Electrospray Ionization Mass
Spectrometry of the Interaction of
Oligonucleotides with Metals,
Small Molecules and Drugs
Janna Anichina
Diethard K. Bohme
York University
Department of Chemistry
Centre for Research in Mass Spectrometry
Toronto, CANADA
1
ASMS 2007
Why Metal Ion – Ligand – Oligonucleotides?
 In vivo processes with DNA are mediated by
interactions with metal ions, small molecules and
proteins.
 The mechanisms of action of many anti-tumor drugs
include the formation of their adducts with strands of
DNA only in the presence of metal ions.
 ESI-MS has been successfully utilized in the study of
interactions between DNA and Pt complexes important
in chemotherapy (Beck et al. Mass Spec. Rev., 2001, 20, 61).
 Systematic ESI-MS studies of metal ion - ligand - DNA
interactions remain insufficient.
2
Binding of oligonucleotides with metal complexes
Na+, Mn2+, Fe2+, Co2+, Ni2+,
Cu2+, Zn2+, Fe3+, Co3+
Bleomycin A2
CH3
+
H3C S
O
AA, CC, GG, TT, CCC, ATAT,
GCAT, GCGC, CATAC,
ACTCG, AGTCTG, TTAGGG,
GCATGC
NH
NH2
H
NH2
NH
NH2
H
N
O
N
H2N
CH3
S
N
CH3 H H O
N
N
H
O
N
HO
CH3
CH
3
H H
H
N
HN
HO
S
N
HH
O
HO
H O
O
H
OH
O
O
HO
O
N
1,10-phenanthroline
NH
OH
O
OH
O
HO
O
N
H2N
H
N
NH2
N
H
Triethylenetetramine
NH2
3
Experimental
 Instruments: MDS SCIEX API 2000 and Q Trap 2000.
 Duplexes of the hexamers were prepared by heating 70 M ss in 70 mM
aqueous NH4CH3COO to 900C for 10 min, neutral pH, and then cooled
down slowly over a 3-hour period.
0.9
 20 M solution prepared in
20:80 (vol/vol) methanol/water
was injected into the ESI
sources of the mass
spectrometers.
3-
[d(5'GCATGC)2 - 3H]
0.8
Relative Intensity
0.7
y = 0.0415x + 1.2798
y =0; x = -30.8 V
0.6
0.5
 Ratios metal cation to
ligand to ss were 5:varied:1,
[ss]0 = 20 M.
0.4
0.3
TV
0.2
0.1
0
0
-5
-10
-15
-20
-25 -30
-35
-40
Laboratory Collision Voltage /V
-45
-50
-55
 Flow rate: 5 L/min; N2 as
the collision gas; collision
voltages : -1 to -100 V in the
4
negative mode.
Formation of duplexes ?!
SS
596.3
100
447 - [d(5'GCATGC) - 4H]4596.3 - [d(5'GCATGC) - 3H]3894.5 - [d(5'GCATGC) - 2H]2-
90
80
70
90
60
50
40
30
20
447
894.5
10
0
400
100
600
800
1000
1200
1400
1600
614.7 [d(5'TTAGGG) m/z
- 3H]3-
1343 - [d(5'GCATGC)3 - 4H]4-
60
50
40
596.3
30
1193.6
20
1343.1
600
800
1000 1200
922.5 m/z
1432.6
1400
1791
1600
1800
90
Relative Abundance (%)
Relative Abundance (%)
70
100
70
60
50
4-
[d(5'TTAGGG) - 4H]
40
2-
461
[d(5'TTAGGG) - 2H]
922.5
80
70
60
50
40
30
20
3-
[d(5'TTAGGG)2 - 3H]
614.7
1229.3
10
10
0
400
1193.6 - [d(5'GCATGC)2 - 3H]3-
0
400
1800
80
20
80
10
90
30
894.5
100
Relative Abundance (%)
Relative Abundance (%)
DS
600
800
1000
1200
m/z
1400
1600
1800
0
400
600
800
1000
1200
m/z
1400
5
1600
1800
MS/MS of the duplexes of the hexamers
[d(5’GCATGC)2 -3H]3- --> [d(5’GCATGC) -H] - + [d(5’GCATGC) -2H]2-
0.9
[d(5'GCATGC)2 -3H]3-
Relative Intensity
0.8
0.7
0.6
0.5
2-
[d(5'GCATGC) -2H]
0.4
0.3
0.2
-
[d(5'GCATGC) -H]
-
0.1
w1
0
-2
-6 -10 -14 -18 -22 -26 -30 -34 -38 -42 -46 -50
Laboratory Collision Voltage /V
[d(5’TTAGGG)2 -3H]3- --> [d(5’TTAGGG) -H] - + [d(5’TTAGGG) -2H]21229
1846
922.5
6
Underlined species were not observed due to the limited mass range
Metallation of ds hexamers
-Q1: 60 MCA scans from Sample 4 (q1ms_Zn(II)_TTAGGGan_neg) of Janna_Feb15_06.wiff
1230.05
Max. 8.1e6 cps.
[d(5’T2AG3)2 - 3H]3-
7.0e5
5:1
6.5e5
6.0e5
5.5e5
I n t e n s it y , c p s
5.0e5
4.5e5
4.0e5
[Zn2d(5’T2AG3)2-7H)]3-
3.5e5
3.0e5
2.5e5
[Nad(5’T2AG3)2-4H)]3-
2.0e5
[Znd(5’T2AG3)2-5H)]3-
1237.15
1.5e5
1251.15
1235.95
1.0e5
1231.85
[Zn3d(5’T2AG3)2-9H)]3-
1271.55
1241.45
1292.55
1263.55
1242.15
1258.05
1273.85
1234.35
1245.85
1274.55
1285.25
1300.05 1306.35 1313.65
1233.65
1260.75
1223.35
0.0
-Q1:
60 MCA scans from Sample 3 (q1ms_Cu(II)_BLM_TTAGGGan) of Janna_Feb5_07.wiff
1220
1230
1240
1250
1260
1270
1280
1290
1300
1310
m/z, amu
1312.85
2.0e5
5.0e4
1217.25
[Cu4d(5’T2AG3)2-11H)]3-
[Cu3d(5’T2AG3)2-9H)]3-
1.9e5
1.8e5
1.7e5
1292.95
15 : 1
1.6e5
1.5e5
I n t e n s it y , c p s
1.4e5
Max. 2.0e6 cps.
[Cu2d(5’T2AG3)2-7H)]3-
1.3e5
1.2e5
1271.15
1.1e5
1.0e5
[Cud(5’T2AG3)2-5H)]3-
9.0e4
8.0e4
7.0e4
1231.25
1251.05
1237.35
1299.85
1251.85
1273.35
1213.15
1241.05 1244.75
1210.15 1221.45
1226.15
1254.75
1228.15
1.0e4
1210
1220
1321.55
1311.55
1343.55
1301.45
3.0e4
0.0
1315.15
1230.45
5.0e4
2.0e4
1272.05
[d(5’T2AG3)2 - 3H]3-
6.0e4
4.0e4
1293.75
1230
1240
1250
1274.951285.05
1264.95
1288.85
1267.05
1260
1270
1280
1290
m/z, amu
1333.75
1345.45
1303.85 1306.95
1369.45
1341.05
1339.55
1355.55
1296.75
1300
1310
1320
1330
1340
1350
1360
1370
7
Dissociation of metallated duplexes
Relative Abundance (%)
100
1212.6
1212.6 - [Cod(5'GCATGC)2 - 5H]3-
90
1231.6 - [Co2d(5'GCATGC)2 - 7H]3-
80
1250.6 - [Co3d(5'GCATGC)2 - 9H]
70
1269.6 - [Co4d(5'GCATGC)2- 11H]
33-
4-
60
1343 - [d(5'GCATGC)3 - 4H]
1231.6
50
1343
40
1250.6
30
20
1269.6
10
0
1200
1250
1300
1350
1400
m/z
0.9
Metallated duplexes
dissociate into two
strands!
3-
[Znd(5'GCATGC)2 - 5H]
Relative Intensity
0.8
0.7
[d(5'GCATGC) - 2H]2-
0.6
0.5
0.4
2-
[Znd(5'GCATGC) - 4H]
0.3
0.2
[d(5'GCATGC) -H]-
0.1
0
-2
-6 -10 -14 -18 -22 -26 -30 -34 -38 -42 -46 -50
Laboratory Collision Voltage /V
8
Dissociation pathways of metallated duplexes
M = Mn, Fe, Co, Ni - pathway (1) dominates
M = Cu, Zn - pathways (1) and (2) are nearly equal
[Mss - 3H]- + [ss- 2H]2- (1)
[Md(5’GCATGC)2 - 5H]3Pathway (3) dominates for all metals
[M2d(5’GCATGC)2 - 7H]3-
[Mss - 4H]2-+ [ss- H]- (2)
[Mss - 3H]- + [Mss- 4H]2- (3)
[M2ss - 5H]- + [ss- 2H]2- (4)
[M3d(5’GCATGC)2 - 9H]3-
[M2ss - 5H]- + [Mss- 4H]2- (5)
Underlined species were not observed due to the limited mass range
9
Tangent voltages for the dissociation of singly, doubly and
triply metallated double-stranded 5’GCATGC3’ trianions.
[Md(5’GCATGC)2 - 5H]3-48
Mn
[M2d(5’GCATGC)2 - 7H]3-
Tangent Voltage /V
Fe
-43
[M3d(5’GCATGC)2 - 9H]3-
Co
Ni
-38
Cu
Zn
-33
No metal present
-28
TV ([d(5’GCATGC)2 - 3H]3-) = -(30.7 0.2) V
10
Metallated Bleomycin A2 adducts with ds hexamers
2+
Kryatov et al. Chem. Rev., 2005, 105, 2175-2226
"peroxide shunt"
O2
[Fe(II)BLM]2+
1e, 1H+
[O2Fe(II)BLM]2+
H2O2
[HOOFe(III)BLM]2+
[Fe(III)BLM]3+
"activated BLM"
substrate
DNA
oxidation products
(DNA strand scission)
11
Chen, J. and Stubbe J. Cur. Op. Chem. Biol.,
2004, 8, 175 - 181
O
S
S
N
H3C
H3C
S
+
O
N
N
N
H
H
Arrows indicate potential hydrogen-bond donors or acceptors. Note the
crescent shape of the fragment
12
CID profiles of [MBLMd(5’GCATGC)2 - 6H]40.8
[MnBLMd(5'GCATGC)2 - 6H]4[d(5'GCATGC) - 2H]2-
-45
-43
0.7
-41
0.6
0.5
0.4
[MnBLMd(5'GCATGC) - 4H]2-
0.3
0.2
Tangent Voltage /V
Relative Intensity
Mn
-39
-37
-35
Ni
-33
TTAGGG
GCATGC
Cu
-31
0.1
-29
0
-27
-2
Zn
Co
-6 -10 -14 -18 -22 -26 -30 -34 -38 -42 -46 -50 -25
Laboratory Collision Voltage /V
[MBLMd(5’GCATGC)2 - 6H]4-  [d(5’GCATGC) - 2H]2- +[MBLMd(5’GCATGC) - 4H]2-
BLM = (Bleomycin A2 - H+); M = Mn, Co, Ni, Cu
13
Special case of Zn(II) containing complex
0.6
Relative Intensity
0.5
[d(5'GCATGC) - 2H]2[ZnBLMd(5'GCATGC)2 - 6H]4-
0.4
[BLMd(5'GCATGC) - 2H]2[ZnBLMd(5'GCATGC) - 4H]2-
0.3
[Znd(5'GCATGC) - 4H]2-
0.2
0.1
0
-6
-10 -14 -18 -22 -26 -30 -34 -38 -42 -46 -50
Laboratory Collision Voltage /V
[d(5’GCATGC) – 2H]2- + [ZnBLMd(5’GCATGC) – 4H]2[ZnBLMd(5’GCATGC)2 – 6H]4-
[Znd(5’GCATGC) – 4H]2- + [BLMd(5’GCATGC) – 2H]2-
Zn2+ has higher affinity for the phosphate groups of DNA compared to
14
BLM!
ESI/CID of 1,10 - phenanthroline-containing complexes
7.E+05
ds3-
[LnComd(5'GCATGC)2 - (2xm+3)H]3with n in the range 1 - 3m, m being 1, 2 and 3
6.E+05
Intensity, cps
5.E+05
Co2+ : Phen : ss
5:5:1
4.E+05
Cods
3-
3.E+05
N
3-
LCods
N
3-
L2Cods
2.E+05
L3Co2ds3Co 2 ds 3-
1.E+05
Co 3 ds
0.E+00
1100
1200
3-
LCo2ds3- L2Co2ds3-
L3Co 3ds
LCo 3ds
1300
L3Cods
0.5
0.4
0.3
0.2
0.1
0
3-
3-
1400
1500
1600
1700
1800
m/z
1.0
[d(5'GCATGC3') -2H]20.9
2[d(5'GCATGC3') -2H]
0.8 [NiL 3d(5'GCATGC3')2 -5H]33[Cod(5'GCATGC3')2 -5H]
0.7
0.6
30.5
3[NiL d(5'GCATGC3')2 -5H]
[CoL d(5'GCATGC3')2 -5H]
0.4 [NiL 2d(5'GCATGC3')2 -5H]3[Ni(5'GCATGC3')2 -5H]30.3
0.2
0.1
0.0
-2 -4 -6 -8 -10 -12 -14 -16 -18 -20 -22 -24 -26 -28 -30
-2 -6 -10 -14 -18 -22 -26 -30 -34 -38 -42 -46 -50
3-
[CoL 2 d(5'GCATGC3')2 -5H]
Relative Intensity
Relative Intensity
0.6
L4Co2ds3L4Co 3ds
L2Co 3ds33-
0.8
0.7
3-
Laboratory Collision Voltage /V
Laboratory Collision Voltage /V
[MLnds - 5H]3-  [MLn-1ds - 5H]3- + L with n = 1-3
15
containing
1, 2 and 3 Phensspecies
Relative stabilities
of Phen-containing
2323L22ssML
Species
dsML
ssML2dsML
ssML23dsML
ssML32logK
dsML1 3logK
dsML2 23logK
dsML3 33log
3
10.4) -(22.40.5) -(26.00.3)
-(22.20.4) -(25.60.3)
-(23.10.4) -(36.20.4)
-(22.40.5) 7.02
-(26.00.3) 6.7
-(25.60.3) 6.28
-(36.20.4) 7.
70.3)
50.4)
Co
-(30.40.4)
Ni
-(25.70.7)
Cu
-(32.20.4)
-(30.10.3) -(34.60.4)
-(24.70.3) -(34.10.3)
-(30.40.4) 8.0
-(32.20.4) 8.0
-(34.60.4) 7.9
-(34.10.3)
-(29.50.2)
-(25.00.4) -(27.00.3)
-(23.50.4) -(36.00.4)
-(25.70.7) 8.82
-(29.50.2) 6.67
-(27.00.3) 5.02
-(36.00.4)
The last 3 columns contain common logarithms for the binding constants of stepwise coordination of
Co(II), Ni(II) and Cu(II) with1,10-phenanthroline at 298 K and ionic strength  =0.1
N
N
N
N

 OEs (in volts) of the dissociation
of 1:1, 1:2
3-
M
-82
N
Tangent
/V /V
Tangent Voltage
Tangent
Voltage
/VVoltage
-72
-77
-67
-72
-82
-62
-67
-77
-57
-62
-72
-52
-57
-67
-47
-52
-62
-42
-47
-57
-37
-42
-52
-37
-47
N
Co
2+
[ds(ML3)3]
and 1:3 d(5’GCATGC)
2 - [M(II)L3]
3complexes
[ds(ML
Ni
3)3]
Co
Ni
M
N
N
N
-77
-82
N
N
Co

Co
Co
N
Co
Ni
Cu
[ds(ML3)3]3-
dsML33-
-(36.20.4) -(34.10.3) -(36.00.4)
dsM2L63-
-(39.0 0.4) -(38.10.6) -(39.90.5)
dsM3L93-
-(79.8  0.5) -(81.30.7) not determined
Ni
Ni
Ni
Cu
Cu
Coggan et al. Inorg. Chem.,
1999, 38, 20, 4496
-
[ds(ML3)2]3-
[ds(ML33)2]3[dsML3]
[dsML ]3-
16
8.
8.
Special case of Trien as the ligand
Trien forms mixed complexes with oligonucleotides only in
the presence of copper (II) !
CID spectrum of [CuTriend(5'GCATGC)2 - 5H]3(1262.6) at Lab Collision Voltage - 40 V
Intensity, cps
2+
[d(5'GCATGC) - 2H]2-
1.2E+03
NH
895
NH
1.0E+03
8.0E+02
[Cud(5'GCATGC)2 - 5H]3-
Cu
6.0E+02
1262.6
1214
4.0E+02
2.0E+02
NH2
NH2
0.0E+00
0
500
1000
1500
m/z
[CudsTrien]3[CudsPhen]3-
-(35.70.3) V
-(29.50.2) V
17
Conclusions
 ESI/CID provides insight into Metal ion - Ligand - DNA
interactions: the stoichiometry and mode of binding, the
dissociation pathway and relative gas phase stabilities.
Future Plans
 Systematic ESI/CID studies of Metal ion - Drug - DNA interactions
are needed to establish general trends in the gas-phase stability,
dissociation mechanisms.
 Investigation of the intrinsic reactivity of metallated biological
ions toward gaseous carcinogens and other harmful compounds
using the Q-trap 2000 and ESI SIFT QqQ instruments.
 Solution and gas-phase experiments with double-stranded
sequences containing a mismatching base pair and various
intercalating species.
18
Acknowledgements
Prof. D. K. Bohme
Greg Koyanagi
Michael Jarvis
Andrea Dasic
Sara Hashemi
Tuba Gozet
Stefan Feil
Mike Duhig
Voislav Blagojevic
$$ NSERC
MDS SCIEX
19