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