Synthetic control of retinal photochemistry and photophysics in solution

Article abstract of DOI:10.1021/ja4121814

Understanding how molecular structure and environment control energy flow in molecules is a requirement for the efficient design of tailor-made photochemistry. Here, we investigate the tunability of the photochemical and photophysical properties of the retinal-protonated Schiff base chromophore in solution. Replacing the n-butylamine Schiff base normally chosen to mimic the saturated linkage found in nature by aromatic amines results in the reproduction of the opsin shift and complete suppression of all isomerization channels. Modification of retinal by directed addition or removal of backbone substituents tunes the overall photoisomerization yield from 0 to 0.55 and the excited state lifetime from 0.4 to 7 ps and activates previously inaccessible reaction channels to form 7-cis and 13-cis products. We observed a clear correlation between the presence of polarizable backbone substituents and photochemical reactivity. Structural changes that increase reaction speed were found to decrease quantum yields, and vice versa, so that excited state lifetime and efficiency are inversely correlated in contrast to the trends observed when comparing retinal photochemistry in protein and solution environments. Our results suggest a simple model where backbone modifications and Schiff base substituents control barrier heights on the excited-state potential energy surface and therefore determine speed, product distribution, and overall yield of the photochemical process.

Full text of DOI:10.1021/ja4121814

Article
pubs.acs.org/JACS
Synthetic Control of Retinal Photochemistry and Photophysics in
Solution
Giovanni Bassolino,† Tina Sovdat,‡ Matz Liebel,† Christoph Schnedermann,† Barbara Odell,‡
Timothy D.W. Claridge,‡ Philipp Kukura,*,† and Stephen P. Fletcher*,‡
†Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1
3QZ, U.K.
‡Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.
S
* Supporting Information
ABSTRACT: Understanding how molecular structure and
environment control energy flow in molecules is a requirement
for the efficient design of tailor-made photochemistry. Here,
we investigate the tunability of the photochemical and
photophysical properties of the retinal-protonated Schiff base
chromophore in solution. Replacing the n-butylamine Schiff
base normally chosen to mimic the saturated linkage found in
nature by aromatic amines results in the reproduction of the
opsin shift and complete suppression of all isomerization channels. Modification of retinal by directed addition or removal of
backbone substituents tunes the overall photoisomerization yield from 0 to 0.55 and the excited state lifetime from 0.4 to 7 ps
and activates previously inaccessible reaction channels to form 7-cis and 13-cis products. We observed a clear correlation between
the presence of polarizable backbone substituents and photochemical reactivity. Structural changes that increase reaction speed
were found to decrease quantum yields, and vice versa, so that excited state lifetime and efficiency are inversely correlated in
contrast to the trends observed when comparing retinal photochemistry in protein and solution environments. Our results
suggest a simple model where backbone modifications and Schiff base substituents control barrier heights on the excited-state
potential energy surface and therefore determine speed, product distribution, and overall yield of the photochemical process.
RPSB in solution have been largely resistant to synthetic or
environmental control. Small variations in both quantum yields
(QYs) and excited state lifetimes (ESLs) have been
reported,16−20 but it has never been possible to mimic the
features observed in opsin photochemistry. The differences in
RPSB reactivity between solution and protein environments are
most dramatically illustrated by the photochemical properties of
the chromophore in the bacterial proton pump bacterioRho-
dopsin (bR)21,22 and in solution2 (Table 1). The red shift of
INTRODUCTION
■
The retinal chromophore is ubiquitously used in nature to
transform light into chemical, mechanical, or electrical energy.1
The desired function is invariably performed by a surrounding
protein but is initiated by an efficient photoisomerization of
retinal covalently bound via a protonated Schiff base linkage.
Although the choice of chromophore is convenient from a
metabolic perspective, its reactivity, spectral overlap with the
solar emission spectrum, and photochemical specificity are poor
for retinal and retinal protonated Schiff bases (RPSBs) in the
absence of a protein environment.2−5 Remarkably, all of these
parameters are dramatically improved in retinal-containing
proteins, which is in line with the idea of optimized
photobiological function. The discrepancy between solution
and protein behavior has been largely attributed to the
complexity of the protein pocket with its three-dimensional
arrangement of amino acids resulting in a unique steric and
dielectric environment.6,7
Table 1. Retinal Protonated Schiff Base Photochemistry in
Protein and Solution
The tunability of absorption spectra, isomerization yield, and
reaction speed makes RPSB the ideal candidate for
investigations aiming to unravel the molecular and structural
origins of efficient photochemistry.6−15 As a consequence,
RPSB has become a paradigm for understanding the origins of
activation and suppression of ultrafast relaxation processes,
which is essential for the rational engineering of photoreactivity.
Despite numerous efforts, however, the reactive properties of
Received: December 6, 2013
© XXXX American Chemical Society
A
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the absorption spectrum in the protein, the so-called “opsin
shift”, has been investigated in detail synthetically, spectroscopi-
cally, and computationally.4,10,23−33 The currently accepted
mechanism of color tuning is that the absorption maximum is
determined by the dielectric environment of the retinal binding
pocket.34−37
Table 2. Chain Substituents for the Backbone-Modified
Retinals
Addressing other aspects of opsin photochemistry is more
complex, and a widely adopted approach has been to prepare
pigment analogues containing retinal derivatives with modified
backbones.8,38−42 Taken together, these studies point toward
protein-induced steric and electrostatic interactions as a
primary determinant in the outcome of the photoreaction.43−47
To the best of our knowledge, only one study on the
photochemistry of a RPSB with a modified retinal backbone
(11-cis-13-demethyl RPSB) in solution has been reported.48 In
our recent exploratory work,49 we demonstrated that addition
of a methyl group to the 10-position of the retinal backbone
dramatically affects RPSB solution photochemistry, reducing
the ESL by almost 1 order of magnitude from 4 to 0.55 ps
toward the lifetime observed in bR (0.5 ps). Together with the
changes in ESL, the modification caused a 50% decrease in the
isomerization yield, suggesting an inverse correlation between
reaction speed and yield, but could only provide an incomplete
picture as to the mechanistic origin of the observed effect.
In this work, we systematically explored the influence of both
backbone and Schiff base substitution on RPSB photo-
chemistry. By appropriate choice of the Schiff base, we
succeeded in reproducing the shift of absorption maximum
observed for RPSBs in the protein environment, with the tail of
the absorption reaching all the way to the near-infrared (700
nm). Addition or removal of polarizable substituents from the
retinal backbone for the natural n-butyl (nBu) protonated Schiff
base allowed us to tune the ESL from 400 fs to 7 ps and achieve
a spread of the overall reaction yield from effectively 0 to 0.55.
We further induced changes in the selectivity of these reactions,
either completely closing secondary isomerization pathways or
obtaining 7-cis or 13-cis isomers rather than the 9-cis isomer
observed with natural RPSB in solution (see the Supporting
Information, Figure SF18). Our results demonstrate that
remarkable tunability of RPSB photochemistry can be achieved
in solution, without a specific spatial, steric, and dielectric
environment as provided by a protein pocket evolved through
natural selection.
(2b−m) (Scheme 1).12 Substituents at C10 of the backbone
(derivatives 2b−f,l) were introduced via coupling of commercially
available β-ionone with the appropriate phosphonate A.50,51 The HWE
reaction inevitably generated a mixture of E/Z-isomers. However,
subsequent reduction to the aldehyde allowed for the isolation of the
desired isomer in all cases. The chain was then extended with (E/Z)-
diethyl (3-cyano-2-methylallyl)phosphonate;50,52,53 again generating a
mixture of isomers. Upon reduction, all-trans C10-substituted retinals
were isolated (2b−f). The methyl group at position 14 of the retinal
backbone (2k,l) was installed using a methylated modification of (E/
Z)-diethyl (3-cyano-2-methylallyl)phosphonate52 prepared from
chloroacetone and diethyl(cyanoethyl)phosphonate. Methylation of
C8 (2g) was implemented through HWE coupling of β-cyclocitral with
diethyl (cyanoethyl)phosphonate. The methyl group at C9 was
introduced via addition of MeLi to the resulting nitrile.54 The polyene
chain was extended in a manner analogous to the C10-substituted
retinal synthesis. Addition of a methyl group to C15 (2m) was achieved
through quantitative 1,2-addition of MeLi to commercially available
all-E-retinal55 followed by oxidation with catalytic TPAP, which was
found to be a more suitable oxidizing agent than MnO2 or Dess−
Martin periodinane.
Demethylated retinal analogues (2h−j) were accessed by stepwise
formation of additional double bonds from the ring toward the
aldehyde functional group so that demethylation at C9 (2h) was
obtained through two consecutive cycles of HWE coupling followed
by DIBAL reduction starting from β-cyclocitral. Likewise, C13 methyl
group removal (2i) employs an analogous strategy starting from the
appropriate aldehyde.
Schiff base formation was achieved by condensing a retinal (2a−m)
with the appropriate amine. Chromophores containing aliphatic
amines were prepared by stirring the two components for 1 h at
0 °C over dry 4 Å molecular sieves in methanol under rigorously dark
conditions. Imines with aromatic amine moieties were prepared at
room temperature with extended reaction times (10−12 h).
Formation of protonated Schiff bases (1a−m and 3−5, see Figure
2) was achieved via the addition of a drop of neat trifluoroacetic acid
(TFA) to a solution of the imine in methanol.56 All photosensitive
materials were handled under dim red light illumination. See the
Supporting Information for details of the preparation, purification, and
characterization of each derivative.
MATERIALS AND METHODS
■
Synthesis. We used the general synthetic approach depicted in
Scheme 1 to prepare a library of retinal derivatives (Table 2). A series
of consecutive Horner−Wadsworth−Emmons (HWE) reactions and
DIBAL-H reductions were employed to prepare the modified retinals
a
Scheme 1. Synthesis of Modified Retinal Chromophores
Ultrafast Spectroscopy. Laser pulses were delivered by a
LightConversion PHAROS-6W amplifier (1030 nm, 180 fs, 5.5 W at
10 kHz). Fifteen milliwatts of the output was used for white light
generation in sapphire to generate broadband probe pulses. The
remainder was used for second-harmonic generation (SHG) in BBO
followed by sum frequency generation (SFG) between second
harmonic and fundamental (BBO).57 The obtained output at 343
nm (500 mW) pumped a white-light seeded two-stage noncollinear
optical parametric amplifier NOPA58 to generate the excitation pulse
(500 nm, 25 fs, 30 mW). The pump was compressed using chirped
mirrors (Layertec) and characterized with SHG frequency-resolved
a
Key: (a) BuLi, A; (b) DIBAL-H; (c) MeLi; (d) BuLi; (e) MeLi; (f)
TPAP, NMO.
B
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Figure 1. Experimental approach to determine RPSB photochemistry. (A) Differential absorbance map as a function of time delay and probe
wavelength recorded for natural nBu RPSB (1a). (B) Time traces at selected probe wavelengths throughout the stimulated emission band. (C)
1
Transient absorption spectra corresponding to the temporal evolution of the data. (D) Typical H NMR spectra before and after irradiation of
natural nBu RPSB.
optical gating (FROG)59 in a 10 μm BBO. A flow cell with path length
of 500 and 120 μm windows was used for the experiments. The flow
speed was adjusted to replenish the sample between consecutive
pulses. The diameters of the pump and probe beams were measured to
be 150 and 50 μm, with the pulse energies adjusted to 20 and 2 nJ,
respectively. Broadband detection was performed by a home-built
single shot prism spectrograph60 equipped with a LW-ELIS-1024A-
1394 detector. We corrected the differential absorbance maps for
probe chirp from solvent only traces to generate transient absorption
traces as a function of probe wavelength and time delay (Figure 1A)
and then fitted the latter via a global optimization routine.61 All
transient absorption data analysis was performed in MATLAB.62 The
dynamics of all compounds could be described by a triexponential
function convolved with a Gaussian instrument response and a step
function (Figure 1B). Typical transient differential absorption spectra
are shown in Figure 1C. The three time constants ranged from 0.2−
0.4, 0.5−3, and 3−10 ps for the first, second, and third constant,
respectively (see the Supporting Information, Table S1 and Figures
SF1−SF17, for details on each compound). The coherent response
due to the overlap of the pump and probe pulse is modeled with a
combination of Gaussian derivatives up to the ninth order.
photoproduct formed was determined by 500 or 700 MHz H NMR
spectroscopy by integration, comparing the average of several starting
material signals with the average of several product signals (Figure
1D). Sample concentration was determined by adding 10 μL of a 1%
v/v solution of an appropriate internal standard to 700 μL of
nonirradiated RPSB and comparing the average area of the RPSBs
1
1
signals with the internal standard in the H NMR spectrum (see the
Supporting Information, Table S2, for details on the internal standards
used for each compound).
For the aromatic amine containing compounds (3−5, Figure 2), the
QY and excited state dynamics were measured in CH3CN or CD3CN.
For this class of compounds no isomerization product was detectable
1
via H NMR spectroscopy.
Photoproduct Determination. Irradiations of the RPSBs were
carried out as described above, adjusting the reaction time to achieve
∼20% conversion. Under these conditions, no new species were
formed as determined by 1H NMR spectroscopy. Photoproducts were
identified by 1D and/or 2D NOESY, TOCSY, and COSY experiments
(see the Supporting Information).63 In some cases, photoproducts
were identified by comparison with a fully characterized synthetic
standard (see the Supporting Information).
Computational Methods. Ground-state optimized geometries for
all molecules studied in this paper were obtained with Gaussian 0964
using the wb97xd/cc-pVDZ potential.65,66 As a test for the optimized
structure to be a local minimum, the second derivatives of the Hessian
matrix were checked to be positive.67 Merz−Kollman (MK) partial
atomic charges68,69 for the ground and first singlet excited state were
evaluated with the wb97xd/6-31+G(d,p)65,70,71 potential at the ground
state optimized geometry. The solvent (methanol) was treated at the
CPCM level.72,73
Determining the Excited State Lifetime. The differential
absorption between 550 and 850 nm was used to evaluate the average
lifetime for each RPSB. From the global optimization routine, each
time constant was retrieved together with a decay associated spectrum
(DAS).61 For all detection wavelengths within the stimulated emission
(SE) band (∼600−850 nm) the average ESL was evaluated as a
weighted average of the decay constants and the absolute value of the
DAS amplitude, according to the following formula:
∑k τk·|DASkλ|
ESLλ =
RESULTS
∑k |DASkλ|
■
(1)
Aromatic Amines. To reproduce the “opsin shift”, we used
an aromatic amine to form the Schiff base, thus introducing
additional electron delocalization to the RPSB system. Three
compounds (3−5) exhibiting a broad range of photophysical
behavior, in addition to natural RPSB (1a), are shown in Figure
2. The red shift of the absorption when using aromatic rather
than aliphatic amines can be seen in the UV−vis absorption
spectra (Figure 2B). It consisted of up to 90 nm (to 534 nm)
for p-phenylenediamine RPSB 5 relative to nBu RPSB 1a. In
addition, the spectra broadened considerably. In contrast to the
uniform trend observed for shifts in the absorption maximum,
The values reported in the Results are the average of the lifetime
values obtained along the SE band (see the Supporting Information,
Table S1, for details on each compound).
Quantum Yield Measurements. A CW diode-pumped solid state
laser (532 nm) was used to irradiate a glass test tube containing 1 mL
of a stirred ∼3 mM (OD ≫ 2) solution of RPSB in CD3OD.
Expanding the beam to a diameter of 0.8 cm at an incident power of
0.2 mW resulted in irradiation in the linear regime. Experiments were
performed in such conditions to ensure less than 10% conversion of
the starting material, so that irradiation times ranged between 15 and
40 min depending on the isomerization yield. The amount of
C
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the ground electronic state for natural nBu RPSB (1a) (Figure
3). A positive sign of this charge difference corresponds to
Figure 3. Difference in MK charges of the excited and ground states
for polyene chain carbons of natural nBu RPSB 1a.
depletion of electron density, while a negative sign to an
increase in electron density for the atom in question. One
would thus expect the effect of backbone substitution to be
greatest on the topography of the excited electronic state when
there is a significant difference in electron density upon
excitation. Based on this reasoning, we focused on two types of
synthetic modifications: addition or removal of methyl groups
from the natural backbone (2b and g−m), and addition of
substituents with different electronic character at the C10
position (2b−f). The former class of compounds allowed us to
probe the effect of the substitution position, while the latter
helped us to understand the importance of the character of the
substitution. Evaluation of the ground state MK partial atomic
charges and the difference in MK charges upon excitation
revealed a pattern for these new derivatives qualitatively similar
to the one found for natural RPSB (1a), but differences were
observed at the position of substitution (see the Supporting
Information, Figures SF19−21).
Figure 2. Modification of the Schiff base. Comparison for RPSBs
obtained with aromatic amines (A) structures, (B) absorption spectra,
(C) decay kinetics, and (D) transient spectra at 1 ps after
photoexcitation with a resonant 25 fs pump pulse. Dashed lines
represent the fits obtained from the global optimization routine.
The results of the photochemical investigation on the
examined compounds are reported in Figure 4. A general
trend for the effects of substitution on the photochemistry
becomes immediately clear: all derivatives with substituents
added to the backbone (1b−g and 1k−m) exhibit faster excited
state decay than natural nBu RPSB 1a, while demethylation
(11h−j) increases the ESL. The only exception is 9-demethyl
RPSB 1h, which exhibits a slightly lower ESL than 1a. The ESL
can be tuned over a range of values smaller than for the
chromophore in bacterioRhodopsin (500 fs), as in the case of
10,14-dimethyl RPSB 1l (400 fs), to more than 1 order of
magnitude larger than in bR, in the case of 9,13-demethyl 1j (7
ps). Addition of substituents at the 10-position always shortens
the ESL, and the effect is correlated with the polarizability of
halogenated substituents (Br > Cl > F). A single demethylation
(1i) extends the ESL, with removal of both methyl backbone
groups (1j) having the largest effect in this sense.
the effect of the extended π-system on the ESLs (Figure 2C)
differed: In two cases, p-methoxyaniline RPSB (4) and p-
phenylenediamine RPSB (5), the lifetime decreased compared
to natural RPSB (1a) (3.4 and 1.2 ps vs 6.8 ps, respectively),
while in the case of the unsubstituted aniline (3) it increased
(12 ps).
A comparison of the transient spectra at 1 ps delay time for
natural nBu RPSB (1a) and the three aromatic derivatives (3−
5) is shown in Figure 2D. The features of the transient
spectrum of natural nBu RPSB (1a) are well-known including a
broad SE band (600→900 nm) overlapping with an excited-
state absorption band (ESA) that results in a distinctive double
peak shape. Another ESA band is present at 580 nm, with the
tail of the ground-state bleach (GSB) visible up to 550 nm.74
The most striking feature for the aromatic derivatives (3−5) is
the separation and reduced spectral overlap of the red ESA and
SE bands. As the absorption maxima for these derivatives is
considerably red-shifted, the GSB is now clearly visible in our
observation window up to ∼600 nm. Remarkably, the
isomerization yield for all RPSBs with aromatic Schiff bases
(3−5) is zero.
Modification of the Retinal Backbone. While extending
the π-system allowed us to reproduce the “opsin shift”, it did
not enable us to change the topography of the excited-state
potential energy surface (PES) and change the QY beyond
completely halting photoisomerization. Prompted by published
work reporting a considerable electron redistribution following
excitation for RPSBs,75,76 we evaluated the difference in MK
partial atomic charges between the first excited singlet state and
A similar trend is observed for the corresponding QY of
isomerization (Figure 4B). Removal of backbone substituents
(1h−j) increases the total isomerization yield, while the
addition of substituents (1b−g and 1k−m) reduces the yield.
The tunability ranges from a barely detectable overall yield
(≪0.02) for 10-chloro RPSB 1d to a total yield of 0.55 for the
13-demethyl isomer (1i). For those species where the
photoproducts identities could be determined, the major
photoproduct was the 11-cis isomer. Addition or removal of
substituents in the 9- and 10-position (1b,c,f) led to an
increased reaction selectivity, with yields as high as 25% in the
case of the 9-demethyl RPSB (1h). In contrast, addition of
methyl-groups to other CC bonds (1g,k,m) activated
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Figure 4. Kinetics and isomerization yields for backbone-modified RPSB derivatives in solution. (A) Excited state lifetimes and (B) QY for
photoproduct formation. The values for bR are taken from refs 21 and 22. (C) Structures of the (de)methylated all-trans isomers and changes to
isomerization yields. Backbone modifications are shown in pink. Isomerizing bonds are colored according to the graph in B. “+” or “−” signs refer to
the change in quantum yield for the corresponding double bond with respect to 1a.
isomerization channels previously silent in the natural backbone
1a. Most notably, these included the activation of the 7,8 CC
isomerization upon methylation of the 8-position (1g), and the
activation of the 13,14 CC channel upon methylation of the
14- and 15- positions (1k and 1m, respectively). The latter is
the only channel active in the isomerization of retinal in the
bacteriorhodopsin proton pump, and this photoisomerization
channel is undetectable in our measurements of natural nBu
RPSB after prolonged irradiation (1a) (see the Supporting
Information, Figure SF18). The changes in isomerization yield,
illustrated as ‘+’ for increasing or opening a photoisomerization
channel, and ‘−’ for decreasing or closing a pathway upon
(de)methylation of the backbone, are summarized in Figure
4C.
the single bond character of CC bonds, and vice versa. This
dramatic change in equilibrium bond lengths upon excitation is
well reflected in the ground-state preresonance Raman spectra
of RPSB that are dominated by C−C and CC stretching
coordinates,77 as expected for strongly displaced modes (Figure
5A).78 Replacing the nBu moiety with an aromatic one has only
a negligible effect on the appearance of the Raman spectrum,
except for a splitting of the CC stretching band (1500 cm−1
region) caused by symmetric and asymmetric combination of
the backbone stretching with the Schiff base stretching modes
(Figure 5A). Calculations of the MK charges for the optimized
ground-state geometry (Figure 5B) and charge differences at
the same point between first excited state and ground state
(Figure 5C) show remarkable similarity independent of the
Schiff base character.
These observations suggest that the nature of the excited
electronic state in the Franck−Condon region, the character of
the initial charge translocation, and the resulting changes in
bond lengths are very similar irrespective of the nature of the
Schiff base. The dramatic suppression of photochemical
reactivity must therefore have its origin outside the Franck−
Condon region. A possibility is that the addition of the
aromatic amine not only serves to extend the π-system but also
to provide an alternative relaxation channel such as a twisted
intramolecular charge transfer (TICT)79 state, from which the
system can decay efficiently without backbone isomerization.
An alternative, but useful, interpretation focuses on the
conversion of photon into nuclear kinetic energy in the form of
vibrational excitations. The latter are critical in determining the
rate constants for the various relaxation channels and thus
branching ratios for ultrafast decay processes that take place on
time scales faster or comparable to vibrational relaxation times.
DISCUSSION
■
Aromatic Amines. Extension of the RPSB π-system by
addition of an aromatic unit as the amine moiety (3−5) has
two major effects on the absorption spectra, red-shifting the
absorption maximum by about 80 nm and allowing for limited
tunability of the latter in the 520 nm region. This bathochromic
shift is accompanied by changes in the ESL with no apparent
trend. The presence of electron-donating groups (4, 5) reduces
the ESL, while it increases in their absence (3) with respect to
natural RPSB (1a). Independent of the effect on the ESL, all
aromatic Schiff bases are completely unreactive.
To understand the origin of the complete lack of
photochemical activity, it is useful to consider the change in
the distribution of the positive charge upon excitation from the
ground state to the first excited singlet state.75,76 For the natural
nBu RPSB, a partial translocation of the positive charge is
observed predominantly toward the β-ionone ring increasing
E
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in the protein pocket could play a similar role to the aromatic
amines used in this work. However, in the protein the effect is
likely opposite, pushing the charge even more toward the
retinal backbone compared to a simple alkane as in natural nBu
RPSB (1a). We attempted to test this hypothesis by preparing
RPSBs using amines with electron-withdrawing character.
Imine formation using aromatic amines bearing strongly
electron-withdrawing groups proved difficult under a variety
of reaction conditions. We found that imine formation was
incomplete, and the products were susceptible to hydrolysis.
Attempts to prepare these compounds are ongoing and provide
exciting prospects for future work. Overall, controlling the
direction of charge translocation and thus nuclear energy flow is
a potential control mechanism for photochemical processes.
Backbone Modification. Although Schiff base modifica-
tion allowed us to completely halt the photoisomerization of
RPSB in solution, we could not investigate the molecular origin
of photochemical reactivity and selectivity. Photoisomerization
of natural all-trans nBu RPSB (1a) in solution produces a
mixture of 11-cis and 9-cis RPSBs, with the former being
dominant. In contrast, the same process in bR produces
exclusively the 13-cis isomer with a 3-fold higher overall yield.
Comparison of parts A and B of Figure 4 shows a clear
correlation between ESL and isomerization yield. In contrast to
the impression created from the comparison of RPSB dynamics
in proteins (bR) and in solution, which suggests that reaction
speed is a prerequisite for efficient photochemistry, these
results clearly demonstrate an opposite trend.
Figure 5. Electronic character in the Franck−Condon region is
unchanged upon substitution of an aromatic Schiff base. (A)
Preresonance Raman spectra of natural nBu RPSB (1a) and p-
methoxyaniline RPSB (4). (B) MK charges for the ground electronic
state. (C) Difference in MK charges between ground and first excited
state computed for different protonated Schiff bases.
In addition, the observed specific activation and deactivation
of isomerization channels depended on the position of
backbone substitutions. Within the range of species studied
here, we observed a wide variety of selectivity profiles and have
induced isomerization for all backbone CC double bonds. In
some cases, we observed almost complete specificity (for
example, 10-methyl RPSB (1b) and 9-demethyl RPSB (1h)); in
other cases, the overall quantum yield approaches that observed
in the bR protein environment (0.55 for 1i vs 0.65 for bR). The
observed trends qualitatively agree with a simple model based
on polarizability, charge stabilization, and concomitant effects
Charge translocation necessarily causes dramatic changes in
equilibrium bond lengths, which in turn causes the deposition
of photon energy in nuclear coordinates that are structurally
coupled to the movement of the charge. Our results suggest
that the direction of energy flow is critical in determining the
photochemical reactivity of retinal. If that energy flow can be
influenced by electrostatic effects, charged amino acid residues
Figure 6. Modification of excited-state barrier heights as a mechanism for tuning RPSB photochemistry. (A) General effect on the addition of
polarizable groups to the retinal backbone on excited-state barriers and location of the surface crossing. (B) Effect of adding a methyl group to the
14-position on the barriers toward excited-state decay using distortions toward the 7,9,11,13-cis products (green, orange, blue and red, respectively).
(C). Structure for all-trans 14-methyl RPSB (1k) and the possible photoproducts. The isomerizing bonds are colored. (D) Multidimensional
representation of the excited-state PES indicating all major decay pathways with differing barrier heights. SP: Stationary Point; CI: Conical
Intersection.
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on barrier heights resulting in different branching ratios (Figure
6).
even though reducing the competitivity of the channel, leads to
a later CI which increases the quantum yield for the naturally
active pathways (9-cis and 11-cis).
The general trend in ESL suggests a simplified potential
energy surface as shown in Figure 6A: addition of polarizable
groups decreases the ESL and vice versa. The effects of change
in substituent polarizability are shown as dashed lines, using
solid lines to qualitatively depict the situation in natural nBu
RPSB (1a). Given that the lifetime is likely controlled by a
barrier in the excited electronic state toward internal
conversion,2,15,19,80−83 it is reasonable to assume that the effect
of the polarizable substituents is to lower that barrier. The
correlation between ESL and isomerization yield suggests this
to be a consequence of the barrier lowering. This observation is
in line with isomerization coordinates at least partially
contributing to excited state decay and a lower barrier leading
to an earlier crossing along the isomerization coordinate, which
in turn results in lower isomerization yield. Such early crossing
agrees with experiments on bacteriorhodopsin that showed how
the initial stationary point reached in the excited electronic
state, after relaxation out of the Franck−Condon region,
corresponds to only a little evolution along the isomerization
coordinate.84 Additional support for this hypothesis has been
recently provided by high-level quantum chemical calculations
that observed the existence of a barrier on the excited electronic
state on the path toward the conical intersection (CI).15,83
Furthermore, the connection between internal conversion and
isomerization coordinate is in agreement with the symmetries
of any vibrational modes coupling the two electronic states of
different symmetry, i.e., a requirement for nontotally symmetric
modes, such as backbone torsions and hydrogen wags. Models
that involve excited-state decay by distortion of the backbone
require that, at least in principle, all CC bonds can mediate
internal conversion and thus appear as potential photoproducts.
The product distribution would then depend on the relative
barrier heights and crossing points and could therefore be
altered from the naturally occurring pathways (9-cis and 11-cis)
by changing the barrier heights by backbone substitution.
As an example, consider 14-methyl RPSB (1k), a substitution
that results in the complete suppression of the 9-cis channel,
reduction of 11-cis yield, and activation of the 13-cis channel
(Figure 6B). All of these observations are in line with the
simple model introduced above. Methylation in general causes
all barriers to drop, lowering the quantum yields of the naturally
occurring channels (Figure 6A). In the case of 14-methyl RPSB
(1k), 9-cis as a product becomes undetectable (yellow, Figure
6B), while the 11-cis yield drops from 0.16 to 0.11 (blue). For
both the 9-cis and the 11-cis channels, the lowering of the
excited state barrier leads to faster decay, but a lower yield as
the surface crossing occurs earlier along the isomerization
coordinate. At the same time, the 13-cis channel is activated
(QY of 0.03) as a result of lowering of the barrier along the 13-
cis isomerization channel sufficiently to generate a detectable
amount of photoproduct (red channel). The C7C8 double
bond, however, being too distant from the point of substitution,
experiences only negligible changes to the excited state barrier
height, remaining inactive. We remark that exactly the same line
of argument can be used to rationalize all changes in QYs
observed in this work. 8-Methyl RPSB (1g), for example, acts in
the same way as 14-methyl RPSB (1k), except that now the
nearby C7C8 double bond is activated while the 9-cis product
becomes undetectable and 11-cis drops. Removal of methyl
groups, instead, raises the barriers for all inactive channels to
such a degree that they remain inactive, while a higher barrier,
To visualize such a highly dimensional PES, it is convenient
to project the individual isomerization coordinates onto
nonorthogonal dimensions (Figure 6C,D). These coordinates
do not represent normal modes of the molecules but simply the
definition of the PES in degrees of freedom related to a
backbone isomerization. Although it may appear counter-
intuitive, in this representation, the system evolves along all
coordinates simultaneously from the stationary point without
requiring a bifurcation of the potential energy surface near the
Franck−Condon region. Taken together, all of these evolutions
represent the overall structural change taking place during
possible isomerization reactions, as opposed to the more
common backbone stretching or torsional degrees of freedom.
At this stage, the barrier heights determine the possible decay
rates as well as the likelihood of isomerization through the
location of the crossing point as discussed for 14-methyl case
(1k) above (Figure 6B).
Although such a model suggests multiexponential decay with
component amplitudes related to the isomerization yield for
each particular decay channel, their experimental observation
by transient absorption spectroscopy is likely to be challenging.
Although in principle additional, lower amplitude components
could be added, features such as vibrational cooling make the
quantification of more than three decay constants challenging
as addition of more parameters tends to improve the fit so that
determining a clear cut-off point is difficult. Further testing of
the proposed model would thus require time-resolved vibra-
tional spectroscopy with high sensitivity, where the growth of
individual photoproducts can be observed and differentiated.
A conceptually simple, but challenging (in terms of thermal
stability of the RPSB), method to obtaining high QY would be
to incorporate a strongly electron-withdrawing Schiff base that
pushes the charge toward the retinal backbone maximizing the
activation of backbone-twisting decay channels and thus the
isomerization yield. Combining such efforts with backbone
modifications such as a 9-demethyl or 10-demethyl RPSB
(1h,b, respectively) that show remarkable selectivity provides
scope for the artificial design of materials with high isomer-
ization yields and specificity to an extent that was thought to
exist only in an evolution-optimized, three-dimensional protein
environment.
The correlation between ESL, QY, and substituent polar-
izability suggests an electronic origin of the excited-state barrier.
As noted by others85 numerous polarizable aromatic residues
are present in the protein pocket and recent spectroscopic37
and computational36 studies have described color tuning by
modulation of the electrostatic environment. Our results
demonstrate that it is possible to control the yield, speed and
selectivity by addition or removal of polarizable groups along
the backbone, or appropriate choice of the amine for Schiff base
formation. In the case of bR and opsin proteins, which exhibit
exquisite control over all of these factors, it remains unclear
how to reproduce the degree of selectivity observed in nature −
but our results point toward a mechanism in which nonbonded
interactions between the retinal chromophore and amino acid
residues in the protein pocket can be used to provide complete
control of retinal photochemistry. Testing the validity of this
hypothesis and unraveling the details of these isomerization
mechanisms offers considerable prospects for future work and
our eventual understanding of these processes.
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Article
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ASSOCIATED CONTENT
* Supporting Information
Synthetic procedure and characterization data for all com-
pounds and details of the analysis on the transient absorption
data, QY measurement, and MK charge analysis. This material
is available free of charge via the Internet at http://pubs.acs.org
■
S
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AUTHOR INFORMATION
Corresponding Authors
philipp.kukura@chem.ox.ac.uk
stephen.fletcher@chem.ox.ac.uk
■
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Giulio Cerullo, Eberhard Riedle, and
Nicolaus Ernsting and their research groups for assistance in
the construction of the ultrafast setup and to Christopher D.P.
́
Duffy and Vincent Liegeois for helpful discussions. We
acknowledge the use of the University of Oxford Advanced
Research Computing (ARC) facility in carrying out this work.
The EPSRC supports P.K. and S.P.F. by Career Acceleration
Fellowships (EP/H003541/1 and EP/H003711/1) and this
research project through standard grant EP/K006630/1
awarded to P.K. and S.P.F.
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