universidad politécnica de valencia instituto de tecnologÍa quimica, upv-csic drug-biomolecule...
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Universidad Politécnica de Valencia
INSTITUTO DE TECNOLOGÍA QUIMICA, UPV-CSIC
Drug-Biomolecule Interactions Drug-Biomolecule Interactions
in the Excited Statesin the Excited States
Miguel A. MirandaMiguel A. Miranda
Israel, April 2010
- Introduction
- Photodamage to DNA
- Photorepair
- Conclusions
Outline
Introduction: Photochemical damage to biomolecules
290 320 400 nm Photobiological spectrum
UVC:UVC: not present in ambient sunlight; it is filtered by stratosphere layer of ozone.
UVB:UVB: overlaps with the DNA and protein absorption spectra and is within the range mainly responsible for pathological effects through direct photochemical damage.
Direct Photochemistry
UVA :UVA : photocarcinogenic and involved in photoaging, but weakly absorbed by DNA and proteins. Produces damages indirectly, through light absorption by other chromophores.
Photosensitization
UVC UVB UVA Visible IR
(Poly)unsaturated fatty acids peroxidationCholesterol oxidationMembrane lysis Phototoxicity
Lipids
- 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 photobindingProtein-protein photocrosslinkingBinding site occupancy
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.
ProteinsModel dyads
Photoallergy
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.
Nucleic acids
Base damage
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.
Thymine dimerization Guanine oxidation 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.
PhotogenotoxicityPhotomutagenicity
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
Stereodifferentiation(chiral drug/nucleosides)
- Introduction
- Photodamage to DNA
- Photorepair
- Conclusions
Encinas, Belmadoui, Climent, Gil, Miranda, Chem. Res. Toxicol. 2004, 17, 857.
Benzophenone photosensitized Interaction with Thd
Thymine dimer formation
33BP*BP*33ThdThd
NH
N N
NH
O
O
CH3CH3
O
O
NH
N N
NH
O
O
CH3CH3
O
O
Triplet-Triplet (T-T) Energy transferTriplet-Triplet (T-T) Energy transferPaterno-Büchi Paterno-Büchi
PhotocycloadditionPhotocycloaddition
Oxetane formation
N
HN
O
O
CH3
OO
OH
HO
BPBP
O
0,0 8 10 120
10
20
0 5 10 20 30 40 500
10
20
30
40
50
60
I, u
.a.
Tiempo de elución, min
Thd + Thd<>Thd
Oxetanes (S)-KP-Thd
Oxetanes DKP-Thd
Oxetanes (R)-KP-ThdHN
N N
NH
O OCH3 CH3
O O
dR dR
N
HN
O
O
CH3
dR
O
H3C
COOH
N
HN
O
O
CH3
dR
O
CH3
Products distribution after photolysis of Thd in the presence of KP
Lhiaubet-Vallet, Encinas, Miranda. J. Am. Chem. Soc. 2005, 127, 12774
Elution Time (min)
Ketoprofen (KP)Ketoprofen (KP)
O CH3
COOH
KP/Thymidine interaction: Laser flash photolysis
300 400 500 600 7000,00
0,01
A,
a.u
.
, nm
3KP
0
1
2
3
4
5
0 0,5 1 1,5 2 2,5 3[Thd], mM
o /
(R)-KP
(S)-KP
0 2 4 6 8 10 12 140,000
0,004
0,008
0,012
A, a
.u
Time, s
EnantiodifEnantiodiffferenerenttiaiatiotion n In theIn the 33KP-ThdKP-Thd interaction interaction
33KP KP Quenching byQuenching by Thd ThdTriplet-Triplet Triplet-Triplet transition oftransition of KP KP detected detected
kS(Thd)= 3.6 108 M-1s-1
kR(Thd)= 5.1 108 M-1s-1kR/kS = 1.4
Lhiaubet-Vallet, Encinas, Miranda. J. Am. Chem. Soc. 2005, 127, 12774
Detection of thymine dimers formation in DNA
Supercoiled Plasmid DNA
Form I
Form IIssb dsb
Form II Form III
Form IElectrophoresis : different mobility
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.
ENX NFX PFX ANFX OFX RFX Cont0
10
20
30
40
50
60
70
% F
orm
II (
from
hv
+ E
ndo
V)
h 15 min 10 min 5 min
DNA-photosensitization
• pBR + FQ (20 M) + UVA (355 nm)• enzymatic treatment with T4 endonuclease V
Form II
Form I
T<>T
No T<>T
T<>T formationnative DNA
Agarose gel electrophoresis
ENX, NFX and PFX sensitize T<>T but ANFX does not!!
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
260
265
270
275
3PFX3NFX
3ANFX
ET(k
J.m
ol-1
)
3T (DNA)
T<>T
267
3OFX
3RFX
3ENX
255
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.
X N
COOH
R1
O
F
N
N
R3
- Introduction
- Photodamage to biomolecules
- Photorepair
- Conclusions
Formation of (6-4) pyrimidine dimers
(6-4) Photoproducts can be formed through a Paterno-Büchi photoreaction
between two adjacent pyrimidines in DNA
HN
N
X
O
R
PN
HN
O
X
R
5́
3́
X = O, NHR = Me, H
h
h´ +
HN
N
X
O
R
PN
HN
O
X
R
5́
3́
HN
N
X
O
PN
N
O
R
5́
3́
R
XH
(6- 4) Photoproduct
Photolyase
PET Cycloreversion of oxetanes in DNA repair
HN
N
X
O
N
N O
R1
R2
OH
H
HN
N
X
ON
HN O
R1
R2
H
O HN
N
X
ON
HN O
R1
R2
H
O
FADH FADH
h
HN
N
X
O
HN
N
O
O
+
R1
H
R2
H
HN
N
X
O
HN
N
O
O
+
R1
H
R2
H
• 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.
Oxidative PET-cycloreversion of oxetanes
O
PhPh
h/ SMeCHO +
Ph
Ph
X
Ph
Ph PhY
S =
A: X = O, Y = BF4B: X = S, Y = OCl4
HO
+MeCN
O
H
PhPh
HO H
HH
HO
PhPh
HO
h/ SMeCN
O
PhPh
HO
O
OPh
Ph
Intramolecular Nucleophilic Trapping
Stepwise cycloreversion of oxetane radical cation via initial O-C2 cleavageSpin 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
Perez-Ruiz, Izquierdo, Miranda, J. Org. Chem., 2003, 68, 10103.
OMe
CN
OMe
Reductive PET-cycloreversion of oxetanes
Perez-Ruiz, Gil, Miranda, J. Org. Chem., 2005, 70, 1376.
(S)Me
OO (S)
(S)
(R)OCN
OMe
(S)Me
OO (R)
(R)
(S)OCN
OMe
6 7
1
~ H5
S
R2
R1
H
Ph
1S
Ph Ph
6
3 + 4 (or 3 + 4 )a) b)
e´)
3 + 4
3a + 4
IMCs
3b + 4
6
IMCs
R1
R2
- e-
b´)
c)
c´)
+ e-
- e-
d)
d´)
e)
f)
f´)
+ e-
- e-
g)
g´)5
3c + 4
3 + 45 + 5´
S
Ph Ph
R2
R1
1a-ca, R1 = Me, R2 = Phb, R1 = Me, R2 = p-C6H4OMec, R1 = Ph, R2 = Ph
SPh
Ph
Ph+
2
S
R2
R1 Ph S
R2
R1 Ph
5´a-b5a-bR2
S
Ph Ph
R1
3a-c 4
Oxidative PET-cycloreversion of thietanes: Ion-molecule complexes
Argüello, Pérez-Ruiz, Miranda, Org. Lett. 2010, 12, 1756.
- Introduction
- Photodamage to biomolecules
- Photoprotection/photorepair
- 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
Conclusions
ACKNOWLEDGEMENTS
PhD STUDENTS
P. Bartovsky P. BonanciaM. Gómez M. Marin G. NardiE. Nuin L. PiñeroJ. RohacovaS. Soldevila
POST-DOCS
R. AlonsoI. AndreuM. C. CuquerellaV. Lhiaubet-Vallet R. Pérez
COLLABORATIONS
UPV-CSICF. BoscáS. EncinasM. J. ClimentM. C. Jiménez M. L. MarinI. M. MoreraR. Tormos
EXTERNALJ. V. CastellJ. Pérez-PrietoT. GimisisC. Chatgilialoglu
FUNDING
European UnionSpanish Government Regional Government UPV and CSICISDINOrganon/Merck