 47.0˚C 52.0˚C 66.5˚C 66.0˚C 52.0˚C 52.0˚C
2 38.0˚C 43.0˚C 48.0˚C 52.0˚C 65.0˚C 67.0˚C 53.5˚C 51.0˚C
3 39.0˚C 43.0˚C 49.0˚C 50.0˚C 66.0˚C 66.0˚C 51.0˚C 53.0˚C
Avg 38.3˚C 42.7˚C 48.0˚C 51.3˚C 65.8˚C 66.3˚C 52.2˚C 52.0˚C
StdDev 0.5˚C 0.5˚C 0.8˚C 0.9˚C 0.6˚C 0.5˚C 1.0˚C 0.8˚C
y1 = -3.0499E-05x + 2.7789E-03
R² = 9.9815E-01 y2 = -2.8445E-05x + 2.7961E-03
R² = 9.9776E-01
y3 = -2.5372E-05x + 2.8355E-03 R² = 9.4206E-01
3.00E-03
3.05E-03
3.10E-03
3.15E-03
3.20E-03
3.25E-03
-15 -14 -13 -12 -11 -10 -9
1/T
m (K
)
ln(Ctot) (M)
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Supplementary Figure 28 - Tm curves of various O4-alkyl-dT mono-adducts in DNA duplexes 5’ GGCTXGATCACCAG 3’ / 5’ CTGGTGATCCAGCC 3’ where X is dT (——), O4-MedT (……), O4-butyl-4-ol-dT for S4 (– • –) and O4-heptyl-7-ol-dT for S7 (– – –). Solutions containing a total strand concentration of 2.8 µM for the duplexes in 90 mM sodium chloride, pH = 7.0, 10 mM sodium phosphate, and 1 mM EDTA buffer, were heated at 0.5°C/min.
0
0.05
0.1
0.15
0.2
0.25
10 20 30 40 50 60 70 80 90
NO
RM
AL
IZE
D A
BS
OR
BA
NC
E (
26
0n
m)
TEMPERATURE (°C)
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Supplementary Figure 29 - CD profiles of various O4-alkyl-dT mono-adducts in DNA duplexes 5’ GGCTXGATCACCAG 3’ / 5’ CTGGTGATCCAGCC 3’ where X is dT (——),O4-MedT (……), O4-butyl-4-ol-dT for S4 (– • –) and O4-heptyl-7-ol-dT for S7 (– – –). Solutions containing a total strand concentration of 2.8 µM for the duplexes in 90 mM sodium chloride, pH = 7.0, 10 mM sodium phosphate, and 1 mM EDTA buffer.
-30
-20
-10
0
10
20
30
40
220 240 260 280 300 320
MO
LA
R E
LLIP
TIC
ITY
(θx1
04)
WAVELENGTH (nm)
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Supplementary Figure 30 - Repair gel of O4MeT, S4 and S7 DNA by hAGT, Ada-C and OGT. Denaturing gel of the repair of 2 pmol of DNA by 10 pmol AGT. Lane 1, Control; lane 2, O4MeT DNA; lane 3, O4MeT DNA + hAGT; lane 4, O4MeT DNA + Ada-C, lane 5, O4MeT DNA + OGT; lane 6, S4 DNA; lane 7, S4 DNA + hAGT; lane 8, S4 DNA + Ada-C, lane 9, S4 DNA + OGT; lane 10, S7 DNA; lane 11, S7 DNA + hAGT; lane 12, S7 DNA + Ada-C, lane 13, S7 DNA + OGT.
Supplementary Figure 31 - Repair of O4-MedT, S4 and S7 by hAGT (red), OGT (green) and Ada-C (blue).
0
10
20
30
40
50
60
70
80
90
100
%R
ep
air
O4-MedT O4-butyl-4-ol-dT O4-heptyl-7-ol-dT
1 2 3 4 5 6 7 8 9 10 11 12 13
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Supplementary Table 1 - Nucleoside Ratio of the Various Modified Oligonucleotides as Observed by Snake Venom Digestion using RP-HPLC
Oligomer Nucleoside Nucleoside ratios
Expected Observed
XLTT4
dC 4.00 4.00
dG 4.00 4.24
dT 6.00 5.86
dA 6.00 6.22
dT-butyl-dT 1.00 1.55
XLTT7
dC 4.00 4.00
dG 4.00 4.18
dT 6.00 5.84
dA 6.00 6.34
dT-heptyl-dT 1.00 1.51
S4
dC 4.00 3.88
dG 4.00 4.00
dT 2.00 1.98
dA 3.00 2.99
O4-butyl-4-ol-dT 1.00 1.06
S7
dC 4.00 3.85
dG 4.00 4.05
dT 2.00 2.00
dA 3.00 2.90
O4-butyl-7-ol-dT 1.00 1.18
Supplementary Table 2 - Kd and stoichiometry of hAGT binding with XLTT4, XLTT7 and control duplex
DNA Duplex Kd (µM) Stoichiometry
Control 30.57±3.00 1.94 ± 0.03
XLTT4 2.45±0.05 1.90 ± 0.11
XLTT7 2.58±0.67 2.09 ± 0.02
Supplementary Table 3 - Kd of hAGT binding to single stranded 5' CGAAAXTTTCG
X Kd approximate (µM)
T >30
G 1-10
C 30
A 30
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Supplementary Table 4 - Kd of hAGT binding to duplexes containing various O4-Alkyl-dT mono adducts and O6MeG
DNA Duplex Kd (µM)
Control 6.90 ± 0.04 O4MeT 2.87 ± 0.19
S4 (O4-Butyl-4-ol-dT) 3.41 ± 0.28
S7 (O4-Heptyl-7-ol-dT) 3.68 ± 0.15
O6MeG 2.86 ± 0.16
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