Download Hydrogen Bonding Promotes Ultrafast

I
P C
Christian-Albrechts-Universität zu Kiel
Hydrogen Bonding Promotes Ultrafast Nonradiative
Electronic Relaxation in DNA Building Blocks
Institut für Physikalische Chemie,
Olshausenstr. 40, D-24098 Kiel, Germany
Nina K. Schwalb and Friedrich Temps
UV Fluorescence Up-Conversion Setup
Motivation & Aims
The DNA encodes the genetic information and is therefore the most
important biomolecule for the reproduction of life. Yet the available
information on the electronic properties and excited state dynamics is still
very limited. Time-resolved data on medium sized building blocks, especially
on hydrogen bonded nucleobase pairs are virtually missing and remain as a
DNA Strands
kind of black box.
Free Nucleosides
Thymine
O
Adenine
Adenine
H
H
N
N
Guanine
mediu
m size
buildin
d
g block
s?
Cytidine
O
H
N
N
N
Cytosine
N
N
N
H
N
O
O P O
O
H
N
N
O
O
H
O
O
H
Guanine
O
Thymine
N
N
N
O
N
H
O P O
O
femtosecond dynamics
dynamics up to nanoseconds
• The excitation wavelength was tuned between 258 nm and 302 nm.
• The emission/detection wavelength was set to 350 nm (~ emission maximum).
• The pump energy was ≤ 0.05 µJ.
Si
• The concentration varried between 0.01 and 1 mM, whereas here the results at 0.1 mM are
presented:
- nucleosides are only soluble in water or alcohols
O
- protic solvents prevent base pairing
O
240
N
N
H
H
N
O
H
C(TBDMSi)2 0.1 mM
G-C
G(TBDMSi)3 0.1 mM
280
300
320
Degree of GC Association
GC(TBDMSi)5 0.1 mM
0.8
- Synthesis of specially protected nucleosides
H
262 nm
nucleosides are now soluble in aprotic solvents
0.8
0.4
0.4
no participation of solvent molecules or hydroxy groups
O
O
G
C
λPUMP
Strategy:
N
260
wavelength / nm
N
H
0.3
0.0
%
N
N
0.6
- formation of nucleoside stacks in protic solvents
N
O
absorbance
Si
C
G
G-C
G plus C
0.9
Problems:
O
UV Absorption Spectra
Fluorescence Decay Profiles of G, C and the GC Watson-Crick Base Pair
Here, we report on the first femtosecond time-resolved experiments on the
the GC Watson-Crick base pair in solution to determine the effect of
hydrogen bonding on the excited state lifetime.
0.0
0.0
of the ribose moiety in hydrogen bonding
Si
-8
no base stacking but defined H-bonded nucleobase
Si O
G(TBDMSi)3 0.1 mM
C(TBDMSi)2 0.1 mM
dimers
283 nm
GC profile after subtraction
of the residual G and C monomers
0.4
0.0
Static FTIR Spectra of G, C and GC
0.8
600
G(TBDMSi)3 0.1 mM
C(TBDMSi)2 0.1 mM
C(TBDMSi)2 10 mM
τ = 0.36(3) ps
GC(TBDMSi)5 0.1 mM
0.8
0.4
0.4
0.0
400
GC dimer
NH (s) stretch
of monomer
296 nm
-1
G(TBDMSi)3 10 mM
ε / L * mol * cm
-1
5 mM
1 mM
0.25 mM
0
concentration / mol L
0.8
Characterisation of the H-bonded Base Pairs
-4
-1
GC(TBDMSi)5 0.1 mM
5 mM
1 mM
0.25 mM
NH (a) stretch
of monomer
200
H bonds
GC dimer
free NH stretch
of G or C dimer
GC(TBDMSi)5 10 mM
H bonds
GG dimer
5 mM
1 mM
0.25 mM
3500
3400
3300
3200
0
1
2
Delay Time / ps
0
0
H bonds
CC dimer
3600
-1
0.0
2
4
0
2
4
0
2
Monoexponential fluorescence decay for the
pure GC base pair is independent of the
pump wavelength.
4
Delay Time / ps
Solvent: CHCl3
3100
-1
wavenumber / cm
• Static FTIR measurements of the G and C monomers, homo- and heterodimers in
a concentration range between 0.25 – 150 mM.
• Multiple Gaussian Band Fitting of each spectral trace.
• Assignments of bands were supported by G3B3 (rel. energies of tautomers) and
DFT-PW91 (frequencies of the free nucleosides and H-bonded dimers)
calculations.
C(TBDMSi)2 0.1 mM
283 nm
As the association in n-hexane is much
stronger than in CHCl3, the formation of
trimers or higher aggregats may prolong the
excited state lifetime.
0.0
NH
D
20 °C
30 °C
40 °C
50 °C
60 °C
D
OD
0.02
2 NH2 (s)
0.00
FTIR Spectra fitted with Gauss Profiles
0.4
data G
linerare regression
0.04
D
0.06 0.12 0.18 0.24 0.30
conc. / OD
KGG = 728 (246) M-1
mOD
KCC =
5
41 (6)
M-1
KGC = 6.1·104 (1.5 ·104) M-1
0
3550 3500 3450 3400 3350 3300 3250 3200 3150 3100
-1
Wellenzahl / cm
1CT
4
4
Electron-proton back transfer
in the excited state
•
(GC)
•
•
hν
200 – 300 fs
WC ground state structure
•
S0
0.0
10
2
2
Single Gaussians
Multiple Gauss Fit
G @ 20 mM in CHCl3
Single Gaussians
Multiple Gauss Fit
G @ 0.2 mM in CHCl3
(G)
π (GC)
(C)
1 ππ*
ππ
1nπ
π*
Single Band Analysis
0.08
0.06
D
D
0.8
0
Conclusion & Discussion
1 ππ*
ππ
Energy / eV
NH2 (s)
NH2 (a)
-15
4
•
6
mOD
∆mOD
D
2
1
2
M
0
0
Delay Time / ps
Coupled electron-proton transfer
in the excited state
amino-oxo GG
M
M
4
amino-oxo G
Temperature dependent FTIR Spectra
M
2
Solvent: n-hexane
1 NH
0
1 NH2 (s)
200
2 NH2 (a)
0
1 NH2 (a)
Intensity
200
C(TBDMSi)2 0.1 mM
0.4
0
Calculated FTIR Spectra of G and GG
C(TBDMSi)2 0.1 mM
0.8
1.0
1.8
1.4
r(NH) / Å
Re-formation
of the original structure
Domcke et al. (2005); Doltsinis et al. (2007); Groenhof et al. (2007)
•
Our results unambigiously demonstrate that
the excited state lifetime is reduced by
complementrary hydrogen bonding in the WC
base pair in solution.
This is the first direct measuremnent of
the effect of H-bond formation.
The effect is stronger on G than on C.
The findings point at a unique ultrafast
optically dark relaxation pathway.
The pathway might be a theoretically proposed coupled electron-protontransfer in the excited state that is exclusively
accessible in the Watson-Crick form, where it
leads to a barrierless ultrafast relaxation back
into the ground state.
Calculated excited state lifetimes agree
very well with our measured lifetime of 0.36(3)
ps.