Universidad Politécnica de Valencia
INSTITUTO DE TECNOLOGÍA QUIMICA, UPV-CSIC
Drug-Biomolecule Interactions
in the Excited States
Miguel A. Miranda
Israel, April 2010
Outline
- Introduction
- Photodamage to DNA
- Photorepair
- Conclusions
Introduction: Photochemical damage to biomolecules
UVC UVB UVA
290 320 400 nm
Visible
IR
Photobiological spectrum
UVC: not present in ambient sunlight; it is filtered by
stratosphere layer of ozone.
UVB: overlaps with the DNA and protein absorption spectra
and is within the range mainly responsible for pathological
effects through direct photochemical damage.
UVA : photocarcinogenic and involved in photoaging,
but weakly absorbed by DNA and proteins. Produces
damages indirectly, through light absorption by other
chromophores.
Direct
Photochemistry
Photosensitization
Lipids
(Poly)unsaturated fatty acids peroxidation
Cholesterol oxidation
Membrane lysis
Phototoxicity
- Boscá, et al., J. Photochem. Photobiol., B: Biol. 2000, 58, 1
- Samadi et al., Photochem. Photobiol.2001, 73, 359
- Miranda et al., Chem. Commun. 2002, 280
- Boscá et al., Chem. Commun. 2003, 1592
- Andreu et al., Org. Lett. 2006, 8, 4597
- Andreu et al., Org. Biomol. Chem. 2008, 6, 860
Proteins
Drug-protein photobinding
Protein-protein photocrosslinking
Binding site occupancy
Model dyads
 Miranda et al. J. Am. Chem. Soc., 1999, 121, 11569.
 Miranda et al. Chem. Commun., 2000, 2257.
 Perez-Prieto et al. J. Org. Chem., 2004, 69, 374.
 Perez-Prieto et al. J. Org. Chem., 2004, 69, 8618.
 Lhiaubet-Vallet et al. J. Phys. Chem. B 2007, 111, 423.
 Vaya et al. J. Phys. Chem. B 2007, 111, 9363.
 Vaya et al. Chem. Phys. Lett. 2010, 486, 147.
Photoallergy
Proteins
 Miranda et al. Chem. Res. Toxicol. 1998, 11, 172.
 Lahoz et al. Chem. Res. Toxicol. 2001, 14, 1486.
 Lhiaubet-Vallet et al. J. Am. Chem. Soc. 2004, 126, 9538.
 Jimenez et al. J. Am. Chem Soc. 2005, 127, 10134.
 Vaya et al. ChemMedChem 2006, 1, 1015.
 Lhiaubet-Vallet et al. J. Phys. Chem. B 2007, 111, 423.
 Vaya et al. J. Phys. Chem. B 2008, 112, 2694.
 Vaya et al. Chem Eur. J. 2008, 14, 11284.
 Montanaro et al. ChemMedChem 2009, 4, 1196.
 Bueno et al J. Phys. Chem. B 2009, 113, 6861.
 Perez-Ruiz et al, J. Phys. Chem. Lett. 2010, 829.
Nucleic acids
Base damage
Photogenotoxicity
Photomutagenicity
Thymine dimerization
Guanine oxidation
 Lhiaubet-Vallet et al. Photochem. Photobiol. 2003, 77, 487.
 Lhiaubet-Vallet et al. J.Phys Chem. B 2004, 108, 14148.
 Bosca et al. J. Am. Chem. Soc. 2006, 128, 6318.
 Lhiaubet-Vallet et al. J. Phys. Chem. B 2007 111, 7409.
 Trzcionka et al. ChemBioChem 2007, 4, 367.
 Lhiaubet-Vallet et al. Photochem. Photobiol. 2009, 85, 861
 Agapakis et al. Photochem. Photobiol. 2000, 71, 499
 Belvedere et al. Chem. Res. Toxicol. 2002, 15, 1142.
 Cuquerella et al. Chem. Res. Toxicol. 2003, 16, 562.
 Lhiaubet-Vallet et al. Toxicol. in vitro 2003, 17, 651.
 Chatgilialoglu et al. Chem. Res. Toxicol. 2007, 20, 1820.
 Paris et al. Org. Lett. 2008, 10, 4409.
Stereodifferentiation
(chiral drug/nucleosides)
 Encinas et al. ChemPhysChem, 2004, 5, 1704.
 Lhiaubet-Vallet et al. J. Am. Chem. Soc. 2005, 127, 12774.
 Encinas et al. Chem. Commun. 2005, 272.
 Belmadoui et al. Chem. Eur. J. 2006, 12, 553.
 Jimenez et al. Org. Biomol. Chem. 2008, 6, 860.
- Introduction
- Photodamage to DNA
- Photorepair
- Conclusions
Benzophenone photosensitized Interaction with Thd
O
BP
Triplet-Triplet (T-T) Energy transfer
3BP*
Paterno-Büchi
Photocycloaddition
3Thd
O CH3
HN
HO
O
O
O
CH3 CH3
NH
N
N
NH
O
O
NH
N
N
O
O
CH3 CH3
NH
O
O
N
O
OH
O
Oxetane formation
Thymine dimer formation
Encinas, Belmadoui, Climent, Gil, Miranda, Chem. Res. Toxicol. 2004, 17, 857.
Products distribution after photolysis of Thd in the presence of KP
O
CH3
O
COOH
Thd +
Thd<>Thd O
I, u.a.
Ketoprofen (KP)
O
CH3 CH3
HN
N
N
dR
dR
O
60
10
O
CH3
0
0,0
HN
50
Oxetanes
(S)-KP-Thd
40
Oxetanes
(R)-KP-Thd
20
NH
O
N
O
8
dR
H3 C
30
O
COOH
20
HN
Oxetanes
DKP-Thd
10
CH3
O
N
O
dR
CH3
0
0
5
10
20
30
40
50
Elutionde
Time
(min) min
Tiempo
elución,
Lhiaubet-Vallet, Encinas, Miranda. J. Am. Chem. Soc. 2005, 127, 12774
10
12
KP/Thymidine interaction: Laser flash photolysis
Triplet-Triplet transition of KP detected
3KP
Quenching by Thd
0,012
0,01
A, a.u
A, a.u.
3KP
0,00
300
400
500
, nm
600
700
0,008
0,004
0,000
0
2
4
6
8
10
Time, s
12
14
Enantiodifferentiation
In the 3KP-Thd interaction
kS(Thd)= 3.6 108 M-1s-1
kR(Thd)= 5.1 108 M-1s-1
Lhiaubet-Vallet, Encinas, Miranda. J. Am. Chem. Soc. 2005, 127, 12774
kR/kS = 1.4
Detection of thymine dimers formation in DNA
Supercoiled Plasmid DNA
Electrophoresis : different mobility
Form I
Form II
ssb
Form II
dsb
Form I
Form III
T<>T detection:
T4 endo V specific of cis-syn T<>T
 formation of a ssb
Bosca, Lhiaubet-Vallet, Cuquerella, Castell, Miranda, J. Am. Chem. Soc., 2006, 128, 6318.
% Form II (from hv + Endo V)
DNA-photosensitization
h
15 min
10 min
5 min
T<>T
70
60
Agarose gel electrophoresis
• pBR + FQ (20 M) + UVA (355 nm)
• enzymatic treatment with T4 endonuclease V
50
40
ENX, NFX and PFX sensitize T<>T
but ANFX does not!!
No T<>T
30
20
10
0
ENX
NFX
PFX ANFX OFX
RFX Cont
Form II
Form I
T<>T formation
native DNA
Bosca, Lhiaubet-Vallet, Cuquerella, Castell, Miranda, J. Am. Chem. Soc., 2006, 128, 6318.
Lhiaubet-Vallet, Cuquerella, Castell, Bosca, Miranda, J. Phys. Chem. B, 2007, 111, 7409.
Triplet state energy of thymine in DNA
275
ET(kJ.mol-1)
3ENX
270
3PFX
N
R1
3NFX
267
265
R3
3T
(DNA)
3ANFX
3OFX
N
X
N
F
260
COOH
O
T<>T
255
3RFX
Photosensitizer with ET > 267 kJ mol-1 = potential photogenotoxic agent
Bosca, Lhiaubet-Vallet, Cuquerella, Castell, Miranda, J. Am. Chem. Soc., 2006, 128, 6318.
Lhiaubet-Vallet, Cuquerella, Castell, Bosca, Miranda, J. Phys. Chem. B, 2007, 111, 7409.
- Introduction
- Photodamage to biomolecules
- Photorepair
- Conclusions
Formation of (6-4) pyrimidine dimers
X
X
X
R
HN
O
N
5´
HN
R
HN
O
P
h
h´ +
Photolyase
N
3´
X = O, NH
R = Me, H
R
R
X
O
HN
X
N
5´
R
HN
O
P
O
XH
N
5´
N
3´
R
N
O
P
N
3´
(6- 4) Photoproduct
(6-4) Photoproducts can be formed through a Paterno-Büchi photoreaction
between two adjacent pyrimidines in DNA
PET Cycloreversion of oxetanes in DNA repair
• PET Cycloreversion of oxetanes is important for the photoenzymatic repair of
(6-4) photoproducts of the DNA dipyrimidine sites by photolyases
• The mode of action involves photochemical transfer of one electron from a reduced and
deprotonated flavin (FADH-) to an oxetane. Subsequently, the oxetane radical anion cleaves to
provide one neutral pyrimidine plus one pyrimidine radical anion.
X
X
X
R1
HN
O
R1
HN
OH
N
N
R1
O

O

O
H
HN
H
N
O
h
N
H
R2
N
R2
+
O
N
H
H
R2
O
R1
HN
N
H
O
N
H
R2
HN
+
O
N
X
R2
HN
O
FADH
O
R1
HN
H
N
N
O
N
FADH
X
O
O
N
H
Oxidative PET-cycloreversion of oxetanes
Intramolecular Nucleophilic Trapping
h/S
MeCN
HO
O
Ph
Ph
h/S
MeCN
Ph
HO
O
Ph
HO
MeCHO +
Ph
+
Ph
O
Ph
O
H
H
Ph
Ph
Ph
O
Ph
HO
H
O
H
Ph
Ph
S =
Ph
X Ph
Y
A: X = O, Y = BF4
B: X = S, Y = OCl4
Stepwise cycloreversion of oxetane radical cation via initial O-C2 cleavage
Spin and charge localized in the oxygen and C2, respectively
Formation of 2,3-diphenyl 4-hydroxytetrahydrofuran by intramolecular
nucleophilic trapping
Izquierdo, Miranda, J. Am. Chem. Soc. 2002, 124, 6532
H
Reductive PET-cycloreversion of oxetanes
Me
O
OMe
CN
O
O
O (S) O (R)
O (R) O (S)
CN
Me
(S)
(S)
OMe
6
CN
Me
(S)
(R)
OMe
7
Perez-Ruiz, Izquierdo, Miranda, J. Org. Chem., 2003, 68, 10103.
Perez-Ruiz, Gil, Miranda, J. Org. Chem., 2005, 70, 1376.
Oxidative PET-cycloreversion of thietanes: Ion-molecule complexes
Ph
R1
R2
S
Ph
Ph
Ph
S
+
1a-c
a, R1 = Me, R 2 = Ph
b, R 1 = Me, R 2 = p-C 6H 4OMe
c, R1 = Ph, R 2 = Ph
1
a)
-
-e
1
R1
R1
R1
Ph
Ph
R2
2
Ph R
R2 Ph
3a-c
4
5a-b
Ph
S
S
5´a-b
2
R1
b)
Ph
b´)
S
S
c)
Ph
3 + 4 (or 3 + 4
c´)
R2
d)
+ e-
d´)
- e-
)
3+4
IMCs
e)
3a + 4
e´)
3b + 4
R1
S
Ph
H
3c + 4
6
5 + 5´
IMCs
3+4
5
f´)
R2
g) + e-
f) ~ H
g´) - e
6
Argüello, Pérez-Ruiz, Miranda, Org. Lett. 2010, 12, 1756.
5
- Introduction
- Photodamage to biomolecules
- Photoprotection/photorepair
- Conclusions
Conclusions
- Solar (and artificial) light may produce both desired
and undesired effects on biological systems
- It is necessary to achieve a satisfactory understanding of
the chemical mechanisms involved in photobiological
effects
- Based on mechanistic knowledge, it is possible to
minimize the adverse effects of light, while enhancing
its beneficial effects
ACKNOWLEDGEMENTS
PhD STUDENTS
POST-DOCS
COLLABORATIONS
P. Bartovsky
P. Bonancia
M. Gómez
M. Marin
G. Nardi
E. Nuin
L. Piñero
J. Rohacova
S. Soldevila
R. Alonso
I. Andreu
M. C. Cuquerella
V. Lhiaubet-Vallet
R. Pérez
UPV-CSIC
F. Boscá
S. Encinas
M. J. Climent
M. C. Jiménez
M. L. Marin
I. M. Morera
R. Tormos
FUNDING
European Union
Spanish Government
Regional Government
UPV and CSIC
ISDIN
Organon/Merck
EXTERNAL
J. V. Castell
J. Pérez-Prieto
T. Gimisis
C. Chatgilialoglu