unravelling the potential of sulfur-substituted dna analogues as uva photosensitizers marvin pollum...

Unravelling the Potential of Sulfur-Substituted DNA Analogues as UVA Photosensitizers

MARVIN POLLUM and CARLOS E. CRESPO-HERNÁNDEZ

American Society for PhotobiologyJune 15, 2014

Department of ChemistryDepartment of Chemistry

N

HN

S

SO

OH

O

PO-

OO

O

OH

O

P

O-

O O

O

OHO

PO-

OO

O

OH

OH

P

O-

OO

O

OHO

ONA

NA

NA

NA

Photosensitizers• Compounds that initiate a chemical reaction following

absorption of light

• Medicinal Uses– Treatment of

various skin diseases– Cancer treatment


2

Alone No Effect Photosensitizer

Alone No Effect Light

+ Chemical ChangePhotosensitizer Light

Photosensitizers• Necessary Properties

1. Strong absorption of light

• For medicinal application longer wavelengths desired


3Figure: Moreira, M.C.; Prado, R.; Campos, Applied Biomedical Engineering. 2011

Photosensitizers• Necessary Properties

1. Strong absorption of light

• For medicinal application longer wavelengths desired

2. Light absorption populates reactive excited states

• Typically lowest energy triplet state because of long lifetime


4

S(*)

T1

S0

3O2

1O2

Biomolecule

Type I

Type II

Thiobases as Photosensitizers


5

• Sulfur-substituted DNA base analogues

• Begin with thiothymine– Change site of sulfur substitution– Change degree of sulfur

substitution

• Impact of sulfur substitutions on:– Absorption properties– Excited-state dynamics

• Propose thiothymines as potential photosensitizers?

HN

NH

O

O

1 2

4

3

5

6

Thymine (T)

HN

NH

S

O

4-thiothymine (4tT)

HN

NH

O

S

2-thiothymine (2tT)

HN

NH

S

S

2,4-dithiothymine (2,4dtT)

Steady-State Absorption


6

HN

NH

O

O

1 2

4

3

5

6

Thymine (T)

HN

NH

S

O

4-thiothymine (4tT)

HN

NH

O

S

2-thiothymine (2tT)

HN

NH

S

S


250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Abs

orba

nce

(au)

Wavelength (nm)

T

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Abs

orba

nce

(au)

Wavelength (nm)

T 2tT 4tT

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Abs

orba

nce

(au)

Wavelength (nm)

T 2tT

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Abs

orba

nce

(au)

Wavelength (nm)

T 2tT 4tT 24dtT

UVC UVB UVA

• Tune absorption over 100 nm

Excited-State Dynamics


7

• Broadband Transient Absorption Spectroscopy– ~200 femtosecond time resolution

Pump Laser Pulse

ProbeBroadband

Pulse

Delay time

I0 I

Sample Detector

~320 to 710 nm

400 500 600 700

0

5

10

15

20

A x

10-3

Wavelength (nm)

400 500 600 700

0

5

10

15

20

Dynamics of 2-thiothymine


8 Pollum, M. and Crespo-Hernández, C. E. J. Chem. Phys. 2014, 140, 071101

HN

NH

O

S

2-thiothymine (2tT)

PBS Buffer, pH 7.4λex = 316 nm

400 500 600 700

0

5

10

15

20

A x

10-3

0 ps

2.5 ps

2.5 ps

250 ps

No change in lifetime of experiment (1000 ps)



9 Pollum, M. and Crespo-Hernández, C. E. J. Chem. Phys. 2014, 140, 071101Taras-Goślińska, K. et al. J. Photochem. Photobio. A. 2014. 89

HN

NH

O

S

2-thiothymine (2tT)


0 200 400 600 800 10000

2

4

6

8

10

12

14

16 2tT

ex

= 316 nm

626 nm345 nm

A x

10

-3

Time (ps)

0.1 1 10 100 10000

2

4

6

8

10

12

14

16 2tT

ex

= 316 nm

626 nm345 nm

A x

10-3

Time (ps)

Thiobase τISC

(ps)Triplet Yield

2tT 0.62 0.9 ± 0.1

4tT

2,4dtT

400 500 600

-6

-4

-2

0

2

4A

x1

0-3

Wavelength (nm)



10

HN

NH

S

O

4-thiothymine (4tT)

Reichardt, C. and Crespo-Hernández, C. E.. J. Phys. Chem. Lett. 2010, 1, 2239-2243


400 500 600

-6

-4

-2

0

2

4

A x

10-3

0 ps

1.5 ps

1.5 ps

80 ps

400 500 600

-6

-4

-2

0

2

4




11 Reichardt, C. and Crespo-Hernández, C. E.. J. Phys. Chem. Lett. 2010, 1, 2239-2243


0.1 1 10 100 1000

1

2

3

4

54tT

ex

= 340 nm

567 nm507 nm

A x

10-3

Time (ps)

HN

NH

S

O

4-thiothymine (4tT)

0 200 400 600 800 1000

1

2

3

4

54tT

ex

= 340 nm

567 nm507 nm

A x

10-3

Time (ps)

Thiobase τISC

(ps)Triplet Yield

2tT 0.62 0.9 ± 0.1

4tT 0.24 0.85 ± 0.15

2,4dtT

Dynamics of 2,4-dithiothymine


12

400 500 600 700-2

-1

0

1

2

3

4

A x

10-3

Wavelength (nm) Pollum, M. and Crespo-Hernández, C. E. (Unpublished). 2014

HN

NH

S

S



400 500 600 700-2

0

2

4

A x

10-3

0 ps

1.5 ps

1.5 ps

100 ps




13 Pollum, M. and Crespo-Hernández, C. E. (Unpublished). 2014

HN

NH

S

S



0 200 400 600 800 10000

1

2

3

ex

= 316 nm

551 nm398 nm

A x

10-3

Time (ps)

2,4dtT

0.1 1 10 100 10000

1

2

3

ex

= 316 nm

551 nm398 nm

A x

10-3

Time (ps)

2,4dtT

Thiobase τISC

(ps)Triplet Yield

2tT 0.62 0.9 ± 0.1

4tT 0.24 0.85 ± 0.15

2,4dtT 0.37 N.D.

Conclusions


14

• Sulfur substitution of thymine results in:

– Red shift of absorption up to 110 nm

– Population of reactive triplet excited states• Ultrafast• High yields• In all three derivatives

Thiobase τISC

(ps)Triplet Yield

2tT 0.62 0.9 ± 0.1

4tT 0.24 0.85 ± 0.15

2,4dtT 0.37 N.D.

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

No

rma

lize

d A

bso

rba

nce

(a

u)

Wavelength (nm)

T 2tT 4tT 24dtT

UVC UVB UVA

Conclusions


15

• Propose 4tT and 2,4dtT as effective UVA photosensitizers

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

No

rma

lize

d A

bso

rba

nce

(a

u)

Wavelength (nm)

T 2tT 4tT 24dtT

UVC UVB UVA

Thiobase τISC

(ps)Triplet Yield

2tT 0.62 0.9 ± 0.1

4tT 0.24 0.85 ± 0.15

2,4dtT 0.37 N.D.

Figure: Moreira, M.C.; Prado, R.; Campos, Applied Biomedical Engineering. 2011

Future Work


16

• Test 4tT and 2,4dtT as a UVA photosensitizer in cells

• Use these spectroscopic techniques to screen for other potential photosensitizers

NH

NH

S S

S

6-thioguanine

NH

NNH

N

NH2

1

23

4

56

7

8

9

2,4-dithiouracil

NH

N

NH2

S

2-thiocytosine

Acknowledgments• Dr. Carlos Crespo

• Group Members

• National Science Foundation (CHE-1255084)


17


18

Experimental Methods


19

– Femto- to millisecond time window

UVA Photosensitizers• 4-thiothymidine shown to be a good

UVA photosensitizer


20Reelfs, O. et. Al. Nucl. Acids Res. 2011, 1. Reelfs, O. Karran, P. Young, A.R. Photochem. Photobio. Sci. 2012, 11, 148.

Incorporation into DNA Cancer Cell Targeting

Type I or Type II Mechanism?


21

Thiobase Singlet Oxygen Yield

4tT 0.17 ± 0.02

2tT N.D.

2,4dtT 0.08 ± 0.01

• Singlet Oxygen Yields– Collaboration with Steffen Jockusch– Direct measurement by 1O2 luminescence

• Relatively Low

22

1.0

0.8

0.6

0.4

0.2

0.0

806040200x10-6

15

10

5

0

x10

3

1320128012401200

1O2= 0.981

1R. Schmidt, C. Tanielian, R. Dunsbach, C. Wolf, J. Photochem. Photobiol. A 1994, 79, 11-17.

Phenalenone

Oobs = 1270 nm

time (µs)

lex = 355 nm

norm

alize

d in

tens

ity

inte

nsity

lex = 355 nm

Singlet oxygen phosphorescence standard

SJ_032814 1

l (nm)

air saturated TRIS buffer (D2O)pH = 7.5

UVA Photosensitizers• Two Modes of

Photosensitization

Type I– Direct Reaction

Type II– Indirect Reaction

• Singlet Oxygen Generation

• Typically from the T1 state because of long lifetime


23

S(*)

T1

S0

3O2

1O2

Biomolecule

Type I

Type II

Site and Degree of Substitution EffectsDepartment of ChemistryDepartment of Chemistry

24

Thiobase τISC

(ps)τSQ

(ps)Triplet Yield

4tT 0.24 84 0.85 ± 0.15

2tT 0.62 49 0.9 ± 0.1

2,4dtT 0.37 35 N.D.

Reichardt, C. and Crespo-Hernández, C. E.. J. Phys. Chem. Lett. 2010, 1, 2239-2243 Pollum, M. and Crespo-Hernández, C. E. J. Chem. Phys. 2014, 140, 071101

0.1 1 10 100 10000

1

2

3

ex

= 316 nm

551 nm398 nm

A x

10-3

Time (ps)

24dtT

0.1 1 10 100 10000

2

4

6

8

10

12

14

16 2tT

ex

= 316 nm

626 nm345 nmA

x 1

0-3

Time (ps)0.1 1 10 100 1000

1

2

3

4

54tT

ex

= 340 nm

567 nm507 nm

A x

10-3

Time (ps)

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Abs

orba

nce

(au)

Wavelength (nm)

Thd

Background


25

Epidermis

Dermis

UVC 0%

UVB 5%

UVA 95%

Ozone Layer

UV Radiation

UVC UVB UVA

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Abs

orba

nce

(au)

Wavelength (nm)

Thd 2tThy 4tThd 2,4dtThy

Background• Ultrafast relaxation of the

natural bases

– Ring puckering at C5 in uracil and thymine


26

HN

NH

O

O

1 2

4

3

5

6

Uracil

HN

NH

O

O

1 2

4

3

5

6

Thymine

Transient Absorption Spectroscopy (TAS)


27

S0

S1

Sn

T1

Tn

Pump Pulse

Wavelength

Δ A

Wavelength

Δ A

Wavelength

Δ A

Probe Pulse White light ~325 to 710 nm

ISC

ICISC

Wavelength

Δ A

WavelengthΔ

A

Time 1

Time 2

Time 3

Time 4

Time 5

Transient Absorption Spectroscopy (TAS)


28

Wavelength

Δ A

Wavelength

Δ A

Wavelength

Δ A

Wavelength

Δ A

WavelengthΔ

A

Wavelength

Abso

rban

ceTime 1

Time 2

Time 3

Time 4

Time 5

Time

Abso

rban

ce

0 1 2 3 4 5

Propose Good UVA Photosensitizers?Department of ChemistryDepartment of Chemistry

29

Thiobase τISC

(ps)Triplet Yield

Singlet Oxygen Yield

4tT 0.21 0.85 ± 0.15 0.17 ± 0.02

2tT 0.69 0.9 ± 0.1 N.D.

2,4dtT 0.52 N.D. 0.08 ± 0.01

HN

NH

S

O

4-thiothymine (4tT)

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

No

rma

lize

d A

bso

rba

nce

(a

u)

Wavelength (nm)

T 4tT 2tT 24dtT

UVC UVB UVA

Background• Photodynamic Therapy (PDT)

• Used to treat:– Cancer– Variety of skin diseases


30 www.photolitec.org/Tech_PDT

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Abs

orba

nce

(au)

Wavelength (nm)

T

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Abs

orba

nce

(au)

Wavelength (nm)

T 2tT 4tT

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Abs

orba

nce

(au)

Wavelength (nm)

T 2tT

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Abs

orba

nce

(au)

Wavelength (nm)

T 2tT 4tT 24dtT

UVC UVB UVA

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Abs

orba

nce

(au)

Wavelength (nm)

T

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Abs

orba

nce

(au)

Wavelength (nm)

T 2tT 4tT

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Abs

orba

nce

(au)

Wavelength (nm)

T 2tT

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Abs

orba

nce

(au)

Wavelength (nm)

T 2tT 4tT 24dtT

Sulfur-Substituted ThymineDepartment of ChemistryDepartment of Chemistry

HN

NH

O

O

1 2

4

3

5

6

Thymine (T)

HN

NH

S

O

4-thiothymine (4tT)

HN

NH

O

S

2-thiothymine (2tT)

HN

NH

S

S


32

UVC UVB UVA

400 500 600 700

0

5

10

15

20

A x

10-3

Wavelength (nm)

400 500 600 700

0

5

10

15

20



33 Pollum, M. and Crespo-Hernández, C. E. J. Chem. Phys. 2014, 140, 071101

HN

NH

O

S

2-thiothymine (2tT)


400 500 600 700

0

5

10

15

20

A x

10-3

0 ps

2.5 ps

2.5 ps

250 ps


400 500 600

-6

-4

-2

0

2

4A

x1

0-3

Wavelength (nm)



34

HN

NH

S

O

4-thiothymine (4tT)

Reichardt, C. and Crespo-Hernández, C. E.. J. Phys. Chem. Lett. 2010, 1, 2239-2243


400 500 600

-6

-4

-2

0

2

4

A x

10-3

0 ps

1.5 ps

1.5 ps

80 ps

400 500 600

-6

-4

-2

0

2

4




35

400 500 600 700-2

-1

0

1

2

3

4

A x

10-3

Wavelength (nm) Pollum, M. and Crespo-Hernández, C. E. (Unpublished). 2014

HN

NH

S

S



400 500 600 700-2

0

2

4

A x

10-3

0 ps

1.5 ps

1.5 ps

100 ps



36

HN

NH

S

O

4-thiothymine (4tT)

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

No

rma

lize

d A

bso

rba

nce

(a

u)

Wavelength (nm)

T 4tT

UVC UVB UVA

Thiobase τISC

(ps)Triplet Yield

4tT 0.24 0.85 ± 0.15


37

Thiobase τISC

(ps)Triplet Yield

4tT 0.24 0.85 ± 0.15

2tT 0.62 0.9 ± 0.1

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

No

rma

lize

d A

bso

rba

nce

(a

u)

Wavelength (nm)

T 4tT 2tT

UVC UVB UVA

HN

NH

O

S

2-thiothymine (2tT)

X


38

Thiobase τISC

(ps)Triplet Yield

4tT 0.24 0.85 ± 0.15

2,4dtT 0.37 N.D.

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

No

rma

lize

d A

bso

rba

nce

(a

u)

Wavelength (nm)

T 4tT 24dtT

UVC UVB UVA

HN

NH

S

S


Future Work


39

• Test 4tT and 2,4dtT as a UVA photosensitizer in cells

• Use these spectroscopic techniques to screen for other potential photosensitizers

• Study the dynamics of thiobases in DNA oligomers

NH

NH

S S

S

6-thioguanine

NH

NNH

N

NH2

1

23

4

56

7

8

9

2,4-dithiouracil

NH

N

NH2

S

2-thiocytosine

N

HN

S

SO

OH

O

PO-

OO

O

OH

O

P

O-

O O

O

OHO

PO-

OO

O

OH

OH

P

O-

OO

O

OHO

ONA

NA

NA

NA

unravelling the potential of sulfur-substituted dna analogues as uva photosensitizers marvin pollum...

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