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
Department of ChemistryDepartment of Chemistry
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
Department of ChemistryDepartment of Chemistry
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
Department of ChemistryDepartment of Chemistry
4
S(*)
T1
S0
3O2
1O2
Biomolecule
Type I
Type II
Thiobases as Photosensitizers
Department of ChemistryDepartment of Chemistry
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
Department of ChemistryDepartment of Chemistry
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
2,4-dithiothymine (2,4dtT)
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
Department of ChemistryDepartment of Chemistry
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
Department of ChemistryDepartment of Chemistry
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)
Dynamics of 2-thiothymine
Department of ChemistryDepartment of Chemistry
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)
PBS Buffer, pH 7.4λex = 316 nm
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)
Dynamics of 4-thiothymine
Department of ChemistryDepartment of Chemistry
10
HN
NH
S
O
4-thiothymine (4tT)
Reichardt, C. and Crespo-Hernández, C. E.. J. Phys. Chem. Lett. 2010, 1, 2239-2243
PBS Buffer, pH 7.4λex = 340 nm
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
No change in lifetime of experiment (1000 ps)
Dynamics of 4-thiothymine
Department of ChemistryDepartment of Chemistry
11 Reichardt, C. and Crespo-Hernández, C. E.. J. Phys. Chem. Lett. 2010, 1, 2239-2243
PBS Buffer, pH 7.4λex = 340 nm
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
Department of ChemistryDepartment of Chemistry
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
2,4-dithiothymine (2,4dtT)
PBS Buffer, pH 7.4λex = 335 nm
400 500 600 700-2
0
2
4
A x
10-3
0 ps
1.5 ps
1.5 ps
100 ps
No change in lifetime of experiment (1000 ps)
Dynamics of 2,4-dithiothymine
Department of ChemistryDepartment of Chemistry
13 Pollum, M. and Crespo-Hernández, C. E. (Unpublished). 2014
HN
NH
S
S
2,4-dithiothymine (2,4dtT)
PBS Buffer, pH 7.4λex = 335 nm
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
Department of ChemistryDepartment of Chemistry
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
Department of ChemistryDepartment of Chemistry
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
Department of ChemistryDepartment of Chemistry
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)
Department of ChemistryDepartment of Chemistry
17
Department of ChemistryDepartment of Chemistry
18
Experimental Methods
Department of ChemistryDepartment of Chemistry
19
– Femto- to millisecond time window
UVA Photosensitizers• 4-thiothymidine shown to be a good
UVA photosensitizer
Department of ChemistryDepartment of Chemistry
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?
Department of ChemistryDepartment of Chemistry
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
Department of ChemistryDepartment of Chemistry
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
Department of ChemistryDepartment of Chemistry
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
Department of ChemistryDepartment of Chemistry
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)
Department of ChemistryDepartment of Chemistry
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)
Department of ChemistryDepartment of Chemistry
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
Department of ChemistryDepartment of Chemistry
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
2,4-dithiothymine (2,4dtT)
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
Dynamics of 2-thiothymine
Department of ChemistryDepartment of Chemistry
33 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)
400 500 600
-6
-4
-2
0
2
4A
x1
0-3
Wavelength (nm)
Dynamics of 4-thiothymine
Department of ChemistryDepartment of Chemistry
34
HN
NH
S
O
4-thiothymine (4tT)
Reichardt, C. and Crespo-Hernández, C. E.. J. Phys. Chem. Lett. 2010, 1, 2239-2243
PBS Buffer, pH 7.4λex = 340 nm
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
No change in lifetime of experiment (1000 ps)
Dynamics of 2,4-dithiothymine
Department of ChemistryDepartment of Chemistry
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
2,4-dithiothymine (2,4dtT)
PBS Buffer, pH 7.4λex = 335 nm
400 500 600 700-2
0
2
4
A x
10-3
0 ps
1.5 ps
1.5 ps
100 ps
No change in lifetime of experiment (1000 ps)
Propose Good UVA Photosensitizers?Department of ChemistryDepartment of Chemistry
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
Propose Good UVA Photosensitizers?Department of ChemistryDepartment of Chemistry
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
Propose Good UVA Photosensitizers?Department of ChemistryDepartment of Chemistry
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
2,4-dithiothymine (2,4dtT)
Future Work
Department of ChemistryDepartment of Chemistry
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